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1st FEMS Congress / Posters 103^505

P1^1 IDENTIFICATION OF A NOVEL LACTOCOCCAL BACTERIOCIN FROM A L. LACTIS SUBSP. LACTIS STRAIN M. Akcelik(1), C. Tukel(2) ° « ° (1) Ankara University, Faculty of Science, Department of fl Biology, Tandogan, 06100, Ankara, Turkey ; (2) Ankara University, Faculty of Agriculture, Department of Food Engineering, D|s°kap|, 06110, Ankara, Turkey 40 di¡erent L. lactis strains were isolated from traditional fermented milk products and screened for their antimicrobial activities. Eight of the isolates showed inhibitory e¡ect against di¡erent indicator bacteria and identi¢ed as L. lactis subsp. lactis. Nisin was characterized in three and lacticin 481 was characterized in four of eight bacteriocinproducing isolates by PCR-techniques. A broad inhibitory spectrum bacteriocin produced by one of the L. lactis subsp. lactis strains didn't show any similarity between any known lactococcal bacteriocins. The production was associated with a 50 kb plasmid. P1^2 BIOGENIC AMINES PRODUCTION BY LACTOBACILLI AND STAPHYLOCOCCI ISOLATED FROM FERMENTED SAUSAGES A. Storti, C. Tutta, C. Andrighetto, G. Marcazzan, A. Lombardi ' Veneto Agricoltura Istituto per la Qualita e le Tecnologie Agroalimentari, via San Gaetano 74, 36016 Thiene, Vicenza, Italy In several ripened or fermented foods biogenic amines, such as hystamine, tyramine, phenyletilamine and triptamine may be present; according to their concentration, to the particular product composition and to the individual health response may cause tossic e¡ect of di¡erent intensity to the human body. Biogenic amines production in fermented sausages is often related to the decarboxylating activity of lactic acid bacteria that play an important role in meat fermentation and ripening processes. To control biogenic amines in fermented meat products, it is very important to have information about the microorganisms that during may contribute to the synthesis or degradation

of biogenic amines. The aim of this work was to study biogenic amines production by lactic acid bacteria and coagulase negative staphylococci isolated from fermented sausages. These strains were evaluated for their ability to produce tyramine, hystamine, cadaverine and putrescine in a di¡erential plating medium containing the corresponding aminoacid. A total of 73 lactobacilli and 23 coagulasenegative staphylococci isolated from traditional fermented meat products were screened for biogenic amine production. Among lactobacilli 8 strains identi¢ed as Lactobacillus curvatus displayed decarboxylase activity (4 strains produced putrescine and 4 putrescine and tyramine); regarding staphylococci, 2 strains identi¢ed as Staphylococcus xylosus were able to produce hystamine and putrescine and 1 strain belonging to the species Staphylococcus carnosus produced hystamine, putrescine and cadaverine. Work is now in progress in order to evaluate the ability of strains of Staphylococcus xylosus to degrade biogenic amines. Strains exhibiting such activity might have a potential to prevent an accumulation of biogenic amines during sausage fermentation. P1^3 GROWTH OF YEAST STRAINS DURING BATCH PRODUCTION OF SINGLE-CELL PROTEIN FROM CHEESE WHEY L ¤ ¤ ¤ B. Asvanyi(1), G. Bugyi(2), L. Daroczi(3), R. Kovacs(1), J. Szigeti(1) and L. Varga(1) (1) University of West Hungary, Faculty of Agricultural and Food Sciences, Institute of Food Science, 15-17 Luc¤ ¤ sony Street, 9200 Mosonmagyarovar, Hungary ; (2) Mik¤ ¤¤ roauto Inc., Cegled, Hungary; (3) Y-Food Inc., Berettyoujfalu, Hungary The objective of this research was to evaluate the suitability of various Kluyveromyces species for use in single-cell protein (SCP) production. The maximum viable cell counts reached by selected strains of K. fragilis, K. lactis, and K. marxianus during batch production of SCP were determined. The Kluyveromyces strains were grown in unfractionated, heat-treated cheese whey, which had a lactose content of 4.5%, thereby providing optimum growth conditions for yeasts. Fermentations were run batchwise in an automated BIOFLO III0 batch/continuous fermenter under identical conditions with respect to pH, aeration, and agitation rate. The parameters set were computer-controlled using Advanced Fermentation Software version

0378-1097 ß 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.

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3.42. Samples were taken at regular preset intervals, their viable cell counts were determined by the pour-plate technique, and the strains were then ranked in order of growth rate. By reaching the highest maximum cell counts of all the Kluyveromyces strains tested within 24 h of fermentation, K. marxianus NCAIM Y.00933 proved to be the strain most suitable for SCP production. An economical technology for the large-scale production of SCP is still to be developed. The use of SCP for animal nutrition might be an alternative to the traditional uses of cheese whey. P1^4 ISOLATION OF MICROORGANISMS FROM LOOGPAENG FOR RICE KOJI PREPARATION S. Attrapadung(1), S. Bovonsombut(2), A. Plikomol(1) and S. Bovonsombut(1) (1) Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand; (2) Department of Food Technology, Faculty of Engineering and Agro-Industry, Maejo University, Chiang Mai 50290, Thailand For improving the quality of Thai rice wine, A number of loog-paeng (Thai traditional rice wine starter) was collected from 8 di¡erent areas in the northern part of Thailand for isolation of useful microorganisms. These isolates were 67 fungi , 55 yeasts and 37 bacteria. Rhizopus spp., Bacillus spp. and Saccharomyces spp. were dominant species of fungi, bacteria and yeast respectively.The types of microorganism were not the same pattern from one location to another according to the formulation of loogpaeng. Whilst the amount of these ones decrease along with the age of loog-paeng. The isolates were tested for amylolytic activity by using starch agar medium. The result showed 83 isolates were able to produce clear zone. Among them, 30 isolates which made a large clear zone were selected for determining amylase activity and some quality attributes on rice koji. P1^5 ANTIMUTAGENICITY OF PROBIOTIC BACTERIUM ENTEROCOCCUS FAECIUM M-74 ¤ ¤ > A. Belicova, L. Krizkova, J. Dobias, L. Ebringer, J. Kraj> > covic ¤ Institute of Cell Biology, Comenius University, Odborarske ¤ nam. 5, 811 07 Bratislava, Slovak Republic Live and killed cells of probiotic bacterium Enterococcus faecium M-74 grown in the presence and absence of sodium selenite pentahydrate (Se) as well as the MRS media with selenium (+Se) and MRS media without selenium (-

Se) extracts and the diethyl ether extracts from unfermented milk and milk fermented by E. faecium M-74 were prepared and their possible protective e¡ects on the mutagenicity of selected mutagens in Salmonella and Euglena gracilis assays were studied. MRS media extract (+Se) after cultivation of E. faecium M-74 showed a signi¢cant higher antimutagenic activity in reducing genotoxicity of o£oxacin, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 5-nitro-2-furyl acrylic acid (NFA) than MRS (-Se) extract in Salmonella Typhimurium TA98, TA100 and TA102. The live cells of the probiotic strain M-74 showed higher antimutagenic activity in inhibiting the mutagens [CM1]than the killed bacterial cells. However, the live bacterial cells grown in the presence of Se showed signi¢cantly higher antimutagenic activity. The diethyl ether extracts isolated from unfermented milk and milk fermented by E. faecium M-74 exhibited a signi¢cant dose-dependent protective e¡ect against MNNG-, nitrovin- (NIT), NFA- and ultraviolet (UV) irradiation-induced mutagenicity on the S. Typhimurium TA97, TA100 and E. gracilis. Overall, the fermented milk extract was the most active against UV irradiation, less active against NIT and MNNG, and the least active against NFA on bacteria. The highest antibleaching e¡ects were observed against MNNG on E. gracilis. The di¡erences between antimutagenic e¡ects from fermented and unfermented milk extracts were statistically signi¢cant. P1^6 IMPORTANCE OF NUTRITION CONTROL OF SACCHAROMYCES CEREVISIAE IN WINEMAKING S. Belviso(1), L. Bardi(2), A. B. Bartolini(3), M. Marzona(1) (1) Dept. Applied General and Organic Chemistry, University of Turin, Via Pietro Giuria 7, 10125 Turin, Italy ; (2) Plant Nutrition Experimental Institute, Via Pianezza 115, 10151 Turin, Italy ; (3) INTEC S.r.l., Via Monti Berici 4, S.Giovanni Lupatoto (Verone), Italy It is well known that sluggish and stuck fermentations are very important problems in winemaking. To prevent them correct yeast nutrition gestion and careful biological processes control during fermentation are required. Among biological processes linked to alcoholic fermentation, lipid metabolism is little studied. Sometimes happens that grape must is a low lipid medium and, as a consequence, during fermentation lipid biosynthesis is activated. Since anaerobiosis condition is present, UFA and sterol biosynthesis which are considered among lipids ``surviving factors'' for Saccharomyces cerevisiae is not possible. The lack of these components can cause the arrest of cell multiplication and then of sugar consumption resulting in stuck fermentation. In our laboratory we carried out fermentations without

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any nutritional apport, fermentations where a lipid source in form of inactivated yeast was added as nutritional apport (Saccharomyces cerevisiae can include fatty acids and sterol from the growth medium in cell membranes), and fermentations where de¢ned volumes of oxygen were added (UFA and sterol biosynthesis need molecular oxygen). In the two last series of fermentations more additions were carried out at established values of sugar concentration in growth medium. We checked: growth kinetics, sugar consumption, yeast cell composition, acetic acid production as symptom of lipid synthesis stress and nitrogen consumption to assure that it was never a limiting growth factor. Results con¢rmed that it was the lack of lipid compounds to cause fermentation arrest. Additions of inactivated yeast and oxygen, in fact, improved fermentation performances. These e¡ects can be very important for winemakers in order to prevent sluggish and stuck fermentations. P1^7 LACTOBACILLUS GASSERI LF221 AND K7 PREVENT LATE BLOWING OF PROBIOTIC CHEESE > >¤ > B. Perko, B. Bogovic Matijasic, R. Marinsek-Logar and I. Rogelj University of Ljubljana, Biotechnical Faculty, Chair of > Dairy Science, Groblje 3, 1230 Domzale, Slovenia Among di¡erent media which have been tested as carriers for probiotic bacteria, cheese has appeared as an attractive one. Human isolates Lactobacillus gasseri K7 and LF221 produce bacteriocins which inhibit several Clostridium tyrobutyricum strains, therefore we tested their ability to prevent late blowing of cheese used as their carrier. A mixture of C. tyrobutyricum 1551 and 1559 spores (2.3Á103/ml) or a silage juice (10 spores/ml) and derivatives of L. gasseri LF221 and K7 strains resistant to rifampicin (107 cfu/ml) were added to cheese milk in di¡erent combinations. Cheese samples were aseptically collected every week, and analysed for n-butyric acid, cfu/g of lactobacilli (Rogosa), clostridia spores (RCM), LF221 and K7 strains (Rogosa with 250 Wg/ml rifampicin). During ripening the level of added probiotics in general remained the same or was slightly increased (¢nal cfu/g was 3.5Á108 ^ 1,1Á109). While in cheeses without probiotics, the number of nonstarter lactobacilli increased up to 6.5Á106 ^ 2.5Á107 cfu/g, LF221 and K7 strains prevailed in cheeses where added. The identity of the colonies grown on agar with rifampicin was con¢rmed by RAPD analysis. The visual inspection and the values of n-butyric acid content in cheese with added clostridia spores (849 mg/kg in cheese with silage juice and 2185 mg/kg in cheese with spores from pure cultures) clearly showed the late-blowing of cheeses without probiotics. On the other hand, the n-butyric acid con-

tent in the cheeses with added probiotic strains, was obviously lower from those of cheeses without probiotics. P1^8 STUDY OF ARGININE DEGRADATION BY LACTIC ACID BACTERIA IN RELATION WITH POSSIBLE APPEARANCE OF ETHYL CARBAMATE IN WINES ¤ A. Bordons, J. Gil, I. Araque, S. Romero, M. C. Masque, ¤ C. Reguant and R. Carrete ¤ Departament de Bioqu|mica i Biotecnologia, CeRTA, Universitat Rovira i Virgili, Tarragona, Catalonia, Spain Ethyl carbamate (EC) is a potential carcinogenic compound sometimes found in wines. EC can appear by reaction of ethanol, at the acid pH of wine, with urea produced by yeasts, or with citrulline or carbamyl phosphate, both produced by lactic acid bacteria (LAB) from arginine. On the other hand, wine LAB such as Oenococcus oeni are well known by the malolactic fermentation (MLF) they carry out. The main positive e¡ects of MLF are the reduction of wine acidity (conversion of l-malic to l-lactic acid) and the modi¢cation of £avor properties. Complete degradation of arginine by LAB occurs via the ADI pathway, leading to ammonia, ornithine, ATP and CO2, but several strains do not degrade it completely and can produce citrulline or carbamyl phosphate. In order to minimize the appearance of EC in wines, we have studied ¢rstly the ability for degrading arginine in a lot of strains of di¡erent species of LAB wines. We have found di¡erent responses in Oenococcus depending on the strain, and that some homofermentative Pediococcus could degrade arginine. Activities of enzymes of the ADI pathway (arginine deiminase [ADI], ornithine transcarbamylase [OTC] and carbamate kinase [CK]) have been measured in cells extracts of di¡erent strains and species. On the other hand, in order to detect the genes of ADI pathway in strains of O. oeni, speci¢c PCR primers have been designed for each gene (ADI, OTC and CK) and have been tested in several strains.

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P1^9 RAPID ASSESSMENT OF PHYSIOLOGICAL STATE OF OENOCOCCUS OENI BY FLOW CYTOMETRY DURING MALOLACTIC FERMENTATION M. Bouix, S. Ghorbal, J. L. Tholozan, J. Y. Leveau ¤ Dept of Industrial Microbiology, Ecole Nationale Superieure des Industries Agricoles et Alimentaires, Massy, France We proposed a simple £ow cytometric method to evaluate the intracellular pH (pHi) and the membrane potential of the cells of Oenococcus oeni during the malolactic fermentation. At the begining of the malolactic fermentation, the pHi of cells was low and equal to the external pH of the medium, and the membrane potential was weak. During the growth, the pHi raised to a maximum value of 6 whatever the external pH (3 or 5), and the membrane potential also raised. When the malic acid was exhausted of the medium, the pHi dropped down to the external pH value, and the cells appeared depolarized. The assessment of these parameters let to understand the cellular physiology of Oenococcus oeni and then to better get under control the malolactic fermentation in the wines. P1^10 THE TECHNOLOGICAL ACCEPTABILITY OF ENTEROCOCCI ISOLATED FROM CHEESES ¤ > ¤ S. Bulajic, Z. Mijacevic Faculty of Veterinary Medicine, University of Belgrade, 11 000 Belgrade, Serbia Enterococci are part of the commensal microbiota of animals and humans. In the area of food microbiology they have been considered as indicators of fecal contamination. Also, they have been regarded as index microorganisms for unhygienic food processing. Additionaly, studies on the micro£ora of traditional cheeses in Mediterranean contries have clearly indicated that enterococci strains display diversity enzymatic activities that may have desirable role in the process of ripening and contribute to typical taste and £avour. However, their presence in food system is still a matter of controversy owing to their pathogenic potential and also potent ability to exchange genetic material comprimases the gene pool of antibiotic resistance. In this study, enterococci strains isolated from cheeses originated from our local market were subjected to technological characterisation regarding their proteolytic activity. Proteolytic pro¢les of investigated strains were examined by electrophoresis. On the basis of our results we

were conducted the selection of the most appropriate cultures to be used for the enchancment of the organoleptic properties of cheese. P1^11 INFLUENCE OF YEAST ON POLYPHENOL COMPOSITION OF WINE A. Caridi(1), A. Cufari(1), R. Lovino(2), R. Palumbo(3) and I. Tedesco(3) (1) Dipartimento di Scienze e Tecnologie Agro-Forestali e Ambientali, Reggio Calabria University, Piazza San Francesco 7, I-89061 Gallina (RC), Italy; (2) Istituto Sperimentale per l'Enologia, Sezione Operativa di Barletta, Via Vittorio Veneto 26, I-70051 Barletta (BA), Italy ; (3) Istituto di Scienze dell'Alimentazione, Consiglio Nazionale delle Ricerche, Via Roma 52A/C, I-83100 Avellino (AV), Italy In red wine production, the type and quantity of polyphenols play a major role in wine quality. Anthocyanins, £avonols, catechins and other £avonoids contribute to the di¡erent characteristics of wine, particularly color and astringency; in addition, recently it has been shown that they possess a wide range of antioxidant and pharmacological e¡ects. The objective of this research was to examine the in£uence of yeast used for winemaking on the type and quantity of phenolic compounds, elucidating possible relationships with the antioxidant capacity of wine. Two strains of Saccharomyces, previously selected for enology and for their di¡erent interaction with grape polyphenols, were employed. A sample of Gaglioppo must from Calabrian black grapes was utilized. This variety was employed because it has limited anthocyan content so it requires careful handling to protect the phenolic compounds. Winemakings were carried out in triplicate using stainless steel vessels of 600 l, containing about 400 l of must with grape skins and seeds. Twenty-seven physicochemical and polyphenolic parameters were determined in the red wines produced. Among the polyphenolic parameters, very signi¢cant (p 6 0.01) di¡erences were observed for color intensity, total polyphenols, and non-anthocyanic £avonoids. Moreover, signi¢cant (p 6 0.05) di¡erences were observed for OD520 and monomeric anthocyans. This research elucidates for the ¢rst time how yeasts interact with speci¢c polyphenols, so varying the wine concentration of these compounds. This behavior modi¢es signi¢cantly the physicochemical and antioxidant characteristics of wines and, probably, also the sensorial and volatile constituents.

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P1^12 COMPARATIVE EFFECTS OF FEEDING CONTAINING FLAVOMYCIN, BIOTEKSIN-L AND DRY YEAST (SACCHAROMYCES CEREVISIAE ) ON BROILER PERFORMANCE M. Denli(1), K. Celik(2), F. Okan(1) ° (1) Cukurova University Animal Sci. Dept., 01330 Adana, ° Turkey ; (2) Canakkale Onsekiz Mart University, Animal ° Sci. Dept., Canakkale 17100, Turkey ° A 6-week study was conducted to determine the e¡ects of feeding diets containing commercial probiotic (BiyoteksinL), antibiotic (Flavomycin) and dry yeast (Saccharomyces cerevisiae) upon would a¡ect performance ,abdominal fat weight, carcass weight and carcass yield of broiler chicks. Four dietary treatments were randomly assigned to four groups of chicks. A control or containing 0,15% commercial probiotic (bioteksin-L), 0,15% antibiotic,(£avomycin) and 0,3 % dry yeast. A signi¢cant increase in body weight gain, feed conversion rate and carcass weight of birds was observed in birds fed £avomycin group and dry yeast group in end of 6-wk period compaired to the control (P 6 0.05).This increase was partly accounted for by increased feed intake. The results obtained in the experiment showed that Biyoteksin-L and Saccharomyces cerevisiae plus supplementation to diets tended to decrease, abdominal fat weight and abdominal fat percentage (P 6 0.5).while having no signi¢cant e¡ects on carcass yield. P1^13 PURIFICATION AND PARTIAL CHARACTERIZATION OF A NOVEL BACTERIOCIN PRODUCED BY A THERMOPHILIC ENDOSPORE-FORMING STRAIN GEOBACILLUS STEAROTHERMOPHILUS 32A K. Pokusajeva, M. Stuknyte, N. Kuisiene, D. Chitavichius Department of Microbiology and Plant Physiology, Vilnius University, Chiurlionio 21/27, Vilnius, LT-2009, Lithuania Aerobic, endospore-forming thermophilic strain Geobacillus stearothermophilus 32A was identi¢ed as a two bacteriocins producer with a bactericidal activity against Geobacillus subterraneus DSM 13552, G. uzenensis DSM 13551, G. thermocatenulatus DSM 730, and G. thermoleovorans DSM 5366. These bacteriocins are produced during log-phase growth and are inhibitory to active growing cells. The antimicrobial activities of these substances di¡er. Twofold dillution tests showed that the activity of one of the bacteriocins appeared later after another one disap-

peared. The antimicrobial activity of the bacteriocins on the sensitive indicatory cells disappeared completely by treatment with proteinase K, which indicates its proteinaceous nature. Bactericidal activity was kept during storage at 4C and was remarkably stable in the wide range of pH. In SDS-PAGE analysis, only two peptide bands displayed antimicrobial activity against thermophilic indicatory strain 35C. The inhibitory peptides had a molecular weight of approximately 12 and 6.8 kDa. Results of this study suggest that these antimicrobial substances produced by the wild type strain of Geobacillus stearothermophilus 32A may be the new bacteriocins. P1^14 PHENOTYPIC, GENOTYPIC AND TECHNOLOGICAL CHARACTERISTICS OF LACTOCOCCI ISOLATED FROM TRADITIONAL FIORE SARDO CHEESE S. Cosentino, M. B. Pisano, C. Piras and A. Corda Department of Experimental Biology, Section of Hygiene, University of Cagliari, S.S. 554, Km. 4,500, 09042 Monserrato (CA), Italy The evolution and composition of dairy micro£ora is of particular interest for PDO cheeses such as Fiore Sardo, that is manufactured with raw ewe's milk without the addition of any natural or starter cultures. In this case, the ripening process relies entirely on the indigenous £ora present in the milk and in the dairy environment. Studies carried out on the micro£ora of Fiore Sardo have shown lactococci and enterococci to be the dominant bacterial population. This paper reports the phenotypic, genotypic and technological characterization of lactococcal strains isolated from 14 batches of artisanal Fiore Sardo cheese, in order to assess the biodiversity within this wild microbial population. Lactococci were isolated from M17 agar plates incubated at 30C and were initially identi¢ed by morphological, physiological and biochemical tests. This identi¢cation was con¢rmed by a polymerase chain reaction analysis with Lactococcus genus specii¢c primers LC1 and LC2. Randomly ampli¢ed polymorphic (RAPD) DNA technique was used for the genetic typing of the isolates. A total of 80 isolates were identi¢ed as Lactococcus lactis. Most strains were able to hydrolyse casein, none produced lipolytic reactions on tributyrin agar and several were good acid-producers. The RAPD patterns from 50 representative strains were analysed by UPGMA dendograms. At a similarity level of 50% two main clusters with several subclusters were distinguished, showing a high heterogeneity in the biotypes. The results of our survey con¢rm that wild bacterial population should be preserved to protect the traditional raw milk cheeses and to select new starter strains for the dairy industry.

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This work was supported by a grant from MURST, Plan ``Agroalimentary products: dairy products'', Cluster 08B, Project n.7. P1^15

P1^16 CULTIVATION OF KOMBUCHA ON SWEETENED ECHINACEA TEA D. Cvetkovic, J. Canadanovic-Brunet, S. Markov

INFLUENCE OF THREE BOTRYTICIDES ON WINE FERMENTATION DYNAMICS AND YEAST POPULATION BIODIVERSITY í > í >> > F. Cus(1), A. Amalietti(2), N. Cadez(2), A. Gregorcic(3) and P. Raspor(2) (1) University of Ljubljana, Biotechnical faculty, Agronomy Department, Chair of Viticulture, Jamnikarjeva 101, 1000 Ljubljana, Slovenia; (2) University of Ljubljana, Biotechnical faculty, Food Science and Technology Department, Chair of Biotechnology, Jamnikarjeva 101, 1000 Ljubljana, Slovenia; (3) Agriculture Institute of Slovenia, Hacquetova 17, 1000 Ljubljana, Slovenia Wine fermentation depends on many viticulture and enological practices. One of them, not yet clearly understood, is a usage of botrtryticides in vineyard and in£uence of their residues on fermentation dynamics and yeast population biodiversity. In our research, three botryticides of di¡erent chemical groups were used: iprodione, pyrimethanil and cyprodinil plus £udioxonil. The grape of the cultivar Rebula (Vitis vinifera L.) was sprayed at the closure of the berries and at the beginning of the grape ripening. At the harvest, the botryticides residues on the grape were below prescribed tolerance measured with a combination of gas chromatography and mass spectroscopy. To preserve indigenous microbial populations grape processing was performed aseptically in three parallels for each treatment. No sul¢te and wine yeasts were added. The must composition during the fermentations was analyzed by HPLC. The yeast population dynamics was followed by colony counting, colony morphotyping and by molecular methods, electrophoretic karyotyping and PCRRFLP of the rDNA. There was a signi¢cant di¡erence in fermentation duration between the treatments: 36 days for the control and iprodione fermentations, 50 days for the cyprodinil plus £udioxonil fermentations and 68 days for pyrimethanil fermentations. The non-Saccharomyces yeasts persisted during the fermentations, with Candida stellata and, to lesser extent, Hanseniaspora uvarum remaining long after the fermentations were either dominated or not by Saccharomyces cerevisiae. This research was supported by the Ministry of Education Science and Sport and by the Ministry of Agriculture Forestry and Nutrition (Project no. V4-0591-01).

Faculty of Technology, University of Novi Sad, Boulevar Cara Lazara 1, 21 000 Novi Sad, Yugoslavia Kombucha is a beverage with special therapeutic properties produced by metabolic activity of yeasts (Schizosaccharomyces pombe, Saccharomyces ludwigii, Saccharomyces cerevisiae...) and acetic acid bacteria (Acetobacter xylinum, Acetobacter aceti, Gluconobacter oxydans...) in cultivation medium (sweetened black tea). The ability of black tea, as a traditional source of nitrogen compounds in cultivation medium, to be replaced with echinacea tea (radix and herba) was investigated. It is well known that Echinacea spp. have proved pharmacological properties. The use of echinacea tea in preparation of kombucha would produce beverage with increased therapeutic properties in comparing to traditional beverage produced with black tea. Fermentation process of sweetened echinacea tea was observed during ¢ve days of incubation by determination of total account of yeast cells and acetic acid bacteria, as well as chemical parameters. The investigations showed that fermentation of sweetened echinacea tea was very e/cient. Also, higher acidity was gained in comparing with traditional kombucha for the same incubation period. Antioxidative capacity of black and echinacea tea and their kombucha beverages was investigated by electron spin resonance (ESR) spectroscopy. Results showed that echinacea tea (especially tea prepared from herba of the plant) have signi¢cantlly higher antioxiadtive properties in model system then black tea. On the other hand, kombucha beverages, produced by fermentation of sweetened black and echinacea tea, showed an aditionally higher antioxidative capacity then the black and echinacea tea. This e¡ect was caused by compounds produced during kombucha culture fermentation and/or their synergestic e¡ect with extracted compounds from teas.

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P1^17 SOME ASPECTS ON THE ACTION AND APPLICATION OF BACTERIOCINS PRODUCED BY LACTOBACILLUS STRAINS ISOLATED FROM FERMENTING TABLE-OLIVES A. Delgado(1), D. Brito(2), M. Caeiro(3) and C. Peres(2) (1) ITQB, Oeiras, Portugal; (2) INIAP/EAN, Oeiras, Portugal; (3) Instituto Piaget, Almada, Portugal Two bacteriocin producers (Lactobacillus plantarum LB17.2b and Lactobacillus pentosus LBB96) were previously isolated from fermenting olive brines, of distinct technological procedures. A brief study on their bacteriocins revealed that the bacteriocin from L. pentosus LBB96 is apparently activated by heat (65 C to 100 C). It also seems to have a broad activity spectrum, including Enterococcus spp. strains that are multiresistant to antibiotics. The in£uence of technologically relevant factors from brine, such as oleuropein and di¡erent NaCl concentrations, was evaluated on growth, on bacteriocin production and on bacteriocin bioavailability. Oleuropein (0.4%) seemed to have a limited in£uence on growth or on bacteriocin production. NaCl enhanced the activity of both bacteriocins but a fall in bacteriocin production, by L. plantarum LB17.2b, was registered for 2% NaCl whereas the growth was not a¡ected. Bacteriocin production by L. pentosus LBB96 seemed to be stimulated by 2 to 4% NaCl. This last strain showed to be more salt-tolerant being able to grow and to produce bacteriocin in MRS broth with 8% NaCl and 0.4% oleuropein. This study supports the hypothesis that LAB bacteriocins can be active in complex food systems and some of them may also have health-care applications. P1^18 ISOLATION, IDENTIFICATION AND CHARACTERISATION OF GLYCEROL-DEGRADING LACTIC ACID BACTERIA FROM SOUTH AFRICAN RED WINES S. J. Krieling, I. S. Pretorius and M. du Toit Institute for Wine Biotechnology, Department of Viticulture and Oenology, Stellenbosch University, Stellenbosch ZA7600, South Africa Two-hundred-and-forty lactic acid bacteria (LAB) were isolated from Pinotage, Merlot and Cabernet Sauvignon grapes and wine samples obtained from ¢ve wineries. In 2001, the LAB population on Cabernet Sauvignon grapes ranged from 102 to 104 cfu/ml. In both 2001 and 2002, the

LAB population on Pinotage and Merlot grapes ranged from 102 to 103 cfu/ml. The Cabernet Sauvignon grapes carried a LAB population of between 103 and 104 cfu/ml in 2002. The number of LAB in Cabernet Sauvignon wine after alcoholic fermentation (AF) ranged from 103 to 105 cfu/ml in 2001 and from 102 to 105 cfu/ml in 2002. After AF, Pinotage and Merlot wines carried a LAB population ranging from 102 to 104 cfu/ml in 2001. In 2002, the number of LAB in Merlot and Pinotage ranged from 102 to 105 cfu/ml. Twenty-eight strains were identi¢ed as Oenococcus oeni, four as Leuconostoc mesenteroides, ¢fteen strains were identi¢ed as Lactobacillus brevis, another ¢fteen strains as Lactobacillus hilgardii by means of speciesspeci¢c primers. Ninety-eight strains were identi¢ed as Lactobacillus plantarum, three as Lactobacillus paraplantarum and 12 as Lactobacillus pentosus by means of a multiplex PCR assay using species-speci¢c primers. Primers speci¢c for Lactobacillus paracasei were used to identify 28 strains. Two strains of the obligately homofermentative group were identi¢ed as Pediococcus acidilactici and 35 strains were taken as Pediococcus spp., based on cell morphology. Twenty-six of the isolated strains were found to contain the glycerol dehydratase gene sequence. Preliminary results suggest that the GD-possessing strains exhibit an inhibitory activity against Gram-positive and Gramnegative bacteria, and that this antimicrobial activity is similar to that of reuterin, which is produced by Lb. reuteri. P1^19 SCREENING, ISOLATION AND CHARACTERISATION OF ANTIMICROBIAL / ANTIFUNGAL PEPTIDES PRODUCED BY LACTIC ACID BACTERIA ISOLATED FROM WINE J. Morgan, M. A. Vivier, I. S. Pretorius and M. du Toit Institute for Wine Biotechnology, Department of Viticulture and Oenology, Stellenbosch University, Stellenbosch ZA7600, South Africa A total of 170 lactic acid bacteria (LAB) were isolated from Pinotage, Merlot and Cabernet Sauvignon cultivars in the Western Cape region. Samples were collected from grapes and during di¡erent stages of the winemaking process and screened for antimicrobial activity. Of the total isolates, 25 showed activity predominantly towards Lactobacillus- and Pediococcus-sensitive strains. Two isolates were selected for further characterisation due to their stability in bacteriocin production and constant high activity against the indicator organism, Lactobacillus plantarum LMG 13556. The two bacteriocin-producing strains were identi¢ed as Lactobacillus paracasei #77 and Lactobacillus brevis #81.1. The bacteriocins were inactivated by proteinase K, K-chymotrypsin and lysozyme, but not by

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catalase. The bacteriocins were heat stable and displayed the highest activity from pH 3 to pH 7. The selected isolates were found both to be bacteriostatic in their mode of action. The highest production of bacteriocin occurred after approximately 16 h of growth at 30C. The molecular weight of the bacteriocins, as determined by tricine SDS-PAGE, was between 6.5 and 14.0 kDa. The LAB isolates were also tested for antifungal activity against Botrytis cinerea. The most predominant activity was seen after 24 h of incubation with germinating spores. This study shows that LAB found in South African vineyards and in the winemaking process produce antimicrobial substances that have the potential to inhibit or out-compete other non-producing or sensitive LAB naturally present in the wine and also to in£uence the proliferation of fungal spores. P1^20 CHARACTERISATION OF WINE-ISOLATED ACETIC ACID BACTERIA FROM SOUTH AFRICAN RED WINES A. Oelofse, M. G. Lambrechts, I. S. Pretorius and M. du Toit Institute for Wine Biotechnology, Department of Viticulture and Oenology, Stellenbosch University, Stellenbosch ZA7600, South Africa Acetic acid bacteria (AAB) isolated from South African wine were screened for the production of antimicrobial peptides, production of extracellular enzymes and characterised by their ability to cause volatile acidity (VA). The supernatant of two isolates exhibited bioactivity against other strains of AAB and other Gram-negative microorganisms at a pH of 6.5 with the agar di¡usion method. By means of biochemical tests and PCR-RFLP analysis of the 16S rDNA, one of the producer strains was identi¢ed as Gluconobacter frateurii. This revealed the occurrence of an AAB species that has not yet been found in the winemaking environment. The antibacterial compound was determined to be a proteinaceous substance, since it lost its activity after proteolytic enzyme treatment. Preliminary characterisation indicated that the antimicrobial substance was stable in a pH range from 3.0 to 8.0 and that it was temperature sensitive, with the activity diminishing slightly with the temperature increase from 4C to 65C and thereafter, all activity was lost. The screening of AAB for extracellular enzymes revealed the production of pectinases, proteases, xylanases, L-glycosisdases, cellulases and amylases. The range of enzymes produced varied with di¡erent isolates of the same species. This study also revealed the ability of some of these aerobic microorganisms to produce large quantities of acetic acid ( s 3.5 g/l) under anaerobic conditions. It was evident that AAB could easily

survive and grow during the strenuous conditions of fermentation and consequently contribute to the wine's volatile acidity. Undesirable levels of volatile acidity a¡ect wine quality negatively and serve as indication of microbial spoilage, mainly by AAB. P1^21 FISH PROTEIN HYDROLYSATES AS NITROGEN SOURCE FOR MICROBIAL GROWTH, PROTEASE AND LIPASE PRODUCTION ¤ N. Souissi(1), L. Dufosse(2), R. Ghorbel(1), Y. Triki-Ellouz(1), F. Guerard(2) and M. Nasri(1) ¤ (1) Unite de Technologie Enzymatique et de Microbiologie, ¤ Ecole Nationale d'Ingenieurs de Sfax, B.P. ``W'' 3038 Sfax, ¤ Tunisie ; (2) Laboratoire de Microbiologie Appliquee, LUMAQ, E.A. 2651, I.U.P. Innovation en Industries Alimentaires, Creac'h Gwen F-29000 Quimper, France Fish protein hydrolysates from low cost ¢sh species (Sardinella aurita) were prepared and tested as nitrogen source for microbial growth and lipase production by Rhizopus oryzae and Staphylococcus simulans. The strains tested exhibited a greater lipase production than that obtained with casein peptone. Higher lipase production was achieved only when cells were grown in media containing defatted meat ¢sh protein hydrolysates, indicating the presence in lipid fraction of some constituents, which may repress lipase synthesis. Protease production by Bacillus subtilis and Bacillus licheniformis was assayed in media containing only ¢sh substrates at a concentration of 10 g/L and compared with control media. The best results were obtained with combined heads and viscera £ours indicating that it was not necessary to add ingredients (such as salts, glucose and yeast extract) to ¢sh medium. Furthermore, it seems that heads and viscera £ours contain bioactive molecules that stimulated enzyme synthesis. The high lipase and protease activities obtained with substrates from cheap ¢sh species clearly indicated that these substrates could be used in industrial fermentation processes.

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P1^22 SEMI-INDUSTRIAL AND INDUSTRIAL BAKER'S YEAST STRAINS IMPROVED FOR USE IN FROZEN DOUGHS F. Dumortier(1), A. Teunissen(1a), M-F. Gorwa(3b), J. Bauer(3c), A. Tanghe(1), A. Lo|ez(3), P. Smet(4), P. « Van Dijck(1,2) and J. M. Thevelein(1) (1) Laboratorium voor Moleculaire Celbiologie and (2) Vlaams Interuniversitair Instituut voor BiotechnologieVIB, Institute of Botany and Microbiology, Katholieke Universiteit Leuven, B-3001 Leuven-Heverlee, Flanders, Bel¤ gium; (3) Lesa¡re Developpement, F-59706 Marcq-enBarÝul, Cedex, France; (4) Algist Bruggeman N.V., B9000 Gent, Belgium. Present addresses : aDepartment of Pharmacology, ErasmusMC,3000 DR Rotterdam, The Netherlands; bDepartment of Applied Microbiology, Lund University/Lund Institute of Technology, S-22100 Lund, Sweden; cBASF-LYNX Bioscience AG, D-69120 Heidelberg, Germany. The possibility to store doughs in the freezer permits the separation of the processes of dough production and baking. However, routine production and storage of frozen doughs are still problematic. Although commercial baker's yeast is highly resistant to environmental stress conditions, it rapidly looses stress resistance during dough preparation due to initiation of fermentation. As a result, the yeast signi¢cantly looses gassing power during storage of frozen doughs. We obtained freeze-tolerant mutants of semi-industrial (i.e. strains used in crossing schemes resulting in improved industrial strains) and industrial strains by selection of UV-mutagenized strains for increased stress resistance. In the ¢rst instance, strains were tested for freeze tolerance by suspending the cells in water and freezing them at -20C for 12 days. Several improved mutant strains were obtained, but when tested in pilot scale, no improved freeze tolerance was observed. Therefore it was decided to carry out the selection in conditions that were more close to reality: selection with frozen doughs. In this way the industrial strain S47 gave rise to two mutant strains (AT25 and AT28) which maintained a better dough-rising capacity during frozen storage of the dough. Further investigation of AT25, the most promising of both, showed that other industrially important properties such as yield, growth rate, nitrogen assimilation and phosphorus content were very similar to those in the original S47 strain. Teunissen et al. (2002). Isolation and characterization of a freeze-tolerant diploid derivative of an industrial baker's yeast strain and its use in frozen doughs. Appl. Environ. Microbiol. 68: 4780-4787.

P1^23 POLYMORPHISM OF LACZ AND LACS GENES OF STREPTOCOCCUS THERMOPHILUS FROM DAIRY ENVIRONMENT AS REVEALED BY SEQUENCE ANALYSIS D. Ercolini, V. Fusco, G. Blaiotta and S. Coppola ' Dipartimento di Scienza degli Alimenti, Universita Federico ' 100, 80055 Portici (NA), Italy II di Napoli, via Universita Streptococcus thermophilus is widely used in food fermentation. Lactose, the principal energy source used by Streptococcus thermophilus for growth in milk, is transported into the cell by a permease (LacS) and then hydrolysed within the cell into glucose and galactose by L-galactosidase. The aim of this study was to investigate the sequence heterogeneity of lacZ and lacS genes of Streptococcus thermophilus in order to di¡erentiate strains arising from different dairy environments. Strains of Streptococcus thermophilus isolated from a variety of dairy products were initially characterised at strain level by RAPD-PCR analysis. Primers were designed from the published sequences of the lacZ and lacS genes and used in PCR experiments in order to obtain fragments of the genes. The primers were shown to work for all the Streptococcus thermophilus strains used. The amplicons from lacZ and lacS genes were digested with restriction enzymes and di¡erences were detected between the strains analysed suggesting the nucleotide sequence of the genes to be di¡erent. Moreover, direct sequencing of the PCR ampli¢ed products was performed. Comparison of the sequences obtained from the wild as well as reference strains showed several di¡erences in nucleotides distributed throughout the genes. Several base changes were also found in lacZ genes of Streptococcus thermophilus by other authors. The corresponding protein sequence was also virtually determined and the di¡erences in aminoacid composition of the enzymes evaluated. Furthermore, the possibility to design strain-speci¢c molecular markers from the polymorphic sequences of the lac genes is currently under evaluation. P1^24 APPLICATION OF MIXED YEAST CULTURES IN BREWING G. Farkas, J. Rezessy-Szabo, A. Hoschke Department of Brewing and Distilling, Szent Istvan University, 1118 Budapest, Menesi ut 45, Hungary Production of low-alcoholic or non-alcoholic beers is based on two methods : by removal of alcohol, or by restriction of alcohol formation. In the latter method the

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application of non-brewer's yeast is one of the possibilities. With use of a yeast strain that does not utilize maltose, the ethanol content of beer will be low, but will also taste sweet. In our work four non-brewer's yeast strains were tested. Saccharomycodes ludwigii, Saccharomyces exiguus, Saccharomyces delbrueckii and Torulaspora delbrueckii are yeast strains which do not ferment maltose. Beside them a well characterized brewer's yeast, Saccharomyces cerevisiae WS34/70 was used. Mono- and mixedculture fermentations in wort were carried out. In the latter ones ratio of brewer's and non-brewer's yeast were 1:1, 1:2, 1:5 and 1:10. Products were analyzed by beer analyzer and gas chromatography. Cell concentrations were determined by Buerker chamber. In mixed-culture fermentations Saccharomyces cerevisiae dominated, regardless of initial cell ratios. Beer analysis showed that degree of attenuation and ethanol concentration was low. Some mixed-culture samples gave favorably reduced alcohol content values. Non-brewer's yeast mono-culture samples provided values that do not match the £avor pro¢le of a beer: low concentration of esters was experienced. Results of mixed-culture fermentation gave values that are within acceptable concentration limits of the £avor active compounds. Although the results of the analysis suggests that the use of non-brewer's yeast in mono-culture fermentation would not provide a fully acceptable product, application of mixed-cultures that includes a brewer's yeast strains with good fermentation characteristics can compensate for insu/ciencies. P1^25 ANTIFUNGAL ACTIVITY OF MONOTERPENES AGAINST TROPICAL FRUITS FUNGI M. Pupo(1), E. S. S. Alves(1), R. B. Santos(2), J. A. Ventura(3) and P. M. B. Fernandes(1) " ¤ ¤ (1) Dept. de Ciencias Fisiologicas and (2) Dept. de Qu|mica, Universidade Federal do Espirito Santo ^ UFES ; (3) " ¤ Instituto Capixaba de Pesquisa, Assistencia Tecnica e Extensao Rural ^ INCAPER < Papaya, Banana and Pineapple are the most consumed tropical fruits in the world, being Brazil one of the main worldwide producers. Diseases caused by fungus that infects plants lead to losses in the production and demand the use of chemical fungicides. This work aimed to evaluate the e/ciency of ¢ve monoterpenes isolated from plant essential oils in the inhibition of the mycelial growth and conidia germination of the three pathogens Colletotrichum gloeosporioides, Colletotrichum musae, Fusarium subglutinans fsp ananas in vitro. The inhibitory activity of the monoterpenes compounds in the concentration ranging from 20 to 100 % was tested against the fungi. The essential oils constituents' citral, citronellal and L-

carvone showed a potent fungicidal activity, but little or no activity was observed from the treatments with Kpinene and isopulegol. Based in the results with the monoterpenes, plants that present greater amounts of citral and citronellal had been selected and their oily extracts have been tested. The oil extracts tested were from the plants: Cymbopogon citratus, Cymbopogon nardus, Eucalyptus citrodora, Eucalyptus globosus and Lippia alba. Those results may be an indication to the use of those essential oils as natural pesticides in the control of fruit diseases. Financial support: finep, cnpq, funcitec P1^26 EVOLUTION OF NATURAL MYCOBIOTA GROWING ON THE SURFACE OF THE SPANISH CURED MEAT PRODUCT CECINA DURING ITS RIPENING PROCESS F. Fierro, F. Laich and J. F. Martin Institute of Biotechnology of Leon (INBIOTEC), Avenida Real 1, 24006-Leon, Spain ¤ Cecina is a cured meat product typical of Leon (NorthWest of Spain) that is elaborated from meat pieces of bovine animals. Throughout the ripening process, which takes approximately one year and involves a smoking stage, di¡erent microorganisms grow on the surface of the piece. The presence of yeasts, bacteria from the family Micrococcaceae and ¢lamentous fungi is considered as a signal of a good curing process. The fungi participate in the ripening process and contribute to the £avour and taste of the ¢nal product. We have carried out a quantitative study of the fungal species growing on the surface of cecina pieces during the ripening process, and the main conclusions are reported here. Isolates were identi¢ed by morphological characters and RAPD analysis. Species of the genus Penicillium are preponderant throughout the process, especially in the ¢rst stages, being the most frequently isolated : P. solitum, P. verrucosum, P. implicatum, P. nalgiovense and P. chrysogenum and P. commune. After the smoking stage, species more tolerant to high salt concentrations and lower water activity appear, among them some species of the genus Eurotium. Together with species considered bene¢cial for the product, as P. nalgiovense, commonly used as surface starter for fermented meats (salamis), other species were isolated which are highly mycotoxigenic, like P. verrucosum. The convenience to use methods of control of the natural mycobiota during the curing process is discussed.

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P1^27 POPULATION DYNAMICS OF ACETIC ACID BACTERIA DURING RED WINE FERMENTATIONS ¤ ¤ A. Gonzalez, N. Hierro, M. Poblet, N. Rozes, A. Mas, J. ¤n M. Guillamo ¤ ¤ Departament de Bioqu|mica i Biotecnolog|a, Unitat d'Enologia del CeRTA, Facultat d'Enologia, Universitat Rovira i Virgili, Tarragona, Spain In oenology, acetic acid bacteria (AAB) have received little attention so far. However, the increase in acetic acid in wines as a consequence of the AAB growth is a common problem for wineries. In order to study the AAB population dynamics during the wine-making process we have used two rapid and reliable molecular techniques such as Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) and Repetitive Extragenic PalindromicPCR (REP-PCR), which proved useful for characterising AAB strains. A total amount of 440 strains originated from 2001 and 2002 vintages and di¡erent fermentation conditions (yeast inoculation, SO2) of red grenache grapemusts were ¢ngerprinted. We detected a high degree of strain diversity in the ¢rst stage of fermentation that decreased throughout the process. However, several strains and species were dominant in the alcoholic fermentation phases. Gluconobacter oxydans dominated the fresh must, while Acetobacter aceti was the only isolated species at the end of the process. Gluconoacetobacter hansenii and Gluconoacetobacter liquefaciens were also isolated in signi¢cant numbers at the beginning of fermentation. P1^28 ANTIMUTAGENICITY OF GREEN TEA ANALYZED ¤ BY USING MICROORGANISMSa TESTS ¤ ¤ ¤ ¤ ¤> R. Hlad|kova(1), I. Marova(1), P. Ptacek(1), A. Mikul¤ (1), M. Nemec(2) > cova (1) Faculty of Chemistry, Brno University of Technology, > Purkynova 118, 612 00 Brno; (2) Faculty of Science, Ma¤ saryk University, Tvrdeho 14, 602 00 Brno Green tea extract contains various antioxidant substances, such as tea polyphenols and ascorbic acid. Numbers of these antioxidants inhibit the formation of mutagenic activity because oxygen free radicals and lipid peroxidation processes are the most important factors in the induction of mutagenesis and carcinogenesis. Aim of this work is a study of antimutagenic and antioxidant e¡ects of tea extracts. Quanti¢cation of active substances in tested extracts was performed using HPLC. Biological e¡ects of extracts were analyzed using four independent tests of

genotoxicity: 1) The Ames test with Salmonella typhimurium TA98, 2) Cytogenetic analysis of peripheral blood lymphocytes (CAPL), 3) test with Saccharomyces cerevisie D7 and 4) test with Euglena gracilis. Extracts were allowed to be positive antimutagens based on their ability to inhibit mutagenic e¡ects of standard mutagens. Antioxidant capacity of extracts was analyzed using ``Randox Total Antioxidant Status'' kit. No toxic e¡ects of green tea extract were found. Green tea extracts exhibited high antioxidant as well as high antimutagenic e¡ects (more than 60 percent of mutagenicity inhibition). Antioxidant and antimutagenic e¡ects (more than 40 percent of mutagenicity inhibition) were proved with standard tea catechins : (-)catechin and (-)catechin-gallate too. P1^29 HYDROPHOBICITY AND ADHESIVE PROPERTIES OF LACTOBACILLI USED IN PROBIOTIC YOGHURTS C. Guigas, U. Schillinger and W. H. Holzapfel Bundesforschungsanstalt fur Ernahrung, Institut fur Hy« « « giene und Toxikologie, Haid-und-Neu-Str. 9, D-76131 Karlsruhe Cell surface hydrophobicity is one of the factors that may contribute to the adhesion of bacterial cells to host tissues. It may play an important role in the interaction of probiotic lactobacilli and other lactic acid bacteria with the epithelial cells of the gastrointestinal tract. The adherence of these organisms to mucosal structures is generally believed to facilitate colonisation and persistence of probiotic strains in the normal intestinal population. 19 Lactobacillus strains isolated from probiotic yoghurts were examined for adhesion to the mucus-secreting human enterocyte-like cell line HT29 MTX and to several extracellular matrices such as collagen, ¢brinogen and ¢bronectin as well as for their cell surface hydrophobicity. The investigations included well-studied probiotic strains such Lactobacillus johnsonii LC-1 and Lactobacillus rhamnosus GG as controls. Strain-speci¢c di¡erences in the adhesive properties were found. Hydrophobicity, however, did not necessarily correlate with the adhesion properties, indicating that adhesion to the host cell is a very complex process mediated by many di¡erent mechanisms.

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P1^30 EFFECT OF BACTERIOCIN-PRODUCING ENTEROCOCCUS FAECALIS BFE 1071 ON THE GUT FLORA OF MALE SPRAGUE-DAWLEY RATS A. Wijaya, Ch. Neudecker, C. M. A. P. Franz and W. H. Holzapfel Bundesforschungsanstalt fur Ernahrung, Institut fur Hy« « « giene und Toxikologie, Haid-und-Neu-Str. 9, D-76131 Karlsruhe Bacteriocin production has been described as a functional trait of probiotic bacteria, and probiotic strains that produce bacteriocins have found application in foods. To date, information on the in£uence of bacteriocin production by probiotic strains on the autochthonous £ora of the host is rare. In this investigation, the in£uence of the bacteriocin-producing and potentially probiotic strain Enterococcus faecalis BFE 1071 on the gut £ora of healthy male Sprague-Dawley rats, was studied. Rats (weight, 125 to 150 g; n = 36,), were separated into 4 groups and fed 25g of each of the following diets per day: group A (n=6) received basal diet (Altromin C1000); group B (n=6) basal diet containing 1% of heat-inactivated bacteriocin-producing E. faecalis BFE 1071 biomass; group C (n=12) basal diet containing 1% bacteriocin-producing E. faecalis BFE 1071 biomass (3.6 x 1011 CFU/g); and group D (n=12) basal diet containing 1% of the bacteriocin-negative mutant E. faecalis BFE 1071/79(-) (1.9 x 1011 CFU/ g). The faeces of all groups was collected daily and analysed. Lactobacilli numbers in faeces ranged from 107 to 108 CFU/g with no noticeable di¡erence in numbers between the diet groups. However, enterococcal numbers in groups C and D were ca. 1 log higher at 109 CFU/g when compared to groups A and B. From the predominant Lactobacillus and Enterococcus representatives, respectively 101 and 139 strains were isolated in total from the four diet groups, and characterised by phenotypic features and RAPD ¢ngerprinting. The predominant lactobacilli from the rat GI tract comprised L. gasseri, L. johnsonii and presumptive L. murinus isolates. Interestingly, the administration of bacteriocin-producing enterococci appeared to promote L. johnsonii and L. gasseri numbers. RAPD ¢ngerprinting proved suitable to recognise the bacteriocin-producing Enterococcus strain or its mutant. Thereby, the predomination of the test strain in the faeces and its survival during passage of the rat intestinal tract could be con¢rmed.

P1^31 TOTAL NUMBER OF MICROORGANISMS IN SHEEP'S MILK AND WHITE ^ SOFT CHEESE IN DIFFERENT PHASES OF TECHNOLOGICAL PROCESS IN MACEDONIA N. D. Hristovski(1), E. Krsteva(2), N. Kozarovski(1), M. K. Arapceska(1) and J. N. Hristovska(1) (1) Faculty of Biotechnical Sciences, 7000 Bitola, Macedonia; (2) Institute for Health protection 7000 Bitola, Macedonia In Pelister's region of Macedonia exists, traditional production of sheep's white ^ soft cheese in home condition. The aim of research was to standardize process of production of this kind of cheese, from raw sheep's milk to ¢nal product with high quality, which can been sold at a market in vacuum form with weight of 0.3 ^ 0.6 kg. During research was conducted monitoring of bacteriological status of raw milk and cheese in di¡erent phases of technological process. The following bacteriological examination were exerted: total number of microorganisms in broth agar in fresh milk: 1000 ^ 400 microorganisms /ml, total number of microorganisms in broth agar in pasteurized milk before curdling: 200 microorganisms /ml, total number of microorganisms in broth agar in fresh cheese: 1000 microorganisms /ml, total number of microorganisms in broth agar in fermented cheese: 20 microorganisms /ml. In technological process of cheese's production were used low pasteurization at 65C with duration of 30 minutes, 3% starter culture, 0.015% CaCl2, cheese ferment and the milk was at the temperature of 30C. The ripening of the cheese was exerted in metal cans in duration of 30 days. After that the cheese was vacuumed and sold at a market as high quality sheep's white ^ soft cheese from Pelister's region of Macedonia. With the holding to prescribed technology was gotten a cheese with high quality according to chemical composition, aroma, £avor and acceptable price, which gives opportunity for pro¢table production of this kind of cheese.

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P1^32 T-RFLP AS A METHOD FOR MONITORING ANTIBIOTIC INDUCED DISTURBANCES IN HUMAN NORMAL MICROFLORA C. Jernberg(1,2), A. Sullivan(2), C. Edlund(2) and J. K. Jansson(3) (1) Sodertorn University College, Department of Natural Sciences, S-141 89 Huddinge, Sweden ; (2) Karolinska Institute, Department Laboratory Medicine, Division of Clinical Bacteriology, S-141 86 Huddinge, Sweden; (3) Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, S-750 07 Uppsala, Sweden Clindamycin is an antibiotic used to treat infections caused by obligate anaerobes such as Bacteroides species. During antibiotic treatment, the natural intestinal micro£ora can be disturbed allowing pathogens, such as Clostridium di/cile, to overgrow and produce toxins. These negative impacts may be counterbalanced by use of probiotics during antibiotic treatment. In this study, changes in the composition of the human faecal bacterial micro£ora of healthy volunteers due to the use of clindamycin and a probiotic was analysed qualitatively and quantitatively, primarily using a conventional culturing method. Since only a small fraction of the intestinal micro£ora can be cultivated, the faecal samples were also analyzed using a culture independent molecular ¢ngerprinting technique, terminal restriction fragment length polymorphism (T-RFLP). It was shown that antibiotic treatment had a signi¢cant impact on several bacterial populations in the intestinal community. Di¡erent populations either increased or decreased in relative abundance in both the placebo and the probiotic treated groups. Furthermore, some populations did not seem to be a¡ected at all. Selected pure isolates were also characterized by T-RFLP. Attempts were made to match terminal restriction fragments (TRFs) of the isolates with speci¢c TRFs in the bacterial community ¢ngerprint pattern. The data resulting from culturing methods and T-RFLP analyses were compared. For example, the culture-based results for the Bacteroides group were in accordance with the T-RFLP results.

P1^33 BOTRYTICIDES INFLUENCE THE YEAST MICROFLORA OF GRAPE BERRIES í í > > K. Jug(1), N. Cadez(1), F. Cus(2), P. Raspor(1) (1) University of Ljubljana, Biotechnical faculty, Food Science and Technology Department, Chair of Biotechnology, Jamnikarjeva 101, 1000 Ljubljana, Slovenia; (2) University of Ljubljana, Biotechnical faculty, Agronomy Department, Chair of Viticulture, Jamnikarjeva 101, 1000 Ljubljana, Slovenia Grapes are a primary source of indigenous yeast £ora, which plays an important role in wine fermentation with either their impact on distinctive wine style, or causing stuck or sluggish fermentation. The composition of the yeast population of grape berries is in£uenced by several factors such as ripening stage of the grape, weather conditions, geographic location, grape cultivar and viticulture practice, in particular the time and intensity of disease and pest management. The aim of this study was to determine the in£uence of three commonly used botryticides such as iprodione, pyrimethanil and cyprodinil + £udioxonil on yeast £ora composition of the grape berries of cultivar Rebula (Vitis vinifera L.) in Primorska vine growing region. The agar plate counting results of yeast populations varied among the treatments from 9x102 to 4x103 CFU per ml. Further, colony morphotypes were described and enumerated. Using the combination of molecular technique (PCR-RFLP of 18S+ITS ribosomal DNA) and standard yeast identi¢cation tests, 18 species among 308 yeasts isolates were identi¢ed. The basidiomyceteous yeasts, such as Rhodotorula glutinis, R. minuta, Cryptococcus luteolus and Sporobolomyces sp. predominated in all samples. Hanseniaspora species, which are mostly predominant species on grapes and at the beginning of wine fermentation, were only detected on grape berries of untreated control. This work was ¢nanced by the Slovenia Ministry of Education, Science and Sport and Ministry of Agriculture, Forestry and Food project No. V4-0591 (CRP).

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P1^34 COMPARISON OF GROWTH AND GLYCEROL PRODUCTION KINETICS OF TWO ENDOGENIC WINE YEAST STRAINS IN DIFFERENT SUBSTRATE MEDIA º S. Karasu Yalc|n, Z. Y. Ozbas° ° Food Engineering Department, Hacettepe University, Beytepe 06532, Ankara, Turkey Glycerol has been known as an important by-product of wine fermentations improving sensory quality of wine. This study was carried out with two endogenic wine yeast strains, Saccharomyces cerevisiae Kalecik 1 and Saccharomyces cerevisiae Narince 3. E¡ects of di¡erent sustrates on growth and glycerol production characteristics of the yeasts were investigated in batch system. The kinetics of growth and glycerol biosynthesis were analysed at various initial concentrations of glucose, fructose, and sucrose which take place in the sugar composition of must. Glucose was found as the substrate which S. cerevisiae Kalecik 1 showed most tendency to use for growth, while it was sucrose for S. cerevisiae Narince 3. For both of the strains, speci¢c glycerol production rate was highest in fructose medium and reached maximum level at 350 g/L of initial concentration. Anyway, S. cerevisiae Narince 3 could not produce glycerol below 100 g/L initial concentration of fructose. No glycerol production was observed for the strain Kalecik 1 below 200 g/L initial sucrose concentration, and very low speci¢c glycerol production rates and glycerol yields were obtained below this concentration. When natural white grape juice was used as fermentation medium, S. cerevisiae Kalecik 1 and S. cerevisiae Narince 3 reached a maximum dry weight of 9.3 g/L and 8.0 g/L, respectively. The strain Kalecik 1 produced glycerol at a maximum concentration of 11.8 g/L while Narince 3 produced 14.1 g/L in grape juice. P1^35 ACID RESISTANCE OF BACTERIOCIN-PRODUCERS ISOLATED FROM BOUZA: A TRADITIONAL EGYPTIAN FERMENTED BEVERAGE A. A. M. Khalil Department of Protein Research, Genetic Engineering and Biotechnology Institute, Mubarak City for Science and Applied Technology, Research Zone, Borg Al-Arab, Alexandria, Egypt. On leave from Department of Biotechnology, Lund University, Lund, Sweden Bouza [Written also bosa, bozah, bouza] as a lactic acid fermented food is produced in many mid-Asian, Middle

East, the Balkans, and African countries. Bouza is a traditional Egyptian beverage made by yeast and lactic acid bacteria fermentation of barely, wheat or millet seed and has high alcohol content (up to 7% by volume). These products have many advantages such as destroying undesirable factors in the raw products, and providing a safer product with high acidity. Ability of any bacterial isolate to grow at low pH values is a precondition for them to be selected for a consequent study for proving their probiotic characteristics. The aim of this study is to examine in vitro the ability bacteriocin-producers isolated from Bouza to tolerate low pH values. Viable counts were determined and resistance to pH (R%-pH) was determined as the percentage of surviving cells after incubation for 1 h at pH of 2.5, 3.0, and 3.5. Lactobacillus fermentum was not signi¢cantly in£uenced by pH values of 3.5 and 3.0 and this strain showed the highest survival percentage within the pH range tested. The R%-pH values obtained were 83.5, 64.9, and 51.3% at pH values of 3.5, 3.0 and 2.5 respectively. Lactobacillus casei showed a signi¢cant decrease in viable cells at all tested pHs. However, Lactobacillus plantarum cells were completely killed at pH values of 2.0 and 2.5. The obtained results proofed that Lactobacillus fermentum is a promising probiotic culture. P1^36 INFLUENCE OF SODIUM CHLORIDE AND SODIUM NITRITE ON ANTIBACTERIAL PROTEIN PRODUCTION BY LACTOBACILLUS SAKEI LB 706 A. A. M. Khalil Department of Protein Research, Genetic Engineering and Biotechnology Institute, Mubarak City for Science and Applied Technology, Research Zone, Borg Al-Arab, Alexandria, Egypt. Present address : Department of Biotechnology, Lund University, Lund, Sweden Fermentation of meats is an ancient and excellent preservation technique which results in stable and safe end products. However, the possibility of using bacteriocin-producing starter cultures is still being questioned since the amount of available bacteriocin in the meat environment appears to be lower than expected. In this study, Lactobacillus sakei Lb 706 was used as a producer of sakacin A with Weissella paramesenteroides DSM 20288 as an indicator organism. A series of in vitro fermentation experiments was performed with MRS broth containing di¡erent concentrations of sodium chloride and sodium nitrite in order to examine the e¡ects of these compounds on both cell growth and the production of sakacin A by L. sakei Lb 706. It was found that sodium chloride and sodium nitrite interfere with cell growth and bacteriocin activity. The growth of L. sakei Lb 706 is sometimes enhanced in the presence of low concentrations of sodium chloride but

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was clearly inhibited in the presence of high NaCl concentrations. Increases in salt concentration decrease the growth rate of L. sakei Lb 706 linearly. Moreover, sodium chloride negatively a¡ects the production of sakacin A by L. sakei Lb 706. On the other hand, sodium nitrite had an insigni¢cant e¡ect on bacteriocin activity. At higher sodium nitrite concentration the sakacin A ceased accompanied with lower cell masses. Production of sakacin A decreases because the amount of biomass formed decreases. It has been suggested that the decrease in bacteriocin production in the presence of salt is due to interference of sodium chloride molecules with binding of the induction factor, which is essential for bacteriocin production, to its receptor. P1^37 CRYORESISTANT LACTATE LEAVEN IN BREADMAKING S. V. Kitaevskaya, O. A. Reshetnik Department of Food Technology, Kazan State Technological University, K. Marks str., 68, Kazan, Russia Recently, the technologies of manufacture of £acky smallpiece products from wheat £our on the base of the frozen semi-products are widely used, however, there are no any processing lines making bread from frozen rye or wheatrye semi-products. Special attention should be devoted to the correct selection of compounding components, lactic acid bacteria and yeast strains, freezing-defrosting regimes and the test semi-products storage conditions. A restorative e¡ect of freezing on the blended rye and wheat £our dough of various moistures has been discovered. The expediency of application of the technology of bread manufacture from such a £our blend on the base of frozen semiproducts was shown. The technological parameters of dough batch, freezing and defrosting of wheat-rye bread semi-products were optimized, aiming the conservation of test micro£ora cells. Research work for selection of cryoresistant lactic acid bacteria strains was carried out. The bringing in of lactic acid bacteria Lactobacillus casei TMB-D on the stage of dough batch was shown as stabilizing the qualities of wheat-rye semi-products during freezing. The positive e¡ect of researched strain metabolites on rheological characteristics of dough and the quality of ¢nished articles was established.

P1^38 MICROMANIPULATION AND FLUORESCENCE IN SITU HYBRIDISATION FOR THE ISOLATION AND IDENTIFICATION OF OENOCOCCUS OENI STRAINS FROM WINE H. Konig, J. Frohlich, S. Hirschhauser « « « Institute of Microbiology and Wine Research, Johannes Gutenberg-University, 55099 Mainz, Germany The Gram positive bacterium Oenococcus oeni plays an important role in winemaking and it is bene¢cial to wine quality. Oenococci possess a greater tolerance of acid and ethanol than many other lactobacilli from wine belonging to the genera Lactobacillus, Leuconostoc and Pediococcus. Oenococcus strains are involved in deacidi¢cation, £avor modi¢cation, and increasing the microbial stability of wine. The malolactic fermentation usually occurs after alcoholic fermentation. The acidity of wine is decreased due to the conversion of malate to lactate and carbon dioxide. Since Oenoccocus strains vary in malolactic activity, ethanol, acid and temperature tolerance and fermentation pattern the identi¢cation of new strains with novel feature for special applications in wine-making is desirable. We have developed speci¢c molecular probes for the rapid identi¢cation of oenococci. In combination with micromanipulation Oenococcus strains were rapidly isolated from di¡erent wines.

P1^39 MANUFACTURE OF HONEY BEER WITH SACCHAROMYCES CEREVISIAE AND SACCHAROMYCES PASTORIANUS L ¤ ¤ R. Kovacs(1), B. Vecseri-Hegyes(2), B. Asvanyi(1), L. ¤ Varga(1), J. Szigeti(1), L. Daroczi(3) and G. Bugyi(4) (1) University of West Hungary, Faculty of Agricultural and Food Sciences, Institute of Food Science, Mosonma¤ ¤ ¤ gyarovar, Hungary ; (2) Szent Istvan University, Faculty of Food Science, Department of Brewery and Distillery, ¤¤ Budapest, Hungary; (3) Y-Food Inc., Berettyoujfalu, Hun¤ gary; (4) Mikroauto Inc., Cegled, Hungary The aim of this research was to produce two di¡erent kinds of honey beer, a high-alcohol and a low-alcohol product with unique sensory properties. First, the ethanol production of two yeast species (Saccharomyces cerevisiae and S. pastorianus) was determined by lab-scale experiments. Fermentations were conducted at 15 and 30C for 14 days using glucose and fructose as substrates in fermentation equipment speci¢cally designed for this pur-

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pose. Glucose, fructose, and ethanol levels were measured with the use of a high-performance liquid chromatography (HPLC) system. Both yeast species proved to be capable of producing enough alcohol to make honey beer from substrates containing 25 to 30% honey. Honey beer was manufactured then under industrial pilot plant conditions using the same two Saccharomyces species. Mixtures of malt and honey were used as raw material. The total carbohydrate content was set at 10%. Honey made up 10 to 50% of total carbohydrates. At the end of the ripening process, the chemical composition, i.e., volatile compounds, diacetyl, ethanol, and residual carbohydrate contents, of beers was determined and their sensory properties were also evaluated. In conclusion, a delicious beer rich in aroma compounds was produced with S. cerevisiae. This product was similar in alcohol content to the traditional beer varieties. In contrast, S. pastorianus was hardly capable of fermenting maltose, thus producing a low-alcohol beer whose aroma pro¢le also fell short of expectations. Therefore, the sensory properties of this latter product should be improved by addition of £avouring substances. P1^40 ANTIBACTERIAL AND ANTIMUTAGENIC ACTIVITY OF PROBIOTIC BACTERIUM ENTEROCOCCUS FAECIUM M-74 AND THEIR MODULATION BY SELENIUM ¤ ¤ > > > L. Krizkova, A. Belicova, J. Dobias, J. Krajcovic and L. Ebringer Comenius University, Faculty of Natural Sciences, Institute ¤ ¤ of Cell Biology, Odborarske nam. 5, 811 07 Bratislava, Slovak Republic The antibacterial activity of the probiotic bacterium Enterococcus faecium M-74 was assessed on De Man, Rogosa, Sharpe (MRS), Todd Hewitt (T-H), M17 (M-17) and Brain Heart Infusion (BHI) media with sodium selenite pentahydrate (+Se) and without sodium selenite pentahydrate (-Se) under aerobic or anaerobic conditions against nine bacterial pathogens. The highest antibacterial activity was found to be in the MRS medium under anaerobic conditions. There were no di¡erences in the antibacterial activity between MRS(+Se) and MRS(-Se) media. The antimutagenic activity of MRS(+Se) and MRS(-Se) extracts after cultured with E. faecium M-74 as well as of live and killed cells of E. faecium M-74 grown in the presence or absence of Se against genotoxicity of o£oxacin (OFL) and acridine orange (AO) was determined in Euglena gracilis assay. The MRS(+Se) extracts showed a signi¢cant higher activity in reducing genotoxicity of OFL and AO than MRS(-Se) extracts. The live cells of the probiotic strain M-74 exhibited higher antimutagenic activity than the killed bacterial cells, but di¡erent depending on

the mutagen used. However, the live bacterial cells grown in the presence of Se showed signi¢cantly higher antimutagenic activity. These results suggest a potential bene¢t for the future development of new Se-enriched probiotics exhibiting higher antimutagenic properties. P1^41 THE MYOELECTRICAL MIGRATING COMPLEX (MMC) OF PIG SMALL INTESTINE EXCITE AUTOLYSIS OF PEDIOCOCCUS PENTOSACEUS î D. Kruszewska, A. Ljungh, P. Podgurniak, M. Da ° bek, and S. G. Pierzynowski Department of Medical Microbiology, Dermatology and Infection, Lund University, Solvegatan 23, 223-62 Lund, Swe« den It is known that some mammalian organs and tissues generate extremely low electromagnetic ¢elds (ELEF). The small intestine MMC generates such ELEF and natural micro£ora is exposed to ELEF. We test if the ELEF having characteristics similar to those produced by the MMC of the pig small intestine a¡ect the production of the peptidoglycan hydrolases of Pediococcus pentosaceus in in vitro culture. The autolytic phenotype of P. pentosaceus strain was evaluated under starvation, in PBS at 300C with or without the action of the MMC. For stimulations, the GGP-3 MMC Simulator (Flow Instruments, Poland) was used. The electric current emitted by the device was transmitted to the tubes using platinum plate electrodes. Released peptidoglycan hydrolases were evaluated by renaturing two-dimensional gel electrophoresis (2-DE). The ¢rst dimension was performed in a pH gradient in the range from 3 to 10; in the second dimension SDSPAGE with P. pentosaceus extracted peptidoglycan was supplemented. The antibacterial activity of P. pentosaceus peptidoglycan hydrolases against lactic acid bacteria (LAB) : Lactobacillus plantarum, L. paracasei, Leuconostoc mesenteroides was evaluated. As a result of autolytic activity the 30% of the P. pentosaceus cells were destroyed in the suspension after 24h. Twenty-¢ve extracellulary proteins were separated. The major activity band was observed to migrate at about 45 kDa. The MMC induced the appearance of a diverse group of hydrolases with lower Mr and di¡erent pI. The antibacterial activity of the MMC stimulated peptidogylycans hydrolases of P. pentosaceus was more variable than those from the untreated cultures.

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P1^42 APPLICATION OF TEMPORAL TEMPERATURE GRADIENT GEL ELECTROPHORESIS TO THE BACTERIAL DIVERSITY OF RAW MILKS V. Lafarge(1), J.-C. Ogier(2), V. Girard(1), V. Maladen(1), O. Son(2), C. Bach(2), A. Delacroix-Buchet(2), J.Y. Leveau(3) ¤ ¤ (1) Agence Francaise de Securite des Aliments, Maisons ° Alfort, France; (2) Institut National de la Recherche Agronomique, Jouy en Josas, France; (3) Ecole Nationale ¤ Superieure des Industries Agricoles et Alimentaires, Massy, France Until now, the bacterial community of raw milk was described by classical microbiological methods. In practice, these methods are long and tedious, and allow only a partial inventory of the bacterial micro£ora. In this study, we applied molecular biological techniques based on the direct analysis of DNA to identify the micro-organisms present in raw milks. The Temporal Temperature Gradient gel Electrophoresis (TTGE) method was adapted to identify the diversity and the evolution of the bacterial £ora in refrigerated and non-refrigerated raw milk samples. TTGE was run under conditions that optimise separation of ribosomal DNA fragments from species bacteria whose genomes have either low or high GC content. Using TTGE, we examined raw milk samples before and after their incubation at 4C for 24 hours. The raw milk samples gave rise to TTGE pro¢les that varied in complexity. A given sample generated a TTGE pro¢le of between 3 and 10 bands. Most samples displayed a dominant band of ``Lactococcus lactis''. After incubation of raw milk samples at 4C for 24 hours, TTGE pro¢les were modi¢ed, with an increase of psychrotroph £ora (Listeria, Pseudomonas £uorescens, Hafnia alvei). P1^43 THE STRUCTURES OF EXOPOLYSACCHARIDES PRODUCED BY STRAINS OF LACTOBACILLUS FERMENTUM 133 AND 108 T. Lipinski, A. Korzeniowska-Kowal, M. Gawlik, M. Strus, P. B. Heczko, C. Jones, A. Gamian Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 53-114 Wroclaw, Poland Bacteria of the genus Lactobacillus are Gram-positive microorganisms, known as natural inhabitants of mammalian gastrointestinal tract. These bacteria can bene¢cially a¡ect the health of the host by improving the indigenous microbial balance. Colonisation of the intestinal mucosa

by probiotic microorganisms is considered to be important for antagonistic activity against enteropathogens, modulation the immune system, and for increased healing of damaged gastric mucosa. Essential role in the adhesion phenomenon play the surface antigens of bacterial cell e.g. polysaccharides and proteins. Exopolysaccharides (EPS) produced by lactic acid bacteria are excreted to the growth medium or remain attached to the bacterial cell wall. Such polysaccharides have growing interest also due to the suitable rheological properties for the dairy industry and the structures of EPS from several Lactobacillus strains have been established. Our research are focused on probiotic properties of Lactocbacillus polysaccharides. Strains 108 and 133 have been isolated from gastrointestinal tract of healthy mice and from mice with experimentally induced IBD (in£ammatory bowel disease) respectively. Comparative studies of exopolysaccharides produced by Lactobacillus found in healthy mice and mice with IBD may help to improve our knowledge on the IBD pathogenesis. Here we report the structures of the polysaccharides isolated from Lactobacillus fermentum strain 133 and 108. P1^44 IDENTIFICATION AND CHARACTERIZATION OF LACTIC ACID BACTERIA AND YEASTS ISOLATED FROM SOURDOUGHS F. Zilio(1), L. Zampese(2), C. Andrighetto(1), A. Lombardi(1) ' (1) Veneto Agricoltura, Istituto per la Qualita e le Tecnologie Agroalimentari, Via San Gaetano 74, 36016, Thiene (VI), Italy ; (2) Bioagro srl, Via San Gaetano 76, 36016, Thiene (VI), Italy Sourdough is a complex fermentation of cereals in which lactic acid bacteria and yeasts are involved. In the present study 78 lactic acid bacteria and 31 yeasts were isolated from sourdoughs used for the production of traditional bread and cakes in Veneto region, Northern Italy. The sourdoughs were produced from Triticum aestivum wheat. Isolates were identi¢ed by conventional physiological and biochemical tests and by molecular methods (RAPDPCR). The results showed di¡erences between sourdoughs regarding their microbial pro¢le and revealed an high diversity at species and strain level. Among lactic acid bacteria Lactobacillus sanfranciscensis was the most frequently found species (60%). Inside this species, RAPD-PCR analysis showed the presence of di¡erent groups of strains within the same sourdough. Other lactic acid bacteria were identi¢ed as Leuconostoc citreum, Lactococcus lactis, Lactobacillus plantarum. Technological relevant properties such as gas production, acetate/lactate production and acidifying activity were evaluated. A good acidifying ability, with pH ranging from 3.60 to 4.10 after 18 hours of

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incubation in a wheat £our-based substrate, was found in all the tested strains. Regarding yeasts, all the sourdoughs contained Saccharomyces barnettii as dominant species; Saccharomyces cerevisiae and S. pastorianus were also isolated but with a lower frequency. P1^45 BIOCHEMICAL CHARACTERIZATION OF LACTIC ACID BACTERIA ISOLATED FROM FRENCH DRY FERMENTED SAUSAGES M. C. Lopez-Mendoza(1), R. Huerta(2), C. M. Mata(1) and R. Jordano(2) (1) Department of Animal Production and Food Science and Technology, Cardenal Herrera-CEU University, Edi¢cio Seminario sn, E-46113 Moncada (Valencia), Spain; (2) Department of Food Science and Technology, University of Cordoba, Campus de Rabanales, Edi¢cio Darwin, Anexo. E-14071, Cordoba, Spain The use of starter cultures to control and run the fermentative process is a usual way of manufacturing sausages in meat industries. Food technologists have developed several types of starter cultures to be used for sausages production, but sometimes these microorganisms are di¡erent than those found in natural fermented sausages. The aim of this study was to check if the strains of lactic acid bacteria present in French dry-sausages were according to the starter added initially. A total of 180 strains of lactic acid bacteria isolated from four batches of two different French dry-sausages types (type A and type B), elaborated in pilot plant, were characterised. The main test used to identify the isolated bacteria were: microscopic-morphologic characteristics, catalase activity, production of gas, growth at 4, 15 and 45 C, fermentation of ¢fteen carbohydrates, growth at pH 3.9, growth in the presence of 7% and 10% (w/v) NaCl, hydrolysis of arginine, gas CO2 production from glucose, production of hydrogen sulphide, production of hydrogen peroxide, production of dextrane and production of acetoin. The isolated species of lactic acid bacteria corresponded, in higher percentages, to those added as starters: Lactobacillus sake (74 % of strains) for dry-sausage type A and Pediococcus pentosaceus (47.5% of strains) for type B. But other species such Lactobacillus curvatus and Pediococcus acidilactici (24.5% and 1.5% of strains respectively) have been also isolated in product A and L. sake, L. curvatus, Pediococcus urinaeequi and P. acidilactici (25%, 20%, 5% and 2.5% of strains respectively) in product B.

P1^46 CHARACTERISATION OF BACTERIOCINS PRODUCED BY NATURAL ISOLATE LACTOBACILLUS PARACASEI SUBSP. PARACASEI BGBUK2-16 AND ITS ANTIMICROBIAL ACTIVITY AGAINST SOME PATHOGENIC STRAINS J. Lozo(1), M. Vukasinovic(2) and Lj. Topisirovic(1) (1) Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444A, P.O.Box 794, 11000 Belgrade, Yugoslavia; (2) Faculty of Technology and Metallurgy, Karnegieva 4, 11000 Belgrade, Yugoslavia The strain Lactobacillus paracasei subsp. paracasei BGBUK2-16 produces two bacteriocins, designated Bac216 and Bac217. Bacteriocin Bac217 is inactivated by the heat treatment at 100C for 60 minutes, whereas Bac216 retained activity after heating at 100C for 90 minutes. Both bacteriocins also retain their activities after storage at -20C for 9 months and at 4C for 4 months. These bacteriocins showed activity within pH range from 3 to 12. After treatment with the various proteolitic enzymes they lost their activity. These results strongly suggested that Bac216 and Bac217 should be proteins. Antimicrobial activity of bacteriocins against some pathogenic strain was tested by using well di¡usion assay. These analyses showed that these bacteriocins exhibited a wide range of antimicrobial activity on many di¡erent pathogenic strains including some spoilage and food-born pathogenic bacteria such as Staphylococcus aureus ATCC25923, Listeria monocytogenes and Bacillus cereus ATCC 11778. The e¡ect of BGBUK2-16 on the growth of S. aureus in mixed culture was observed. Treatment of S. aureus with 1600 AUml-1 of each bacteriocins led to considerable decrease in CFUml-1 and after 7 hours of treatment all S. aureus cells were killed. In the case when the initial inoculum of S. aureus was lower all viable cells were killed after 4 hours. These results showed that Lactobacillus paracasei subsp. paracasei BGBUK2-16 could be used as a co-protective culture in food fermentation.

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P1^47 BROAD AND COMPLEX ANTIFUNGAL ACTIVITY OF LACTIC ACID BACTERIA J. Magnusson(1), K. Strom(1), J. Sjogren(2) and J. « « Schnurer(1) « (1) Department of Microbiology, Swedish University of Agricultural Sciences, P.O.Box 7025, S-750 07 Uppsala, Sweden; (2) Department of Chemistry, Swedish University of Agricultural Sciences, P.O. box 7015, S-750 07 Uppsala, Sweden More than 1200 isolates of lactic acid bacteria isolated from di¡erent environments were screened for antifungal activity in a dual-culture agar plate assay. Approximately 10% of the isolates showed inhibitory activity and 4% showed strong activity against the indicator mould Aspergillus fumigatus. A smaller number of isolates was studied further to elucidate the nature of the antifungal e¡ect. By di¡erent separation protocols, it was possible to purify and characterize several di¡erent antifungal compounds of both proteinaceous, and low molecular weight nature from these isolates. Examples are antifungal cyclic dipeptides, phenyllactic acid and various fatty acids. Addition of glycerol to the growth medium strongly enhanced the antifungal e¡ect of Lactobacillus coryniformis. The current study shows that lactic acid bacteria from di¡erent environments and from di¡erent genera and species can exhibit antifungal activity against a number of common spoilage moulds and yeasts. The inhibitory activity is caused by several di¡erent compounds with varying inhibitory spectra and degree of e¡ect. Complex synergistic interactions occur between the primary fermentation products lactate and acetate, and the more speci¢c antifungal compounds. P1^48 METABOLIC PATHWAYS INVOLVED IN THE PRODUCTION OF CHEESE AROMA L. Marilley, H. Sollberger, J. O. Bosset and M. G. Casey Swiss Federal Research Station, Schwarzenburgstr. 161, CH-3003 Bern, Switzerland Cheese manufacture begins with the adjunction of starter bacteria, which initiate the fermentation process. During the ripening stage of Swiss semi-hard and hard cheeses, which are produced from raw milk, a second bacterial population, composed mainly of mesophilic facultatively heterofermentative lactobacilli reachs cell densities of 107109 cfu /g cheese. This second micro£ora also in£uences the quality of cheeses by producing £avour compounds. The objective of this study was to determine the aroma-

relevant end-products of the lactic acid bacteria metabolism, and thus to determine the metabolic pathways involved in the production of cheese aroma. The production of aromatic volatile compounds was measured by gas chromatography followed by mass spectrometry (GCMS) using a purge and trap enrichment system. The aldehydes 3-methylbutanal, 2-methylbutanal, 2-methylpropanal, the secondary alcohols 3-methylbutanol, 2-methylbutanol, 2-methylpropanol (catabolism of branched-chain amino acids), and the methyl ketones 2-pentanone, 2-heptanone and 2-nonanone (putative incomplete L-oxidation) ranked among the main compounds. The production of these volatiles di¡ered between the strains analysed, and thus some strains may have a stronger ability to in£uence the aroma of cheeses. Swiss Emmentaler cheeses were manufactured with adjunctions of di¡erent facultatively heterofermentative lactobacilli. In those manufactured with Lactobacillus casei adjunct cultures, chemical analysis showed an increased concentration of the above-mentioned aldehydes and secondary alcohols. A better understanding of these metabolic routes is a prerequisite to the biotechnological development of new high quality cheese products. P1^49 CONTROL OF FOOBORNE PATHOGENS AND SPOILAGE BACTERIA BY STARTER CULTURES AND BIOPRESERVATIVES IN SPANISH DRY FERMENTED SAUSAGE ¤ C. M. Mata-Anguiano and M. C. Lopez Department of Animal Production and Food Science and Technology, Cardenal Herrera-CEU Universitiy, Edi¢cio Seminari s/n, E-46113, Moncada (Valencia), Spain. ¤ ``Salchichon'' is a Spanish dry fermented sausage produced by the hurdles technology being implicated the ¢ve factors following: nitrite and salt as preservatives, decreased of Eh, competitive lactic £ora, and lowered pH and aw. Lactic acid bacteria (LAB) produces the reduction of pH and gives place to the appearance of some metabolites with antimicrobial action (lactic acid, hydrogen peroxide and bacteriocines). Also the addition of starter cultures or biopreservatives to these meat products try to inhibit the growth of foodborne pathogens and spoilage bacteria. The aim of this study was to observate the e¡ects of di¡erents starter cultures and biopreservatives on the growth of foodborne pathogens (coliforms, enterococci, Escherichia coli, Staphylococcus aureus, Salmonella spp and Listeria) in sausage during ripening. Five di¡erents batch of sausage were prepared each one with a di¡erent starter culture (batch D and E) and biopreservatives (batch B and C). Control is batch A. The samples with starter cultures added showed a signi¢cantly smaller

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counts of coliforms and enterococci than the other batch (p 6 0,001), and the sample with preservative 1 (batch B) presented signi¢cantly smaller coliforms and enterococci counts than the control and batch C (p 6 0,001). In the same way, the sausage added with biopreservatives presented a signi¢cantly higher S. aureus counts than those added with starter cultures (p 6 0,001). E. coli, Salmonella spp and Listeria was not found in any sample. P1^50 BIFIDOBACTERIUM BIFIDUM B7.1 ISOLATED FROM HUMAN ORIGIN IS A PROMISING PROBIOTIC STRAIN A. Mayer, J. Rezessy-Szabo, B. Kondas, A. Hoschke Szent Istvan University, Faculty of Food Sciences, Department of Brewing and Distilling, Menesi ut. 45, Budapest, Hungary Probiotic dairy products have a market share of around 70% on the functional food market. Several probiotic foods containing bi¢dobacteria are commercially available. One of the most important criteria of a probiotic strain is human origination. Therefore, our aim was to isolate bi¢dobacteria from human sources, which have good probiotic (produce SCFA, adherence capability, antagonistic e¡ect to enteropathogens) and moreover technological (acid and oxygen tolerance) features. The strains isolated from healthy adults and infants were identi¢ed with physiological, biochemical and molecular methods. From infants mainly B. bi¢dum and B. breve, from adults B. longum were obtained. Adherence capability to human tissue culture and antagonistic e¡ects to enteropathogens of the isolated strains were tested. The resistance of the most promising strains was examined against gastric and bile acid and digestive enzymes in test tube methods. Furthermore they were tested in a simulator of the human gastrointestinal tract. The strain B. bi¢dum B7.1 proved to be the best among the tested strains, it was able to survive the stomach-small intestine model in 80% and had good e¡ects on the microbiota and the production of SCFA in the colon model. Elaboration of reliable mass propagation of this strain is one of the guarantees for its application as functional food ingredient and/or supplement. To achieve this it was cultivated in TPY broth and soymilk in two-litre laboratory fermentor. After the 24-hours incubation time we got approx. 109 cfu/ml concentration. After separation of the ferment liquor we got a product with 1010 cfu/ml concentration.

P1^51 THE CAPABILITY OF SPIRULINA PLATENSIS CELLS TO ACCUMULATE COPPER AND MANGANESE A. A. Nalimova, V. V. Popova, N. A. Pronina Institute of Plant Physiology, Russian Academy of Sciences, 127276, Botanicheskaya, 33, Moscow, Russia Growth and accumulation of Cu and Mn in Spirulina platensis cells, cultivated on media enriched with these metals have been investigated. It was shown that growth of cyanobacteria was stimulated by low (up to 2.55 mg/l Cu, 138.9 mg/l Mn) and inhibited by high concentrations of the elements in cultivation media. 5 mg/l Cu, 280 mg/l Mn were lethal for cells. Maximal value of intracellular Cu concentration was observed in S. platensis cells during the ¢rst hours after addition of CuSO4 following by copper decrease in the course of cell cultivation. It was the result of intracellular reduction Cu2+ to Cu1+ with the secretion of latter into the medium. On the contrary, continuously increase in the intracellular level of Mn was detected during cultivation of S.platensis in the presence of MnCl2. S. platensis incorporated Cu and Mn preferably in the fraction of soluble proteins (above 55% for Cu, above 90% for Mn). The accumulation of Cu is light dependent and was not detected in the dark. On the contrary, Mn was accumulated in higher concentration on the dark as compared to light conditions. P1^52 MICROBIAL ECOLOGY OF DAIRY PRODUCTS BY TEMPORAL TEMPERATURE GEL ELECTROPHORESIS (TTGE) AND DENATURING GRADIENT GEL ELECTROPHORESIS (DGGE) J.-C. Ogier(1), V. Lafarge(2), A. Rault(2), V. Maladen(2), O. Son(1), C. Bach(1), J.-Y. Leveau(3) and A. Delacroix-Buchet(1) (1) Institut National de la Recherche Agronomique, Jouy en ¤ ¤ Josas, France; (2) Agence Francaise de Securite des Ali° ¤ ments, Maisons Alfort, France; (3) Ecole Nationale Superieure des Industries Agricoles et Alimentaires, Massy, France Numerous micro-organisms, including bacteria, yeasts, and moulds, are present in dairy products, forming a complex ecosystem. Our goal is to characterise the complex bacterial ecosystem of the raw milk micro£ora and to follow its evolution during dairy processing. We applied Temporal Temperature Gradient Electrophoresis (TTGE) and Denaturing Gradient Gel electrophoresis (DGGE) to identify the bacterial species present in dairy products.

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Total DNA was extracted from dairy products, and a variable region (V3) of the 16S rDNA gene was ampli¢ed by PCR with universal bacterial primers, resulting in a mixture of amplicons separable via TTGE or DGGE. We developed an original approach to rapidly identify the bacteria present in an unknown dairy ecosystem; each band was assigned to a bacterial identity by comparing against an exhaustive bacterial reference database (V150 species) that includes useful dairy micro-organisms (lactic acid bacteria), spoilage bacteria (e.g., Pseudomonas and Enterobacteria) and a few pathogenic bacteria (e.g., Listeria monocytogenes and Staphylococcus aureus). We used TTGE to distinguish between bacteria species with a low GC V3 sequence ( 6 55%), whereas DGGE was more effective in separating bacterial species with a high GC V3 sequence ( s 55%). Some related species could not be differentiated using the V3 region. We therefore compared di¡erent regions, either within the 16S rDNA region (V6, V8), or within other functional genes (i.e., tmRNA and rpoB) to improve resolution. We also developed speci¢c primers to detect pathogens (e.g., Listeria). TTGE and DDGE have proven to be powerful and simple methods to rapidly identify the main bacterial species within dairy products by using a combination of universal primers, or to detect contaminants by using speci¢c primers. P1^53 INFLUENCE OF ETHANOL QUANTITY IN A NUTRITIVE MEDIA ON SACCHAROMYCES CEREVISIAE D. J. Pejin, J. D. Pejin, O. S. Grujic and S. D. Kocic Faculty of Technology, University of Novi Sad, Boulevar Cara Lazara 1, 21 000 Novi Sad, Yugoslavia The aim of this study was to determine in£uence of ethanol on metabolic activities and content of cytochromes, alcohol dehydrogenase, isocitrate lyase and malate dehydrogenase in yeast cells. From the obtained results it can be concluded that the increase of ethanol concentration in a nutritive media in£uences cytochromes concentration in yeast. Most signi¢cant decrease is observed in content of cytochromes aa3 though the content of cytochromes b and cytochromes c also decreased. This decrease can be explained by the ethanol concentration increase that disturbs cytohromes formation and thus the intensity of yeast cell breathing. From the results obtained the conclusion can be drawn that ethanol concentration exceeding 7.94 vol % decreases the metabolic activity as the ability of yeast to respire glucose is reduced about three times at the ethanol concentration of 11.45 vol %. The ability of yeast to utilize oxygen at 11.45 vol % when ethanol is the media is also lower. The activity of malate dehydrogenase is found to be depressed by augmentation of ethanol concentration in the

initial media. For example, malate dehydrogenase activities are twice as low in the medium containing 11.45 vol % compared to the value obtained with the ethanol concentration of 7.94 vol %. Alcohol dehydrogenase activity remains high in the cells even at the ethanol concentration of 11.45 vol %, i.e. it does not essentially change. The activity of isocitrate lyase is also slightly lower when the ethanol concentration of 11.45 vol % is used in comparison to that of 7.94 vol %. It is necessary to perform more investigations of the mentioned metabolic and enzymatic activities in the media with the initial amount ranging from 7.94 vol % to 11.45 vol %. Besides, dependence of the activity of malate synthetase upon ethanol concentration in the initial media is to be established. P1^54 PRELIMINARY CHARACTERISATION OF LACTOBACILLUS PLANTARUM STRAINS ISOLATED FROM SOURDOUGHS O. Pepe, G. Blaiotta, M. Anastasio, D. Ercolini and F. Villani ' Dipartimento di Scienza degli Alimenti, Universita degli Studi di Napoli Federico II, 80055 Portici, Italy Twenty-nine Gram-positive rods, non-producing gas from glucose, were isolated from sourdoughs and recognised as Lactobacillus spp. Twenty-eight of them were identi¢ed as belonging to the species Lactobacillus plantarum by both phenotypic (API System) and genotypic (PCR speci¢c ampli¢cation) methods. Moreover, a high degree of discrimination was obtained by pulsed-¢eld electrophoresis (PFGE). In fact Lactobacillus plantarum strains were gathered in 12 di¡erent genomic groups indicating a remarkable genetic polymorphism within the species. This microbial diversity was con¢rmed by the technological performances of fourteen Lactobacillus plantarum strains, associated with maltose positive or maltose negative Saccharomyces cerevisiae strains, tested in dough making experiments. The microbial contents and fermentation properties of the starter cultures were greatly in£uenced by the interaction between LAB and the type of associated yeasts. The time of leavening of doughs prepared with Lactobacillus plantarum and yeast strains appeared shorter than those prepared with the yeast alone, in spite of the detrimental e¡ect on the yeast growths. However, requiring a shorter production time from 7 to around 5 hours, it is worth noting that the association with the maltose positive Saccharomyces cerevisiae can be regarded as more e¡ective. Moreover, the presence of Lactobacillus plantarum strains in the starter enhanced the acidi¢cation activities. The results showed as some technological properties are dependent on the type of the LAB strains, besides the type of species, included in the starter.

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P1^55 THE OLIVE MILL WASTEWATER: A CULTURE MEDIUM FOR THE GROWTH OF LACTIC ACID BACTERIA ' N. Rozez(1), Ma M. Oliveira(2), L. Catulo(2) and C. Peres(2) (1) ITQB, Oeiras, Portugal; (2) INIAP/EAN, Oeiras, Portugal Highly pollutant olive mill wastewaters (OMW) are still produced by a large number of Portuguese units that use traditional olive oil extraction processes. OMW is rich in organic compounds such as polyphenols, fats and sugars but poor in nitrogen. Several dilutions of OMW supplemented with di¡erent nitrogen levels were inoculated with lactic acid bacteria isolated from brined olives. Liquid and liquid/solid systems were tested in order to maximise degradation/growth. Performance was also evaluated by comparing the results of trials that were carried out under aseptic and non-aseptic conditions. The presence of a nitrogen source such as tryptone and/or yeast extract was indispensable for growth. Degradation of polyphenols was observed to be dependent on nitrogen source and its concentration. Also, OMW compounds were used as carbon and energy source. Lactate production increased with the augmentation of tryptone and OMW levels. The cells incorporated oleic and linoleic acids present in OMW and synthesised dihydrosterculic acid from oleic acid and so increase the degree of unsaturation of the plasmatic membrane. Unfortunately OMW could not be fermented without any dilution. P1^56 GROWTH AND ADAPTATION OF LACTOBACILLUS PLANTARUM TO OLIVE FERMENTATION CONDITIONS ' N. Rozes(2), T. Baptista da Silva(2), Ma M. Oliveira(1) and C. Peres(1) (1) INIAP/EAN, Oeiras, Portugal; (2) ITQB, Oeiras, Portugal The lactic acid fermentation of plant materials is presented from an ecological perspective emphasising microbial interactions and their in£uence on production of fermented plant foods. In olive fermentation lactobacilli and yeasts are the most important genera involved in the processes. Among the lactic acid bacteria (LAB) found on olives there appears to be a predominance of homolactic LAB, with Lactobacillus plantarum being the most frequently occurring species with an important role on lactic olive

fermentation. The e¡ect of some parameters of olive brine ecosystem on the growth, physiology and cell composition of L. plantarum isolated from traditional homemade olive brines were determined. Oleuropein is the most important phenolic compound of olives. Sodium chloride is already present in olive brines. Results indicated that the speci¢c growth rate was not modi¢ed by the glucose concentrations, but in the presence of fructose it was lower. Concentrations of sodium chloride had a strong e¡ect on the speci¢c growth rate and it decreased with increasing NaCl amounts. When oleuropein was the only source of carbon L. plantarum exhibited growth and synthetised lactic acid. Nevertheless, both compounds oleuropein and NaCl displayed greater inhibition and showed a bactericidal e¡ect whatever the growth temperatures used. Growth was delayed at 15C and rapid at 37C. However the antibacterial e¡ect of oleuropein was more pronounced. The fatty acid pro¢les and phospholipid composition were weakly a¡ected by growth conditions. This study allowed some new aspects of olive fermentation to be shown. P1^57 THIAMINE REQUIREMENTS IN RELATION TO PYRUVIC ACID PRODUCTION FOR YARROWIA LIPOLYTICA O. A. Perevoznikova, S. V. Kamzolova, I. G. Morgunov G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, pr-t Nauki 5, Pushchino, Moscow region, Russia 142290 In this study, to a pyruvate producing yeast strain Yarrowia lipolytica YKM-Y 2407 and variations in their thiamine requirements with regard to the productivity of pyruvic acid under growth on di¡erent substrates (glucose or glycerol) are presented. With various carbon sources, the limitation of yeast growth by thiamine was shown to be the main condition of the production of pyruvic acid by Y. lipolytica which are enable to synthesize the pyridine part of thiamine molecule and requires addition of this vitamin to the medium. Excretion of pyruvic acids starts simultaneously with a decrease in the growth rate, when the thiamine content in proliferating cells, and, hence, the activity of thiamine-dependent enzymes involved in metabolism is drastically decreased. The decrease in the activity of pyruvate dehydrogenase stimulates the production of pyruvic acid. The threshold concentration of thiamine at which Y. lipolytica ceased to synthesize pyruvic acid was 2.0 and 4.5 Wg/l on glucose and glycerol, respectively. Based on the results obtained under yeast cultivation in £asks, the optimal condition for pyruvic production by batch culture of Y. lipolytyca were proposed; under glycerol as substrate of carbon and energy and the limiting thiamine (2.0 mg/l) pyruvic acid production was as high

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as 67.0 g/l ; speci¢c rate of pyruvic acid synthesis reached 85mg pyruvic acid (g cells h) -1, mass yield coe/cient was 0.70 P1^58 EFFECT OF THE ADDITION OF AUTOCHTHONOUS STARTER CULTURES ON THE MANUFACTURING OF FIORE SARDO CHEESE M. B. Pisano, M. Deplano, M. E. Fadda, A. Senis and S. Cosentino Department of Experimental Biology, section of Hygiene, Univ. of Cagliari, SS 554, Km 4.500, 09042 Monserrato (CA), Italy In order to improve the quality of Fiore Sardo cheese productions, still maintaining its traditional organoleptic characteristics, 8 batches were elaborated, in a pilot dairy plant, using raw milk with and without the addition of autochthonous starter cultures. The lactic acid bacteria used as starters were previously isolated from traditionally made Fiore Sardo cheese. The evolution of chemical and microbial characteristics as well as the sensory parameters were determined for each batch of cheese at di¡erent stages of ripening. No signi¢cant di¡erences in total solid or in the mean content of NaCl, fat, protein and total nitrogen were found in cheeses made with both manufacturing processes. Nevertheless lower values of standard deviations were observed in cheeses manufactured with the addition of starters. As for the microbiological parameters, at 3 months of ripening the mean count of Gramnegative bacteria increased at 48h of ripening, then decreased to levels of 3 log units lower in cheeses manufactured with starters. A similar trend was observed for Staphylococci. Lactic acid bacteria reached a maximum at 48h of ripening and then remained at high levels during ripening with mean counts at least 2 log units higher in cheeses made with starters. As for sensory analysis, cheeses made with starters received higher scores, particularly for openings, texture and taste. These preliminary results seem to con¢rm the potential use of autochthonous £ora as adjunct cultures in the manufacturing of Fiore Sardo cheese in a attempt to standardize the production of this artisanal ewe's product. This work was supported by a grant from MURST, Plan ``Agroalimentary products: dairy products'', Cluster 08B, Project n. 7.

P1^59 INITIAL LOADING OF SACCHAROMYCES CEREVISIAE DICTATES BIOPROCESS KINETICS IN SINGLE AND COMPOSED CULTURES WITH NONSACCHAROMYCES YEAST K. Povhe Jemec and P. Raspor Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia The dynamics of three non-Saccharomyces strains (Hanseniaspora uvarum, Candida stellata and Metschnikowia pulcherrima) in the combination of Saccharomyces cerevisiae at di¡erent starting concentrations in the inoculated Malvasia grape must have been studied. The yeast population of each strain has been evaluated by morphological examination. Stability of S. cerevisiae karyotype was followed during the fermentation process. The quantity of S. cerevisiae population in the ¢rst ¢ve days of fermentation has been evaluated on the selective and non-selective media. After one day of fermentation in all fermenters H. uvarum took over 50% of whole yeast population, even in fermenters with highest concentration of S. cerevisiae cells. As S. cerevisiae population was in the progress, H. uvarum population was in the regression. Population of M. pulcherrima yeasts was even more inhibited by S. cerevisiae and rising concentration of the ethanol, whereas C. stellata population showed to be more ethanol tolerant. At the thirty-¢rst day in all fermenters, no matter on the S. cerevisiae cell starting concentration, S. cerevisiae was, as the only one species, detected in the must. The highest density of yeast population counted 3x107 cfu/ml in the must inoculated only with S. cerevisiae while in the fermenters inoculated with combined inoculum has not exceeded 2x107cfu/ml. Conversion of the sugars to the ethanol was more e/cient in the fermentation processes conducted by higher concentration of S. cerevisiae cells. Glucose was almost completely exhausted in all fermenters, nondependent on the type of the inoculum used, while in fermenters with lower S. cerevisiae starting concentration, more of residual fructose was detected. This study was supported in part by the Ministry of Science and Technology of the Republic of Slovenia (Project no. L4-8849-490). K. Povhe Jemec is grateful to the Ministry of Science and Technology of the Republic of Slovenia for grant (no.: S 38-490-007/18126/97). We are grateful to Vinakoper wine industry to kindly supplying grape í must, and to M. Sergan for some chemical analyses and K. Matastik for her technical assistance.

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P1^60 IDENTIFICATION OF LACTOCOCCUS LACTIS SUBSPECIES USING REP-PCR FINGERPRINTING J. Prodelalova, A. Spanova, B. Rittich Department of Microbiology, Faculty of Science, Masaryk University, Tvrdeho 14, 602 00 Brno, Czech Republic Bacteria of the species Lactococcus lactis are important members of lactic acid bacteria. Two Lactococcus lactis subspecies, lactis and cremoris, are widely used in the manufacture of cheese and other fermented dairy products. L. lactis subsp. hordniae is not used in dairy technology. Different procedures of DNA isolation for DNA ampli¢cation were tested. Repetitive element sequence-based PCR (rep-PCR) was used to identify three subspecies of Lactococcus lactis mentioned above. Single primer LL-REP1 [1] was used in ampli¢cation reaction. Ampli¢ed fragments were resolved by 1,5% agarose gel and stained with ethidium bromide. The use of LL-Rep1 primer resulted in a banding pattern containing approximatelly 6-10 visualised PCR products. The size of the DNA fragments obtained after ampli¢cation using LL-Rep1 primer ranged between 300-2000 bp. Obtained data indicate that used method is suitable for distinguish between subspecies of bacteria Lactococcus lactis. The ¢ngerprinting rep-PCR was used for subspecies identi¢cation of di¡erent Lactococcus lactis strains collected in Czech Republic. [1] E. Urbach et al (1998). FEMS Microbiol. Lett. 162: 111-115. P1^61 THE EFFECT OF FRUCTOOLIGOSACCHARIDES AND POLYFRUCTANS ON THE DEVELOPMENT OF BIFIDOBACTERIA M. Bekers, M. Marauska, P. Semjonovs, M. Grube, A. Rapoport, R. Ionina Institute of Microbiology and Biotechnology, University of Latvia, Kronvald Blvd., 4, LV-1586 Riga, Latvia The production of functional food products increases rapidly. Probiotics and prebiotics as well as dietary ¢bers are signi¢cant components of functional foods. Methods of biotechnology can be used for production of the abovementioned functional food components. Polyfructan ^ levan was produced by Zymomonas mobilis in a laboratory scale experiments. Levan ^ levansucrase complex was obtained from the cell-free culture liquid with ethanol and further used as biocatalyst for fructooligosaccharides (FOS) production in sucrose syrup. Obtained fructan syr-

up (FS) containing FOS and levan was investigated and compared with glucose, commercial FOS and inulin preparations as carbon sources for cultivation of Bi¢dobacterium lacticum 12. It was established that Bi¢dobacterium at physiological conditions could not directly utilize inulin and levan. However, levan was hydrolyzed to FOS by lactic acid. FOS was a good carbon source for Bi¢dobacterium. Inulin was more inert component as compared with levan if yeasts (invertase) as hydrolyzing agent were used. FS was an excellent medium component for Bi¢dobacterium. FS contais 65% of total carbohydrates including 20-25% FOS and 6-7% dietary ¢ber ^ levan. Tests on animals (mice) proved that FS is a non-toxic product with cholesterol lowering properties. FS has a pleasant honeylike taste and reduced (30%) energetic value taking into account the total carbohydrate content. A good quality yogurt was produced from fat-free milk with 5-10% FS additive. P1^62 MICROBIAL ECOLOGY OF PORTUGUESE EWE'S CHEESE H. I. Santos(1,2), J. J. Figueiredo Marques(1,3), L. Raposo(1), R. Tenreiro(4), M. T. Barreto Crespo(1) # (1) IBET/ITQB, Apartado 12, 2781-901 Oeiras, Portugal; (2) Universidade Agostinho Neto, Luanda, Angola; (3) ¤ Estacao Agronomica Nacional, INIA, Oeiras, Portugal; °< ¤ (4) FCUL, Campo Grande, Edif|cio C2, Piso 4, 1700 Lisboa, Portugal Traditional cheeses constitute a group of fermented food products that are highly appreciated due to their nutritional and organoleptic properties. Portuguese ewe's cheese, produced from unpasteurized milk, depends on the environmental microbial innocula to develop its characteristic texture and £avour, as no starter cultures can be added to these traditional products. The maturation period, when well succeeded, leads to an exquisite ¢nal product. The knowledge on the microbial succession in such a process is therefore very important to preserve the same quality standards. Many of the published works on the microbiology of traditional cheese mainly focus on the culturable microorganisms in usual laboratory media. Part of the information on the microbial evolution can than be lost if non-culturable strains are present, as well as, information on the sources of environmental innocula. This work will present results of the microbial ¢ngerprints of the manufacturing areas, utensils and raw material and ¢ngerprints on the microbial succession along the maturation process. Axial distribution of the microbial community along the cheese will also be accessed. The evaluation of those populations will be performed using total DNA extracts from samples, ampli¢cation of 16SrDNA and

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subsequent temperature gradient gel electrophoresis (TGGE). Culturable microorganisms of the same samples will be obtained by traditional plating methods, and will be used for comparison and identi¢cation of the members of the community. P1^63 THE INFLUENCE OF NEW GROWTH REGULATOR OF MICROORGANISMS ``HYDROPTERINE'' ON THE LACTIC ACID FERMENTATION PROCESSES M. Shamtsyan(1), O. Pantyuk(1), A. Bochkova(2) (1) St. Petersburg State Institute of Technology (Technical University), St. Petersburg, Russia ; (2) All-Russian Institute of Food Acidulates, Aroma and Colors, St. Petersburg, Russia The in£uence of hydropterine, a novel, chemically synthesized growth regulator on the lactic acid fermentation processes of Lactobacillus delbrueckii and Streptococcus bovis was studied. It was determined, that this substance can be easily dissolved in water, is not toxic for human and animals use and is ecologically safe.The experiments prove high e/ciency of this regulator. Its concentrations of 106 , 10-5 g/l were signi¢cantly stimulating the processes of biomass accumulation and lactic acid production of both producers. Addition of hydropterine to the fermentation media at the beginning of the process was causing the reduction of fermentation period of L. delbrueckii for 1115% and for 18-20% for S. bovis. Daily addition of hydropterine to the fermentation media was valuably more affective. The in£uence of this substance on several key enzymes of lactic acid fermentation was also, studied. P1^64 DEHYDRATION OF PROBIOTIC STRAINS LACTOBACILLUS GASSERI K7 AND LF221 ¤ > >¤ S. Stojkovic, B. Bogovic Matijasic, I. Rogelj University of Ljubljana, Biotechnical Faculty, Zootechnical > Department, Chair of Dairying, Groblje 3, 1230 Domzale, Slovenia Freeze-drying and spray drying were used to dehydrate Lactobacillus gasseri K7 and LF221 strains. In freeze-drying process various protective media (1: MRS + 20 % glycerol; 2: 10 % NFDMS (CaCO3)-non-fat dry milk solids (cultivated in MRS broth supplemented with 1 g/l CaCO3); 3: 10 % NFDMS + 5 % glycerol; 4: 10 % NFDMS + 10 % sucrose + 0,5 % ascorbic acid + 0,5 % NH4Cl) were applied. The best survival rates determined after freeze-drying and storage, especially at room temper-

ature were obtained in the protective medium 4. The addition of Ca2+ increased the survival rate of both strains during 1 month storage at room temperature. In general, survival at room-temperature was the worst, while there were no di¡erences between results of the storage at 4C and at ^ 20C. Bacteriocin production ability of K7 strain was not altered in any of protective media during freezing, but later (after drying) it decreased substantially in medium 1, while remained unchanged in other media. Spray drying was conducted in a laboratory-scale spray dryer at constant inlet (170C) and outlet (95C) air temperature. Heat pre-adaptation (52C for 15 min) improved the survival of K7 cells and their bacteriocin production ability during 14-days-storage at 4C, while it had negative e¡ect on the survival of LF221 cells. P1^65 VARIETY OF SOYBEAN ON THE QUALITY OF THAI FERMENTED SOYBEAN ``THUA-NAO'' INOCULATE WITH BACILLUS SUBTILIS L. Sutthirangkun and M. Sukchotiratana Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand Thai fermented soybean from the northern provinces, locally called ``Thua-Nao'' is comparable to ``natto'' but the inoculum is natural. The product is subsequently non sticky. The varieties of soybean might have some e¡ect on PGA production. Therefore, four stains of Bacillus subtilis i.e. B. subtilis IFO16449 and B. subtilis natto from Japan, B. subtilis SS9 and B. subtilis SS11 isolated from thua-nao which were found to produce Q-polyglutamic acid (PGA) were used as inoculum for 5 varieties of soybean i.e. Chiang Mai 3(CM 3), Chaing Mai 60(CM 60), SJ.2 , SJ.4 and SJ.5 and incubated at 45C for 48 hours. The amount of PGA produced was then detected by colorimetry method at 520 nm. B. subtilis SS9 was found to give maximum PGA from all the varieties of soybean. The amount of PGA produced on the soybean substrate was CM 3, 7.30 mg/ml ; CM 60, 7.64 mg/ml; SJ.2, 7.10 mg/ml; SJ.4,7.20 mg/ml; SJ.5, 7.15 mg/ml respectively. B. subtilis SS9 also produce highest PGA in PGA producing medium.

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P1^66 Q-POLYGLUTAMIC ACID PRODUCTION BY BACILLUS SUBTILIS FROM THUA-NAO M. Sukchotiratana and P. Noisuwan Microbiology Section, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand Forty isolates of bacteria were obtained from Thua-nao, fermented soybean of Northern Thailand. Twenty-six isolates were found to produce Q- polyglutamic acid (PGA) detected by Colorimetry method at 520 nm when they were grown aerobically in PGA producing medium containing 2 % glucose and 1% ammonium sulfate as carbon and nitrogen sources respectively. The isolate SS11 from Thua-nao in Sansai district, Chiang Mai which was identi¢ed to be Bacillus subtilis produced the maximum PGA of 9.63 mg/ml. The spore suspension was inoculated in PGA producing medium for optimizing the PGA productivity condition. The medium containing 2% glucose and 3% ammonium sulfate incubated at 45 OC pH 8.0 for 48 hr with shaking at 200 rpm gave the maximum PGA of 10.44 mg/ml. Two other isolates from Thua-nao i.e. isolate SS2 and MC18 which were Lactobacillus sp. and Micrococcus sp. were found to produce PGA hydrolase enzyme, endo-type speci¢city, in PGAH medium. This enzyme caused the unstickiness of Thua-nao in contrast to the sticky Japanese natto. P1^67 SYNBIOTIC EFFECT OF LACTOBACILLUS ACIDOPHILUS M92 í > ¤ >¤ J. Suskovic, B. Kos, S. Beluhan, J. Frece and S. Matosic Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10 000 Zagreb, Croatia The scienti¢c basis for the development of probiotics, prebiotics or their combination (synbiotics) stems from the knowledge that the gastrointestinal micro£ora is involved in protecting the host (man or animals) against colonization of the intestinal tract by non-indigenous microorganisms. The use of probiotics (live bene¢cial microorganisms) and prebiotics (non-digestible oligosaccharides) as food/feed supplements bene¢cially a¡ect the host by improving its intestinal microbial balance. The probiotic strain Lactobacillus acidophilus M92 was examined for its ability to assimilate the various kinds of prebiotic substrates (sorbitol, manitol, lactulose, ra/nose, oligofructose and inulin). Bacterial cell yields in the exponential growth

phases was higher on prebiotic substrates than on glucose. Calculated parameters of metabolic activity of Lactobacillus acidophilus M92 have shown slightly better assimilation of polyols and lactulose than the other tested prebiotics. Increase of the cell number and stimulation of metabolic activity of examined probiotic strain can lead to enhanced e¡ect, termed the synbiotic e¡ect. P1^68 CHARACTERIZATION OF LACTOBACILLUS SPP. STRAINS ISOLATED FROM DAIRY PRODUCTS P. Svec(1), V. Drab(2), E. Durnova(3), P. Roubal(4) and I. Sedlacek(1) (1) Masaryk University, Faculty of Science, Czech Collection of Microorganisms, Tvrdeho 14, 602 00 Brno, Czech Republic; (2) Dairy Research Institute, Sobeslavska 841, 390 02 Tabor, Czech Republic; (3) Institute of Hygiene, Partyzanske nam. 7, 728 92 Ostrava, Czech Republic; (4) Dairy Research Institute, Ke Dvoru 12, 160 00 Praha 6, Czech Republic A series of twenty biotechnologically usable Lactobacillus strains with unclear taxonomical position were analysed and characterized by biochemical tests as well as by ribotyping. All strains were characterized by using API 50CH kit and by conventional tests key for this bacterial group. Biochemical test results assigned 17 strains to the species L. acidophilus ; two strains were identi¢ed as L. rhamnosus and one strain were identi¢ed only as Lactobacillus sp. Ribotyping with EcoRI and a probe complementary to 16S and 23S rRNA divided all strains analysed into a few groups. Both L. rhamnosus strains as well as one Lactobacillus sp. strain were di¡erentiated in the full agreement with biochemical test results. Ribotyping revealed intraspecies diversity among seventeen L. acidophilus strains and divided this group into four clusters. The most numerous cluster including eleven strains and the type strain L. acidophilus CCM 4833T. Obtained results suggested that ribotyping as applied in this study could be useful for di¡erentiation of L. acidophilus complex. This study was supported by Ministry of Agriculture project QE 1382.

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P1^69 USE OF REP-PCR TECHNIQUE TO CLASSIFY AND IDENTIFY A WIDE RANGE OF LACTIC ACID BACTERIA F. Feutry, M. Oneca, P. Torre ¤ Laboratorio de lactologia, Area de Nutricion y Bromatolo¤a, Departamento Ciencias del Medio Natural, Universidad g| ' Publica de Navarra, 31006 Pamplona, Spain This study was performed to evaluate the suitability of the REP-PCR (Repetitive Extragenic Palindromic-Polymerase Chaine Reaction) for classifying and identifying a wide range of lactic acid bacteria. The primer pair REP1R-Dt and REP2-D was employed. First, the possibility of working directly with colonia without extraction of DNA was successfully attempted. In a second step, the REP-PCR was applied to some reference strains belonging to di¡erent species of the genera Lactococcus, Leuconostoc, Enterococcus, Lactobacillus, Streptococcus and Pediococcus. The dendogram generated after cluster analysis showed that the REP-PCR technique was enable to discriminate lactic acid bacteria at the genera, at the species and at the subspecies level. The method was then extended at identi¢ed strains isolated from food. It resulted that each one of the strains studied was located in a distinct cluster including the corresponding reference strain and was even enable to distinguish them at the strain level. In conclusion, REPPCR was proven to be a useful gentotypic tool for rapid and reliable screenning, speciation and strain detection of a wide range of lactic acid bacteria from food. P1^70 THE ROLE OF YEAST AND LACTIC ACID BACTERIA ON THE ACCUMULATION OF BIOGENIC AMINES IN WINE F. Gardini(1), S. Torriani(2) and G. Suzzi(3) (1) Dipartimento di Protezione e Valorizzazione Agroali' mentare, Universita di Bologna, Via Fanin 46, 40127 Bologna, Italy ; (2) Dipartimento Scienti¢co Tecnologico, Uni' versita di Verona, Strada Le Grazie 15, 37134 Verona, Italy ; (3) Dipartimento di Scienze degli Alimenti, Univer' sita di Teramo, Mosciano Sant'Angelo, Teramo, Italy Biogenic amines (BA) are basic compounds whose presence in foods can be a cause of disease for consumers. They are usually produced by microbial decarboxylation of amino acids and their presence can be relevant in fermented foods in which the desired growth of microorganisms can be associate with their accumulation. The presence of such compounds in wine is particularly dangerous

because ethanol inhibits the amine oxidases, which are the enzymes responsible for their detoxi¢cation. Recently, a particular attention has been posed on the role of malolactic bacteria on the accumulation of BA. Nevertheless, also yeasts can contribute to the production of these amines, either directly or in£uencing the metabolism of lactic acid bacteria. This work was aimed to the study of the e¡ects of the yeast strains and some wine-making procedures on the accumulation of BA in wines subjected to malolactic fermentation by using a strain of Oenococcus oeni. The results underlined the potential role of yeasts in the production of some amine (putrescine and cadaverine), while bacteria were responsible for the accumulation of 2phenylethylamine. In addition, a key role of the type of must and of the length of alcoholic fermentation was evidenced, as well as the presence of precursors in must and wine. P1^71 APPLICATION OF DGGE IN PROFILING MICROBIAL COMMUNITIES OF TRADITIONAL FERMENTED FOODS S. Torriani, S. Fasoli, V. Gatto, M. Marzotto, L. Rizzotti and F. Rossi ' Dipartimento Scienti¢co e Tecnologico, Universita degli Studi di Verona, Strada le Grazie 15, 37134 Verona, Italy In this research, we applied the Polymerase Chain Reaction Denaturing Gradient Gel Electrophoresis (PCRDGGE) technique to study the microbial communities of several traditional fermented foods, i.e. fermented milks, cheeses, sourdoughs, sausages and wines. For each product, a speci¢c DNA extraction protocol was developed to recover ampli¢able DNA from the main microbial groups associated with these complex matrices. Several sets of universal and taxon-speci¢c primers, targeting rDNA genes, were selected on the bases of their ability to di¡erentiate the species of interest. A large number of type strains belonging to the bacterial and yeast species commonly found in such foods were used to optimise the assays and to generate reference ladders. Using the selected sets of primers, di¡erent composite DGGE pro¢les were obtained for each food. The majority of bands in DGGE pro¢les were correlated to a de¢ned species either by using the reference ladders or by sequencing. The dynamics of the microbial population were monitored during manufacturing processes of some foods. Di¡erences in the composition of the microbial communities were observed, as indicated by the presence and intensity of the obtained bands. Generally, no discrepancies were found when the results of the PCR-DGGE were compared with conventional plate counts integrated by molecular identi¢cation of single isolates. The use of such culture-independent ap-

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proach should enable microbiologists to gain, in a short time, a reliable overview of the microbial components that more likely determine overall quality of the food products. P1^72 EXOPOLYSACCHARIDES PRODUCTION BY LACTOBACILLUS PLANTARUM EP56: PHYSIOLOGICAL AND BIOCHEMICAL ASPECTS R. Tallon(1), P. Bressollier(1,2), and M. C. Urdaci(1) ¤ (1) Ecole Nationale d'Ingenieurs des Techniques Agricoles ¤ ¤ de Bordeaux (ENITAB), 1 cours du General de Gaulle, ¤ 33175 Gradignan cedex, France; (2) IUT de Limoges, De¤ nie Biologique, Allee Andre Maurois, 87000 ¤ ¤ partement Ge Limoges, France Exopolysaccharides (EPS) produced by lactic acid bacteria present a growing interest because of their rheological properties and health bene¢ts. To ¢nd new EPS producers, a total of 32 Lactobacillus plantarum strains were screened for mucoid phenotypes on MRS-agar plates. One strain, EP56, was isolated and was grown in a chemically de¢ned medium (CDM). Two EPS fractions were isolated: 1) a cell-bound EPS fraction (EPS-b) composed of a single high molecular weight polymer containing glucose, galactose, N-acetyl galactosamine and traces of glycerol; 2) a released EPS fraction (EPS-r) composed of the EPS-b polysaccharide and a second low molecular weight polymer containing glucose, galactose, rhamnose and traces of glycerol. Presence of phosphate (1.6% w/w) in both EPS-b and EPS-r, contributes to give a negative net charge to the polymers. Studies on polysaccharides production kinetics and location showed that both polysaccharides are synthesized during the exponential growth phase and that capsular material i.e. high molecular mass polysaccharide is progressively released into the culture medium during stationary growth phase. EPS production by L. plantarum EP56 is in£uenced by environmental parameters. When incubation temperature was reduced from the optimal value of 37C to 18C, the amount of polysaccharide is fourfold increased. EPS-producing properties of L. plantarum EP56 may present interests for potential applications in fermented food and probiotics.

P1^73 TRACE ELEMENT ACCUMULATION BY SPIRULINA PLATENSIS (CYANOBACTERIA) AND ITS DIETARY IMPLICATIONS ¤ L. Varga, J. Szigeti and N. Molnar Institute of Food Science, Faculty of Agricultural and Food Sciences, University of West Hungary, 15-17 Lucsony ¤ ¤ Street, 9200 Mosonmagyarovar, Hungary The purpose of this study was to investigate the accumulation of trace elements by the cyanobacterium Spirulina platensis, which was cultivated for 8 days in arti¢cial culture media containing KI, ZnCl2, and Na2SeO3Á5H2O at concentrations ranging from 0.03 to 30 mg/L. Iodine, zinc, and selenium levels in the Spirulina dry matter (DM) were then determined. The maximum iodine intake of S. platensis was found to be 450 mg/kgDM. This was observed when the initial KI level of the culture medium was 30 mg/L. A concentration of 0.03 mg/L KI in the culture medium resulted in a 370-fold accumulation of iodine in the cyanobacterial DM. As for zinc, its level was slightly less than 100 mg/kgDM when 10 mg/L ZnCl2 was present in the culture medium. The lowest ZnCl2 concentration tested (0.3 mg/L) caused the highest accumulation of zinc (47-fold) in the Spirulina DM. Selenium content of the cyanobacterial biomass was 330 mg/kg when S. platensis was cultivated in a medium containing 30 mg/L Naselenite. Similarly to what was experienced with the other two minerals, the lowest level (0.03 mg/L) of Na2SeO3Á5H2O in the culture medium resulted in the highest accumulation of selenium (58-fold) in the Spirulina biomass. The cyanobacteria accumulating trace elements in their cells are highly suitable for human consumption because minerals are present in the Spirulina cells in organic or complex bonds, thus trace elements have an increased absorption rate and their bene¢cial e¡ects are further improved by the proteins, vitamins and other bioactive substances of cyanobacteria. P1^74 THE TECHNOLOGY OF PRODUCTION OF DRY DAIRY STARTER ON THE BASIS LACTOBACILLUS ACIDOPHILUS OL4 STRAIN FROM NON-COMMERCIAL DAIRY PRODUCT G. V. Yamborko, L. V. Kaprelyants, V. O. Ivanytsya I. I. Mechnikov Odessa National University, Odessa, Ukraine From the result of screening biological and technical characteristics of 40 Lactobacillus strains isolated from non-

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commercial fermented products manufactured in the Odessa region, the strain L. acidophilus OL4, perspective for obtaining starter has been selected. This strain has more intensive antagonistic activity than some other industrial Lactobacillus strains, polyresistance to antibiotic drugs used in medical practice, and saves viability in model conditions of a gastrointestinal tract. The technology of obtaining of a starter contains the following operations: the growth of cells of L. acidophilus OL4 in the optimized nutrient medium ; the concentrating bacterial mass with using ultra¢ltrational membrane and desiccating of a bacterial concentrate in a £uidized layer on the inert stu¡. The usage of the given technology allows to lower energy output in the process of obtaining of a dry bacterial concentrate with the high quality and activity in 2 times. The dry bacterial concentrate L. acidophilus OL4 is powder acquiring the following characteristics : the total quantity of lactobacteria in 1 gram ^ 2,0 ¾ 109 CFU, the level of humidity ^ 3,0 %, the index of dissolubility ^ 0,5 þ 0,2 ml of raw sediment, the speed of acidoformation ^ 12,6 þ 1,4 oT/h, the speed of milk fermentation ^ 3,8 þ 0,5 h, the acidity of a clot ^ 100 þ 4 oT. The dry bacterial concentrate L. acidophilus OL4 has the capacity to correct normal biota of intestine of experimental animals caused by antibiotic therapy, and it colonizes vaginal ecosystem, supporting constant population of lactobacilli. The bacterial concentrate may be used for manufacturing dairy products of preventive assigning, and it can be applied as a separate treatment for correction of disbacteriosis. P1^75 USE OF RIBOTYPING AND PROTEIN PROFILE ANALYSIS IN THE CHARACTERIZATION OF BEER CONTAMINANTS í A. Yansanjav(1), I. Hollerova(2), P. Svec(3) and M. fl mec(1) Ne (1) Department of Microbiology, Faculty of Science, Masaryk University, Tvrdeho 14, 60200 Brno, Czech Republic; (2) Research Institute of Brewing and Malting, Plc., Lipova 15, 120 44 Praha, Czech Republic; (3) Czech Collection of Microorganisms, Faculty of Science,Masaryk University, Tvrdeho 14, 60200 Brno, Czech Republic Mankind began brewing beer in Mesopotamia around 2000 BC without any knowledge of microorganisms or enzymes. Hop compounds were presumably introduced into beer as a strong preservative of quality. Nevertheless, unwanted microorganisms still grow in beer, including a few lactic acid and Gram negative bacteria and wild yeasts which are able to grow even in ¢nished beer with a low concentration of nutrients and oxygen. These contaminants bother consumers and cause economic losses to breweries. The major contaminants of Czech beer are lac-

tic acid bacteria. An API 50 CH kit was used to identify lactic acid bacteria but the method gave doubtful results. Therefore, this work at ¢rst was focused to try to di¡erentiate beer spoilage strains from non-spoilage strains using ribotyping and protein pro¢le analysis using SDSPAGE. Secondly, to make clear the classi¢cation of some closely related strains such as Lactobacillus collinoides and Lactobacillus brevis. Identi¢cation of isolates was made on the basis of locations of the type strains on the clusters. Ribotyping with EcoRI restriction enzyme and protein pattern analysis was shown to be a good tool to di¡erentiate closely related taxons such as L. brevis and L. collinoides. No correlation was observed between the ability to spoil beer and their ribotypes or respective protein pro¢les of tested strains. P1^76 BIOLOGICAL PROPERTIES OF LACTOBACILLI ISOLATED FROM HEALTHY CHILDREN N. O. Yelinska, I. V. Fabiyanska, V. O. Ivanitsa Odessa National University, Microbiology and Virology Department, Dvoryanskaya St., 2, Odessa, 65 026, Ukraine From contents of gastric-intestinal tract of healthy children at the age of 1-7 years which are habitants of Odessa and were prophylactic examined has been isolated 67 strains of lactobacilli bacteria. The isolated strains have been classi¢ed as genus Lactobacillus on the grounds of studying morphological, cultural and physiological-biochemical properties. The species identi¢cation of the isolated strains has shown that the majority (15,1%) are L. delbrueckii subsp. bulgaricus. It has been determined a nature of antagonistic activity of the isolated strains against opportunistic bacteria and yeast-like fungi. It has been discovered that lactic acid decreasing medium pH, play predominant role in depression of the test-culture growth. Insigni¢cant role peroxide of hydrogen and bactericidal substances in depression pro- and eukaryotic microorganisms has been shown. It has been demonstrated that the investigated strains of lactobacilli were characterised by strong resistance to the most drugs ^ to polyenilike antibiotics, phusidine, imidasoles, chinolines, polymixine and phtorchinolines. More low resistance to macrolides, tetracyclines, cephalosporines has been noted. The isolated strains were sensitive to ryphampicines, levomycitines, penicilines and other L- lactamic antibiotics. The strains of lactobacilli have strong antagonistic activity to opportunistic microorganisms and high resistance to antibiotics and may be recommended as the basis of probiotic preparations for intestinal microbiocenosis have been selected.

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P1^77 COMPARATIVE STUDY OF BACTERIOCIN PRODUCTION OF TWO LACTOBACILLUS GASSERI PROBIOTIC STRAINS í > > > M. Zoric, H. Holo, I. F. Nes, A. Canzek Majhenic, B. > Matijasic and I. Rogelj ¤ Bogovic Chair of Dairying, Zootechnical Department, Biotechnical > Faculty, University of Ljubljana, Groblje 3, 1230 Domzale, Slovenija The production of similar or even identical bacteriocins by di¡erent lactic acid bacteria is not rare, especially if the strains colonise the same ecological niche. Lactobacillus gasseri LF221 and K7 are isolates from faeces of two babies and are producing similar Class II bacteriocins with a wide antimicrobial activity.The structural genes of K7 bacteriocins that have been sequenced are shown to be identical with corresponding parts of LF221 A and LF221 B acidocin genes of LF221 strain which suggests that the bacteriocins from the two strains are identical. The genes are in both strains chromosomally located. In the present study, the missing region of orf* sequence, located upstream of orf69 (acidocin LF221 A gene) was obtained. The complete sequence of orf* was revealed to encode a 79 amino acids long peptide (orf79), the putative complementary peptide, required for the acidocin LF221 A activity. The upstream region of acidocin LF221 B genes (the structural bacteriocin and immunity genes) revealed an orf with strong homology with ABC-transporter of other LAB bacteriocin encoding gene clusters. At the same time, the corresponding DNA regions in K7 were sequenced as well and the same gene organisation was found in that bacteriocin system, too. However, both strains differ in some phenotypical characteristics, such as the level and optimal conditions for growth and bacteriocins production, RAPD and plasmid pro¢le and adhesion to Caco-2 cells. Additional gene and expression studies might explain the di¡erences observed in the bacteriocin production between K7 and LF221 strains. P2^1 ANTIBACTERIAL ACTIVITY OF LACTOBACILLUS SAKEI ISOLATED FROM TRADITIONAL DRY SAUSAGE S. Ammor, I. Chevallier and E. Dufour ¤ ¤ Unite de Recherche Typicite des Produits Alimentaires, ENITA-CF, Site de Marmilhat, 63370, Lempdes, France Traditional dry sausages rely on natural contamination by environmental £ora. Therefore, an important role of the

lactic acid bacteria is to inhibit the competing natural £ora which includes spoilage bacteria and, occasionally, pathogens such as Staphylococcus aureus and Listeria monocytogenes. In most cases, the formation of lactic and acetic acids from carbohydrates and the resulting pH decrease, are responsible for the antagonistic e¡ect. However, a pathogenic bacterium such as Listeria monocytogenes is able to survive both the fermentation and drying stages of sausage-manufacturing process because of its ability to survive acidic conditions and its tolerance to considerable amounts of sodium and nitrite. The use of bacteriocinogenic starter cultures and/or co-cultures may o¡er promising solution which minimizes the risks associated with the growth of undesirable bacteria. A total of 20 Lactobacillus sakei isolated from traditional dry sausages have been screened for bacteriocins production towards some indicator organisms belonging to pathogenic (Listeria innocua, Staphylococcus aureus and Escherichia coli), spoilage (Pseudomonas £uorescens, Pseudomonas putida, Hafnia alvei and Enterococcus faecium) and aromatic (Staphylococcus xylosus and Staphylococcus carnosus) bacteria. Obtained results showed the capacity of one strain to inhibit the growth of Listeria innocua, Staphylococcus aureus, Pseudomonas £uorescens and Enterococcus faecium, without inhibition of the growth of the aromatic staphylococci. P2^2 ENTEROCOCCI IN FOOD PRODUCTS CONSTITUTE A RESERVOIR FOR TRANSFERABLE TETRACYCLINE RESISTANCE GENES S. R. Andersen, A. Wilcks and T. R. Licht Institute of Food Safety and Nutrition, Danish Veterinary and Food Administration, MÖrkhÖj Bygade 19, DK ^ 2860 SÖborg, DK A signi¢cant amount of tetracycline resistant faecal enterococci can be isolated from Danish food product samples collected at retail level. Among randomly collected isolates of E. faecalis from various uncooked food sources, approximately 30% were resistant to tetracycline with MIC values exceeding 8 Wg/ml of this drug. We studied the transferability in vitro on agar surfaces and in broth of tetracycline resistance from food-borne E. faecalis isolates to a known E. faecalis recipient strain. Our study shows that transfer does indeed take place under laboratory conditions. The recipient E. faecalis strain received the tetracycline resistance phenotype in approximately 75% of the mating experiments (app. 70% on agar surfaces and app. 43% in broth). Further analysis by PCR revealed that tet(M) was present in approximately 73% of the donor isolates and that Tn916 was present in several, but not all of these investigated donor isolates. Results will be

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presented further revealing the genetic basis for the transferability of the tetracycline resistance phenotype. Our data provide a measure for potential human exposure to transferable tetracycline resistance genes deriving from indicator faecal enterococci in raw food shortly before it enters the consumer kitchen. P2^3 MICROBIAL CONTAMINATION OF TROUT AND POND ENVIRONMENTS C. Armbrust and H.-D. Werlein Universitat Hannover, Institute of Food Science, Wunstorfer « Str. 14, 30453 Hannover, Germany In recent years the proceeds of ¢shery decline. Intensive aquacultures became important to satisfy the increasing demand for ¢sh and ¢sh products. Aquaculture is currently one of the fastest growing food production sectors in the world. Fish is generally regarded as safe, but products from aquaculture have sometimes been associated with certain food safety issues. Increasing stocking rates e.g. can lead to higher microbial contaminations of the ¢sh. Own studies were undertaken to investigate three trout farms of greater Hannover, Germany for a period of three month. The experimental approach consisted of enumerating aerobic psychrotrophic and mesophilic bacteria and anaerobic bacteria in pond water, sediment samples and on the skin and in ¢sh £esh of trout. In addition all samples were assayed for Pseudomonas, Enterobacteriaceae, Salmonella, Aeromonas, yeasts and molds, Clostridium and Clostridium spores. The objective was to elucidate the relationship between the obtained results and the pond environment, temperature of water, stocking rate and supply of water. It could be detected that the microbial contamination of water was nearly changeless in all ponds over the whole period although the total viable counts of sediment and ¢sh samples varied. Stocking rate and supply of water predominantly in£uenced the incidence of Enterobacteriaceae in sediment and ¢sh samples. A decreasing water temperature and stocking rate tend to result in decreasing microbial contamination of ¢sh samples. Salmonella could not be detected in any of the samples. Clostridium was always present in the sediment samples. If Clostridium could be detected in water samples it mostly occurred in the ¢sh samples too.

P2^4 VIT GENE PROBE METHOD FOR THE RAPID AND SPECIFIC DETECTION OF LISTERIA MONOCYTOGENES IN FOOD SAMPLES B. Becker(1), A. Sabrowski(2), M. Lohneis(2), W. H. Holzapfel(1) (1) Institut fur Hygiene und Toxikologie der Bundesfor« schungsanstalt fur Ernahrung, Haid-und-Neu-Str. 9, D« « 76131 Karlsruhe ; (2) Chemisches und Veterinar-Untersu« chungsamt, D-76131 Karlsruhe On account of the high morbidity ( s 30%) experienced with listeriosis in humans, and the wide distribution of Listeria species in the food environment, the exact and rapid identi¢cation of food-borne Listeria strains is of utmost importance. Among the recently developed accelerated detection and identi¢cation methods for Listeria, and especially for L. monocytogenes, those involving gene probes promise special advantages. Bene¢ts may be expected in their application in routine control procedures and in support of quality and safety assurance operations in the food industry. The objective of this model study was to assess the applicability of the VIT (``Vermicon Identi¢cation Technology'') System (Vermicon, Munchen, Ger« many) for the detection of L. monocytogenes in 30 commercial samples of vacuum packaged smoked salmon. The o/cial method according to 35 LMBG served as reference procedure. The VIT-system was developed to enable the application of FISH on a routine basis. Fluorescence labelled gene probes are applied directly in the sample. After reaction with target bacteria, these are not removed by subsequent washing. Upon stimulation with high energy light source, Listeria spp. show a green shine and L. monocytogenes both green and red. Using the o/cial reference method, 15 (50%) of the 30 samples studied were identi¢ed as Listeria positive, and were also con¢rmed positive by the VIT system. Moreover, 13 samples could be identi¢ed as L. monocytogenes positive. For 12 samples, the Listeria numbers were 6 102 cfu/g, and ranged for 3 samples between 150 and 1.9 x 103 cfu/g. The direct application of VIT to food samples appears feasible, provided the cell numbers exceed 103 cfu/g. As Listeria numbers are generally 6 102 cfu/g, a pre-enrichment step seems necessary. Application of the VIT procedure requires 3-4 h. A particular advantage is the speci¢c and simultaneous detection of bacteria of the genus Listeria and of L. monocytogenes in a single test.

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P2^5 OCCURRENCE OF MICROBES OF FOOD SAFETY IMPORTANCE IN SOILS TREATED WITH SEWAGE SLUDGE ¤ ¤ J. Beczner(1), B. Biro(2), Sz. Janko(3) (1) Central Food Research Institute, P.O. Box 393, 1537 Budapest, Hungary ; (2) Research Institute for Soil Science and Agricultural Chemistry HAS, Herman Otto u. 15, 1022 Budapest, Hungary ; (3) Budapest University of Technology and Economy, Gellert ter 4, 1111 Budapest, Hungary A number of food-borne human pathogens are naturally present in soils. Stabilised, composted and pollutant-free sewage sludge can be utilised in the agriculture as nitrogen-, phosphor-, microelement- and organic matter supplements. Biological treatments of the sludge generally reduce the abundance of human pathogens, nevertheless absolutely pathogen-free status can rarely be reached. Four representative Hungarian soil-types (calcareous chernozem-, acidic forest-, calcareous sandy- and acidic sandysoils) were loaded with two di¡erent sewage-sludge forms at di¡erent concentrations for three consecutive years. Soil-samples were quantitatively investigated (in the last two years) for the presence of the total count, moulds, coliforms, E. coli, sulphite-reducing clostridia, Salmonella, Listeria spp., Bacillus cereus by standard food microbiological methods. No essential di¡erence was found between the e¡ect of sludge-types on the microbiological status of the soils in general. The number of most microbes investigated increased as a function of increasing sludge doses. Salmonellae were eliminated during sludge treatment, however microbes indicating faecal contamination survived (coliforms, Enterobacteriaceae). Moulds preferred acidic soils. The abundance of sulphite-reducing clostridia increased as a function of sludge dose, more in the sandy than in the loamy soils. Spores of Bacillus cereus occurred in all soil types. In spite of Listeria spp. occurring in every soils and treatments, neither the presence nor the absence of L. monocytogenes could be unanimously proved. The sludge-doses increased the microbial abundance of the soils, however the cell counts indicated relatively fast elimination of the introduced microbes probably due to the activity of indigenous competitive micro-£ora, or some other environmental factors. The work was supported by Ministry of Education: NKFP-4/0028/2002, Ministry of Agriculture and Regional Development: KF-78/7/2001, and COST 838.

P2^6 ENDOTOXINS ON GRASSES INFLUENCED BY DIFFERENT INTENSITY LEVELS OF MANAGEMENT U. Behrendt(1) and A. Ulrich(2) Centre for Agricultural Landscape and Land Use Research Muncheberg (ZALF e.V.), Institute of Primary Production « and Microbial Ecology (1) Gutshof 7, D-14641 Paulinenaue and (2) Eberswalder Str. 84, D-15374 Muncheberg, Ger« many Measures of extensi¢cation for sustainable pasture management are accompanied by a reduced frequency of utilization and a lower intensity of fertilization. Especially the delay of the ¢rst use from May until mid July, which is required within the framework of bird protection programmes, goes along with microclimatic changes in the ¢eld as well as with changes in the physiological status of the host plant. A consequence can be a structural and quantitative alteration of microbial communities that in£uence the formation and enrichment of microbial toxins on over-matured herbages. The endotoxins (lipopolysaccharides) of Gram-negative bacteria are of special interest since increased concentrations in feed-stu¡s of ruminants can trigger certain endotoxin associated diseases. Thus, the content of bioactive endotoxin in herbages that di¡er in senescence, plant species composition and history of management was determined by LAL (Limulus amebocyte lysate) testing. Furthermore, the bacterial community structure of the phyllosphere was characterized by cultural and culture-independent analyses (selective media; terminal restriction fragment length polymorphism of 16S rDNA) in order to ¢nd relations considering the enrichment of endotoxins. Results of this study revealed the senescence of plant material to be the main cause for increased endotoxin concentrations on grasses. In over-matured plants of mid July, contents were found that are probable to a¡ect animal performance. Additionally, seasonal changes in the microbial community were a further factor in£uencing endotoxins on herbages. P2^7 SCREENING FOR PLANT ANTIMUTAGENS WITH MICROBIAL TESTS T. Beric-Bjedov, J. Knezevic-Vukcevic, B. Vukovic-Gacic, D. Mitic, B. Nikolic, J. Stanojevic, S. Stankovic, D. Simic Chair of Microbiology, Faculty of Biology, University of Belgrade, Belgrade, Yugoslavia The naturally occurring dietary constituents might modulate the mutagenesis and possibly carcinogenesis process.

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In the study of plant antimutagens, microorganisms provide a powerful experimental tool. For screening of plant antimutagens and for detection of mechanisms of antimutagenesis, we used E. coli K12 assay (Simic at al., 1998). The test consist of: Test A ^ repair pro¢cient strain and its uvrA counterpart for detection of induced mutations; Test B -isogenic mutator strains (i) de¢cient in methyl ^ directed mismatch repair for detection of spontaneous mutations due to replication errors and (ii) de¢cient in removing 8-oxo-G for detection of spontaneous mutations due to oxidative damage; Test C ^ isogenic repair pro¢cient strain constitutive for alkaline phosphatase carrying s¢A: :lacZ fusion for measuring the level of SOS induction (induction of mutagenic SOS repair); Test D ^ strains carrying di¡erent recA alleles and two non-overlapping deletions in duplicated lac operon for measuring intrachromosomal recombination. The results obtained in E. coli are compared with standard S. typhimurium (Ames) and S. cerevisiae D7 tests. We screened more than 25 di¡erently prepared extracts of wild and cultivated sage (Salvia o/cinalis L.) and basil (Ocimum basilicum L.), as well as their pure constituents. Monoterpenes from cultivated sage and basil inhibit UV mutagenesis in bioantimutagenic manner. Protective e¡ect of essential oils and pure monoterpenes is due to modulation of DNA repair, i.e. by enhancement of the error-free (excision, recombination) and by inhibition of the error-prone (SOS mutagenic) repair pathways. P2^8 MULTIPLEX-PCR AS A CONVENIENT TOOL IN FOOD SAFETY TO DETECT SIMULTANEOUSLY DIFFERENT FUSARIUM SPECIES IN WHEAT B. Birzele, J. Bunker, J. Kramer and A. Prange « « Dept. of Agricultural and Food Microbiology, University of Bonn, Meckenheimer Allee 168, 53115 Bonn, Germany Mycotoxin producing and phytopathogenic fungi of the genus Fusarium infect wheat during the vegetation period. Their mycotoxins, e.g. deoxynivalenol (DON), are predominantly produced in the ¢eld, but also after harvest when storage conditions are impropriate or when grains have to be harvested at too high moisture contents and immediate drying of grains is impossible. When storing wheat under suboptimal conditions further mycotoxin increase is possible lowering wheat quality and food safety. In order to investigate the in£uence of suboptimal storage conditions on the ability of Fusarium spp. to survive and to produce DON, di¡erent storage trials were conducted with moisture contents of 17% and 20% and temperatures of 15C and 20C. DON contents were determined by ELISA. A multiplex-PCR assay was designed to detect the four Fusarium species, which are most frequently iso-

lated from grains (grown in the Rhineland [1]) by cultural methods. By this PCR assay a precise and simultaneous detection of F. graminearum, F. culmorum, F. poae and F. avenaceum on the basis of established PCR reactions [2,3] was achieved thus o¡ering a time e/cient and convenient means of quality control to detect Fusarium species in food. To validate the PCR results, Fusarium species were also isolated by incubating 200 grains per sample on selective media and were subsequently di¡entiated microscopically. [1] B. Birzele et al. (2002) Eur. J. Plant Pathol. 108: 667673. [2] A.G. Schilling et al. (1996) Phytopathol. 86: 515522. [3] D.W. Parry and P. Nicholson (1996) Plant Pathol. 45: 383-391. P2^9 TOXOPLASMOSIS IN SERBIA : A DOMINANTLY MEAT-BORNE INFECTION B. Bobic, A. Nikolic and O. Djurkovic-Djakovic Toxoplasmosis Research Laboratory, Institute for Medical Research, P.O. Box 102, 11129 Belgrade, Yugoslavia No program of prevention of congenital toxoplasmosis has ever been implemented in Serbia. To de¢ne the subgroups of the women of generative age at particular risk of Toxoplasma gondii infection during pregnancy, a study to identify the risk factors for infection was conducted. The series involved 2936 women 15-45 years of age from throughout Serbia serologically tested for anti-Toxoplasma antibodies (Sabin-Feldman dye test) during the 1988-1997 period. Analysis of the data collected by means of an epidemiological questionnaire showed undercooked meat consumption to be the single predictor of infection (of those usually considered) in the whole series (P=0.003). Undercooked meat consumption signi¢cantly (P=0.000) contributed to infection in the Belgrade suburban area, as opposed to both urban and rural settings. Its in£uence continuously decreased over the study period and paralleled the decrease in the prevalence of infection from 85.9% in 1988 to 39.1% in 1997. Analysis of food meats by animal species showed an association between infection and beef consumption (P=0.002), which triggered preliminary studies of the infection rate in various food animals in Serbia currently underway. Consumption of meat and meat products out of home also contributed to infection (P=0.001 and P=0.000, respectively). The same analysis performed in women grouped according to obstetric history showed a higher infection rate in women with a history of pathological pregnancies who aknowledged undercooked meat consumption than in those who denied it, while there was no such di¡erence among women with no pathological pregnancies regardless of eating habits (P=0.420).

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P2^10 THERMAL INACTIVATION OF LISTERIA INNOCUA IN A RANGE OF SOUS VIDE MODEL PRODUCTS : SOME EFFECTS OF PRIOR HEAT STRESS AND ACID ADAPTATION P. Bourke, D. O' Beirne Food Science Research Centre, University of Limerick, Castletroy, Limerick, Ireland Thermal inactivation D- and z- values of Listeria innocua were determined in a range of sous vide processed model products: sliced potato, broccoli £orets, and beef and salmon pieces. The thermal inactivation of populations subjected to prior heat stress or acid adaptation was also examined in broccoli and potato model products. Two commonly used commercial processes for sous vide foods: 70C for 2 minutes and 85C for 11 minutes were also challenged with acid adapted populations. The e¡ects of some post processing storage conditions were also investigated. D-values were a¡ected by product type, with signi¢cantly higher D-values in salmon and beef products than in potato and broccoli samples. Prior heat stress or acid adaptation of the inoculum resulted in greater heat resistance in potato samples (p 6 0.05). Similar e¡ects were observed for the broccoli model product but were not always signi¢cant. The commercial pasteurisation process of 70C for 2 minutes resulted in 6 log reductions in both acid adapted and non-adapted populations, but Listeria innocua was still detected using conventional plate count methods. Following a process of 11 minutes at 85C, the organism was detected by enrichment procedures and was found in all model products. Subjecting samples to freezing (liquid nitrogen) followed by frozen storage at -40C reduced populations compared with samples refrigerated (8C) post processing. Commercial processes need to address e¡ects of product and stresses on survival of pathogens in products subjected to sous vide processing.

P2^11 MICROBIAL INSPECTION OF ICE-CREAM PLANT FOR MINIMIZING THE RISK OF CONTAMINATIONS S. Bovonsombut(1), K. Kongsoonthorn(1), S. Buabarn(1) and S. Bovonsombut(2) (1) Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50202, Thailand; (2) Department of Food Technology, Faculty of Engineering and Agro-Industry, Maejo University, Chiang Mai, 50290, Thailand This article describes a microbial inspection carried out on ice-cream plant in Chiang Mai province (north of Thailand). The samples obtained were the intermediate products of ice cream from suspected equipments in the process line, the ¢nal products and water. And the swab test conducted on the surface of the equipments as such and the hands of some employees. Using classical methods, aerobic plate count, coliform, faecal coliform, Escherichia coli and Staphylococcus aureus were enumerated. S. aureus were the main species found throughout the experiment due to the cross contamination by the human. Water supply system still gives a good quality of drinking water according to the law. P2^12 CHARACTERISATION OF LISTERIA MONOCYTOGENES, SEROTYPE 1/2b, USING IN VIVO PATHOGENICITY TESTS AND AMPLIFICATION PROFILES ANALYSIS OF THE VIRULENCE GENES iap AND inl P. Cabrita(1), S. Ferreira-Dias(2) and L. Brito(1) ¤ (1) Laboratorio de Microbiologia, DBEB and (2) Centro ¤ ¤ de Microbiologia e Industrias Agr|colas DAIAT, Instituto Superior de Agronomia, Tapada da Ajuda 1349-017 Lisboa, Portugal Listeria monocytogenes, a foodborne pathogen, is capable of causing serious invasive disease in both humans and animals. The iap and inl genes of L. monocytogenes, which code for important virulence proteins present variable regions among strains belonging to the same serovar. In this work, a total of 39 L. monocytogenes isolates from milk, smoked meat and chicken carcass, belonging to serotypes 1/2a, 1/2b, 3b, 4a and 4c, were analysed by a combination of PCR and restriction enzyme analysis (REA) of the gene inl and by PCR ampli¢cation with speci¢c primers for the gene iap. The same isolates were typed by RAPD ampli¢cation with three di¡erent primers. Serial dilutions of cell suspensions, from isolates belonging to serotype 1/2b, were

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also tested for pathogenicity by intraperitoneal injection into immunocompetent mice. The population of L. monocytogenes necessary to kill about 50% of the mice (LD50) in each test, set within 7 days, ranged from 104 to 106 cfu. The association among pathogenicity, ampli¢cation pro¢les cleavage patterns and origin of the strains was evaluated using multivariate data analysis (Principal Component Analysis, Cluster Analysis and Discriminant Analysis). According to cleavage patterns and ampli¢cation pro¢les, strains belonging to the same serovar may be divided into di¡erent groups, which may di¡er in virulence potential. We thank M.Guerra for providing the isolates of L. monocytogenes. P2^13

tococcus lactis producing or non-producing mutants. We are attempting to develop suspension array beads immunoassay applicable to fast £ow cytometric analysis. It is unlikely that degraded nisin has any in£uence on the natural intestinal £ora, since the biological activity is lost in the gastro-intestinal tract. The level of nisin and its degradation fragments in the intestine will be signi¢cantly lower than observed in pure culture supernatant, and is expected to be below the bioassay detection limit but above the immunochemical detection limit. P2^14 HYGIENIZATION OF CHICKEN EGGS BY IONISING RADIATION LEADS TO SAFETY EGGS S. Cabo Verde(1), R. Tenreiro(2) and M. L. Botelho(1)

IMMUNOCHEMICAL DETECTION OF NISIN FROM LACTOCOCCUS LACTIS IN BIOLOGICAL SAMPLES C.-H. Brogren, I. Badiola, A. Johansen. B. Jelle and F. K. Vogensen Department of Dairy and Food Chemistry, Centre of Advance Food Sciences, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark The bactericin nisin A is used as a natural antibiotic additive in dairy products to inhibit growth of Gram-positive pathogens, e.g. Listeria monocytogenes. It is not known whether nisin impacts on the natural intestinal £ora in vivo. As it is produced by the non-probiotic Lactococus lactis, no in situ intestinal colonisation is expected, and any nicin in the intestine must derive directly from oral intake. Proteolitic enzymes such as alpha-chymotrypsin can cleave nisin and abolish its activity. It is not known if the digestion fragments have any in£uence on the normal intestinal £ora. This study concerns immunochemical detection of nisin and its degradation product. Polyclonal antiserum was raised in rabbit against the intact puri¢ed nisin conjugated to KLH. The no-nonsense antibodies speci¢c to nisin were puri¢ed by a/nity chromatography on nisin-ECH-Sepharose columns. Pure antibody or antigen was used to coat ELISA plates for competitive and noncompetitive immunoassays, respectively. A detection limit of 5 ng/ml was obtained, compared to about 1 ug/ml in the bioassay. This is, to our knowledge, the most sensitive immunoassay so far described. Although biological activity is abolished after alpha-chymotrypsin digestion or in the presence of faecal matrix, fragments of nisin could still be detected by the immunochemical methods. Proteolytic degration was studied further by gel electrophoreses and immunoblotting. The sensitive immunoassays will now be applied to faecal samples obtained from in vivo experiments in rats given oral challenge with pure nisin or Lac-

¤ (1) Instituto Tecnologico e Nuclear, Estrada Nacional 10, ¤ Apartado 21, 2686-953 Sacavem, Portugal; (2) Departa¤ mento de Biologia Vegetal e Centro de Genetica e Biologia " ncias da Universidade de LisMolecular, Faculdade de Cie boa, 1749-016 Lisboa, Portugal The purpose of this study is to develop the application of irradiation technology to chicken eggs in order to get a product free of pathogenic microorganisms. Bioburden values of eggs from chicken with di¡erent ages (n=150) were found to be no signi¢cantly di¡erent (p 6 0.05). An average value of (2.0 þ 0.3).105 cfu/egg was obtained for the shell. Two major morphological types were characterized in eggs natural microbiota, but no Salmonella and Campylobacter were detected. HACCP studies indicated the feed as a critical point. Dosimetry studies were carried out in gamma and electron beam facilities to ¢nd the best geometries and dose rate for irradiation. Whole eggs were arti¢cially contaminated with control strains of Salmonella typhimurium and Campylobacter coli and irradiated in the Q facility at sub-lethal doses (0.2 to 1 kGy) with a dose rate of 1.0 kGy/h. Dvalue varied between 0.31 kGy and 0.26 kGy in S. typhimurium and between 0.21 kGy and 0.18 kGy in C. coli, for shell and yolk+white respectively. Using sub-lethal doses up to 5 kGy, the Dvalue of natural microbiota in whole eggs was 1.29 kGy for gamma radiation and 1.42 kGy using the electron beam. Surviving microbiota after egg irradiation includes the same two major morphological types, although a selection of original pigmented strains seems to occur. As no Salmonella and Campylobacter were also detected and bioburden was e¡ectively reduced, results point out that low irradiation doses could guarantee egg safety.

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P2^15 DETECTION OF MICROBIAL SEQUENCES INSERTED INTO GENETICALLY MODIFIED PLANTS í K. Cankar, T. Dreo, K. Gruden, M. Ravnikar and J. Zel National Institute of Biology, Dept. of Plant Physiology and > Biotechnology, Vecna pot 111, 1000 Ljubljana Products from GM soybeans, corn and oilseed rape are on the European market for some years. The products that contain genetically modi¢ed organisms (GMO) in concentration above 1% must be labelled in order to guarantee the consumer's choice between GM and non-GM products. Methods for GMO detection are predominantly based on detection of DNA sequences inserted into genetically modi¢ed plants. At the moment four transgenic lines of corn, one transgenic line of soybean and 4 lines of rapeseed are approved for use in food and feed. Promoters and terminators used in genetically modi¢ed plants originate from plant associated viruses and bacteria of which 35S promoter from cauli£ower mosaic virus (CaMV) and NOS terminator from Agrobacterium tumefaciens are most frequently used and enable screening of above described maize and soybean lines. In our laboratory qualitative screening methods have been adapted for Real-time PCR, thus minimising the possibility of contamination with PCR products and increase of reliability of the method. The major disadvantage of 35S and NOS screening is the possibility of false positive results in case of presence of CaMV virus or bacterium Agrobacterium tumefaciens. Positive results must be therefore con¢rmed and GMO content quanti¢ed by construct speci¢c ampli¢cation methods. To di¡erentiate between virus infected plants and genetically modi¢ed plants a system for Real-time PCR was designed that allows recognition of virus coat protein gene. This assay is especially important for introduction of detection methods for GM rapeseed, since rapeseed is susceptible to CaMV virus infections.

P2^16 INACTIVATION OF SUSPENDED AND ATTACHED TO FOOD CONTACT MATERIALS SALMONELLA AND LISTERIA MONOCYTOGENES CELLS BY HEAT AND COMMERCIAL DISINFECTANTS ¤ J. Carballo, R. Marra and A. B. Araujo L ¤ ¤ Area de Microbiolog|a, Departamento de Biolog|a Funcional y Ciencias de la Salud, Universidad de Vigo, Facultad de Ciencias, Campus de Ourense, As Lagoas s/n, 32004 Ourense, Spain Bacterial adherence and colonization of surfaces may cause problems in the food industry. Food can be contaminated with spoilage and/or pathogenic bacteria because attached cells become more resistant to cleaning and disinfection procedures. Salmonella and Listeria monocytogenes are two pathogenic bacteria which are able to adhere to surfaces.The objective of this study was to compare the e¡ect of di¡erent concentrations of two commercial sanitizers, heat (85 C, 15 minutes) and their combinations on Salmonella spp. and L. monocytogenes cells in suspension and attached to food contact materials (stainless steel, polytetra£uorethylene and rubber). Only attached L. monocytogenes cells survived heat treatment, although the percentage of survival was very low ( 6 1%). Attached bacteria survived the treatment with the concentrations recommended by the manufacturers of two disinfectants with di¡erent composition. Concentrations double and quadruple than that recommended by the manufacturer of the sanitizer based on alquyldiethylenediamineglicyne and di-alquyldiamineethylglicyne reduced in 1 or 2 log cycles, respectively, the surviving population to the recommended one. Sanitizer containing quaternary ammonium compounds used at double concentration reduced attached bacteria by 2-3 log cycles and no attached bacteria survived the treatment with the concentration quadruple than the recommended, irrespective to the surface to which bacteria were adhered. Bacteria were eradicated from the surfaces with the combination of heat treatment with each of the sanitizers at recommended concentrations. Thus, combining heat treatment with chemical treatment would be useful to improve the desinfection of food industry surfaces without increasing the in use concentrations of sanitizers.

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P2^17 INHIBITORY ACTION OF NISIN AGAINST LISTERIA MONOCYTOGENES ON SURFACES ¤ J. Carballo, P. Morais and A. B. Araujo L ¤ ¤ Area de Microbiolog|a, Departamento de Biolog|a Funcional y Ciencias de la Salud, Universidad de Vigo, Facultad de Ciencias, Campus de Ourense, As Lagoas s/n, 32004 Ourense, Spain Listeria monocytogenes are pathogenic bacteria that can contaminate food and cause listeriosis (a food-borne disease with high mortality rate). These bacteria were isolated from walls, £oors and equipment of the food industry. Their ability to attach to di¡erent surfaces and form bio¢lms increases their resistance to cleaning and desinfection operations. It was demonstrated that nisin adsorbed on surfaces reduced L. monocytogenes adherence to them and inhibited the growth of this pathogen in food when adsorbed on food packaging ¢lms. The objective of this study was to investigate the e¡ect of nisin suspensions with di¡erent concentrations against planktonic and attached (to stainless steel, polyethyleneterephthalate and rubber) L. monocytogenes cells. Attached cells were more resistant than their planktonic counterparts. Planktonic cells in populations of 105 and 108 CFU were eradicated with concentrations of nisin of 5 and 50 Wg/mL (depending on the strain) and 1000 Wg/mL, respectively. On the contrary, amounts of attached cells between 6x106 CFU (case of strain ES25 attached to rubber) and 9x107 CFU (strain ES24 adhered to stainless steel) survived the treatment with 10.000 Wg/mL of nisin, although the degree of survival was very low. Reductions in 3-4 log cycles were achieved, independently of the material to which bacteria were attached. The treatment of surfaces with nisin could contribute to control L. monocytogenes in surfaces of the food industry. P2^18 DEVELOPMENT AND VALIDATION OF A METHOD TO DETECT CRYPTOSPORIDIUM PARVUM ON RASPBERRIES: TOWARDS A STANDARD METHOD N. Cook(1), N. Wilkinson(1), K. Paton(2), K. Barker(1), R. Nicholls(2) and H. V. Smith(2) (1) Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK; (2) Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Balornock Road, Glasgow G21 3UW, UK A method was developed based on elution of C. parvum oocysts from raspberries by rolling in 1 M glycine pH 5.5,

concentration of oocysts by centrifugation, IMS separation, labelling of oocysts with FITC-MAb / DAPI, and microscopic identi¢cation and enumeration. In the originating laboratories the recovery e/ciency of the method was 41.0 þ 13.0 % (n = 30). The method was subjected to interlaboratory collaborative trial, involving eight expert laboratories in the United Kingdom. The trial involved eight expert laboratories in the United Kingdom. Samples comprised 60 g raspberries. They were inoculated at three levels : low (8.5 ^ 26.8 oocysts), medium (29.7 ^ 65.7 oocysts), and high (53.9 ^ 131.3 oocysts). Blank, or uninoculated, samples were also tested. The method had a overall sensitivity (correct identi¢cation of all inoculated samples) of 90.9 %, and a speci¢city (correct identi¢cation of uninoculated samples) of 83.3 %. The total mean percentage recovery (from all inoculated samples) produced by the method was 49.2 þ 28.3 %. Testing all samples whether inoculated or blank, the overall accordance, or repeatability, was 80.4 %. The overall concordance, or reproducibility, was 80.2 %. The qualitative performance aspects of the method were just as reproducible between laboratories, as repeatable within a laboratory. The results of the collaborative trial indicate that the assay may be con¢dently applied in analytical microbiological laboratories. The work was supported by the United Kingdom Food Standards Agency. P2^19 DEVELOPMENT AND VALIDATION OF A METHOD TO DETECT CRYPTOSPORIDIUM PARVUM ON LETTUCE: TOWARDS A STANDARD METHOD N. Cook(1), N. Wilkinson(1), K. Paton(2), K. Barker(1), R. Nicholls(2) and H. V. Smith(2) (1) Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK; (2) Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Balornock Road, Glasgow G21 3UW, UK A method was developed based on elution of C. parvum oocysts from lettuce by stomaching with 1 M glycine pH 5.5, concentration of oocysts by centrifugation, IMS separation, labelling of oocysts with FITC-MAb / DAPI, and microscopic identi¢cation and enumeration. In the originating laboratories the recovery e/ciency of the method was 59 þ 12 % (n = 30). The method was subjected to interlaboratory collaborative trial, involving eight expert laboratories in the United Kingdom. Samples comprised 30 g lettuce. They were inoculated at three levels : low (8.5 ^ 14.2 oocysts), medium (53.5 ^ 62.6 oocysts), and high (111.3 ^ 135.0 oocysts). Blank, or uninoculated, samples were also tested. The method had an overall sensitivity (correct identi¢cation of all inoculated samples) of 89.6 %, and a speci¢city (correct identi¢cation of uninoculated samples) of 85.4 %. The total mean percentage recovery

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(from all inoculated samples) produced by the method was 40.0 þ 26.3 %. Testing all samples whether inoculated or blank, the overall accordance, or repeatability, was 80.1 %. The overall concordance, or reproducibility, was 79.5 %. The qualitative performance aspects of the method were just as reproducible between laboratories, as repeatable within a laboratory. The results of the collaborative trial indicate that the assay may be con¢dently applied in analytical microbiological laboratories. The work was supported by the United Kingdom Food Standards Agency. P2^20 A VALIDATED PCR ASSAY FOR DETECTION OF LISTERIA MONOCYTOGENES : TOWARDS AN INTERNATIONAL STANDARD M. D'Agostino(1), M. Wagner(2), J. A. Vazquez-Boland(3), J. Hoorfar(4), R. Karpiskova(5), S. Novella(3), J. Ellison(1), A. Murray(1) and N. Cook(1) (1) DEFRA Central Science Laboratory (CSL), Sand Hutton, York, YO41 1LZ, UK; (2) Institute for Milk Hygiene, Milk Technology and Food Science, Veterinaerplatz 1, Vienna 1210, Austria ; (3) University Complutense de Madrid, 28040 Madrid, Spain; (4) Department of Bacteriology, Danish Veterinary Institute, 27 Bulowsvej, Copenhagen 1790, Denmark; (5) National Institute of Public Health, Palackeho 1-3, Brno 61242, Czech Republic Successful validation of any method should be necessary for its adoption as a standard. We report a PCR assay for Listeria monocytogenes. It has a detection limit of less than 10 bacterial cells per reaction. When tested against 38 L. monocytogenes strains and 52 non-target strains (29 nonmonocytogenes Listeria spp. and 23 non-Listeria spp.), the assay had a high diagnostic accuracy, being 100 % inclusive and exclusive. It was evaluated in an international ring trial involving 12 participating European laboratories (from Austria, Czech Republic, Denmark, Germany (3 participating laboratories), Greece, Ireland, Poland, Spain (2 participating laboratories), and Sweden). In this trial, the method was tested against an additional 14 L. monocytogenes strains and 12 non-monocytogenes Listeria strains. Here, the inclusivity was 100 % and the exclusivity was 99.4 %. The PCR assay was just as reproducible between laboratories, as repeatable within a laboratory, and thus may be con¢dently applied in analytical microbiological laboratories.

P2^21 A VALIDATED PCR-BASED METHOD FOR DETECTION OF LISTERIA MONOCYTOGENES IN RAW MILK : TOWARDS AN INTERNATIONAL STANDARD M. D'Agostino(1), J. Hoorfar(2), and N. Cook(1) (1) DEFRA Central Science Laboratory (CSL), Sand Hutton, York, YO41 1LZ, UK; (2) Department of Bacteriology, Danish Veterinary Institute, 27 Bulowsvej, Copenhagen 1790, Denmark An enrichment / PCR method for the detection of Listeria monocytogenes in raw milk was tested in an international collaborative trial, involving thirteen European laboratories (in Austria, Czech Republic (2), Denmark, France, Germany, Greece, Ireland, The Netherlands, Poland, Portugal, Slovakia, and Spain) in December 2002 and January 2003. Samples of raw milk inoculated at approximately 2, 20 and 200 cells per 25 ml were enriched in half-Frasers's broth, and sent to each participant. The participants incubated the samples in the secondary enrichment broth LEM2 and then applied the PCR. The accuracy parameters ^ sensitivity and speci¢city ^ of the method were determined. Sensitivity is the percentage of inoculated samples that gave a positive result. Speci¢city is the percentage of uninoculated samples that gave a negative result. Repeatability and reproducibility were determined by calculating accordance and concordance values. Accordance is de¢ned as the percentage chance of ¢nding the same result (i.e. both positive or negative whether correctly or not) from two identical inoculated samples analysed in the same laboratory under standard repeatability conditions. Concordance is de¢ned as the percentage chance of ¢nding the same result (i.e. both positive or negative whether correctly or not) from two identical inoculated samples analysed in two di¡erent laboratory under standard repeatability conditions. The concordance odds ratio was calculated in order to assess the degree of between-laboratory variation in results. The completed results of the trial and evaluation of the method will be available at the congress.

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P2^22 EFFECT OF THE ADDED STARTER CULTURE ON THE SURVIVAL OF LISTERIA MONOCYTOGENES IN DRY SAUSAGES L. De Zutter, K. Houf Department of Veterinary Food Inspection, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Traditional dry sausages are frequently contaminated with L. monocytogenes. The present paper reported the behaviour of L. monocytogenes during the production and storage of such sausages inoculated with 2 di¡erent starter cultures (SM272 and TSC150). For the production of all sausages one batch of meat was used. After comminuting the meat and addition of the adjectives, one part was inoculated with SM272 and the other with TSC150. Each portion was further divided in 3 parts : 1/ no addition of L. monocytogenes (controls), 2/ addition of 102/g and 3/ addition of 105 L. monocytogenes/g. All sausages were produced and stored in the same conditions as the commercial ones. From each type of sausages a portion was individually packaged in MAP after 7 days.The control sausages were initially L. monocytogenes positive. During production and storage less sausages were found positive containing the starter culture SM272. For inoculated sausages a larger reduction was observed with the starter culture SM272 than with TSC150: in low level inoculated sausages (102/g) reduction levels of 103 and 101 and in high level inoculated sausages (105/g) reduction levels of 105 and 102 were stated respectively. In MAP packed sausages L. monocytogenes levels stabilized during the second half of the storage period. In conclusion these challenge tests showed that the reduction of L. monocytogenes depended on the starter culture used and the number of L. monocytogenes initially presented. At the moment that such sausages are commercialized (7 days after production), absence of L. monocytogenes cannot be guaranteed. P2^23 SURVEY OF ANTIBIOTIC SUSCEPTIBILITY IN LACTIC ACID BACTERIA ISOLATED FROM CABRALES, A TRADITIONAL BLUE-VEINED SPANISH CHEESE ¤ A. B. Florez, S. Delgado and B. Mayo ¤ Instituto de Productos Lacteos de Asturias (CSIC), 33300Villaviciosa, Asturias, Spain At present, there is a great concern about the spread of antibiotic resistance determinants by bacteria from the food chain [1]. Thus, strains intended to be used on

food systems have to be systematically examined for antibiotic susceptibility. Species of lactic acid bacteria isolated from Cabrales cheese were investigated for their antibiotic susceptibility. It seems clear that the use as starters of antibiotic resistant lactic acid bacteria should be avoid [1]. Ninety-¢ve lactic acid bacteria strains isolated from di¡erent batches of artisanal starter-free Cabrales cheese were tested against 15 antibiotics with the ``Sensitrite Anaero3'' kit (Treck Diagnostic System). MICs to some antibiotics have to be completed on Mueller-Hinton plates by the NCCLS procedure. Cefoxitin and metronidazole were not e¡ective against this group of bacteria. Lactococcus and Enterococcus strains displayed antibiotic resistance pro¢les di¡erent from those of Lactobacillus and Leuconostoc ones. Penicillins MICs were always higher for Lactobacillus and Leuconostoc species. However, in clinical terms, none of them was resistant. MICs to tetracycline were also higher for the Lactobacillus and Leuconostoc isolates. All Leuconostoc isolates and all but four Lactobacillus were resistant to vancomycin, a resistance that was only found in two related Enterococcus isolates, whereas it was absent in Lactococcus. Enterococci were more resistant and/or presented higher MICs to most antibiotics than lactococci. Moreover, three Enterococcus strains presented a kind of multiresistance. In spite of this, a few Lactococcus isolates were resistant to tetracycline and chloramphenicol, a fact that justify this analysis. [1] Teuber, M., L. Meile, and F. Schwarz. 1999. Acquired antibiotic resistance in lactic acid bacteria. Antonie van Leeuwenhoek 76:115-137. P2^24 ANTIBIOTIC SUSCEPTIBILITY OF LACTOBACILLUS AND BIFIDOBACTERIUM SPECIES FROM THE HUMAN GASTROINTESTINAL TRACT S. Delgado(1), L. Otero(2) and B. Mayo(1) ¤ (1) Instituto de Productos Lacteos de Asturias (CSIC), ¤ 33300-Villaviciosa; (2) Servicio de Microbiolog|a del Hos¤ pital de Cabue·es, 33394-Gijon, (Asturias), Spain At present, there is a great concern about the spread of antibiotic resistance determinants by bacteria from the food chain [1]. Thus, strains intended to be used on food systems have to be systematically examined for antibiotic susceptibility. We are characterizing several Lactobacillus and Bi¢dobacterium species from the human gastrointestinal tract that could eventually be used as probiotics. The absence of antibiotic resistance is one of the criterium to select presumptive useful strains. Seventy strains of Bi¢dobacterium and Lactobacillus species have been tested against 15 antibiotics with the ``Sensitrite Anaero3'' kit (Treck Diagnostic System). MICs to some antibiotics were completed on Mueller-Hinton plates by

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the NCCLS procedure. All strains were sensitive to chloramphenicol, imipenem and the group of penicillins. On the other hand, most of the strains were resistant to metronidazole. All bi¢dobacteria isolates were susceptible to cefoxitin, whereas about half of the lactobacilli were resistant. Single strains of Bi¢dobacterium infantis/longum, B. pseudocatenulatum, Lb. acidophilus and Lb. rhamnosus were resistant to erythromycin and/or clyndamicin. Approximately, ¢fty per cent of the Bi¢dobacterium isolates were resistant to tetracycline, as well as two Lb. acidophilus strains and one Lb. plantarum. None of the tested Bi¢dobacterium infantis/longum isolates was resistant to vancomycin, while around ¢fty per cent of the lactobacilli were found to be resistant. Most of the observed resistances seemed to be intrinsic and were similar to those reviewed on the literature [1, 2]. [1] Teuber, M., L. Meile, and F. Schwarz. 1999. Acquired antibiotic resistance in lactic acid bacteria. Antonie van Leeuwenhoek 76:115-137. [2] Gasser, F. 1994. Safety of lactic acid bacteria and their occurrence in human clinical infections. Bull. Inst. Pasteur 92:45-67. P2^25 PROBIOTIC ENTEROCOCCI IN ANIMAL FEEDS: PROPOSED METHODS FOR THE OFFICIAL QUALITY ASSESSMENT K. J. Domig(1), A. Weiss(1), H. K. Mayer(1), R. G. K. Leuschner(2) and W. Kneifel(1) (1) Department of Dairy Science and Microbiology, BOKU ^ University of Natural Resources and Applied Life Sciences, Vienna, Austria ; (2) Central Science Laboratory, Department for Environment, Food and Rural A¡airs, York, UK The enumeration and identi¢cation of probiotic enterococci strains has become an important item within quality control of probiotic animal feeds. As an important part of the EU project ``Methods for the o/cial control of probiotics (microorganisms) used as feed additives (SMT4-CT98-2235), protocols for the selective enumeration of enterococci and molecular biological procedures for the identi¢cation on strain level were developed and validated. Aiming at the selective enumeration of enterococci either present as a single component or in combination with other microorganisms (bacilli, bi¢dobacteria, lactobacilli, pediococci, yeasts), varieties of culture methods applying selective and non-selective agar media were screened. Bile Esculin Azide Agar was shown to be the most suitable medium. The enumeration method was validated within a collaborative study involving 20 laboratories from 12 European countries. For identi¢cation of probiotic strains, the performance of molecular biological typing methods based on Polymerase Chain Reaction

(PCR) and Pulsed Field Gel Electrophoresis (PFGE) were investigated using 74 enterococcal test strains. In contrast to the other typing methods, which vary regarding their reproducibility and discriminatory properties, PFGE is currently considered to be the ``golden standard'' for sub-typing enterococci. A proven PFGE protocol was slightly modi¢ed and examined with nine restriction endonucleases. It could be demonstrated that this method shows a pronounced e/cacy. The PFGE protocol was proposed as a standard method that is capable of checking the identity of enterococcal strains used as probiotic feed additives. P2^26 COMPROMISED PLANT HEALTH INCREASES THE RISK OF OPPORTUNISTIC COLONISATION BY HUMAN PATHOGENIC BACTERIA B. Du¡y Swiss Federal Research Center for Fruit Production, Viticulture and Horticulture (FAW), CH-8820 Wadenswil, « Switzerland Human disease outbreaks traced to foodborne Salmonella and E. coli O157:H7 are increasing worldwide. These bacteria opportunistically colonize a variety of plants, with some degree of host speci¢city. Here we demonstrate that host health in£uences colonization. S. enteriditis, S. typhimurium and E. coli O157:H7 populations established at low levels on healthy wheat roots (103 cfu/g root), tomato leaves (103 /g leaf) and strawberry (102 cfu/fruit). Human pathogen colonization was signi¢cantly increased on hosts infected with a fungal pathogen. Populations on wheat roots infected by Rhizoctonia solani or Gauemannomyces graminis, on tomato leaves infected by Botrytis cinerea, and on strawberry infected by B. cinerea averaged 104-105 cfu/g host tissue depending on the bacterium-disease combination. None of the human pathogens a¡ected fungal growth or plant disease development. Human pathogenic bacteria were not detected on conidia of B. cinerea sporulating on tomato leaves. These results indicate that increased risk of contamination of diseased crops should be considered in designing intervention strategies to reduce produce-linked outbreaks.

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P2^27 ANTIMUTAGENIC EFFECT OF HIGH PRESSURETREATED AND UNTREATED BRASSICA OLERACEA EXTRACTS ON HETEROCYCLIC AMINES-INDUCED SALMONELLA TYPHIMURIUM MUTANTS E. Edalucci(1), M. Benincasa(2), P. Rovere(3) and M. Tamaro(1) (1) Department of Biomedical Sciences, Microbiology Section, University of Trieste, via Fleming 22, I-34127 Trieste, Italy ; (2) Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, via Giorgeri 1, 34127 Trieste, Italy ; (3) SSICA, viale Tanara 31/A, I-43100 Parma, Italy The protective e¡ect exerted by vegetables in the diet against various toxic substances is well known. Several epidemiological studies indicate that the consumption of vegetables might deter the occurrence of cancer. The Ames test which has been extensively carried out to identify mutagens potential carcinogens is increasingly being used to evaluate the antimutagenic activity of potential anticarcinogens. In this study we have assayed the in£uence of di¡erent methods of food stabilisation, high pressure (HP) vs. pasteurisation (P), on the antimutagenic response to mutagenicity induced by heterocyclic aromatic amines (HAAs). HP-treated Brassica oleracea extracts displayed high inhibitory activity against mutagenicity induced by heterocyclic aromatic amines, as well as untreated samples; whereas conventional pasteurisation process reduced the antimutagenic properties of B. oleracea extracts. Protective e¡ects of the consumption of Brassica vegetables are related to the presence of a variety of molecules and/or enzymatic proteins, i.e. peroxidase. In this study has been showed that the peroxidase activity was present in both HP-treated and untreated samples. High pressure treatment of food as a means of preservation can guarantee high quality, elevated microbiological safety and the preservation of the organoleptic properties of the product. When the consumption of fresh vegetables is not achievable, the use of vegetable products treated with non-conventional methods of preservation, such as HP, should not be dismissed as a viable alternative, since in our study this method has been shown to maintain the biological properties of the fresh product.

P2^28 INHIBITORY EFFECT OF ENTEROCIN EJ97 AGAINST BACILLUS MACROIDES/B. MAROCCANUS ISOLATED FROM SPOILED VEGETABLE PUREE ¤ ¤ ¤ A. Galvez, Ma T. Garc|a, R. Lucas, N. Ben Omar, R. Per¤ ez-Pulido, and M. Mart|nez-Ca·amero L ¤ Area de Microbiolog|a, Facultad Ciencias Experimentales, ¤ ¤ Universidad de Jaen, 23071-Jaen, Spain Enterocin EJ97 is an antimicrobial peptide isolated from Enterocococus faecalis EJ97. This bacteriocin is active against food-borne pathogenic and/or spoilage bacteria (Listeria monocytogenes, Bacillus coagulans, B. macroides/ B. maroccanus, Staphylococcus aureus), a reason why it shows great interest for biopreservation. The peptide sequence and the genetic determinants of EJ97 have been elucidated. The puri¢ed bacteriocin showed bactericidal activity against strains of B. macroides/B. maroccanus isolated from spoiled zucchini puree. At low concentrations, enterocin EJ97 killed all viable cells from strain INRA P53-2 within 4 h of incubation at 37C, 24 h at 15C or 48 h at 4C. The bactericidal e¡ect was more pronounced at pH 7.0 compared to pH 9.0 or pH 5.0, and was potentiated by sodium nitrite. Inhibition of B. macroides/B. maroccanus INRA P53-2 in a commercial vegetable puree required a ten-fold higher bacteriocin concentration. The rate of bacterial killing by EJ97 in a vegetable puree increased as the incubation temperature was higher in the range of 4C to 37C. These results suggest that enterocin EJ97 may have a potential for use in the prevention of food spoilage caused by endospore-forming bacteria. P2^29 PATHOGENS BEHAVIOUR IN LOW-ACID FERMENTED SAUSAGES TREATED BY HIGH PRESSURE ¤ M. Garriga, T. Aymerich, B. Mart|n, B. Marcos and M. Hugas Institute for Food Research and Technology (IRTA), Meat Technology Centre, 17121 Monells, Spain Low-acid fermented sausages form a group of traditional Mediterranean products with a great variety within the di¡erent regions. Because of its physico-chemical characteristics, mainly a high pH, several pathogens of concern like Salmonella, Listeria monocytogenes and Staphylococcus aureus, could survive during the ripening process and even multiply. High hydrostatic pressure is an emergent technology with potential application for the extension

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of shelf life of several meat products and is already being applied in sliced cooked meat products. However, few investigations have been done with fermented meat products. The purpose of this study was to assess the application of high pressure processing for the safety of low-acid fermented sausages ripened at 12C. Two di¡erent products ``fuet'' and ``chorizo'' were manufactured and inoculated (103 cfu/g) with a cocktail of Salmonella, L. monocytogenes and S. aureus. Four batches were designed, 2 of them with additional inoculation of a mixture of selected lactic acid bacteria and staphylococci. The level of pressure (200 MPa, 10 min., 16C) applied before ripening gave the correct appearance for colour and texture of both products but was not e¡ective in diminishing the pathogens inoculated, compared with the non treated products. The inoculation of a mixture of selected strains gave better results than high pressure processing, specially regarding L. monocytogenes. No di¡erences between treatments were recorded for Salmonella, which diminished in treated and not treated products below the detection limit ( 6 10 cfu/g). When the non treated ripened products were treated at higher pressure (400 MPa), absence of salmonella in 25 g was achieved. P2^30 BIFIDOBACTERIA AS FAECAL INDICATORS: DETECTION AND IDENTIFICATION OF STRAINS ISOLATED FROM RAW MILK AND IN ENVIRONMENT OF FARMS ¤ F. Gavini, C. Grare, H. Ramboainiaina and M. Ca¡e Institut National de la Recherche Agronomique, BP 39, 59650 Villeneuve d'Ascq, France Within the framework of an European project (QLK1-CT2000-00805) the bi¢dobacteria are studied as new standard for ensuring hygienic conditions through the food chain (raw milk and meat products). Their use as indicators of faecal contamination in food is particularly interesting because : (1) they are an important part of the micro£ora in human and animal faeces; (2) the dominant Bi¢dobacterium species are di¡erent in human and in animal £ora; (3) they are anaerobes and thus cannot multiply in most foods. Bi¢dobacteria were isolated from 83% of 300 samples of raw milk at the teat from farms in North of France and in Belgium. Bi¢dobacteria were detected in all samples of manure tested and in air of 3 farms.The bi¢dobacteria were detected in 97% of cow dung (30 samples) and in 48% of faeces from other animal species (52samples) on farm. The same major species , B. pseudolongum (subspecies not identi¢ed yet), was isolated in all animal faeces that present bi¢dobacteria, as in cow dung, except for pigeons. The second isolated species was B. thermophilum in 20% of the faecal samples containing bi¢dobacteria in

animal species other than cow. The species B. pseudolongum was also isolated from 96% of raw milk at the teat samples that contained bi¢dobacteria, followed by B. thermophilum (in 4% of the raw milk at the teat samples, 4% of tanks). These results show that the main faecal contamination origin of raw milk is animal and likely cow dung. P2^31 LACTIC ACID BACTERIA ISOLATED FROM SOURDOUGH EXHIBIT ANTIFUNGAL PROPERTIES M. Giesova(1), L. B. Bullerman(2), J. Chumchalova(1) and M. Plockova(1) (1) Department of Dairy and Fat Technology, Institute of Chemical Technology, Technicka 5, 166 28 Prague 6, Czech Republic; (2) Department of Food Science and Technology, University of Nebraska, Lincoln, Nebraska, 68583-0919, USA In this study 60 Lactobacillus strains were isolated from 2 sourdough cultures of di¡erent origin. These strains were screened for their antifungal activity using Penicillium verrucosum as an indicator strain. Based on these preliminary results, 8 sourdough Lactobacillus strains together with Lactobacillus casei 154 and Lactobacillus rhamnosus VT1 were used for further studies. These strains were screened for their antifungal activity against 14 mold strains from the genera Aspergillus, Penicillium, Rhizopus, Cladosporium, Geotrichum and Fusarium. Each of Lactobacillus strains was inoculated into MRS or modi¢ed MRS (mMRS) agar, after solidi¢cation MRS (or mMRS) agar was overlaid with soft potato dextrose agar (PDA) (0.75 %w/w of agar). Suspension of mold spores (103) was spotted onto the PDA surface. Plates were incubated 21 days at 30C, colony diameter was measured every day. Aspergillus niger, Penicillium roqueforti, Penicillium digitatum and Geotrichum candidum were found to be the least inhibited mold strains and their growth and sporulation was delayed for 1-4 days. Aspergillus ochraceus, Penicillium commune, Cladosporium cladosporoides and Fusarium proliferatum were found to be the most inhibited mold strains. The highest inhibitory e¡ect was caused by Lactobacillus strains originating from ``original'' sourdough culture, while growing on MRS agar. Lactobacillus casei 154 exhibited the highest inhibitory e¡ect while growing on mMRS agar.

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P2^32 MICROBIOLOGICAL QUALITY OF SLOVENIAN CHEESE MADE FROM RAW COW'S MILK > K. Godic Torkar, M. Smolej, S. Golc Teger Laboratory for Dairying, zootechnical Dept., Biotechnical > Faculty, University of Ljubljana, Groblje 3, 1230 Domzale, Slovenia The presence of pathogen and indicator microorganisms in 14 semi-hard cheeses after one month ripening made from raw cow's milk by individual Slovenian producers was studied. Salmonella spp., Listeria spp., Proteus, sulphitereducing clostridia and Campylobacter spp. were not detected in cheese samples. E. coli was found in 4 (30 %) of samples on levels from 10 to 3400 CFU/g of cheese. Coagulase positive staphylococci were present in 9 (64 %) of samples ranged from 100 to 50 000 CFU/g of cheese. High number of enterococci (from 3.103 to 15.107 CFU/g) and coliforms (from 10 to 19.105 CFU/g) were detected. The same microorganisms were established in total of 98 samples taken during milking and processing of cheeses mentioned before: swabs from udders and milking machine, fresh raw and mixed milk from vat, whey and salt water. Listeria spp. was isolated from four cow's udders, one swab from milking machine, two milk and one whey samples, while none of examined samples were positive to presence of Salmonella spp. and Campylobacter spp. Proteus was present in 7 (7 %) cases of milk and whey. Clostridia were detected in 10 (10 %) samples (swabs, raw milk, whey). E. coli was isolated from 12 (12 %) samples of swabs, raw and mixed milk, whey and salt water. Because of improper milking and processing hygiene conditions, which are expressed also with high number of enterococci and coliforms, three (21 %) of tested cheese samples did not correspond to the microbiological criteria according valid regulations. P2^33 ASSESSMENT OF UNCERTAINTY OF MEASUREMENT IN PLATE COUNT METHOD ON 21C WITH PREINCUBATION > M. Gosnjak Institute of Public Health, 3000 Celje, Slovenia No measurement is perfect because it is always a¡ected by many factors. Uncertainty of measurement (u.m.) quanti¢es the range within the true value is likely to fall. Laboratories need to produce the estimated uncertainties where it is required by client or by the test speci¢cation or where the estimated uncertainty is necessary for the validity or

application of the result. The most important factors that may contribute to the uncertainty of measurement in microbiology are uncertainty of the inoculum volume, uncertainty due to the random scatter of the particles and uncertainty caused by human factor. We assessed the uncertainty of the plate count method at 21C with preincubation. The factors that could a¡ect the quality of the result were determined, their in£uence on the ¢nal result was calculated or assessed. The results of calibrations of pipettes were the base for the calculation of their uncertainty. Their in£uence was considered in the process of the preparation of test sample (0.24% of combined u.m.) and in the preparation of dilutions (0.81% of combined u.m.). We considered also the uncertainty of measurement arising from the non uniform distribution of micro-organisms in the sample (Poisson distribution) which represents 22.7% of combined u.m.. Technologist to technologist variation during the sample preparation accounted for 66.4% and 8.4% of combined u.m. during the reading of results. We estimated also the personal error of the analyst at the reading of results (1.45% of combined u.m.). P2^34 COMPARISON OF PCR-BASED TECHNIQUES TO DISTINGUISH FUNGI ON HAZEL NUTS M. Grube(1), K. Pelant(1) and M. Stelzl(2) (1) Institute of Botany, Holteigasse 6, 8010 Graz, Austria; (2) Hygienicum, Andritzer Reichsstrasse 44, A-8045 Graz/ Austria Food spoilage fungi produce a range of toxic compounds and thus represent a risk to human health. The detection of toxins usually involves chromatographic and other techniques for direct risk assessment associated with a food item. However, fungi often remain undetermined in such assays, which ignores the biological diversity of toxin production in di¡erent fungal strains. Classic determination of food fungi involves phenotypic or physiological characters, such as morphology expressed on various culture media. Beside this, numerous molecular typing techniques based on PCR have been developed for recognition of fungal strains. Some of the genetic markers amplify variable portions of speci¢c genes, which are then sequenced for strain comparison. Other approaches distinguish strains by the presence of PCR-fragments after ampli¢cation with toxin speci¢c genes or with primers for anonymous loci which may be present in the entire genome. We applied various PCR-techniques to distinguish strains of fungi isolated from hazel nuts and present a comparison of these approaches.

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P2^35 HEAT, ACID AND OSMOTIC SHOCK AND NISIN RESISTANCE IN LISTERIA MONOCYTOGENES M. M. Guerra(1), F. A. Bernardo(2), and M. Adams(3) (1) Escola Superior de Hotelaria e Turismo do Estoril, ¤ ¤ Portugal; (2) CIISA/Laboratorio de Inspeccao Sanitaria, °< ¤ ria, Universidade Tecnica ¤ Faculdade de Medicina Veterina de Lisboa, Portugal; (3) School of Biomedical and Life Sciences, University of Surrey, UK Food-borne pathogens are commonly stressed during food processing. This may reduce the e¡ectiveness of food preservation hurdles and food safety may be compromised if pathogens develop stress resistance as a response. The effect of the application of several preliminary stress shocks (heat, acid and osmotic) on the resistance of Listeria monocytogenes Scott A to nisin action was investigated. A sublethal dose of acid (HCl, pH 5.5) or NaCl (7%, wt/vol) was added to Listeria monocytogenes broth cultures in both the exponential and stationary phases of growth, and the cells were allowed to grow for 1h. Cells in both phases were also heat shocked at 45C for 1h. The stress-adapted cells were then exposed to 100 IU/ml of nisin for 90 minutes. Viable counts of the pathogen were determined at time intervals throughout the experiment. The preliminary stresses increased resistance to the antilisterial activity of the bacteriocin only in stationary phase cells ( s 1 log di¡erence in the inactivation of nonshocked and shocked cells). This di¡erence decreased over time for acid and osmotic shocked cells. Acid shock provided the highest initial protection to stationary phase cells, but heat shock was the one that provided a more prolonged resistance. These results clearly show that the sensitivity of Listeria monocytogenes to nisin will depend on the cell's previous history. This could have important pratical implications on how best to use bacteriocons in multifactorial food preservation systems. (age of culture phase growth, the type of stress, time of exposure to nisin). P2^36 THE MICROBIAL QUALITIES OF MILK AND ITS BY-PRODUCTS IN TIRANA CITY Z. Haxhiaj and Y. Xhumari Institute of Public Health, Rruga Alexander Moisiu, No. 80, Tirana, Albania The transition from a centralized state controlled system towards a free market based one comported drastic changes in the food production and distribution system of Albania. The lack of incentives in the former system,

gradually installed a chronicle insu/ciency of availability for food products. The adopted initial policy for ¢lling this gap was an unconditional support of all free initiatives within the food system. This policy, created within a short time the expected abundance of food products on the market, but it was associated to the creation of considerable problems on food safety and the consequent impairment of public health. The aim of the present study is to perform an assessment of the microbial qualities of milk and its by-product and to identify dangerous products on public health. Samples related products bought randomly on the Tirana market. The samples (about 350) were analytically examined for the following parameters as well : Aerobe Meso¢le microorganisms, Coliforms, Escherichia coli, Salmonella, Staphylococcus aureus, Molds. The microbiological quality of milk and its products is not in needed level. The results over the norms are: Aerobe Meso¢le 53.4 %, Coliforms 54 %, E.coli 45 % , Molds 43.1%. All the kind of analysed products presented high values for previous parameters, The faecal contamination is primary in general contamination. Staphylococcus aureus is founded in one sample of butter and two samples of curd. Salmonella and Shigella are not present in examined samples. At the end of our study we have given some recommendations for improvement of the situation. P2^37 GROWTH OF SALMONELLA SEROVARS IN HENS' EGG ALBUMEN AS AFFECTED BY STORAGE W. Messens, L. Duboccage, K. Grijspeerdt, M. Heyndrickx and L. Herman Ministry of the Flemish Community, Agricultural Research Centre-Ghent, Department of Animal Product Quality and Transformation Technology, Brusselsesteenweg 370, B-9090 Melle, Belgium Salmonella enterica serovar Enteritidis is a common foodborne pathogen, the transmission of which is primarily associated with the consumption of contaminated shell eggs. There is no agreement about the growth of Salmonella when deposited in egg albumen near room temperature. The objective of our study was to aid solving the problem of con£icting results of growth/no growth of Salmonella in fresh and stored albumen at room temperature. Di¡erent S. enterica serovars, including serovar Enteritidis, were tested for growth at 20C in separated albumen, both fresh and up to 3 weeks stored, upon inoculation with approx. 31 cells per ml. Shell eggs were stored as such or the albumen was stored separately. The serovar Enteritidis did not behave di¡erently compared to the other serovars. A pronounced growth occurred more frequently and up to a one-log unit higher level in fresh than in stored albumen. Since growth in the albumen

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was una¡ected by the storage condition, we have no indication that nutrients or some factors negating the inhibitory properties of the albumen leak out from the yolk during the 3 weeks storage at 20C. A better growth in fresh albumen was also observed when inoculating approx. 8 cells in the albumen of whole shell eggs. In this case, growth occurred up to higher levels than when separated albumen was inoculated possibly due to migration of Salmonella towards the yolk. It was proven that the enhanced growth in fresh albumen compared to the stored albumen was at least partly caused by a pH e¡ect. P2^38 ANTIMICROBIAL, ANTIOXIDANT AND ANTIMUTAGENIC ACTIVITY OF PLANT EXTRACTS CONTAINING PHENOLIC COMPOUNDS ¤ ¤ ¤ ¤ M. Hnilova, R. Hlad|kova and M. Drdak Department of Food Technology and Biotechnology, Faculty of Chemistry, Brno University of Technology, Purky> nova 118, 61200 Brno, Czech Republic Plant phenolics, especially £avonoids, are currently of growing interest owing to their supported functional properties in promoting human health. The plant extracts pre¤ pared from Daisy (Bellis perennis), Sheperas purse (Cap¤ s tail (Equisetum articum), Sage sella Bursa-pastoris), Colta (Salvia o/calis), Chamomile (Matricaria chamomilla), Plantain (Plantago lanceolata), Green tea (Camellia sinensis) and standards of £avonoids were used to evaluate their antimicrobial, antioxidant and antimutagenic activities. Antimicrobial screening of plant extracts and standards of £avonoids against selected foodborne microbes (Bacillus subtilis, Micrococcus luteus, Escherichia coli, Saccharomyces cerevisiae, Zygosaccharomyces rouxii, Hansenula anomala, Pichia farinosa, Candida vini) was conducted in this study. The tests were carried out using di¡usion methods ^ Hole Di¡usion Assay (HDA) and Minimal Inhibition Concentration (MIC). The plant extracts showed an entire antimicrobial spectrum, especially extract prepared from Daisy (Bellis perennis) had excellent antimicrobial activity against common foodborne microorganisms and on growth on Bacillus subtilis, Micrococcus luteus, Saccharomyces cerevisiae and Zygosaccharomyces rouxii exhibited comparable inhibition as a chemical preservative, potassium sorbate. The antioxidant activity of plant extracts and standards of £avonoids was determinated by a radical (DPPH) decoloration assay and by a Randox test. Observed antioxidant activity was compared with a total content of £avonoids in plant extracts. The antimutagenic activity of plant extracts and standards of £avonoids was determinated by Ames test on auxotri¢c mutant Salmonella typhimurium TA98. All studied plant extracts showed slight antimutagenic activity.

P2^39 THE INDICATION OF CHROMIUM (TRIS) PICOLINATE AS DNA DOUBLE STRAND BREAKER S. Jenko-Brinovec(1), A. Plaper(2), M. Golob(2), J. Kos(2,3), and P. Raspor(1) (1) University of Ljubljana, Biotechnical Faculty, Ljubljana Slovenia; (2) KRKA d.d., Research Dept. of New Entities, Novo mesto, Slovenia; (3) Jozef Stefan Institute, Dept. of Biochemistry and Molecular Biology, Ljubljana, Slovenia Trivalent chromium is an essential mineral important in the metabolism of fats and carbohydrates. Its dietary intake is suboptimal and therefore chromium supplements are used frequently. Among them chromium picolinate (Cr(pic)3) is the most popular. Recently, questions have been raised about the safety of Cr(pic)3, especially with regards to its mutagenic and clastrogenic e¡ects. Our work demonstrates that Cr(pic)3, in the presence of cellular reductant ascorbate and H2O2, cleaves DNA. Both single-strand and double-strand DNA breaks are formed. The cleavage is probably the consequence of hydroxyl radicals formed during the reoxidation of reduced form of Cr(pic)3 with H2O2, since the addition of DMSO, an e¡ective àOH scavenger, completely prevented the formation of nicked or linearized plasmids. Interestingly, chromium chloride in the same system produced only single strand DNA breaks. It seems that the same picolinate ligands which enable trivalent chromium to pass the cell membranes more easily also make the chromium more dangerous once it gets inside the cell. Besides the formation of ROS and consequently oxidative DNA damage, Cr(pic)3 also inhibited the gyrase relaxation activity in vitro. As Cr(pic)3 does not bind to the DNA the inhibition is most likely due to the binding of Cr(pic)3 to the enzyme, and in this way preventing its enzymatic activity or its binding to the substrate. Chromium picolinate also had citotoxic e¡ect on mammalian cells in the cell culture. Obviously, the risk-bene¢t ratio of Cr(pic)3 has not yet been adequately characterized and further studies should be employed before taking chromium supplements on daily basis. We thank the Ministry of Science and Technology of the Republic of Slovenia (Project no. : J4-7454-490) for ¢nancial support and S.J.B. would like to thank to the Ministry of Education, Science and Sport of the Republic of Slovenia for grant (S4-490-007/21462/2000).

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P2^40 CYTOTOXICITY ASSESSMENT OF AEROMONAS HYDROPHILA ON VERO CELL CULTURES N. R. Karabasil Department for Food Hygiene and Technology of Animal Origin, Faculty of Veterinary Medicine, University of Belgrade, 11000 Belgrade, Bulevar JNA 18. Serbia and Montenegro Aeromonas hydrophila, introduced as a ``new'' foodborne pathogen, are environmental bacteria, ubiquitous in water, soli, and in many foods. The purpos of this paper is to provide data for cytotoxic acitvity of Aeromonas hydrophila. Six Aeromonas hydrophila strains were used, ¢ve food-derived strains previosly isolated from freshwater ¢sh obtained on local retail market place and one type strain (CCM 4528). Cytotoxicity were estimated on Vero cells monolayers, 45 minutes to 24 h at 37C after ¢ltrates of A. hydrophila added. In e¡orts to obtain is it heat-labile or heat-stable cytotoxin, ¢ltrates of A. hydrophila were heated during 30 minutes at 56C and 5 minutes at 100C. Treated ¢ltrats were added to Vero cell monoleyers and cytotoxicity were estimated after 24 h at 37C. Tested ¢ltates of Aeromonas hydrophila caused cytotoxic e¡ect at the Vero cells, but the intensity of changes di¡ered in dependance of the ¢ltrate. In all tested strains, cytotoxic e¡ect was induced with heat-labile cytotoxin. P2^41 OLIGODYNAMIC WATER DISINFECTION METHOD R. R. Khaydarov and R. A. Khaidarov Scienti¢c Devices Designing Department, Institute of Nuclear Physics, 702132, Ulugbek, Tashkent, Uzbekistan The purpose of this work was to improve existing oligodynamic water disinfecting method: destroying impacting low concentrations of metal ions on bacteria in the water. Experiments have shown that the maximum disinfecting e¡ect is obtained by using the composition of 4 various metal ions with Ag as the main component. It was investigated dependence of bacteria killing time against e¡ective concentration of metal ions in the water, initial bacteria concentration (from 103 to 1012 CFU/L) and their types, in£uence of di¡erent cations and anions (Cl-, SO42-, S2-, Fe2+, Fe3+, etc.) in the water on disinfecting process. It was shown that when the concentration of metal ions in treated water does not exceed American drinking water regulation limit, the killing time of Legionella-pneumophila and typhoid-paratyphoid is 60 minutes, salmonellas and

cholera is 30 minutes when their concentration is 1000 1/ liter. Besides during 20 days there was not bacteria growth in treated water samples. Increasing the initial bacteria concentration causes increasing the bacteria killing time in accordance with given above formula. Spores are more resistant to destruction than vegetative bacteria. Combination of described method with ultraviolet and electric ¢eld was used for killing Bacillus subtilis as the acceptable analogue spores. Bacteria destruction time was 2 hours at an initial concentration of 1000 CFU/litre. P2^42 INFLUENCE OF METHODOLOGICAL CRITERIA ON THE RESULTS OF ANTIBIOTIC SUSCEPTIBILITY TESTING OF LACTIC ACID BACTERIA B. Kogler(1), K. Domig(1), H. P. Lettner(2), W. « Kramer(2) and W. Kneifel(1) (1) Department of Dairy Research & Bacteriology, Agricultural University, Gregor Mendel-Str. 33, A-1180 Vienna, Austria ; (2) Lactosan Starterkulturen GMBH & Co KG, Industriestr. West 5, Kapfenberg, Austria Known as safe starter cultures lactic acid bacteria (LAB) have not received much attention in regard to antimicrobial resistance. Recently, the susceptibility of LAB to transferable antibiotic resistance has become a subject of concern. Therefore, antibiotic susceptibility testing programmes gain in signi¢cance. The present study demonstrates that there are many variables a¡ecting the results of antimicrobial susceptibility tests. Di¡erences in media, inoculum density, incubation conditions and testing methods in£uence the interpretation of antibiotic breakpoints. In the current study several lactobacilli strains were tested for susceptibility and resistance against thirteen antibiotics by the agar dilution method and the broth microdilution method as suggested by the National Committee for Clinical Laboratory Standards (NCCLS). Modi¢cations of the standardized NCCLS methods concerned the culture medium, the inoculation density and the incubation conditions. An obvious increase of the MIC value was noted when an unde¢ned medium (e.g., MRS or BHI) and an inoculum density (e.g., 106 CFU/spot) was used. The same tendencies in MIC di¡erences were seen upon aerobic and anaerobic conditions as well as agar and broth method. There is still a lack of an international agreement on a standardized antimicrobial susceptibility testing procedure and on breakpoints. Consequently, the establishment of a standardized antimicrobial resistance surveillance system of LAB, at least within Europe, would facilitate the comparison of resistance data among di¡erent laboratories.

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P2^43 EXTREMOPHYLIC MICROSCOPIC FUNGI FROM THE SOUTHERN SLOPES OF THE CAUCASUS AS A PRODUCERS OF STABLE ENZYMES G. I. Kvesitadze, L. I Kutateladze, T. I. Alexidze, T. A. Sadunishvili, E. G. Kvesitadze Durmishidze Institute of Biochemistry and Biotechnology, Academy of Sciences of Georgia, 380059 Tbilisi, Georgia Extremophilic microorganisms attract special attention because of their ability to produce enzymes stable to critical conditions. Such strains are also suitable for the cloning of genes of stable enzymes. With this aim, 3996 micromycete cultures were isolated from di¡erent soil-climatic zones of the Caucasus: 15% were toxic, 6% thermophilic, 1.5% alkaliphilic, 0.8% acidophilic and above 2% halophylic. The collection was screened on the ability to produce enzymes stable to extreme conditions. Among the cultures of extremophiles, over 30 strains producing stable enzymes widely used in food processing, such as cellulases and xylanases, heat and acid-stable glucoamylases, acid-stable protease and some other enzymes has been revealed. The majority of these enzymes were puri¢ed and their basic physical-chemical properties determined. Stability, kinetic and thermodinamic characteristics of these enzymes allows their wide application in food processing. For instance, the increase of the cultivation temperature of Allesheria terrestris from 40 to 48-490 C resulted in the formation of a new endoglucanase which was inactivated only by 20% at 650C for 6h in the absence of substrate. The molecular weight of the heat-stable enzyme is 69 kD; the isoelectric point (pI) is 6.4 and the Michaelis constant (Km) is 6.6 g/ liter. The thermostable endoglucanase of Allescheria terrestris reacts with polyclonal antibodies against the unstable endoglucanase of Trichoderma reesie. The physiology of the strain-producers have been investigated. Using mutagens (UV irradiation, ethylenimine, and nitrosomethylurea), 12 mutant strains synthesizing considerable amounts of extracellular cellulases have been created. Protoplasts fussion of obligately thermophilic (Allesheria terrestris) and mesophilic (Aspergillus niger) strains resulted in a fusant displaying a higher thermal stability of endolgucanase compared with the parents. Some strains (Aspergillus, Trichoderma, Allesheria, etc) are being screened on their growth inhibition by plant preparation which based on current investigation (INTAS-Food 00-0727) could be successfully applied for food spoilage prevention to improve its quality and extend shel£ife.

P2^44 CHARACTERISATION OF STAPHYLOCOCCUS EPIDERMIDIS ENTEROTOXIN TYPE C FROM A FOOD OUTBREAK S. Loncarevic, T. Mathisen, R. Kristensen, L. M. Rorvik Section for Feed and Food Microbiology, National Veterinary Institute, P.B. 8256 Dep., 0033 Oslo, Norway Although Staphylococcus aureus intoxication is a common food-borne illness, no case of S. epidermidis food-borne intoxication has been reported. However, S. epidermidis producing staphylococcal enterotoxins (SET) has been recovered from sheep and goat milk in some investigations. In 2001, a series of suspected staphylococcal intoxications occurred in a hotel in Norway. Foodstu¡s obtained at the three di¡erent occasions showed the presence of di¡erent staphylococcal species. SET was recovered only from few bacterial cultures and not directly from the implicated food samples. In the ¢rst outbreak only one isolate of S. aureus produced SET A, in the second two isolates of S. epidermidis isolates produced SET C and in the third no SET or staphylococcal bacterial isolates were detected from food samples. Two isolates from meat pudding and cheesecake, con¢rmed by API and additional biochemical tests as S. epidermidis, were shown to produce SET C. Reversed passive latex agglutination (SET-RPLA) was used for detecting the SET A-D production, while multiplex PCR-technique was used to reveal genes coding for SET types A-E and G-J. Furthermore, molecular typing of the S. epidermidis isolates by Pulsed Field Gel Electrophoresis (PFGE) con¢rmed that they shared identical pattern. In order to compare these S. epidermidis SET C genes with published S. aureus SET C genes, sequencing will be performed. The results will give more information about SET produced by S. epidermidis and possible involvement of S. epidermidis in food poisoning.

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P2^45 MOLECULAR IDENTIFICATION OF TRICHOSPORON CASEORUM SP. NOV. AND TRICHOSPORON LACTIS SP. NOV. ISOLATED FROM CHEESES K. Lopandic(1), T. Sugita(2), W. J. Middelhoven(3), H. Prillinger(1), M. Herzberg(4), J. W. Fell(5) (1) Institute of Applied Microbiology, University of Agriculture, Muthgasse 18, A-1190 Vienna, Austria ; (2) Department of Microbiology, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo, 204-8588 Japan; (3) Laboratory of Microbiology, Wageningen University, P.O. Box 8033, 6700 EJ, Wageningen, The Netherlands; (4) PhilipsUniversity Marburg, Karl von Frisch Str., 35043 Marburg, Germany ; (5) University of Miami, Rosenstiel School of Marine and Atmospheric Sciences, 4600 Rickenbacker Causeway, Key Biscayne, FL 33149, USA During the studies of yeast contaminants associated with milk products two strains from unripened soft and fresh cheese (Salzburg region Austria) were isolated showing phylogenetic relatedness to the genus Trichosporon. The extensive analysis included biochemical and physiological characterisation, partial sequencing of 26S rDNA and complete sequences of 18S rDNA, ITS1/ ITS2 and IGS1 regions as well as RAPD-PCR ¢ngerprinting. The results demonstrated that the strains represent two new species and in reference to the source of isolation the strains are named T. caseorum and T. lactis. The new species are closely related to the human pathogens T. ovoides and T. inkin on the phylogenetic trees based on 26S and 18S rRNA encoding genes and the internal transcribed spacer (ITS) regions. The length and sequences of the IGS1 regions as well as the RAPD-PCR patterns are remarkable di¡erent in comparison to those of the closest phylogenetic relatives. Both species could be distinguished phenotypically and show strong ability to decompose mutagenic and toxic compounds such as phenol, hydroquinone, cresole and cinnamate. The two new Trichosporon species did not grow well in liquid media below pH 5.5 that made a deviation from standard physiological tests necessary. The failure to assimilate inositol and growth on D-glucosamine can be used to distinguish both species from T. inkin and T. ovoides.

P2^46 ANTIBIOTIC RESISTANCE IN DAIRY ENTEROCOCCI: WHAT SHOULD WE FEAR? ¤ F. S. Lopes(1), T. C. Ribeiro(1), A. Eugenio(1), M. Abrantes(1), R. Tenreiro(2), M. T. Barreto Crespo(1) (1) IBET/ITQB, Apartado 12, 2781-901 Oeiras, Portugal; ¤ (2) FCUL, Campo Grande, Edif|cio C2, Piso 4, 1700 Lisboa, Portugal Enterococci are part of the comensal microbiota of humans and animals, and are present in the environment, namely in some food products, like milk and cheese. In recent years they have become a subject of increasing concern due to their multiple antibiotic resistance to the majority of antibiotics used in the health care system. The emergence of multiple resistant strains is, in part, a consequence of their capability to exchange genetic material, namely through plasmids and transposons. Studies conducted in the last decades have been concerned mostly with clinical isolates and the environmental contribution to this serious antibiotic problem has been neglected. Therefore, some antibiotics, for which resistance is usually associated with mobile genetic elements, namely gentamycin, erythromycin, vancomycin and tetracycline, have been a subject of study in 200 enterococci isolated from milk and cheese in our laboratory. High-level resistance to gentamycin has not been detected in these isolates, and MIC values for this antibiotic are still low. Nevertheless, erythromycin resistance is widely disseminated in these dairy isolates, but not transposon Tn917. However, this resistance is easily lost, together with resistance to chloranphenicol, cipro£oxacin, spiramycin, nor£oxacin, o£oxacin and ceftriazone, in the presence of the curing agent acridine orange, indicating a mobile element mediated transfer. Therefore, studies of transfer of resistance of these resistances are also being performed. P2^47 RAPID DETECTION OF BACILLUS CEREUS FROM FOODS BY PCR ANALYSIS M. Manzano, C. Giusto, L. Iacumin, C. Cantoni and G. Comi Department of Food Science, University of Udine, via Marangoni 97, 33100 Udine, Italy B. cereus, a well known food poisoning organism, has been identi¢ed as the causative agent in a number of food poisoning outbreaks many of which unreported because confused with those of other pathogens. Improving microbial safety and extending the shelf-life of industrial

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food products has always been an important concern to the food industry. There is still confusion regarding how many di¡erent enterotoxins are produced by B. cereus. In fact it produces a high variety of toxins. Furthermore enterotoxins could be produced also by non-B. cereus species. Isolates of B. circulans, B. lentus, B. mycoides and B. thuringiensis, which are strictly related to B. cereus, demonstrated positive results using RPLA assay. Some authors proved di/culties to distinguish between B. cereus, B. mycoides and B. thuringiensis because they referred as B. cereus also the B. mycoides and B. thuringiensis. In this work we developed a couple of speci¢c primers for B. cereus and speci¢c DNA extraction techiques to obtain PCR products from food without any previous enrichment. Salad, meat, rice, co¡ee and milk samples were used to verify the procedure. The gyrB gene used as target for the primer design was useful to obtain ampli¢ed products only for B. cereus also in the presence of a low number of B. cereus cells and a high number of other natural contaminant microrganisms. Moreover positive results were obtained using the PCR detection of B. cereus directly in food samples, and the data are con¢rmed by classical microbiological analyses. P2^48 CHARACTERIZATION OF S. JORGE CHEESE, A PORTUGUESE RAW COW'S MILK CHEESE: EVOLUTION OF MICROBIAL AND PHYSICOCHEMICAL PROFILES J. Marcelino Kongo(1,2) and F. X. Malcata(2) (1) CIRN ^ Universidade dos Acores, Rua Mae de Deus, < ° 9500 Ponta Delgada, Acores-Portugal; (2) Universidade ° ¤ Catolica Portuguesa ^ Escola Superior de Biotecnologia, Rua Dr. Antonio Bernardino de Almeida, 4200-072 PortoPortugal S. Jorge cheese is a Portuguese dairy product, which has been manufactured for more than 350 years in the Azores island with the same name from cow's raw milk, in the absence of any commercial starters; this cheese was granted an AOC status in 1984. Emerging market rules have enforced stricter quality and safety requirements, so a full characterization of the Portuguese artisanal cheese sold in largest numbers is in order, especially because such a cheese is the main economic support of S. Jorge islanders. The aim of this work was thus to produce experimental data that would characterize S. Jorge cheese. Toward such goal, the artisanal technology used in the production of the cheese was monitored for six months. Milk and cheese samples were collected every three weeks at seven di¡erent factories, and standard plating on such media as KAA, Rogosa, MSE, VRBGA, M17 and OGYEA was done so as to ascertain the evolution of

the microbial pro¢le, followed by tentative identi¢cation via API galleries. In addition, plating on XLDN, PALCAM, OXFORD, MSA and VRBGA media was done so as to grasp the safety of actual consumption of said cheese, namely in terms of Listeria, Enterobacteriaceae, Staphylococcus and Salmonella spp. The physicochemical and microbiological data produced were subject to multivariate statistical analysis, in attempts to correlate them. P2^49 DETECTION OF HISTIDINE DECARBOXYLASE GENE IN LACTIC ACID BACTERIA ISOLATED FROM PORTUGUESE WINES A. P. Marques(1), M. C. Leitao(1), V. Basto(1), R. Ten< reiro(3), M. V. San Romao(1,2) < ¤ (1) Instituto de Biologia Experimental e Tecnologica/Insti¤mica e Biologica ^ Universidade ¤ tuto de Tecnologia Qu| Nova de Lisboa. Apt, 12, 2781-901 Oeiras, Portugal; (2) ¤ Estacao Vitivin|cola Nacional, 2565-191 Dois Portos, Por°< tugal; (3) Departamento de Biologia Vegetal/Centro de ¤ " Genetica e Biologia Molecular, Faculdade de Ciencias, Universidade de Lisboa, Campo Grande, 1740-016 Lisboa, Portugal Biogenic amines (BA) are basic nitrogenous compounds found in wine and in other fermented food products. Histamine is one of the major BA in wine and one of the causative agent of physiological distress such as hypotension and digestive disturbances. Lactic acid bacteria are considered the main responsible for the BA formation. Some of these bacteria contain enzymatic systems that allow them to metabolise the peptides accumulated after alcoholic fermentation of wine, during yeast autolysis, with the subsequent formation of amino acids that can, after decarboxilation, originate BA. Some Portuguese wines, special red wines, present histamine contents ranging the 8 mg/L. It is therefore important to ¢nd the microorganisms responsible for histamine formation in Portuguese wines. The aim of this work was the detection of the histidine decarboxylase (HDC) gene in strains of lactic acid bacteria (LAB) isolated from Portuguese wines. The detection of HDC gene was done by PCR and colony hybridization with non-isotopic DNA probe. DNA hybridization is a convenient way to detect undesirable histamine-forming strains. Speci¢c primers designed from hdcA gene of Lactobacillus buchneri (DSMZ 5987) were used to amplify the internal part of the HDC gene. Ampli¢ed DNA was puri¢ed and sequenced. The sequence showed highest degree of homology with sequences of HDC gene in the database of Genbank. DNA probe was generated by PCR and labelled with digoxigenin (DIG High Prime DNA Labelling and Detection Starter

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Kit II ^ Roche). The HDC gene was detected in some of the tested strains. This work was partially supported by Agro Medida 8.1 ¤ Program, Project n. 33 ^ Formacao de aminas biogenicas °< ¤ gicos existenno vinho ^ caracterizacao de produtos enolo °< tes no mercado. P2^50 º ° º BRUCELLOSIS IN MUNICIPAL OF KERCOVE DURING THE PERIOD 1983-90 I. Z. Mehmedi and M. R. Mehmedi The Department of Infective Diseases, Medical Center Kichevo, Kichevo 6250, M.Tito bb, Republic of Macedonia Objective: to present clinical features and serological data of brucellosis in the municipal of Kercove, Republic of Macedonia. Methods : patients with brucellosis followed between January 1983 ^ December 1990 were retrospectively evaluated. Initial evaluation included complete blood count urinalysis and serological tests (standard tube Brucella agglutination test-STA). Diagnosis of brucellosis was made by following criteria : a) history of exposure, b) compatible clinical picture of disease, c) brucella serum agglutination titer more then 1/160. Results: In the above mentioned period were registered 66 cases with brucellosis, 14 of which are female and 52 male. The average age was 41þ30 years. The most frequent features in the patients disease of acute Brucellosis are: fever 46(69,69%); joints pain( mostly in the spine and in the hips) 53 (80,30%); sweat 45(68,18%); loss of appetite 30(45,45%); headache 34(51,51%); hepatosplenomegaly 7 (10,60%). In the diagnosis of human brucellosis the best results were shown with the Rose Bengal test, which was positive 100% of patients with acute brucellosis, then with the Coombs test positive in 96,96%. The test by Wright with 93,93%. Conclusion : The most frequent features in the patients disease of acute Brucellosis are: joint pain(80,30%), fever (69,69%), sweat (68,18%). In the diagnosis of human brucellosis the best results were shown with the Rose Bengal test, which was positive 100% of patients with acute brucellosis.

P2^51 EFFECT OF TEMPERATURE AND VISCOSITY ON BACTERIAL INACTIVATION BY HIGH PRESSURE HOMOGENIZATION A. Diels, E. Y. Wuytack and C. W. Michiels Department of Food and Microbial Technology, Katholieke Universiteit Leuven, Leuven, Belgium High pressure homogenization is a unit process used in the food and pharmaceutical industry primarily to make or stabilize emulsions or suspensions. The development of applications at increasingly higher pressure has created an interest in taking bene¢t from the microbial inactivation that occurs during this process. We have studied the inactivation of E. coli by high pressure homogenization (100 ^ 300 MPa) in bu¡ered suspensions adjusted to di¡erent viscosities with polyethylene glycol, and at di¡erent initial temperatures of the cell suspensions (5 ^ 50C). The results indicate that (i) bacterial inactivation increases with initial temperature and decreases with viscosity of the suspension; (ii) at least up to 35C, the increased inactivation due to temperature can be entirely explained by the reduced viscosity of the suspensions at elevated temperature; (iii) above a certain temperature, bacterial inactivation exceeds the inactivation that would be predicted by the reduced viscosity e¡ect, suggesting that thermal e¡ects start to contribute to cellular inactivation in addition to mechanical e¡ects. These experiments should contribute to a better understanding of the bactericidal mechanisms of high pressure homogenization, and to the development of novel applications. P2^52 IMPROVING THE MICROBIOLOGICAL SAFETY OF SEEDS AND SPROUTS BY LOW-DOSE GAMMA IRRADIATION L ¤ ¤ Cs. Mohacsi-Farkas, E. Andrassy, A. Bruckner, V. Gergely « Department of Microbiology and Biotechnology, Szent Ist¤ ¤ ¤ van University, Somloi ut 14-16, 1118 Budapest, Hungary Consumption of raw seed sprouts has been linked to several outbreaks during the last decade in countries around the world. Most of these foodborne outbreaks were due to contamination with Salmonella spp. or Escherichia coli O157:H7. In order to avoid these outbreaks, treatments to reduce the pathogenic microorganisms on sprouts or on seeds are needed. In the frame of an IAEA Co-ordinated Research Project our aim was to investigate the e¡ect of low dose gamma-radiation on natural micro£ora and germination of selected seeds and sprouts. Furthermore, an

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optimal dose that provides their microbiological safety was also to be determined. Alfalfa and radish seeds were irradiated using 60Co gamma-radiation source and were treated with doses of 1, 2 and 3 kGy. After the treatment, seeds germination or yield ratio and changes of microbial load were analysed. To investigate the e¡ect of irradiation on pathogenic microorganisms, seeds were inoculated with a suspension of avirulent strains of E. coli O157 and Listeria monocytogenes. Results showed that irradiation with 2 kGy dose decreased the total aerobic plate count by 2-3 log cycles on seeds. The yield ratio decreased by 30%. Total aerobic plate count of sprouts germinated from irradiated seeds achieved the same level on all samples. 1 kGy dose decreased the number of L. monocytognes by 11.5 log cycles on seeds, and 3 log cycles on sprouts. E. coli O157 proved to be much more radiation -sensitive than L. monocytogenes. On the seeds 1 kGy dose reduced the number of E. coli O157 by 3 log cycles, and on sprouts a 100,000-fold reduction was achieved. P2^53 FREQUENCY OF LISTERIA MONOCYTOGENES IN DAIRY AND MEAT PRODUCTS J. Nowroozi(1), A. J. Negad(2), S. Shahidi(2) (1) Iran University of Medical Sciences and (2) Islamic Azad University, Tehran, Iran Listeria minocytogenes is a Gram positive, non-sporeforming, rod shaped bacterium and has often been associated with raw and pasteurized milk, dairy products, such as soft cheese, meat products and vegetables. The aim of this study was to ¢nd the frequency of L. monocytogenes in dairy and meat products distributed in Tehran city and detection of plasmids by electrophoresis. Samples of dairy products (such as cheese, butter, cream), milk; sausage and frankfurt was added to TSYEB and was refregrated (cold enrichment method). Then, two selective medium (Palcam agar and Listeria Selective agar) was used for growth and biochemical tests for identi¢cation. Bacterial plasmids were isolated by alkali lysis and has been electrophoresis through agarose gel, then, observed and photographed. From 61 dairy products, 18 samples (29.5%) and from 52 sausage and frankfurt, 8 samples (15.38%) were contaminated with L. monocytogenes. From 26 L. monocytogenes isolates, 5 (19.23%) of them contained plasmid DNA with 2.3 kbp molecular weight. Contamination of dairy products and meat products (sausage and frankfurt) by L. monocytogenes can occur during preparation, transport and distribution. So, because of the high consumption of these products in our country, it is necessary to surveillance the production and distribution to prevent of incidence of listeriosis in susceptible people.

P2^54 COMBINED ACTIVITY OF BACTERIOCINS AND HIGH PRESSURE TREATMENTS ON THE SURVIVAL OF STAPHYLOCOCCUS AUREUS IN RAW MILK CHEESE ¤ ¤ J. L. Arques(1), E. Rodr|guez(1), P. Gaya(1), M. Medina(1), B. Guamis(2) and M. Nu·ez(1) ¤ (1) Tecnolog|a de Alimentos, INIA, Carretera La Coru·a km 7, Madrid, 28040 Spain; (2) CeRTA, Universitat ' Autonoma de Barcelona, Bellaterra,08193 Spain. Staphylococcus aureus is one of the most common causes of food-borne illness. The combined activity of bacteriocin-producing lactic acid bacteria used as protective cultures and high pressure treatments on the survival of S. aureus during ripening of raw milk cheese was investigated. S. aureus was inoculated at approximately 105 cfu/ml into raw milk. Milk cultures of ¢ve strains of lactic acid bacteria producing di¡erent bacteriocins were added at 0.1%. Semi-hard cheese was manufactured according to usual procedures. High pressure treatments of 300 MPa (P300) during 10 min at 10C or 500 MPa (P500) during 5 min at 10C were applied to 2-day-old cheeses. On day 3, S. aureus reached 6.46 log cfu/g in control cheese. Counts were 0.16-0.46 log units lower in cheeses with bacteriocin producers, and 0.45 and 2.43 log units lower respectively in P300 and P500 cheeses without bacteriocin producers. Synergistic inhibitory activity was detected in treatments with P500 and bacteriocin producers, the highest inhibitory activity being recorded for the combined treatment P500 and nisin A. S. aureus was not detected on day 20 in any P500 cheese. On day 60, counts of S. aureus were 5.30 log cfu/g in control cheese, and 0.02-0.81 log units lower in cheeses with bacteriocin producers. Counts in P300 cheeses without bacteriocin producers were 2.84 log cfu/ ml, and 0.05-0.58 log units lower with bacteriocin producers.

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P2^55 MYCOLOGICAL AND MYCOTOXICOLOGICAL INVESTIGATIONS DURING MICROMALTING PROCESS OF BARLEY M. Skrinjar(1), O. Grujic(1), I. Peric(1), J. Pejin(1), V. Krstanovic(2) and S. Kocic(1) (1) Faculty of Technology, University of Novi Sad, Bulevar Cara Lazara 1, 21000 Novi Sad, Yugoslavia; (2) Faculty of Food Technology, University of J. J. Strossmayer in Osijek, F. Kuhaca 18, 31001 Osijek, Croatia Mycological and mycotoxicological analyses were undertaken of raw materials (unsorted barley, ¢rst class barley), semi-products (green malt, dried malt) and ¢nal products (malt), for brewing industry. Water samples used for barley steeping were also analyzed. The total count of moulds in 1g/ml of sample was determined, followed by isolation and identi¢cation of the mould. Mycotoxicological analysis by TLC enabled qualitative and quantitative determination of a£atoxins B1 and G1, ochratoxin A and zearalenone. Determination of the total count of moulds per 1g of barley before and after germination, as well as malt was carried out by grains disposal (10) directly to Saubouraud maltose agar containing chloramphenicol and oxytetracyclin, while in 1ml of steeping water it was determined by the application of the standard Koch's method. Average count of moulds per 1g of unsorted barley before malting process was 2.5¾102, and after ¢rst, second and third day of steeping this was 3.7¾102, 5.6¾102 and 2.1¾103, respectively. First class barley contained 2.6¾102 moulds per g, and after the ¢rst, second and third day of steeping 3.5¾102, 4.1¾102 and 1.4¾103, respectively. 1 ml of water taken from steeping vessel before process of malting contained 81.5 moulds, and after the ¢rst, second and third day of barley steeping 1.0¾102, 2.8¾102 and 1.2¾104, repectively. After germination, the total count ranged from 3.6¾105 (unsorted barley) to 5.6¾105 (¢rst class barley) moulds per g. In 1g of malt with roots, the total count ranged from 8.7¾104 (unsorted barley) to 2.5¾104 (¢rst class barley). Similarly, in malt this ranged from 3.4¾104 (unsorted barley) to 2.4¾104 (¢rst class barley) moulds per g. Between 65 and 80% of moulds isolated from barley were from the group Dematiaceous Hyphomycetes. After steeping, their share in mycopopulations increased to 9095%. During the third day of steeping and during the period of germination, signi¢cant presence of Penicillium species was registered. In the course of malt drying, Penicillium spp. and Zygomycetes were registered again at high levels. None of the samples investigated was contaminated by toxins.

P2^56 A BIOSENSOR SYSTEM WITH PARAMECIUM CAUDATUM FOR THE ASSESSMENT OF POTENTIALLY TOXIC FUNGAL METABOLITES B. Pernfuss, J. Stemer, R. Poeder and H. Strasser Institute of Microbiology, Leopold-Franzens University of Innsbruck,Technikerstrasse 25, 6020 Innsbruck, Austria In the course of the EU-funded project QLK1-2001-01391 ``Risk Assessment of Fungal Biological Control Agents (BCA's)'' it is investigated by a multidisciplinary European consortium if fungal compounds enter the food chain and if they pose a health risk. Besides the extraction, identi¢cation and quanti¢cation of fungal metabolites from BCA's information on intra-species and inter-species variability in the production of selected metabolites, as well as data on their spatial-temporal distribution and their rate of decomposition will be provided. Fast and accurate screening and testing protocols should be developed taking into account existing regulatory testing programmes and the need to integrate endpoints. One of the key issues of the project is the development of in vitro toxicity assays such as sensitive cell lines (i.e. biosensors) to detect selected metabolites. Plant and animal cell cultures should act as a substitute for animal testing, and must facilitate high throughput screening for detecting toxicity and in vitro toxicity. In this context we have optimised conditions for the assessment of the ciliate Paramecium caudatum as a ``biosensor''. This sensitive test organism can detect fungal secondary metabolites in an inexpensive testing system and give an indication of their toxic potential. E¡ective dose data (ED50) of P. caudatum with oosporein, one of the secondary metabolites produced and excreted by the entomopathogenous anamorphic fungus Beauveria brongniartii, indicate that this biosensor system is even more sensitive than hamster tumour cells, as well as selected human and insect cell lines. P2^57 THE INFLUENCE OF COOKING ON THE GENOTOXIC POTENTIAL OF MEAT AND MEATSTUFFS CONTAINING FOOD ADDITIVES AND MEGATERIN V. Y. Ponomarev, O. B. Ivanchenko, O. A. Reshetnik Dep. of Food Technology, Kazan State Technological University, K. Marks str., 68, Kazan, Russia Traditionally, microbial enzymes are widely used as a research tools in genetic engineering, medicine and molecular biology. Last time, microbial protease was used in food industry for improving the quality of meat. The animal

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meat and model meat products containing food additives and microbial enzyme of Bacillus megaterium (commercially known as ``megaterin'') were investigated for genotoxicity before and after cooking under high temperature. The liquid procedure was used for assessing the potency of meat extracts to cause DNA damages in DNA-repair test. The repair-pro¢cient and repair-de¢cient (recA-, polA-, uvrA-) Escherichia coli trp auxotrophs were used. Water extracts from meat and meat products were tested with and without microsomal activation mixture. The animal meat and the multicomponent salting mixture, containing sodium nitrite, phosphates, chlorides, showed no genotoxic properties. Enzyme of B. megaterium appears to possess DNA damaging activity in uvr A- and pol A- mutants of E. coli strains. Perhaps, the growth inhibition was related with proteolytic activity of enzyme. The genotoxicity of meat with food additives increased after addition of megaterin. The considerable DNA-damaging e¡ect on E. coli test strains was shown. Cooking at the high temperatures led to the loss the DNA-damage activity of model systems. However, the extracts from roasted meat and model meat products induced DNA-damage after metabolic activation in vitro with enzymes of rat liver microsomal fraction. P2^58 PREVALENCE OF LISTERIA MONOCYTOGENES IN BROILER MEAT AT RETAIL LEVEL IN ESTONIA K. Praakle, J. Lunden, M. Lindstrom, M.-L. Hanninen and « « H. Korkeala Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, P. O. Box 57, FIN-00014 University of Helsinki, Finland Listeria monocytogenes is a ubiquitous pathogen that has been implicated within the past decade as the causative organism in several outbreaks of foodborne disease. It is well established that any fresh food product of animal or plant origin may harbour varying numbers of L. monocytogenes. However, there are no studies on the occurrence of L. monocytogenes done in Estonia. Therefore we have studied the prevalence of L. monocytogenes in broiler meat at retail level in Estonia. A total of 92 samples of broiler thigh meat produced in Estonia and 87 samples imported to Estonia (56 from Denmark, 21 from Hungary, and 10 from other countries) were examined. For the detection of L. monocytogenes the ISO 11290-1:1996 method was used. The method includes pre-enrichment in half-Fraser broth and selective enrichment in Fraser broth. Both enrichment media were plated on selective palcam agar and LMBA (Listeria monocytogenes Blood Agar). Typical colonies were further streaked on blood agar plates and veri¢ed as L. monocytogenes by Gram-reaction, catalase test, and

API LISTERIA. The contamination rates for L. monocytogenes were 87% (80) for the broiler meat produced in Estonia, 38% (21) for the meat imported from Denmark, and 76% (16) for the broiler meat imported from Hungary. The characterization of the isolates by genotypical method is in progress. P2^59 TRANSFER OF ANTIBIOTIC RESISTANCE GENES IN DAIRY ENTEROCOCCI ¤ T. C. Ribeiro(1), F. Lopes(1), A. Eugenio(1), M. Abrantes(1), R. Tenreiro(2), T. Crespo(1) (1) IBET/ITQB, Apartado 12, 2781-901 Oeiras, Portugal; ¤ (2) FCUL, Campo Grande, Edif|cio C2, Piso 4, 1700 Lisboa, Portugal Enterococcus are ubiquitous bacteria frequently found in the environment and they are part of the commensal microbiota of humans and animals. They can occur in several traditional cheeses made with raw milk. Enterococcus are considered as emerging human pathogens and they are often associated with hospital-acquired infections. A reason for the rise of nosocomial infections related to enterococci is their ability to develop resistance against most antibiotics currently used. The rapid spread of antibiotic resistances in a wide variety of bacteria is mainly due to the location of antibiotic resistance genes on mobile genetic elements such as plasmids and transposons. Food-borne enterococci, like those from milk and cheese, are able to rapidly horizontally transfer their antibiotic resistance genes to other members of the genus and potentially to other bacteria of clinical importance. To evaluate this risk, conjugation phenomena are being studied for antibiotics for which resistance is probably associated with plasmids, such as kanamycin, tetracycline, erythromycin, ampicillin and vancomycin. In order to identify the plasmid(s) carrying these resistance determinants, PFGE and southern blot hybridisation are being used. These ¢lter mating assays are being performed using Enterococcus faecalis FA2-2, 13 food isolates previously cured of erythromycin resistance and 1 cured of tetracycline resistance (acridine orange, 45 Wg/mL) as recipient strains. Enterococci with vancomycin resistance genotype (vanA, vanB and vanC), already determined, are being used as donor strains.

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P2^60 SEROLOGICAL IDENTIFICATION OF LISTERIA MONOCYTOGENES ISOLATED FROM FOOD USING SLIDE AGGLUTINATION TEST í ¤ T. Rupel, T. Majstorovic, M. Sedlbauer, N. Trunk Institute of Public Health of the Republic of Slovenia, Centre for Environmental Health, Department of Sanitary > Microbiology, Grabloviceva 44, 1000 Ljubljana, Slovenia Listeria monocytogenes (LM) is subdivided into serotypes on the basis of somatic (O) and £agellar (H) antigens. Thirteen serotypes of LM have been identi¢ed. Because the vast majority of cases of human disease are caused by only three serotypes (1/2a, 1/2b and 4b), serotyping is only minimally helpful in epidemiologic investigations. Strains of LM isolated from food have been collected at Department of Sanitary Microbiology, since 1994. Pure cultures of Listeria isolated from foods were con¢rmed with the api-Listeria test (bioMerieux). Serological slide agglutination tests were done on all isolates of LM, using commercially prepared antisera Bacto- Listeria O Antisera Types 1,4 and Poly (Difco). The 56 strains were subdivided into 2 serovars. The 46 strains of LM were serotype 1, six strains were serotype 4 and the test for 4 strains was negative. The goal of subtyping LM isolates was to determine which serotypes were the most frequently isolated from food samples. P2^61 A MODIFIED NESTED PCR METHOD TO DETECT NON-CULTURABLE E. COLI CELLS IN DRINKING WATER A. Rust, A. J. B. Zehnder and W. Koster « Swiss Federal Institute For Environmental Science and Technology, Ueberlandstr. 133, CH-8600 Duebendorf, Switzerland In order to guarantee safe drinking water, the microbial quality is controlled routinely by analysing water samples for bacterial parameters. A simple, inexpensive but time consuming culturing method for the detection of E. coli has been used for many decades. We developed a faster polymerase-chain-reaction (PCR)-method with two subsequent runs to detect E. coli in drinking water. A DNAse digestion step was introduced in the course of the sample preparation to avoid false positive results by free DNA of dead bacteria. The PCR method was evaluated as alternative to the classical culture method. We spiked drinking water samples with E. coli cells at low concentrations and stored them at cold temperature intending to simulate en-

vironmental conditions. Tests with both methods were performed at intervals ranging from days up to weeks. At early time points, the two methods delivered comparable results. However, at later time-points, the E. coli cells became non-culturable, whereas the results obtained by PCR showed no signi¢cant decrease of ampli¢able DNA. The PCR gives positive results for all E. coli cells containing chromosomal DNA inside an intact cell envelope, also for those cells which are in a non-culturable state. At the moment we do not recommend the application of this PCR technique for routine analysis but see its bene¢ts in environmental water research. P2^62 QUANTITATIVE RISK ASSESSMENT FOR SALMONELLA IN FEED FOR FATTENING PIGS IN SWITZERLAND I. Sauli(1), J. Danuser(1), A. H. Geeraerd(3), K. Bogli« Stuber(1), B. Bissig-Choisat(1), K. D. C. Stark(1) and C. « Wenk(2) (1) Swiss Federal Veterinary O/ce, Schwarzenburgstrasse 161, 3003 Bern, Switzerland; (2) Swiss Federal Institute of Technology, Institute for Animal Sciences, Universitat« strasse 2, 8092 Zurich, Switzerland ; (3) BioTeC ^ Biopro« cess Technology and Control, Department of Chemical Engineering, Katholieke Universiteit Leuven, W. de Croylaan 46, B-3001 Leuven, Belgium In pig production, the carrier pig, contaminated feed and environment are currently considered the most signi¢cant sources of infection. Pigs rarely show clinical signs, thus undetected Salmonella carriers can enter the food production chain. In a ``Farm to Fork'' food safety concept, the production of safe feed is the ¢rst step for ensuring safe foods and is therefore crucial. The aims of this study were (1) to investigate the prevalence of Salmonella throughout the production of feed for fattening pigs in Switzerland, and (2) to perform a quantitative risk assessment for estimating the risk that Salmonella-contaminated batches of feed are produced in Swiss mills. Samples of major ingredients were collected at delivery, and samples of ¢nished feed were collected in the storage bins. Dust samples were moreover collected at critical points in one feedmill. A model for simulating the behaviour (growth, survival or inactivation) of the pathogen throughout the production steps was build using predictive microbiology tools. Various scenarios were created considering the production processes commonly implemented in Switzerland. Using the results of the prevalence survey as input data in the simulation model, the risk that Salmonella-contaminated batches of feed are produced in Swiss mills was assessed. Preliminary results of the survey revealed a low prevalence of Salmonella in both ingredients and ¢nished feed. The

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risk that Salmonella-contaminated batches of feed are produced depended on the production process, but was low for all scenarios. Final results of the prevalence survey and of the quantitative risk assessment will be presented. P2^63 OCCURENCE, FACTORS INFLUENCING THE RESISTANCE AND SAFETY IMPLICATIONS OF HIGHLY HEAT RESISTANT SPORES P. Scheldeman(1,2), K. Goossens(1), A. Pil(1), L. Herman(1), P. De Vos(2), O. Guillaume-Gentil(3), J. Hansen(4), S. Foster(4) and M. Heyndrickx(1) (1) DVK-CLO, Dept. Animal Product Quality, Brusselsesteenweg 370, 9090 Melle, Belgium ; (2) Universiteit Gent, Laboratory for Microbiology (WE10V), K. Ledeganck¤ straat 35, 9000 Gent, Belgium ; (3) Nestle Research Center, P.O. Box 44, CH-1000 Lausanne 26, Switzerland ; (4) University of She/eld, Dept. Molecular Biology and Biotechnology, Firth Court, Western Bank, She/eld S10 2TN, UK Highly heat resistant spores (HRS) of Bacillus sporothermodurans may survive sterilizing and ultra high temperature (UHT) treatment of milk. Spores originating from the dairy farm display variable but lower heat resistances than spores freshly isolated from UHT-treated dairy products. REP-PCR and ribotyping indicated that the majority of these B. sporothermodurans isolates from UHT- products belong to a clonal lineage (HRS-clone), whereas clearly more heterogeneity was found among dairy farm isolates. Also, HRS-clone spores grown under lab conditions seem to gradually lose their heat resistance upon successive culturing. It was investigated if these features of clonal origin but di¡erent heat resistances were correlated with the in£uence of sublethal stress conditions. Indeed, a sublethal H2O2 treatment resulted in a signi¢cant heat resistance increase of HRS-clone spores. To evaluate the importance of this experience, the intrinsic heat resistance of spores from a variety of Bacillus species present at the dairy farm was studied next to the question whether an increased heat resistance following peroxide stress is restricted to B. sporothermodurans or if other spore forming bacteria such as Bacillus cereus can also show this. Understanding the mechanism of this heat resistance induction is of great importance to accommodate industrial heat- and/ or other treatments in the light of food safety and quality. Therefore, a fundamental study of endospore properties important for heat resistance was initiated, aiming to compare the chemical structure (spore peptidoglycan composition and mineralization) of B. sporothermodurans, Paenibacillus sp. and B. cereus spores displaying di¡erent heat resistances under di¡erent stress conditions, and to determine any correlation between these parameters and the spore resistance properties.

P2^64 EFFECTS OF GROWTH CONDITIONS ON BACILLUS CEREUS GROWTH AND TOXIN PRODUCTION P. Schmitt, T. Clavel, M. Jobin and C. Duport ¤ ¤ ¤ UMR A408 INRA/Universite d'Avignon ``Securite et Qual¤ ¤ ¤ ite des Produits d'Origine Vegetale'' INRA Domaine St Paul 84914 Avignon cedex 9, France The objective of this presentation is to report and discussed about our results and those cited in literature on the e¡ects of growth rate, physico-chemical and nutritional parameters in£uencing Bacillus cereus growth and toxins production. B. cereus occurs widely in the environment and is frequently isolated from cooked chilled food and vegetable. B. cereus is responsible for two types of food associated illness: the emetic syndrome and the diarrheal syndrome. The former is due to a small molecular weight cyclic toxin, cereulide. The diarrheal syndrome results from enterotoxins production by B. cereus growing in the small intestine of host following ingestion of viable cells and/or spores, making this condition a true toxicoinfection. Some contradictories results had been published resulting from (i) the high diversity among B. cereus strain (ii) use of rich or poor media, with or without various glucides (iii) di¡erent physico-chemical conditions such. In addition, majority of experiments were performed on uncontrolled batch cultures. Conversely to batch culture, chemostat permits cells to grow inde¢nitely at a de¢ned growth rate and biomass concentration, in an environment of stable substrates and products. For this reason, B. cereus was cultivated in regulated conditions and in chemostat. Our results shown that in aerobiosis, the maximum HBL amount was detected during the middle of exponential growth phase, whereas it took place at the early stationary growth phase for anaerobic growth cultures. The greatest HBL production occurred under anaerobiosis In this condition, experiments carried out in chemostat shown that the production of toxin was regulated by the growth rate and glucose: HBL production decreased as growth rate increased and was inhibited by high glucose concentrations (300 mM, 5.4 % w/v).

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P2^65 RISK ASSESMENT OF FUNGAL BIOLOGICAL CONTROL AGENTS ^ BEAUVERIA BRONGNIARTII AND ITS METABOLITE OOSPOREIN: NEEDS AND DEEDS C. Seger(1), D. Erlebach(2), U. Griesser(1), S. Sturm(1), H. Stuppner(1), and H. Strasser(2) (1) Institute of Pharmacy, Department of Pharmacognosy, Leopold-Franzens University of Innsbruck, Innrain 52, A6020 Innsbruck; (2) Institute of Microbiology, LeopoldFranzens University of Innsbruck, TechnikerstraÞe 25, A6020 Innsbruck Much has been investigated in the ¢eld of mycotoxins produced on feed and fodder however ecological studies on the formation, signi¢cance and fate of secondary metabolites produced by fungi in the environment are scarce. In order to perform a responsible risk-assessment of fungal biological pest control agents investigations on the environmental enrichment and the signi¢cance of secondary metabolites released by entomopathogenic fungi are needed. It is the major aim of the EU-RTD-project RAFBCA (QLK1-CT-2001-01391) to obtain information on the temporal-spatial distribution of metabolites in the biocontrol agents as well as in the applicated environment. Beauveria brongniartii (Ascomycota, anamorph of Clavicipitales) is known to be a very selective and highly virulent entomopathogenic fungus used in the control of Melolontha melolontha (chockchafer; coleoptera, scarabaeidae). Although there have been numerous experiences in the use of this fungus in infested areas since the seventies only few data have been published on the ecotoxicological impact and relevance of metabolites synthesised and secreted by B. brongniartii. The major metabolite ^ the bis-dihydroxybenzoquinone derivative oosporein is poorly characterized and its fate in biological matrices is unknown (i.e. soil and crop). Data on the physico-chemical characterization of oosporein will be presented and their impact on the risk assessment process will be discussed. P2^66 ENTRY OF ESCHERICHIA COLI INTO CORN PLANT VIA THE ROOT SYSTEM S. Sela(1), N. Bernstein(2), and R. Pinto(1) (1) Department of Food Science ; (2) Institute of Soil Water and Environmental Sciences, Agricultural Research Center, The Volcani Center, Beth-Dagan, Israel The incidence of human bacterial pathogens on fresh fruits and vegetables has been a growing concern in industrial-

ized countries. Contamination of crops might occur at any stage of the food chain. In several cases, manure or contaminated irrigation water has been suspected as the primary source of contamination. Recent studies have demonstrated that E. coli and Salmonella enterica can enter the plants via the root system, translocate to the shoot, and survive in the internal tissues of lettuce and tomato, respectively. Since conventional washing treatments are ine¡ective in removal of bacteria from inner tissues, such contaminated plants might pose a health hazard for consumers. To verify that other plants are susceptible to this route of contamination we have examined the uptake of antibiotic-resistant E. coli by corn plants. Young corn seedlings were grown hydroponically in nutrient solution supplemented with E. coli. Plants were divided into three groups, one with intact roots, another with root-tip-removed (damaged) and the third group with the entire root system removed. The upper plant parts were tested for bacterial internalization at 48 hrs after inoculation. We found that E. coli was indeed capable of internalization via the root system of both intact and root-damaged corn seedling. The uptake of bacteria was signi¢cantly higher in plants with damaged or without roots compared to intact plants. These results support the notion that human pathogens might contaminate fresh vegetables pre-harvest by internalization via the root system. P2^67 ISOLATION AND IDENTIFICATION OF XEROTOLERANT YEASTS FROM FRUIT YOGHURT AND QUARK SAMPLES º S S °. °enses, Z. Y. Ozbas° Food Engineering Department, Hacettepe University, Beytepe 06532, Ankara, Turkey Xerotolerant yeasts have been known to be the major spoilage £ora in high-sugar foods. In order to prevent high ¢nancial losses caused by xerotolerant yeasts, isolation and identi¢cation of this group of yeasts should be performed, properly. Additionally, recent developments in the medical ¢eld have resulted in a number of emerging pathogens, also including some foodborne strains. In this study, swollen packages of 50 fruit yoghurt and 4 quark samples were collected from retail markets and investigated for the presence of xerotolerant yeasts. During the study, Dichloran 18% Glycerol (DG18) agar, Tryptone Glucose Yeast Extract (TGY) agar, Malt Extract Yeast Extract 50% Glucose (MY 50G) agar and Malt Extract Yeast Extract 40% Sucrose (MY 40S) agar which has been modi¢ed by us, were used as isolation media. In total, 13 yeast strains were isolated from the samples. For the identi¢cation of these isolates, ID 32C strips and some other identi¢cation tests were performed. 13 strains of asporog-

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enous yeasts were identi¢ed as eight species belonging to two genera. The major species identi¢ed were; Candida famata, C. guilliermondii, C. glucosophila, C. kefyr, C. krusei, C. lambica, C. parapsilosis, Rhodotorula glutinis. Using the ID 32C, 31 di¡erent carbon assimilations were investigated for the isolates. All isolates were capable of glucose assimilation, whereas only 77% of them assimilated sucrose, 69% maltose, 69% galactose, 54% ra¢nose and 15% lactose. Approximately 69% of the isolates were found as xerotolerant yeasts. Also approximately 84% of the isolates were found to ferment glucose. P2^68 THE INHIBITION OF GROWTH OF VARIOUS HUMAN AND PHYTHOPATHOGENIC FUNGI BY TEA EXTRACTS IN VITRO M. Shamtsyan, A. Komolov, N. Zolnikova St. Petersburg State Institute of Technology (Technical University), St. Petersburg, Russia Microscopic fungi are the main pathogens of agricultural plants and are causing valuable losses of the harvest. Also they can produce various mycotoxins, which are the reason of heavy disfunctions of animal and human organisms. Our studies were focused on the e¡ect of green and black tea extracts on the growth of microscopic fungi, belonging to 20 di¡erent genera. Micromycetes were cultivated on the agar media with or without addition of tea extracts. It appears, that the addition of de¢nite amounts (0.05 ^ 0.5 %) of tea extracts to the growth media causes a signi¢cant slowing in the growth of all of the species of tested micromycetes. It was also estimated, that the fungistatic e¡ect of green tea extracts was higher than of the extracts obtained from black tea. Green tea extracts caused the most pronounced e¡ect in growth reduction on the tested species from the genera of Fusarium (growth was reduced for 55 ^ 75 % for di¡erent species), Alternaria (60 ^ 70 % reduction), Aspergillus (60 ^ 65 % reduction) and Botritis (55 ^ 60% reduction). Investigations on the in£uence of green tea extract on the morphology of fungi were carried out. The obtained results indicate the possibility for the utilization of green tea extracts as a natural fungistatic product. One that can protect plants, animal feed and food from the pathogenic fungi and act as a natural antifungal preservative, and also in research practices for the isolation of micromycetes and their puri¢cation.

P2^69 EFFECTS OF LACTATE AND DIACETATE ON VIABILITY OF LISTERIA MONOCYTOGENES AND CHANGES IN ITS CYTOPLASMIC PROTEINS E. Mbandi and L. A. Shelef School of Science, Wayne State University, Detroit, Michigan, U.S.A. The antilisterial e¡ects of sodium lactate, diacetate, and their combination in ready-to-eat meats, pH 6.3, were studied using Listeria monocytogenes Scott A (serotype 4b). Each of the salts alone (2.5% lactate and 0.2% diacetate) delayed growth of the pathogen during refrigerated (5C) and abuse (10C) storage for up to 45 days. Combination of the salts was most inhibitory, resulting in listericidal e¡ects at 5C and listeriostatic e¡ects at 10C. A proteomic approach was used to study changes in the cytoplasmic proteins of the organism. Cells were treated for 6 h with each salt and their combination as above in a chemically de¢ned medium, pH 6.3.SDS-PAGE gels showed similar band patterns in untreated and treated samples. Data from two-dimensional electrophoresis showed that diacetate caused the highest changes in total number of proteins (198 vs. 131 in control, and 150 in lactate treatment), highest unmatched proteins (124 vs. 53 in lactate), highest increase in expression (20 vs. 5 in lactate), and highest number of novel protein (90 vs. 45 in lactate). The highest number of repressed proteins was observed in the combination treatment (41 vs. V30 in the single salt treatment). No di¡erence showed in the expression level of prfA protein, the regulator of virulence genes of the pathogen, using Western blot analysis. Results suggest that survival of L. monocytogenes in the presence of lactate and diacetate is similar to survival mechanisms during NaCl or acid stress conditions, and that expression of virulence proteins may not be a¡ected by treatment with these salts.

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P2^70 HIGH DOSES OF PROBIOTICS (DP16, DP28, DP38, DP66, AND DP88) HAVE NO ADVERSE EFFECT ON THE HEALTH OF MICE A. Liu(1), Y. Gao(1,3) and Q. Shu(2) (1) Mt Albert Health Research Laboratory and (2) Microbial Science Research Group of the New Zealand Institute for Crop and Food Research Limited, Private Bag 92 169, Auckland, New Zealand ; (3) Instititute of Food, Nutrition and Human Health, Massey University, Auckland, New Zealand The safety of probiotic strains DP16, DP28, DP38, DP66, DP88, and LA1 (a commercial probiotic strain used as a positive control), was studied in mice (BALB/c) by feeding at a high dose. Mice (6V8 week-old) were acclimatized on a skim milk powder based diet for 14 days prior to being randomly allocated into 7 groups (n=8). Following acclimatization, mice were orally administered DP88 [Group (G) 7], DP66 (G6), DP38 (G5), DP28 (G4), DP16 (G3), and LA1 (G2) (5 x 1010 CFU/mouse/day) from day 0 to day 7. The negative control mice (G1) did not receive any probiotic organisms. The e¡ect of the probiotics on the health of mice was assessed by monitoring the health appearance, behavior, presence of diarrhoea, feed intake, water intake, and liveweight gain of the animals. On day 14, mice were euthanased by an iso£uorance overdose and bacterial translocation to mesentery lymphatic node (MLN), spleen, liver, kidney, and blood were also measured. No abnormal clinical signs were observed in any of the groups during the period of the experiment. Also, there was no signi¢cant di¡erence in feed intake, water intake, and liveweight gain between the control and other groups (P s 0.05). No bacteria were detected in blood, kidney, liver, and spleen of animals from any group. There were no bacteria in the MLNs of the negative control mice. However, small numbers of bacteria were found in the MLNs of some animals from G2 (2/8), G3 (1/8), G4 (1/8), G5 (2/8), G6 (1/8) and G7 (1/8); The numbers of bacteria in the MLNs were positively correlated with the faecal numbers of lactic acid bacteria (P 6 0.01, r=0.6). Interestingly, DNA ¢nger printing analysis showed that the bacteria isolated from the MLNs were di¡erent from the strains used in this study. These results demonstrated that probiotic strains DP16, DP28, DP38, DP66, and DP88 did not translocate to systemic tissues and had no adverse e¡ect on the health of mice.

P2^71 MYCOLOGICAL AND MYCOTOXICOLOGICAL QUALITY OF SOME AGRICULTURAL PRODUCTS TAKEN FROM ``HEALTHY FOOD'' STORES M. Skrinjar, V. Injac, I. Sedej and S. Kocic University of Novi Sad, Faculty of Technology, Boulevard Cara Lazara 1, 21000 Novi Sad, Yugoslavia Mycological and mycotoxicological investigations of some agricultural products (corn £akes, integral rice, dried grapes, wheat £akes, oats £akes) were performed. The products were taken from ``healthy food'' shops. Five samples of every product were tested. In all samples the total number of moulds in 1g was examined, as well as identi¢cation of isolated species. Investigation of the presence of a£atoxin B1 (AB1), G1 (AG1), ochratoxin A (OA) and zearalenone (ZEA) have been involved in mycotoxicological analysis. Two media were used for isolation of moulds: Sabouraud maltose agar (SMA) and MY50G medium for xerophiles. Qualitative and quantitative determination of mycotoxin was done applying the TLC method according to A.O.A.C. The greatest number of investigated samples were contaminated with moulds. In 1g of corn £akes from 3 to 9,6¾102 , integral rice from 10 to 40, dried grapes from 2 to 7 and in wheat £akes from 2 to 2,8¾102 moulds were found. The most frequent species belonged to genera Cladosporium, Alternaria, Eurotium, Aspergillus and Penicillium. OA was established in 1 sample of corn £akes (40.0 Wg Á kg -1) and integral rice (80.0 Wg Á kg -1), in 2 samples of dried grapes (40.0 and 56.0 Wg Á kg -1 ) and wheat £akes (80.0 and 160.0 Wg Á kg -1) and in 3 samples of oats £akes in traces up to 160.0 Wg Á kg -1. ZEA was found in 1 sample of corn £akes and wheat £akes at concentration of 192.0 Wg Á kg -1 and in 2 samples of oats £akes (384.0 and 480.0 Wg Á kg -1). No sample was contaminated with AB1 and AG1. P2^72 VANCOMYCIN RESISTANT ENTEROCOCCI FROM NORWEGIAN POULTRY FARMS ESTABLISHED AFTER THE BAN OF AVOPARCIN M. SÖrum(1), G. Holstad(1), A. Lillehaug(1) and H. Kruse(2) (1) National Veterinary Institute, Oslo, Norway; (2) Norwegian zoonotic centre, Oslo, Norway An association has been documented between the use of avoparcin as a growth promoter and the occurrence of vancomycin resistant enterococci (VRE) in poultry production. Studies have shown that VRE have persisted up

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to 3-4 years after avoparcin was banned in Norway in 1995. VRE have also been shown to survive on the farms. The aim of this study was to examine the prevalence of VRE in poultry at Norwegian poultry farms established after the ban of avoparcin was implemented. Faecal samples from 39 farms established after 1995 (sample farms) and from 25 farms previously exposed to avoparcin (control farms) were examined. The concentration of VRE and generic enterococci were determined. One colony from each positive sample was selected, identi¢ed, and tested for presence of the vanA gene by PCR. The susceptibility to narasin was also examined. VanA-type VRE were detected in samples from 25 out of 39 sample farms (64%) compared to 23 out of 25 of the control farms (92%). There seemed to be higher concentration of VRE in the control samples. No resistance towards narasin was detected. This study shows that VRE are present at poultry farms previously not exposed to avoparcin. The proportion of VRE positive farms was lower for recently established farms than for farms previously exposed to avoparcin. The concentration of VRE at the recently established farms seems to be lower than at farms previously exposed to avoparcin. P2^73 MYCOLOGICAL AND MYCOTOXICOLOGICAL PICTURE OF SOME CATEGORIES OF WHEAT KERNELS T. Stojanovic(1), M. Skrinjar(2), M. Saric(2) (1) High School of Food Technology, 18400 Prokuplje, Yugoslavia; (2) Faculty of Technology, 21000 Novi Sad, Yugoslavia According to the type and degree of infection, all samples of wheat were classi¢ed into four groups: healthy, dark germed, little fusarious and very fusarious kernels. They all derived from the two experimental localities, 1 and 2, and they were of the following sorts : Kg56S and Evropa 90 from the experimental locality 1 and Kg56S, and Nora from the experimental locality 2. Sixteen samples from each group were analysed. Mycotoxicological examination (a£atoxin B1 and G1, ochratoxin A and zearalenone) were carried out by using mycotoxin method which was described by Balzer et al. (1978). A total number of moulds per kernel was as folowing: in healthy kernels from 0.75 to 1.21, dark germed from 1.52 to 2.51, little fusarious from 2.12 to 2.94, and in very fusaruous kernels between 2.20 and 3.21. Mould isolated from all wheat samples were classi¢ed into 13 genera and 34 species. The following genera of mould were isolated: Acremonium, Alternaria, Aspergillus, Botrytis, Chaetomium, Cladosporium, Fusarium, Gilmaniella, Helminthosporium, Monilia, Penicillium, Verticillium and Ulocladium. A£atoxin B1 isolated from

very fusarious wheat kernels deriving from the experimental locality 1 at the concentration to 2.3 mkg/kg. Contamination by ochratoxin A was more frequent, and the concentrations ranged from 11 mkg/kg at the healthy kernels to 48 mkg/kg at the very fusarious ones. The most severe infections were caused by zearalenone which was found in as many as 75% of the analysed samples. Its concentrations ranged from 160 mkg/kg to 500 mkg/kg. P2^74 MEASUREMENT OF STAGES DURING GERMINATION AND OUTGROWTH OF INDIVIDUAL SPORES OF CLOSTRIDIUM BOTULINUM S. C. Stringer, M. D. Webb and M. W. Peck Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK Clostridium botulinum is a group of spore forming anaerobic bacteria that produce the extremely potent botulinum neurotoxin. It is vital to prevent growth of this organism in foods as consumption of as little as 30 ng of the toxin can result in severe illness or death. Clostridium botulinum is a particular concern in heat-treated products as competing vegetative £ora is eliminated. In such products growth is likely to initiate from just a few spores so the distribution of time to growth in packs will re£ect the heterogeneity of times to growth from individuals. Knowing the times to germination and growth from individual spores rather than just time to growth from a population will allow more accurate calculation of the risk of toxin production in a pack. Additionally, although the time to growth of an individual cannot be predicted from knowledge of the behaviour of the entire population, the time to growth of any size of population can be predicted if the underlying distribution of times to growth of individuals is known. An image analysis system and phase contrast microscopy has been used to follow individual spores of nonproteolytic Clostridium botulinum strain Eklund 17B from dormancy, through germination and emergence, to cell division. Frequency distributions ¢tted to the data show there is considerable variability within the spore population and there is poor correlation between germination and subsequent growth events such as emergence and cell division.

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P2^75 SUBLETHAL INJURY AND DETECTION OF GRAMNEGATIVE BACTERIA AND YEASTS ¤ ¤ ¤ ¤ ¤ E. Svirakova, L. Horn|kova and M. Plockova Institute of Chemical Technology Prague, Department of Dairy and Fat Technology, 166 28 Prague 6, Czech Republic. This work was directed to study the e¡ect of physical (heating, freezing) and chemical (EDTA, mixture of organic acids, NaCl, nisin) stress factors to growth and viability of Gram-negative bacteria (Escherichia coli DMF 7502, Pseudomonas sp. DMF 9010) and yeasts (Candida sp. DMF 1021, Kluyveromyces marxianus var. marxianus DMF 1005), and to possibilities of detection of sublethally injured bacteria and yeasts. The maximal inhibitive e¡ects to growth of bacteria and yeasts were reached with the EDTA e¡ect (20 mmol l-1) (99% reduction of both types of microorganisms relative to their original cells populations). Both bacterial strains showed to direct inhibitive e¡ect of nisin (0,1; 1; 10; 100 IU ml-1) lower sensitivity than the sublethally (physically or chemically) damaged bacteria. Both yeast strains showed to direct inhibitive e¡ect of nisin (0,1; 1; 10; 100 IU ml-1) only slight sensitivity ; the growth of sublethally injured yeasts was stimulated by nisin (100 IU ml-1). A thorough determination of the behaviour of sublethally injured bacteria and yeasts can be achieved using a combination of detection methods, for example, for bacteria, spectrophotometry together with selective and non-selective media, and for yeasts, £ow cytometry together with some selective and non-selective media. P2^76 INFLUENCE OF LACTIC ACID BACTERIA ON SOME FOOD-BORNE PATHOGEN MICRO-ORGANISMS K. Szeker, J. Beczner Dept of Microbiology, Central Food Research Institute, Herman Otto ut 15, H-1022 Budapest, Hungary Modern consumers nowadays demand natural food products. The traditional food preservation processes (for example thermal processing at high temperature, sugaring, pickling, drying) although kill pathogen bacteria, often decrease the nutritional value and taste of food products as well. Instead of drastic preservation processes more attentions are being given to physical and chemical methods alone or in combinations in low doses in order to keep the freshness of food products (minimal processing). These

novel, mild technics are for example the microwave treatment, intensive light impulse, high pressure treatment, electrical and magnetic pulses, bacteriocins and bacteriocin producing cultures (mainly lactic acid bacteria). The in£uence of these novel treatments on pathogen micro-organisms are not completely known yet, so every new combination must be examined previously, whether the new methods ensure the microbiological safety of the food product. The bacteriocin production of 23 lactic acid bacterium strains against vegetative cells of Bacillus cereus T and Listeria monocytogenes 4ab was tested. The in£uence of lactic acid bacteria on the growth and survival of pathogen bacteria in co-culture was examined. The changes of the CFU were followed also by impedance measurement (Malthus System V). Selective media were needed to detect the lactic acid bacteria and the test microbes derived from the co-culture selectively quantitatively. MRS was found as suitably selective medium for lactic acid bacteria and polymyxin B-containing PCA (PBPCA) for Bacillus cereus. Lactococus lactis subsp. lactis (106 cm-3) in co-culture did not inhibit the growth of B. cereus (104 cm-3) in milk at 30 0C. The work was supported by the Hungarian National Scienti¢c Research Foundation: OTKA T038215, Ministry of Education: NKFP-4/0028/2002, and Ministry of Agriculture and Regional Development: KF-79/7/2001. P2^77 A STUDY OF MICROBIAL CHARACTERS OF SAFFRON IN IRAN F. Tabatabaee Department of Food Science and Technology, Faculty of Agriculture, Ferdowsi University, Mashad-Iran Sa¡ron (Crocus sativus) is an important traditional plant from the Iridaceae Family that is produced a lot in the eastern area of Iran. It is a bene¢cial plant that has red color, suitable £avour and fragrance, that is used in food, cosmetic and drug industries and a lot of this product is exported every year. In this research, microbial characters of sa¡ron were tested by standard microbiological methods and determined coliform bacteria as the most important £or in Iranian sa¡ron, especially when the sa¡ron has high wet. Also were determined the total count factor in sa¡ron of Salmonella typhi, Bacillus reus, and Clostridium perferingens.

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P2^78 STUDY OF ANTIMICROBIAL EFFICACY OF SOME PLANT INHIBITORS M. Tediashvili, T. Koberidze, K. Porchkidze, N. Janelidze, G. Tsertsvadze, K. Akhvlediani G. Eliava Institute of Bacteriophage, Microbiology and Virology, 3 Gotua str., Tbilisi 380060, Georgia Screening of potential antibacterial plant compounds in 64 strains of di¡erent species of food pathogenic bacteria and 4 phages was carried out. Evaluation of MIC of antibacterial substances for each group of test bacteria was performed by dilution and disc di¡usion method. Bacteriophage susceptibility towards Inhibitor T3 was assessed by Phage Plaque assay. Transmission Electron Microscopy was used to register morphological changes under in£uence of tested inhibitors. The highest inhibitory activity of T3, proportional to the dose of inoculated substances, was observed for S. aureus, Sh. sonnei, Sh. £exneri, P. vulgaris and P. mirabilis. T1, T2, T3, preparations as well as Green Tea extract exhibited a higher activity against gram- positive bacteria, S. aureus, in particular. Obvious e/cacy was revealed against meticillin-resistant S. aureus (MRSA) strains. EM studies demonstrated cell damage and aggregation in S. aureus cell culture after 24 hours incubation with T3 preparation. For suppression of growth of some gram-negative bacteria such as S typhimurium and enterohemoragic E. coli, as well as Pseudomonas sp. inoculation of more then 1000 mkg/ml of T3 was necessary. On the contrary, these concentrations of plant compounds do not a¡ect food bene¢cial and probiotic bacteria like B. bi¢dum, Lb. plantarum, Lb. delbruki etc. Anti -viral potential of plant antibacterial substances was demonstrated by inhibitory e/cacy of T3 preparation towards Un phage leading to 3 log decrease of phage titer after 24 hours exposition with 500 mkg/ml of the inhibitor. P2^79 ASSESSMENT OF MUTAGENICITY AND CARCINOGENICITY EFFECTS OF PLASTICS BAGS AND DISPOSABLE FOOD CONTAINERS IN THE SALMONELLA/MICROSOME TEST M. Tohidpour, S. Mehrabian, M. Emtyazjoo, H. Assempour Dept. of Microbiology, Faculty of Basic Sciences, University of Azad Islamic, Branch of North of Tehran, Iran Nowadays, plastics bags and disposable food containers made of high and low-density polyethylene have found increasing use in Iran. As there is a direct relation between

the safety of such a type a product with the society's health, in this research the mutagenicity and carcinogenicity of these products have been investigated using Ames test and Salmonella typhimurium (TA100, TA104). As the result of the e¡ects of mutation, these bacteria strains have no potential to produce histidine on culturing in a minimal glucose agar medium. In general, carcinogen materials will cause a reverse mutation of these bacteria and results in production of histidine by the bacteria in the MGA medium for the Ames test. To investigate the mutagenicity of the PE bags and containers, we faced the pieces of these products on a test plate S. typhimurium (TA100, TA104) and the results of the experiments were these compared with MGA medium containing sodium azide as the positive control and distilled water as the negative control. The comparison of the colonies in the negative control, which are produced spontaneously, with those of the investigating PE samples a way to prove the mutagenicity of the experimental samples. The result showed that the HDPE grades as the raw material and their products made in form of disposable glasses and containers, drinking bottles and milk bags don't possess the mutagenicity property. The thin ¢lm as plastics bags caused reverse mutation of S. typhimurium of these products. If these products coated by liquid or solid oils were found to have much more a¡ects on mutagenicity of the ¢lms. Following the above tests, rat liver tissue microsomes it was later obtained under sterile conditions and was added to a MGA medium including the suspected PE samples. The results obtained from the latter experiments con¢rmed that the PE thin bags used for packaging of the food products in Iran su¡er from carcinogenicity. P2^80 OCCURRENCE OF ANTIBIOTIC-RESISTANCE IN THE PRODUCTION CHAINS OF ITALIAN DAIRY AND MEAT COMMODITIES S. Torriani(1), L. Rizzotti(1), F. Clementi(2), L. Aquilanti(2), F. Biavasco(3), C. Vignaroli(3), P. S. Cocconcelli(4), S. Gazzola(4), B. Cenci-Goga(5) and F. Dellaglio(1) ' (1) Dipartimento Scienti¢co e Tecnologico, Universita di Verona, Italy ; (2) Dipartimento di Biotecnologie Agrarie ' e Ambientali, Universita di Ancona, Italy ; (3) Istituto di ' Microbiologia, Facolta di Scienze MM. FF. NN. e di Me' dicina, Universita di Ancona, Italy ; (4) Istituto di Micro' di Agraria, Universita Cattolica del Sacro ' biologia, Facolta Cuore di Piacenza, Italy ; (5) Dipartimento di Scienze degli ' Alimenti, Universita di Perugia, Italy Antibiotic resistance (AR) in bacteria is a concrete threat to human health, emerging in the last decades as consequence of the large-scale use of antibiotics as growth pro-

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moters and in human and veterinarian medicine. The phenomenon of acquired resistance becomes more worrisome as these genes normally are on mobile genetic elements and this makes their intra-, inter-speci¢c and inter-generic transfer possible. AR bacteria, belonging to the genera Enterococcus and Staphylococcus and the lactic acid bacteria group, are increasingly isolated from foods with a large consumption. To deepen the knowledge of the role that foods play as AR vectors, seems therefore of great interest. The present research surveys the di¡usion and distribution of AR genes and microorganisms in some production chains of food of animal origin (breedingmilk-cheese ; breeding-fresh meat/processed meat) in di¡erent Italian geographic areas. The presence of genes codifying for the most important resistances (e.g. vancomycin, methicillin, erythromycin, gentamycin) was investigated using speci¢c PCR assays performed directly on DNA from food matrices. We found that several samples of animal faeces, feedstu¡s, dairy and meat products gave positive ampli¢cation reactions. Bacteria, resistant to antibiotics and belonging to the above-mentioned groups, were isolated from these positive samples. Their characterisation by means of molecular techniques allowed the monitoring of single strains along the production chain. The ¢ndings of this study indicate a frequent occurrence of AR in food-associated bacteria at all levels of the food production chains. The usefulness of molecular ¢ngerprinting to individuate points in which contamination might occur was emphasised. P2^81 PHARMACEUTICAL MICROBIOLOGY ON GUARD DRUGS QUALITY S. Tyski National Institute of Public Health, Warsaw, Poland Microbiology is very important in assessing the quality of pharmaceutical preparations. There are several steps in pharmaceutical industry, where control of microbial contamination is crucial. Monitoring of production environmental in clean areas during aseptic manufacturing, involve measurement of viable microorganisms content in the air, on the surfaces and on personal gloves. The usage of disinfecting products with wide antimicrobial spectrum, dedicated to the appropriate surfaces is recommended to limit the bioburden level. Microbial status (bacteria and fungi contamination) of starting materials and water used for production are essential for microbiological quality and safety of ¢nal product. It is di/cult to understand that microbiological requirements for drinking water are higher than for puri¢ed pharmacopoeial water. Microbial quality of pharmaceutical preparations, which are on the market, is described in Pharmacopoeias. There is a strict

borderline between sterile products and those, which can contain permitted level of microorganisms. Preparations for topical use, for oral and rectal administration as well as herbal medicinal products have limited quantity and quality of contaminating microorganisms described in Pharmacopoeias. Sometimes antimicrobial preservatives may be added, to prevent proliferation or to limit microbial contamination, which during normal conditions of storage and use, particularly multidose containers could occur in the product. It may present a hazard to the patient from infection and spoilage of the preparation. The e/cacy of an antimicrobial preservative may be demonstrated by the pharmacopoeial test with bacterial and fungal test strains. Proper test microorganisms are used also for determination of antibiotics and antiseptics activity. P2^82 THE EVALUATION OF MICROBIAL POPULATION OF PIZZA CHEESE IN REFRIGERATOR AND FREEZER CONDITIONS A. Valaei, J. Fooladi and S. Mehrabian Azzahra University, Vanak St. Tehran, Iran Cheese is produced by the lactic fermentation of milk. Mozzarella is an Italian soft cheese made of cow's milk. One type of this cheese which has more fat and less water is pizza cheese ; the amount of fat and water are 24 % and 48 %, respectively (compared to normal mozzarella at 18 % and 52 %, respectively). In this research, 280 samples of pizza cheese at +4C and -20C at these times of examination 0, 1, 3, 5, 7, 10, 14, 30, 60 days and 10-1 10-2 dilutions, were studied. Microbial contamination of pizza cheese was examined for 5 bacteria: E. coli, coliform, S. aureus, Enterococcus spp. and Cl. perfringenes. Pizza cheese was not found to be contaminated by E. coli, indicating that the milk was pasteurized. The result of 0 time showed that there was no E. coli, but coliforms (Citrobater freundii, Aerobacter aerogenes ), Cl. perfringenes, Enterococcus and S. aureus were observed above the standard level. The result for +4C in survival of bacteria showed that coliforms, Cl. perferingens and Enterococcus increased in temperature of refrigerator compared to 0 time, but S. aureus decreased. During the ¢rst days, the amount of Enterococcus and Coliforms increased in the temperature of -20C. From ¢fth day, bacteria decreased gradually, but from fourteenth day bacteria decreased strongly, but S. aureus at the ¢rst days decreased. According to the results obtained, the following suggestions are o¡ered : 1) Pizza cheese should be kept 15 days of storage after production in -20C freezer then be contributed to the market; 2) Quality control producing factories should be sampled after 15 days of production storage in fridge -20C; 3) For home usage, pizza cheese should be packed in small

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sizes, so that the whole package would be consumed after freezing; 4) Pizza cheese should be used cooked to kill micro-organisms in this cheese. P2^83 COMPARISON OF FOOD AND CLINICAL ISOLATES OF ENTEROCOCCUS FAECIUM AND ENTEROCOCCUS FAECALIS L. Vannini(1), G. Suzzi(2), P. S. Cocconcelli(3) and M. E. Guerzoni(1) (1) Dipartimento di Protezione e Valorizzazione Agroali' ' mentare, Facolta di Agraria, Universita degli Studi di Bologna Via Fanin 46, 40127 Bologna, Italy ; (2) Dipartimen' ' to di Scienze degli Alimenti, Facolta di Agraria, Universita degli Studi di Teramo, Via Spagna 1, Mosciano S. Angelo, ' Teramo, Italy ; (3) Istituto di Microbiologia, Universita Cattolica del Sacro Cuore, Via Emilia Parmense 84, 29100 Piacenza, Italy Enterococci are ubiquitous microorganisms that can colonise di¡erent niches and may serve as indicators of the sanitary quality of foods. Enterococci commonly occur in vegetables and fermented foods of animal origin. In fermented meats, enterococci can be detrimental because they can cause spoilage. On the contrary, they have important implications in the dairy industry as they contribute to the development of organoleptic characteristics during cheese ripening. However, enterococci have recently assumed major importance in clinical microbiology due to their enteric habitat, transmission through the food chain, antibiotic resistance and their possible involvement in food-borne illness or environmental contamination in hospitals due to the presence of virulence factors. The aim of this work was to study isolates of Enterococcus spp. of food and clinical origin for their physiological and molecular characteristics. In particular they were isolated from fermented foods and cheeses, from blood, catheters, endocarditis, meningitis and sepsis. All the isolates were subtyped with the Automated RiboPrinter System, screened for resistance to di¡erent antibiotics, investigated for plasmid analysis, the presence of virulence factors and growth ability at di¡erent Aw and pH values. The comparison of the ribotype patterns evidenced the existence of di¡erent pro¢les between the isolates of the two main species considered (E. faecium and E. faecalis). However, no ribotypes speci¢cally associated with strains isolated from foods or from clinical sources were observed. This could indicate a possible transmission of such isolates along the food chain to humans. Moreover, the ability of the isolates of clinical origin to grow in environmental stress conditions suggests that some of the isolates from nosocomial infections may have food origin.

P2^84 SEARCH FOR NEW PROBIOTIC CANDIDATES : SAFETY AND QUALITY CRITERIA A. Weiss(1), H. K. Mayer(1), H. P. Lettner(2), W. Kramer(2) and W. Kneifel(1) (1) Department of Dairy Research and Bacteriology, University of Agricultural Sciences, Gregor-Mendel-Strasse 33, A-1180 Vienna, Austria ; (2) Lactosan Starterkulturen GmbH & Co KG, Industriestrasse West 5, A-8605 Kapfenberg, Austria Over the past decades probiotic micro-organisms have gained a great market potential for both human and animal nutrition. In parallel, the demand for new, more suitably performing probiotic strains has grown. Before a given strain is incorporated into a product, several safety and quality (functional and technological) aspects have to be considered. At ¢rst, strains should be isolated prevailingly from the site of the intended application and should have a history of being non-pathogenic. The safety properties of strains as intrinsic properties, pharmacokinetics and interactions between probiotic strain and host have to be assessed. It is of utmost importance that strains do not carry transmissible antibiotic resistance genes in order to forestall the spread of antibiotic resistance. In addition to the proper identi¢cation, in the present study the probiotic strains were tested concerning several quality criteria to allow the prediction of the tolerance of the manufacturing process, the shelf life and the passage through the intestinal tract: tolerance of acid, bile, sodium chloride and enzymes of the gastrointestinal tract, tolerance of oxygen, autoaggregation, fermentation of carbohydrates, growth stimulation by prebiotic carbohydrates, enzyme pro¢le, antagonistic potential against potentially pathogenic micro-organisms, fermentation pro¢les and -products as well as incubation temperature. Exemplary results of these tests will be presented. P2^85 THERMAL INACTIVATION OF SALMONELLA SENFTENBERG W775 ON MUNGBEAN SEEDS USED FOR SPROUT PRODUCTION A. Weiss and W. P. Hammes Institute of Food Technology, University of Hohenheim, 70593 Stuttgart, Germany Seed sprouts have frequently been implicated as vehicle in food borne disease outbreaks. The sprouting process provides optimal conditions for bacterial growth and therefore, the absence of pathogens on seeds can be de¢ned as

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the critical control point in sprout production. The Food and Drug Administration recommended a 5 log units reduction of pathogens to minimise the risk of food poisoning, and for this purpose a treatment with 20.000 ppm calcium hypochlorite is in use. For the production of organic food in general, the use of disinfectants is commonly not accepted. To design an easy and economic alternative we studied the applicability of thermal processing. Mungbean seeds were inoculated with 1 x 108 cfu/g Salmonella Senftenberg W775 by immersion. This strain is well known as extremely heat resistant organism. The seeds were dried and stored at 2 C. The Salmonella counts on the dried seeds remained unchanged for 6 weeks at a level of 1-5 x 108 cfu/g. The contaminated seeds were treated in a water bath at 55, 58 and 60 C for 0,5-16 minutes. The experiments were repeated 3 times. The D-values were determined for 55, 58 and 60C and the z-value of 6,2 (r2= 0,99) was calculated for the inactivation of S. Senftenberg W775 on the mungbean seeds. The thermal treatment at time/ temperature regimes of 55C/20 min, 60C/10 min, 70C/5 min and 80C/2 min reduced the pathogens for more than 5 log units without a¡ecting sprouting. P2^86 COMPARISON OF DRYCULT0 TPC, PETRIFILM AND STANDARD METHOD FOR THE DETERMINATION OF MICROBIAL QUALITY IN WATER, JUICES AND SOFT DRINKS H.-D. Werlein and C. Armbrust University of Hannover, Institute of Food Science, Wunstorfer Strasse 14, D-30453 Hannover, Germany DryCult0 TPC (Orion Diagnostica, Espoo Finland) is a new simple system to investigate the microbial quality of water and other liquid samples on location. Dry Cult0 consists of a dehydrated medium which absorbs 1 ml of the liquid sample and should show comparable results to the standard methods. Samples of water, mineral water, beer, milk, orange-, apple- and grape juice were examined. Some of the samples were arti¢cially inoculated with different microorganisms. DryCult0 was compared with the standard and the petri¢lm method. DryCult0 and Petri¢lm were treated as described in the protocols of the suppliers, respectively the standard method. Di¡erent investigations carried out with E. coli, S. aureus, Saccharomyces cerevisiae and naturally contaminated samples showed, that DryCult0 TPC is able to detect di¡erent kinds of microorganisms. Water, mineral water, beer and soft drinks are suitable products. Milk and some juices are only limited suitable. The results are comparable to the standard methods and to the petri¢lm method. The DryCult0 TPC system is very easy to handle. The DryCult0 method shows a tendency to lower results compared to the

standard methods. It was remarkable if the sample is highly contaminated no growth is visible. P2^87 DETECTION OF AFLATOXINOGENIC FUNGI USING PCR METHOD ¤ > ¤ ¤ I. Zachova(1), J. Vytrasova(1), M. Pejchalova(1), G. > ar-Kalcher(2) Tavc (1) Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, í Pardubice, Czech Republic, Strossova 239, CZ-530 03; (2) University of Ljubljana, Veterinary Faculty, Institute for > Hygiene and Pathology of Animal Nutrition, Gerbiceva 60, 1000 Ljubljana, Slovenia This work covers the possibility of using PCR method for acceleration and more accurate identi¢cation of the a£atoxinogenic fungi isolated from feed and foodstu¡s. The method was optimalized on pure cultures (Aspergillus £avus CCM F-108 and Aspergillus parasiticus CCM F/550). The speci¢city of the optimalized PCR method was veri¢ed using various fungal strains. 50 samples of feed were examined, of which 18 were positive for the presence of the a£atoxinogenic fungi on AFPA medium. Isolated Aspergillus strains were examined using PCR method. The results obtained almost always agreed with the results gained on the AFPA medium. This method is a possible starting point for accelerating the detection of the a£atoxinogenic fungi, but it will be necessary to solve certain non-speci¢c reactions, which are caused by a complex sample matrix. The results obtained are an initial step for further research. The PCR technique has proved itself in the detection of a£atoxinogenic fungi isolated from feed. This study was supported by Research Project msmt No. 253100002 and Project kontakt me 241. P2^88 COAGULASE-POSITIVE STAPHYLOCOCCI : ENTEROTOXIN PRODUCTION AND ANTIBIOTIC RESISTANCE P. Zangerl(1), M. Gonano(2), A. Rammelmayr(3), M. Wagner(2) (1) Federal Institute of Alpine Dairying, A-6200 Rotholz; (2) University of Veterinary Medicine, A-1210 Vienna; (3) Austrian Agency for Health and Food Safety, A-3261 Steinakirchen am Forst, Austria To study the enterotoxigenicity of coagulase-positive staphylococci, 247 dairy isolates (121 and 126 isolates

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from raw milk and raw-milk cheeses, respectively), 91 isolates from mastitis milk and 48 clinical human isolates were analysed for the production of the enterotoxins (SEs) A, B, C, D, and E by a commercially available ELISA test kit (Tecra). Additionally PCR-assays for the detection of the enterotoxin genes sea, seb, sec, sed, see, seg, seh, sei, and sej were carried out for the mastitis and human strains, together with 91 strains selected from the dairy isolates. These and further 43 human strains (91 human strains in total) were analysed for the resistance against 14 antibiotics by an agar di¡usion test. The ability to produce SEA-SEE was strongly dependent on the origin of strains. While 41.7% of the human isolates were found to produce SEA-SED, dairy and mastitis isolates showed much lower frequency of enterotoxin production (13.0% and 10.8%, respectively). Irrespective of origin, SEC was found to be the predominant enterotoxin and none of the strains harboured the see gen. The genes encoding the recently characterised enterotoxins G-J were detected much more frequently. The most frequent combination of enterotoxin genes was seg/sei. Antibiotic resistance testing revealed that 44.6% of the dairy isolates, 67.3% of the mastitic isolates and only 2.7% of the human isolates were susceptible to all antibiotics tested. Penicillin resistant strains exhibited utmost importance since 10.9% of the mastitic isolates, 35.5% of the dairy isolates and 60.1% of the human isolates were resistant to penicillin G. P2^89 HUMAN CALICIVIRUS OUTBREAKS IN SLOVENIA IN 2000-2002 > > > J. Zimsek(1), M. Poljsak-Prijatelj(1), D. Barlic-Magan> evar-Grom(3) ja(2), A. Hoc (1) Institute of Microbiology and Immunology, Medical Faculty, University of Ljubljana, Slovenia; (2) Veterinary Faculty, University of Ljubljana, Slovenia; (3) Institute of Public Health of the Republic of Slovenia, Ljubljana Noroviruses, members of the family Caliciviridae, are small non-enveloped viruses with single-stranded polyadenilated genome of positive polarity. The way of transmission for these viruses is via faecal-oral route; viruses can spread between people by direct contact or through contaminated food and water, causing sporadic and epidemic gastroenteritis in children and adults. We have investigated the incidence of noroviruses associated with outbreaks of acute nonbacterial gastroenteritis in Slovenia, since 2000. Stool specimens from outbreaks were tested during this period. After ultracentrifugation stool samples were examined by direct electron microscopy. RNA extracted from stool specimens with SV Total RNA Isolation system (Promega) was used as the template for reverse transcription, followed by ampli¢cation in a single tube, two-en-

zyme system. Gene fragments were ampli¢ed using generic primers targeting the polymerase region of the genome. The identity and the speci¢city of amplicons belonging to genogroup II were con¢rmed by hybridisation with biotin labelled capture probes. Genogroup II dominated among the specimens of infants, children and adults. During 2000 most frequent genotype were Lordsdale and Hawaii, during 2001 genotype Lordsdale and Melksham; in 2002 genotype Lordsdale and multiple genetic type were detected. During 2002 the strains, which could not be detected before increased and were further preceded for sequence analysis. Most outbreaks occurred in canteens, homes for the elderly, holiday lodges and schools. P3^1 HALOBACILLUS KARAJENSIS SP. NOV., A NEW MODERATELY HALOPHILIC SPECIES ISOLATED FROM SALINE SOIL M. A. Amoozegar(1), F. Malekzadeh(1), K. A. Malik(2), P. Schumann(2) and C. Sproer(2) « (1) Department of Biology (Microbiology unit), Faculty of Science, University of Tehran, Tehran, Iran; (2) DSMZ ^ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1 B. 38124 Braunschweig, Germany A moderately halophilic, Gram-positive, spore-forming bacterium was isolated from surface saline soil of the region of Karaj, Iran. The strain, designated MA-2 was strictly aerobic, rod-shaped, occurring singly, in pairs or short chains, contained L-orn-D-asp type peptidoglycan, and the major respiratory lipoquinone was MK-7. It was non-motile, having ellipsoidal endospore located centrally or sub-terminally. Growth occurred at 10-49 C and the pH range was 6-9.6. Strain MA-2T grew at salinities 1-24% (w/v) NaCl, showing optimal growth at 10% (w/v). The guanine-plus-cytosine content of the DNA was 41.3 mol%. Phylogenetic analysis based on 16S rRNA sequences showed that strain MA-2T was associated with rRNA group 1 Bacillus. The microorganisms showing the closest phylogenetic relationship to strain MA-2T were members of the genus Halobacillus: Halobacillus litoralis and Halobacillus trueperi. On the basis of phenotypic and chemotaxonomic characteristics, 16S rRNA sequence analysis and DNA-DNA similarity data, it is proposed that strain MA-2T should be placed in the genus Halobacillus as a new species, Halobacillus karajensis sp. nov.

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P3^2 GENETIC POPULATION ANALYSIS OF PSEUDOMONAS AERUGINOSA BY MULTILOCUS SEQUENCE TYPING I. Vernez, P.M. Hauser, D. S. Blanc University hospital of Lausanne, Lausanne, Switzerland In order to study the population genetics structure of Pseudomonas aeruginosa, we developed a multilocus sequence typing (MLST) scheme. The sequences of internal fragments of seven housekeeping genes were obtained for 34 P. aeruginosa isolates from patients hospitalized in 5 di¡erent European cities. Twenty six di¡erent allelic pro¢les were identi¢ed. The mean allelic diversity was 0.854 (range: 0.606 to 0.978), which was about 6X greater than the results obtained with the multilocus enzyme electrophoresis (MLEE) method. Linkage disequilibrium was measured with the index of association. An index of 1.95+0.24 was calculated when all the strains were taken into consideration. This index was 1.76+0.27, when only one strain per sequence type was taken into consideration. Both results are di¡erent from 0, indicating that there is an association within loci, which means that the population structure of P. aeruginosa is clonal. These results are in contradiction with a previous study based on MLEE data, which showed that this structure was non-clonal. P3^3 THE FUNCTIONAL EVOUTION OF STREPTOKINASE DOMAINS IN PLASMINOGEN ACTIVATION D. M. Kim, J. H. Chung and S. M. Byun Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 305-701 Daejon, South Korea Streptokinase is a human plasminogen activator, secreted from pathogenic bacteria, Streptococcus species. Random mutagenesis of streptokinase was carried out to determine functional amino acid residues in plasminogen activation. Fourteen streptokinase mutants without plasminogen activation activity on skim milk-plasminogen overlay plate were selected and sequenced. Six mutants (D41C, S44K, S44P, R45P, H48T and D220G) showed substantial amidolytic activities as compared with that of wild type. Moreover, ¢ve point mutations in Asp41-His48 region of a streptokinase plays an important role in binding to a substrate plasminogen. Because of the structural and sequence similarity of SKK domain to those of staphylokinase, the chimeric proteins substituted SKK domain with staphylokinase were examined for their activity of plas-

minogen activation. The kinetic data showed that these fusion proteins bound to human plasmin for plasminogen activation. Additionally the N-terminus of staphylokinasestreptokinase fusion protein was substituted by the N-terminus of streptokinase. The serial substitution showed that the non-hydrophilic residues at the N-terminus of the fusion proteins without SKQ domain did not a¡ect the formation of the active plasmin-activator complex. However, the fusion mutants with Q domain shortened the lag times in kinetic data. These results propose that streptokinase requires the N-terminal non-hydrophilic residues and Q domain for formation of the active streptokinase-plasminogen complex. Recombinant bovine plasminogen activator also activated human plasminogen by staphylokinase-like activation mode. Because the activator has homology with streptokinase, the bovine plasminogen activation activities of streptokinase fragments were examined. Streptokinase fragment without Q domain among them activated bovine plasminogen. These data suggest that the Q domain of streptokinase determines plasminogen species necessary for activation, and converses ability of substrate recognition to human species. P3^4 CLONAL DIFFUSION AND HORIZONTAL TRANSMISSION OF MEC DETERMINANT IN METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS ISOLATED IN ITALY F. Campanile(1), M. Santagati(1), D. Bongiorno(1), S. Borbone(1), M. Cassone(2) and S. Stefani(1) (1) Department of Microbiological and Gynaecological Science, University of Catania; (2) Department of Clinical Medicine, Rome, Italy The key genetic component of methicillin-resistance ^ the mecA determinant ^ is not native of Staphylococcus aureus. The origins of the major MRSA clones are still poorly understood; previous reports have suggested a common origin for all MRSA from a single ancestral S. aureus strain that acquired the mec complex. Recent studies have shown that some MRSA strains are very divergent, implying that mecA has been transferred among S. aureus lineages. Multilocus-sequence typing (MLST) of S. aureus has been used to probe population biology of bacterial pathogens and to predict ancestral genotypes and evolutionary descendent within groups of related genotypes. Studies carried out in our laboratory revealed the presence of ¢ve MRSA clones in Italy: the archaic clone, the multiresistant Iberian and Brazilian clones and two new ones, classi¢ed as Italian and Rome clones. The purposes of this study were to explore the evolution of early and contemporary MRSA isolates in a selected sample of strains; to compare their clonal type and their genetic backgrounds

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with main Italian MRSA clones, re-examined for their genetic relatedness by MLST. Our results show that the Rome clone have undergone an evolution due to the acquisition of two copies of Tn554 and their modi¢cation of mecA polymorphism II to I. A closed correlation among four previously classi¢ed clones (Archaic, Iberian, Brazilian and Rome) has been showed by the same allelic pro¢le (3-3-1-12-4-4-16). Surprisingly, the Italian clone belongs to a totally di¡erent genetic background (1-4-1-4-12-24-29), suggesting horizontal mecA transfer among di¡erent S. aureus ancestral lineages. P3^5 TRANSPOS-ID : Tn231LM A PECULIAR STRUCTURE OF

P3^6 DISTRIBUTION OF IS231-LIKE SEQUENCES AMONG BACILLUS THURINGIENSIS SEROVARS " ¤ K.-B. Joung and J.-C. Cote Agriculture and Agri-Food Canada, 430 Gouin Blvd., SaintJean-sur-Richelieu, QC, J3B 3E6, Canada Insertion sequences (IS) are the simplest transposable elements, and usually contain only the genes responsible for their transposition. Some IS (IS231, IS232, IS233, IS240, ISBT1 and ISBT2) and two class II transposons (Tn4430 and Tn5401) have been isolated from a limited number of Bacillus thuringiensis strains. IS231 are found closely associated with cry genes and are believed to play a role in their multiple locations and genetic mobility, and in the generation of novel speci¢city. We set to analyze the distribution of IS231-like sequences among Bacillus thuringiensis serovars. A total of 95 bacterial strains were tested in the present study. These include the 82 known B. thuringiensis serovars type strains, ¢ve more B. thuringiensis intra-serovar strains, kurstaki HD-1, subtoxicus, dendrolimus, tenebrionis and sandiego, a non-motile hence non-serotypeable strain, B. thuringiensis var wuhanensis, six B. cereus strains, and the B. mycoides type strain. A 723-bp HaeII conserved fragment from IS231M has been used as a probe against EcoRI-digested B. thuringiensis total DNA to yield serovar-speci¢c hybridization pro¢les. The approach was useful at revealing the extent of distribution of IS231-like sequences between and within strains. Of the 88 B. thuringiensis strains tested, 70 showed hybridization banding patterns that comprised between one and 20 distinct bands. These 70 B. thuringiensis strains were grouped based on banding patterns similarities. Interestingly, intraserovar strains did not necessarily cluster together. P3^7 PATTERNS OF 16S-23S INTERGENIC TRANSCRIBED SPACER (ITS) AND DISTRIBUTION OF A 16-BP MOTIF, BOX X, PRESENTS IN THE 5' CONSERVED ITS IN BACTERIA. " ¤ D. Xu and J.-C. Cote Agriculture and Agri-Food Canada, 430 Gouin Blvd., SaintJean-sur-Richelieu, QC, J3B 3E6, Canada Nucleotide sequence comparisons done in our lab, between and within bacterial species, show that the bacterial 16S-23S intergenic transcribed spacer (ITS) can be divided into four regions: I, II, III and IV. Region I, which corresponds to the 5' end of 16S-23S ITS, is the foremost inter-allelically conserved region within species ; Region

" ¤ D. Xu and J.-C. Cote Agriculture and Agri-Food Canada, 430 Gouin Blvd., SaintJean-sur-Richelieu, QC, J3B 3E6, Canada Composite transposons contain a central region £anked by 2 Insertion sequences (IS). The central region, unconnected to transposition, usually carries drug resistance. Both IS can be found in same or in inverted orientations. We report here the peculiar structure of a novel composite element, which we tentatively named Tn231LM. It was isolated from Bacillus thuringiensis strain M15. This composite transposon is delimited by two IS elements, IS231L and IS231M. Both IS are found in the same orientation. Each IS contains 18-bp inverted terminal repeats (IR) and generates 9-bp direct repeats (DR) at each end. The sequence from the right IR through the right DR of IS231M complements that of the left IR and left DR of IS231L. This complementary sequence extends an additional 9-bp, named inner extended IR (ieIR). The sequence of the right IR and right DR of IS231L perfectly complements that of the left IR and left DR of IS231M. This complementary sequence extends an additional 27-bp, named outer extended IR (oeIR). The oeIR ends in an 8-bp DR. Based on the structure, two composite transposons are possible. One, in which the central region is 448-bp between both oeIR ends. Alternatively, should the transposon be considered ending at these 8-bp DR, the central region of the transposon would be an autonomously replicated megaplasmid. In the latter case, Tn231LM can be considered having the capacity of autonomous replication. We thus propose a new name for this special apparatus: ``transposid''. In the strain M15, there are about 20 copies of IS231 sequences. But the Tn231LM remains unique based on a result of Southern hybridization with a 5' end £uoresceinlabeled probe with the sequence of 27-nt oeIR.

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II is variable; Region III is inter-allelically conserved; and Region IV, at the 3' end of 16S-23S ITS, is variable. Some ITS contain all 4 regions, others contain regions I, II and III, and others contain only regions I and III. An analysis of 197 16-23S ITS sequences from 115 ¢rmicute and mollicute species revealed that a 16-nt conserved motif located near the 3' end of Region I is present in all ITS. We called this motif ``box X''. Part of the 3' end sequence of box X could form a hairpin structure with the downstream sequence. An analysis of the RNA secondary structure shows that box X and its £anking sequences can form a double-stranded structure with part of the leader region of the 16S rRNA gene. Presumably, this structure could serve as an RNA double-stranded processing site (DsPS) for RNase III. The variable Region II may contain tRNA genes in some of the alleles. This Region can be absent in others. Region III contains, near its 5' end, the conserved box A ^ antitermination sequence. Region IV is variable and is not present in all species. Interestingly, in Bacillus halodurans, as many as three copies of box X can be found in tandem in Region I. This suggests that box X may play an important role, in addition to the formation of DsPS, possibly during the transcription of the rrn operons. P3^8 METHICILLIN RESISTANCE IN STAPHYLOCOCCUS SCIURI I. Couto(1,2,3), S. Wei Wu(2), A. Tomasz(2) and H. de Lencastre(1,2) ¤ ¤ (1) Laboratorio Genetica Molecular, Instituto de Tecnolo¤mica e Biologica (ITQB/UNL), Rua da Quinta ¤ gia Qu| Grande, 6, Apartado 127, 2780-156 Oeiras, Portugal; (2) Laboratory of Microbiology, The Rockefeller University, New York, NY 10021, USA; (3) Centro de Recursos Mi¤ " crobiologicos, Faculdade de Ciencias e Tecnologia (FCT/ UNL), Quinta da Torre, 2829-516 Monte da Caparica, Portugal. Resistance to beta-lactam antibiotics in staphylococci is associated with the presence of the mecA gene that encodes PBP2A, a penicillin-binding protein with greatly reduced a/nity to these antibiotics. While studying the origin of mecA, which is exogenous to S. aureus and to other clinically relevant staphylococci, we identi¢ed a genetic element closely related to it in the animal commensal species S. sciuri. The S. sciuri mecA homologue is native to this species, being found in all S. sciuri isolates tested to date ( s 160). Despite the similarities between the S. sciuri mecA and the methicillin-resistant S. aureus (MRSA) mecA, the great majority of S. sciuri isolates showed no appreciable resistance to beta-lactam antibiotics. We now describe two unusual S. sciuri strains isolated from hu-

mans that showed resistance to methicillin associated with high rates of transcription of the S. sciuri mecA homologue and production of a protein resembling PBP2A. In one of these strains increased transcription of the mecA homologue was related to the insertion of IS256 upstream mecA while the other strain had single nucleotide alterations in the promoter region of the gene. A third methicillin-resistant human isolate of S. sciuri that carries both the native mecA homologue and a MRSA type mecA was shown to be unstable in the absence of drug selection, segregating antibiotic susceptible cells, which had lost the MRSA-type mecA. These observations illustrate the remarkable variety of strategies available to bacteria to acquire mechanisms of drug resistance in the in vivo environment. P3^9 ACINETOBACTER PARVUS SP. NOV., A SMALL COLONY FORMING SPECIES ISOLATED FROM HUMAN SPECIMENS A. Nemec(1), L. Dijkshoorn(2), I. Cleenwerck(3), T. De Baere(4), D. Janssens(3), T. J. K. van der Reijden(2), P. > Jezek(5) and M. Vaneechoutte(4) (1) National Institute of Public Health, Prague, Czech Republic ; (2) Department of Infectious Diseases, Leiden University Medical Center, The Netherlands; (3) BCCM/ LMG Bacteria Collection, University of Ghent, Gent, Belgium; (4) Department of Clinical Chemistry, Microbiology and Immunology, Gent, Belgium ; (5) Department of Clin>| ical Microbiology, Pr¤bram, Czech Republic The taxonomic status of seven glucose non-acidifying, non-proteolytic Acinetobacter strains characterized by forming small colonies (from 0.1 to 0.7 mm in diameter after 24 h of incubation at 30 C on di¡erent kinds of agar media). With one exception, all strains were from human specimens. They could be distinguished from all described Acinetobacter (genomic) species by their ability to grow on ethanol and acetate as sole sources of carbon but not on 22 other substrates tested including D,L-lactate or D,L-4aminobutyrate. DNA-DNA hybridization studies, 16S rRNA gene sequence analysis, ampli¢ed ribosomal DNA restriction analysis, and DNA polymorphism analysis by AFLPTM showed that these strains represent a hitherto unknown species for which the name Acinetobacter parvus sp. nov. is proposed. The type strain is LMG 21765T (= CCM 7030T) isolated from an ear of an outpatient. The EMBL accession number for its 16S rDNA sequence is AJ293691 and its DNA G+C content is 41,8 %.

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P3^10 APPLICATION OF AMPLIFIED RIBOSOMAL DNA RESTRICTION ANALYSIS TO THE IDENTIFICATION OF ENVIRONMENTAL ACINETOBACTER ISOLATES REVEALS A NEW RESTRICTION PATTERN AND SEVERAL UNDESCRIBED PROFILES L. Dolzani, F. Gombac, M. Bellino, C. Lagatolla, R. Bressan, E. Edalucci, E. Tonin, and C. Monti-Bragadin ' Dipartimento di Scienze Biomediche, Universita di Trieste, via Fleming 22, 34127 Trieste, Italia The genus Acinetobacter, including both environmental and clinically relevant organisms, comprises more than twenty DNA genomic groups or genomospecies, delineated by DNA/DNA hybridization. Genomic species identi¢cation, however, may still be problematic. Among the available methods, Ampli¢ed Ribosomal DNA Restriction Analysis (ARDRA) showed a good correlation with DNA/DNA hybridization (System. Appl. Microbiol.21,33-39, 1998), but, although developed as a general method, its usefulness was mainly tested on clinical isolates. In this study, we evaluated the usefulness of ARDRA for the identi¢cation of 46 environmental Acinetobacter isolates, collected during the year 2000 from samples of soil and water. Isolation strategy included enrichment in Baumann medium followed by plating on LAM selective medium. Identi¢cation at the genus level was con¢rmed by the transformation assay of Juni. ARDRA results showed the following: only 26 (57%) isolates gave already described restriction patterns and patterns combinations, and could therefore be identi¢ed to the genomospecies level. Six isolates showed a new RsaI pattern. All of them had identical restriction pro¢les with the other endonucleases and performed homogeneously in a series of biochemical tests, so they are likely to belong to the same genomospecies. The remaining isolates showed undescribed combinations of already described patterns. Phenotypic characterization of the isolates that could be identi¢ed by ARDRA gave a concordant identi¢cation in most cases, while it was unconclusive for the other strains. Overall results indicated that the diversity of environmental Acinetobacter strains is higher than previously described and that ARDRA is at present of limited usefulness for the identi¢cation of such strains.

P3^11 PHYLOGENY OF LACTIC ACID BACTERIA BASED ON recA AND 16S rDNAS SEQUENCE ANALYSIS G. E. Felis, F. Dellaglio, E. Knij¡ and S. Torriani ' Dipartimento Scienti¢co e Tecnologico, Universita degli Studi di Verona, Strada le Grazie 15, 37134 Verona Italy Current delineation of the evolutionary relationships among bacteria and, in particular, among Lactic Acid Bacteria (LAB) is largely stated on 16S rRNA sequence analysis. Based on branching patterns in the 16S rRNA trees, LAB belong to the Gram-positive low GC division (Firmicutes) and are separated in several genera, phylogenetically intermixed (Lactobacillus and Pediococcus) and/ or internally sub-divided (Lactobacillus). In addition, they exhibit di¡erent morphology, GC content, metabolic and phenotypic traits, that only partially correlate with their phylogenetic placement. Such premises support the opportunity of a deeper investigation on genealogical inter-relationships among LAB, to depict a more robust taxonomic scheme for these bacteria of relevant ecological and food interest. The present study addressed a comprehensive phylogenetic analysis of several LAB genera and species by integrating the information deriving from di¡erent molecular markers, i.e. recA gene and 16S rDNAs. Partial sequences of RecA protein and its coding gene have successfully been used to perform phylogenetic analysis of many bacterial genera, but rarely on LAB. Complete recA primary structures, available from databases or directly sequenced, were analysed for a major number of species belonging to the genera Lactobacillus, Pediococcus, Enterococcus, Leuconostoc, Oenococcus, Lactococcus, Carnobacterium and Weissella. A comparison of 16S rDNAs and recA phylogenetic trees obtained by the same bioinformatic methods was carried out. Statistical robustness of the deduced trees, in£uence of genome GC content on tree reconstruction and correlation among phylogenetic, phenotypic and genotypic classi¢cation schemes of LAB were discussed in a polyphasic scenario.

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P3^12 DEVELOPMENT OF AN INSECT MODEL SYSTEM FOR THE EVOLUTION OF VIRULENCE AND THE ROLE OF VIRULENCE FACTORS IN THE IMPORTANT HUMAN PATHOGEN STAPHYLOCOCCUS AUREUS V. M. Fleming and E. J. Feil Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, BA2 5AB, England, UK. Staphylococcus aureus represents an increasing public health burden and is a major cause of community-acquired and nosocomial sepsis. Although commonly regarded as an opportunistic pathogen, recent evidence has suggested that virulence may be a cumulative e¡ect related to the total number of bacterial virulence determinants present in the genome of a given strain. In order to examine this hypothesis, we have developed an in vivo model of virulence based on the insect larvae Manduca sexta (the Tobacco Horn Worm). Preliminary research on this model suggests an LD50 of approximately 104 cells of S. aureus at 37C, with viable S. aureus cells readily recovered from the cadavre, suggesting that in vivo colonisation has occurred. Through the use of a well-characterised bacterial strain collection, we have also demonstrated consistent di¡erences in virulence (as measured by weight change in the larvae) between bacterial strains, and noted correlations with the strain-speci¢c virulence gene-pro¢le. Furthermore, those strains which have most commonly been recovered from cases of serious disease in humans also tend to show the greatest virulence in the M. sexta model. This model is being further developed in order to investigate the conditions under which S. aureus is able to adapt to the in vivo caterpillar environment, assay the relative ¢tness of strains through competition experiments, and index the relative gain in virulence potential through measurements of Manduca weight loss or death. Once strains of di¡ering pathogenic potential have been identi¢ed, it may be possible to track changes in putative virulence loci, or metabolic genes that may be linked to such loci, by the generation of nucleotide sequence data.

P3^13 CRYOCOLA ANTIQUUS GEN. NOV., SP. NOV., ISOLATED FROM SIBERIAN PERMAFROST E. Yu. Gavrish(1,2), S. G. Karasev(3), N. E. Suzina(2), D. A. Gilichinsky(4) and L. I. Evtushenko(1,2) (1) Pushchino State University, Pushchino, Moscow Region, 142290, Russia ; (2) All-Russian Collection of Microorganisms (VKM), G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms RAS, Pushchino, Moscow region, 142290 Russia; (3) Kuban State University, Krasnodar, 350040, Russia ; (4) Institute of Physicochemical and Biological Problems of Soil Science RAS, Pushchino, Moscow region, 142290, Russia Two yellow-pigmented actinomycetes (strains VKM Ac2070T and VKM Ac-2278) were isolated in the course of studying the microorganisms from frozen Late Pliocene samples 1.8-3.0 million years old (Kolyma lowland, Russia). The bacteria were characterized by non-spore-forming, straight or curved long cells (0.3 ^ 0.4 x 3.0 ^ 4.5 Wm) fragmenting into shorter motile rods (0.3 ^ 0.5 x 0.7 ^ 1.2 Wm) with perithrichous £agella. The growth temperature range was 4 ^ 26C; thermal optimum 18 ^ 20 C. The strains contained 2,4-diaminobutyric acid in their peptidoglycan (B2Q type), and rhamnose as the predominant cell wall sugar. The major menaquinones were MK-11 and MK-12. The principal phospholipids were phosphatidylglycerol and diphosphatidylglycerol, predominant fatty acids were anteiso-15:0, anteiso-17:0 and iso-16:0. The G+C content of DNA was approximately 65 mol%. The phylogenetic analysis based on 16S rDNA sequences revealed that the permafrost isolates formed a separate branch within the Microbacteriaceae phylogenetic cluster, which is closely linked to the Cryobacterium psychrophilum and exhibited 96.3-96.6% 16S rDNA binary sequence similarity to this species. However, the isolates clearly differed from Cryobacterium psychrophilum in the cell morphology and pigmentation, growth temperature range and optimum, and the composition of menaquinones and fatty acids. On the basis of both phenotypic and molecular genetic ¢ndings, we propose a new genus and species, Cryocola antiquus gen. nov., sp. nov. in the family Microbacteriaceae.

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P3^14 PHYLOGENY OF PHOTOTROPHIC GREEN SULFUR BACTERIA AND DIVERSITY IN MARINE HABITATS B. Alexander, J. F. Imho¡ Institut fur Meereskunde, Dusternbrooker Weg 20, D-24105 « « Kiel, Germany Phototrophic green sulfur bacteria represent a separate phylogentic line of the eubacteria. They are common within the light £ooded anoxic marine environment, in particular under conditions of low light intensities and high sul¢de concentrations. Little is known about the diversity of green sulfur bacteria in marine habitats and a limited number of green sulfur bacteria have been isolated from the marine environment. Therefore, their phylogeny was studied on the basis of gene sequences of the 16S rRNA and of the Fenna-Matthews-Olson (FMO) protein. Primers were designed that allowed speci¢c ampli¢cation of green sulfur bacterial 16S rDNA and of the major part of the fmoA gene. The largely congruent phylogenetic relationship of gene sequences of the fmoA gene and of 16S rDNA obtained by pure culture studies facilitated the comparison of results with both sets of primers with natural samples. Marine strains were found to form clusters separate from freshwater strains (Alexander et al., 2002). The analysis of samples from coastal habitats from geographical distant areas showed that so far known green sulfur bacteria occur around the world at habitats that are suitable and typical for green sulfur bacteria. Their natural diversity in marine habitats is relatively low and the major phylogenetic lines were known from the analysis of available cultures of green sulfur bacteria. P3^15 COMPARISON OF THE PHYLOGENETIC PLACEMENT OF OCHROBACTRUM AND BRUCELLA ¤ C. Teyssier(1), M. Simeon de Buochberg(1), H. Marchandin(2), J. L. Jeannot(1) and E. Jumas-Bilak(1) ¤ ¤ (1) Laboratoire de Bacteriologie, Faculte de Pharamacie, 15, av. C. Flahault, BP 14491, 34093 Montpellier cedex 5, ¤ France; (2) Laboratoire de Bacteriologie, Hopital Arnaud de Villeneuve, Montpellier, France The bacteria belonging to the genus Ochrobactrum are likely members of the microbiota of soil more and more frequently isolated from immunocompromised patients. The genus Ochrobactrum include 4 species: Ochrobactrum anthropi, Ochrobactrum intermedium, Ochrobactrum tritici and Ochrobactrum grignonense and is the closest known

relative of Brucellae. Oddly enough, 16S rRNA based phylogeny placed the species O. intermedium in an intermediate position between O. anthropi and Brucella spp. We constructed a phylogenetic tree based on 16S rDNA sequences of 11 clinical isolates of O. anthropi (N=5) and O. intermedium (N=6) and 7 selected type strains representative of the genera Ochrobactrum and Brucella. The tree con¢rmed that O. intermedium isolates are closer relative from Brucella spp. than from O. anthropi. This paradoxical branching was also found when we performed a phylogenetic analysis based on 23S rDNA sequences. Finally, we ampli¢ed and sequenced the rpoB gene of Ochrobactrum isolates and type strains. In the rpoB-based tree constructed, the genus Brucella was clearly branched (bootstrap value of 91%) out of the genus Ochrobactrum. Thus, the paradoxical grouping of Ochrobactrum and Brucella in the rRNA-based trees was not con¢rmed by rpoB phylogeny. These results highlighted the need of di¡erent markers to assess the phylogenetic placement of an organism, especially in groups where the rRNA polymorphism is too low to clearly resolve the relationships between species. P3^16 GENOME SIZING AND RRN COPY NUMBERING IN SOME REPRESENTATIVE MEMBERS OF THE SPOROMUSA SUB-BRANCH ¤ H. Marchandin(1), C. Teyssier(2), M. Simeon de Buochberg(2), H. Jean-Pierre(1) and E. Jumas-Bilak(2) ¤ " (1) Laboratoire de Bacteriologie, Hopital Arnaud de Villeneuve, 371 Avenue du Doyen Gaston Giraud, F-34292 ¤ Montpellier Cedex 5, France; (2) Laboratoire de Bacter¤ iologie, Faculte de Pharmacie, 15 Avenue Charles Flahault, BP 14491,F-34093 Montpellier Cedex 5, France Chromosome number, genome size and rRNA copy number are parameters that displayed great variability among bacteria. Although several members of the Sporomusa subbranch are considered as putative human pathogens, the genome organization and structure of the bacteria belonging to this sub-branch of the Gram positive bacteria phylum has yet never been investigated. We performed the ¢rst genomic investigations for 31 representative strains of ¢ve genera belonging to this phyletic group: Acidaminococcus, Anaeroglobus, Dialister, Megasphaera, Selenomonas, and Veillonella. Number, size, and topology of bacterial chromosome, as well as 16S rRNA gene copy number were determined using a combination of Pulsed-Field Gel Electrophoresis of full-length or I-CeuI restricted chromosomal DNA and hybridization approaches. All strains were shown to possess one circular chromosome ranging in size from approximately 1380 kb for Dialister pneumosintes to 2580 kb for Selenomonas sp. Copy number of the

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rrn loci varied from 3 in the genus Acidaminococcus to 7 for Megasphaera elsdenii. Mapping analysis revealed that one of the rrn operon was inverted in orientation with respect to the others and low-resolution maps could be established for 3 Veillonella type strains. These data are the ¢rst available for members of the Sporomusa subbranch and although no correlation seemed to exist between chromosome size and rRNA genes number in bacteria, we observed that all the strains we have studied displayed the smallest genome sizes described according to their rrs copy number. P3^17 REVEALING OF COLLAGEN-LIKE SEQUENCES IN THE MICROORGANISMS OF DIFFERENT TAXONOMIC GROUPS E. V. Karpova, O. V. Alekseeva , T. S. Kalebina, I. S. Kulaev Moscow State University, Moscow, Russia Collagens and collagen-like sequences are widely represented in higher eukaryotes. While much is know about animal collagens, these proteins have not been found in plants yet. The distribution of collagen-like proteins among microorganisms is poorly investigated. Collagens are the proteins characterized by a speci¢c primary structure, and the presence of the unique triple helical domain formed by three polypeptide chains. Earlier we have detected the genomic DNA fragments in prokaryotes (Micrococcus luteus and Nocardia sp.) and yeasts (Candida utilis and Candida maltosa) homologous to the chicken A1(1) collagen-encoding cDNA. In present work we used the highly puri¢ed speci¢c enzyme ^ collagenase from Clostridium histolyticum for the detection of collagen-like sequences in the proteins of di¡erent microorganisms. Using the selective biotinilation by NHS-LC-Biotin, electrophoresis and electron microscopy we have identi¢ed the proteins hydrolyzed by this enzyme in Halobacterium salinarium, Candida utilis and Hansenula polymorpha cells. We have shown that predominantly collagenase-sensitive proteins are revealed the cell wall localization and, evidently, serve as structural proteins. The data obtained allow us to suggest that collagen-like sequences are widely spread among microorganisms of di¡erent taxonomic groups and collagens, apparently, are evolutionary ancient proteins. This work has been supported by grants 00-04-48356, 0104-06571 and 00-15-97851 of Russian Foundation for Basic Research and 01-0583 INTAS.

P3^18 gyrB AS A PHYLOGENETIC MARKER FOR SPECIES IDENTIFICATION H. Kasai, K. Watanabe, and S. Harayama Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi, Japan The current progress in bacterial systematics can be traced back to the introduction of 16S rDNA-based techniques. By using the techniques for 16S rDNA cataloguing and sequencing, prokaryotic taxa can be ordered hierarchically among the ranks of genera and kingdoms. Determination of inter- and intraspecies relatedness has been facilitated by the techniques for protein-coding gene sequencing, DNA pro¢ling and DNA microarrays. On 1995, Yamamoto and Harayama developed the universal primers to amplify the gyrB genes of various bacteria. Because the evolutionary rate is faster than the gene for 16S rDNA, it can be applied for identi¢cation and classi¢cation of closely related bacteria. From the results of comparative studies between the gyrB-based phylogenetic classi¢cation and DNA-DNA hybridization, it is likely that the gyrB is useful to classify the bacteria at the genomic species level. Then, we have established the gyrB database for identi¢cation and classi¢cation of bacteria since 1998. The database contain the pages for the gyrB sequence data, for the methods for ampli¢cation of the gyrB, for the multiple alignment of the gyrB, for the blast search tools, for recent topics on the gyrB, and so on. Recently, we have released nearly 2,000 gyrB sequence data from the type strains of various taxa. From the accumulated data, we would like to introduce the primer sets for amplify the gyrB fragment e/ciently. In parallel, we would show the results of comparative studies between the gyrB phylogeny and DNADNA hybridization on several. P3^19 APPLICABILITY OF BOX-PCR FINGERPRINTING TO DIFFERENTIATE STREPTOMYCES SPECIES B. Lanoot(1), M. Vancanneyt(1), P. Dawyndt(2), M. Cnockaert(1), H. Ying(3), Z. Liu(3) and J. Swings(1) (1) BCCMTM / LMG Bacteria Collection, K.L. Ledeganckstraat 35, 9000 Gent, Belgium ; (2) Laboratorium voor Microbiologie, Universiteit Gent, K.L. Ledeganckstraat 35, 9000 Gent, Belgium ; (3) Institute of Microbiology, Chinese Academy of Sciences, P.O. Box 2714, Beijing 100080, P.R. China The type strains of 476 validly described Streptomyces species were screened using the rep-PCR ¢ngerprint tech-

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nique. The aim of the study was to evaluate the taxonomic relevance of the rep-PCR technique as a tool for species delineation within Streptomyces systematics. Of the primers tested BOX, (GTG)5 and primer pair REP, the BOX primer yielded the less complex and more robust patterns if applied under highly standardised conditions. A good correlation was observed between rep-PCR ¢ngerprinting and DNA-DNA pairing studies. Species sharing nearly identical rep patterns were correlated with DNA-DNA homology values above 70%. These data clearly demonstrate that the genus is largely overspeciated and that at least 38 species should be reclassi¢ed as junior synonyms. Emended descriptions are proposed for eight species. However BOX-PCR does not allow one to reveal all synonymous taxa. Within a species the dispersion of the repetitive BOX element over the genome can however be very diverse as strains sharing a DNA-DNA homology value at or above 70% can have di¡erent rep patterns. The taxonomic level of discrimination is quite high namely between intra-species and species level depending on the species analysed. We concluded that rep-PCR is a reproducible and easy-to-perform technique valuable for speciation and typing of streptomycetes which will help in the establishment of a genomically based classi¢cation of all validly described Streptomyces species. P3^20 DESCRIPTION OF A NOVEL VIBRIO SPECIES ISOLATED FROM SEA BREAM, BIVALVES AND SEA WATER M. C. Macian(1,2), M. J. Pujalte(1,2), P. A. D. Grimont(3) and E. Garay(1,2) (1) Department of Microbiology and Ecology, University of Valencia; (2) Cavanilles Institute of Biodiversity and Evolutive Biology (ICBBE), Burjassot E-46100, Valencia, Spain; (3) Unity of Emergent Pathogen Bacteria Biodiversity, Institut Pasteur, Paris, France A group of 18 strains that behaved as typical Vibrio species has been subjected to a polyphasic taxonomic study, including numerical taxonomy, oxidation pro¢le (Biolog), ribotyping (RiboPrinter0), DNA-DNA hybridization (S1 nuclease/trichloroacetic acid method) and phylogenetic analysis of the 16S rRNA gene sequences. Three strains were isolated during an annual survey of Vibrio species from bivalves and marine water on the Spanish Mediterranean coast in the 80's. More recently, a group of 15 strains were isolated from kidney of diseased Sparus aurata (sea bream) cultured at Spanish ¢sh farms at the Mediterranean coast. The isolates were ¢rst phenotypically characterized, and the results revealed a very high degree of phenotypic similarity between them. Thereafter, other tests were performed. Oxidation pro¢le obtained with the

Biolog system was characteristic and di¡erent from those pro¢les included in the system. Ribotypes obtained with two restriction endonucleases showed a moderate variability within the strains. The almost complete 16S rDNA sequences of ¢ve isolates were determined, and the comparative analysis con¢rmed their a/liation to the genus Vibrio, with levels of sequence similarity between strains higher than 98 %, and of 95.5-96.5 % with other Vibrio species. Phylogenetic analysis performed by applying three alternative treeing methods situated our strains in the vicinity of the V. £uvialis-V. furnissi cluster. DNA-DNA hybridization results con¢rmed the independence of the isolates in a new genospecies. All data support a very close relationship between the 18 strains and suggest that they could constitute a new species within genus Vibrio. P3^21 NOVEL HALOARCHAEAL ISOLATES FROM SLOVENIAN AND CROATIAN SALTERNS í >¤ M. Crnigoj, B. Herzog-Velikonja, N. Poklar and L. Pasic > Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia Until recently, the presence of halophilic archaea in hypersaline habitats in eastern Adriatic coast has not been investigated. Among numerous habitats, the Slovenian salt> erns in city of Secovlje and Ston salterns, located at >ac peninsula, were chosen. The salterns difCroatian Peljes fer in ground foundation. The ground foundation of Se> covlje ponds is made out of arti¢cial gypsum crust rich in green algae known as ``petola'', while the ground forma> tion of Ston salterns is cemented. Both Secovlje salterns and Ston salterns were repeatedly sampled from august 2000 to august 2002. Inoculation of ¢ltered hypersaline samples into standard halophilic media led to isolation of more then 50 strains in pure culture. Several phenotypic characteristics, which include antibiotic susceptibility, gelatine, casein, and starch hydrolysis, ability to degrade DNA, production of acids from sugars, catalase and oxidase activity, were tested. A number of strains were found to produce proteinaceous compounds which, like halobacterial halocins, inhibited the growth of Halobacterium halobium. Based on phenotypic characteristics tested we presumptively identi¢ed these isolates as the members of the Halobacteriaceae family. Based on partial sequence of 16S rDNA, isolates were classi¢ed as Haloarcula, Haloterrigena, Halobacterium, Haloferax and Natrialba, respectively.

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P3^22 NUCLEOTIDE SEQUENCE ANALYSIS AND MOLECULAR COMPARISON OF THE NOVEL INCJ CONJUGATIVE TRANSPOSON-LIKE CTNR391 AND THE SXT ELEMENTS: MOBILE DNA REPAIR CASSETTES ? J. T. Pembroke(1), J. O'Halloran(1), D. Boeltner(2), A. M. Osborn(2), P. Strike(3), C. MacMahon(1), and B. McGrath(1) (1) Molecular Biochemistry, University of Limerick, Ireland; (2) Department of Biological Sciences,University of Essex, UK; (3) Donnan Laboratories, University of Liverpool, UK The prototype IncJ group mobile genetic element CTnR391, originally classi¢ed as a plasmid, has been shown to integrate speci¢cally in E. coli into the prfC gene and behave as a conjugative transposon. This element is related to a small group of other IncJ elements pMERPH, R997 and R748 with apparently a very restricted global distribution. Nucleotide sequence analysis of the 89-kb element has revealed some 96 ORFs with 30 ORFs that could not be assigned function. Much of the sequence showed homology to the mobile SXT element of Vibrio. Comparative analysis has revealed a high level of evolutionary conservation in respect to nucleotide sequence and gene order. We have identi¢ed a number of putatitive insertions into the core of each element di¡ering in length and sequence that appear in identical loci in both elements, which in the main encode unknown ORFs. Functional analysis of both CTnR391 and SXT reveals a mosaic structure consisting of bacteriophage, plasmid and transposable elements. The CTnR391 integrase resembles phi-80 like integrases while there are a series of transfer modules which resemble the transfer system of the Salmonella plasmid R27. Most interestingly analysis has revealed that both SXT and CTnR391 possess a large number of DNA repair like genes involved in induced mutagenesis (rumAB), UV sensitisation (ORF13,14,15), repair ATPases (ORF's 25,26) recombination (ORF's 68,75), DNA binding (ORF's 4,67), repair induction (lon ORF31). These functions are conserved in both elements and suggest that the element may have evolved as a mobile repair cassette.

P3^23 DEVELOPMENT OF INTERACTIVE IDENTIFICATION SYSTEM OF UNIQUE AND EXTREMOPHILIC MICROORGANISMS ``SINIDEM'' IN INTERNET I. Sorokin, N. Galchenko and V. F. Galchenko Institute of Microbiology of RAS, Prospekt 60-let Oktjabrja, 7/2, 117312 Moscow, Russia The full-function microbiological database program sinidem and the setup version used for installation of this program to a computer with Microsoft Windows 95, 98, 2000, XP operating systems have been created. The software/hardware platform for sinidem database was assembled and tested. The ¢rst version of sinidem that allows the viewing of properties, values, and descriptions of microorganisms using a web-browser and provides the base possibilities of The interactive identification was started up and published in Internet (http://uniqem.ru). Thus, the microbiological database program sinidem permits the fast search of information on microorganisms described in the database. sinidem will provide probabilistic attribution by speci¢ed properties of microorganism that is not described in the database to speci¢c microorganism or the group of similar ones described in the database; that is, it gives opportunity of The interactive probabilistic identification of microorganisims. The vocabulary of main properties used for description in sinidem was created and prepared for publication in Internet. The complex of sinidem software tools makes it possible to add new properties and their values after the reviewing by specialists. P3^24 MOSAICISM OF THE LARGE NATURAL ESCHERICHIA COLI PLASMID PRK100 M. Starcic Erjavec(1,2), W. Gaastra(2), J. van Putten(2) í and D. Zgur-Bertok(1) (1) Department of Biology, Biotechnical Faculty, Univer> sity of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia; (2) Department of Infectious Diseases and Immunology, Utrecht University, P.O. Box 80.165, 3508 TD Utrecht, The Netherlands In search for the evolutionary origin of the conjugative Flike plasmid pRK100, we determined the plasmid's functional replication region(s) and performed targeted genetic analysis (hybridization, PCR, DNA sequencing) of the plasmid. Construction of minireplicons via ligation of Tn1725 with plasmid fragments and targeted cloning of putative replication regions, followed by sequence analysis

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indicated two functional replication regions, a plasmid Flike RepFIB and a plasmid R1-like RepFIIA replication region. Partial nucleotide sequencing of regions of the plasmid revealed genes that encode a putative enterochelin iron uptake system previously associated with an Escherichia coli pathogenicity island including genes with similarity to PAI III536-like enterochelin and the pColV-like aerobactin genes. In addition, a homologue of the plasmid R100-like RmoA gene was found that exhibits strong similarity to the Hha/YmoA class of modulators of gene expression. PCR and hybridization experiments further demonstrated that pRK100 harbors multiple IS2 and IS3 insertion sequences that may have facilitated in the acquisition of elements from other plasmids. These data together with the previous identi¢cation of a F-like tra region and a pColIa-like colicin Ia, indicate that pRK100 has a highly mosaic structure with elements derived from many di¡erent known large natural plasmids and the chromosome. P3^25 CONSERVATION OF THE MOSAIC STRUCTURE OF THE FOUR INTERNAL TRANSCRIBED SPACERS AND LOCALIZATION OF THE RRN OPERONS ON THE STREPTOCOCCUS PNEUMONIAE GENOME ' V. Giannino, M. Santagati, G. Guardo, C. Cascone and S. Stefani Department of Microbiological and Gynaecological Sciences, University of Catania, Italy The detection of heterogeneity of the 16S-23S ribosomal intergenic transcribed spacer (ITS) region has become rather common for identi¢cation and typing purposes of bacteria. ITS not only varies in sequence and length, but also in number of alleles per genome and in their position on the chromosome, allowing discrimination of several species within a genus. Also variation in ITS sequences between the multiple rrn operons present within a genome may be as high or greater than between strains of the same species or subspecies. The aim of this study was: i) to test the polymorphism of the spacer by PCR-ribotyping for typing and identi¢cation purposes in Streptococcus pneumoniae; ii) to characterise the molecular structure of the ITS sequence; iii) to determine the number and localization of rrn operons in S. pneumoniae by PFGE of I-Ceu I restriction fragments of entire genome. Our results show that the genome of S. pneumoniae contains 4 ribosomal operons, localised in the same place on the chromosome, each containing a single ITS allele of 241bp: thus, the PCR-ribotyping in S. pneumoniae is not useful as a molecular typing method, but can be use only for identi¢cation purposes. The ITS sequence presents a mosaic organ-

isation of blocks highly conserved intra and inter-species, giving no possibility to mutations to arise. The observation that the sequence blocks of the ITS mosaic of S. pneumoniae are sharing with oral streptococci localised in a di¡erent branch in the phylogenetic tree, leads to consider that the causative recombination events are ancient. P3^26 WHAT IS THE VALUE OF CHEMICAL DATA IN THE TAXONOMY OF THE FAMILY HALOMONADACEAE? C. Belloch and B. J. Tindall DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH., Mascheroder Weg 1b, D-38124 Braunschweig, Germany Members of the family Halomonadaceae have been chosen as a model system for study, in order to appreciate the signi¢cance of genetic, chemical and biochemical/physiological data in the current taxonomy. Previous work showed that the genera Halomonas and Deleya did not form clear coherent groups and led to the proposal to combine these two genera. Subsequent work has relied heavily on 16S rDNA squence data as the basis for the current taxonomy. At present the family Halomonadaceae comprises the genera Halomonas, Cobetia, Chromohalobacter, Zymomonas, and Carnimonas. Although there are currently some clear groupings within the family, as more species are added it is becoming more di/clut to assign new taxa to existing groups or to clear distinguish the current groupings based on the 16S rDNA data alone. In the present study we have returned to the chemical data, which initially showed that the genera Halomonas and Deleya were not coherent groups, with a view to examining the relationship between the 16S rDNA and chemical data as a means of resolving some of the present problems in the taxonomy of this family. P3^27 ANCIENT PROKARYOTES ^ WHAT IS THEIR SIGNIFICANCE IN PROKARYOTIC SYSTEMATICS? B. J. Tindall DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH., Mascheroder Weg 1b, D-38124 Braunschweig, Germany In recent years there have been a number of reports of organisms associated with geologically ancient material (rock salt or amber). Although there are some doubts

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about the source of isolated strains or 16S rDNA sequences from such ancient material, these reports have a number of consequences in the interpretation of the evolutionary history of the organisms concerned. One of the major problems is that some of the logical consequences of the hypothesis that the organisms or the sequences associated with them are ancient have not been fully explored. An interesting paradox is that if we accept that the biological material examined really is ancient then some of our current theories and hypotheses on which our understanding of prokaryotic evolutionary history and systematics is based may not be supported. P3^28 RECONSTRUCTING THE PAST ^ TAKING A CLOSER LOOK AT THE COMPLEXITY B. J. Tindall DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH., Mascheroder Weg 1b, D-38124 Braunschweig, Germany The goal of modern prokaryotic systematics is to group organisms according to their evolutionary relationships. One of the major di/culties is that there is no reliable fossil record for prokaryotes which allows us to trace their evolution over geological time. This applies to morphology and molecular (protein and DNA sequence) data. Thus we tend to extrapolate backwards using a set of assumptions, some of which may not re£ect the true picture. When two data sets are compared one of the most important aspects is that they meet certain criteria, in particular that homologous parameters are being analysed. Over the past 30 years concepts, such as orthology, parology, xenology, paraxenology, and plerology have been formulated. Unfortunately, such concepts are usually neglected in modern prokaryotic systematics. Taking a closer look at the way these concepts a¡ect our interpretation of data provides an interesting insight into the di/culties associated with unravelling the evolutionary history of prokaryotes.

P3^29 HOW IMPORTANT IS THE PHENOTYPE IN EVALUATING 16S rDNA DATA? C. Belloch and B. J. Tindall DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH., Mascheroder Weg 1b, D-38124 Braunschweig, Germany The analysis of the small subunit rRNA molecule (or its gene sequence) has become a routine method in microbiology. The way in which we interpret the signi¢cance of the sequence data is strongly in£uenced by concepts developed in the mid-1980's. However, recent advances in the study of the ribosome, including the study of RNA and protein interactions in intact ribosome particles, has led to the availability of important information of signi¢cance to the way in which the rRNA sequence data may be interpreted. Using a model set of organism and taking the structure and function of the 16S rRNA molecule into consideration (including both rRNA primary and secondary structure, together with protein interactions) it is possible to show a number of novel aspects in the way in which this data may be interpreted. This approach is not only important in understanding the relevance of this molecule in determining taxonomic groupings in prokaryotes, but it also helps to appreciate the evolutionary constraints on this molecule. P3^30 CHARACTERIZATION OF PSEUDOALTEROMONAS SP. STRAIN CP76: AN EXTRACELLULAR PROTEASE PRODUCER ¤ ¤ C. Sanchez-Porro, E. Mellado, S. Mart|n, D. R. Arahal, M. ¤ C. Marquez and A. Ventosa Department of Microbiology and Parasitology, University of Sevilla, 41012 Sevilla, Spain During a large screening for the isolation of moderately halophilic bacteria (able to grow optimally in media containing between 3 and 15 % NaCl) showing hydrolytic activities, we isolated a total of 26 proteolytic moderately halophilic bacteria from several hypersaline environments (salterns and saline soils) in South Spain. One of the isolates, designated as strain CP76, was selected as the best producer and most appropriate strain to carry out further studies. The extracellular protease was puri¢ed by a two steps process and characterized biochemically. This enzyme showed optimal activity over a wide range of salt concentrations, from 0 to 20 % NaCl, and at 55C and pH 8.5; The molecular mass was estimated to be of 38 Kda.

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We sequenced its N-terminal amino acid sequence and it showed high similarity to several metalloproteases. The genetic characterization of the gene involved in the production of this enzyme is under way. Besides, we have characterized taxonomically strain CP76. Phenotypic data as well as the comparison of 16S rRNA complete sequences showed that this bacterium belongs to the genus Pseudoalteromonas. The highest 16S rRNA similarity with most Pseudoalteromonas species is 95.6% or lower, except for the recently described species Pseudoalteromonas ruthenica, to which it was closely related (99% similarity). P. ruthenica has been described very recently (2002) and is a slight halophile (optimal growth at 1-3 %NaCl) isolated from marine invertebrates. Althoght there are several phenotypic di¡erences between this species and strain CP76, DNA hybridization studies will determine if our isolate constitutes a separate species of the genus Pseudoalteromonas. P4^1 CHARACTERIZATION OF PSYCHROTROPHIC BACTERIA ISOLATED FROM FISH AND SEA WATER OFF THE ICELANDIC COAST, BASED ON 16S GENE ANALYSIS AND BIOCHEMICAL REACTIONS ¤ E. Benediktsdottir(1) and V. Th. Marteinsson(2) (1) Institute of Biology, Microbiology Laboratory, University of Iceland, Armuli 1A, IS-108 Reykjavik, Iceland; (2) Prokaria, Gylfa£ot 12, IS-112 Reykjavik, Iceland Thirty-six sea samples and samples taken from skin, gills and intestines of 19 individual ¢sh of ¢ve species, were cultivated at 15C on Marine agar, TCBS agar and in alkaline peptone water supplemented with minerals. All samples were taken from pelagic ¢sh and water o¡ the Icelandic coast, with water temperatures at or below 11C. One hundred thirty-three strains isolated from ¢sh and 193 strains isolated from sea water were presumptively identi¢ed as Vibrio and Photobacteria based on Gram reaction, oxidative/fermentative ability, oxidase reactions and inability to grow on CLED agar. These strains and selected reference strains were subjected to biochemical tests and numerical taxonomy. Forty-six of the strains were subjected to partial or complete 16S ribosomal analysis. According to the 16S gene sequencing, the isolates belonged to 6 genera, most strains belonged to the Vibrio genus, but Photobacterium, Enterovibrio, Psychromonas, Moritella and Shewanella were also represented. Nine strains belonged to unknown species, with 97 % or less identity to strains of known species: Three of these belonged to the Vibrio genus, 2 to the Psychromonas genus, 2 to the Shewanella and 2 to a possible new genus. The results of the biochemical tests clustered strains into four

main clusters, V. splendidus-like cluster, V. logei-like cluster, Photobacterium-like cluster, and an unreactive group including the Moritella and Shewanella strains. The relevance of the di¡erent tests for psychrotrophic bacteria will be discussed. P4^2 PRIMARY STRUCTURE OF S-LAYER PROTEINS FROM MESOPHILIC, THERMOPHILIC AND HYPERTHERMOPHILIC METHANOCOCCI H. Konig(1), E. Akca(1), H. Claus(1), B. Schlott(2), T. « Debaerdemaeker(3) and J.-P. Declercq(4) (1) Institut fur Mikrobiologie und Weinforschung, Johannes « Gutenberg-Universitat, 55099 Mainz, Germany ; (2) Insti« tut fur Molekulare Biotechnologie (IMB), 07745 Jena, Ger« many; (3) Sektion Rontgen- und Elektronenbeugung, Uni« versitat, 89069 Ulm, Germany; (4) Laboratoire de Chimie « ¤ et de Cristallographie, Universite Catholique de Louvain, 1348 Louvain-la-Neuve, Belgium Cells of methanococci are covered by a single layer of protein subunits (S-layer) in hexagonal arrangement, which are directly exposed to the environment and cannot be stabilized by cellular components. We have isolated Slayer proteins from cells of Methanococcus vannielii (Topt.= 37 C), Methanococcus thermolithotrophicus (Topt.= 65 C) and Methanococcus jannaschii (Topt.= 85 C). The primary structure of the S-layer proteins was determined by sequencing the corresponding genes. According to the predicted amino acid sequence the molecular masses of the Slayer proteins of the di¡erent methanococci are in a small range between 59064 Da and 60547 Da. Compared to its mesophilic counterparts the observation is noteworthy that in the S-layer protein of the extreme thermophile Mc. jannaschi the acidic amino acid Asp is predominant, the basic amino acid Lys occurs in higher amounts and Cys and His are only present in this organism. Despite the di¡erences in the growth optima and the predominance of some amino acids, the comparative total primary structure revealed a relatively high degree of identity (38 ^ 45 %) between the investigated methanococci. This observation indicates that the amino acid sequence of the S-layer proteins is signi¢cantly conserved from the mesophilic to extremely thermophilic methanococci.

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P4^3 MODERATELY ACIDOPHILIC, THERMOPHILIC ANAEROBIC BACTERIA FROM KAMCHATKA HOT SPRINGS I. V. Kublanov, M. I. Prokofeva, I. V. Subbotina, T. P. Tourova and E. A. Bonch-Osmolovskaya Institute of Microbiology, Russian Academy of Sciences, Prospekt 60 Let Oktyabrya 7 bldg 2, 117312, Moscow, Russia Acidic hot springs are quite common in volcanic environments, but most known thermoacidophilic prokaryotes are either aerobes, or organisms with a di¡erent type of anaerobic respiration. Thermoacidophiles with tentatively fermentative type of metabolism are represented by Archaea of genera Thermoplasma and Acidilobus and by the only representative of Bacteria ^ Thermoanaerobacterium aotearoense, which is a moderate thermophile and moderate acidophile. 15 samples of water and sediment from Kamchatka hot springs from 35 to 80C and from pH 2.0 to 4.0 were used for inoculation of anaerobic medium with di¡erent sugars as growth substrates. pH of the medium and incubation temperature were the same as in sampling sites. Four very active enrichment cultures growing on sucrose or starch at 60C at pH 3.5 were obtained, and from two of them dominating organisms were isolated in pure culture and characterized. Strain 761 had rodshaped spore-forming cells, motile with one £agellum. It grew optimally at 55C and pH 5.7; lower pH limit of growth was 3.2. Isolate 761 was able to ferment a wide range of mono-, di- and polysaccharides; when thiosulfate was added, the formation of elemental sulfur was observed. G+C content of DNA was 33.8 mol.%. Analyses of 16S rDNA sequence placed the new isolate in genus Thermoanaerobacterium. Cells of strain 711 were also spore-forming motile rods. It grew optimally at 60C and pH 5.0 and was able to ferment many sugars. Thiosulfate, when added, stimulated the growth on fermentable substrates, being reduced to hydrogen sul¢de. Hybridization with a molecular probe speci¢c for genus Thermoanaerobacter revealed strain 711 as a member. Thus, both new isolates were organisms with fermentative metabolism, moderate thermophiles and moderate acidophiles, belonging to bacterial genera that included (with the exception of T. aotearoense) only neutrophilic species.

P4^4 NOVEL THERMOPHILIC BACTERIA FROM DEEPSEA HYDROTHERMAL VENTS M. L. Miroshnichenko(1), S. L'Haridon(2), O. Nersessian(2), S. Spring(3), P. Schumann(3), E. Stackebrandt(3), E. Bonch-Osmolovskaya(1), C. Jeanthon(1) (1) Institute of Microbiology, Russian Academy of Sciences, 117312 Moscow, Russia; (2) UMR 6539, Institute Universitaire Europeen de la Mer, 29280, Plouzane, France ; (3) German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, 38124 Braunschweig, Germany Five new genera of moderately thermophilic bacteria were isolated from the deep-sea hydrothermal habitats. New isolates were found to be diverse both phylogenetically and phenotypically, possessing chemolithoautotrophic, chemoorganoheterotrophic, or mixotrophic types of metabolism. Epsilon-Proteobacteria isolated from the East Paci¢c Rise and Mid-Atlantic Ridge were represented by anaerobic thermophiles Nautilia lithotrophica gen. nov., sp. nov. and Caminibacter profundus sp. nov., growing chemolithoautotrophically with molecular hydrogen in the process of sulfur reduction. The ability of chemolithotrophic growth was also shown for the new representatives of the family Thermaceae. These are Oceanothermus profundus gen. nov., sp. nov. and Vulcanithermus mediatlanticus gen. nov., sp. nov. Both microorganisms were able to microaerophilic growth with a wide range of substrates, including molecular hydrogen. They could also grow anaerobically in the presence of nitrate. From the hot vents of Mid Atlantic Ridge Caldithrix abyssi gen. nov., sp. nov. was isolated. This strictly anaerobic moderate thermophile, capable of mixotrophic growth, represented a new phylum. It was able to grow by means of fermentation of proteinaceous substrates, or chemolithoheterotrophically by oxidizing molecular hydrogen in the course of reduction nitrate to ammonium. Marinobacillus modestocaldus gen. nov., sp. nov. was found to be able to oxidize organic substrates in the presence of nitrate or grew chemolithoheterotrophically with molecular hydrogen as electron donor and nitrate as electron acceptor.

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P4^5 DETECTION OF HYPERTHERMOPHILIC CRENARCHAEOTA OF THE GENUS DESULFUROCOCCUS BY HYBRIDIZATION WITH OLIGONUCLEOTIDE PROBES A. A. Perevalova, A. V. Lebedinskii, N. A. Chernyh, and E. A. Bonch-Osmolovskaya Institute of Microbiology, Russian Academy of Sciences, 117312 Moscow, Russia The goal of this study was to design oligonucleotide primers and probes that would allow chemio£uorescent detection and identi¢cation of representatives of the genus Desulfurococcus in pure cultures, enrichments, and natural samples. The Crenarchaeota-speci¢c primer pair that we designed, Cren 7F (5'-TTCCGGTTGATCCYGCCGGACC-3') and Cren 518R (5'- GCTGGTWTTACCGCGGCGGCTGA-3'), was used in order to obtain PCR products on the DNA of pure cultures, enrichments, and natural samples. The PCR products were hybridized with oligonucleotide probes targeting the genus Desulfurococcus (Dco. 198, 5'-CGTTAACYCCYGCCACACC-3') and its species Dco. mobilis (Dco_mob 198, 5'-CGTTAACCCCTGCCACACC-3') and Dco. amylolyticus (Dco_amy 198, 5'-CGTTAACCCCCGCCACACC-3'). With the use of these primers and probes, four new strains of hyperthermophilic archaea were identi¢ed as members of the species Desulfurococcus amylolyticus. We also managed to detect representatives of the genus Desulfurococcus in marine natural samples, where they had not earlier been found. P4^6

ples. The genus-level probe Tab 827 (5'-GCTTCCGCDYCCCACACCTA-3') was suggested, as well as probes for three phylogenetic groups of genus Thermoanaerobacter: TabI 844 (5'-TTAACTACGGCACGRAATGCTTC-3') speci¢c for T. wiegelii, T. siderophilus, T. ulfurophilus, T. brockii, T. kivui, T. ¢nii, T. ethanolicus, T. acetoethylycus and T. thermohydrosulfuricus ; TabII 424 (5'-CACTAMYGGGGTTTACAACC-3') speci¢c for T. thermocopriae, T. mathranii and T. italicus ; TabIII 184 (5'-TCCTCCATCAGGATGCCCTA-3') speci¢c for T. tencongensis, T. yonseiensis and Carboxydobrachium pacificum (the latter organism is related to the genus Thermoanaerobacter according to its 16S rRNA sequence). Speci¢city and sensitivity of all probes were evaluated by hybridization of DIG-11-dUTP labelled probes to 16S rDNA PCR products of bacterial strains on nylon membrane. Optimum conditions for the hybridization were determined. The probes were speci¢c for the target Thermoanaerobacter spp. and did not hydridize to the rDNA of non-target bacteria. Eight pure unidenti¢ed cultures and ¢ve enrichments were analyzed. All cultures were phenotypically similar to members of the genus Thermoanaerobacter. On the basis of the hybridization results it was concluded that seven of the 13 samples contained representatives of Thermoanaerobacter. One of the strains was isolated from a deep-sea hydrothermal habitat, where this genus was not previously found. Thus, our method enabled identi¢cation of representatives of the genus Thermoanaerobacter without traditional culture-based and microscopic techniques. P4^7 ALKALIPHILIC ANAEROBES FROM SODA LAKES T. N. Zhilina and G. A. Zavarzin

DETECTION OF MEMBERS OF THE GENUS THERMOANAEROBACTER USING OLIGONUCLEOTIDE PROBES I. V. Subbotina, N. A. Chernyh, A. V. Lebedinskii, E. A. Bonch-Osmolovskaya Institute of Microbiology, Russian Academy of Sciences, 117312 Moscow, Russia The genus Thermoanaerobacter comprises Gram-positive thermophilic anaerobic bacteria which can chemoorganotrophically reduce thiosulfate, sulfur and iron as electron acceptors. These organisms have been isolated from a wide variety of environments including hot springs, oil reservoirs and anaerobic digestors, but their distribution has not been thoroughly investigated. We designed oligonucleotide probes that target the genus Thermoanaerobacter and applied these for detection and identi¢cation of these organisms in pure cultures, enrichments and natural sam-

Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia Soda lakes represent an extreme example of terrestrial athalassophilic environment. Due to the evaporation these lakes accumulate carbonate salts and have high mineral content. Microbial community in this habitat is subjected to extreme alkalinity and osmotic stress. High productivity of soda lakes is caused mainly by cyanobacteria with anoxigenic phototrophic bacteria as the main secondary producers, closing pathway of decomposition. Anaerobic decomposers perform metabolic pathways between these two groups. Alkaliphilic microbial community of soda lakes is enormously rich in representatives. Isolated alkaliphilic anaerobes belong to new genera of most diverse phylogenetic a/liation and comprise complete trophic network. In total we have described 9 new genera and 14 species from di¡erent functional groups making preliminary overview of trophic interrelations in this complicated community.

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Among alkaliphilic anaerobes are described: sulfate reducers Desulfonatronovibrio and Desulfonatronum; haloanaerobes: Natroniella, Halonatronum; acetogens and ammoniefers Tindallia, Natronincola ; saccharolytic Anoxynatronum, Alkalibacterium, species of spirochaetes; aerotolerant formiate producing bacilli Amphibacillus sibiricum and A. fermentum, fumarate producing gliding Alkali£exus. P4^8 A STUDY OF MICROBIAL POPULATIONS AND THEIR CONTROL IN LIBYAN OILFIELD M. Gaja, H. Ben Hussein and N. Sharaa Production Technologies and processing research Department, Petroleum Research Centre, P.O. Box 6431, Tripoli, Libya This study aim to assess and enumerate various trouble microbial groups; such as general aerobic bacteria, sulphur-oxidizing bacteria, sulphate-reducing bacteria and iron related bacteria in injection water and produced water at Libyan oil ¢eld. Results showed that bacterial levels were high however e¡ective biocides were used. In addition, bacterial populations were also evaluated at reservoir and upstream temperatures in these commingled waters in presence and absence of biocides. This study also discusses failure reasons of currently used biocides in these studied conditions. P4^9 LIGNOCELLULOLYTIC MICROORGANISMS IN A COMPOSTING ENVIRONMENT: UBIQUITY OF LIGNOCELLULOSE-DEGRADING ENZYMES AND POTENTIAL USE ¤ ¤ G. Guisado, M. J. Lopez, M. C. Vargas-Garcia, F. SuarezEstrella and J. Moreno ¤ ¤ Area de Microbiolog|a, Departamento de Biolog|a Aplicada, ¤a, La Ca·ada de San CITE II-B, Universidad de Almer| ¤ Urbano, 04120 Almer|a, Spain The capacity of microorganisms to assimilate organic matter during composting depends on their ability to produce the enzymes needed for degradation of the substrate. Plant wastes are the most usual materials for composting. Those materials are mainly composed by cellulose, hemicellulose, and lignin. Thus, e/cient degradation during composting means that biodegradation of lignin is also needed together with cellulose and hemicellulose. Lignocellulolytic microorganisms may increase substrate bioavailability and improve humi¢cation processes. Compost is a naturally

enriched environment in microorganisms showing these activities and may be viewed as a natural source of these interesting microorganisms. In this study, lignocellulolytic activities were analysed in 211 microorganisms isolated from composting piles, including bacteria (173 strains) and fungi (38 strains). These microorganisms were selected using a screening procedure according to their abilities to grow from cellulose, lignin and hemicellulose. Several enzymatic activities related to lignocellulose degradation were evaluated. Hemicellulolytic activity was more often found than cellulolytic or ligninolytic activities. Most of the isolates showed peroxidase and oxidase activities. The number of strains with laccase activity ranged from 10 to 30% depending on the detection method used. Tyrosinase and polyphenoloxidase were the less usual activities with a 5% of the microorganisms analysed being positives. Some activities related to lignin degradation typically found in white rot fungi were also detected in several bacteria. According to the results obtained in this work, lignocellulosic enzymatic activities are more widespread than usually assumed. The analyses carried out allowed the selection of several microorganisms according to their enzymatic features. These microorganisms may serve as inoculants for composting improvement, biomass treatment operations or e¥uents depuration. P4^10 DIVERSITY AND EXPRESSION OF CATABOLIC GENES INVOLVED IN CATABOLISM OF PHENOL AND p-CRESOL IN PSEUDOMONADS ISOLATED FROM THE POLLUTED AREA A. Heinaru, M. Merimaa, M. Lehiste, J. Truu and E. Heinaru Institute of Molecular and Cell Biology, Tartu University, 23 Riia Street, Tartu 51010, Estonia Three di¡erent catabolic types were revealed for the biodegradation of phenol and p-cresol in bacteria isolated from the river water continuously polluted with phenolic compounds of oil shale leachate. Phenol and p-cresol were degraded via catechol meta (type I strains like P. mendocina PC1), p-cresol via protocatechuate ortho, and phenol either via catechol ortho (type II strains like P. £uorescens PC24) or catechol meta pathway (type III strains like P. £uorescens PC18). Catabolic potential of the 39 strains were studied on the basis of genetic diversity of phenol hydroxylase, p-cresol methylhydroxylase and catechol 2,3-dioxygenase genes. It was found that 11 strains possessed almost identical single-component phenol monooxygenase gene pheA and the remaining 28 strains had multicomponent phenol hydroxylases. The phylogenetic analysis of the partial amino acid sequences of the largest subunit of this enzyme among these strains showed strong

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similarity to the MopN and PoxD. The partial nucleotide sequences of catechol 2,3-dioxygenases of the 26 strains clustered predominantly with xylE DK1 and nahH genes. Phenol hydroxylase and catechol 2,3-dioxygenase genes did not cluster with dmp, phl and phh genes which were expected to be most characteristic for pseudomonads. Despite the fact that the activity of p-cresol methylhydroxylase was detected in both strains PC18 and PC24, only phenol in strain PC18 was an inducer of p-cresol methylhydroxylase. The nucleotide sequences of the corresponding gene clusters pchC-pchX-pchF revealed a high similarity rate (97, 94 and 86% of corresponding genes), being much closer to each other than the corresponding genes in the strain NCIMB9866 (sharing only 61-76% similarity rate). P4^11 IN SITU IDENTIFICATION OF OIL-DEGRADING RHODOCOCCI USING IMMUNOFLUORESCENT TECHNIQUE I. B. Ivshina(1), M. S. Kuyukina(1), C. J. Cunningham(2) and J. C. Philp(3) (1) Institute of Ecology and Genetics of Microorganisms, Russian Academy of Sciences, 13 Golev str., Perm 614081, Russia; (2) Contaminated Land Assessment and Remediation Research Centre (CLARRC), The King's Buildings, Edinburgh EH9 3JN, Scotland, UK; (3) School of Life Sciences, Napier University, 10 Colinton Road, Edinburgh EH10 5DT, Scotland, UK Indirect immuno£uorescence (iIF) appeared to be e/cient for studying in situ diversity of the genus Rhodococcus bacteria belonging to mycolic acid-containing actinobacteria. Polyclonal immune sera against most valid Rhodococcus species were used. High sensitivity of the iIF method, short sample processing time (1.5-2.0 hrs) and species speci¢city of anti-rhodococcal sera used allowed study of a great number of soil and subsurface samples from oil¢elds and oil-contaminated sites. Individual Rhodococcus species were shown to be associated with hydrocarbon accumulations. R. ruber dominated in subsurface samples from oil¢elds. The predominant components of biocenoses of oil-contaminated soil were represented by R. erythropolis, R. opacus and R.``longus''. Rhodococci were detected in numbers 103-104 cells g-1 in the presence of 106-108 cells g-1 of attendant microorganisms. The iIF speci¢city was tested by immuno£uorescent staining of Kocuria rosea, Micrococcus luteus and Pseudomonas spp., isolated and identi¢ed as predominant components of alkanotrophic rhodococci-associated micro£ora. Rapid detection of rhodococci in natural samples allowed us to develop a ¢eld method for microbiological prospecting of oil deposits. This method is currently used in ¢eld-scale bioremediation

of oil-contaminated soil for monitoring indigenous and introduced oil-degrading bacteria. This work was supported by INTAS grant 2001-2151. P4^12 CLASSIFICATION OF NAPHTHALENE DEGRADATION IncP-9 PLASMIDS OF FLUORESCENT PSEUDOMONAS STRAINS T. Yu. Izmalkova, S. L. Sokolov, I. A. Kosheleva, A. M. Boronin Institute of Biochemistry and Physiology of Microorganisms RAS, Pushchino, Russia The collection of naphthalene degrading £uorescent Pseudomonas strains isolated from di¡erent coal tar- and oilcontaminated sites was tested on the presence of IncP-9 plasmids. Seventeen strains of twenty-¢ve IncP-9 plasmidbearing pseudomonads were selected for the further study. Fifteen strains are P. putida, one (37NF) ^ P. £uorescens and one (8909N) ^ P. aeruginosa. Most of plasmids were placed into IncP-9 L-subgroup, while four plasmids (pBS216, pSN11, pNF142 and pBS1145) ^ into N-subgroup. Plasmid pBS2 contains fused replicons IncP-9 and IncP-7. According to the RFLP patterns generated by EcoRI digestion, the plasmids were divided into three groups (A, B, C). IncP-9N subgroup plasmids and pBS2 belong to the group C. Thus, plasmid replicon organization correlates with the whole plasmid structure. PCR ampli¢cation of nahAc DNA fragment of these plasmids and subsequently digestion of amplicons with HaeIII has demonstrated that nahAc sequences are highly conserved. Comparison of nahG restriction patterns revealed that nahG from P. £uorescens 37NF is similar to nahG from archetypal plasmid NAH7, while the other studied strains contain nahG alike one from pDTG1. Ampli¢cation of the regulatory gene nahR was observed only in the case of B and C plasmid groups. Group A plasmids did not give ampli¢cation of nahR fragment. Earlier we have demonstrated the coexistence of two di¡erent salicylate hydroxylase genes in P. putida strains BS202, BS3701 and BS3790 ; one of these has no homology with classic nahG. All representatives of group A plasmids are suggested to have clustered genetic structure. This work was supported by INTAS, grants NN 99-1481 and 01-2383.

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P4^13 DIRECT AND SPECIFIC DETECTION OF AIRBORNE THERMOPHILIC ACTINOMYCETES FROM COMPOSTING FACILITIES P. Kampfer(1), R. Schafer(1), C. Beimfohr(3), A. « « Neef(2) (1) Institut fur Angewandte Mikrobiologie, Justus-Liebig« Universitat Giessen, Heinrich-Bu¡-Ring 26-32, D-35392 « Giessen, FRG; (2) GBF ^ Gesellschaft fur Biotechnologi« sche Forschung, Bereich Mikrobiologie, Mascheroder Weg 1, D-38124 Braunschweig, FRG; (3) vermicon AG, EmmyNoether-Str. 2, D-80992 Munchen, FRG « The high number of biowaste treatment facilities like composting plants has led to an increased attention for the detection of potentially pathogenic microorganisms from the airborne state. Certain groups of compost inhabiting thermophilic actinomycetes (TPAs) which can be emitted from treated biomaterials into the atmosphere are of hygienical importance. For detection and identi¢cation of di¡erent TPAs, nowadays the time consuming cultivation-based approach is most often used. Application of FISH (Fluorescence in situ hybridisation) o¡ers a more rapid and speci¢c identi¢cation of TPAs. Speci¢c 16S rRNA-targeted oligonucleotide probes were developed and evaluated in a FISH format with agar-grown whole cells for the detection of the species Saccharopolyspora rectivirgula, the genera Thermoactinomyces and Saccharomonospora as well as for the groups of Nocardiopsis / Thermobi¢da and certain thermophilic Streptomyces. Conditions for cell wall permeabilisation and hybridisation stringency were optimised independently for all groups of organisms. The probes proved to be speci¢c for the target organisms and enabled the detection of ¢laments and spores. E/zient permeabilization of the cells was achieved via combined acetic acid/lysozyme or hydrochloric acid treatments prior to the hybridization step.

P4^14 FUNCTIONAL AND SPECIES DIVERSITY OF SOIL OIL-OXIDIZING BACTERIA IN CONTRASTING CLIMATIC ZONES E. V. Karaseva(1), S. G. Karasev(1), I. E. Girich(1), V. V. Koksina(1), I. B. Ivshina(2), M. S. Kuyukina(2) and J. C. Philp(3) (1) Biology Faculty, Kuban State University, 7 Stavropolskaya str., Krasnodar 350040, Russia; (2) Institute of Ecology and Genetics of Microorganisms, Russian Academy of Sciences, 13 Golev str., Perm 614081, Russia ; (3) School of Life Sciences, Napier University, 10 Colinton Road, Edinburgh EH10 5DT, Scotland, UK Crude oil-contaminated soils of di¡erent types and geographical locations, particularly ``podzol'' from Middle Urals and ``chernozem'' from Kuban region were studied. A common characteristic of the oil-degrading bacteriocenoses was that representatives of several actinobacteria genera, e.g. Rhodococcus and Gordonia predominated in heavily (above 10 % w/w) oil-polluted soils, with some isolated strains according to the morphological, cultural and chemotaxonomical properties being accommodated to the genera Arthrobacter, Nocardia, Nocardioides, Streptomyces. However, as natural oil degradation proceeded, the microbial community of soils studied shifted towards an increased proportion of proteobacteria, represented by various Pseudomonas species, and to relatively high number of micrococcii among soil actinobacteria. Seasonal dynamics in oil-oxidising bacteriocenoses of Kuban soils displayed an increase in spore-forming bacteria at temperatures above 35C and prolonged periods of desiccation. A speci¢c feature of oil-degrading bacteriocenoses of Urals soils was found to be a lack of sharp seasonal £uctuations in number of Rhodococcus spp. The average level of these actinobacteria, determined in situ by indirect immuno£uorescent microscopy, was 103-104 cells g-1 soil. Physiological properties and ecological behaviour of actinobacterial species (G. rubropertincta, G. terrae, R. erythropolis, R. opacus, R. ruber), dominant in oil-oxidising bacteriocenoses, were examined. Isolated bacterial strains ^ active biodegraders of petroleum (aliphatic, aromatic, polycyclic) hydrocarbons were deposited in the Regional Specialized Alkanotrophic Microorganism Collection IEGM (www.ecology.psu.ru/iegmcol/). The information on these strains as potential bioaugmentation agents for oil-contaminated soil remediation is included into the IEGM Collection database. This work was supported by INTAS grant 2001-2151.1.

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P4^15 CHANGING OF MICROBIOLOGICAL VARIETY IN OIL-CONTAMINATED SOILS N. A. Kireeva, E. M. Tarasenko, M. D. Bakaeva Biology Department, Bashkir State University, Ufa, Russia Getting in soils, the oil ambiguously in£uences all complex of the microorganisms describing the given ecosystem. During laboratory and ¢eld experiments it was obtained, that the number of geterotrophic microorganisms in oilcontaminated wood gray soils at high pollutant concentrations is much lower than in uncontaminated control soil. At low concentrations of contamination the number of microorganisms is increased. Species variety however is considerably creased. As the result of oil a¡ecting the atypical strains are widely presented. Thus the number of phytotoxic forms is increasing and it leads to soil phytotoxic to plants. By the increasing of time of oil staying in the soil the number of hydrocarbon-oxidizing microorganisms is proportionally increased. The increasing of hydrocarbon-oxidizing microorganisms number in oil contamination conditions promotes an intrinsic self-cleaning of soil. On the other hand, population ``£ash'' leads to a degradation of environment, where the nutritious substances are exhausted. In ¢eld conditions the highest values of number are found out for chronic oil contaminated soil. It is connected to increasing of hydrocarbon-oxidizing funguses number, which one are Fusarium sp., Aspergillus fumigatus, Aspergillus niger. Screening of hydrocarbon-oxidizing bacteria's has shown their accessory to genus's Arthrobacter, Pseudomonas, Rhodococcus. Including hydrocarbon-oxidizing micromycetes and bacteria, the biological preparation for bioremediation of oil-contaminated soils is created. P4^16 BACTERIAL GUT MICROFLORA OF WOODLICE PORCELLIO SCABER AS HEAVY METAL IMOBILISATION AGENTS A. Lapanje, D. Drobne, M. Rupnik > Biotechnical Faculty, Department of Biology, Vecna pot 111, 1000 Ljubljana, Slovenia Metal pollution is a serious environmental problem. The toxicity of metals is primarily dependant on its bioavailability. Metal bioavailability is changed by chemical and biotic transformations. Bacterial activity is responsible for metal bioavailability in a great deal. Sulfate-reducing bacteria (SRB) are well known to precipitate metals. The SRB can produce H2S, which precipitate the metal and reduce

its bioavailability. The other type of bacterial activity related to metal transformations is production of more bioavailable and toxic metal forms. Our research on mercury uptake, elimination and transformation in a terrestrial isopod Porcellio scaber provide evidence on transformation of inorganic mercury into methyl-mercury in the isopod gut (Jereb et al., 2003). This fact triggered our research to explore presence of SRB in the emptied gut of P. scaber. Namely, SRB have a distinctive capability of transformation of mercury into methyl-mercury. We used culture dependant as well as molecular methods for identi¢cation of SRB in the emptied gut. We found at least three di¡erent morphotypes of colonies of SRB and we isolated one species. In the gut of P. scaber we found also some other bacterial species, which could in£uence metal bioaviability. They are from the genus Shewanella. Some of them belonged to the genus Paracoccus which members can degrade organic contaminants. These bacterial species were detected with primers which were basically designed for detection of Desulfoccocus, Desulfonema and Desulfosarcina phylogeneticaly distinct group. The speci¢ty of primer pairs designed by Daly et al. (2000) is discussed. We also discuss the presence of strictly anaerobic bacteria in the gut of P. scaber, which was unexpected because of unfavourable physico-chemical properties of the gut lumen. P4^17 MOLECULAR MONITORING OF MICROBIAL DIVERSITY IN ANAEROBIC WASTEWATER TREATMENT SYSTEMS WITH AN IDENTIFICATION DNA ARRAY K. Roest, H. G. H. J. Heilig, H. Smidt, A. D. L. Akkermans, A. J. M. Stams and W. M. de Vos Laboratory of Microbiology, Wageningen University, Hesselink van Suchtelenweg 4, NL-6703 CT Wageningen, The Netherlands Wastewater treatment in Up£ow Anaerobic Sludge Blanket (UASB) bioreactors is a commonly applied technology, especially for industrial wastewater streams with high organic matter content. Although these anaerobic systems are working highly e/cient, only limited knowledge is available on the microbial processes that take place inside the reactors. Microbial diversity of sludge from anaerobic wastewater treatment systems was studied using both cultivation techniques and 16S rRNA/DNA analysis methods. Besides Denaturant Gradient Gel Electrophoresis ¢ngerprinting, cloning and sequencing, and Fluorescent In Situ Hybridisation, a macro array for detection of molecular diversity in UASB sludge was developed. Approximately 80-bp fragments of the V6 variable region of cloned and identi¢ed 16S rDNA sequences from sludge were ampli¢ed, ¢xed on nylon membranes, and subjected

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to reverse hybridisation with 32P-labelled 16S rDNA from a selection of the initial clones. To circumvent cross-hybridisation, the highly conserved primer sites were removed by enzymatic cleavage of introduced phosphorothioate-bonds, or blocked with anti-sense probes before hybridisation. Furthermore, di¡erent target/probe ratios were used. The microbial diversity of sludge samples from di¡erent anaerobic wastewater treatment systems was screened with the macro array and compared to other available molecular techniques. This showed the capacity and strength of the array. To further improve reliability and taxonomic resolution of the array, additional variable regions of sequenced clones will be used for the development of a multiple probe array. The developed macro array will be miniaturised to an identi¢cation DNA micro array and used for rapid molecular detection of microbial diversity in sludge of wastewater treatment samples. P4^18 COMETABOLISM OF ANILINE AND ITS CHLORINATED DERIVATIVES IN AZOTOBECTER SP. CULTURE MEDIUM S. Russel Department of Soil Environmental Sciences, Division of Agricultural Microbiology, Warsaw Agricultural University, 02-528 Warsaw, ul. Rakowiecka 26/30, Poland Aromatic amines are established intermediary products during the decomposition of numerous pesticides. Aniline and its derivatives are used as a substrate for chemical synthesis of dye, explosives, synthetic polymers etc. All these compounds are degraded during microbial metabolism to anilines, but the fate of intermediates in nature is only partially clari¢ed. Several di¡erent transformation products of the halogenated anilines have been found, but a pathway of complete degradation, i.e., decomposition into mineral products, has not yet been elaborated. In this presentation, the activity of an Azotobacter sp. isolated from soil, capable of transforming various anilines under aerobic condition is described. Approximately 30 bacterial strains were isolated from soil. The bacterium which was most active in metabolism of 4-chloroaniline was an Azotobacter sp. and this strain was selected for further study. The bacterium was grown in a nitrogenfree medium at 280C under aerobic conditions. The ¢nal concentration of various anilines in the growth medium was 20 ppm. After 2 days of incubation the Azotobacter isolate decomposed 100% unsubstituted aniline and 73 to 85% dichlorinated its derivatives. Clear evidence of the ability of the Azotobacter sp. to metabolize anilines was obtained with GC and TLC analysis. Using radioactive 4chloroaniline it was possible to ¢nd in bacterial culture medium 3 metabolites which were named M-1, M-2 and

M-3. Gas chromatography indicated production by Azotobacter sp. four metabolites. Based on its chromatographic characteristics, melting point, UV- and mass-spectra, metabolite M-1 was identi¢ed as 4-chloroacetanilide and M-2 as 4-chloropropionanilide. The infrared spectra of chemically synthesized 4-chloroacetanilide and 4-chloropropionanilide and isolated metabolites were identical. P4^19 BASIC REPLICONS OF BIODEGRADATION PLASMIDS OF PSEUDOMONAS STRAINS S. L. Sokolov(1), I. A. Kosheleva(1), N. P. Kovalenko(1), A. M. Boronin(1), K. Smalla(2) and C. M. Thomas(3) (1) Institute of Biochemistry and Physiology of Microorganisms RAS, Pushchino, Russia ; (2) Federal Research Centre for Agriculture and Forestry, Braunschweig, Germany; (3) University of Birmingham, School of Biosciences, Birmingham, UK Since biodegradative plasmids are of environmental significance, the studying of their replicons is important in cataloguing the diversity and phylogeny of these plasmids. The pathway for the catabolism of polycyclic aromatic hydrocarbons is often found in £uorescent Pseudomonas on large conjugative catabolic plasmids. Screening of laboratory collection revealed that most studied plasmids for biodegradation of naphthalene belong to Pseudomonas P-9 and P-7 incompatibility groups. Using two pairs of primers speci¢c for IncP-9 replicons: korA3Fa-rep3Rc and mpfA1Fa-korA2Ra, it was shown that HaeIII digested amplicons of catabolic/resistance plasmids vary and fall into three subgroups. Resistance plasmids such as pM3 and pMG18 belong to K-subgroup, when caprolactam degradation plasmids pBS265, pBS267 and pBS268 ^ to Q-subgroup. Majority of tested naphthalene biodegradation plasmids and toluene degradation plasmid pWWO di¡er in the structure of their backbone segments from plasmids determining the antibiotic resistance and degradation of caprolactam and were placed in L-subgroup of IncP-9 plasmids. However, it has been shown that in the case of some naphthalene degradation plasmids belonging to IncP-9 group the PCR product with RepA-RepB primers was not obtained, and these plasmids can be referred to the fourth, N-subgroup. A mini-replicon of N-plasmid containing all necessary genes for plasmid replication and maintenance was constructed by ligation of 6 kb plasmid fragment with Tcr-cassette. In order to develop molecular tools for classi¢cation of the IncP-7 plasmids and to determine the sequence diversity within IncP-7 family the mini-replicon of pBS213 was constructed. This work was supported by INTAS, grants NN 99-1481 and 01-2383.

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P4^20 INOCULATION OF COMPOSTING WINDROWS WITH SELECTED MICROORGANISMS ¤ ¤ ¤ F. Suarez-Estrella, M. C. Vargas-Garc|a, M. J. Lopez, G. Guisado and J. Moreno ¤ ¤ Area de Microbiolog|a, Departamento de Biolog|a Aplicada, ¤a, La Ca·ada de San CITE II-B, Universidad de Almer| ¤ Urbano, 04120 Almer|a, Spain In the past decades, the role of intensive agriculture in the economy of the South of Spain has become determinant. This important expansion involves negative aspects, such as the production of enormous amounts of horticultural wastes. Di¡erent solutions have been proposed in relation to this concern, being composting one of the most interesting. This wastes treatment alternative allows the elimination of potentially dangerous wastes at the same time as an interesting product is obtained, compost, which is used as an organic amendment in agriculture. The aim of this work was to study the e¡ect of inoculation with microorganisms on the ¢nal product quality and the duration of a composting process of melon plant wastes. Microorganisms used for inoculation were selected by their aptitude to grow on solid wastes at 40 and 60C, as well as by their ability to produce great quantities of biomass on ordinary liquid media in a short time. A total of 23 isolates from horticultural wastes were screened. This experiment allowed the selection of six bacterial strains that showed the adequate characteristics. To test the ability of these microorganisms to persist in the windrows, as well as their in£uence on the properties of the ¢nal product, a composting assay was achieved. Melon plants were arranged in seven 1,56 m3 piles (Dimensions: length 2,5 m; height 0,8 m; base 1,10 m). Each windrow was inoculated with a bacterial suspension to reach a concentration between 107-108 cfu/g of waste, being the last windrow used as uninoculated control. Samples were extracted and tested in relation to strain persistence. Physical and chemical characteristics of compost were also evaluated.

P4^21 DIVERSITY OF THE EPIPHYTIC BACTERIAL COMMUNITY ASSOCIATED WITH THE GREEN ALGA ULVA LACTUCA N. A. Tujula(1,2), T. L. Skovhus(2,3), C. Holmstrom(1,2), J. S. Webb(1,2), P. Steinberg(2) and S. Kjelleberg(1,2) (1) School of Microbiology and Immunology, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney 2052, Australia ; (2) Centre for Marine Biofouling and Bioinnovation, University of New South Wales, Sydney 2052, Australia; (3) Department of Microbial Ecology, Aarhus University, DK-8000 Aarhus, Denmark Pseudoalteromonas tunicata is a dark green-pigmented bacterium that produces anti-fouling compounds against invertebrate larvae, algal spores, bacteria, fungi, protozoa and diatoms. It was originally isolated from the surface of the tunicate Ciona intestinalis and has since been reported in marine environments across the world. In Australia it has been isolated from the marine alga Ulva lactuca. Both C. intestinalis and U. lactuca remain relatively free from fouling organisms in the ¢eld. Recent qualitative analysis of the distribution of Pseudoalteromonas spp. on living surfaces in the marine environment support the notion of a host speci¢c association. It has been proposed that some living surfaces may be colonised by antifoulantproducing bacteria (like P. tunicata) which can help in protecting the host organisms from biofouling. The aim of our research is to examine the ecological role of the inhibitory bacterium P. tunicata within the epiphytic bacterial community on U. lactuca. In this study we report the ¢rst molecular bacterial community analysis of U. lactuca. A Eubacterial 16S rDNA clone library was constructed from U. lactuca samples collected from Shark Point, Sydney, Australia. Two hundred clones were initially screened by using the DNA ¢ngerprinting method of restriction fragment length polymorhisms (RFLP). The result demonstrated 70 di¡erent RFLP patterns, indicating a high level of diversity on the plant surface. A representative from each class of the di¡erent RFLP patterns was sequenced. The majority of the clones belong to the Proteobacteria. Futhermore, denaturing gradient gel electrophoresis has been used to assess spatial variablity of the epiphytic community of U. lactuca.

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P4^22 MATHEMATIC MODEL OF MICROBIOLOGICAL PROCESSES IN THE OIL-CONTAMINATED SOIL V. V. Vodopyanov, N. A. Kireeva, E. M. Tarasenko Biology Department, Bashkir State University, Ufa, Russia In resent years the study of microbiological processes, which are taking place in oil-contaminated soils was carried out by methods of mathematical models. Oil in£uencing on kinetics of loss and growth of microorganisms, growth and progressing of microscopical funguses, kinetics of enzymatic reacting was studied. The special attention was given on a problem: how the microbiological processes, which are taking place in oil-contaminated soils, in£uence speed of destruction of oil in soil. By methods of mathematical models was found out, that per the ¢rst year after contamination the number of microorganisms in soil practically irrelevant with dynamics of oil destruction. It was pointed, that in soil per the ¢rst year after pollution, there is destruction of oil by two factors: aborigine microbiota, existing in soil before its contamination, physicochemical factors. It was found out, that under operating of the physicochemical factors in ground is decomposed about 20 % of oil. In view of variation of fractional composition of oil during degradation and di¡erent in£uencing of the imported biological components in soil (fertilizers, hydrocarbon-oxidizing microorganisms association, etc.) on di¡erent fractions of oil the multicomponent model of oil degradation was studied. The obtained model describes the dynamics of oil degradation in di¡erent situations well. P4^23 INFLUENCE OF VARIOUS CONCENTRATION OF 2,4,6-TRINITROTOLUENE ON STRATEGY OF ITS TRANSFORMATION BY BACILLUS SUBTILIS SK1 G. Yu. Yakovleva and B. M. Kurinenko Department of Microbiology, Laboratory of Engineering Enzymology, Kazan State University, Kazan, 420008, Russia The in£uence of various 2,4,6-trinitrotoluene (TNT) concentrations (from 20 to 200 mg/l) on the viability of the Bacillus subtilis SK1 and the strategy of transformation of the xenobiotic has been studied. It is shown that all the TNT concentrations under consideration are found toxic for this strain. The toxic action of the xenobiotic results in suppression of culture growth, reduction of the glucose utilization, amount of reduced piridine and oxidated £avine cofactors. We have found that the quantity of toxic

e¡ect depends on the TNT concentration. At low concentrations of xenobiotic (20 mg/l), B. subtilis SK1 saves its active metabolism and viability ; after the TNT elimination it begins the active growth. The high concentration of TNT suppresses the growth of the strain and reduces the number of viable cells. Although the toxic e¡ect of the TNT concentration from 20 to 200 mg/l is essentially different, it does not in£uence on the speed of the TNT transformation during the ¢rst two hours of the experiment. But the strategy of the TNT transformation at low and high concentration is found to be di¡erent for this strain. At low concentrations of xenobiotic (20 mg/l), B. subtilis SK1 reduced nitro groups of TNT producing the products of nitroreduce, at high concentrations (100-200 mg/l) ^ eliminated nitro groups from the ring with accumulation nitrites in the medium. To our knowledge, two di¡erent mechanisms of TNT transformation for a single strain have been observed for the ¢rst time. P4^24 RHIZOSPHERE-ASSOCIATED MICROORGANISMS: DIVERSITY, ANTAGONISTIC POTENTIAL AND APPLICATIONS IN BIOCONTROL G. Berg(1), J. Lottmann(1), S. Bruckner(2) and K. « Smalla(3) (1) University of Rostock, Institute for Molecular Physiology and Biotechnology, Albert-Einstein-Str. 3, D-18051 Rostock, Germany; (2) Prophyta Biologischer P£anzenschutz GmbH, D-23999 Malchow/Poel, Germany ; (3) Federal Biological Research Centre for Agriculture and Forestry, Messeweg 11/12, D-38104 Braunschweig, Germany The study of rhizosphere-associated microorganisms and their antagonistic potential is important not only for understanding their ecological role and the interaction with plants and plant pathogens but also for any biotechnological application. The interface between soil and plant roots ^ the rhizosphere ^ is a microbial hot spot where root-colonizing antagonistic bacteria play an important role in plant health and growth. The fungus Verticillium dahliae Kleb. responsible for high yield losses in many crops worldwide was chosen as model organism for antagonism studies. In comparison to other microenvironments of plants, the abundance, diversity and antagonistic activity in the rhizosphere of ¢eld grown potato plants was enhanced. In another study analyzing three di¡erent potential Verticillium host plants (potato, oilseed rape and strawberry) the abundance, the phenotypic and genotypic diversity of rhizobacteria, which showed antagonistic activity towards Verticillium was strongly plant species dependent. Altogether more than 7,000 isolates belonging to 81 di¡erent bacterial species identi¢ed by their fatty acid pro¢les (FAME) and/or sequencing the 16S rRNA were

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analyzed. To allow a cultivation-independent analysis, 16S rDNA fragments ampli¢ed by PCR from soil or rhizosphere bacterial DNA and analyzed by denaturing gradient gel electrophoresis (DGGE) con¢rmed the plantspeci¢city of rhizosphere-associated microorganisms. To assess the potential of bacterial strains in biocontrol (from this and other studies), a screening strategy using di¡erent in vitro and ad planta steps from laboratory to ¢eld trials was used. Altogether, ¢ve biocontrol agents were found. Based on them two biocontrol products (RhizoStar0, RhizoVit0) were developed. P4^25 IDENTIFICATION AND CHARACTERISATION OF PSEUDOMONAS SPECIES ISOLATED FROM SEMI ARID REGION OF UZBEKISTAN D. Egamberdiyeva, J. Dilafruz and D. Kakhramon Institute of Microbiology, A.Qadiriy str.7 B, 700128 Tashkent, Uzbekistan The purpose of this study was to investigate the Pseudomonas species isolated from the Rhizosphere of di¡erent agricultural crops grown in ¢eld location in semi arid region Uzbekistan and to characterize their functions and physiological activity. Pseudomonas species were isolated from the Rhizosphere of cotton, wheat, alfalfa, melon, tomato and maize grown at the ¢eld location in Surchandarya region Uzbekistan. Isolated strains identi¢ed using fundamental selection methods, the standard battery of biochemical and physiological characterization tests. It was found that the most commonly genera were Pseudomonas denitri¢cans, P. mendocina, P.rathonis, P. alcaligenes, P. aurantiaca, P. £uoro-violaceus. and P.stutzeri. These strains were well distributed among the isolates from the wheat, maize, alfalfa, cotton and tomato. Bacterial strains hydrolyzed Tween 20, Tween 60, and lecithin, produced Auxin. The isolated strains produced B group vitamins: thiamine, pantothenic acid, pyridoxal, nicotinic acid and biotin, di¡erent amino acids: lysine, asparagine, histidine, leucine, valine, glutamine and enzymes : lipase, nitratreductase, amylase, and arginine dihydrolase. P. alcaligenes PsA15, P. denitri¢cans PsD6 reacted antagonistically to soil- borne plant pathogens (e.g. Fusarium culmorum, Verticillum loteritum). Several strains had distinctive patterns of antibiotic resistance that are potentially useful as genetic markers. All of the strains oligonitrophil, and they have ability to survive N-de¢cient soils. Most of Pseudomonas species were salt tolerant, being able to grow in media containing 7% NaCl. P. alcaligenes PsA15, P. denitri¢cans PsD35, P.rathonis PsR47 survived under high temperature (50C) conditions. These abilities contributed the isolated bacteria to survive in the environmentally stressed conditions.

P4^26 THE INFLUENCE OF SOME HUMAN ACTIVITIES ON THE OCCURRENCE OF THE ECTOMYCORRHIZAL FUNGI AND ECTOMYCORRHIZAE T. Grebenc, H. Kraigher Slovenian Forestry Institute, Vecna pot 2, SI-1000 Ljubljana, Slovenia The community of ectomycorrhizal fungi can re£ect the natural or human-in£uenced changes in the forest ecosystems. In our study the in£uence of the new felled gap (30m diameter, no natural regeneration) in the mature beechsilver ¢r forest stand was compared with the naturally occurring gap of about the same size and with abundant regeneration. Sporocarps of ectomycorrhizal fungal species were mapped and types of ectomycorrhizae were collected in a transect line through the gap and identi¢ed after anatomical characteristics and with PCR-ITSRFLP and sequence analysis of as yet undescribed types. In two years samplings the occurrence of the sporocarps in the natural regenerating gap was not signi¢cantly di¡erent from the undamaged part of the research plot while the number of types of ectomycorrhizae only decreased in samples collected on a footpath. The analysis of ectomycorrhizae revealed some not yet described types of ectomycorrhizae, predominantly on silver ¢r, which were partially determined with sequence analysis. In the centre of the newly felled gap the abundance of ectomycorrhizal fungi (sporocarps and ectomycorrhizae) decreased to almost zero due to the complete removal of the symbiotic partner indicating unsuitable in£uence to the forest ecosystem. P4^27 EFFECT OF NODULATION AND MYCORRHIZATION ON MICROBIAL COMMUNITIES IN MEDICAGO TRUNCATULA RHIZOSPHERE C. Mougel(1), L. Ranjard(1), T. Corberand(1), D. Merdinoglu(2) and P. Lemanceau(1) ¤ (1) INRA ^ CMSE, UMR `Microbiologie et Geochimie des Sols', 17 rue de Sully, B.P. 86510, 21065 Dijon Cedex, ¤ ¤ ¤ France ; (2) INRA ^ Laboratoire de Genetique et d'Amelioration des Plantes, 28 rue de Herrlisheim, B.P. 507, 68021 COLMAR Cedex, France Plant root provide suitable habitats for the growth of microorganisms, as indicated by the high number of di¡erent microbial populations associated with the roots. Among the plants ^ microbes interactions occurring in the rhizosphere, symbiotic associations are known to improve plant

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growth in low fertility conditions. The model plant Medicago truncatula has the ability to develop symbiotic association with rhizobia and glomalean fungi. Mutants of M. truncatula a¡ected in their ability to establish symbiotic association with one or both symbionts are available. The e¡ect of M. truncatula wild-type and mutants on bacterial and fungal communities was analyzed on DNA directly extracted from rhizospheric soil and root tissue. The community structures were assessed by RISA (ribosomal intergenic spacer analysis) ¢ngerprinting obtained on automatic sequencer. Variations among the structures of bacterial (B-ARISA) and fungal (F-ARISA) communities were processed using principal component analysis. These analyses indicated that the communities had di¡erent genetic structure according to the compartments sampled (bulk soil, rhizospheric soil and root tissue). Furthermore, in each rhizospheric compartment (rhizospheric soils, root tissues), the genetic structure of the microbial communities varied between the genotypes of M. truncatula (wild-type and mutants). The biological meaning of the shifts in the structure of the microbial communities associated with roots nodulated and/or mycorrhized compared to roots impaired in their ability to develop symbiotic relations are discussed. P4^28 EFFECTS OF MYCORRHIZATION AND NODULATION ON THE STRUCTURE AND DIVERSITY OF FLUORESCENT PSEUDOMONADS ASSOCIATED WITH MEDICAGO TRUNCATULA ROOTS A. Robin(1), T. Corberand(1), C. Robin(2), E. Benizri(2), Ph. Lemanceau(1) and C. Mougel(1) ¤ (1) UMR INRA/Universite de Bourgogne `Microbiologie et ¤ ochimie des Sols', INRA CMSE, BP 86510 21065 Dijon Ge Cedex, France; (2) UMR `Agronomie et Environnement', INPL (ENSAIA) ^ INRA, BP 172, F54505 Vandoeuvre' les-Nancy, France Despite the importance of mycorrhizae and Rhizobia on plant growth and health of leguminous plants, little is know on their interaction with other rhizospheric microorganisms. The aim of our study was to assess the e¡ect of mycorrhization and nodulation on the structure and diversity of indigenous populations of £uorescent pseudomonads. The study strategy consisted in comparing the populations of £uorescent pseudomonads associated with the roots of M. truncatula wild-type J5, mycorrhized and nodulated (Nod+Myc+), to that of mutants impaired in their ability to establish symbiotic relations: mutant TRV48 (Nod-Myc+) and mutant TRV25 (Nod-Myc-). The phenotypes of the wild-type and mutants of M. truncatula were characterized (dry weight matter, mycorrhization rate, presence/absence of nodules). Fluorescent pseu-

domonads were numerated and isolated from three compartments (rhizospheric soil, root tissues and uncultivated soil). These populations were characterized by AFLP (Ampli¢ed Fragments Length Polymorphism). Comparison of densities of pseudomonads con¢rmed the rhizosphere e¡ect and indicated that the carrying capacities for £uorescent pseudomonads of the rhizosphere of the three plant phenotypes were similar. The root tissues of the mutants Nod- harbored lower £uorescent pseudomonads populations than those of the wild-type (Nod+) suggesting an analogy of recognition process between plant/Rhizobia and plant/pseudomonads. Statistic analyses of the data showed (i) that the frequency distributions of populations associated with roots and soil signi¢cantly di¡ered and (ii) that the diversity of the rhizospheric populations was higher than that of the soil populations. However, the data yielded did not allow the identi¢cation of populations of £uorescent pseudomonads preferentially associated with mycorrhized and/or nodulated roots. P4^29 STRUCTURE OF MICROBIAL COMMUNITY IN RHIZOSPHERE OF DIFFERENT WHEAT SPECIES A. I. Melentiev, L. Yu. Kuzmina, N. F. Galimzianova and T. F. Boyko Institute of Biology, Ufa Research Center RAS, prospekt Oktiabria, 69, Ufa, 450054, Russia The growth and development of micro£ora in the plant rhizosphere promotes the nutrient uptake, inhibits the development of plant pathogens and in any cases meets the requirements in phytogormones. The microbial community of plant rhizosphere is formed from aboriginal micro£ora of a soil. According some data the structure of rhizosphere microbial community is speci¢c for each genera of plant. The in£uence of genome composition of wheat (Triticum L.) on the structure of forming rhizosphere microbe community in typical chernozem soil was studied. The experiments were carried out in ¢eld conditions with the next wheat: T. monococcum (Ab), T. timopheevii (AbG), T. dicoccum (AuB), T. durum (AuB) and T. aestivum (AuBD). As a result it was ascertained that the main di¡erences of microbial community in rhizosphere are located in the population density of some groups of microorganisms. The number of heterotrophs and number of nitrogen transforming bacteria in rhizosphere of wheat is increased in the order T. timopheevii, T. dicoccum, T. aestivum, T. monococcum, T. durum. The population density of these bacterial groups di¡ered in 1000 and 100 times accordingly between the extremes. At the same time, the di¡erences in number of spore-forming bacteria, fungi and actynomycetes were not revealed in rhizosphere of investigated wheat.

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P4^30 NITROGEN-FIXING ACTIVITY OF RHIZOBIUM GALEGAE STRAINS SPECIFIC FOR GALEGAE ORIENTALIS LAM. AND GALEGAE OFFICINALIS L. B. Milicic, Dj. Kuzmanovic, D. Josic and A. T. Vidojevic Institute for Soil Science, Belgrade, Yugoslavia In two parallel experiments conducted in test tubes on agreded Jansen's substrate, the nitrogen-¢xing activity of three strains of Rhizobium galegae (speci¢c for G. orientalis Lam.) was tested on G. orientalis and two indigenous strains (speci¢c for G. o/cinalis L.) on G. o/cinalis. Inoculation of G. orientalis with its speci¢c strains 801, 802 and 803 in the form of a `single strain inoculum' caused a 2.3 ^ 2.9 fold increase in the yield of dry shoot mass and a 6.0 ^ 8.6 fold increase of N content in it as compared with the control. The percentage of ¢xed N in these strains was 83.5 ^ 88.4%. Autochthonous strains 821 and 822 of G. o/cinalis prepared as a single strain inoculum caused a 1.7 fold increase yield of dry shoot mass and 4.6 ^ 5.0 fold increase of N content in it compared with the control. The percentage of ¢xed N in these strains was 78.4 and 79.8%. An inoculum composed of a mixture of strains 801 + 802 (`double strain inoculum') speci¢c for G. orientalis prompted the roots of G. o/cinalis to from inactive nodules ( Nod + Fix-) that do not ¢x N from the atmosphere. In the same way, an inoculum composed of a mixture of strains 821+822 (double strain inoculum) speci¢c for G. o/cinalis elicited formation of likewise inactive nodules ( Nod + Fix-) on roots of G. orientalis. These results obtained in our investigation clearly indicate that strains of R. galegae are strictly speci¢c for plant species of the genus Galega (G. orientalis and G. o/cinalis). P4^31 DIVERSITY OF phlD GENE POOLS OF THE WHEAT RHIZOSPHERE IN A RICE-WHEAT CROPPING SYSTEM (MIDDLE GANGA PLAIN, INDIA) G. Imfeld(1), D. Roesti(1) N. Shani(1), S. Sharma(2), R. Gaur(2), K. Jeet(2), M. Aragno(1), B. N. Johri(2) and P. Rossi(1) (1) Laboratory of Microbiology (LAMUN), University of " " Neuchatel, 11 rue Emile-Argand, 2007 Neuchatel Switzerland; (2) G.B. Pantangar University of Agriculture and Technology, Department of Microbiology, Pantnagar, India Among the antifungal compounds synthesized by biocontrol strains, the 2,4-diacetylphloroglucinol (DAPG) is a key metabolite associated with disease suppression in several pathosystems. The phlD gene is involved in the bio-

synthesis of a DAPG precursor and is used as a genetic marker to detect putative DAPG producing strains. The goal of this study was to assess the impact of fertilization level and yields of three wheat ¢elds on the diversity of phlD gene pools of the wheat rhizospheric soil and rhizoplane/endorhizosphere fractions. Diversity of putative DAPG producers was assessed after combining RFLP analysis of the phlD gene from DNA of isolated strains and environmental samples. In parallel, RFLP analysis was also performed on the 16S-23S rDNA Intergenic Spacer Region of the isolated strains in order to improve the strain characterization. Operating Taxonomic Units obtained by the restriction patterns lead to the distinction of several dominant groups among the phlD positive bacteria that were correlated with the speci¢c ¢eld characteristics. The combined approach allowed a better understanding of the phlD population structure of the studied agricultural system. Moreover, this study constitutes a preliminary basis for time and spatial follow-up of multiple samples as well as for gene expression analysis in order to enhance potential information concerning phlD population dynamics and their role in agricultural systems. P4^32 INFLUENCE OF DIFFERENT PLANT SPECIES ON THE DIVERSITY OF BURKHOLDERIA STRAINS AND SELECTION OF ANTAGONISTIC ISOLATES J. F. Salles(1), P. Garbeva(1), J. A. van Veen(2), J. D. van Elsas(1) (1) Plant Research International, P.O. Box 16, 6700 AA Wageningen, NL; (2) NIOO-CTO, P.O. Box 40, 6666 ZG Heteren, NL The genus Burkholderia has great potential for agricultural use such as in biocontrol, due to the production of antibiotic compounds and ability to colonise the rhizosphere of many plants in high population density. The aim of this work was to evaluate the diversity of Burkholderia species in the rhizosphere of di¡erent crops, as well as the ability of these to select Burkholderia strains with antagonistic activity against the soilborne pathogen Rhizoctonia solani. Therefore, various crops were planted in pots containing soil obtained from areas with di¡erent history. Burkholderia isolates were obtained by plating in selective medium and further checked for antagonistic activity in dual-culture assay. The diversity was evaluated by PCR-DGGE with speci¢c primers, allowing the assessment of Burkholderia populations. Furthermore, in order to determine the part of the Burkholderia community responding to crop plants, root exudates were collected and added to soil in the presence of bromodeoxyuridine (BrdU). DNA from the active population (BrdU-labelled) was extracted by immunocapture and used for PCR-DGGE. The results

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showed that although crop history had a major impact on Burkholderia diversity, plant species played an important role as well, since the communities associated with maize and grass, or oat and barley were similar. The analysis of DGGE patterns obtained from total and culturable populations showed that the presence of speci¢c bands on DGGE could be correlated to those crops. Although no di¡erences in the selection of antagonistic isolates were observed between treatments, the e¡ect of root exudates on the organisms will be shown. P4^33 DIVERSITY AND FUNCTIONS OF THE BACTERIA ASSOCIATED WITH THE CLUSTER ROOTS OF WHITE LUPIN. EFFECTS OF ROOT EXUDATES ON THE SURROUNDING MICROFLORA L. Weisskopf(1,2), N. Fromin(1), E. Abou-Mansour(3), E. Martinoia(2), N. Tomasi(2), R. Tabacchi(3) and M. Aragno(1) " (1) Laboratory of Microbiology, University of Neuchatel, " CH-2007 Neuchatel, Switzerland ; (2) Laboratory of Plant Physiology, University of Zurich, CH-8008 Zurich, Switzerland; (3) Laboratory of Analytical Organic Chemistry, " " University of Neuchatel, CH-2007 Neuchatel, Switzerland White lupin (Lupinus albus) is a non-mycorrhized leguminous plant which is able to grow on soils with sparingly available phosphate by producing cluster roots (so-called proteoid roots). These cluster roots exude large amounts of organic acids, mainly citrate and malate, and secondary compounds such as phenolics, causing fast and considerable changes in their proximity. These compounds, in addition to their role in phosphate acquisition, are very likely to in£uence the surrounding micro£ora. In contrast to the physiology of cluster roots, which has been intensively studied in Lupinus albus, little attention has been paid so far to the associated micro£ora, with respect to its diversity as well as to its potential promoting or deleterious e¡ect on root growth and nutrition. Here we report the diversity and functions of bacteria associated to the cluster roots of L. albus. We investigated the diversity of bacterial communities by ¢ngerprinting methods based on PCRDGGE analysis of the V3 region of 16S rDNA and 16S rRNA. We also assessed the functions of isolated strains by testing some PGPR traits in vitro, like the ability to produce auxins or siderophores, to solubilize inorganic phosphate or to inhibit the growth of pathogenic fungi. Since our ¢rst results showed a strong impact of the plant on the bacterial micro£ora, we then analyzed the e¡ects of di¡erent root exudates components on the isolated strains in order to elucidate chemical components of the plant bacteria communication.

P4^34 INTERSPECIES AND SUBSPECIES DIVERSITY AMONG LEPTOSPIRES IN TERMS OF ECOLOGY Yu. V. Ananyina, A. P. Samsonova Gamaleya Instititute for Epidemiology and Microbiology of the Russian Academy of Medical Sciences, Gamaleya str.18, 123098, Moscow, Russia Pathogenic leptospires (Leptospira interrogans sensu lato) are known worldwide to play an important role in human and animal infectious pathology. Some Leptospira agents (for example, of copenhageni, icterohaemorrhagiae and canicola serovars) can cause severe diseases with high case fatality rates or resulting in late clinical sequelae in humans a¡ected. Meanwhile others, though less virulent (grippotyphosa, pomona serovars, etc.), are able to induce cluster or outbreak, mainly water-borne, morbidity involving dozens and even hundreds of human beings. At present pathogenic leptospires are classi¢ed as belonging to at least seven genomospecies and more than 230 serovars have been so far recognized. Experimental evidence testi¢es that leptospires of di¡erent species and subspecies taxons display not only substantial diversity in pathogenic potential, but also ecology. These spirochaetes are highly variable in terms of host speci¢city (speci¢c mammalian host range), selective tropism to renal and nervous tissues as well as ability to compete and persist in vivo as well as under environmental conditions. Experimental data on interspecies and subspecies biodiversity among leptospires will be presented and analyzed in relevance to leptospirosis epidemiological and clinical patterns. P4^35 IDENTIFICATION, BASIC GENETIC CHARACTERISATION AND REGIONAL DISTRIBUTION OF VIRULENT LACTOCOCCAL BACTERIOPHAGES DETECTED IN POLISH DAIRIES ¤ A. Szczepanska, W. Krysa and J. Bardowski Department of Microbial Biochemistry, IBB PAS, Pawinskiego 5a, 02-106 Warsaw, Poland Here presented are the results of our research on virulent bacteriophages infecting dairy lactococcal starter cultures. The type, morphology and geographical distribution of lactococcal bacteriophages, which are a serious problem in dairy plants, were investigated. After detection and isolation of phages from industrial whey samples DNA was extracted from each isolated phage and characterised with the use of restriction endonucleases. Detected phages were assigned into several types, regarding the restriction pat-

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tern and the genome size. The phage host range was determined against industrial lactococcal strains as well as against two model strains of Lactococcus lactis ^ IL1403 and MG1363. Subsequently, the identi¢ed phages were classi¢ed into three major genetic groups ^ P335, 936 and C2 ^ by multiplex PCR analysis. Several phages showed speci¢city to more than one genetic group which could indicate either presence of a mosaic phage or coisolation of a prophage. This result needs further examination. Additionally, from distribution studies we could conclude that one type of phage was equally disseminated throughout the regional network of dairies. The remaining phages exhibited speci¢city to certain geographical regions. Overall, we have isolated and characterised over 150 individual phages from 14 whey samples, which form di¡erent restriction groups. Majority of them belong to the c2 and 936 genetic groups. These studies allowed us to assess the type of phages a¡ecting the fermentation processes. Furthermore, we were able to determine that not one but several phages dominate in the analysed industrial dairy samples. This work was supported by the KBN grant No 3 P06T 051 23. P4^36 POPULATION GENETICS OF IXODES RICINUS TICKS AND BORRELIA BURGDORFERI SENSU LATO: PRELIMINARY ASPECTS S. Casati(1), M. V. Bernasconi(1), L. Gern(2), and J.-C. Pi¡aretti(1) (1) Istituto Cantonale di Microbiologia, Via Mirasole 22A, 6500 Bellinzona, Switzerland ; (2) Institut de Zoologie, ¤ " " Universite de Neuchatel, Rue Emile-Argand, 2000 Neuchatel, Switzerland The present work is inserted into a major project in progress aimed at analysing at the genetic level both the vector (the tick) and the micro-organisms, they may carry. We intend to identify a possible correlation between the vector and the pathogen. The study will consider a collection of about 1500 Ixodes ricinus ticks and the pathogens Borrelia burgdorferi s.l., Babesia sp. and TBE virus. A ¢rst step involves 600 ticks collected from vegetation and animals " in Neuchatel and in Ticino. By means of PCR and direct sequencing (using 162 bp of the recA gene) we tested the presence and the identity of Borrelia burgdorferi s.l. 32% " of the ticks of Neuchatel and 15% of the ticks of Ticino were positives The following genospecies have been identi¢ed: B. afzelii, B. garinii, B. valaisiana, B. lusitaniae and B. burgdorferi s.s.. The intra-speci¢c variation ranged from 1.8 to 9.2%. Thus, we found a considerable amount of inter- and intra-speci¢c variation : di¡erent lineages of Borrelia have been met in limited area. A second step

investigates the genetic variability within I. ricinus (from Switzerland and Scandinavia) using 12S and 16S rDNA genes. Both genes show a low variability (2-3%) precluding the discrimination of unique geographic populations and hence also a correlation between the vector and the pathogens. In conclusion, the inter- and intra-speci¢c variability found in the Borrelia strains re£ects the variability found in Europe. Whereas, the I. ricinus ticks from Europe seem to be extremely homogeneous from a population genetics point of view. P4^37 CLONAL ANALYSIS OF SOME MULTIPLE ANTIBIOTIC RESISTANT AND HEAVY-METAL TOLERANT E. COLI STRAINS ISOLATED FROM POLLUTED WATERS R. Cernat, V. Lazar, C. Balotescu, E. Coipan, C. Cojocaru and C. Bucur Dep. Microbiology-Immunology, Faculty of Biology, University of Bucharest, Aleea Portocalelor 1-3, Sect.5, Bucharest 77206, Romania Self-transmissible plasmids conferring multiple antibiotic resistance are wide-spread in coliforms populations. In soil and water, multiple antibiotic resistance is clearly associated with resistance/tolerance to heavy-metals (Hg2+, Cu2+, Pb2+, Zn2+, Cd2+). For di¡erent genera the genes for heavy-metals resistance are often plasmid encoded. Since these genes are clustered on the same plasmids, heavy-metals and drugs can serve as selective pressure factors for the populations of these plasmid-harboring bacteria. The aims of this preliminary study were (1) to ¢nd possible correlation between resistance genotype determined by genetic analysis and antibiotic and heavy-metal resistance patterns and (2) to assess the genetic diversity of several E. coli strains isolated from chronically polluted waters. Antibiotic susceptibility testing was performing for aminoglycosides (amikacin, gentamycin, and kanamycin), L-lactams (ampicillin, imipenem), amoxicillin/clavulanate, cephalosporins (ceftazidime, cefotaxime and cephalotin), quinolones (cipro£oxacin, nor£oxacin and nalidixic acid), tetracycline and chloramphenicol, as described by KirbyBauer disk di¡usion method following NCCLS recommendations. MICs values of antimicrobials and heavymetal salts (CuSO4, CdCl2, Co(NO3)2, Cr(NO3)3, HgCl2, NiCl2 and ZnSO4) were determined by dilution method. For the data analysis NCCLS breakpoints for resistance and sensitivity were used. Plasmid DNA was isolated from E.coli strains by an alkaline lysis method and digested to completion with EcoRI enzyme. Genetic similarity and clustering were calculated using NTSIS program. The phenotypic data shows the direct association between multiple antibiotic and heavy-metal resistance for E.coli strains in

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polluted waters. DNA ¢ngerprinting with plasmid DNA RFLP suggested that, depending of source of isolation, E. coli strains could be grouped in distinct populations with a di¡erent plasmid diversity. P4^38 GENETIC DIVERSITY OF BORRELIA BURGDORFERI SENSU LATO IN IXODES RICINUS TICKS COLLECTED FROM TWO AREAS IN SLOVAKIA AND CZECH REPUBLIC M. Derdakova(1), L. Beati(3), B. Petko(1), M. Stanko(2) and D. Fish(3) (1) Parasitological Institute, Slovak Academy of Science,Hlinkova 3, Kosice, Slovak Republic ; (2) Institute of Zoology, Slovak Academy of Science, Kosice, Slovak Republic; (3) School of Epidemiology and Public Health, Yale University, New Haven, USA The causative agent of Lyme borreliosis belongs to the Borrelia burgdorferi sensu lato complex. At least 5 distinct genospecies has been detected in Ixodes ricinus ticks from Europe: B. afzelii, B. garinii, B. burgdorferi sensu stricto, B. valaisiana, B. lusitaniae and B. bissetti. Geographic distribution of distinct genospecies in Europe varies. The heterogeneity of B. burgdorferi s.l. in I. ricinus populations from two geographically distinct areas was compared in this study. The genetic variability of B. burgdorferi s.l. was established by PCR-SSCP analysis of the rrfA-rrlB intergenic spacer. By this method we were able to detect also di¡erences within single genospecies and 16 di¡erent genotypes were described. The overall prevalence of infection was similar in both areas. 20.3% and 25.7% of I. ricinus ticks were found to be infected with B. burgdorferi s.l. in Ceske Budejovice, Czech republic and Kosice, Slovakia respectively. B. afzelii, B. garinii, B. valaisiana, B. burgdorferi s.s. were present in both sites. In addition, the presence of genomic group A14 S was detected in Ceske Budejovice. The predominant genospecies in Ceske Budejovice was B. afzelii (56.5%) in contrast to Kosice where B. burgdorferi s.s. was detected in 39.6% of positive ticks. Higher occurence of mixed infection of two and in one case three di¡erent Borrelia genospecies was detected in ticks from Kosice. Di¡erences were also detected among Borrelia population according to the developmental stage and sex of I. ricinus from both study sites.

P4^39 BACTERIAL COLONIZATION OF THE DOLPHIN FOREGUT ¤ M. G. Dom|nguez-Bello(1,2), G. Godette(3), P. Gueneau(1), C. Bonaventura(3), A. Read(3), D. Gannon(3), W. McLellan(4) (1) Laboratorio de Fisiologia Gastrointestinal, IVIC, Caracas, 1020A, Venezuela; (2) University of Puerto Rico, San Juan, Puerto Rico; (3) Duke Marine/Freshwater Biomedical Center, Duke University, Beaufort, NC 285169721, USA; (4) University of North Carolina Willmington, USA The mammalian digestive tube, even the hostile acid stomach, has been colonized by microorganisms. Forestomach compartmentalization allows ruminants to nourish on fermentation products and microbial biomass. Bacterial protein can be digested thanks to a gastric acid resistant lysozyme ruminants have recruited as a digestive enzyme. In herbivores, evolution of forestomachs appear to have been driven by the selective pressure of dietary cellulose. The only known case of gastric compartmentalization not associated with herbivorous diet is that in cetaceans. In this paper we have performed a preliminary characterization of bacteria colonizing gastric chambers of bottlenose dolphins and determined expression of gastric lysozyme in the acidic pouch. Bacteria were cultured anaerobically from frozen forestomachs of dolphins, and identi¢ed according to culture characteristics and membrane fatty acids analysis. Detection of Archaea in forestomach contents and Helicobacter spp in the acidic sac was performed by speci¢c PCR ampli¢cations. Bacteriolytic activity of gastric protein extracts and proteins puri¢ed with cationic exchange chromatography, was assayed in lysoplates with M luteus. Proteins were electrophoresed on 8-25% SDS gradient gels. We found anaerobic cultivable bacteria, most closely related to the proteolytic Clostridium, Eubacterium, and also to Enterobacter and Streptococcus strains. There was no ampli¢cation of Archaebacteria, in forestomach contents. Most (14/17) dolphins were colonized by gastric Helicobacter spp. We report for the ¢rst time, the expression of gastric lysozyme in Cetaceans. We propose that the major function of the cetacean foregut is the digestion of dietary compounds that would otherwise be poorly digested, such as chitin and wax ethers. As cellulose for ruminants, chitin may have exerted a selective pressure for the evolution of a complex foregut in crustacean feeder Cetaceans.

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P4^40 MOLECULAR IDENTIFICATION OF BIFIDOBACTERIA FROM SMALL INTESTINE OF RATS FED WITH RAW KIDNEY BEAN (PHASEOLUS VULGARIS L.) > L. Fanedl, F. V. Nekrep and G. Avgustin University of Ljubljana, Biotechnical Faculty, Zootechnical > Dept., Groblje 3, SI-1230 Domzale, Slovenia During a raw kidney been feeding trial, which was set in order to study the e¡ect of antinutrients i.e. phytohemagglutinin (PHA) on rat gut microbial community, several Gram-positive bacteria, phenotypically identi¢ed as bi¢dobacteria were isolated from rat small intestines. The molecular investigation of their relatedness to each other and to type strains of 20 bi¢dobacterial species by RAPD technique revealed, that they fall into two main groups and that none is related to any of the analysed type strains. Representative strains from the larger group of isolates were phylogenetically analysed by comparative sequence analysis of the PCR ampli¢ed 16S rRNA genes. All sequences clustered together and formed a coherent group within the phylogenetic subgroup Bi¢dobacterium lactis as de¢ned by the Ribosomal Database Project. Their closest relatives were found to be the bacteria from B. pseudolongum subsp. pseudolongum and B. pseudolongum subsp. globosum, generally with less than 97% sequence homology. The similarity levels among 16S rRNA sequences of isolated strains also showed considerable genetic variability, ranging from 91.4 to 95.8%. In conjunction with other molecular, phenotypic and ecological data, the 16S rRNA sequence analysis supports the proposal for novel bi¢dobacterial species inhabiting the rat small intestines. Their role during the antinutrient i.e. lectin administration to the rat gut, still remains hidden. P4^41 PLASMID pC PRESENT IN SALMONELLA ENTERICA SEROVAR ENTERITIDIS INFLUENCE PHAGE RESISTANCE D. Gregorova(1), M. Pravcova(1), A. Sebkova(1), R. Karpiskova(2) and I. Rychlik(1) (1) Veterinary Research Institute, Hudcova 70, 621 32 Brno, Czech Republic; (2) National Institute of Public Health, Center for Food Chain Hygiene, Palackeho 1-3, 612 42 Brno, Czech Republic Salmonella enterica serovar Enteritidis (S. Enteritidis) possesses plasmids of di¡erent sizes and roles. Besides the serovar-speci¢c virulence plasmid present in most ¢eld strains, S. Enteritidis can harbour plasmids of low molec-

ular mass whose biological role is poorly understood. We therefore identi¢ed and sequenced the most frequent low mass plasmids in S. Enteritidis. Using DNA hybridisation we found that there are at least three distinct groups of plasmids, which had negative cross hybridisation. We therefore sequenced 5 representative plasmids from each group and we concluded that they belong to ColE1, ColE2 and rolling circle replicating plasmids. From ColE1 group, plasmid pC present in S. Enteritidis strains belonging to phage type PT14b, was sequenced. The size of plasmid was determined to be 5 269 bp and it was predicted to encode 4 ORFs. The ¢rst two ORFs were found (initial 3230 bp) to be highly homologous to ORFs on ColE1 plasmid of E. coli. Proteins encoded by other ORFs were 99% homologous to a restriction methylase and restriction endonuclease encoded by plasmid pECO29 of a ¢eld strain of E. coli. Using insertional mutagenesis we con¢rmed experimentally the function of the plasmid pC encoded restriction modi¢cation system. The presence of the plasmid encoded restriction modi¢cation system explains the high resistance to phage infection of S. Enteritidis PT14b strains. P4^42 DIVERSITY OF MESOPHILIC SHEWANELLA ISOLATED FROM THE NORTH-WESTERN PART OF THE PACIFIC OCEAN E. P. Ivanova(1), T. Sawabe(2), N. V. Zhukova(3), N. M. Gorshkova(4), O. I. Nedashkovskaya(4), G. M. Frolova(4), A. F. Sergeev(5), V. V. Mikhailov(4), R. Christen(6), D. V. Nicolau(1) (1) Industrial Research Institute Swinburne University of Technology PO Box 218, Hawthorn, Vic 3122, Australia; (2) Graduate School of Fisheries Sciences, Faculty of Fisheries, Hokkaido University, 3-1-1 Minato-cho, Hakodate 041-8611, Japan; (3) Institute of Marine Biology of the Far-Eastern Branch of the Russian Academy of Sciences, 690032, Vladivostok, Palchevskogo St. 17, Russia ; (4) Paci¢c Institute of Bioorganic Chemistry of the Far-Eastern Branch of the Russian Academy of Sciences, 690022 Vladivostok, Pr. 100 Let Vladivostoku 159, Russia ; (5) Paci¢c Oceanological Institute of the Far-Eastern Branch of the Russian Academy of Sciences, Baltiiskaya Str., 43, 690017, Vladivostok, Russia; (6) UMR 6078 CNRS & ¤ Universite Nice Sophia-Antipolis, Bat. J. Maetz, F06238 Villefranche sur mer cedex, France Although bacteria of the genus Shewanella belong to one of the readily cultivable group of Q-Proteobacteria, little is known about the occurrence and abundance of these microorganisms in marine ecosystem. Studies revealed that of 654 isolates obtained from marine invertebrates (ophiuroid Amphiopholis kochii, sipuncula Phascolosoma japoni-

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cum, and holothurian Apostichopus japonicus, Cucumaria japonica), seawater and sediments of the North-Western part of the Paci¢c Ocean (i.e. the Sea of Japan and i. Iturup, Kurile Islands), 10.7% belonged to genus Shewanella. The proportion of viable Shewanella species varied from 4% to 20% depending on the source of isolation. From the isolation study, representative strains of di¡erent phenotypes (out of seventy presumptive Shewanella strains) were selected for detailed characterization using phenotypic, chemotaxonomic, and phylogenetic testing. 16S rDNA sequence-based phylogenetic analysis con¢rmed the results of tentative identi¢cation and placed the majority of these strains within only a few species of the genus Shewanella, namely S. japonica, S. colwelliana, and two novel species S. ¢delis and S. waksmanii. Numerically dominant strains of S. japonica were metabolically active and produced proteinases (gelatinases, caseinases), lipases, amylases, agarases, and alginases. Shewanella strains studied demonstrated weak antimicrobial and antifungal activities that might be an indication of their passive role in colonization of living and non-living surfaces. P4^43 CHARACTERIZATION OF RHIZOBIUM GALEGAE STRAINS BY PLASMID PROFILES, INTRINSIC ANTIBIOTIC RESISTANCE AND RAPD D. Josic, Dj. Kuzmanovic, B. Milicic, B. Misic Institute for Soil Science, Belgrade, Yugoslavia Five Rhizobium galegae strains ^ three strains speci¢c for Galega orientalis Lam. (801, 802 and 803) and two indigenous strains speci¢c for Galega o/cinalis L. (821 and 822) were characterized by intrinsic antibiotic resistance (IAR), plasmid pro¢le and RAPD. Characterization of strains by plasmid pro¢le analysis and IAR showed high similarity of strains. Plasmid pro¢les of strains 821 and 822 were identical, as well as plasmid pro¢les of strains 801 and 803. All strrains were found to carry one plasmid band (size 200-330kb). IAR tests for eight antibiotics (Ampicilline, Tetracycline, Penicilline, Neomycine, Streptomycine, Erythromycine, Rifampycine and Chloramphenycol) and RAPD pro¢les showed di¡erences between three strains speci¢c for G. orientalis. However, strains 821 and 822 speci¢c for G. o/cinalis had identical IAR patterns as well as plasmid pro¢les, since RAPD analysis showed the di¡erences between them.

P4^44 IDENTIFICATION OF DOMINANT RHIZOBIUM LEGUMINOSARUM BV. TRIFOLII ISOLATED FROM DIFFERENT TYPES OF SOIL IN SERBIA D. Josic, B. Milicic, A. Terzic-Vidojevic Institute for Soil Science, Belgrade, Yugoslavia Rhizobium leguminosarum bv. trifolii is microsymbiont Trifolium pratense and Trifolium repens, very important legumes in Serbia. Since nitrogen is often the limiting nutrient in soil, the use of appropriate strains of Rhizobium to supply nitrogen to the plant is economically and ecologically justi¢ed. That is why the natural nodulating population of those bacteria was collected during 2002 year. We planed to estimate biodiversity distribution by monitoring dominant genotypes of these bacteria. The population of Rhizobium leguminosarum bv. trifolii were colected from 50 marked locations of 11 types of soil in Serbia. 437 natural isolates, rescued from nodules of Trifolium repens or Trifolium pratense, were analysed by phenotypic approache. We obtained 162 di¡erent isolates on the basis of di¡erences in their IAR ^ intrinsic antibiotic resistance (¢ve antibiotics) and HMT- heavy methal tolerance (¢ve heavy methal). We investigated 32 dominant isolates with more than three di¡erences in IAR-HMT patterns by plasmid pro¢les analysis and RAPD ¢ngerprinting. The results showed vide diversity of dominant Rhizobium leguminosarum bv. trifolii ¢eld isolates and o¡ered the possibility to assess their changes on marked locations during time and under di¡erent environmental conditions. P4^45 THE RELATIONSHIP BETWEEN HLYA AND SHEA GENES OF EXTRAINTESTINAL ESCHERICHIA COLI ¤ ¤ ¤ ¤ M. Kerenyi, I. Batai, G. Mestyan, L. Emody and T. Pal ¤ Department of Medical Microbiology, Pecs University, ¤ cs, Szigeti u. 12. H-7643, Hungary Pe Extraintestinal Escherichia coli most frequently causes urinary tract infection. It can also be isolated from blood culture and abdominal wound. The alpha-hemolysin, which is one of the most important virulence factors, is present in about 60 % of isolated strains from urinary tract infection. The exact role of silent hemolysin in virulence has not been determinated. In this study hemolytic phenotype and occurrence of hlyA and sheA genes were determined and compared in 520 clinical isolates of E. coli. The hlyA gene carrier strains do not cause hemolysis, if their secretory mechanism is defected. 13 strains of clinical

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isolates carried structure hemolysin gene, but hemolysis zone were not developed around their colonies in de¢ciency of secretion. In spite of lack of hemolysin structure gene seven strains were hemolytic on blood agar. These strains were pozitive for sheA gene. This suggests that increased expression of silent hemolysin or its releasing is responsible for hemolysis. These hemolysis zones around colonies are similar to the ones caused by alphahemolysin. The alpha-hemolysin is coded on chromosome (most often on PAI) of E. coli in human. SheA gene was also determined on chromosome in lab strains, in animal, and human isolates. GC % content in hlyA and sheA genes is similar, but it is di¡erent from E. coli genom. A further question if there is incompatibility between these genes on the chromosome and what is responsible for it. We conclude that hemolysis around colonies of E. coli was caused by other than alpha-hemolysin. P4^46 PHYLOGENETIC AFFILIATION OF BACTERIA ATTACHED TO THE HINDGUT CUTICLE OF TERRESTRIAL ISOPOD PORCELLIO SCABER í > > R. Kostanjsek(1), J. Strus(1), G. Avgustin(2) (1) University of Ljubljana, Biotechnical Faculty, Biologi> cal department, Vecna pot 111, 1000 Ljubljana, Slovenia; (2) University of Ljubljana, Biotechnical Faculty, Zooteh> nical department, Groblje 3, 1234 Domzale, Slovenia Porcellio scaber is a terrestrial isopod crustacean involved in decomposition of decayed plant material. Observations of P. scaber tube-like hindgut revealed the presence of ¢lamentous bacteria attached to spine-shaped protuberances formed by cuticular lining of the inner gut surface. In order to determine the phylogenetic position of the attached bacteria the 16S rRNA gene sequences were retrieved directly and analysed. The phylogenetic analysis of attached bacteria revealed that they form a single cluster within Mycoplasmales group, clearly separated from other mycoplasmas. The nearest sequences clustered in so called group `A' consisting of bacterial 16S rRNA sequences retrieved directly from bovine, pig and human intestine. The phylogenetic analysis further showed that the 16S rRNA sequences of attached bacteria from P. scaber gut and of group `A' are monophyletic and deeply branched groups positioned between Spiroplasma and Acholeplasma-Anaeroplasma group. A/liation of attached bacteria from P. scaber to the mollicutes was further con¢rmed with electron microscopy observations, which revealed the absence of cell wall. The microscopy also revealed the presence of spherical shaped attachment structure at the end of bacterial cells. In situ hybridisation with speci¢c oligonucleotide probe con¢rmed the presence of the targeted sequences to attached bacteria in P. scaber

hindgut. Given the speci¢c host, unique attachment structure and phylogenetic distance we propose that the ¢lamentous bacteria attached to the cuticular spines of P. scaber hindgut represent a novel bacterial taxon within mollicutes. P4^47 COMPARISON BETWEEN RAPD-PCR AND RAPPCR IN OENOCOCCUS OENI STRAINS T. Lechiancole, D. Messina and G. Salzano Dept. of Biology, Univerity of Basilicata, Campus Macchia Romana, 85100 Potenza, Italy Oenococcus oeni, formely Leuconostoc oenos is the species of lactic acid bacteria most frequently associated with the malolactic fermentation in wine. O. oeni strains are members of a genomically homogeneous species, in spite of remarkable divergences in their genomic organization. as supported by chromosomal DNA homology studies, 16S and 23S rRNA sequencing, and more recently, sequence analysis of the 16S-23S rDNA intergenic spacer region. Results of several studies on genotypic diversity among strains of O. oeni, carried out using di¡erent molecular techniques (DNA ¢ngerprinting, PFGE, RAPD-PCR) suggest that this species is also genomically homogeneous. PCR-based methods, such as RNA arbitrarily primed PCR (RAP-PCR), have recently been developed to identify di¡erentially expressed genes in eukaryotic organisms However, to our knowledge, there is only one report on successful identi¢cation of di¡erentially expressed genes in bacteria using RAP-PCR. In this work two molecular tools were applied to discriminate among O. oeni strains isolated from Aglianico wines coming from Basilicata : RAPD-PCR (Random Ampli¢ed Polymorphic DNAPolymerase Chain Reaction) and RAP-PCR (RNA Arbitrarily Primed-Polymerase Chain Reaction). Our results demonstrate that RAP-PCR gives the best results. In this work we demonstrated that the analysis on expressed genes is useful tool to evaluated biodiversity among isolates belonging to the same species, in particular O. oeni strains isolated from wines coming from the same region.

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P4^48 NOVEL SOURCES OF ACTINOMYCETE DIVERSITY FOR DETECTION OF ANTIMICROBIAL AGENTS WITH PHARMACEUTICAL APPLICATIONS E. M. H. Wellington(1), F. Marinelli(2), H.-P. Fiedler(3), L. Dijkhuizen(4), J. Vater(5), M. Goodfellow(6), S. A. Fotinos(7), and A. D. Karagouni(8) (1) Department of Biological Sciences, University of Warwick ; (2) Biosearch Italia SpA; (3) Mikrobiologisches Institut, Universitat Tubingen; (4) Microbiology, University « « of Groningen; (5) Max-Volmer-Institut fur Biophysikali« sche Chemie und Biochemie, Technische Universitat Berlin; « (6) Department of Agricultural and Environmental Science, University of Newcastle upon Tyne; (7) Lavipharm S A; (8)Department of Biology, University of Athens Actinobacteria represent a signi¢cant proportion of the terrestrial and aquatic bacteria in oligotrophic environments. The biosynthetic potential of actinobacteria is far from being fully realized due to problems of selective isolation and cultivation. The key aspect of this project (actapharm qlrt-2000-01783 2001-2004) is the combination of expertise for detection and recovery of highly diverse actinobacteria populations through isolation and cultivation approaches and avoiding culture by provision of environmental gene libraries, with advanced detection technology for identi¢cation of bioactive metabolites. In order to provide an improved strategy for the detection and exploitation of microbial metabolic diversity, the project was divided into work packages that include the development of molecular tools to detect genetic diversity of actinobacteria and the application of novel isolation techniques for recovery of actinobacteria from natural habitats. As a further step, the development of hosts and vectors will allow the construction and expression of BAC environmental gene libraries, so that genetic diversity and biosynthetic potential of uncultured actinobacteria will be captured. Finally, characterization of the chemical diversity within metabolite pro¢les of isolates and clone libraries and development of a data-base to combine chemical and biological activity data with genetic diversity data is held to enable discovery and exploitation of novel bioactive compounds.

P4^49 MYXOBACTERIA AS POTENTIAL SOURCE OF NEW ANTI-INFECTIVES F. Gaspari(1), Y. Paitan(2), D. Losi(1), E. Z. Ron(2) and F. Marinelli(1) (1) Biosearch Italia S.p.A., Via R. Lepetit 34, Gerenzano (VA), Italy ; (2) Faculty Of Life Sciences, Tel-Aviv University, Ramat Aviv, Tel-Aviv, Israel Pharmaceutical industry is constantly searching for new biologically active molecules. One strategy is to seek them among microbial metabolites, and to set out the e¡orts for new potential producer microorganisms. Myxobacteria are known to be proli¢c producers of a variety of bioactive secondary metabolites including antibacterial and antifungal compounds. About 80 basic structures and 450 structural variants have been described up to now, most of them being exclusively produced by this microbial group. Even though these microorganisms are attractive sources of new compounds, they have not been extensively exploited in pharmaceutical screening because their isolation is time consuming and they are quite di/cult to handle and cultivate. During the collaboration between Biosearch Italia and Tel Aviv University, 100 myxobacteria belonging to 5 of the 12 described genera, were isolated from 36 soil and 14 tree bark samples collected in di¡erent areas inside the State of Israel. Four isolation methods, previously reported in the literature and based on the peculiar metabolic and cell cycle aspects of myxobacteria, were combined with puri¢cation procedures and optimisation of cultivation conditions and medium composition. 97 out of 100 were fermented and screened against a panel of clinically relevant pathogens including fungi and bacteria. High frequency of antibacterial and antifungal activities produced by these isolates con¢rms the attractiveness of mixobacteria for screening projects. Typical mixobacterial antibiotics such as Mixovirescin have been identi¢ed as well as other anti-infective molecules usually produced by actinomycetes as in the case of Althiomycin.

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P4^50 MOLECULAR TYPING ANALYSIS OF LACTIC ACID BACTERIA FROM BEER BY USING PCR-BASED METHODS AND PULSED-FIELD GEL ELECTROPHORESIS M. Marino, M. Maifreni and G. Rondinini ' Dipartimento di Scienze degli Alimenti, Universita degli Studi di Udine, via Marangoni 97, 33100 Udine, Italy Beer production carries a risk of microbial contamination at all stages of the process. Only a few species of the lactic acid bacteria, i.e., Lactobacillus and Pediococcus, are su/ciently resistant to the antibacterial properties of beer to grow at any stage of the process, causing turbidity and on o¡ £avour, often attributable to diacetyl. Superattenuation by starch-fermenting lactobacilli and slime formation by pediococci are other possible faults. The growth of bacteria is possible in beers because of a normal pH range of 4-5 and a good content of utilisable nutrients. Identi¢cation of brewery bacterial isolates has traditionally been accomplished biochemically by determining the assimilation and fermentation patterns of a number of carbohydrates and nitrogen sources. However, biochemical identi¢cation is not accurate for determining the genotypic di¡erences of microorganisms. Alternatively, there are many recently developed approaches for the molecular ¢ngerprinting of bacteria. Ribotyping is the only molecular technique which has been successfully used to characterize di¡erent brewery contaminants. Pulsed-¢eld gel electrophoresis also allows an overall clear identi¢cation potential at the strain level. Alternative simple and faster genotypical approaches involve the use of PCR for molecular ¢ngerprinting. In this work a preliminary characterization of lactic acid bacteria isolated from artisanal beers was performed using RAPD analysis, REP-PCR techniques and pulsed-¢eld gel electrophoresis. P4^51 GENETIC CHARACTERIZATION OF THE NITRATEREDUCING COMMUNITY IN FRENCH SOILS ¤ L. Philippot, E. Mounier, D. Cheneby, S. Piutti, S. Hallet, F. Martin-Laurent, J. C. Germon ¤ UMRA 111, INRA ^ CMSE, Microbiologie des Sols- Geosols, 17 rue Sully, B.V. 86510, 21065 Dijon Cedex, France Microbial respiratory nitrate reduction is the ¢rst step of the denitri¢cation and dissimilatory reduction of nitrate to ammonium pathway, which are considered as important processes since they contribute to the global cycling of nitrogen. This ability of facultative anaerobes to respire

nitrate has been ascribed mainly to the activity of a membrane bound nitrate reductase encoded by the narGHJI operon. In this study, we employed direct PCR and cloning of narG gene fragments to determine the phylogenetic a/liation of nitrate-reducing bacteria occurring in di¡erent soils. The clones from each soil speci¢c library were grouped by restriction fragment length polymorphism, and representatives from each group were sequenced and analyzed. Phylogenetic analysis of narG sequences separated the clone families into two main groups that represent the Gram-positive and Gram-negative nitrate-reducing bacteria. Novel narG lineages that branch distinctly from all currently known membrane bound nitrate-reductase encoding genes were detected within the Gram-negative branch for most of the studied soils. In addition each soil exhibited some speci¢c narG gene clusters. Altogether, our results revealed a more complex nitrate-reducing community than did previous culture-based studies. P4^52 DIVERSITY OF STREPTOMYCETES FROM INDOOR DUST H. Rintala, A. Hyvarinen, A. Nevalainen « National Public Health Institute, Department of Environmental Health, P.O. Box 95, 70701 Kuopio, Finland Streptomycetes are Gram-positive, ¢lamentous bacteria of the order Actinomycetales. They are common in soil, but also present in aquatic habitats, composts and fodder. During their vegetative growth, they form extensive branching mycelium, but as soon as the environmental conditions worsen, spores are formed. Streptomycetes are able to produce thousands of di¡erent secondary metabolites having a broad spectrum of biological activities. In buildings they can colonise moist building materials, grow and proliferate there, and thus can produce volatile secondary metabolites, such as geosmin, and spores into the indoor air. Spores of streptomycetes isolated from moisture-damaged buildings have been shown to induce in£ammatory responses in cell cultures and laboratory animals. In mice, also systemic e¡ects similar to those induced by anthracyclines have been observed. Indoor dust consists of biological and non-biological material originating from both indoor and outdoor sources. However, it re£ects the microbial condition of a building. We isolated 14 Streptomyces -strains from dust collected from regular vacuum cleaner dust bags of the occupants of 12 moisturedamaged residences that were located in eastern Finland. The 16S rRNA gene was partially sequenced and a phylogenetic tree was constructed. The strains clustered in four groups, the largest group consisting of eight strains a/liated with Streptomyces anulatus and Streptomyces halstedii, which are members of the largest species cluster of ISOLATED

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streptomycetes, the Streptomyces albido£avus cluster. That cluster also contains many producers of secondary metabolites. We also screened the strains for genes coding for the biosynthesis of secondary metabolites. P4^53

P4^54 PCR DETECTION OF MEMBERS OF GEODERMATOPHILUS AND NOCARDIACEAE IN DNA OBTAINED FROM STONE SURFACES O. Salazar, A. Valverde, A. Villalba and O. Genilloud

DIVERSITY OF THE NITRATE REDUCTASE GENES IN THE PSEUDOMONAS GENUS L. Roussel-Delif(1), S. Tarnawski(1), J. Hamelin(1), L. Philippot(2), M. Aragno(1) and N. Fromin(1) ¤ " (1) Laboratoire de Microbiologie, Universite de Neuchatel, " CP 2, CH-2007 Neuchatel, Switzerland; (2) INRA CMSE, Laboratoire de Microbiologie des sols, 17 rue Sully, F21034 Dijon cedex, France Denitri¢cation is the reductive respiration of nitrate to gaseous N compounds. This function is present in di¡erent phyla including Q-Proteobacteria. The nitrate reductase enzymes catalyse the ¢rst step of denitri¢cation pathway. Two isoenzymes were described for nitrate reductase: the periplasmic form encoded by nap genes and the membrane-bound form encoded by nar genes. We investigated nitrate reduction and denitri¢cation in Pseudomonas populations in the rhizosphere of two perennial grasses di¡ering in trophic requirements: Lolium perenne and Molinia coerulea under ambient or elevated atmospheric CO2 content, at ¢ve sampling dates. 1246 Pseudomonas strains were checked for nitrate reduction and denitri¢cation activities. 40% and 20% of Pseudomonas strains isolated from L. perenne and M. coerulea respectively, were able to reduce nitrate only to nitrite and 13% were complete denitri¢ers for both plants. The proportions of both nitrate reducing and denitrifying Pseudomonas were generally higher in soil fraction and under elevated CO2. At the gene level, the occurrence and diversity of napA and narG genes among nitrate reducing Pseudomonas were assessed by using PCR-RFLP analysis. We detected a large diversity of narG and napA genes. The proportion of the membrane-bound form was higher in root fractions and under elevated CO2. These results are discussed in relationship with the structuration of Pseudomonas populations in rhizosphere environments.

¤ ¤ Centro de Investigacion Basica de Espa·a (CIBE), Merck Research Laboratories, Merck Sharp & Dohme de Espa·a, S. A. Josefa Valcarcel 38, 28027 Madrid, Spain Actinomycetes of the genus Geodermatophilus and the family Nocardiaceae have been recovered from stone surfaces of rocks and altered surfaces of monuments [1-3] Nevertheless, they only represent a minor group of the actinomycetes that can be obtained from diverse rock environments using standard isolation methods [4]. Given that their isolation frecuency can be biased with these procedures, we have focused on the development of molecular tools that could provide us with a deeper insight on their natural occurrence in the di¡erent habitat studied. Frequently, the wide range of morphologies and phenotypes of actinomycetes, and the frequent absence of sporulation in laboratory conditions di/cult the current identi¢cation of many wild type isolates and make necessary the use of alternative molecular tools for identi¢cation. We present here the design of two pairs of speci¢c oligonucleotides for the rapid detection of members of Geodermatophilus and Nocardiaceae by speci¢c ampli¢cation of 16S rDNA region. The speci¢city of these primers was validated with type strains and these primers were used to detect the presence of representatives of these taxa directly from rock surfaces DNA collected in di¡erent Spanish locations. This study presents the usefulness of the application of these identi¢cation tools to assess the occurrence of members of these taxa directly from this alternative isolation source as well as from other environments considered of high interest for microbial isolation programs. [1] Eppard et al. (1996) Arch. Microbiol 166, 12-22. [2] Urzi et al. (2001) Environmental Microbiology 3, 471479. [3] Groth et al. (1999) J. Microbiol. Met. 36, 115¤ 122. [4] Gonzalez et al (2003). Accompaning poster.

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P4^55 DIVERSITY OF AUTOTROPHIC BACTERIA HARBOURING cbbL GENES IN DIFFERENT AGRICULTURAL SOILS D. Selesi, I. Pattis, M. Schmid and A. Hartmann GSF National Research Centre for Environment and Health, Institute of Soil Ecology, Ingolstadter Landstrasse « 1, 85764 Munchen-Neuherberg, Germany « Autotrophic bacteria harbouring the Calvin cycle enzyme ribulose-1,5-bisphosphate carboylase/oxygenase (RubisCO) may play a signi¢cant part in the conversion of carbon dioxide into organic matter and microbial biomass and may thus contribute to the global carbon cycling. To gain an insight into the genetic diversity of CO2-¢xing bacteria in soil habitats we developed PCR-based assays targeting the large subunit gene cbbL of the form I RubisCO. Based on the calculation of phylogenetic relationships we designed di¡erent primer sets having strong speci¢city for `red-like' and `green-like' cbbL sequences of selected terrestrial autotrophs. Bulk genomic DNA was isolated from agricultural soils with rye crop at di¡erent long-term fertilization as well as from a soil under clover/ gras cover. The RFLP and phylogenetic analysis of the ampli¢ed cbbL sequences indicated a high diversity of `red-like' and a low diversity of `green-like' cbbL sequences in these soils. Additionally variations in community composition on the basis of cbbL genes could be observed in the di¡erently managed soils. P4^56 AN UPDATE OF VIBRIONACEAE TAXONOMY F. L. Thompson and J. Swings Laboratory for Microbiology and BCCMTM/LMG Culture Collection, Ghent University, K.L. Ledeganckstraat 35, Ghent 9000, Belgium The family Vibrionaceae (Gammaproteobacteria) is probably one of the best-documented bacterial groups from the aquatic environment. Six main groups of vibrios are delineated on the basis of their 16S rDNA sequences i.e. the Vibrio core group, the psychrophilic Vibrio species, V. hollisae, Enterovibrio and Salinivibrio. Vibrios are ubiquitous and abundant in the aquatic environment. These microbes were initially supposed to represent the dominant marine micro£ora as deduced from plate counts. Recent studies based on cultivation independent approaches have shown vibrios compose about 4 % (V108 cells liter-1) of the Bacteria in aquatic environments. High abundance of vibrios is also detected in tissues and/or organs of certain

marine animals e.g. ¢sh, squids, abalones, bivalves, shrimps, copepods and corals. The Vibrionaceae harbours a wealth of diverse genomes as revealed by AFLP genomic analysis of 506 strains. It was found that 236 isolates distributed in 31 clusters have genomes unrelated to any type strain and are thus potential new taxa. Among these unknown isolates, a novel genus i.e. Enterovibrio norvegicus and ¢fteen novel species i.e. V. barceloniensis, V. brasiliensis, V. chagasii, V. coralliilyticus, V. fortis, V. gallicus, V. kanaloae, V. neptunius, V. pomeroyi, V. pacinii, V. probioticus, V. rotiferianus, V. superstes, V. tasmaniensis and V. xuii have been described. Several of these new species have ubiquitous occurrence, but their exact ecological role is unknown at present. Acknowledgement: F.L.T. has a Ph.D. Scholarship from CNPq, Brazil. P4^57 CULTURE COLLECTION IBSO AND ELECTRONIC COLLECTION ``BIOLUMBASE'': STUDY AND MAINTAINANCE OF LUMINOUS BACTERIA DIVERSITY G. A. Vydryakova, S. E. Medvedeva, A. M. Kuznetsov, D. A. Kotov, J. V. Chugaeva, and E. K. Rodicheva Institute of Biophysics SB RAS, 660036, Krasnoyarsk, Russian Federation There are di¡erent bacterial species able to emit light. The Culture Collection of the Institute of Biophysics of the Siberian Branch of the Russian Academy of Sciences (CC IBSO) deposits about 700 strains of marine luminous bacteria and genetically modi¢ed E. coli strains with marker lux-gene. Luminous bacteria are producers of many enzymes: L-ornithine-, L-arginine-, L-lysine-decarboxylases, neuraminidase, chitinase, cellulase, alginate lyase, endonucleases of restriction. The most interesting are luciferase, oxidoreductase and dehydrogenases. While relatively little is known about the biological signi¢cance of bacterial light emission, much has been published about the biochemistry and regulation of bioluminescence in luminous bacteria. But real structure of emitter is unknown. Luminescence spectra of luminous bacteria (Vibrio ¢scheri, Vibrio harveyi, Photobacterium phosphoreum, Photobacterium leiognathi) and recombinant E. coli (with lux-genes from P. leiognathi) have been studied in our research. It was shown that V. harveyi, P. phosphoreum, P. leiognathi wavelength spectrum peaked at about 475 nm, where as E. coli and V. ¢scheri wavelength spectrum peaked at about 490 nm. So, it is interesting to note di¡erences at wavelength spectrum peaks of E. coli (with lux-genes from P. leiognathi) and P. leiognathi. As we can assume such differences possible are dependent on reaction microenvironment. There is the work version of electronic collection

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HBiolumBaseg of natural and transgenic luminous microorganisms that are maintained in microbial collections of IBP SB RAS (http://lux.ibp.ru). It contains information about their main morphological, physiological-biochemical and genetic features and bioluminescent systems of di¡erent bacteria. This work was supported by RFBR (grant 00-07-9011). P4^58 RFLP rDNA CHARACTERISATION OF THE GENUS CLADOSPORIUM ISOLATED FROM BATHROOMS M. Butala, P. Zalar and N. Gunde-Cimerman University of Ljubljana, Biotechnical Faculty, Biology De> partment, Vecna pot 111, 1000 Ljubljana, Slovenia Saprotrophic fungi from the genus Cladosporium are usually the dominant microoorganisms in outdoor as well as in indoor air. They are not known as agents of the ``sick building syndrom'', which was assigned mainly to the presence of airborne Penicillium and Stachybotrys spp, but are sometimes described as agents of pulmonary and cutaneous infections. Inside of every building, not necessary sick building, there are places where occasionally warm and moist atmosphere develops. Bathrooms and kitchens represent such special habitats, which are quickly occupied by moulds. In our study we sampled 26 bathrooms all over Slovenia with the aim to study the presence and a species composition of the genus Cladosporium. Different species from this genus were present in 80 % of the sampled bathrooms. Isolates were compared morphologically and with the molecular characterisation ^ RFLP of rDNA (partially SSU, whole ITS and partially LSU). Reference strains for common species, type and neotype material where described, and strains isolated from plant material were included in the study. According to the speci¢c restriction pattern, obtained by selection of speci¢c restriction enzymes, fungi were identi¢ed to the species level. By this method, a quick identi¢cation tool for the members of the most common saprotrophic species from the genus Cladosporium was established. The prevailing species was Cladosporium sphaerospermum, which was present in 70 % of sampled bathrooms, followed by C. cladosporioides and C. herbarum, present in 22 and 8 % of sampled bathrooms, respectively.

P4^59 MOLECULAR IDENTIFICATION OF HANSENIASPORA AND KLOECKERA SPECIES í > N. Cadez(1), M. Th. Smith(2), P. Raspor(1) (1) University of Ljubljana, Biotechnical Faculty, Department of Food Science and Technology, Jamnikarjeva 101, 1000 Ljubljana, Slovenia; (2) Centraalbureau voor Schimmelcultures, Yeast Division, P.O. Box 85167, 3508 AD Utrecht, The Netherlands The yeast genus Hanseniaspora and its anamorph Kloeckera are morphologically characterized as apiculate yeasts with bipolar budding. The Hanseniaspora / Kloeckera yeasts are frequently isolated from various natural sources such as soil, fruits and insects as well as from fermented foods and beverages. As predominant inhabitants on the surface of grape berries and in starting wine fermentations, these genera have been intensely studied to determine their role on the quality of the ¢nal fermentation product. For monitoring the presence apiculate yeasts in wine fermentations the rapid and accurate identi¢cation method is required. The identi¢cation of Hanseniaspora and Kloeckera species using traditional, physiological testing is hampered by the low number of positive growth characters and moreover, inconsistencies in identi¢cation results. The recent development of molecular techniques for yeast identi¢cation has substantially reduced time needed for identi¢cation and has improved the reliability through the direct analysis of DNA, thus identi¢cation does not vary with the physiological state of organism. In our study three molecular methods, PCR ¢ngerprinting, electrophoretic karyotyping and RFLP of the PCR-ampli¢ed ITS regions (ITS1, ITS2 and the intervening 5,8S rDNA), were studied for accurate identi¢cation of 92 strains of Hanseniaspora and Kloeckera isolated from di¡erent sources as well as from geographically distinct regions. Of these three methods PCR-RFLP analysis of ITS regions with three restriction enzymes is proposed as rapid and accurate identi¢cation technique in a form of a two-step molecular identi¢cation key to Hanseniaspora and Kloeckera species. This study was partly supported by a FEMS fellowship.

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P4^60 INJURIES OF GRAPE BERRIES HAS IMPACT ON BIODIVERSITY OF YEAST POPULATION ISOLATED FROM GRAPE BERRY SURFACES í > > > D. Miklic Milek, N. Cadez, S. Smole Mozina, P. Raspor University of Ljubljana ; Biotechnical faculty, Chair of Biotechnology, Jamnikarjeva 101, 1000 Ljubljana, Slovenija Yeasts are widespread in nature and are found in soils, on surfaces of vegetables and in the digestive tract of animals. Yeasts of di¡erent genera, species and strains found on the surface of grapes and associated with surfaces of winery equipment are the main microorganisms responsible for wine fermentations. The aim of this study was to determine the in£uence of geographic locations and health condition on the indigenous yeast £ora of grape berries. Samples of grape berries were collected from grape cultivar Modra frankinja, which is one of the main vine cultivars for production of red vine Cvicek in Slovenia. The healthy and slightly injured grapes were collected during the ripening in September 1999 on ¢ve di¡erent locations of Dolenjska vine growing region. The grape berries were homogenized and plated at serial dilutions on media. The microbial population that consisted mostly of yeasts and ¢lamentous fungi was enumerated. In order to identify the yeast colonies on surfaces of di¡erent grapes a combination of restriction analysis of PCR ampli¢ed rDNA-ITS region and morphological and physiological tests were used. Yeast population on healthy grapes was numerically lower than the population of yeasts isolated from damaged grape berries. Further, sampling location had great impact on yeast number and on diversity of yeast species. In most samples the following yeast species predominated: Hanseniaspora uvarum, Rhodotorula glutinis, Aureobasidium pullulans, Metschnikowia pulcherrima and Cryptococcus spp.. Saccharomyces cerevisiae was not found. This work was ¢nanced by the Slovene Ministry of Education, Science and Sport (project No. L4-8849-98). P4^61 TYPING OF DEBARYOMYCES HANSENII AND KLUYVEROMYCES LACTIS STRAINS FROM FIORE SARDO CHEESE M. E. Fadda, V. Mossa, M. B. Pisano and S. Cosentino Department of Experimental Biology, section of Hygiene, University of Cagliari, S.S 554, Km 4,500, 09042 Monserrato (CA), Italy Over the last decade various methods based on molecular biological techniques to characterize yeasts isolated from

food products have been developed. An extensive study carried out on the natural £ora of Fiore Sardo showed the di¡use presence, throughout ripening, of several yeast species. Since Debaryomyces hansenii and Kluyveromyces lactis were found to be predominant, in this study we investigated the genetic heterogeneity among the isolates of these species. 20 D. hansenii and 15 K. lactis strains isolated from Fiore Sardo cheese were typed by RAPDPCR with the M13 primer and by the restriction pattern of 18S rDNA digested with two restriction enzymes (HaeIII and HpaII). DNA banding patterns were analysed using the Gel Compar software package, version 2.5 (Applied Maths, Kortrijk, Belgium). Similarities among isolates were estimated using the Pearson coe/cient and clustering was based on the UPGMA method. The species D. hansenii and K. lactis originated distinct RAPD patterns and therefore were clearly separated in the dendogram. At a 70% level of similarity 2 clusters were identi¢ed for both D. hansenii and K. lactis. Restriction enzyme analysis with HaeIII and HpaII showed the presence of one pro¢le within K. lactis and D. hansenii strains. Both techniques were found useful for the characterization of yeasts. PCR ampli¢cation with M13 gave good results for the identi¢cation at species level and also presented a good discrimination power at subspecies level. In comparison, rDNA restriction analysis was useful for discrimination at species level but it was not satisfactory for identi¢cation at subspecies level. This work was supported by a grant from MURST, Plan ``Agroalimentary products : dairy products'', Cluster 08B, Project n.7. P4^62 EXTRACHROMOSOMAL DNAs IN YEASTS ISOLATED FROM SPRING SAPWOOD FLUXES ¤ A. Fejfar(1), J. Kucsera(1), W. I. Golubev(2) (1) Department of Microbiology, University of Szeged, P.O.Box 533, Szeged, Hungary, H-6701; (2) Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Russia Exudate of deciduous trees in the spring provides a carbohydrate-rich media for yeast species. The three dominant species of this habitat are Nadsonia fulvescens var. elongata, Trichosporon pullulans and Xanthophyllomyces dendrorhous. Their extrachromosomal DNA elements were investigated during this study. The existence of DNA plasmids was revealed in all of the examined species. Two strains of N. fulvescens var. elongata contained three or four types of DNA plasmids (strain VKM Y-268: 4.70, 1.09 and 0.97 kb; strain VKM Y-1653 : 5.20, 4.34, 0.94 and 0.83 kb, respectively), Trichosporon pullulans VKM Y-273 harboured one species of it (4.1 kb), while a number of

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were identi¢ed in ¢ve strains of X. dendrorhous with sizes 6.5-2.0 kb. All the plasmids were sensitive to exonuclease treatment what indicates their linear structure. The plasmids of N. fulvescens var. elongata and X. dendrorhous strains had buoyant density similar to that of the nuclear DNA. Contrary plasmid from T. pullulans copuri¢ed with the mitochondrial DNA on CsCl gradient, showing lower G+C content than the nuclear DNA. Plasmids from Nadsonia and Trichosporon strains were absent from puri¢ed organelles suggesting their possible cytoplasmic location; X. dendrorhous plasmids were located in the mitochondrial fraction. Plasmids of Nadsonia and Trichosporon strains are cryptic while DNA plasmids of X. dendrorhous could take part in the mitochondrial genome organisation. This work was supported by FKFP 0091/2001. P4^63 MOLECULAR ASSESSMENT OF YEAST DIVERSITY ì AT THE SAO DOMINGOS MINE (PORTUGAL), A METAL POLLUTED ACIDIC ENVIRONMENT IN THE IBERIAN PYRITE BELT M. Gadanho and J. P. Sampaio ¤ Centro de Recursos Microbiologios (CREM), Seccao °< ¤ " Autonoma de Biotecnologia, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal The Iberian Pyrite Belt (IPB) is a vast mining area in the South of Portugal and Spain and one of the most important pyrite regions in the world. At several sites in the IPB, like the S. Domingos abandoned mines, Acid Rock Drainage is responsible for the contamination of soils and groundwater, giving rise to acidic reddish-brown waters with high concentrations of metals. Since yeasts are well adapted to grow in acidic conditions, this study attempted to address their occurrence, diversity and potential in bioremediation. At S. Domingos mine, four sites were investigated. The pH of the water samples ranged from 1.7 to 2.7 and the concentration of Fe2+ was 0.069-25.9 g/l. Water samples (5 liters) were collected in di¡erent seasons of the year and yeasts were investigated using pure culture-independent approaches. Total DNA was extracted and PCR-TGGE (Temperature Gradient Gel Electrophoresis) was employed. Since yeasts appeared to be present in low densities, the same approach was used in enrichment studies. Nutrients were added to the water samples and yeasts were monitored between the ¢rst and 15th day of incubation. DNA from various species was detected and identi¢cation was achieved by sequence analysis. Pure culture studies were performed in parallel using di¡erent growth conditions and culture media employing water from the sites under investigation. The most frequent taxa belonged to new species of the genera Rhodosporidi-

um and Cryptococcus. The temporal and spatial distribution of yeast populations was characterized and the results obtained in the molecular and traditional approaches were compared. M.Gadanho was supported by a grant N SFRH/BD/ 1170/2000. P4^64 INTERPRETATION OF mtDNA POLYMORPHISMS OF ASPERGILLUS TUBINGENSIS STRAINS L ¤ A. Juhasz, H. Engi, Zs. Hamari, F. Kevei Department of Microbiology, Faculty of Sciences, University of Szeged, P.O. Box 533, H-6701 Szeged, Hungary In previous studies considerable inter- and intraspeci¢c mtDNA polymorphisms were detected among black Aspergilli. Aspergillus tubingensis strains proved to be highly variable they could be divided into six groups (labeled 2a2f) based on their mtDNA pro¢les. To study the reason of mtDNA variability physical and partial functional maps were developed. Physical map were constructed by four restriction enzyme (EcoRI, EcoRV, BglII, HindIII) applying reciprocal digestion technique (each fragment was isolated and re-digested by another enzyme). Functional maps were developed by sequence analysis of certain restriction fragment clones and size determination of PCR products. The precise size of mitochondrial genes was established by the size of PCR products ampli¢ed using forward primers designed to match the start point of A. nidulans and/or A. niger mitochondrial genes and reverse primers designed to match the end of these genes. Gene order and size of the intergenic regions between two neighboring genes were also determined by PCR using various combinations involving forward primers designed to match the end of the mitochondrial genes and reverse primers designed to match the start of those same genes. The basic organisation of mtDNAs was highly similar but intergenic sequences and optional intron content showed some alterations. The results will be discussed in detail. This work was supported by grants (T 037584 F032704 and) of Hungarian Scienti¢c Research Foundation (OTKA).

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P4^65 STUDIES ON THE HETEROGENEITY OF THE CAROTENOGENIC YEAST RHODOTORULA MUCILAGINOSA FROM PATAGONIA, ARGENTINA D. Libkind(1), M. Gadanho(2), M. van Broock(1) and J. P. Sampaio(2) ¤ ¤ (1) Laboratorio de Microbiolog|a Aplicada y Biotecnolog|a, Universidad Nacional del Comahue, Centro Regional Universitario Bariloche (CRUB) ^ CONICET (Consejo Na¤ ¤ cional de Investigaciones Cient|¢cas y Tecnologicas), Bar¤o Negro, Argentina; (2) Centro de Recursos iloche, R| ¤ ¤ Microbiologicos, Seccao Autonoma de Biotecnologia, Fac°< " ncias e Tecnologia, Universidade Nova de Lisuldade de Cie boa, 2829-516 Caparica, Portugal The basidiomycetous red yeast Rhodotorula mucilaginosa is a ubiquitous species and can be found both in natural and in human-related environments. Due mostly to similar physiological pro¢les, several species are presently regarded as synonyms of Rh. mucilaginosa, although very few have been investigated using molecular methods. In this study, forty-¢ve Rh. mucilaginosa isolates obtained from diverse natural and arti¢cial environments (lakes, soil, nectar, wild fruits, grape surfaces) from Patagonia, Argentina, were studied. The methods employed encompassed morphological and physiological characterisations, DNA ¢ngerprinting using MSP-PCR (mini/micro-satellite primed-PCR), single nucleotide sequencing (SNS) and DNA sequence analysis of the D1/D2 and ITS regions. Preliminary MSP-PCR experiments using primer (GTG)5 revealed considerable heterogeneity in Rh. mucilaginosa. Subsequent studies with primer M13 allowed the formation of three groups, one of them encompassing the type strain and 84% of the studied strains. Physiological and morphological results correlated with the presence of the three groups. No sexual compatibility reactions were observed between or within these groups. Selected isolates were investigated by sequence analysis. In contrast with the D1/D2 region, the less conserved ITS region revealed di¡erences between the tested strains of the di¡erent groups. However, no correlations were found between the three groups and the geographic origin of the isolates, or their isolation source. The observed heterogeneity is presently interpreted as intraspeci¢c variability. Future work will expand the set of studied isolates, will address the molecular relationships of the current synonyms of Rh. mucilaginosa and will conceivably allow a better understanding of the heterogeneity presently reported.

P4^66 COMPLEX COMPOSITION OF THE YEAST ZYGOFABOSPORA / KLUYVEROMYCES LACTIS: GENETIC AND MOLECULAR DIFFERENTIATION OF SUBPOPULATIONS G. I. Naumov, N. N. Sukhotina and E. S. Naumova State Institute for Genetics and Selection of Industrial Microorganisms, I-Dorozhnyi, 1, Moscow 117545, Russia Our new experimental and literature data (Naumov, 1986; Sidenberg and Lachance, 1986; Fuson et al., 1987 ; Naumov and Naumova, 2002) are discussed in the light of heterogeneity of the Z. lactis species. It includes both wild lactose-negative geographic populations, having partial genetic isolations, and domesticated lactose-positive dairy strains. Using di¡erent molecular markers we di¡erentiated eight wild populations of Z. lactis: ¢ve NorthAmerican (including the known varieties Z. lactis var. drosophilarum and Z. lactis var. phaseolospora), one European (Z. lactis var. krassilnikovii), one South-African (Z. lactis var. vanudenii), and one Far East Asian. The results of genetic hybridisation, PCR analysis and sequencing rDNA will be presented. P4^67 GENETIC VARIABILITY IN THE SPECIES RHIZOPUS STOLONIFER ASSESSED BY RAPD ANALYSIS L ¤ « T. Papp(1), H. Heinrich(2), K. Acs(2) and Cs. Vagvolgyi(2) (1) HAS-USZ Microbiological Research Group, University of Szeged, Faculty of Sciences, Department of Microbiology, P.O. Box 355, H-6701, Szeged, Hungary; (2) University of Szeged, Faculty of Sciences, Department of Microbiology, P.O. Box 355, H-6701, Szeged, Hungary Rhizopus stolonifer is one of the most important members of the class Zygomycetes. Recent taxonomy based on morphological and growth characteristics reduces the number of species di¡erentiating only two varieties (var. stolonifer and re£exus) within this species. The objective of this study was to examine this situation with molecular markers. Twenty-nine R. stolonifer strains isolated from various locations and substrates were characterized by random ampli¢ed polymorphic DNA (RAPD) analysis. The numerical analysis of the RAPD data revealed four main clusters with extremely high dissimilarity values, at the same time, low or moderate variabilities were observed within these groups. R. stolonifer var. stolonifer, R. stolonifer var. re£exus and R. niveus showed species level genetic distances with this method. These results suggest

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higher genetic heterogeneity in the case of R. stolonifer, than as could be expected on the basis of the recent species concept. P4^68 DNA POLYMORPHISM FOUND IN THE INTERNAL TRANSCRIBED SPACER (ITS) REGION OF UDENIOMYCES PYRICOLA M. Takashima(1), T. Nakase(2), J. Tornai-Lehoczki(3), T. Deak(4) and T. Kudo(1) (1) Japan Collection of Microorganisms, Bioscience Technology Center, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama, Japan; (2) National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Bangkok, Thailand; (3) National Collection of Agricultural and Industrial Microorganisms, ¤ Szent Istvan University; (4) Department of Microbiology, ¤ n University, Budapest, Hungary Szent Istva Udeniomyces pyricola, an anamorphic basidiomycetous yeast, was isolated from Switzerland, Canada, Japan, New Zealand and Tasmania, Australia, but has not been isolated from the tropical or subtropical area of southeastern Asia (Nakase, T. 2000. J. Gen. Appl. Microbiol. 46: 189-216). Of a total of 100 ballistoconidium-forming yeasts isolated from plants in Hungary, 10 strains from 10 plants were identi¢ed as U. pyricola by their morphological, physiological and biochemical characteristics. In addition, 9 strain maintained at the Japan Collection of Micoorganisms (JCM), from Switzerland (JCM 2958, type strain), Canada (neotype strain of Bullera grandispora JCM 8249 plus two isolates), Japan (3), New Zealand (1) and Tasmania (1), were used in this study. The sequence of the D1/D2 region of 26S rDNA of 16 strains including the type strain, JCM 2958, were identical, and one base di¡erence was found in the remaining three strains. Based on the sequences of the internal transcribed spacer (ITS) region, they were divided into three clusters. JCM 2958 and strains isolated from Hungary (7 out of 10), Canada, New Zealand and Tasmania composed cluster 1, and three Japanese and three Hungarian isolates each made a distinct cluster. In cluster 1, four or one base insertions/deletions were detected at two positions, respectively, near the 3' end of ITS2 region. Based on these insertions/deletions, the Hungarian strains were divided into three groups, and the Canadian strains belonged one of these ITS2 type group.

P4^69 POLYKETIDE SYNTHASE GENE SEQUENCES IN OCHRATOXIN-PRODUCING ASPERGILLUS SPECIES ¤ ¤ J. Varga, K. Rigo, S. Kocsube and B. Farkas Department of Microbiology, University of Szeged, Faculty of Sciences, P.O.Box 533, H-6701 Szeged, Hungary Ochratoxin A (OA) is a pentaketide dihydroisocoumarin derivative linked to an L-phenylalanine moiety. OA is frequently encountered in various foods including cereals, co¡ee, spices and wine, and exhibits nephrotoxic, immunosuppressive, teratogenic and carcinogenic properties. Although several e¡orts have been made, none of the enzymes or genes responsible for OA production has been isolated or characterized up to date. Our aim was to identify polyketide synthase (PKS) gene sequences in ochratoxin producing Aspergillus species (A. ochraceus, A. niger, A. muricatus, A. albertensis) using primer pairs developed for the ketosynthase (KS) domain of fungal PKSs. Ketosynthase domain probes ampli¢ed a single DNA fragment of about 700 bp in each examined isolate. Sequences of these domains were aligned and analyzed by phylogenetic methods. The KS domain sequences were highly diverse indicating that they most probably represent PKSs responsible for di¡erent functions. A. albertensis and A. niger KS domain sequences clustered together with sequences of genes required for pigment biosynthesis (wA) in A. nidulans and P. patulum, the KS sequence of N. muricatus was most closely related to an A. parasiticus wA type domain sequence, while those of the A. ochraceus isolates were highly similar to an A. terreus naphthopyrone synthase gene. Since some A. fumigatus isolates are also able to produce OA, KS domain sequences were also used to search the TIGR A. fumigatus genomic database for PKS related sequences. At least 12 putative PKS genes were identi¢ed, two of which also carry a C-methylation domain which was suggested earlier to be part of a hypothetical ``ochratoxin synthase'' gene. Further studies are in progress to see whether the identi¢ed KS sequences are part of the PKS gene responsible for the make-up of the isocoumarin skeleton of OA.

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P4^70 EVOLUTIONARY RELATIONSHIPS AMONG ASPERGILLUS TERREUS AND ITS RELATIVES ¤ ¤ ¤ J. Varga(1), B. Toth(2), K. Rigo(1), S. Kocsube(1), B. ¤ ren(3) Farkas(1) and J. Te (1) Department of Microbiology, University of Szeged, Faculty of Sciences, P.O.Box 533, H-6701 Szeged, Hungary ; (2) Cereal Research non-Pro¢t Company, P.O. Box 391, H-6701 Szeged, Hungary ; (3) Animal Health and Food Control Station, P.O. Box 446, H-6701 Szeged, Hungary Aspergillus terreus is a ubiquitous fungus in our environment. This fungus is an opportunistic human pathogen, and economically important as the main producer of lovastatin, a cholesterol lowering drug. Lovastatin has recently also been suggested to have potent anti-cancer effects. Our aim was to examine the genetic variability of A. terreus and closely related species (22 isolates representing 12 species) using molecular and analytical techniques. Lovastatin production was examined by HPLC. Lovastatin was produced by some isolates belonging to the species A. terreus, A. niveus and A. carneus. RAPD analyses were carried out using 20 di¡erent random decamers. Neighborjoining analysis of RAPD data (245 characters) let us cluster the isolates into distinct groups. Since previous studies indicated that isolates of A. terreus and closely related species have identical 28 S rRNA gene sequences, we sequenced the intergenic spacer region and the 5.8 S rRNA gene of the isolates. Phylogenetic analysis of sequence data let us classify the isolates into di¡erent clades. A. terreus and its subspecies and A. allahabadii formed one clade, another clade included A. £avipes, A. iizukae and an A. niveus isolate, a distinct clade included A. carneus isolates and A. obscurus, while the other examined species (A. janus, A. anthodesmis, A. ambiguus) formed distinct branches on the tree. The related species A. £avipes was more heterogeneous than A. terreus isolates, indicating that isolates currently assigned to the species A. £avipes possibly represent a number of undescribed species.

P4^71 WALLEMIA : THE GENUS OF OSMOPHILIC FUNGI INHABITING SALTERNS P. Zalar(1), G. S. de Hoog(2) and N. Gunde Cimerman(1) (1) University of Ljubljana, Biotechnical Faculty, Biology > Department, Vecna pot 111, 1000 Ljubljana, Slovenia; (2) Centraalbureau voor Schimmelcultures, P.O.Box 85167, 3508 AD Utrecht, The Netherlands Wallemia is an important genus of xerophilic fungi involved in the spoilage of low water activity food commodities. It is only occasionally described from natural low water activity environments. Up to now only one species, W. sebi was recognised in this genus and a number of synonyms proposed. For several strains of the species W. sebi the production of considerably toxic metabolites walleminol and walleminon were reported, which were shown in bio-experiments to be of comparable toxicity with citrinin and pennicillic acid and it thus represents a potential health hazard. In the past also medical cases were reported which indicated its possible pathogenic role to humans. Therefore it is very important to understand the natural ecology of this fungus. In our study of halophilic fungi living in the hypersaline water of salterns worldwide, fungi from the genus Wallemia were repeatedly isolated. On the basis of ITS rDNA variability of a set of strains including reference strains, and comparing their morphological and physiological characters, we propose division of the genus Wallemia into three species. In order to determine their phylogenetic position, SSU rDNA domains of the proposed species representatives were sequenced and compared with the other representatives of the fungal kingdom. According to this it is placed among Basidiomycetes. P4^72 CHARACTERIZATION OF THE BACTERIOPHAGES OF THE PT-SERIES RELATED TO PS. AERUGINOSA N. Chanishvili(1), M. Merabishvili(1), T. Glonti(1), M. Vaneehautte(2) (1) George Eliava Institute of Bacteriophage, Microbiology and Virology, Tbilisi, Georgia; (2) Department of Clinical Chemistry, Microbiology and Immunology, University Hospital, University of Gent, 185, De Pintelaan, 9000, Gent, Belgium Spread of bacterial drug-resistance in modern hospital settings put into discussion a potential application of bacter-

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iophages as alternatives of antibiotics and/or disinfectants. Phage cocktails have been successfully used in medical practice in the FSU for longer than 70 years. Nevertheless, the genetic characteristics of the therapeutic bacteriophages is poorly known. The goal of this study was to ¢ll in some gaps regarding the phages related to Ps. aeruginosa. Five phages have been chosen for these studies: PT-1, PT-2, PT-4, PT-5 and PT-8. The AFLP has been applied as a tool for gene typing of these phages. Obtained results showed that the phage clones : PT-1, PT-4 & PT-8 have the same genetic patterns. The electron microscopic studies and serological studies proved this similarity as well. These facts are especially interesting, since these phages have been isolated in various times and in di¡erent ecological niches. The phage clone PT-1 is one of the components of the traditional commercial preparation `` Intesti- bacteriophage'', the clone PT-4 has been isolated from the river Mtkvari and the clone PT-8 from the Lake Lisi in Tbilisi, Georgia. Despite of the morphological similarity, the genomic patterns of the phages PT-2 and PT-5, signi¢cantly di¡er from each other and the rest of the chosen phages. The phages of PT series have been screened against 200 strains. They showed high e/ciency towards the strains of Ps. aeruginosa, including imipinemeresistant and mucoid bacteria. These results prove genetic diversity of the therapeutic bacteriophages applicable for combating drug-resistant bacteria. P4^73 GENETIC CHARACTERIZATION OF STREPTOCOCCUS THERMOPHILUS BACTERIOPHAGES ISOLATED FROM RAW MILK SAMPLES Zh. P. Dimitrov

tested as hybridization probes with purpose to detect lysogeny among Streptococcus thermophilus strains. Some of primers were used for fast PCR detection of the phage content in milk samples. P4^74 UZBEK COLLECTION OF AGRICULTURAL MICROORGANISMS: ITS DEVELOPMENT D. Egamberdiyeva and K. Davranov Institute of Microbiology, A.Qadiriy str. 7 B, 700128 Tashkent, Uzbekistan Uzbek National Collection of Agricultural microorganisms has been established in order to improve the native microbial resources of Uzbekistan, to study the diversity of native microbial population and to preserve a valuable microorganisms. The main objectives of our Collection are deposit of agriculturally useful microbial strains from Scienti¢c community, their preservation and distribution for education, research, training courses, isolation, identi¢cation of strains. At present we have salt tolerant, heat resistance, plant growth promoting bacterial strains, nitrogen ¢xers, fungi, plant pathogenic fungi, some plant viruses. Because of limited ¢nancial resources there were lack of chemicals and equipment's for preservation of microorganisms. We are grateful for Society of General Microorganisms (SGM, UK) for supporting the development of Uzbek Culture Collection. With this support we will use also freeze dried ampoules for preservation, develop strain data management and publish ¢rst catalogue Uzbek Culture Collection. P4^75

ELBY Bulgaricum, Research Department, 12-a Malashevska Str. 1202 So¢a, Bulgaria The bacteriophage attacks against starters for milk products can lead to serious problems in dairy industry. Streptococcus thermophilus is widely used as a component of dairy starters in the manufacture of yoghurt and several kinds of cheeses. Two bacteriophages active against Streptococcus thermophilus strains were isolated from 50 raw milk samples with di¡erent geographic origins in Bulgaria. Considering very weak phage absorption onto the cells in conventional broth medias the protocol for propagation of these phages was optimized. Restriction analysis with different restriction endonucleases was carried out in order to estimate the molecular weight and to compare the restriction pro¢les with published ones. Applying partially digestion approach the physical maps of the both phage DNA-s was created. Both phages had cohesive ends. Using several known primer pairs for PCR, di¡erent phage DNA fragments were ampli¢ed. After labeling these fragments were

CYANOBACTERIAL BUILDINGS

DIVERSITY

ON

HISTORIC

C. A. Crispim(1), P. M. Gaylarde(1), C. C. Gaylarde(1), B. A. Neilan(2) (1) Federal University of Rio Grande do Sul, Porto Alegre, Brazil ; (2) University of New South Wales, Sydney, Australia Algae and cyanobacteria, together with fungi, are largely responsible for biodeterioration of external surfaces of buildings. Traditional and molecular techniques were used to analyse cyanobacterial populations in bio¢lms on outer walls of historic buildings in Brazil. In mature bio¢lms, members of cyanobacterial sub-sections 1 and 2 were generally the major biomass; occasionally ¢lamentous genera from the botanical families Scytonemataceae, Microchaetaceae and Rivularaceae were dominant. Tradi-

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tional culture techniques, however, resulted in the isolation of mainly ¢lamentous organisms of sub-sections 3 and 4. Sub-section 1 organisms were found growing endolithically. PCR products were obtained from morphologically identi¢ed organisms using 16S rDNA primers, 271F (universal) and 408R (cyanobacteria speci¢c). The sequences of these DNA fragments were compared with known deposited sequences using the BLAST facility. Phylogenetic analysis indicates that all the cyanobacteria sequenced in this study corresponded to their appropriate group. However, sequence homologies with deposited sequences were, on the whole, low and the positions of the isolates in the dendrogram showed that they were deeplyrooted. For our analysis, we used only deposited sequences for which morphological identi¢cations were available. These were mainly aquatic cyanobacteria from environments very di¡erent from our dry, sun-exposed walls, where cells often had thick pigmented sheaths. One of our isolates was very closely related phylogenetically to a specimen obtained from a high, hot dessert (Arches National Park, Utah), where survival strategies are similar to those in the extreme environments of external walls. None of our isolates showed close similarity to extremophiles from very cold or hypothermal regions.

P4^76 CONSTRUCTION OF METAGENOMIC LIBRARIES OF UNCULTURED ELECTROCHEMICALLY ACTIVE MICROORGANIMS J. H. Back(1), H. Cho(1), I. S. Chang(2), B. H. Kim(2) and Y. S. Han(1) (1) Biomedical Research Center, Korea Institute of Science and Technology, P. O. Box 131, Cheongryang, Seoul, Korea; (2) Water Environment Research Center, Korea Institute of Science and Technology, P. O. Box 131, Cheongryang, Seoul, Korea We constructed metagenomic libraries that would be the basis to identify the genes related electron transfer. Metagenomic libraries are a powerful tool for exploring uncultured microorganisms. Traditional methods for culturing microorganism limit analysis to those that grow under laboratory condition. Given the profound utility and importance of uncultured electrochemically active microorganisms in water industries, methods are needed to explore their unique characteristics using the metagenome. This study is to develop a strategy for accessing the genetic diversity and identify the electron transfer mechanism of electrochemically active microorganisms. Also it is aiming to characterize electrochemically active microbes in a mediator-less microbial fuel cell. Metagenomic libraries will be a good resource for discovering new and useful genes among the uncultivated electrochemically active microorganisms. P4^77 A NEW CRYOPRESERVATION METHOD FOR FUNGI USING PERLITE L. Homolka, L. Lisa, I. Eichlerova and M. Tomsovsky Institute of Microbiology AS CR, Videnska 1083, 142 20 Prague 4, Czech Republic E/cient microbiological work requires a reliable source of cultures, which is ensured by its safe storage. Routine subculturing does not prevent genetic and physiological changes. Various storage methods have been developed in order to eliminate these disadvantages. The storage in liquid nitrogen is a safe and perspective method of a longterm maintenance of most fungal species, especially those not amenable to freeze-drying. A new alternative method using perlite as a particulate solid carrier in the growth medium with a cryoprotectant was tested for cryopreservation of several basidiomycete species from di¡erent genera (Armillaria, Pleurotus, Pluteus, Polyporus) which failed to survive or retain their properties in routinely used cry-

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opreservation procedures. Frozen basidiomycete strains were kept in cryovials submerged in liquid nitrogen and were after a 12-month storage thawed and checked for viability, purity and changes in growth, morphology and biochemical characteristics. All cultures survived the cryopreservation procedure and no negative e¡ects of cryopreservation by this method have been observed. As the method turned out to be successful, it was used for cryopreservation of further several hundreds di¡erent basidiomycete strains and also several micromycete strains. A more detailed study was performed with selected strains from the genus Trametes. This method, useful for both sporulating and non-sporulating fungal cultures, has several other important advantages: protection of cultures from contamination (reduced number of transfers) and the saving of time, work and room. This work was supported by grant no. 526/02/1216 from the Grant Agency of the Czech Republic and by Institutional Research Concept no. AV0Z5020903. P4^78 COMPLETE GENOMIC SEQUENCE OF THE LYTIC BACTERIOPHAGE P1203 OF CORYNEBACTERIUM GLUTAMICUM CCRC 18238 W. H. Hsu(1), T. Y. Pan(1), C. L. Chen(1), C. S. Sheu(2) and H. Y. Hu(3) (1) Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan ; (2) Vedan Enterprise Co., Taichung 433, Taiwan; (3) Department of Food Science and Nutrition, Hung Kuang Institute of Technology, Taichung 433, Taiwan Coryneform bacteria are widely used in the industrial production of amino acid and various other biotechnological application such as bioconversion. Bacteriophage infection leads to the lysis of cells and thereby interrupts the production of amino acid. Despite their economic importance, genomic sequences of corynebacteriophage are not available to provide valuable insight regarding the phage-host interaction and bacteriophage evolution which will undoubtedly be necessary for developing long-term phageresistant strains. Lytic corynebacteriophage P1201 was isolated from Corynebacterium glutamicum CCRC18238, a glutamic acid hyperproducing strain, that had become contaminated during an industrial fermentation. P1203 belongs to the Styloviridae family (B1 morphotype) with an isometric head of 52 nm wide and a long non-contractile and striated tail of 348 nm. It had a narrow host range and adsorption to its host was enhanced in the presence of Mg2+ ion. The nucleotide sequence of the 70-kb double stranded DNA genome was determined. A total of 44 open reading frames were predicted from the nucleotide sequence, and analysis of the corresponding proteins was

used to construct a functional map. The Sau3A1 digested genomic DNA was cloned into a promoter probed vector with lac Z as a reporter gene and expressed in Escherichia coli. Several clones with high speci¢c activity of L-galactosidase were found, indicating that these promoters were controlled by the host RNA polymerase. This is the ¢rst corynebacteriophage genome sequence to be reported. P4^79 MOLECULAR TOOLS APPLIED FOR DIVERSITY STUDIES OF CULTURABLE BACTERIAL POPULATIONS IN OIL-CONTAMINATED RHIZOSPHERE M. M. Jussila, L. Suominen and K. Lindstrom « Department of Applied Chemistry and Microbiology, Viikki Biocenter, P.O. Box 56, FIN-00014 University of Helsinki, Helsinki, Finland A culture collection of 52 indigenous meta-toluate tolerating bacteria isolated from oil-contaminated rhizosphere of Galega orientalis was characterized and identi¢ed by classical and molecular biological methods. The phylogenetic diversity was indicated by the presence of ¢ve major lineages of the Bacteria domain, while gram-positive bacteria was the most dominating group. A TOL plasmid-speci¢c xylE-PCR was developed in order to detect both active and potential degraders of m-toluate. The ability to degrade m-toluate in the presence of the gene xylE was detected only within the genus Pseudomonas. The genetic diversity was indicated by cluster analysis of the data, revealing 13 16S rDNA ribotypes and 23 (GTG)5-genotypes among various bacterial isolates ranging from similar strains to di¡erent genera. Generally, the 16S-ribotype and the (GTG)5-genotype corresponded to each other very well and grouped the strains at the species level. 16S rDNA PCR-RFLP ribotyping and (GTG)5-PCR genomic ¢ngerprinting methods combined with partial sequencing of 16S rRNA genes of representatives of the main clusters was used to construct a reference dendrogram in order later to rapidly group and search for new and interesting bacterial species from oil-contaminated rhizosphere.

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P4^80 THE INFLUENCE OF NATIVE, HYDROPHOBIZED AND DIMERIC FORM OF RIBONUCLEASE BACILLUS INTERMEDIUS ON THE LYSOSOME-PHAGOSOME FUSION IN PERITONEAL MACROPHAGES OF RAT N. V. Kalacheva, B. M. Kurinenko Department of Microbiology, Laboratory of Engineering Enzymology, Kazan State University, Kazan, 420008, Russia The e¡ect of native RNAse Bac. intermedius depends on the concentration: comparatively low concentrations (0.5 ^ 50 Wg ml-1 ) stimulate the phagosome-lysosome fusion whereas high concentrations (above 70 Wg ml-1 ) inhibit this process. RNAse modi¢ed by surfactant oxanol-KD6 and dimeric form RNAse possesses only the inhibitory e¡ect that appears at concentrations considerably lower than those of native enzyme. The stimulatory e¡ect of native RNAse and the inhibitory e¡ect of hydrophobized derivative do not depend on the catalytic activity. The results obtained allow us to conclude that the stimulatory e¡ect of RNAse is caused by weak irritation of the cytoplasm tic membrane leading to trigging o¡ cell activation mechanism. But the inhibitory e¡ect is connected with a stronger action of compounds on cellular membranes and it may be caused by the modi¢cation of properties both of phagosome and lysosome membranes. Thus, the common peculiarity of the e¡ect of native, hydrophobized and dimeric form of RNAse on phagocytic cells is the ability to inhibit the fusion between lysosomes and phagosomes. This property of RNAse and its modi¢ed derivatives may be used in anti-in£ammatory therapy and for decrease of cytotoxic reactions dependent on phagocytosis. P4^81 THE ROLE OF MICROBIAL AUTOREGULATORS FROM ALKYL HYDROXYBENZEN GROUP IN THE FORMATION OF HYPOMETABOLIC FORMS OF BACTERIA A. B. Margulis, O. N. Ilinskaya, A. I. Kolpakov Microbiology Department, Kazan State University, Kremlevskaya 18, Kazan, 420008, Russia It is well-known fact, that in response to unfavourable conditions the microorganisms can stop their division and begin to form dormancy (anabiotic) or hypometabolic forms with high resistance to environmental in£uences. The autoregulatory d1 factors of microorganisms may induce the transition of vegetative microbial cells into the

anabiotic state, when they acquire the higher resistance to unfavourable conditions. The present investigation showed that long incubation of gram-positive and gram-negative bacteria in distilled water maintained the viability of bacteria (Bacillus subtilis and Escherichia coli) and their colony formation ability during 12 weeks. Character of these processes under starvation conditions was similar both for gram-positive and gram-negative bacteria. However, after 4th week of incubation a part of bacteria in variant containing 50 Wg/ml of hexylresorcinol (the microbial growth regulator from alkyl hydroxybenzen group) transited into hypometabolic state. Hexylresorcinol induced the formation of hypometabolic forms of gram-negative bacteria after 8 weeks of starvation. It was registered by the decrease of number of colony forming units accompanied with some increase of bacterial suspension density. In Gram-positive bacteria hexylresorcinol induced the formation of new morphotype (small untypical colonies mostly consisted of the cells without endospores). It was found, that about 47% of Bacillus cells after 8 weeks of starvation with hexylresorcinol had no response on addition of exogenic nutrient substrate, i.e. were in hypometabolic state. P4^82 STUDIES ON STABILITY OF BREWERS' STRAINS OF SACCHAROMYCES CEREVIASIAE YEASTS DURING LONG-TERM PRESERVATION IN IAFB CULTURE COLLECTION A. Misiewicz, J. Czuba, I. Sikorska, B. Sieliwanowicz, K. Baranowski Institute of Agricultural and Food Biotechnology, Warsaw, Poland Brewer's strains of Saccharomyces cerevisiae yeasts, top and bottom fermentation are one of the group of microorganisms preserving in Culture Collection of Industrial Microorganisms. Stability of morphological, physiological and biotechnological properties after 50-years preservation were studied. Morphological properties were documented using digital image analysis (images from light and electron microscopy). Physiological features were checked by traditional tests as well as API test with computer software. Biotechnological properties of strains were checked using HPLC and GC techniques. All conducted tests con¢rm that preserving strains conserve their quality after 50 years.

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P4^83 SEROLOGICAL DIAGNOSIS OF LEISHMANIA DONOVANI WITH IIF METHOD L. Puzderliska, M. Dimitrova, L. Karakerezova

P4^85 EXPANDING THE BIODIVERSITY OF MICROORGANISMS IN JAPAN COLLECTION OF MICROORGANISMS M. Takashima, Y. Benno, and T. Kudo

Department of Preventative Health, Stip, Macedonia Leishmania donovani is a provocator of a very serious transmissible parasitic disease also called visceral leishmaniosis. This parasite has a container (dog), a transmitter Phlebotomus papatassi and ¢nal consument sick person. In our examination we would like to present the presence and attendance of local visceral leishmaniosis on the territory of Republic of Macedonia. The total number of examined sera was 74, 67 of which were statistically elaborated, because up to 74 with repeated positive controls. From 67 examined sera with IIF, 19 sera were with new infection of Leishmania donovani or 28.3% and 71.7 were negative. Conclusion : In the presence of prolonged febrile conditions that don't react on antibiotic therapy to take into consideration for possible parasitic with Leishmania donovani. P4^84 RUSSIAN NATIONAL COLLECTION OF INDUSTRIAL MICROORGANISMS ^ MICROBIAL GENETIC RESOURCES FOR BIOTECHNOLOGY S. P. Sineoky Russian National Collection of Industrial Microorganisms, FGUP GosNII genetika, 1-st Dorozhny proezd 1, 113545, Moscow, Russian Federation Russian National Collection of Industrial Microorganisms (VKPM) is a largest Russian service collection for nonpathogenic microbial strains. Over 15 000 strain are maintained for application in fundamental and applied investigations. At the present time, VKPM is functioned as National Bioresource center and is involved in the decision of various problems essential for successful development of Biotechnology. Among such problems ^ biosafety for natural and genetically modi¢ed strains, developing normative base for strains deposition and distribution, protection of intellectual rights in biotechnology and methodology of the strains identi¢cation using PCR technique. VKPM activity in these areas will be presented in the communication. Japan Collection of Microorganisms, Bioscience Technology Center, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama, Japan To keep pace with the progress in research on microbial diversity, the sphere of microorganisms that must be managed in culture collection is expanding. The Japan Collection of Microorganisms, which was established in 1980, works to contribute to domestic, regional, and global improvements in the conservation of microbiological resources in cooperation with other culture collections and institutions, and to supply authentic microorganisms to researchers in the ¢elds of life sciences and biotechnology. In answer to user requests, JCM has developed an e/cient anaerobic chamber to cultivate the extremely oxygen-sensitive (EOS) anaerobes, and is maintaining these microorganisms including the genera Treponema, Prevotella and Tannerella. So-called extremophiles such as methanogens, hyperthermophiles, and subterraneous and piezophilic bacteria are also introduced. As of the end of March 2002, a total of 10,354 strains (6,589 bacteria, 205 archaea, 3,539 ¢lamentous fungi and yeasts, and 21 others) were listed in JCM. Study is continuing on the unknown, that is to say, the undescribed, not yet isolated, or yet-to-be cultured microorganisms using molecular phylogenetic techniques in addition to the usually applied morphological, physiological, biochemical and chemotaxonomic methods. Also, identi¢cation of microorganisms that have been in the collection for some time, of which the taxonomic position at the species level has not been determined, is being made and newly collected strains examined.

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P4^86 A NOVEL APPROACH FOR MOLECULAR CHARACTERIZATION OF BACTERIAL POPULATIONS FROM NATURAL STARTER CULTURES E. Soldati(1), V. Corich(1), G. Poletto(1), C. Andrighetto(2), A. Lombardi(2) and A. Giacomini(1) ' (1) Dipartimento di Biotecnologie Agrarie, Universita di ' , I-35020 Legnaro (PD), Italy ; Padova, viale dell'Universita ' (2) Veneto Agricoltura ^ Istituto per la Qualita e le Tecnologie Agroalimentari, via San Gaetano 74, I-36016 Thiene (VI), Italy Natural starter cultures used in cheese manufacturing are complex bacterial communities with unknown composition in terms of species and strains, whose identi¢cation, possibly directly within the matrix, has great technological importance. A natural whey starter culture from Grana Padano cheese production was used to develop such an identi¢cation system. A preliminary characterization at species level by ARDRA on the culturable fraction and by TGGE on 16SrDNA target sequence from DNA extracted from whey, revealed a population composed by Lactobacillus helveticus (90%) and Lactobacillus delbrueckii subsp. lactis (10%). A characterization at strain level was carried out by PCR-based techniques, one exploiting the presence of particular DNA sequences to originate genetic polymorphism (RAPD-PCR with primers M13 and D8635) and a second newly developed approach (SauPCR) which uses a primer based on the restriction enzyme Sau3AI sequence to amplify genomic DNA digested with the same enzyme. Analysis of electrophoretic pro¢les evidenced two main groups of isolates including 12 minor subgroups. Ten representatives from di¡erent subgroups were screened for the presence of useful polymorphic DNA sequences. Published sequences for aminopeptidases and proteases highly speci¢c for L. helveticus were examined, along with highly strain-conserved fragments from RAPD analyses. The aim of the research was to ¢nd out sequences either highly (for identi¢cation at species level) or poorly (identi¢cation at strain level) conserved among the isolates. Data presented report the results of such analysis.

P4^87 INVESTIGATION OF THE YEAST MICROFLORA OF ``TOKAJ ESSENCE'' H. Csoma, M. Sipiczki Department of Genetics, University of Debrecen, Debrecen, Hungary Tokaj is one of the major European wine regions, where botrytized wines can be produced. Due to the unique climatic conditions of the region, the grapes infected by Botrytis cinerea develop what is called noble rot. The noblerotted berries can be harvested selectively from the infected bunches and stored in containers where the weight of the grapes presses out some of the juice. This juice that drains away freely from the stored botrytized grapes is called ``essence''. The collected essence is placed in wooden barrels for fermentation and maturation (usually at temperatures below 15C). Because of the low temperature and the very high sugar content of the juice, occasionally over 50%, fermentation occurs very slowly and often produces little more than 5 to 7 % alcohol before termination. To investigate the micro£ora, we took samples from fermenting essence in four wineries and isolated yeasts from the samples. The molecular analysis of the isolates (electrophoretic karyotyping, PCR-RFLP and sequencing of the ITS1-5S-ITS2 region of rDNA) revealed a high degree of heterogeneity in the fermenting populations, but most isolates proved to be strains of Zygosaccharomyces, Candida stellata, Torulaspora and Saccharomyces sensu stricto species. The composition of the populations changed with time, usually in favour of Saccharomyces. Di¡erences were also detected between samples taken from the bottom and the surface of the fermenting essence. P4^88 Withdrawn.

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P4^89 IDENTIFICATION OF CULTIVABLE BACTERIA FROM ACTIVATED CARBON FILTERS USED FOR WATER TREATMENT ¤ > A. Magic-Knezev, B. Wullings and D. van der Kooij Kiwa Research and Consultancy, P.O. Box 1072, 3430 BB Nieuwegein, The Netherlands Biological activated carbon ¢ltration (BACF) is frequently used in water treatment for the removal of undesirable organic compounds. Optimisation of the biological activity during BACF may reduce both the high costs and environmental burden associated with thermal regeneration of activated carbon. Bacteria were isolated from BACF ¢lters at several Dutch water treatment plants as part of a study aiming at acquiring information about the nature of the bacterial activity in BACF. Microorganisms were removed from the surface of activated carbon by de¢ned ultrasonic treatment and cultured at solid and liquid media. Although the cultivability was higher in the liquid media, less than 1% of bacteria was cultivated. A total of 260 obtained isolates were grouped by the analysis of repetitive extragenic palindromic DNA (REP) patterns in 12 major clusters. From 60 representative isolates, for which the sequence of 16S rDNA has been determined, 28 were identi¢ed as representatives of the family of Comamondacaea. Within this group 19 isolates were closely related (99%) with uncultured bacterial clones from polluted groundwater or soil. Smaller numbers of isolates were either related to the genus Hydrogenophaga or Sphingomonas, or matched with the sequence of MTBE degrading bacterium. Other isolates were related to unidenti¢ed bacterial clones. These data show that even with molecular techniques it is di/cult to identify cultivable bacteria, which in turn represent less than 1% of the total number of bacteria in BAC ¢lters. Further studies with selected pure cultures will be conducted to elucidate essential physiological properties of these bacteria. P4^90 MOLECULAR CHARACTERIZATION OF LACTIC ACID BACTERIA INVOLVED IN NATURAL FERMENTATION OF TURNIP M. Maifreni, M. Marino and G. Rondinini ' Dipartimento di Scienze degli Alimenti, Universita degli Studi di Udine, via Marangoni 97, 33100 Udine, Italy Lactic acid fermentation, an ancient preservation method, is nowadays especially favoured as a ``natural'' process to increase the shelf-life of various products (dairy, meat,

vegetable). Most vegetables can be lactic acid-fermented, so far cucumber, cabbage and olives are the only vegetables that are fermented in large volumes for human consumption. However, the are agricultural, nutritional, sensory and preservation reasons for evaluating lactic acid fermentation as a potential process for making new products from other vegetables. Lactic acid bacteria are the main responsible for the fermentation of vegetable such as cabbage, carrots, beets, but the indigenous LAB £ora varies as a function of the quality of the raw material, tempertaure and harvesting conditions. Spontaneus fermentation thus leads to variations in the sensory properties of the products. Brovada is an ancient traditional product from the North-East Italian region, included in the list of Italian typical products, which is obtained making the turnips (Brassica rapa) ferment naturally. At present, there is few informations on the spectrum of microrganisms associated with the fermentation of Brassica rapa and the development of £avour compounds during the process. These knowledge are essential for the development of the product with improved quality. The present study reports on the characterization of lactic acid bacteria associated with the natural fermentation of Brovada using PCR-based molecular techniques. Application of RAPD and REP-PCR allowed the grouping of lactic acid bacteria population in Lactobacillus spp. (L. hilgardii, L. plantarum, L. coryniformis) and Pediococcus parvulus. P4^91 BIODIVERSITY AND TECHNOLOGICAL PROPERTIES OF LEUCONOSTOC SP. ISOLATED FROM TRADITIONAL DAIRY PRODUCTS B. Martinez, J. I. Sanchez, T. Delgado and A. Rodriguez Instituto de Productos Lacteos de Asturias (IPLA ^ CSIC), Crta. In¢esto s/n, 33300 Villaviciosa (Asturias), Spain Leuconostoc sp. are commonly used in mesophilic starters for the production of fresh or soft cheeses and fermented milks due to their ability to produce £avours and to modify the appearance and texture of the product. Since these microorganisms are relatively inert in milk they are always cultured together with an acid producing Lactococcus lactis strain. The aim of this work was to determine the technological ability of eleven wild strains of Leuconostoc isolated from traditional dairy products and to select those which present the best properties in order to include them in mixed starter cultures. Molecular characterization of the strains was initially carried out by Pulse Field Gel Electrophoresis (PFGE) and RAPD-PCR techniques. Production of dextran, organic acids and volatile compounds were studied in order to check the texturing and £avouring properties of the strains. The resistance to di¡erent param-

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eters such as nisin, acid and salt concentration as well as the viability after freezing was also determined. Generally, a large variability was found among the isolated strains, mainly regarding the above mentioned resistances. Finally, analyses of the metabolite pro¢le and biomass production in batch co-cultures of Leuconostoc and L. lactis are in progress to de¢ne the right ratio for large-scale mixed starter production. The obtained results clearly showed that natural environment provides a good source of strains with appropriate technological properties. P4^92 INFLUENCE OF BACTERIA IN RIPENING ENVIRONMENTALS OF PECORINO FILIANO CHEESE AND STUDY OF BACTERIAL EVOLUTION DURING RIPENING D. Messina, T. Lechiancole and G. Salzano Dept. of Biology, Univerity of Basilicata, Campus Macchia Romana, 85100 Potenza, Italy It is well know that the Pecorino is a hard cheese manufactured in di¡erent areas of Italy. Pecorino Filiano is a traditional hard cheese produced in Basilicata (Southern Italy) without addition of selected or natural starter culture. The fermentation and ripening processes of this cheese are entirely performed by the indigenous micro£ora present in the milk. Ripening of this cheese occurs in the cave, this is special environment at controlled temperature and humidity. Air microrganisms can represent surface cheeses micro£ora and they have a strong impact on the appearance, £avour and texture development of the cheeses, and usually lead to shorter ripening periods. It is well known that bacteria play an important role during the ripening of cheeses. In this work two Pecorino Filiano cheeses, one is manufactured by using raw whole ewe's milk with addition of 10% raw whole goat's milk (Pecorino A); the other is manufactured by using only raw whole ewe's milk (Pecorino B), ripened into a cold store (reference frame) and in a cave were studied. RAPD-PCR is an important tool for a rapid and reliable screening of a lot of strains. This molecular technique was applied on all isolated bacteria to evaluate interaction between air bacteria and surface cheese bacteria and also to study bacterial evolution of these two cheeses. In Pecorino Filiano cheese interaction between air bacteria and surface cheeses bacteria occur, especially during ripening in the cave. Moreover during ripening of two cheeses there is a very strong heterogeneity of bacterial micro£ora.

P4^93 DIFFERENTIATION OF YEASTS ISOLATED FROM ARTISANAL EWE'S CHEESES USING RAPD-PCR ANALYSIS V. Mossa, M. E. Fadda, M. Deplano and S. Cosentino Department of Experimental Biology, section of Hygiene, University of Cagliari, S.S 554, Km 4,500, 09042 Monserrato (CA), Italy The yeasts, with their metabolic properties, are increasingly considered as important agents in the maturation process of several cheeses. In a previous study we have shown that yeast population from artisanal Sardinian ewe's cheese, was mainly represented by Debaryomyces hansenii, Kluyveromyces lactis, Candida lambica, Candida zeylanoides and Geotrichum candidum species. In the present work RAPD-PCR with the core sequence of the phage M13 as primer was used for typing yeast strains belonging to this predominant species. DNA banding patterns were analysed using the Gel Compar software package, version 2.5 (Applied Maths, Kortrijk, Belgium). Similarities among isolates were estimated using the Pearson coe/cient and clustering was based on the UPGMA method. At a similarity level of 50%, the ¢ve prevalent species were well separated in six clusters, one of which was represented by a single strain. RAPD-PCR analysis showed good capacity to produce species-speci¢c patterns, in fact all the isolates were grouped with the type strains of the species considered, thus con¢rming the results of previous classi¢cation based on phenotypic traits. This analysis have also showed a certain degree of genetic polymorphism among the strains: in fact a similarity of about 55% for the strains of the species C. zeylanoides, K. lactis and C. lambica, and 65% for D. hansenii and G. candidum was observed. Our results indicate the usefulness of RAPD-PCR analysis for di¡erentiation of yeast species. This work was in part supported by a grant from MURST, Plan ``Agroalimentary products: dairy products'', Cluster 08B, Project n.7.

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P4^94 DIVERSITY IN THE TEHNOLOGICAL PROPERTIES OF YOGHURT BACTERIA ISOLATED FROM HOME MADE YOGHURT K. Pashova-Baltova(1), M. Michailova(1), M. Fukui(2) (1) ELBY Bulgaricum, Research Department, 12A Malashevska Str., 1202 So¢a, Bulgaria ; (2) Japan Internetional Cooperaton Agency, JICA The characterization of monocultures is the ¢rst step in the development of new successful starter culture for fermented products. Bulgaria is famous with its traditional product ^ Bulgarian yoghurt. The purpose of this study was to isolate lactic acid bacteria from home made yoghurt from ecological areas and to perform analysis of important technological properties of the collected monocultures. 73 strains L. bulgaricus and 114 strains S. thermophilus were isolated from home made yoghurt. After 10 times of reinoculation strains from both species were tested for: fermentative activity after incubation at 16hr and 40hr (at 370C); osmotic tolerance in 15% sugar syrup; antibiotic tolerance against traces of penicillin, viability of strains during preservation for 20 days at 50C; kinematic and visual viscosity. The results showed that the technological properties of the collected strains of yoghurt bacteria are quite variable. The created database permitted classi¢cation of the tested strains into groups with high, medium and low values of the analyzed technological parameters. The large variations in the properties of the tested strains presents a good potential for the development of new starters for fermented milk products with desired characteristics. P4^95 PHENOTYPIC AND GENETIC VARIABILITY OF KLOECKERA APICULATA STRAINS OF WINE ORIGIN P. Romano, C. Fiore, A. Capece, M. Caruso and M. Paraggio Dipartimento di Biologia, Difesa e Biotecnologie Agro' Forestali, Universita degli Studi della Basilicata, Campus Macchia Romana, 85100 Potenza, Italy The species of the genera Hanseniaspora (and its anamorph form Kloeckera) are frequently isolated from various source as soil, fruits and insects, as well as from fermented foods and beverages. As predominant inhabitants on the surface of grape berries and in early must fermentation, apiculate yeasts perform biochemical activities, producing some important reactions, thus in£uencing pos-

itively and/or negatively the ¢nal wine quality. During the years, we have collected a great number of apiculate yeasts and in this paper we report a study on about two hundreds K. apiculata wine strains. The strains were tested for enzymatic activities of interest in winemaking, such as Lglucosidase, L-xylosidase and protease, and for the production of fermentation metabolites, a¡ecting wine £avour, such as higher alcohols, carbonyl compounds and acetic acid. A signi¢cant variability was found, with the individuation of di¡erent phenotypes. Fifty strains, as representatives of the di¡erent phenotypes, were further analysed for genetic polymorphism, by using RAPD analysis and ARDRA technique. The RAPD analysis showed a low degree of similarity between the strains, whereas no di¡erences were recorded by ARDRA technique, con¢rming that strains belonged to the species K. apiculata. Our results emphasized the existence of a considerable biodiversity among wine apiculate strains and this variability is of technological interest as these yeasts, contrary to a general opinion that they are wine spoilage micro-organisms, constitute £avour potential producers. In this context, it becomes advantageous to select, for each wine, also suitable strains of K. apiculata to use in mixed or sequential cultures with Saccharomyces cerevisiae. P4^96 HIGH THROUGHPUT SCREENING ON FLAVOUR FORMATION BY BACTERIAL CULTURES TO MAP BIO-DIVERSITY AMONG LACTIC ACID BACTERIA B. A. Smit, W. J. M. Engels, J. T. M. Wouters and G. Smit NIZO Food Research, Department of Flavour, Nutrition and Ingredients, PO-box 20, 6710 BA Ede, The Netherlands Flavour is a very important characteristic of foods and drinks. In fermented products like cheeses, sausages and alcoholic beverages formation of odour and taste active compounds by micro-organisms contributes considerably to this £avour. The process of £avour formation is investigated intensively to improve production processes, develop new varieties and accelerate £avour formation while maintaining balanced product characteristics. Flavour formation can be enhanced in several ways, e.g. by the use of alternative (strains of) starter micro-organisms, by the use of adjunct cultures (working additional, or synergetic), by changing of process parameters, or by modi¢cation of strains. In practice, genetically modi¢ed strains are not used in food applications, thus exploration of the natural bio-diversity among bacteria is a powerful alternative. To explore this biodiversity new, fast and £exible screening methods on functional characteristics like production of £avour compounds, relevant enzyme activities, and so on are necessary. In or lab, automated 96-well enzyme and bioassays, in combination with direct mass- spectrometric

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methods for £avour analysis have been developed. These techniques enable fast screening on various enzyme activities in relation to overall £avour production by (a mixture of) strains. An example is the screening on branched chain aldehyde production by lactic acid bacteria. This screening revealed large di¡erences in the actual production and the levels enzyme activities involved. With this knowledge we can select lactic acid bacteria for production of high aroma formation levels. P4^97 CORK STOPPERS INDUSTRY: PENICILLIUM DIVERSITY COLONIZING CORK SLABS G. A. M. Soares(1), A. C. Oliveira(1), M. C. Basilio(1), R. Tenreiro(2), M. V. San Romao(1,3) < ¤ (1) Instituto de Biologia Experimental e Tecnologica/Insti¤mica e Biologica-Universidade Nova ¤ tuto de Tecnologia Qu| de Lisboa, Apt. 12, 2781-901 Oeiras, Portugal; (2) Depar" tamento de Biologia Vegetal-Faculdade de Ciencia da Universidade de Lisboa, Lisboa, Portugal; (3) Estacao Viti°< ¤ vin|cola Nacional, 2565-191 Dois Portos, Portugal Cork stoppers are made from the bark of the cork oak tree, Quercus suber L., widely found in Portugal, Spain and in other Mediterranean countries. Nevertheless Portugal is the world's largest producer of cork stoppers for the wine industry. The manufacturing process of cork stoppers includes a stabilization period during which moulds grow on the cork slabs. This process, in which cork slabs are traditionally considered to be of good quality for stopper manufacturing when they are completely covered with moulds, has been used for several decades. A study intending to obtain information on the mycobiota associated with the Portuguese cork throughout the manufacturing process of stoppers was carried out. Chrysonilia sitophila showed to be the most dominant fungus, followed by a signi¢cant occurrence of the genus Penicillium. This work concerns the identi¢cation at species level of the isolates belonging to the genus Penicillium. The strains were grown for 7 days in CYA at 5, 25 and 37C, in MEA at 25C and in 25% glycerol nitrate agar at 25C. Fungal DNA was also isolated and molecular methods were used to con¢rm the identities of some strains. The ribosomal DNA region containing spacers ITS1, 5.8S and ITS2 was PCR-ampli¢ed and the products analysed. The restriction patterns of the PCR products were con¢rmed by using reference strains. The results show the diversity of mould species colonizing the cork slabs and the use of restriction length polymorphism of the rDNA as a potential tool to con¢rm the identity of Penicillium species. < This work was partially supported by amorim & irmaos. Sta. Maria de Lamas. Portugal.

P4^98 RAPID IDENTIFICATION AND TYPING OF LACTOBACILLUS PLANTARUM STRAINS IN DAIRY PRODUCTS BY USING POLYMERASE CHAIN REACTION AND RANDOMLY AMPLIFIED POLYMORPHIC DNA M. Oneca, F. Feutry, M. Ortigosa, A. Irigoyen and P. Torre ¤ ¤ Laboratorio de Lactolog|a, Area de Nutricion y Bromato¤a, Dpto. C.C. del Medio Natural, Universidad Publica ¤ log| ¤a s/n, 31006 Pamplona, Navde Navarra, Campus Arrosad| arra, Spain Roncal Cheese is a ripened uncooked cheese made from ¤ raw eweas milk in the Autonomous Community of Navarra (Spain) and it has the Guarantee of Protected Origin. The dairies that manufactured this cheese take the milk from the three main cattle raising areas of Navarra. Recent research carried out in this cheese show that di¡erent species of Lactobacilli are present in high number (108 CFU/g), although they are not added to the cheese in the starters. One of the main species found is Lb. plantarum. This species included in the group of Non Starter Lactic Acid Bacteria (NSLAB) contribute to the ¢nal properties of this cheese. The aim of this study is the identi¢cation and typing of Lb. plantarum strains in milk and cheese with the Guarantee of Protected Origin and to search if the strains present in cheeses come from the original milk. The PCR speci¢c reaction to Lb. plantarum was performed using the speci¢c primers Lb Pl1/Lb Pl2 and the semi-universal primers Lb1/Lb2. The RAPD reactions used the primers OPA-3 and P1. Six clusters at the similarity level of 50% were obtained. Comparing the strains, it was observed that the main part of the strains found in cheese didn't come from the original milk. P4^99 EXPLORING MICROBIAL DIVERSITY IN BALTIC SEA SEDIMENTS A. Edlund(1), T. Soule(2) and J. K. Jansson(3) (1) Sodertorn University College, Department of Natural « « Sciences, 141 89 Huddinge, Sweden; (2) University of Idaho, Department of Computer Science, Janssen Engineering Building, Moscow, ID 83844-1010, USA; (3) Swedish University of Agricultural sciences, Department of Microbiology, Box 7025, 750 07 Uppsala, Sweden The aim of this study was to conduct a survey of the dominant prokaryotic species in Baltic Sea sediment, a previously unexplored environment. Sampling took place

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during September 2002 and started in the inner parts of the southern Stockholm archipelago. Triplicate sediment cores were collected from areas which were highly polluted with heavy metals and PCBs. Samples were also collected from eutrophied areas which are strongly a¡ected by the release of sewage treatment e¥uents. References were collected from an area with relatively low levels of pollutants. Total DNA was extracted from the sediment cores at different depths throughout the pro¢les. Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis was performed using primers speci¢c for eubacterial or archael 16S rRNA genes and the products were digested with three restriction enzymes. To visualise the changes in community structures between the sampling areas and between di¡erent depths, relative abundance values and speciesspeci¢c base pair values were correlated using non-parametric multivariate analyses. Principal component analyses were performed to test correlations between environmental variables such as pollutant levels, depths and community structures. Our results suggest that species abundance changes according to depths and according to environmental parameters. For bacteria, the species abundance decreased in the areas with heavy metal pollutants when compared to the reference site. The T-RFLP results were compared to plate counting on di¡erent media. Putative identities of individual terminal restriction fragments (TRFs) were estimated using the Ribosomal Data Base Project II and by comparison to TRFs of isolates. P4^100 MICROBIAL ECOLOGY OF TNT-CONTAMINATED SOILS AND ANAEROBIC TNT BIODEGRADATION PROCESSES L. Eyers, B. Stenuit, S. El Fantroussi and S. N. Agathos Unit of Bioengineering (GEBI), Catholic University of Louvain, Place Croix du Sud 2/19, B-1348 Louvain-la-Neuve, Belgium 2,4,6-trinitrotoluene (TNT) is one of the most common explosives. In spite of its known toxicity and mutagenicity for many organisms, soils and groundwater are still frequently contaminated at manufacturing, disposal and TNT-destruction sites. Fifteen soil samples collected from two TNT-destruction ¢elds were investigated for their concentrations of TNT and its derivatives. They contained TNT at various concentrations, ranging from 4 to 114 g TNT/kg soil. Various metabolites were also detected, including 2,4,6-trinitrobenzaldehyde, 4-amino-2,6-dinitrotoluene, 2-amino-4,6-dinitrotoluene and 2,4-dinitrotoluene. Microbial communities of these soil samples were characterized by means of a 16S rRNA PCR-DGGE technique using bacterial universal primers. The DGGE patterns of these soil communities were compared with non-

contaminated soils found at the two TNT-destruction sites. Non-polluted soils revealed complex ¢ngerprints of microorganisms while the contaminated samples showed the presence of dominant bands, indicating that a strong selection had occurred. Interestingly, some of these contaminated soils contained a dominant band matching the one of the previously isolated TNT-degrading strain Pseudomonas sp. JLR 11 (Esteve-Nunez A. and J. L. Ramos, Env. Sci. Technol. 1998, 32, 3802-3808). The biodegradation capacities of these polluted soils were evaluated by enrichment cultures with TNT as sole N-source under anoxic conditions. Nitrite release together with growth of the consortia suggest that anoxic denitration activities occurred. TNT biodegradation activities under Fe-reducing, sulphate reducing and methanogenic conditions are currently under investigation. P4^101 DIVERSITY OF BENZENE-DEGRADING BACTERIA IN A CONTAMINATED SANDSTONE AQUIFER A. Fahy(1), A. S. Ball(1), T. J. McGenity(1), A. J. Hart(2), K. N. Timmis(1) (1) Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, UK; (2) Environment Agency, Solihull B92 7HX, UK The diversity of aerobic benzene-degrading bacteria from a contaminated sandstone aquifer was evaluated in a microcosm experiment. The aquifer, situated below a large chemical plant, is the focus of research on natural attenuation. Contaminants include BTEX (benzene, toluene, ethylbenzene, xylene), polyaromatic hydrocarbons and chlorinated aliphatic hydrocarbons. Benzene is the most abundant and persistent pollutant, and groundwater samples were collected from the four most contaminated monitoring wells. Degradation occurred under di¡erent conditions in three groundwater samples: it was stimulated by the addition of nitrate in one sample, both phosphate and nitrate in another, but appeared to be inhibited by nitrate in a third sample. No degradation occurred in a fourth sample.Microbial community dynamics of the groundwater in which benzene degradation occurred were monitored using T-RFLP (terminal restriction fragment length polymorphism), and compared to the communities found in situ. The inhibition or stimulation of benzene degradation was re£ected in the community structures. The main bacterial populations were identi¢ed by cloning and partial sequencing of 16S rDNA. One community was dominated by beta Proteobacteria (90% of the clone library), another by gamma Proteobacteria (54%); the third comprised 47% beta Proteobacteria and 43% Firmicutes. Four of the dominant benzene-degrading bacteria were isolated on minimal medium, and were most closely related to the

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following organisms : Hydrogenophaga £ava (98.5% identity), Pseudomonas anguilliseptica (97.9%), and two di¡erent Rhodococcus erythropolis strains (100 and 99.8%). P4^102 DIVERSITY OF METHANOGEN DRAINED FINNISH PEATLAND ARCHAEA IN

P. E. Galand(1), H. Juottonen(1), H. Fritze(2) and K. Yrjala(1) (1) Department of Biosciences, Division of general microbiology, P.O. Box 56, FIN-00014 University of Helsinki, Finland; (2) Finnish Forest Research Institute, Vantaa Research Center, P.O. BOX 18, FIN-01301 Vantaa, Finland. Methane (CH4) is an e¡ective greenhouse gas produced during anaerobic microbial processes by methanogen Archaeae. Wetlands (bogs, fens, etc) are the main source of natural CH4 emission. Possible changes in CH4 production in these habitats will have an impact on the global climate change. Peatlands originally constituted a third of the land area in Finland, before large areas were drained for forestry plantations in the 1960's-70's. Ash fertilization in peat lands has been found to promote tree growth and has been used in Finland to enhance reforestation. Fertilisation of peatland is been thought to decrease CH4 emissions but its e¡ects on the community of methane producers have never been studied. We report results from a study on the impact of ash fertilization on the methanogen Archaea community in Finnish peatland soil. Possible changes in the community structure were examined in relation to the potential CH4 production of the soil. The methanogen diversity was studied by using molecular methods (PCR-DGGE, cloning and sequencing) targeting the functional methyl coenzyme M reductase gene. P4^103 DIVERSITY OF ACTINOMYCETES FROM STONE SURFACES ISOLATED

ily Nocardiaceae has previously been described from deserts and rock surfaces highly exposed to UV radiation [15]. We have attempted the use of di¡erent stones as an alternative source for the isolation of novel microorganisms to be tested in natural products screening programs. We have studied the diversity of the actinomycetes population isolated from 5 di¡erent samples of stones, including limestone, slate and granite rocks collected in Mallorca island and in the Madrid mountains (Spain). Samples were treated according to two general isolation methods coupled to eight selective isolation media and including the use of di¡erent bacterial growth inhibitors. To assess the diversity of the microbial population obtained following the di¡erent procedures, the 360 isolates were initially identi¢ed to the genus level on the basis of their micromorphology. Among these strains were identi¢ed representatives of the genus Streptomyces, as well as members of the families Nocardiaceae, Geodermatophilaceae, Pseudonocardiaceae and Micromonosporaceae. As many as 270 Streptomyces isolates were further characterized chemotaxonomically on their fatty acid composition. In this study we evaluate the diversity of the actinomycete isolates obtained from stones according to the di¡erent nature of the rock and their taxonomic position. [1] Eppard et al. (1996) Arch. Microbiol 166, 12-22. [2] Urzi et al. (2001) Environmental Microbiology 3, 471479. [3] Groth et al. (1999) J. Microbiol. Met. 36, 115122. [4] Schumann et al. (1997) Int J Syst Bacteriol 47, 278-83. [5] Salazar et al (2003). Accompanying poster at 1st Congress of European Microbiologists. P4^104 OCCURRENCE OF OLIGONITROPHILIC YEASTS IN ACID FOREST SOILS G. GorzaTa and S. Russel Department of Soil Environmental Sciences, Division of Agricultural Microbiology, Warsaw Agricultural University, 02-528 Warsaw, ul. Rakowiecka 26/30, Poland It is estimated that soil microorganisms constitute 25% of the total biomass on earth. Despite their wide occurrence and extensive role in soils, only about 40,000 species of soil microorganisms have been cultured and identi¢ed. Yeasts are one of the most important groups of soil microorganisms, although their ecological role is still insu/ciently recognized. The term "oligonitrophilic yeasts'' designates a physiological group of yeasts able to grow in an environment containing trace amounts of nitrogen. The yeasts of genera Lipomyces are the best-known representatives of oligonitrophilic organisms. This work studied the occurrence and distribution of oligonitrophilic yeasts in selected types of acid, forest soils collected in White Forest about 70 km north-east Warsaw. Sample were tak-

¤ I. Gonzalez, M. I. Cercenado, M. J. Gregorio, A. Villalba, and O. Genilloud ¤ ¤ Centro de Investigacion Basica de Espa·a (CIBE), Merck Research Laboratories, Merck Sharp and Dohme de Es¤ pa·a, S. A. Josefa Valcarcel 38, 28027 Madrid, Spain It is well known that actinomycetes are one of the major communities of the microbial population present in soil but they can also be isolated from a variety of sources such as microbial mats, marine sediments, seaweeds, lichens or stone surfaces among others. The isolation of actinomycetes of the genus Geodermatophilus and the fam-

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en ¢ve-times within vegetation season from six soil types: pseudogley soil, rusty soil, muck soil, gley-podzol soil, gley soil and black earth. The total number of oligonitrophilic yeasts was counted by plate method using nitrogen-free medium amended with cycloheximide and streptomycin.The plate cultures were incubated for 7 days at 280C, then 7 days at room temperature. Yeast colonies were observed under the microscope, counted and isolated for further characterization. Oligonitrophilic yeasts are concentrated near the surface of the pro¢le. The highest number of yeasts (respectively 312 and 251 CFU per g od d.m. of soil) was observed in AM horizon of muck soil and in A horizon of pseudogley soil. In all soils, the highest amounts of yeasts were found in the upper layers, with the exception of litter layers where the content of yeasts ranged from 0 CFU (genetic horizon Ofh of gley soil) to just 84 CFU (genetic horizon Ofh of gley-podzol soil) per g of d.m. of litter. The concentration of oligonitrophilic yeasts in upper horizons of soil pro¢les is to a large extent the result of the greater abundance of readily available organic matter. Below 50 cm, yeasts occur sporadically. 20 pure strains of nitrophilic yeasts were isolated and partially characterized. All strains probably belong to Lipomyces, with the exception of 1 strain that intensively produces pseudomycelium. P4^105 DIVERSITY OF BACTERIA AND ARCHAEA IN BALTIC SEA SEDIMENT fi F. Hardeman and S. Sjoling « Section for Natural Sciences, Sodertorns hogskola, 141 89 « « « Huddinge, Sweden Marine prokaryotes play a signi¢cant role in remineralization of organic matter within the aquatic ecosystem and are vitally important for the biogeochemical cycling and eventual degradation of pollutants in the Baltic Sea sediments. However, the role of bacterial and archaeal diversity in the Baltic Sea benthic ecosystems is still a ``black box'' as no empirical data are available. Therefore, 16S rDNA analysis was used to assess the prokaryotic diversity and community structure of Baltic Sea sediment from the Asko archipelgo. Ampli¢ed rDNA restriction analysis « (ARDRA) was performed on 16S rDNA clone libraries ( s 103 unique clones/library) of di¡erent sediment depth. Results indicate a high bacterial diversity of 16S rDNA sequences. Approximately 80 % of the screened bacterial clones showed unique OTUs (restriction patterns). The diversity of Archaea was lower. Seasonal and spatial distribution is discussed.

P4^106 METHANOTROPHIC BACTERIA IN BOREAL FOREST SOIL : LONG-TERM EFFECTS OF PRESCRIBED BURNING AND ASH FERTILIZATION K. Jaatinen(1), C. Knief(2), P. Dun¢eld(2), K. Yrjala(3) « « and H. Fritze(1) (1) Finnish Forest Research Institute, Vantaa Research Centre, P.O. Box 18, FIN-01301 Vantaa, Finland; (2) Max-Planck-Institut fur terrestrische Mikrobiologie, Karl« von-Frisch-Strasse, D-35043 Marburg, Germany; (3) Department of Biosciences, Division of General Microbiology, P.O. Box 56, University of Helsinki, FIN-00014 Helsinki, Finland Methane (CH4) is an e¡ective greenhouse gas and its concentration in the atmosphere has been increasing at a rate of 1% per year. Forest soils are a major terrestrial sink for atmospheric methane. Methane oxidizing bacteria (MOB) are responsible for the atmospheric methane consumption. MOB can use methane as a sole source of carbon and energy. Little is known, however, about the high-a/nity MOB inhabiting soil, and factors that in£uence their activity and diversity in boreal forests. Prescribed burning and wood ash fertilization are common forestry practises used for improving the acid neutralization capacity of naturally acidic soils. These practises have been shown to a¡ect soil microbial community structure and activity. Ash fertilization (amounts of 1000, 2500 and 5000 kg ha1 ) and prescribed burning experiments were established in 1990, in a 100 yr old Scots pine (Pinus sylvestris) stand in Central Finland. Mineral soil samples were taken in 2002 and potential atmospheric CH4 oxidation rates were measured. MOB were characterized by PCR and DGGE with primer sets targeting the pmoA gene, coding for the a-subunit of the particulate methane monooxygenase. Even though the treatments increased soil pH, there was no linear correlation with methane oxidation rates. The community structures of MOB were very similar compared to the control plots. A majority of the pmoA sequences obtained from DGGE bands were only distantly related to known methanotrophs.

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P4^107 CHARACTERISTICS OF CELLULOLYTIC ACTINOMYCETES ISOLATED FROM SELECTED FOREST SOILS ¤ D. JabTonska-GorzaTa and S. Russel Department of Soil Environmental Sciences, Laboratory of Agricultural Microbiology, Warsaw Agricultural University, Rakowiecka Str. 26/30, 02-526 Warsaw, Poland Cellulose is a major structural component of plant cell walls and the most abundant polysaccharide in the biosphere. It is a linear polymer built from 100 to 14,000 glucose molecules linked by -1,4-glycosidic bonds, and is a highly recalcitrant substrate for enzymatic action. In soil, its degradation by di¡erent systematic groups of cellulolytic microorganisms, e.g. bacteria, actinomycetes and fungi, represents a signi¢cant part of the carbon cycle. Although soil actinomycetes, including members of Streptomyces, Micromonospora, Streptosporangium and Thermomonospora, are one of the most active groups of cellulolytic microorganisms, they remain poorly characterised. The aim of this study was to evaluate the quantitative and qualitative occurrence of cellulolytic actinomycetes in morphologically di¡erent soil types in White Forest located 70 km north-east of Warsaw. The soil samples were collected ¢ve times within vegetation season from six soil types: pseudogley soil, rusty soil, muck soil, gley-podzol soil, gley soil and black earth. The total number of actinomycetes in the soil samples was analyzed by plate method: using mineral medium containing ¢lter paper as a sole carbon source. Isolated strains were classi¢ed by morphological, physiological and biochemical characteristics. The activity of enzymes degrading ¢lter paper (FP-ase) and carboxymethyl cellulose (CMC-ase) was assayed by Mandels method. For electron microscopy of cellulosomes, the cytochemical technique with cationionized ferritine CF was used. The highest amounts of actinomycetes were found in muck soil. The greatest concentration of actinomycetes population was observed in the organic, upper horizons of soils. 25 strains of cellulolytic actinomycetes were isolated and characterized as belonging to genera Streptomyces, Micromonospora and Streptosporangium. Electron microscopy showed the presence of cellulosome resembling structures which appear as spherical units on the surface of bacterial cells. It was possible to observe penetration of actinomycete mycelium through the cellulose ¢bres using the laser confocal scanning microscope.

P4^108 CHARACTERIZATION OF METHANE-OXIDIZING BACTERIAL COMMUNITIES IN UPLAND SOILS WITH MOLECULAR METHODS C. Knief and P. F. Dun¢eld Max-Planck-Institute for Terrestrial Microbiology, Karlvon-Frisch Str., 35043 Marburg, Germany We characterized the community of methane-oxidizing bacteria (MOB) in di¡erent upland soils that displayed atmospheric methane uptake. For molecular characterization, soil DNA was extracted and the gene of the particulate methane monooxygenase subunit A (pmoA), a useful phylogenetic marker for MOB, was ampli¢ed by PCR. Mixed PCR-products were separated by denaturing gradient gel electrophoresis and individual bands were sequenced. Comparative sequence analysis to a pmoA-database revealed that some detected sequences were closely related to sequences of the genera Methylocaldum, Methylosinus and Methylocystis. Further sequences belonged to two di¡erent phylogenetic clusters for which there are no known cultured representatives. The ¢rst cluster was previously detected in acidic upland soils and is related to pmoA-sequences of K-Proteobacteria. In several pH-neutral upland soils we found a second cluster of sequences distantly related to pmoA-sequences of the Q-Proteobacteria (upland soil cluster Q). Evidence that not only K-Proteobacteria but also Q-Proteobacteria are involved in the process of atmospheric methane uptake in soils was given by incubating selected soils with 13C-labeled methane at a mixing ratio below 50 ppmv. All selected soils contained pmoA-sequences of the upland soil cluster Q but no other pmoA-sequences related to Q-Proteobacteria. In some soils pmoA-sequences related to K-Proteobacteria were also present. In all soils phospholipid fatty acids 14:0, 16:1g7c and 16:0, characteristic for methane-oxidizing QProteobacteria, were labeled with 13C. This suggests that QProteobacteria contribute to the process of atmospheric methane oxidation and that the organisms containing pmoA sequences of the upland soil cluster Q are indeed methanotrophic. P4^109 DYNAMICS OF ARCHAEAL COMMUNITIES IN ARABLE SOILS A. Gattinger(1), M. Schloter(1), M. G. Ho£e(2) and M. « Labrenz(2) (1) GSF ^ Forschungszentrum fur Umwelt und Gesundheit « GmbH, Institut fur Bodenokologie, 85764 Neuherberg, Ger« «

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many; (2) GBF ^ German National Research Centre for Biotechnology, 38124 Braunschweig, Germany In an interdisciplinary research project interactions between organic matter and microorganisms in arable soils from three di¡erent research sites are investigated. The soil in Scheyern, a Gleyic Cambisol is integratedly managed, whereas the Orthic Luvisol in Merzenhausen is managed conventionally at a normal intensity level. In Bad Lauchstadt three di¡erent fertilisation levels of a Haplic Phae« zoem (HP) are investigated (no fertilisation, mineral and mineral+organic fertilisation). Phospholipid fatty acid (PLFA) pro¢ling as well as single strand conformation polymorphism (SSCP) of samplings taken in spring, summer and autumn revealed that microbial community composition were di¡erent in the three soil types according to soil type, fertilisation level and soil depth. Moreover, Archaea-speci¢c phospholipid etherlipids (PLEL) with distinctive pro¢les were predominantly detected in samples from organically fertilized HP. Molecular analyses of archaeal 16S rDNA extracted from SSCP-gels revealed that these sequences belonged to the Euryarchaeota (Thermoplasma and methanogens) as well as to uncultured Crenarchaeota. However, sequences clustering with Thermoplasma were only distantly related to cultivated strains or environmental clones (82 ^ 84 % similarity). In samples without organic enrichment signi¢cantly lower PLEL concentrations were measured indicating that archaeal abundance is rather in£uenced by fertilisation than by soil type. P4^110 ESTIMATION OF PSEUDOMONAS POPULATIONS IN SOIL G. Lloyd-Jones(1), A. Tizzard(2), A. D. Laurie(1) (1) Landcare Research, PO Box 69, Lincoln 8152, New Zealand; (2) Lincoln Technology, Lincoln Ventures Limited, PO Box 133, Lincoln, Christchurch, New Zealand Pseudomonas are fast growing and nutritionally versatile bacteria that are able to utilise a wide variety of carbon sources. The abundance of the genus has been highlighted by conventional microbiology and the genus is well represented in collections of cultured bacteria. We have evaluated the culturability of the Pseudomonas population by comparing culture-dependent with culture-independent approaches for estimating populations in two New Zealand soils. Four di¡erent incubations, a beech-forest soil and a permanent pasture soil incubated at room temperature under constant moisture conditions and also by exposure to toluene vapour, were used to corroborate our observations. Total Pseudomonas populations were enumerated using quantitative £uorogenic PCR (Taqman) to target a 63-bp amplicon within the 16S gene that is highly speci¢c

for the £uorescent Pseudomonas (sensu stricto) group of bacteria, and the culturable £uorescent Pseudomonas population by plating dilutions of soil extracts onto Pseudomonas-speci¢c agars (King's B and Gould's S1) and counting £uorescent colonies. Incubation of these soils led to population changes manifested as a signi¢cant increase in the total Pseudomonas population as estimated using the culture-independent method, while the culturable population showed at least a 10-fold decrease. The culturable Pseudomonas population (a genus thought highly culturable) represented only a small fraction (V0.1%) of the total Pseudomonas population present in these soils. New isolation approaches to target previously uncultured members of the genus should provide a rich source of material with potentially useful properties for biotechnological applications. P4^111 EFFECTS OF TETRACYCLINE ANTIBIOTICS ON SOIL MICROBIAL COMMUNITY PROFILES L. Macovei, G. Jandl, S. Thiele-Bruhn Institute of Soil Sciences and Plant Nutrition, University of Rostock, Justus-von-Liebig Weg 6, 18059 Rostock, DE Antibiotic pharmaceuticals administered to livestock are for the most part excreted. With the contaminated manure used as fertiliser, they are spread onto agricultural land. Consequently residual concentrations of antibiotics in soils were reported. Since antibiotics are highly e¡ective substances even at low concentrations, they a¡ect soil microorganisms. This was determined for various activity parameters, total microbial biomass and the concentration of ergosterol. The aim of this study was to identify in more detail the e¡ects of tetracycline antibiotics on the community pro¢le of soil microorganisms. For this purpose phospholipid fatty acid (PLFA) patterns of two different soils were analysed, following incubation after soil amendment with di¡erent tetracycline compounds of varied. P4^112 SPATIAL LINKAGE BETWEEN NirK DIVERSITY AND DENITRIFICATION ACTIVITY IN THREE ARABLE FIELDS S. M. Mitchell, R. E. Wheatley, J. Squires and T. J. Daniell Plant Soil Interactions, Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, Scotland, UK Denitri¢cation is an anaerobic process in which nitrogen

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compounds are utilised as alternative electron acceptors for respiration. Incomplete denitri¢cation can lead to the release of nitrogen oxide gases which are potent greenhouse gases. Biological denitri¢cation is widespread in prokaryotic systems with a wide range of bacterial and archaeal species capable of the process. Nitrite reductase is a key enzyme in denitri¢cation catalysing the conversion of nitrite (NO2-) to nitric oxide, a form which is no longer available to most biological systems. A high throughput sequence approach was utilized to analyse the sequence diversity of a nitrite reductase gene in soil sampled from three Scottish arable ¢elds. It has been previously observed that the gene encoding the copper containing nitrite reductase is dominant in terrestrial systems. A fragment of the NirK gene was ampli¢ed by PCR, cloned and approximately 1000 clones have been sequenced. Rarefaction analysis suggests that this sampling has exhausted all dominant types from this system. Sequence information generated has been used to design a T-RFLP strategy allowing for high throughput analysis of NirK relative abundance. Potential and actual nitri¢cation and denitri¢cation rates have been measured in one of the three ¢elds and shifts have been observed both temporally and spatially at a range of scales. Work is in progress to link activity measures for denitri¢cation and the diversity of the NirK gene in order to examine if £uctuations in activity re£ect shifts in the structure of the denitrifying community. P4^113 A METAGENOMIC FOSMID LIBRARY OF WADDEN SEA SEDIMENTS M. MuÞmann, J. Kuver, B. Meyer, A. Ellrott, R. I. Amann « Max Planck Institute for Marine Microbiology, Celsiusstr. 1, 28359 Bremen, Germany The metabolic properties of environmentally abundant but uncultivated bacteria are mostly unknown. Recent investigations have demonstrated the power of the metagenomic approach in elucidating potential activities of these bac¤ teria (Beja et al. 2000). Therefore we established a metagenomic fosmid library (V 34,000 clones) from wadden sea sediment DNA with average insert sizes of 38 kb. The library was screened for adenosine 5'-phosphosulfate reductase (APS-reductase), a functional and phylogenetic marker gene of putative sul¢de oxidizing and sulfate reducing bacteria. Flanking regions of the APS-reductase gene were sequenced and annotated to look for encoding functional genes. The diversity of the APS gene in PCRand non-PCR-based libraries was compared with the 16S rDNA diversity of PCR based clone libraries. The environmental relevance of putative sul¢de oxidizing and

sulfate reducing bacteria was shown by £uorescence in situ hybridization (FISH). ¤ Reference : Beja et al., Nature 2000 P4^114 MYXOBACTERIA OF THE SOUTH-WEST OF UKRAINE O. L. Rakhimova, V. O. Ivanitsa Odessa National University, Microbiology and Virology Department, Dvoryanskaya St. 2, Odessa, 65 026, Ukraine 24 myxobacteria strains were isolated from the soils of Ukraine south-west and Bleak sea contact zones. Isolated strains were identi¢ed as species Myxococcus fulvus, Myxococcus xanthus, Myxococcus stipitatus, Archangium gephyra, Cystobacter fuscus, Polyangium vitellinum, Nannocystis exedens. The isolated strains have wild spectrum of lytic enzymes and can produce antagonistic substances. Certain myxobacteria strains were included to Ukrainian Collection of Microorganisms (UCM). Long term preservation possibility of these strains was studied. It was shown that addition of antioxidants: cysteamine, ionol, K-tokopherol to protective medium (sucrose-gelatin agar) doesn't in£uence on the viability of myxobacteria lyophilizated cells. It was found the possibility for using of the accelerated storage test in the case of lyophilizated M. xanthus UCM 10041 and P. cellulosum UCM 10043 viability predicting. The biological rhythms in vital activity of myxobacteria strains M.xanthus UCM 10041, P.cellulosum UCM 10043 was found. Part of viable cells and ability to form fruiting bodies are £uctuating cyclic. The e¡ect of heavy metals on physiological activity, gliding motility, fruiting body formation was estimated. Ability of M. xanthus strains to accumulate heavy metals was estimated too. It was shown that M. xanthus strains are able to accumulate zinc, cadmium, nickel, chromium, copper, lead in the quantity close to their dry weight. Each from the mentioned heavy metals at certain concentration is toxic for the studied M. xanthus strains. Toxic e¡ect was manifested as depression of growth, worsening of motility, breaking of fruiting body formation, reducing of respiration and biomass synthesis. However, active adsorption of heavy metals occurs even under such conditions when heavy metals have concentrations, which are toxic for these strains.

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P4^115 MICROBIAL POPULATIONS IN DEEP MARINE SEDIMENTS H. Sass, B. Engelen and H. Cypionka Institute for Chemistry and Biology of the Marine Environment, University of Oldenburg, P.O. Box 2503, D-26111 Oldenburg, Germany Only a small percentage of the prokaryotes present in marine sediments can be cultured in traditional media. Most sediment bacteria are classi¢ed as `unculturable', although they have thrived in their natural environment. Aim of study is to enhance the cultivation success of marine sediment bacteria. Our approaches involve some new aspects designed to increase the cultivation success. These include the use of non-selective but de¢ned media with di¡erent carbon sources, like polymers and monomers, all at low concentrations. Other variations are the use of substrate gradients, the variation of the incubation temperature, and the addition of particles (e.g. FeS precipitates). Promotion of syntrophic interactions is achieved by addition of background bacteria to MPN series. In our media the bacteria cannot grow to high densities. Therefore growth analysis has to make use of sensitive detection techniques, like counting of grown bacteria after staining with £uorescent DNA stains, FISH or other molecular biological techniques like PCR and DGGE. These di¡erent cultivation techniques were applied to sediment samples from the North Sea taken from depths down to 6 m and to sediments from the Eastern Mediterranean Sea with ages of up to 200 000 years. The di¡erent cultivation approaches were compared with respect to the cultivation success and the dominating phylogenetic and physiological types obtained. P4^116 BIODIVERSITY OF THE MICROORGANISMS NEAR AN OUTCROP OF GAS HYDRATES OF LAKE BAIKAL O. V. Shubenkova(1), T. I. Zemskaya(1), O. M. Khlystov(1), S. M. Chernitsina(1), B. B. Namsaraev(2), O. P. Dagurova(2) (1) Limnological Institute SB RAS, Box 4199, 664033, Irkutsk, Russia ; (2) Institute of general and Experimental Biology of Siberian Branch of RAS, Ulan-Ude, Russia Bacterial biodiversity of the sediments sampled in the Southern Basin of Lake Baikal (at the water depth of 1400 m) near the gas hydrates outcrop was studied. Earlier, existence of gas hydrates here was predicted by nu-

merous studying (Rensbergen et al., 2001). Area near the outcrop was determined by geophysics data (M. De Batist et al., 1998, 1999, 2000; Klerx et al., 2000). Sediments were analyzed with using complex methods of geochemistry and microbiology. The crystals are situated in silty gray clays. Microbiological studying was realized with using classic and molecular biology methods. Bacteria growing were noted on media with adding methane in anaerobic and aerobic conditions. Total bacterial DNA was extracted from sediments and its concentrations were measured. It was 1.17 Wg/g sediment for surface sediment. In deeper sediment horizons the concentration was lower. Possibilities of spectrophotometric method were limited by equipment sensitiveness. However polymerase chain reaction was successful that suggests of DNA presence. Methanotrophic bacteria were found in the surface sediment. Archebacreria and sulfatereducing bacteria were found in dipper horizons. This biodiversity is compounded on the data of 16S RNA. Analysis of lipids con¢rms it. Studying of bacterial biodiversity of this area is continuous. P4^117 IDENTIFICATION OF INTRONS STRAINS ISOLATED FROM SOIL IN BACILLUS

S. Stankovic(1), V. Lazarevic(2), T. Beric-Bjedov(1), J. Knezevic-Vukcevic(1), D. Simic(1) (1) Faculty of Biology, University of Belgrade, Yugoslavia; (2) Institut de Microbiologie Fundamentale, Batiment de Biologie, Universite de Lausanne, Switzerland Collection of Bacillus sp. strains isolated from soil was screened by low stringency PCR, using oligonucleotides corresponding to the insertion sites of the three intervening sequences in the B. subtilis 168 SPL prophage ribonucleotide reductase genes bnrdE and bnrdF. In 3 (SS6, SS72 and SS114) out of 212 strains tested strong bands were generated. The sequencing of the PCR products revealed one (SS114), two (SS72) and three (SS6) group I introns in the bnrdE-bnrdF tandem. The introns that are inserted at the analogous positions have the same length and exhibit over 97% identity. SS6 bnrdE-I1, SS72 bnrdE-I1 and their 168 homologue are 252 nt long and contain no ORF. SS6 bnrdE-I2, SS72 bnrdE-I2 and SS114 bnrdE-I span 280 nt and also contain no ORF. bnrdF-I from strain SS6 contains a putative HNH endonuclease ORF that is 98% identical to its counterpart. In B. subtilis 168 the nrdE and nrdF genes are separated by 17 nt. The bnrdE-bnrdF intergenic spacers in the three strains are much longer, ranging from 230 to 602 nt. The bnrdE-bnrdF intergenic region in the strain SS6 failed to disclose any protein coding sequences. In contrast, bnrdE-bnrdF intergenic regions in strains SS72 and SS114 contain highly similar ORFs

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(orf435) whose deduced products have no homologues in databases. The in vivo intron splicing was demonstrated by reverse transcription, followed by PCR ampli¢cation of synthesized cDNA. The exact exon-intron junctions were con¢rmed by cloning and sequencing of the RT-PCR products. P4^118 DIVERSITY OF SULFATE-REDUCING BACTERIA FROM THE ROMANIAN BLACK SEA BOTTOM SEDIMENTS E. Stoica(1), J. Kuever(2), M. Dragan-Bularda(3) (1) National Institute for Marine Research and Development ``Grigore Antipa'', Bd. Mamaia 300, Constanta RO8700, Romania; (2) University of Bremen, Center for Environmental Research and Technology, Institute for Soil Science, Leobener Strasse, D-28359 Bremen, Germany ; (3) Babes°-Bolyai University, Department of Plant Biology, 3400 Cluj-Napoca, Romania Seven strains (Et1, But1, But2, But3, Lac1, Isob1, Benz1) of Gram negative, mesophilic, nonsporing sulfate-reducing bacteria were isolated from bottom sediments of the Romanian Black Sea coast. Analysis of partial 16S rDNA sequences obtained from pure cultures of isolated by PCR, revealed that all strains belonged to the N ^ subclass of Proteobacteria. Three strains (But1, But3, Lac1) were morphologically and nutritionally similar. According to their 16S rDNA sequences, the isolates were a/liated with the following species: Desulfofrigus fragile (But1, But3, Lac1, 97.9 ^ 98% similarity), Desulfovibrio acrylicus (Et1, 98% similarity), Desulfobacterium autotrophicum (But2, 99% similarity), Desulfobacterium niacini (Isob1, 99% similarity). This is a ¢rst description of sulfate-reducing bacteria diversity from the Romanian Black Sea coast, after the recent changes of environmental conditions from the NW shelf of the Black Sea as a consequence of eutrophication.

P4^119 DIVERSITY OF nosZ GENE FRAGMENTS IN NATIVE AND CULTIVATED SOIL B. Stres(1,3), I. Mahne(1), G. Avgustin(2), J. M. Tiedje(3) (1) Dept. of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia ; (2) Zootechnical Dept., Biotechnical Faculty, University of Ljubljana, Groblje 3, 1234 Domzale, Slovenia; (3) Michigan State University, Center for Microbial Ecology, PSSB 540, East Lansing, MI -48824, USA Denitri¢cation in conjunction with nitri¢cation leads to loss of plant available nitrogen compounds in soil habitats. Agricultural soil at Kellogg Biological Station, MSU, USA, has been subjected to de¢ned long-term ¢eld experiments to evaluate the e¡ects of land use on various soil properties (http://lter.kbs.msu.edu). In the frame of this experiment the two most divergent plots, i.e. cultivated (standard chemical input, crop rotation, conventional tillage) and native (unspoiled reference site, early successional community, never tilled), were selected to analyze indigenous nosZ gene diversity. The gene nosZ codes for nitrous oxide reductase, the last enzyme in denitri¢cation chain and is believed to be present in majority of denitrifying bacteria. Rarefaction analysis of more than 550 nosZ clones revealed major di¡erences between the two communities and T-RFLP analysis of ampli¢ed indigenous nosZ fragments gave similar results. However, further phylogenetic analysis of 48 selected clones representing major groups of clones and some randomly selected clones revealed less drastic di¡erences in the two communities, pointing to rather minor changes caused by the two treatments and raising an interesting question of suitability of restriction pro¢ling to describe complex microbial communities. P4^120 HETEROTROPHIC BACTERIAL DYNAMICS AND DIVERSITY IN ANNECY, BOURGET AND GENEVA LAKES U. Dorigo, S. Jacquet and J. F. Humbert UMR CARRTEL, INRA, BP 511, 74203 Thonon-les-Bains cedex, France A comparative study of the dynamics and diversity of heterotrophic bacterioplankton of three Alpine lakes in France was undertaken. These lakes are characterised by di¡erent nutrient loads and in particularly by the presence (lake Geneva and lake Bourget) or absence (lake of Ann-

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ecy) of toxic proliferations of the cyanobacterium Planktothrix rubescens. Each lake was sampled at least once a month and at di¡erent water depths in order to establish relationships between toxic proliferations and changes in the bacterioplankton community, which may be responsible of the lysis of the cyanobacterial cells and toxins degradation. PCR-Denaturing Gradient Gel Electrophoresis (DGGE) and sequencing of 16S rDNA were performed to examine the bacterioplankton community composition and to observe changes in the numerically dominating populations. The DGGE-patterns were analysed in relation to lake characteristics, season and dynamics of Planktothrix rubescens. The phylogenetic a/liation of the predominant members of the microbial communities (Proteobacteria, Cytophage-Flavobacterium-bacteroides) was also inferred by £uorescence in situ hybridisation (FISH). Flow cytometry was used to establish the total number of heterotrophic bacteria and to discriminate major populations. First results indicate that the diversity and dynamics of the bacterioplankton vary in relation to the lake and the period of study. Temporal di¡erences in community composition seem to be greater than the spatial di¡erences during either season. P4^121 MICROBIAL DIVERSITY OF MID-ATLANTIC RIDGE HYDROTHERMAL VENTS : FROM CULTURE TO COMMUNITY MOLECULAR FINGERPRINTS M. Gadanho(1), S. Chaves(2), T. Tenreiro(2), J. P. Sampaio(1) and R. Tenreiro(2) ¤ (1) Centro de Recursos Microbiologios (CREM), Seccao °< ¤ " Autonoma de Biotecnologia, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, ¤ Portugal; (2) Centro de Genetica e Biologia Molecular, " Departamento de Biologia Vegetal, Faculdade de Ciencias de Lisboa, Rua Ernesto Vasconcelos, Ed. C2, Piso 4, Campo Grande, 1749-016 Lisboa, Portugal On August 2002, ¢ve hydrothermal sites (Menez Gwen, Rainbow, Lucky Strike, Saldanha Mount and Menez Hom) located on the Mid-Atlantic ridge were visited during the Portuguese mission SEAHMA-1. Deep-sea sampling was performed using the submersible VICTOR 6000 on board of the French research vessel L'Atalante. Samples obtained from animals, sediment, chimneys and water were immediately processed on-board for enrichment, in several culture media, under aerobic and anaerobic conditions, and at incubation temperatures ranging from 10 C to 85 C. Crude samples were also frozen at ^ 70C for further analysis. A total of nearly 500 isolates belonging to the Domains Bacteria and Archae and to the Kingdom Fungi (yeasts) were isolated in pure culture from

the on-board enrichments. DNA was extracted from all the isolates and M13-PCR ¢ngerprinting was performed for similarity grouping. From each M13-cluster one isolate was chosen and 16S or 26S rDNA sequencing was performed for phylogenetic allocation. Preliminary results indicated that anaerobic isolates obtained at the highest temperatures were mainly members of the Thermococcales, Thermotogales and Clostridiales. Yeast isolates were obtained at 20-25C. In parallel, total DNA was extracted from the crude samples and from all the enrichments. After ampli¢cation with primers for 16S and 18S rDNA, the amplicons were separated by Temperature Gradient Gel Electrophoresis (TGGE) generating molecular community ¢ngerprints. Cluster analysis of these pro¢les and identi¢cation of common and speci¢c organisms by direct sequencing of TGGE bands, followed by comparison with the culture-based approach, allowed the assessment of microbial diversity of sampled hydrothermal vents. This work was supported by FCT, Project SEAHMA PDCTM/C/MAR/15281/99. P4^122 GENUS EUROTIUM ^ HALOPHILIC FUNGAL INHABITANTS OF HYPERSALINE WATERS IN THE SALTERNS N. Gunde-Cimerman(1), J. C. Frisvad(2), L. Butinar(3) and P. Zalar(1) (1) Biology Department, Biotechnical Faculty, University > of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia; (2) BioCentrum-DTU, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark; (3) National institute of Chemistry, Department of Biotechnology, Hajdrihova 19, 1000 Ljubljana, Slovenia In the course of the mycodiversity study of hypersaline environments, di¡erent species of the known food-borne xerophilic genera, such as Wallemia, Penicillium, Aspergillus, and its teleomorphic stage, Eurotium as well as halophilic black yeasts were isolated from the man made salterns. Genus Eurotium was represented by ¢ve species: E. amstelodami, E. herbariorum, E. repens, E. rubrum and E. chevalieri. Strains of E. amstelodami were most consistently isolated from the Slovenian salterns and later as well in other salterns (Spain, Israel, Dominican Republic, Namibia), while E. herbariorum, E. repens and E. rubrum were isolated frequently. Therefore these species probably contribute to the indigenous fungal community in hypersaline environments. To determine their halophilic adaptation to long-term survival in hypersaline environments, spores of E. amstelodami, E. herbariorum, E. repens and E. rubrum were in vitro exposed to prolonged suspensions in water with di¡erent salt concentrations. They survived

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from 0-30% NaCl for three months and more. Only E. chevalieri spores did not survive the long-term exposure to NaCl concentrations from 20-30% and were recovered as well from the hypersaline waters just once. Therefore E. chevalieri is probably a temporal inhabitant of brine at lower salinities. P4^123 BLACK HOLES OF SOUTH ANDROS, THE BAHAMAS : WHAT THEY ARE AND WHY THEY ARE BLACK R. A. Herbert(1) and S. Schwabe(2) (1) Division of Environmental and Applied Biology, Biological sciences Institute, University of Dundee, Dundee DD1 4HN, Scotland; (2) International Blue Holes Foundation, 7/121 Sir Fred Schonell Drive, St. Lucia, Brisbane, Queensland, Australia 4067 Black holes are vertical limestone cave systems which have no known lateral passages. One particularily spectacular black hole in the Bahamas is the ``Black Hole of South Andros which has an opening of 300m diameter with a water depth of 47m. The water column is strati¢ed with an upper oxic low salinity water mass overlying a deeper saline layer with a salinity of 33-35psu. Temperature pro¢les show an almost uniform 290 C in the upper water layer (17.8m) with a very sharp increase at the pycnocline to 360C.Co-incident with the pycnocline is a 1m thick layer of phototrophic purple sulfur bacteria with a cell density of V107/ml. Assuming a uniform cell density this equates to a dry weight biomass of 5 tonnes. Classical taxonomy and 16S rDNA sequencing has identi¢ed the dominant phototrophic bacteria as belonging to the genera Allochromatium and Thiocapsa. Both bacteria are characterised by the presence of bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series. Spirilloxanthin absorbs maximally at 550nm which prevents light scattering which would explain why from the air the water surface appears black. Both bacteria have a low e/ciency (20-30% ) in channelling captured light energy to the photosynthetic reaction centres. We postulate that the excess captured light is dissipated as heat which would account for the sharp increase in temperature recorded at the pycnocline. These data will be discussed with respect to the sulfur cycle operating in these unique ecosystems.

P4^124 LACK OF GENETIC DIFFERENTIATION IN BENTHIC AND PELAGIC SUB-POPULATIONS OF MICROCYSTIS AERUGINOSA IN A FRENCH STORAGE RESERVOIR J. F. Humbert(1), D. Duris-Latour(2), B. Le Berre(1) and M. J. Salencon(3) ° (1) UMR CARRTEL, INRA, BP 511, 74203 Thonon-les¤ Bains Cedex, France; (2) Universite J. Monnet, Lab. de ¤ e, 42023 St Etienne, France; Biologie Animale et Applique (3) EDF, Laboratoire National d'Hydraulique et Environnement, Site de Chatou, 6, Quai Watier, 78401 Chatou Cedex We compared the genetic diversity of the ITS1 of the rRNA gene in benthic and pelagic sub-populations of Microcystis aeruginosa isolated at two di¡erent sampling stations and at di¡erent sampling time (winter and summer) in the French storage reservoir of Grangent. For each sampling point, a gene clone library was constructed after PCR ampli¢cation of the large ITS1 fragment. Genetic diversity was estimated by random sequencing of several clones per library. 66 ITS1 sequences could be obtained. Nucleotide diversity of all the sampling sub-populations was in the same range (average number = 0.022) whatever their origin revealing that di¡erent clones are involved during the summer bloom event and contribute to the high biomass production. By phylogenetic study and by analysis of molecular variance (AMOVA), we also found no genetic di¡erentiation between benthic and pelagic subpopulations. Our result con¢rm data on population dynamics for these two sub-populations and show that life cycle of M. aeruginosa in temperate area is characterized by a benthic phase in winter and spring that allows the survey of this organism in unfavorable environmental conditions, followed by a pelagic phase in summer and autumn when environmental conditions permits their growth.

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P4^125 FEMTO-, PICO- AND ULTRAPLANKTON DYNAMICS AND DIVERSITY IN THE 3 LARGEST NATURAL FRENCH ALPINE FRESHWATER ECOSYSTEMS: ANNECY, BOURGET AND GENEVA LAKES S. Jacquet(1), U. Dorigo(1), J.-F. Humbert(1) and I. Biegala(2) (1) UMR CARRTEL, Station INRA d'Hydrobiologie Lacustre, 75 Avenue de Corzent, 74203 Thonon, France; (2) ¤ Station Biologique de Rosco¡, Equipe Phytoplancton Oceanique, Place Georges Tessier, 29680 Rosco¡, France To date, almost nothing is known about microbial communities inhabiting the three largest and deepest French alpine lakes, i.e. Annecy, Bourget and Geneva lakes. Using a combination of techniques and methods such as analytical £ow cytometry and sorting, epi£uorescence microscopy, PCR-DGGE, culture enrichment, TSA-FISH, a ¢rst attempt was made to observe and compare the composition and dynamics of viral, heterotrophic bacterial and picophytoplanktonic communities within these 3 ecosystems. The poster will present our ¢rst results and research perspectives. P4^126 DESIGN AND EVALUATION OF 16S rRNA OLIGONUCLEOTIDE PROBES TO ASSESS THE STRUCTURAL COMPOSITION OF THE ARCHAEAL COMMUNITIES THRIVING IN HOT BIOTOPES O. G. Nercessian(1), M. I. Prokofeva(2), A. V. Lebedinskii(2) and C. Jeanthon(1) (1) UMR 6539, Centre National de la Recherche Scienti¤ ¢que and Universite de Bretagne Occidentale, Institut Uni¤ en de la Mer, 29280 Plouzane, France; (2) ¤ versitaire Europe Institute of Microbiology, Russian Academy of Sciences, Prospect 60 Let Oktyabrya 7/2, 117811 Moscow, Russia Hyperthermophilic cultured Archaea and several new uncultivated lineages are usually encountered in hot biotopes. However, because little e¡ort has been made to study the structural composition of these microbial communities, molecular tools such as 16S rRNA-based oligonucleotide probes remain poorly available. We developed and validated by dot-blot hybridizations sixteen archaeal oligonucleotide probes targeting thermophilic microorganisms and environmental clades frequently detected in hightemperature ecosystems. These probes were designed to target the sulfur-reducing heterotrophic Thermococcales, the sulfate-reducers Archaeoglobus, the lithoautotrophic methanogens Methanococcales and Methanopyrus, the De-

sulfurococcales, the Pyrodictiaceae and Ignicoccus, the thermoacidophiles Sulfolobales and Thermoproteales. We also designed probes targeting uncultivated archaeal groups so far detected in hydrothermal ecosystems: these included the Korarchaeota and the groups 2 and 8 of Deep-sea Hydrothermal Vent Euryarchaeotes. The probes allowed us to determine the structure of archaeal community associated with colonization devices deployed at 13N (East Paci¢c Rise) and chimney and sediment samples originating from geographically distant deep-sea hydrothermal ecosystems (9N on the East Paci¢c Rise and 36N on the Mid-Atlantic Ridge). The Pyrodictiaceae, Sulfolobales, Thermoproteales and Korarchaeota were not detected but the remaining probes gave positive signals in most samples. The richness of hyperthermophilic Archaea in colonization devices and chimney fragments was higher than in sediments. Interestingly, uncultivated groups DHVE2 and DHVE8 were mostly detected associated with colonization devices and chimney fragments suggesting a thermophilic way of life. This extensive set of archaeal 16S rRNA-based oligonucleotide probes proved to be useful to compare the structural composition of archaeal communities in numbers of environmental samples. P4^127 ANTARCTIC LAKES ^ `HOT-SPOTS' FOR MICROBIAL DIVERSITY AND BIOTECHNOLOGICAL SCREENING R. De Wit(1), P. Dyer(2), O. Genilloud(3), E. Got« tlich(4), D. Hodgson(5), S. de Hoog(6), B. Jones(7), J. Laybourn-Parry(2), F. Marinelli(8), E. Stackebrandt(9), J. Swings(10), B. J. Tindall(9), W. Vyverman(10), A. Wilmotte(11) (1) Biological Oceanography, University of Bordeaux 1, 33120 Arcachon, France; (2) Biological Sciences, University of Nottingham, Loughborough LE12 5RD, UK; (3) Merck-Sharp-Dohme Espana, 28027 Madrid; (4) IWW, 45476 Mulheim, Germany ; (5) British Antarctic Survey, « CB3 OET Cambridge, UK; (6) Centraalbureau voor Schimmelcultures, 3740 AG Baarn, Netherlands; (7) Genencor, PO Box 218, 2300 AE Leiden, Netherlands; (8) Biosearch Italia SPA, 21040 Gerenzano (VA), Italy ; (9) DSMZ, 38124 Braunschweig, Germany; (10) Microbiology/Protistology, University of Gent, 9000 Gent, Belgium ; ' ' (11) Botany B22, University of Liege, 4000 Liege, Belgium The EC project MICROMAT (BIO4-98-0040) has been the ¢rst multi-disciplinary study on the diversity of a broad spectrum of microorganisms in benthic microbial mats of Antarctic lakes using conventional and molecular methods. It has arisen from a cooperation between European bacteriologists, protistologists, mycologists, microbial ecologists, paleolimnologists and industrial microbiol-

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ogists from 3 companies involved in biotechnological and pharmaceutical research. We have characterised the diversity of bacteria, cyanobacteria, algae, protozoans and fungi in microbial mat samples of several Antarctic lakes by conventional (microscopy, cultivation) and genotypic (clone libraries and DGGE based on the SSU rDNA from the samples) methods. Isolation of strains yielded 1500 bacterial, 60 cyanobacterial, 230 fungal, 91 algal and 50 protozoan isolates from 3 to 24 lakes. The genotypic data comprised 470 prokaryotic and 300 eukaryotic SSU rDNA sequences and showed the usual discrepancy with cultivated diversity for all organisms, except for the cyanobacteria. A low eukaryotic diversity, dominated by a few highly specialised and often endemic taxa was observed. In contrast, the prokaryotic diversity was extremely high and numerous novel phylotypes were discovered. These newly discovered `hot-spots' of microbial biodiversity have stimulated the interest of biotechnological companies who have screened 1700 strains for new cold enzymes and antimicrobial compounds. P4^128 DESCRIPTION OF BIODIVERSITY AND ENZYME ACTIVITY OF MICROBIAL COMMUNITY OF LAKE BAIKAL WATER MASS V. V. Parfenova, N. L. Bel'kova, J. R. Zakharova, S. J. Maksimenko Limnological Institute SB RAS P.O. Box 4199, 664033, Irkutsk, Russia Lake Baikal is the most ancient freshwater lake of the world, which is characterized by its unique ecological conditions. The peculiarities of hydrological and hydrochemical factors such as big depths, low water temperature, high content of oxygen and low concentration of organic matters, stipulate the speci¢c conditions of vital functions of microorganisms. We isolated 1000 strains of psychrophylic and oligotrophyc bacteria from the water mass and determined their lipase, phosphatase and protease activity. These organisms possessed high enzyme activity. The results of research of biodiversity of the studied lake microorganisms were treated with the help of methods both of classical and molecular biology. The taxonomic composition of cultivated and non-cultivated strains was analyzed. The diversity of cultivated strains is presented mainly by the species, which were observed also in a number of other reservoirs. The microorganisms of known and described species were mainly cultivated. They are Acinetobacter, Artrobacter, Alcaligenes, Bacillus, Flavobacterium, Caulobacter, Micrococcus, Pseudomonas, Sarcina. The majority of bacteria are known not to be cultivated on certain nutrient mediums. The analysis of nucleotide sequences of fragments of the gene 16S of mtDNA revealed a wider

diversity of non-cultivated microorganisms, which belonged to di¡erent phylogenetic groups. However, the overwhelming majority of sequences possess a low homology with the known data bank and form clusters at constructing phylogenetic trees in which there are sequences revealed by the similar methods from other natural ecosystems. The data obtained allow to make a conclusion that the microbial community of the lake is unique consisting both of known species observed in other reservoirs and new ones not studied yet and probably distributed only in Lake Baikal. Moreover, taking into account the low concentration of organic matter in the lake, the bacteria possess high degree of enzyme activity. P4^129 BACTERIA OF GENUS PSEUDOMONAS IN MICROBIAL COMMUNITY OF LAKE BAIKAL O. N. Pavlova, V. V. Drucker Limnological Institute of SB RAS, P. O. Box 4199, 664 033, Irkutsk, Russia The distribution and species variability of bacteria of genus Pseudomonas in microbial community of Lake Baikal known as an oligotrophic and deep water lake was studied. 382 water samples and bottom sediments, collected in di¡erent basins of Lake Baikal, were analyzed during 1998 ^ 2002. 277 pure cultures of genus Pseudomonas was isolated and identi¢ed. Species P. aeruginosa, P. alcaligenes, P. cepacia (Burkholderia cepacia), P. caryophylli (Burkholderia caryophylli), P. diminuta (Brevundimonas diminuta), P. £uorescens, P. mendocina, P. putida, P. stutzeri were found more often. Is established that bacteria of genus Pseudomonas dominate in heterotrophic community of microorganisms in Lake Baikal. While studying seasonal dynamics of quantitative development of bacteria in the shallow zone of Southern Lake Baikal it was stated that pseudomonades are prevailed in MarchMay and in October-November periods. It was studied protease, lipase and phosphatase enzyme activities of isolated cultures of Pseudomonas genus. The most active strains are isolated from shallow water zone and from bottom sediments of Lake Baikal. P4^130 MICRO-MAR: A MARINE PROKARYOTES DATABASE TO CORRELATE HABITAT WITH TAXONOMY ¤ F. Rodr|guez-Valera and J. C. Alba ¤ ¤ Division de Microbiolog|a, Campus de San Juan, Universi¤ ndez, Alicante, Spain dad Miguel Herna

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The last 15 years a large amount of 16S rDNA sequences from microbes has become available. Many if not most correspond to PCR products directly ampli¢ed from natural samples and then cloned and sequenced. The number of sequences retrieved from marine environments only exceeds 5000. On the other hand, oceanographic and ecological databases provide a wealth of information about environmental features at di¡erent locations. Here we describe a public data base where both types of information sources have been combined with sequence based taxonomy to provide a grid of geographic and habitat characteristics for every taxon of marine prokaryotes including non-cultured types known only by sequence. P4^131 ACTINOMYCETES IN MICROBIAL COMMUNITY OF LAKE BAIKAL I. A. Terkina, V. V. Drucker Limnological Institute of SB RAS, P. O. Box 4199, 664033, Irkutsk, Russia The distribution, numbers and species diversity of actinomycetes dominant genera in Lake Baikal are inverstigated for the ¢rst time. It was revealed that actinomycetes in water, in bottom sediments, and in sponges are presented by Streptomyces, Micromonospora, Nocardia and Arthrobacter. There are heterotrophic, olygotrophic and psycrophillic strains among the genera. Actinomycetes can be found in the waterbody of Southern, Middle and Northern Baikal, beginning from the depth of 400 m. Nocardia and Arthrobacter in the deep water regions can be found only up to the depth of 400 m, Streptomyces and Micromonospora inhabit also in the near bottom layer like in lithoral zone but at the greater depths. Nocardia can be met occasionally in the near bottom layers, bottom sediments and in sponges of shallow water zone of Lake Baikal. From 65 strains identi¢ed up to species belonging to Streptomyces and Micromonospora the most often available in bottom sediments, waterbody and sponges in Lake Baikal are Streptomyces globisporus and Micromonospora purpurea. Laboratory studies showed that a lot of representatives Streptomyces and Micromonospora are able to produce various hydrolytic enzymes, such as proteases, lipases, phosphatases and cellulases at low temperature. Besides, these actiomycetes exhibit remarkable biological activity against another bacterial strains. Thus, we can suggest, actinomycetes can be participants of a number of important degradation processes and in£uence on structure and functioning of microbial community in lake Baikal.

P4^132 BACTERIOPLANKTON COMMUNITY COMPOSITION OF NEIGHBOURING EUTROPHIC SIBERIAN RESERVOIRS M. Yu. Trusova, M. I. Gladyshev Institute of Biophysics of Siberian Branch of Russian Academy of Sciences, 660036, Krasnoyarsk, Russia Bacterioplankton are among the most abundant and important components of aquatic ecosystems. The taxonomic composition of bacterial assemblages and their spatiotemporal dynamics in freshwater lakes and reservoirs are likely to be of major importance in determining the role of bacteria in aquatic food web and biogeochemistry. The study of bacterial diversity in water habitats has strongly advanced with the recent introduction of molecular techniques. Due to cultivation-independent methods for identi¢cation of microorganisms a large number of previously unknown taxa were detected. The existence of globally distributed taxa has been discovered. The rRNA approach has provided a powerful instrument to study interactions of bacteria with other components of aquatic ecosystems, and to relate community composition data to environmental data and trophic status of the ecosystem. Using 16S rRNA partial gene sequence analyses we have investigated the bacterial diversity of winter bacterioplankton of two neighbouring eutrophic Siberian reservoirs. The reservoirs are similar in phytoplankton community composition in spring and autumn but tend to di¡er in summer in exhibiting cyanobacterial bloom. Forty-eight unique partial 16S rRNA gene sequences retrieved from two libraries were mostly a/liated with the class Actinobacteria, beta subdivision of the class Proteobacteria, and the phylum Cytophaga-Flavobacterium-Bacteroides. The clone library of the reservoir exhibiting summer cyanobacterial bloom showed more diversity in sequence composition. A signi¢cant number of bacterial 16S rRNA gene clones were closely related to freshwater bacteria previously found in di¡erent aquatic ecosystems. This ¢nding con¢rms the assumption that some bacterial clades are globally distributed. P4^133 GENOMIC DIVERSITY OF HETEROTROPHIC BACTERIA ISOLATED FROM ANTARCTIC MICROBIAL MATS S. Van Trappen(1), J. Mergaert(1) and J. Swings(1,2) (1) Laboratorium voor Microbiologie, Vakgroep Biochemie, Fysiologie en Microbiologie, Universiteit Gent, Belgium; (2) BCCM/LMG Bacteria Collection, Universiteit Gent, Belgium

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The genomic diversity of heterotrophic bacteria, isolated from microbial mats in 10 lakes in 3 di¡erent Antarctic regions (Vestfold Hills, McMurdo Dry Valleys and Larsemann Hills) was further investigated. In a previous study [1], 746 strains have been assigned to 41 clusters by numerical analysis of their fatty acid pro¢les, and 16S rDNA sequence analysis of representative strains showed that they belong to the alpha-, beta- and gamma-subclasses of the Proteobacteria, the high and low percent G+C Gram-positives and to the Cytophaga-Flavobacterium-Bacteroides branch. The aim of this study was to investigate in more detail the genomic diversity of 451 strains belonging to 20 fatty acid clusters, of which representative strains were phylogenetically related to the Proteobacteria. Repetitive extragenic palindromic DNA (rep)-PCR ¢ngerprinting was performed on these clusters and the wealth of di¡erent rep-¢ngerprints obtained, illustrates that the genomic diversity of heterotrophic bacteria in Antarctic microbial mats is extremely high. To investigate the genomic relatedness between the di¡erent rep-PCR groups, DNADNA hybridisations between representative strains are being carried out. Several fatty acid clusters contain or represent di¡erent hybridisation groups and 16S rDNA sequence analysis shows that these groups often represent new species related to recently described species from cold environments. [1] S. Van Trappen et al. Diversity of 746 heterotrophic bacteria isolated from microbial mats from ten Antarctic lakes, Systematic and Applied Microbiology. In press P4^134 NEW PROKARYOTIC GENE DIVERSITY IN ANTARCTIC DRY VALLEYS S. J. Whiting(1), J. M. Ward(1), K. D. Bruce(2) and D. C. Cowan(3) (1) Dept. of Biochemistry and Molecular Biology, Darwin Building, University College London, Gower St., London WC1E 6BT, UK; (2) Department of Life Sciences, Franklin-Wilkins Building, King's College London, 150 Stamford Street, London SE1 9NN, UK; (3) Department of Biotechnology, University of the Western Cape, Bellville 7535, Cape Town, South Africa The Dry Valleys of South Victoria Land, Antarctica contain some of the most extreme biotopes on Earth and have long been considered as valid Martian analogues. Life within the Antarctic desert soils, where water contents range from 0.2-2.0% w/w and mean annual temperatures fall below 6 -20C , must additionally contend with desiccating winds, diurnal freeze-thaw cycles, and high seasonal UVA/UVB radiation. Molecular phylogenetic analysis of Antarctic desert mineral soils have revealed this biotope to support a wide diversity of bacterial species,

the majority of which have no close cultured representatives. Divisions represented in 16S rDNA clone libraries include the Proteobacteria, Actinobacteria, Acidobacteria and Verrucomicrobia along with candidate division TM7. Analysis of Archaeal 16S rDNA sequences has identi¢ed the presence of non-thermophilic Crenarchaeota in the Dry Valley environment that cluster within Group I.b of the uncultured Crenarchaeota, a group represented by clones recovered from forest and agricultural soils. Our investigation of prokaryote diversity is currently being extended through the incorporation of cultivation techniques and additionally, in the generation of a soil metagenomic DNA library from which to probe for 16S rDNA sequences. Further to the characterization of microbial diversity, we are also examining functional gene diversity focussing on the recovery of integrons and their associated gene cassettes. Integrons are genetic elements that permit gene acquisition and expression through the capture of mobile gene cassettes and they are well recognised for their role in the dissemination of antibiotic resistance. Using a PCRbased strategy, we have evidence of integrons and their associated genes in pristine Antarctic environments. P4^135 ANTIFUNGAL SUBSTANCES OF ANTAGONISTIC BACTERIA BACILLUS SP. 739 G. E. Aktuganov, A. I. Melentiev, A. V. Shirokov Institute of Biology, Ufa Research Center RAS, Prospekt Oktiabria, 69, Ufa, 450054, Russia Three groups of extracellular antifungal substances were detected in cultural broth of Bacillus sp. 739 ^ the strain, known as antagonist toward several phytopathogenic fungi. The ¢rst group contains hydrolytic enzymes including chitinase (EC 3.2.1.14), chitosanase (EC 3.2.1.132) and L1,3-glucanase (EC 3.2.1.39). Other two groups of the antifungal substances are low-molecular proteins (MwV10-20 kD) and peptide or lipo-peptide antibiotics, respectively. Chitinase and L-1,3-glucanase from Bacillus sp. 739 caused degradation of intact and dead mycelium a lot species of soil fungi. Besides, these enzymes were respond for mycolytic e¡ect of Bacillus sp. 739 toward Helminthosporium sativum and Fusarium culmorum in mixed culture. However, partially puri¢ed enzyme preparations did not in£uence on the mycelium growth or fungal spore germination in vitro. Three low-molecular protein and peptide fungicides were found in cultural broth of Bacillus sp. 739 by thin-layer and paper chromatography. Preparative isolation of the components was performed by sulfate ammonium saturation, acetone extraction and gel-chromatography on Toyopearl HW-40. Amino acid analysis of total antifungal fraction indicated high content of alanine, valine, leucine and phenylalanine among sixteen amino acids

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found in acid hydrolysis products. Two antibiotic components were identi¢ed as polypeptides (MwV1-2 kD), while third one was a protein (MwV14 kD). Protein inhibited spore germination only, whereas low-molecular antibiotics caused mass formation of spheroplasts in hyphae of growing H. sativum mycelium and its lysis. Minimal inhibiting concentration of protein 14 kD was about 100-150 Wg/ml. Presence of di¡erent mechanism of fungal growth inhibition provided by Bacillus sp. 739 indicates a complex nature of interactions between antagonistic bacteria and soil fungi. P4^136 PHYLOGENETIC ANALYSIS OF CHITIN SYNTHASE GENES FROM PHAEOMONIELLA CHLAMYDOSPORA A. Alves(1), A. J. L. Phillips(2) and A. Correia(1) ¤ (1) Centro de Biologia Celular, Campus Universitario de Santiago, Departamento de Biologia, Universidade de Aveiro, 3810-193 Aveiro, Portugal; (2) Centro de Recursos ¤ " Microbiologicos, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2829-516, Caparica, Portugal Esca syndrome is one of the most destructive diseases of grapevine. It is widespread in most countries where vines are grown, and has been already identi¢ed in several Portuguese regions. Apparently it results from the action of several fungi acting in combination or in succession. The primary pathogens in this succession are mitosporic fungi of the genera Phaeoacremonium (Pm) and Phaeomoniella (Pa). The taxonomy of these genera is still subject to discussion and is not fully de¢ned. In this work, 14 fungal strains isolated from grapevines a¡ected by esca, and belonging to the species Phaeomoniella chlamydospora (= Phaeoacremonium chlamydosporum), Phaeoacremonium aleophilum, Pm. angustius and Pm. viticola were studied. Primers speci¢c for class I and class II chitin synthase genes were used. A chitin synthase DNA fragment was ampli¢ed, cloned and sequenced for all Pa. chlamydospora strains. In all other species it was not possible to detect homologues of this fragment. The nucleotide and deduced amino acid sequence analysis revealed that all the fragments belonged to a class I chitin synthase. Class II chitin synthase genes were not detected. The phylogenetic analysis based on the deduced a.a. sequences revealed that Pa. chlamydospora forms a group distinct from the other species analysed. Nevertheless, among these Pa. chlamydospora seems more closely related to A. niger and A. nidulans, ascomycetes of the genus Emericella (Eurotiales), B. dermatitidis and H. capsulatum ascomycetes of the genus Ajellomyces (Onygenales). These results con£ict with phy-

logenetic analysis based on ITS sequences which places Pa. chlamydospora in the Chaetothyriales. P4^137 IS PICHIA ANOMALA THE ONLY YEAST SPECIES INHIBITING MOULD GROWTH DURING AIRTIGHT STORAGE OF MOIST CEREAL GRAIN? º U. A. Druvefors, J. Schnurer « Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, 750 07 Uppsala, Sweden Penicillium roqueforti is the most important spoilage fungi in airtight stored cereal grain. We have previously shown that the biocontrol yeast Pichia (Hansenula) anomala strain J121 can reduce growth of P. roqueforti both in vitro and in high-moisture cereal grain in a test-tube version of a malfunctioning storage system. The ability of P. anomala J121 to prevent growth of inoculated P. roqueforti and other moulds has been validated using 0.21 m3 pilot scale silos for outdoor airtight storage of 160 kg batches of moist grain during 14 months. We have now investigated whether the inhibiting e¡ect on mould growth in airtight storage is a unique ability of P. anomala J121. More than 85 strains from 34 species from di¡erent yeast genera were evaluated. All strains of P. anomala had high biocontrol activity, including all tested haploid strains. The absolute majority of other yeast species, including several strains of Saccharomyces cerevisiae, Debaromyces hansenii, and Cryptococcus albidus had no or very limited biocontrol activity. Hypopichia burtonii and Pichia guillermondii inhibited P. roqueforti, but not to the same extent as P. anomala. Where several strains of the same species were evaluated no di¡erences between strains were detected. P4^138 BIOLOGICAL CONTROL OF FUNGAL STRAWBERRY DISEASES BY THE CHITINOLYTIC SERRATIA PLYMUTHICA STRAIN HRO-C48 G. Berg(1), S. Kurze(1), J. Frankowski(1), A. Wolf(1), R. Dahl(2) and H. Bahl(1) (1) University of Rostock, Microbiology, Albert-EinsteinStr. 3, D-18051 Rostock, Germany ; (2) Strawberry Farm Rovershagen, D-18182 Purkshof, Germany « The rhizobacterium Serratia plymuthica C48 was previously selected as an antagonist to phytopathogenic fungi. The antifungal activity of the strain mainly based on an e/cient chitinolytic system. One chitinase (E.C. 3.2.1.14) CHIT60 and one N-acetyl-Þ-1,4-D-hexosaminidase (E.C. 3.2.1.52) CHIT100 were puri¢ed and characterized.

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The nucleotide sequence of the gene encoding the chitinase CHIT60 was highly similar to that of chitinase A from Serratia liquefaciens. The puri¢ed CHIT100 (100 Wg ml-1) inhibited the spore germination and germ tube elongation of the phytopathogenic fungus Botrytis cinerea by 28 % and 31.6 % respectively. With CHIT60 (100 Wg ml-1) the e¡ect was more pronounced: 78 % inhibition of germination and 63.9 % inhibition of germ tube elongation. In ¢ve consecutive vegetation periods, ¢eld trials were carried out in soils naturally infested by the soilborne pathogens. The root application of S. plymuthica prior planting reduced Verticillium wilt compared with the nontreated control by 0 to 37.7%, with an average of 24.2%, whereas the increase of yield ranged from 156 to 394%, with an average of 296%. Additionally, in£uence of the biocontrol agent on the bacterial communities (non-target microorganisms) of the strawberry rhizosphere was monitored by analysis of PCR-ampli¢ed fragments of the 16S rRNA genes of the bacterial community after separation by denaturing gradient gel electrophoresis (DGGE) and by analysis of the in£uence on fungal and bacterial diversity. Under ¢eld conditions, the strain survived at approximately log10 3-7 CFU g-1 root in the strawberry rhizosphere at 14 months after root application. P4^139 EVALUATION OF OXILITE AS AGAINST OIL FIELD BACTERIA A BIOCIDE

P4^140 MOLECULAR TYPING OF MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS ISOLATED FROM GOATS AND CATTLE IN NORWAY B. DjÖnne(1), P. Ahrens(2), I. Pavlik(3), P. Svastova(3), and G. Holstad(1) (1) National Veterinary Institute, P.O. Box 8156 Dep., N0033 Oslo, Norway; (2) Danish Veterinary Laboratory, Bulowsvei 27 DK-1790, Copenhagen, Denmark; (3) Veterinary Research Institute, Hudcova 70, Brno 621 32, Czech republic In Norway, clinical paratuberculosis has been frequently diagnosed in goats, while cattle have been almost free of the disease. This di¡erence in disease prevalence between goat and cattle, has led to speculations about the existence of a Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) isolate that is not pathogenic for cattle. There is little available information about genotypic variations of M. a. paratuberculosis isolated from animals in Norway. The aims of the present study were to obtain genotypic information of 58 isolates from goats and four isolates from cattle in Norway by use of ampli¢ed fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) analysis. All M. a. paratuberculosis isolates from cattle and 91% of the isolates from goats belonged to the same AFLP type (type G). The other ¢ve isolates belonged to four di¡erent AFLP types. It was also demonstrated that the isolates from cattle and 76% of the isolates from goats had the same RFLP pattern, B-C1, but some RFLP patterns not previously detected were also found. No genotypic variation that could explain a di¡erence in virulence was found between the isolates from cattle and the majority of the Norwegian goat isolates. This indicates that the most common M. a. paratuberculosis isolates in Norway are able to infect both cattle and goats.

M. Gaja, H. Ben Hussein and K. El-Barouni Production Technologies and processing research Department, Petroleum Research Centre, P.O. Box 6431, Tripoli, Libya Laboratory experiments were carried out to determine the biocidal activities of Oxilite for treatment applications in water systems of oil producing and drinking water. Oxilite (oxidizing agent) is cost e¡ective product that is known to be non-toxic mixture of all individually oxidants in ionic and radical form. The biocidal e¡ects of Oxilite on harmful micro-organisms such as sulphate-reducing bacteria (SRB) in 103D and Augila oil¢eld injection waters and against Escherichia coli (E. coli) in local drinking water have been studied by measurement of their cell numbers at 37C versus time. It is apparent that such product showed bactericidal or killing e¡ect (100%) against SRB in 103D injection water at all tested dilutions. However Oxilite in Augila injection water generally showed bacteriostatic activity with disappearance of ferrous sulphide precipitation by SRB. In drinking water, Oxilite was found (100%) e¡ective to kill E. coli cells. The laboratory results showed that Oxilite can be considered as e¡ective biocides in water systems.

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P4^141 ISOLATION OF CLOSTRIDIUM DIFFICILE FROM THE PATIENTS, OBJECTS OF HOSPITAL ENVIRONMENT AND ANIMALS (REPUBLIC OF BELARUS) N. V. Lebedkova(1), L. P. Titov(1), A. Yu. Finogenov(2), N. N. Androsik(2), E. P. Schesljenok(1), I. Grigorovich(1), V. F. Korotkina(3), J. Silva(4) (1) Research Institute for Epidemiology and Microbiology, Minsk, Belarus ; (2) S. N. Vyshelesski Institute of Experimental Veterinary Medicine, Minsk, Belarus ; (3) Hospital of Infectious Diseases, Minsk, Belarus; (4) Division of Infectious and Immunologic Diseases, University of California, Davis Medical Center, Sacramento, California 958174 The infections caused C. di/cile are registered in many countries of the world. This microorganism is isolated from the patients and carriers, objects of hospital environment and animals. The purpose : to establish distribution C. di/cile at the patients, on objects of hospital environment and at animals. Materials and methods. Samples faeces of the patients, animals and wash-out from objects of hospital environment. Specimens were inoculated onto cycloserine-cefoxitin-fructose agar. The samples were incubated anaerobically for 48-72 h at 37 0C. Identi¢cation C. di/cile carried out by cultural and biochemical properties. C. di/cile isolates were tested for the presence of genes toxins A and B by polymerase chain reaction (PCR). Results. From the patients we obtained 22 isolates C. di/cile. The frequency of isolation C. di/cile were from dogs 4,0% (n=151), foals 2,8% (n=106), cats 1,6% (n=63). C. di/cile are isolated from objects of hospital environment in 2,5% (n=1700) of cases. Basically, this pathogen was found out on objects of hospital environment of intensive care units (ICU)-5,7% (n=700). Most contaminated were bed-pans 22,1% (n=77), £oors 3,1% (n=160), wash-stands 2,2% (n=225), night-tables 2,0% (n=151), walls 1,7 % (n=239), tidying up instruments 1,7% (n=237). All isolates C. di/cile expressed fragments tox genes A and B. Conclusion. Thus, toxigenic isolates C. di/cile are isolated from the patients, objects of hospital environment and animals. Most contaminated were objects of hospital environment of ICU, which can be the factors of transmission, this pathogen.

P4^142 LACTIC UNLIKE OTHER BACTERIA, PROLIFERATE UNDER ATMOSPHERE CONTAINING CARBON-MONOXIDE ONLY U. Maor(1), N. Shaklai(1), N. Gollop(2) and V. A. Tsemakhovich(1) (1) Department of Human Genetics and Molecular Medicine, Faculty of Medicine, Tel-Aviv University, P.O. Box 39040, Tel-Aviv 69978 ; (2) Dept. of Food Science, The Vulcani Center ARO, P.O. Box 6, Bet-Dagan, 50250, Israel It has been long known that carbon-dioxide inhibits of anaerobic bacterial growth. The role of carbon-monoxide, an inert molecule, in anaerobic bacterial proliferation is less clear. The literature contains some information suggesting that carbon monoxide can inhibit anaerobic bacterial growth as well but the picture is not clear. We tested the involvement of, carbon-monoxide, in growth of facultative/anaerobic bacteria. We compared the growth of E. coli (386) on liquid and solid nutrient-rich media under three atmospheres containing; air, nitrogen or carbonmonoxide. We found as expected slower growth under nitrogen in compared to air. However, growth under carbon-monoxide was much inhibited as compared to nitrogen. Exchange of carbon-monoxide by nitrogen resulted in accelerated growth reaching the original level of growth under nitrogen. This data suggested that binding of carbon-monoxide to an iron containing protein site essential for the metabolism of this bacteria is involved. We next tested growth of several lactic bacteria (Lactobacillus plantarum Lactobacillus leichmanii, Lactobacillus casaei, Lactobacillus delbrukii, Leuconostock mesenteroides) under the above atmospheres. We found practically no di¡erence in growth of all above lactic bacteria under the three atmospheres. These data can be explained by lack of iron containing, carbon-monoxide binding sites on key proteins / enzymes in the lactic bacteria. In correlation, lactic bacteria have long been shown as the sole bacteria with no need of iron as an essential element. P4^143 STUDY ON BIASES IN TEMPLATE-TO-PRODUCT RATIOS OF MULTI -TEMPLATE PCR ¤ M. Palatinszky, M. Nikolausz, R. Sipos, K. Marialigeti ¤ Department of Microbiology, Eotvos Lorand University, « « ¤ zmany P. setany 1/C, Hungary ¤ ¤ ¤ Budapest 1117, Pa Molecular ¢ngerprinting methods are widely used in microbial diversity examinations of environmental samples. However any conclusions drawn about the relative abun-

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dance of certain taxons within a community should always take the limitations of the applied techniques into consideration. Among molecular ¢ngerprinting methods TRFLP (Terminal Restriction Fragment Length Polymorphism) is a semi-quantitative technique, based on the separation of multitemplate PCR amplicons according to their size by high-throughput capillary electrophoresis. The preceding steps (DNA isolation, PCR) carry a potential bias distorting template ratios, therefore the T-RFLP quantitation of a given community only applies to the relative abundance of the multi-template PCR products and not to the original composition of the environmental sample. Our aim was to explore how multitemplate PCR alters DNA concentration ratios within the community DNA sample. We have constructed a model community of ¢ve collection strains of bacteria (Aeromonas hydrophila, Bacillus cereus, Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas £uorescens) with di¡erent genome sizes, G+C content and rrn copy number. Following DNA isolations the amount of DNA from each strain was determined by spectrophotometric quantitation so that the ratio of the 16S rDNA copy number could be set at prede¢ned values. The distortion of the relative abundance of the di¡erent amplicons, acquired from the PCR performed on the di¡erent template mixtures, was measured by T-RFLP. Our results show that both the genomic properties of the model bacteria and the conditions of the PCR itself (cycle number, annealing temperature, touch down protocol) in£uence the predicted amplicon ratios. P4^144 DEVELOPMENT OF A HIGH THROUGHPUT DIVERSITY DNA ARRAY FOR THE HUMAN INTESTINAL MICROBIOTA M. Rajilic, H. G. H. J. Heilig, L. Rigottier-Gois, W. M. de Vos, E. E. Vaughan Laboratory of Microbiology, Wageningen University, 6703 CT Wageningen, The Netherlands The human gastrointestinal tract is inhabited by a diverse microbial community termed microbiota, which contains up to 1014 total bacteria that have a profound in£uence on human health. The composition and activity of microbiota is host dependent, but still a¡ected by environmental factors such as diet, age, lifestyle, and may also be altered due to intestinal or other diseases. For following changes in microbiota on a large-scale, high throughput methods such as DNA arrays are required. We have developed a diversity macroarray for the detection of the numerically dominant human intestinal bacteria. The array is made by spotting speci¢c 16S ribosomal DNA based probes from over 100 bacterial species on a nylon membrane. Valida-

tion was performed by hybridizing the array with the 16S rDNA from various bacterial species present on the array, labelled by incorporation of radioactive [K-32P] dATP. The results indicated speci¢c hybridisation suggesting the method has a potential for the development of a microbiota microarray. Furthermore, DNA and RNA isolated from faecal or mucosal samples will be tested to investigate the diversity and activity of the microbiota within the di¡erent niches in the human intestinal ecosystem. P4^145 CURRENT STATE OF ANTIMICROBIAL RESISTANCE OF S. PYOGENES (GAS) IN RUSSIA : RESULTS OF PROSPECTIVE MULTICENTER STUDY (PEHASus-I, PHASE''B'') O. V. Sivaja(1), R. S. Kozlov(2), L. S. Stratchounski(2) and PEHASus Project Group (1) Department of Clinical Pharmacology of Smolensk State Medical Academy, Smolensk, Russia ; (2) Institute of Antimicrobial Chemotherapy, Smolensk, Russia The study was conducted in 16 cities (Chelyabinsk, Ekaterinburg, Irkutsk, Jakutsk, Jaroslavl, Kazan, Krasnodar, Moscow, Novokuznetsk, Saint-Petersburg, Smolensk, Stavropol, Tjumen, Tomsk, Rjazan, Voronezh) in Russia in 2001-2002. The total of 683 non-duplicate clinical isolates of Streptococcus pyogenes (GAS) were included in this study. Identi¢cation of the strains was done on the basis of colony morphology, Gram stain, bacitracin (0.02 IU) susceptibility and latex agglutination tests. Susceptibility testing to penicillin G (PEN), erythromycin (ERY), azithromycin (AZI), clarithromycin (CLA), midecamycin (MID), clindamycin (CLI), telithromycin (TEL), levo£oxacin (LEV), tetracycline (TET), chloramphenicol (CHL), vancomycin (VAN) and linezolid (LIN) was performed centrally by broth microdilution method. Breakpoints were those of NCCLS (2002), except for TEL (9 0.5; 1; v 2 mg/L), SPI (9 1; s 4 mg/L) and MID (9 1; s 4 mg/L). There were no resistance detected to PEN, TEL, LEV, VAN and LIN. Percentage of non-susceptible (intermediate and highly resistant) to macrolides and lincosamides isolates was as follows : ERY ^ 8% (MIC-0.06 mg/ L), AZI ^ 9% (MIC-0.25 mg/L), CLA ^ 7% (MIC-0.125 mg/L), MID 1% (MIC-0.5 mg/L), and CLI ^ 1% (MIC0.03 mg/L). The highest non-susceptibility was observed to CHL (51%) and TET (47%). PEN remains 100% active against all GAS isolates. High resistance to TET and CHL compromises their usage in streptococcal infections. 16-membered midecamycin possessed the highest activity again all strains. Obtained date can be helpful for choice of appropriate antimicrobial for streptococcal infection therapy.

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P4^146 DIVERSITY OF NANOBACTERIA AND PROVOKED MINERAL DEPOSITS IN CALCIFIED PLACENTA T. N. Abashina(1) and R. M. Agababov(2) (1) Pushchino State University, Pushchino, 142290 Russia ; (2) Central Municipal Hospital No3, Donetsk, 83014 Ukraine Calci¢cation of tissues is a common case of disorders in placenta. Current knowledge of the calci¢cation is still limited and controversial. Potential relations between appearance of the mineral deposits and bacterial factors are unknown. Up to present, calci¢cation of placental tissues has never been shown in relation with some bacterial dissemination. Our electron microscopy of placental calci¢ed sites discovered: micro-cavities in the tissues; nanobacteria; mineral micro-deposits of the calci¢cation. The microcavities were of 1 x 6 Wm size, they served as a basic place of localisation of the nanobacteria and the mineral microdeposits. The revealed new and unstudied nanobacteria were presented with small cells (0.15-0.20 Wm in diameter) containing RNA and DNA and coated with cellular membranes, the cell wall was absent. Low electron density of the micro-cavity in comparing with the surrounding tissues testi¢es to the fact that the cavities were ful¢lled with solution and were favourable for accumulation of dissolved inorganic salts, i.e. for deposition of calcium as mineral storage. Redox potential on membrane surface of the nanobacteria could stimulate the process. Thus, our ¢nding con¢rms the early proposed mechanism of placenta calci¢cation : rapid formation of apatite mineral in placenta means existence of supersaturated environment and catalysts of the process (i.e. nanobacteria). Thus, the present study is the ¢rst demonstration of a relation between the placenta calci¢cation and nanobacteria.in micro-cavities in tissues. The authors are grateful to Dr.P.Schwartsburd and Dr.N.Suzina (Pushchino Research Centre RAS) for their support during the investigations.

P4^147 TWO GAMMA-PROTEOBACTERIA GROW IN PROTOZOA AND REQUIRE CHARCOAL FOR GROWTH IN LABORATORY MEDIA. DESCRIPTION OF AQUICELLA LUSITANA GEN. NOV., SP. NOV. AND AQUICELLA SYPHONIS, SP. NOV. P. Santos(1), I. Pinhal(1), F. A. Rainey(2), J. Costa(3), ¤ N. Empadinhas(3), B. Fields(4), R. Benson(4), A. Ver|ssimo(1) and M. S. da Costa(3) " (1) Departamento de Zoologia and Centro de Neurociencias, Universidade de Coimbra, 3004-517 Coimbra, Portugal; (2) Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA; (3) De¤ partamento de Bioqu|mica, Universidade de Coimbra, 3001401 Coimbra, Portugal; (4) Respiratory Diseases Branch, Centers for Disease Control and Prevention, Mailstop G03, 1600 Clifton Road, Atlanta, GA 30333, USA Several isolates, belonging to two new species of the same novel genus of Gamma-Proteobacteria, were recovered from borehole and spa water at Sao Gemil in Central < Portugal. These organisms are allied to the strictly intracellular species of the genus Ricketsiella, which cause desease in arthropods, and to the facultatively intracellular species of the genus Legionella, some of which cause Legionnaires' desease and Pontiac fever. These organisms only grew on bu¡ered charcoal yeast extract (BCYE) medium because, like the species of the genus Legionella, they require activated charcoal for growth. Unlike, the vast majority of the strains of Legionella, the new isolates do not require L-cysteine or pyrophosphate for growth. Strains SGT-39T and SGT-56 grew consistently between 30 and 43C, while strains SGT-108T and SGT-109 grew between 30 and 40C. The pH range for growth of these organisms was surprisingly narrow; strain SGT-39T and SGT-56 grew between pH 6.3 and 7.3, while strain SGT108T and strain SGT-109 grew between pH 6.3 and 7.0. Both organisms infected and proliferated in the amoeba Hartmannella vermiformis but did not grow in U937 human cells. On the basis of 16S rRNA sequence analysis, physiological, biochemical and chemical analysis we are of the opinion that strain SGT-39T represents a new species of a novel genus for which we propose the name Aquicella lusitana, while strain SGT-108T represents a second species of the same novel genus for which we propose the name Aquicella syphonis.

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P4^148 TEPIDIMONAS AQUATICA SP. NOV., A NEW SLIGHTLY THERMOPHILIC BETA-PROTEOBACTERIUM ISOLATED FROM A HOT WATER TANK M. Freitas(1), F. A. Rainey(2), M. F. Nobre(1), A. J. D. Silvestre(3) and M. S. da Costa(4) " (1) Departamento de Zoologia and Centro de Neurociencias, Universidade de Coimbra, 3004-517 Coimbra, Portugal; (2) Department of Biological Sciences, Louisiana State University, Baton Rouge, La 70803, USA; (3) De¤ partamento de Qu|mica, Universidade de Aveiro, 3810-193, Aveiro, Portugal; (4) Departamento de Zoologia, Universidade de Coimbra, 3004-517 Coimbra, Portugal A bacterial isolate, with an optimum growth temperature of about 50C, was recovered from a domestic hot water tank in Coimbra. Phylogenetic analysis using 16S rRNA gene sequence indicated that strain CLN-1T is a member of the beta-Proteobacteria and represents a new species of the genus Tepidimonas. The major fatty acids of strain CLN-1T are C16:0 and 17:0 cyclo. Ubiquinone 8 is the major respiratory quinone, the major polar lipids are phosphatidylethanolamine, and phosphatidylglycerol. The new isolate is aerobic and facultatively chemolithoheterotrophic. Thiosulfate and tetrathionate are oxidized to sulfate in the presence of a metabolizable carbon source. Heterotrophic growth of strain CLN-1T occurs on amino acids and organic acids, but this organism does not assimilate carbohydrates. Glycerol is the only polyol assimilated. Another slightly thermophilic organism, designated DhA-73 that, based on 16S rRNA gene sequence analysis, belongs to an undescribed species of the genus Tepidimonas, is also unable to assimilate carbohydrates and polyols. This organism was isolated years ago was capable of degrading tricyclic diterpenes, such as abietic acid, dehydroabietic acid and palustric acid derived from conifer resin. Resinic acids, namely abietic acid, dehydroabietic acid and isopimaric acid are not degraded. On the basis of the phylogenetic analyses, physiological and biochemical characteristics, we propose that strain CLN-1T represents a new species for which we o¡er the name Tepidimonas aquatica.

P4^149 AKKERMANSIA MUCINIPHILA, GEN. NOV., SP. NOV., A NOVEL INTESTINAL MUCIN-DEGRADING BACTERIUM M. Derrien(1), E. E. Vaughan(1,2), C. M. Plugge(1) and W. M. de Vos(1,2) (1) Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands; (2) Wageningen Centre for Food Sciences, Wageningen, The Netherlands The gastro-intestinal tract (GI-tract) harbours a diverse and abundant microbiota. Recent studies based on 16S rDNA (16S ribosomal DNA) revealed that up to 60 to 80% of the intestinal microbiota has not yet been cultivated. Intestinal mucus, mainly composed of glycoproteins named mucins, forms a crucial intermediate layer between host and microbe since it provides protection against pathogens on one hand and constitutes an important energy source for both commensal and potentially pathogenic micro-organisms on the other. The aim of this study was to monitor the diversity of mucin-degrading bacteria from feces by both a 16SrDNA based approach, and to quantify them by enrichment and dilution. In the present study we describe the isolation and characterisation of a novel intestinal mucin-degrading organism. It was isolated by combining enrichment in liquid and soft agar basal media containing mucin as sole carbon source. It is an ovalshaped, Gram-negative organism, strictly anaerobic, nonmotile and non-spore forming. A striking characteristic was the presence of ¢laments arising from the cells, which appeared to connect cells together leading to aggregates in the mucin-containing medium. The16S rDNA gene sequence analysis revealed that the strain is distantly related to the previously described genera Verrucomicrobium and Prosthecobacter. On the basis of physiological and molecular properties the isolate is considered to represent a new genus and a new species, for which the name Akkermansia muciniphila gen. nov., sp. nov. is proposed in honour of a renouned dutch microbiologist Antoon D.L Akkermans.

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P4^150 ALKALIBACTERIUM SACCHAROVORANS GEN. NOV., SP. NOV. ^ A NEW ALKALIPHILIC SACCHAROLYTIC ANAEROBIC BACTERIUM FROM CELLULOLYTIC COMMUNITY OF LAKE NIZHNEE BELOE (SOUTH-EASTERN SIBERIA, RUSSIA) E. S. Garnova, T. N. Zhilina, T. P. Tourova Laboratory of Relict Microbial Communities, Institute of Microbiology RAS, Prospect 60-letiy Octyabrya, 7/2, 117312 Moscow, Russia Alkaliphilic anaerobic microbial community of epicontinental soda lakes represents an example of natural system where organic matter is completely decomposed (Zavarzin et al., 1999). Saccharolytic pathway, which connects hydrolytic and secondary anaerobic bacteria, is one of the main metabolic routes in the community. Saccharolytic anaerobic alkaliphilic microorganisms have been isolated from geographically di¡erently-located soda lakes and involve a variety of phylogenetic groups: spirochetes (Zhilina et al., 1996), haloanaerobes (Zhilina et al., 2001a), bacilli (Zhilina et al., 2001b) and clostridia strains (Jones et al., 1998). Central Asian soda lakes represent an extreme type of prairie lakes with considerable input of cellulose material. During the study of anaerobic cellulose decomposition in these lakes (Kevbrin et al., 1999) several strains of anaerobic alkaliphilic microorganisms that grew on glucose were isolated (Tourova et al., 1999). One of them was a member of clostridia cluster XI where it represented a new genus named Anoxynatronum sibiricum gen.nov., sp.nov. (Garnova et al., in press). The other three strains had the high level of DNA-homology (96-100%). Strain Z-79820 was studied in further detail. It was Gram positive asporogenous non-motile short rod. The new isolate was true alkaliphile : growth occurred from pH 7.2 to 10.2 with an optimal pH of 9.0. Strain Z-79820 obligately depended on Na+ but needed neither HCO3-/CO3- nor Cl-. It was halotolerant and mesophile, which ¢tted the geological and climatic conditions of its habitat. The organism fermented mono- and disaccharides with production of acetate, ethanol, formate, H2 and CO2. The new bacterium fell into the clostridium cluster XV where it formed an individual branch. Among the representatives of this cluster, it was the ¢rst alkaliphilic organism. According to growth characteristics and genotypic traits of strain Z-79820 we proposed the new genus and species with the name Alkalibacterium saccharovorans.

P4^151 NOVEL GROUPS OF CULTURABLE BACTERIA ARE NUMERICALLY DOMINANT IN THE PORCINE GASTROINTESTINAL TRACT O. Hojberg, L. L. Mikelsen and B. B. Jensen Department of Animal Nutrition and Physiology, Danish Institute of Agricultural Sciences, PO Box 50, DK-8830 Tjele, Denmark Fast elucidation of bacterial diversity by 16S rDNA cloning and sequencing reveal the gaps in our culture collections and expedite the quest for important, yet uncultured groups of bacteria. We analysed V2000 porcine gastrointestinal tract isolates and classi¢ed them into so far V120 operational taxonomic units (otus) on the basis of morphology, physiology and 16S rDNA sequences. Despite high diversity, 75% of the isolates were covered by eighteen otus (each constituted s 1%) representing twelve genera. This could be due to selectivity of culturing, but is actually in accordance with a comprehensive 16S rDNA clone library (Appl. Environ. Microbiol., 2002, 68:673690), where 23 of 375 de¢ned otus ( s 1% each) made up 52% of 4270 clones. Seven of our eighteen dominant otus showed less than 97% similarity to identi¢ed species in the 16S rDNA databases, however most isolates matched porcine bacterial clone sequences. Clearly, some dominant clone library otus were not among our isolates. On the other hand, one of our dominant species (4% of colon isolates) belonging to the Sporomusa subgroup showed less than 97% similarity to any database sequence. The latter as well as other unknown species were major producers of butyrate and lactate. These metabolites are considered crucial for preventing pathogen invasion and development of colon cancer. To our knowledge, the present work is one of the most comprehensive studies on the culturable microbiota of monogastrics and indicates that major groups of so far unidenti¢ed bacteria potentially involved in stabilising a healthy gut can actually be cultured and characterised.

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P4^152 ORANGE-PIGMENTED YEASTS FROM THE PHYLLOPLANE: TAXONOMIC CHARACTERISATION AND DIRECT MOLECULAR DETECTION ON LEAVES L ¤ J. Inacio, A. Fonseca and I. Spencer-Martins ¤ Centro de Recursos Microbiologicos (CREM), Biotechnology Unit, Faculty of Sciences and Technology, New University of Lisbon, 2829-516 Caparica, Portugal An investigation of the mycobiota on leaves from selected plant species (Acer monspessulanum, Quercus faginea, Cistus albidus, Pistacia lentiscus and Osyris quadripartite) col¤ lected at the ``Arrabida Natural Park'', an ecosystem of Mediterranean characteristics in Portugal, yielded about 830 yeast isolates. Five percent of these isolates (42 strains) produced orange-pigmented colonies. They were assigned to the Tremellales lineage of the Hymenomycetes using a combination of phenotypic and molecular methods, which included PCR ¢ngerprinting and rDNA sequence analysis. According to the molecular data, part of the phylloplane isolates was related to species of Dioszegia, whereas others clustered in di¡erent clades within the Tremellales. Among those representing undescribed species, Cryptococcus cistialbidi sp. nov. was notable for its exclusive occurrence on Cistus albidus at high frequencies. A speci¢c oligonucleotide probe, based on a region of the 26S rRNA gene, was designed for this yeast, and £uorescently labelled for direct detection and quanti¢cation of Cr. cistialbidi sp. nov. on the phylloplane by in situ hybridisation. J.I. receives a PhD grant (Praxis XXI/BD/19833/99) from " ``Fundacao para a Ciencia e a Tecnologia'', Portugal. °< P4^153 ISOLATION AND CHARACTERIZATION OF A HIGHLY XYLANOLYTIC RUMEN BACTERIAL STRAIN Mz5, TYPE STRAIN OF A NEW SPECIES PSEUDOBUTYRIVIBRIO XYLANIVORANS M. Zorec(1), J. Kopecny(2), Y. Kobayashi(3), F. V. Nekrep(1) and R. Marinsek-Logar(1) (1) University of Ljubljana, Biotechnical Faculty, Zootechnical Department, Domzale, Slovenia; (2) Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Prague, Czech Republic; (3) Hokkaido University, Graduate School of Agriculture, Sapporo, Japan Several bacterial isolates were obtained from the rumen £uid of a cow using the medium with oat spelts xylan. Strain Mz5 showed the highest cell-associated xylanolytic

activity, it was curved rod with one subpolar £agellum, stained gram-negative, was anaerobic, non-spore-forming and produced butyrate as main fermentation product on di¡erent carbohydrates. Other fermentation products were lactate and hydrogen gas and during growth acetate was utilized. High activities of xylanase, proteinase, pectin hydrolase and DNase were determined. The complete 16S rDNA sequence was obtained and phylogenetic relationships were determined. On the basis of other comparative phenotypic and genotypic analyses (FAMES, RFLP, 16S rDNA sequences, mol% G+C) for the group of 17 closely related strains assignment to a new species was proposed ^ Pseudobutyrivibrio xylanivorans with Mz5 as a type strain. Xylanolytic activity of Mz5T was mainly cell-associated probably due to the production of extracellular polysaccharides that hinder di¡usion of the enzymes. Mz5T produces 11 electrophoretically multiple xylanases (MW ranging from 27 to 146 kDa). Xylanase activity was strongly inducible with oat spelts xylan (130-times) and 146 kDa xylanase was proved to be contitutive. Strain Mz5 could be used as a probiotic in animal feed because of its high xylanolytic activities and production of butyrate that has bene¢cial e¡ects on colonocytes. Competitive ¢tness is furthermore assured by the ability of fermentation of di¡erent substrates and fast adaptability. P5^1 CLONING AND STABLE EXPRESSION OF THE aACETOLACTATE DECARBOXYLASE GENE FROM BACILLUS LICHENIFORMIS IN YEAST X. Bao, Y. Qin, H. Zheng, A. Hou, G. Yang, and D. Gao State Key Laboratory of Microbial Technology, Department of Microbiology, Shandong University, Jinan, P.R. China 250100 The genomic library of Bacillus licheniformis was constructed using plasmid pUC19 in Escherichia coli. The gene encodinga-acetolactate decarboxylase(a-ALDC) was isolated from the library by directly detecting enzyme activity on the agar plate. The nucleotide sequence of a 1.6kilobase DNA fragment containing the a-acetolactate decarboxylase gene was determined. An open reading frame (GenBank accession number AF428095) that may encode a protein composed of about 265 amino acids was found. The a-acetolactate decarboxylase from B. licheniformis was puri¢ed and characters of the enzyme were studied, the results indicate that its pI was 4.5 and the enzyme showed optimal activity at pH 6 and 4. The DNA fragment coding for a-acetolactate decarboxylase was placed under the control of the phosphoglycerate kinase (PGK) promoter and terminator of the Saccharomyces cerevisiae. Then the aALDC gene expression cassette was ligated to the multiple integration plasmid pMIRY2 which has the rDNA region

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as the integrating site. The new recombinant plasmid pMIRY2-ALDC, which was linearized at the SmaI site in the rDNA region, was co-transformed into the beer yeast industrial strain together which a plasmid containing G418 gene. The multiple integration of the plasmid in the transformants was proved by Southern blot. Yeast cells containing pMIRY2-ALDC plasmids showed a-acetolactate decarboxylase activity. The result of laboratory -scale fermentation reveals that the diacetyl concentration in wort was signi¢cantly lower by recombinant strain than by the host strain. P5^2 ARCHICTECTURAL PROPERTIES OF ARAR, THE KEY REGULATOR OF ARABINOSE UTILIZATION IN BACILLUS SUBTILIS ¤ I. Franco(1), R. G. Saraiva(1) and I. de Sa-Nogueira(1,2) ¤ ¤ (1) Instituto de Tecnologia Qu|mica e Biologica, Universi¤ blica, Apartado 127, dade Nova de Lisboa. Avenida de Repu " 2781-901 Oeiras, Portugal; (2) Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2825 Monte de Caparica, Portugal The transcriptional factor AraR plays an important role in carbohydrates catabolism in Bacillus subtilis. AraR is the key regulator of arabinose utilization controlling transcription from the araABDLMNPQ-abfA metabolic operon and the araE/araR divergent unit by di¡erent mechanisms, which allow a tight and £exible control of the system. Additionally, AraR regulates the utilization of xylose and galactose since the main transporter of arabinose AraE is a non-speci¢c permease also responsible for the transport of those carbohydrates into the cell. The amino acid sequence of AraR suggests a chimeric organization for the protein. A small N-terminal domain homologous to the GntR family of bacterial repressors is involved in DNA-binding, whereas a larger C-terminal region typical of the LacI/GalR family of bacterial regulators binds the inducer and is also responsible for oligomerization. Recently, a subdivision of the GntR family according to the structural, phylogenetic and functional characteristics of the C-terminal domain failed to include AraR suggesting the emergence of a new sub-family typi¢ed by AraR. In this work, a collection of AraR mutants displaying single amino acid substitutions was obtained by random mutagenesis of the araR allele and in vivo screening for defects, either associated with a constitutive phenotype or ability to respond to the inducer arabinose. These mutant proteins were characterized by in vivo regulation studies. De¢nition of the AraR functional domains and its implications in the mechanistic mode of action of the repressor will be discussed.

P5^3 REGULATORY MECHANISMS OF THE ARABINANDEGRADING COMPLEX IN BACILLUS SUBTILIS ¤ J. M. Inacio(1), M. P. Raposo(1), L. J. Mota(1a) and I. ¤ -Nogueira(1,2) de Sa ¤ ¤ (1) Instituto de Tecnologia Qu|mica e Biologica, Universi¤ blica, Apartado 127, dade Nova de Lisboa. Avenida de Repu " 2781-901 Oeiras, Portugal; (2) Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2825 Monte de Caparica, Portugal. aCurrent Address: Biozentrum der Universitat Basel, Klingelbergstr. 50-70 CH« Basel, Switzerland. Hemicellulose is composed of xylans, arabinans, galactans and mannans, which contribute to the texture of plant tissues as structural components of the cell wall. Bacillus subtilis produces three di¡erent types of enzymes involved in degradation of the homoglycane arabinan, an endo-arabanase and two arabinofuranosidases, capable of releasing arabinosyl oligomers and arabinose from plant cell walls. These arabinan-degrading activities are able to disintegrate potato tissue and to solubilize sugar beet protopectina. The two arabinofuranosidases are most probably encoded by the last gene of the arabinose metabolic operon araABDLMNPQ-abfA, and the xsa gene, located 23-kb downstream from the operon. The abnA gene, positioned immediately upstream from the metabolic operon, most probably encodes an endo-arabanase. In vivo RNA analysis indicates that abnA and xsa are monocistronic, and transcribed from sigma-A like promoters. Furthermore, transcriptional studies showed that expression of the arabinases is induced by arabinose and arabinan, and repressed by glucose. The induction mechanism is mediated by the key regulator of arabinose metabolism, AraR. However, the regulatory mechanisms of these three genes appear to be di¡erent and its physiological implications will be discussed. This work was supported by grant POCTI/AGR/36212/ from FCT to I. S-N. J. I. was the recipient of fellowship 034/BIC/2001.

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P5^4 GENE CLONING AND CHARACTERIZATION OF CITROBACTER FREUNDII L-METHIONINE -QLYASE I. V. Manukhov(1), S. M. Rastorguev(1), G. B. Zavilgelsky(1), D. V. Mamaeva(2), T. V. Demidkina(1) (1) State Scienti¢c Center (GosNIIgenetika), 1st Dorozhnii pr. 1, 117545, Moscow, Russia ; (2) Engelhardt Institute of Molecular Biology, Vavilov str. 32, Moscow 119991, Russia L- methionine-Q-lyase (MGL) catalyzes Q-elimination of Lmethionine. The enzyme was puri¢ed from Citrobacter freundii cells. Its N-terminal sequence was determined. Using oligonucleotide primers constructed on the basis of Nterminal sequence and sequences of active center of MGL from di¡erent bacteria we obtained 500 kb PCR-product. It was cloned in pUC19. Sequences of some clones were determined. One clone contained a fragment (500 bp) with a sequence homologous to that of Pseudomonas putida MGL. EcoR1 genomic library of Citrobacter freundii was constructed in pUC18 and screened by 500 bp fragment. Two 3.0 kb identical fragments showed MGL activity while expressed in E. coli. The sequence of the 3.0 kb fragment contained two ORF. Region of 1,194 nucleotides has 56% and 61 % sequence identity with MGLs from Pseudomona putida and Fusobacterium nucleatom and thus encodes the MGL gene. It was subcloned and homogeneous MGL was obtained. Kinetic parameters of Q-elimination reactions for the wild type and recombinant enzymes were determined and proved to be identical. Comparison of Citrobacter freundii MGL kinetic parameters with those of MGLs from di¡erent bacteria revealed that enzymes do not di¡er in their a/nity to substrates but di¡er in rates of Q-elimination reactions. P5^5 REDOX BALANCING IN RECOMBINANT XYLOSE UTILISING SACCHAROMYCES CEREVISIAE E. Rintala, L. Salusjarvi, L. Ruohonen and M. Penttila VTT Biotechnology, Technical Research Centre of Finland, P.O. Box 1500, FIN-02044 VTT, Finland Lignocellulosic biomass is an attractive substrate to be used in the production of ethanol as a renewable energy source to replace fossil fuels. Hexose sugars of lignocellulosics are readily fermented by many microorganisms, whereas pentoses, the major constituent hemicellulose are a challenge in the process to make it economically feasible. S. cerevisiae, a well-known fermentation process organism,

cannot naturally utilise xylose although it is able to ferment xylulose, an isomer of xylose. Introduction of Pichia stipitis genes encoding xylose reductase (XR) and xylitol dehydrogenase (XDH) gives S. cerevisiae the ability to utilise xylose. Over-expression of its own xylulokinase-encoding gene (XKS1) further enhances xylose consumption [1]. The XR and XDH reactions generate a cofactor imbalance into the cell because XR has a preference for NADPH, whereas XDH is speci¢c for NAD+. One attempt to solve this imbalance has been the introduction of a transhydrogenase cycle into the cells, to convert NADP+ and NADH, the products of XR and XDH, to NADPH and NAD+, the substrates for XR and XDH [2]. We have studied a of transhydrogenase cycle, which relies on overexpression of malic enzyme. We have also used genome wide approaches to study the physiology of xylose utilisation in recombinant S. cerevisiae. We have made studies both on proteomic and genomic level to reveal novel and unpredictable changes in the metabolism of xylose fermenting yeast [3,4]. [1] Toivari et al. (2001). Metab.Eng.3, 236-249. [2] Aristidou et al.(1999). Patent Application. PCT/FI99/00185. [3] Salusjarvi et al.(2002). Yeast 20,295-314. [4] Salusjarvi et al. Manuscript in preparation. P5^6 PEROXISOME FORMATION AND REGULATION OF PEROXISOME HOMEOSTASIS IN CANDIDA PSEUDOTROPICALIS DURING VARIOUS CARBON SOURCE UTILIZATION I. Rusyn(1), O. Kulachkovsky(2), O. Moroz(2), S. Gudz(1) (1) Department of Microbiology and (2) Interdepartment Laboratory of Electron Microscopy, Biological Faculty, Ivan Franko National University of Lviv, Hrushevskoho St. 4, UA-79005 Lviv, Ukraine Microbodies (peroxisomes, glyoxysomes) play important role in yeast cell metabolism. Peroxisomes contain many of enzyme systems, through which cells are capable to metabolize various carbon compounds. Quantity of peroxisomes, their fermentative maintenance changes according to change of cultivation medium. Regulation of peroxisome homeostasis realize by means of induction of peroxisome biogenesis and catabolite regulation: repression of microbody formation and also peroxisome degradation. Peroxisome formation and regulatory mechanisms of their homeostasis in C. pseudotropicalis were insu/ciently investigated. Aime of this work was investigation of microbody formation in presence of various carbon compounds and regulatory mechanisms of their homeostasis in C. pseudotropicalis. Induction of microbody biogenesis during ethanol, ethylamine, lactose, oleate and hy-

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drocarbons utilization was showed. Microbodies were revealed on each growth phase during lactose utilization in aerobic conditions. Results demonstrated noncomplete glucose catabolite repression of this organelles biogenesis. De¢cient provide of culture with oxygen was the cause of limited utilization of derived from hexoses ethanol. In these conditions of nutrient deprivation despite on presence in cultivation medium of ethanol (as inductor) microbodies biogenesis slow down and their autophagic degradation take place. Ethylamine utilization only as nitrogen source but not carbon source, which is result repressive ``ammonium e¡ect'', was showed. Cells needed addition of carbone source ^ glucose to cultivation medium for ethylamine utilization. Large peroxisomes were revealed in yeast cells during oleate and ethylamine (in presence of glucose) utilization. C. pseudotropicalis are capable to utilize hydrocarbons. Small peroxisomes were revealed in cells during hydrocarbon utilization. P5^7 STRUCTURAL AND FUNCTIONAL ANALYSIS OF NITRILE DEGRADATION CLUSTER FROM RHODOCOCCUS RHODOCHROUS M8 L. Ryabchenko, E. Kotlova, G. Larikova, A. Novikov, D. Podchernjaev, G. Chestukhina, A. Yanenko, V. Debabov Institute of Genetics and Selection of Industrial Microorganisms, 1-st Dorozhny proezd, 1, 113545, Moscow, Russia R. rhodococcus M8 is used as a biocatalyst for industrial production of acrylamide. E/ciency of the catalyst is dependent upon two enzymes ^ cobalt-dependent nitrile hydratase (NH), catalysing transformation of acrylonitrile to acrylamide, and amidase (AM), hydrolysing amide to acrylic acid. NH and AM were isolated from strain M8 and its mutants producing enzymes in great amounts. NH is a heteromer of two types of subunit (26 and 23 kD), while AM comprises 4 identical subunits (Mb 42 kDa). The best substrates for NH and AM are short-chained aliphatic nitriles and amides. NH and AM are completely inhibited by Hg and Ag ions, which are typical reagents on sulfohydrylic group. Activity of NH, in contrast to AM, was decreased in presence of PMSF ^ an inhibitor of serine proteinases. Maximum activity of NH and AM was observed at 45C and 55-60C, respectively. We cloned and sequenced the chromosomal loci that control synthesis of both enzymes in R. rhodochrous M8. One of them, designated NH locus, contains genes coding subunits of NH and regulatory genes controlling induction of NH and AM. The amidase gene was found in the other locus which was not linked to NH genes. The AM gene from M8 had a high level of homology with aliphatic amidases found in Pseudomonas aeruginosa. Thus, the absence of operon structure for NH and AM genes is the key feature

of the strain M8. Expression of NH genes was shown in Rhodococcus erythropolis, and expression of AM gene in E. coli. We introduced amino acid changes into the Cobinding site of NH. Although these mutations did not a¡ect positions which directly interact with Co, all mutants were inactive. The mutant strain M33, which possesses high constitutive synthesis of NH and is defective in AM synthesis, has all regulatory genes in the NH locus deleted. M33 now will be utilized as an industrial biocatalyst to attain 50 % solutions of acrylamide, which are applicable for polymerization. without additional concentration. P5^8 CLONING AND EXPRESSION OF MYO-INOSITOL OXYGENASE FROM THE YEAST CRYPTOCOCCUS NEOFORMANS W. A. Schroeder, S. C. McFarlan and P. M. Hicks Biotechnology Development Center, Cargill Inc., P.O. Box 5702, Minneapolis, MN, 55440-5702, USA The oxidation of myo-inositol to D-glucuronate, the ¢rst dedicated step in myo-inositol catabolism is catalysed by the enzyme myo-inositol oxygenase (MIO). This enzyme could be used to develop novel biosynthetic routes to vitamin C, D-glucaric acid, and D-glucurono-Q-lactone directly from D-glucose or myo-inositol by fermentation in yeast or bacteria. The 35 kDa enzyme was puri¢ed from the yeast Cryptococcus terreus by a combination of anion exchange chromatography, a/nity chromatography, and 2-D gel puri¢cation. A peptide ¢ngerprint of the puri¢ed protein was used to identify mio homologs and ESTs in tissues of fungi, higher plants, and in kidney tissues of mammals. Homologs of mio were cloned from human, rat, Arabidopsis thaliana and Cryptococcus neoformans cDNA libraries. Recombinant MIO was produced in yeast, bacterial and insect cells. Several groups have identi¢ed mammalian homologs of these genes that were expressed in the cortex of kidneys and that repression of mio was associated with acute renal failure, diabetic nephropathy and incorrect embryonic nephrogenesis. In plants, mio expression has been found in response to environmental stresses and during seedling development. Thus, in addition to its value in providing novel routes to fermentation products from D-glucose, myo-inositol oxygenase may be useful in the treatment of kidney disease and in the development of crops with improved agronomic traits.

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P5^9 DISRUPTION OF THE GENE ENCODING TRYPSINLIKE ENZYME FROM STREPTOMYCES RIMOSUS í ¤ > M. Staudohar Kozjan(1), H. Petkovic(2), M. Legisa(3) (1) Krka d.d., Novo mesto, Slovenia; (2) Biotica Technology Limited, Cambridge, United Kingdom; (3) National Institute of Chemistry, Ljubljana, Slovenia Bacterium Streptomyces rimosus is known to produce at least ¢ve extracellular proteases. A 0.4 kb DNA fragment of a trypsin-like gene was ampli¢ed using primers designed on the basis of the sal gene from Streptomyces lividans. Genomic DNA from S. rimosus was used as a template in the polymerase chain reaction. The sequence of the fragment showed 72 % similarity to trypsin-like gene from Streptomyces griseus. The 0.4 kb fragment was used to construct a vector for gene disruption experiment. Four S. rimosus thiostrepton resistant transformants, SRT2, SRT3, SRT5 and SRT6, displaying wild type morphology were selected. Southern hybridization analysis revealed that transformants SRT2 and SRT3 shared the same restriction/integrational pattern, but subsequently lost thiostrepton resistance.The plazmid had integrated by multiple copies into 19 kb SstI fragment of the recipient strain. In the case of SRT5 transformant, the plasmid or at least its part apparently integrated into 3,2 kb SstI fragment of the recipient strain. No large-scale DNA ampli¢cation/rearangement could be detected on the gel electrophoresis due to genetic instability that was well documented for a number of other gene disruptions in streptomycetes. The total proteolytic activity of SRT5 was lower than in the parent strain and SRT2, SRT3, SRT6. P5^10 OPTIMISATION OF THE TRANSCRIPTION TUNING CONCEPT FOR LACTOSE INDUCTION G. Striedner, V. Scherrer, A. Landberg, K. Durrschmid, M. « Cserjan-Puschmann, F. Potschacher, J. Kern and K. Bayer « Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Nussdorfer Lande « 11, 1190 Vienna, Austria Optimal exploitation of the metabolic potential of the cell factory provides an e¡ective approach to obtain high yield of recombinant protein in bacteria. Since the thereto used strong expression vectors overburden host cell metabolism, adaptation of recombinant gene expression to the capabilities of the synthesis machinery is essential. Therefore a novel concept of transcription rate control based on continuous supply of limiting amounts of inducer in a

constant ratio to biomass was developed and implemented in a fed batch process with carbon limited exponential feed regime. As a whole, this approach proved to be a very e/cient strategy for the optimisation of recombinant protein production processes due to increased yield, enhanced process reproducibility and controllability. Under these conditions the substitution of IPGT by lactose is enabled, thereby complying with regulatory issues of the production of recombinant proteins on industrial scale. However, the concept of tuning transcription rate, which was developed for induction with IPTG has to be adapted to the use of lactose, whereby the main issue is maintaining the appropriate induced state versus lactose consumption. To gain optimal yields the behaviour of the lac based T7 expression system during lactose induction and the interaction of L-galactosidase, which is controlled by the lac promotor as well must be investigated. Therefore mRNAs of T7-RNA polymerase, L-galactosidase and of the target protein were quanti¢ed by real time PCR, L-galactosidase was quanti¢ed by ELISA and an activity assay. These data provide signi¢cant information for optimised process design using lactose as inducer. P5^11 HYDROGENASES OF THE MARINE FILAMENTOUS, NON-HETEROCYSTOUS, CYANOBACTERIUM LYNGBYA MAJUSCULA CCAP 1446/4 ^ BIOTECHNOLOGICAL APPLICATIONS E. Leitao(1), F. Oxelfelt(1), P. Oliveira(2), A. M. Bur< ja(3), P. C. Wright(4) and P. Tamagnini(1,2) (1) IBMC ^ Cellular and Applied Microbiology Unit, University of Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal; (2) Dept. Botany, Fac. Sciences, Univ. Porto, Rua do Campo Alegre 1191, 4150-181 Porto, Portugal; (3) Dept of Chemical and Process Engineering, Heriot-Watt University, Edinburgh EH14 4AS, UK; (4) Biological and Environmental Systems Group, Dept. Chemical and Process Engineering, University of She/eld, She/eld S10 2TN, UK Molecular hydrogen has been purported as a potential energy source and carrier for the 21st century. Hydrogen can be produced using both chemical and biological means. The use of various groups of microorganisms, such as cyanobacteria, for H2 production has received increased attention within the past decade. The majority of research within this ¢eld has focussed on heterocystous cyanobacteria, to the detriment of both unicellular and ¢lamentous non-heterocystous strains. Here we report preliminary research focussed on hydrogenases structural and accessory genes within the marine, ¢lamentous, non-heterocystous cyanobacterium, Lyngbya majuscula CCAP 1446/ 4. L. majuscula was found to contain the structural genes

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(hupSL) encoding an uptake hydrogenase, the gene (hoxY) encoding the small subunit of the hydrogenase moiety of the bi-directional enzyme and the accessory genes hypD and hypF. In silico analysis highlighted two sets of long repetitive sequences within the hupSL intergenic region. 5'RACE experiments revealed the presence of a transcription start site, putatively for the hupSL operon, 59 bp upstream of the hupS start-codon. Hydrogen uptake activity was con¢rmed via H2 electrode measurements. In parallel, large scale production of L. majuscula biomass was performed, as a future lead to integrated growth and production of hydrogen and other natural products. P5^12 STRUCTURE AND FUNCTIONAL ANALYSIS OF A LYCOPENE L-MONOCYCLASE GENE ISOLATED FROM A UNIQUE MARINE BACTERIUM PRODUCING MYXOL M. Teramoto(1), S. Takaichi(2), Y. Inomata(1), H. Ikenaga(1) and N. Misawa(1) (1) Marine Biotechnology Institute, Kamaishi-shi, Iwate 026-0001, Japan ; (2) Nippon Medical School, Biological laboratory, Kosugi-cho, Nakahara, Kawasaki-shi 211-0063, Japan Cyclization of the linear, pink-colored carotenoid lycopene (i,i-carotene) is a pivotal branching point in the divergent carotenoid biosynthetic (crt) pathways. In the present study, we found for the ¢rst time a gene coding for lycopene L-monocyclase, which is responsible for the synthesis of Q-carotene (L,i-carotene) from lycopene. The crt gene cluster including this lycopene L-monocyclase gene (designated crtYm) was isolated from a unique marine bacterium, strain P99-3, that produces myxol. The sequence analysis revealed that this cluster contained the genes homologous to the known crtI, crtB, crtZ, crtY, and crtA genes in addition to two unknown open reading frames, and that the crtA homolog was divergently organized relative to the others in a tail-to-tail orientation. CrtYm, a CrtY homolog, metabolized lycopene to Q-carotene in addition to a small amount of L-carotene (L,L-carotene), which was con¢rmed by deletion/expression analysis of the crtYm and by subsequent analysis of the metabolites from lycopene based on the retention times on high-performance liquid chromatography, spectral data through UV-visible absorption, and mass spectrometry. CrtYm activity was markedly inhibited by coexpression of orf1, which was localized upstream of crtYm, suggesting that the myxol synthetic pathway is controlled by an unknown Orf1-mediated mechanism in strain P99-3. A phylogenetic analysis showed the lycopene L-monocyclase from strain P99-3, CrtYm, to be distant not only from the lycopene L-

cyclases of higher plants and a cyanobacterium but also from lycopene L-cyclases of other eubacteria. P5^13 HYDROGEN PHOTOPRODUCTION BY PURPLE BACTERIA. HAS IT A PRACTICAL POTENTIAL? A. A. Tsygankov Institute of Basic Biological Problems RAS, Pushchino, Moscow Region, 142290, Russia Hydrogen can be produced by purple bacteria under the light via nitrogenase system. Optimal conditions for hydrogen photoproduction (temperature, pH, light intensity, electron donor excess, nitrogen de¢ciency, and anaerobic conditions) are already de¢ned. The rates of the reaction as high as 3.6 l per l of matrix in one hour by immobilized cells were measured. However, before the practical consideration of photobiological hydrogen production many scienti¢c and technological problems should be solved. Some of them are: low e/ciency of light energy bioconversion; sensitivity of key enzymes (nitrogenase and hydrogenase) to oxygen; low saturating light intensity comparing with sun light ; low speci¢c rates of hydrogen photoproduction; decrease of speci¢c rates during scale-up procedure. A numerous reviews and research articles were published last decades. Simultaneously, the development of cultivation regimes, immobilization procedures, and photobioreactors brings some methods from the state-of-the art to the technology. In this presentation the application of modern technologies for solution of problems shown above is discussed.

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P6^1 ANTIMICROBIAL RESISTANCE PHENOTYPES IN MRSA STRAINS ISOLATED FROM INVASIVE INFECTIONS IN ROMANIA ^ 1998 ^ 2001 I. Codita(1), V. Dumitrescu(1), L. Grigore(1), E. Moldoveanu(1), O. Dorobat(2), I. Nistor(3), R. Papagheorghe(4), N. Popescu(4), M. Ghita(5), E. Mitache(6) and T. Turcu(7) (1) Cantacuzino Institute, Splaiul Independentei 103, Bucharest, R-70.100 Romania; (2) Infectious and Tropical Diseases Clinical Hospital `` Victor Babes'', Bucures°ti; (3) Emergency Pediatric Clinical Hospital `` Gr. Alexandrescu'', Bucures°ti; (4) Clinical Hospital ``Coltea'', Bucures°ti; (5) Clinical Institute Fundeni, Bucures°ti; (6) ``Matei Bals'' Institute for Infectious Diseases, Bucures°ti; (7) Infectious and Tropical Diseases Clinical Hospital, Iasi 40 strains were isolated from bloodcultures and identi¢ed as methicillin resistant Staphylococcus aureus (MRSA), of which 20 strains were isolated and characterised in 1998 and 20 in 2001.The study of antimicrobial resistance phenotypes using the main antimicrobials active on staphylococci revealed the following: (1) 12 from the 20 strains isolated in 1998 and 8 from those isolated in 2001 showed high resistance to methicilline (Thomasz 1991); (2) of the strains isolated in 1998, 2 were classi¢ed in the ``reduced susceptibility'' or ``GISA candidate'' MRSA (CMI = 4 mg/L) category. All strains isolated in 2001 had CMI values 9 2 mg/L ; (3) 8 of the strains isolated in 1998 and 6 of those isolated in 2001 showed inductible resistance to erythromycin ( ER My S phenotype); one strain isolated in 2001 showed intermediate phenotype to erythromycine (E) and clyndamycine (My) ; (4) one strain in each year showed an KR TS GS / APH (3'') III amynoglicoside phenotype, while the rest of the strains harboured the KR TR GR / AAC (6') ^ APH (2'') phenotype, which is responsible for resistance to all aminoglicosides ; (5) 18 strains in each of the two groups were resistant to £uoroquinolones. This study which examined a greater number of strains isolated from invasive infections yielded information on the susceptibility level and on the prevalence of the acquired resistance to the main classes of antimicrobial substances that are active on MRSA strains. These data can be used both for de¢ning local S/I/R critical values and for de¢ning the activity spectra and the indications for the use of these classes of antimicrobials in invasive infections, in Romania. Identifying the rare resistance phenotypes can help orient the research in order to clarify the resistance mechanisms before resistance spread and for adopting a more appropriate regementation looking to the politics of antimicrobial substances utilisation.

P6^2 ANTIMICROBIAL RESISTANCE AMONG DIARRHEAGENIC ESCHERICHIA COLI IN SERBIA N. Galic(1) and M. Cobeljic(2) (1) Institute of Public Health of Serbia, Belgrade, Yugoslavia ; (2) Military Medical Academy, Belgrade, Yugoslavia The objective of this study was to determine the frequency of antibiotic resistance among diarrheagenic E. coli isolated from feces of patients with diarrhea and healthy persons in Serbia from 1982 to 1999. In total, 884 E. coli strains were identi¢ed and all of them were tested for the presence of all known virulence factors of diarrheagenic E. coli. Among them, 235 (26.6%) strains of enterotoxigenic (ETEC), 26 (2.9%) strains of enteropathogenic (EPEC) and 48 (5.4%) strains of di¡usely adherent (DAEC) E. coli were detected. Enteroaggregative, shigatoxin producing and enteroinvasive E.coli were not found. The remaining E. coli strains did not express virulence factors. All the strains were tested for antimicrobial resistance by disc-di¡usion method. Resistance to one or more antibiotics was found in 113 (48%) ETEC, 26 (100%) EPEC, 38 (79%) DAEC and 575 (60%) strains nonpathogenic E. coli, what was signi¢cantly di¡erent when comparing these groups of strains. Resistance was not detected to quinolones and third generation cephalosporins in diarrheagenic E. coli. ETEC, EPEC and DAEC strains were resistant to two or more antibiotics at a similar rate (84%, 96% and 92%, respectively). The most frequent resistance patterns were ApRClR and StRApRClR in ETEC, StRApRClR and StRTcR in EPEC, StRApRTcRT/SR and ApRClR in DAEC. Common ¢nding of multiple antibiotic resistance among diarrheagenic E. coli in this study may be due to antimicrobial selective pressure and stability of antibiotic genes in the environment. P6^3 ANTIMICROBIAL SUSCEPTIBILITIES OF HAEMOPHILUS INFLUENZAE AND HAEMOPHILUS PARAINFLUENZAE RESPIRATORY TRACT ISOLATES IN A LARGE GREEK HOSPITAL S. Kanavaki, E. Faviou, S. Karambela, M. Makarona, E. Eustathiadou, E. Alexandraki, P. Koumantakis, A. Pefanis Sotiria General Hospital, Athens, Greece A total of 247 isolates of Haemophilus spp [119 H. in£uenzae (HI), and 128 H. parain£uenzae (HP)] were recovered in our hospital laboratory from sputum cultures, from September 2001 to September 2002, and were char-

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acterized with respect to beta-lactamase production, and the in vitro activities of ten antimicrobial agents, by applying the Kirby-Bauer method. The overall percentages of HI isolates found to be resistant to amoxicillin, amoxiclav, cephalothin, cefuroxime, cefotaxime, tetracycline, erythromycin, cotrimoxazole, chloramphenicol, and cipro£oxacin were as follows : 11, 0, 2.5, 0, 0, 4, 50, 27, 0, and 0, respectively. Regarding HP the respective percentages were: 7, 0, 3, 0, 0, 1, 58, 9, 0, and 0. For HI the prevalence of beta-lactamase production was 11%, while for HP was 7%. All beta-lactamase positive isolates were resistant to ampicillin. None of the beta-lactamase negative isolates were resistant to ampicillin (BLNAR). This is in contrast with reports from Spain and USA, where BLNAR strains represent 9.3% and 2.5% of the isolated HI strains. Our ¢ndings demonstrate that antimicrobial resistance pro¢les of HI and HP di¡er between Greece and other EU countries. Most of the EU countries (with the exception of Germany and Italy) report higher percentages of resistance to amoxicillin, amoxiclav, and cotrimoxazole. In contrary, the frequency of erythromycin resistance in Greece, is very high. P6^4 ANTIMICROBIAL SUSCEPTIBILITY OF VIBRIO PARAHAEMOLYTICUS IN KOREA, 1997 TO 2002 J. Kim, S. Kim, H. Jang, H. Nam, Y. Kang, B. Lee Lab. of Enteric Infections, Dept. of Microbiology, National Institute of Health, Nokneon-Dong, Eunpyeong-Gu, Seoul, Korea 122-701 Vibro parahaemolyticus is a major agent of diarrheic disease in Korea. In this study we reviwed the antimicrobial susceptiblility patterns of V. parahaemolyticus to L-lactams, and quinolones. A total of 3,319 isolates of V. parahaemolyticus were received by the national public health network of Korea from 1997 to 2002. Allowing for epidemiological properties, 250 strains among these isolates were selected. Their susceptibility to nalidixic acid (NA), nor£oxacin (NFX), or£oxacin (OFX), spar£oxacin (SPX), and cipro£oxacin (CIP) was tested by agar dilution method. The disk di¡usion method was adopted for the susceptibility to ampicillin (AM), ampicillin/sulbactam (SAM), cefoxitin (FOX), cephalothin (CF), amoxicillin/ clavulanic acid (AMC), azteonam (ATM), ceftriaxone (CRO), ticarcilli (TIC), ceftazidime (CAZ), cefotxime (CTX) and trimethoprim/sulfamethoxazole (SXT). The susceptibility of tested V. parahaemolyticus was 42.9% to AM, 98.2% to SAM, 98.6% to FOX, 95.2% to CEP, 98.0% to AMC, 66.7% to ATM, 98.0% to CRO, 14.3% to TIC, 98.6% to CAZ , and 98.6% to CTX. DNA sequence analysis of the quinolone resistance determining regions (QRDR) of gyrA and parC genes was carried out for the strains which is weakly susceptible to £uoroquinolones.

The V. parahaemolyticus isolates showed a marked decrease in susceptibility to SPX measured by geometricmean MICs, reduced from 57.9% in 1997 to 0% in 2002, and other £uoloquinolones showed similar tendency. Many tested strains have a silent mutation in gyrA and parC gene, but no amino acid substitution was observed. And no isolates showed decrease in susceptibility to SXT. In this study we have not observed an important resistance pattern to all the tested antibiotics, but we may predict the emergence from the decreasing pro¢le of susceptibility, emergence of resistant strain is predicted. P6^5 MUTATION IN gyrA, gyrB, parC AND parE GENES OF QUINOLONE RESISTANT SALMONELLA SPP. ISOLATED IN KOREA J. Kim, S. Kim, H. Jang, Y. Kang, B. Lee Lab. of Enteric Infections, Dept. of Microbiology, National Institute of Health, Nokneon-Dong, Eunpyeong-Gu, Seoul, Korea 122-701 Salmonella spp. strains obtained during 1997 to 2001 were tested for their susceptibilities to quinolones. Among the tested strains, 6 quinolone-resistant strains were con¢rmed (Salmonella Typhi; 1 strain, Salmonella Enteritidis; 5 strains). They were highly resistant to nalidixic acid (MIC, 320 to 1280Wg/ml) and cipro£oxacin (MIC, 0.4 to 0.8Wg/ml). In order to characterize the quinolone-resistant isolates, sequencing of the quinolone resistance determining regions (QRDR) of gyrA, gyrB, parC and parE genes was done. gyrA gene in six quinolone-resistance strains had three mutations in di¡erent amino acid substitution codon serine-83, aspartate acid-87 and glutamate-133. The amino acid substitution in serine-83 was TCC (Ser) ! TTC (Phe). The amino acid substitutions in aspartate-87 were GAC (Asp) ! AAC (Asn) and GAC (Asp) ! GCC (Gly). The amino acid substitution in glutamate-133 was GAA (Glu) ! GGA (Gly). No mutations were found in the QRDR regions of gyrB, parC and parE genes. These ¢ndings indicated this mutation in gyrA plays an important role in acquisition of quinolone-resistance in clinical isolates of Salmonella spp

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P6^6 CHARACTERIZATION OF MULTIDRUG RESISTANCE SHIGELLA SONNEI IN KOREA 1998 TO 2002 S. Kim, J. Kim, H. Jang, Y. Kang, B. Lee

P6^7 REVIEW OF ANTIMICROBIAL RESISTANCE OF SHIGELLA FLEXNERI ISOLATED IN KOREA DURING 1998 TO 2002 S. Kim, J. Kim, H. Jang, Y. Kang, B. Lee

Lab. of Enteric Infections, Dept. of Microbiology, National Institute of Health, Nokneon-Dong, Eunpyeong-Gu, Seoul, Korea 122-701 During the last few years, there has been a continuous increase in the incidence of Shigella infection in Korea. Although the antibiotic therapy may reduce the duration of symptoms and excretion of Shigella, it may increase the risk of developing antibiotic resistance. This study was conducted in order to monitor resistant rates of Shigella sonnei isolates and explore multi-drug resistance pattern. A total of 5,542 S. sonnei strains obtained from 1998 to 2002 were tested by disk di¡usion method for their antibiotic susceptibility to ampicillin (AM), amikacin (AN), ampicillin / sulbactam (SAM), cephalothin (CF), chloramphenicol (C), cipro£oxacin (CIP), ceftriaxone (CRO), cefoxitin (FOX), gentamicin (GM), kanamycin (K), nalidixic acid (NA), streptomycin (S), trimethoprim / sulfamethoxazole (SXT), ticarcillin (TIC), tetracycline (TE), and amoxicillin / clavulanate (AMC). The type speci¢c PCR was used for the classi¢cation of ESBL encoding genes, and genes related to tetracycline and streptomycin resistance. Strains tested showed high resistance to S, TE, SXT (876%, 94.6%, 94.7%), and were susceptible to CIP, C, FOX, AN. 86.9% of tested strains were resistant to 4 or more drugs. 35.2% of strains had resistance to NA, TE, S and SXT. 1,030 strains showed resistance to AM, SAM, K, S, SXT, TIC, TE and AMC. 24 ESBL (TEM, CTX-M, and CMY types) producing strains were con¢rmed. The tetA and strA were common genes related to tetracycline and streptomycin resistance, respectively. Some of the tested NA resistant isolates have a mutation, which causes substitution of Ser-83 in gyrA. This suggests that the decreased quinolone susceptibility was due to the mutation of gyrA gene. Even though the increase of antibiotic resistance over time was not remarkable during this period, decrease in susceptibility to cephalosporins and quinolones was noticeable. These results suggest the limitation of the commonly used antibiotics, and it may emerge as a serious threat to the public health in Korea.

Lab. of Enteric Infections, Dept. of Microbiology, National Institute of Health, Nokneon-Dong, Eunpyeong-Gu, Seoul, Korea 122-701 Recently, shigellosis has reemerged as a major infectious disease in Korea. The large scale outbreaks have been in 1998 through 2002. Shigella £exneri is the second major cause of shigellosis in Korea. In this study, the patterns of antimicrobial susceptibility of S. £exneri were reviewed. A total of 396 S. £exneri strains obtained from 1998 to 2002 were tested for their antibiotic susceptibility to ampicillin (AM), amikacin (AN), ampicillin/sulbactam (SAM), cephalothin (CF), chloramphenicol (C), cipro£oxacin (CIP), ceftriaxone (CRO), cefoxitin (FOX) gentamicin (GM), kanamycin (K), nalidixic acid (NA), streptomycin (S), trimethoprim/sulfamethoxazole (SXT), ticarcillin (TIC), tetracycline (TE), and amoxicillin/clavulanate (AMC) by disk di¡usion method. Tested S. £exneri isolates showed high resistance to penicillins ; AM 96.6%, TIC 90.5%, but in combination with beta-lactamase inhibitor, showed low resistance, especially to AMC, but the resistance to AMC is increasing over time (1998, 0% to 2001, 47.1%). No ESBL producing strain was found from the tested strains. Similar resistance pattern was observed to quinolones. Although resistance rate was low ^ NA (8.6%) and CIP (0.6%), the resistance is increasing year by year. Two CIP resistant strains were isolated in 2002. And the resistance to TE and S was high (89.6% and 96.9%), but on the other hand it was low to AN (0.3%), GM (0.9%), K (2.4%), and CRO (0%). The multi-resistance patterns of S. £exneri are quite di¡erent from S. sonnei. A 96.2% of tested strains were resistant to 4 or more antibiotics, 16.9% of strains have resistance to AM, C, S, SXT, TIC and TE, and 10.1% have resistance to AM, SAM, C, S, SXT, TIC and TE. These observations of emergence of the resistant strains to commonly used antibiotics can be a serious threat to the public health in Korea.

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P6^8 SUSCEPTIBILITY OF TOXIGENIC AND NON-TOXIGENIC CORYNEBACTERIUM DIPHTHERIAE ISOLATED IN 1997-2002 TO COMMONLY PRESCRIBED ANTIMICROBIALS: A CAUSE FOR CONCERN? R. S. Kozlov(1), M. Warner(2), C. Kelly(3), M. Sukhorukova(1) and A. Efstratiou(3) (1) Institute of Antimicrobial Chemotherapy, Smolensk State Medical Academy, P.O. Box 57, 28 Krupskaya Street, Smolensk, 214019, Russian Federation; (2) Antibiotic Resistance Monitoring & Reference Laboratory, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, UK; (3) PHLS Respiratory & Systemic Infection Laboratory, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, UK Diphtheria re-emerged in countries of the former USSR in the 1990s with over 170,000 cases and 4,000 deaths reported to WHO during 1990-1998. The disease still remains endemic in many countries of South-East Asia, Africa and South America. The epidemiology of C. diphtheriae is changing as there have been many documentations of signi¢cant increases in the number of both invasive and non-invasive diseases caused by non-toxigenic strains. WHO currently recommends penicillin as the drug of choice and erythromycin as an alternative for patients with diphtheria and their close contacts. Macrolide resistance in corynebacteria has been previously documented. The susceptibility to penicillin G, amoxicillin / clavulanate, erythromycin, clindamycin, telithromycin and levo£oxacin was determined by agar dilution method on 89 toxigenic and non-toxigenic C. diphtheriae isolated from patients during the period 1997-2002. C. diphtheriae biotype mitis NCTC 11397, Staphylococcus aureus ATCC 29213, ATCC 25923 and Enterococcus faecalis ATCC 29212 were used as controls. In comparison by MIC90, telithromycin was the most active agent (MIC90 = 0.004 mg/l), followed by erythromycin (MIC90 = 0.015 mg/l), clindamycin (MIC90 = 0.125 mg/l), levo£oxacin (MIC90 = 0.125 mg/l), penicillin G (MIC90 = 0. 25 mg/l) and amoxicillin / clavulanate (MIC90 = 0.5 mg/l). Penicillin G and erythromycin retain their activity against C. diphtheriae and should be considered as a ¢rst choice in patients with diphtheria and close contacts. Telithromycin, clindamycin, levo£oxacin and amoxicillin/clavulanate demonstrated favourable in vitro activity and may be considered as the alternatives based upon relevant data from clinical trials.

P6^9 SEROTYPE DISTRIBUTION AND ANTIMICROBIAL RESISTANCE IN STREPTOCOCCUS PNEUMONIAE CAUSING INVASIVE AND OTHER INFECTIONS IN THE FAR EAST OF RUSSIA (VLADIVOSTOK) A. V. Martynova, V. B. Turcutiyucov, I. V. Strizhak, N. M. Kondrashova Epidemiology Department, the Medical University of Vladivostok, Russia Streptococcus pneumoniae continues to be a major cause of morbidity and mortality throughout the world and the Far East of Russia is not an exception. The emerging resistance to some common antibiotics compounds this problem. There arises a need to monitor the resistance pattern and map serotype distribution in di¡erent geographic locations. The present study was undertaken to determine the serotype prevalence and antibiotic susceptibility of clinically signi¢cant S. pneumoniae isolated from the patients of young age (18-20 years) with pneumonia in the State Navy Hospital (Vladivostok, Russia). A total of 120 clinical isolates from invasive and other clinically signi¢cant pneumococcal infections were serotyped and screened for susceptibility to commonly used antibiotics by the NCCLS standards and modi¢ed laboratory procedures. The majority (59.3%) of the isolates belonged to one or other of the serotypes 1, 6, 19, 5, 23 and 7. Serotype 1 was the commonest isolate from patients of meningitis and empyema followed by pneumonia. Nineteen isolates (12.6%) were non-vaccine type. Eleven (7.3%) isolates were relatively resistant to penicillin (minimum inhibitory concentration was between 0.1 and 1 microgram/ml) and 16,4% were resistant to one or more antibiotics. Resistance was distributed equally among the predominant serotypes. In conclusion : the common serotypes responsible for signi¢cant infections were similar to those reported in some other studies from Russia, with minor variations. Resistance to cotrimoxazole and tetracycline was predominant followed by chloramphenicol. Low level resistance to penicillin was observed but no isolate had absolute resistance. This calls for monitoring of resistance and mapping of serotype distribution from various parts of Russia.

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P6^10 INTEGRON PATTERNS AND SELECTED ANTIBIOTIC RESISTANCE GENES OF DT104 AND NONDT104 STRAINS OF SALMONELLA TYPHIMURIUM IN HUNGARY ¤ ¤ ¤ ¤ ¤ N. Nogrady(1), I. Gado(2), J. Paszti(2) and M. Kiraly(2) (1) Veterinary Medical Research Institute of the Hungarian Academy of Sciences, Budapest; (2) National Center of Epidemiology, Budapest The integron pattern (IP) and resistance genes of 44 DT104 and 35 non-DT104 Salmonella Typhimurium strains isolated in Hungary in 1997-1999 were studied. Di¡erent IPs were determined by 5'CS/3'CS PCR in case of the two groups. An IP system was set up which partially di¡ered from the published systems, di¡ering mainly in respect of the non-DT104 strains. The genetic background of Amp and Chl resistances of both groups was established by PCR. Characteristically : pse-1 and £o at DT104 strains, oxa-1 and cat at non-DT104 strains were found with a few exceptions. Tetracyline resistance was coded by TetB in case of non-DT104 strains which gene was not found in DT104 strains. Str/Spc resistance was coded by genes aadA in both groups.The resistance cassette content of the main integrons and their positions were determined by sequencing in case of some representative strains. The sequences of the amplicons covering the aadA genes were blast analysed at http://www.ncbi.nlm.nih.gov. In case of DT104 strains 96-100% identity was found with the aadA2 gene (accession No. AF071555) and the non-DT104 showed 96-100% identity with the aadA1 gene (accession No. AJ009819). PCR applying RH50/RH51 primers resulted in amplicons only in the presence of aadA2 gene, because the binding site of RH51 in the gene aadA1 was inactive. This fact gave the opportunity of a quick discrimination between aadA1 and aadA2. P6^11 MICROBIAL-MONITORING IN POULTRY SLAUGHTERHOUSES WITH SPECIAL CONSIDERATION OF ENTEROCOCCI S. Schmidt, W. Sixl, A. Eisner, G. Wust, G. Feierl, J. « Posch, E. Marth Hygiene Institut University of Graz, Universitatsplatz 4, « 8010 Graz, Austria Fighting infections the medicine nowadays is confronted with the problem of resistance against antibiotics more and more. Enterococci belonging to the physiological £ora

are only facultative pathogenous, however, if anti-infective treatment is required there are only few options due to natural resistance. Therefore resistance against vancomycin is of special relevance, which was described for the ¢rst time in 1988 and is seen in Austria since 1991. Especially because of possible transfer of the van-genes into isolates of Staphylococcus aureus making them resistant to vancomycin this problem is of great importance. In this connection the in£uence of resistant Enterococci in animals is discussed controversially. During HACCP investigation in a slaughterhouse for poultry the spectrum of microorganisms was examinated during half a year. In all samples Enterococci were found, nine of which were resistant to vancomycin and teicoplanin, another nine isolates of E. faecium demonstrated lack of sensitivity to Quinupristin / Dalfopristin. Furthermore we could ¢nd campylobacter sp. in 70% and salmonella sp in 25% of the samples. This decrease of salmonella in poultry is in contrast to a high level of reported cases in humans. Our investigations demonstrates the need of microbiological monitoring of food animals in connection with development and spread of antimicrobial resistance. P6^12 STREPTOCOCCAL SEROGROUPS AND SEROTYPES CIRCULATING IN ROMANIA (1999-2002). MLS RESISTANCE PHENOTYPES fl V. Ungureanu, I. Ciuca, M. Gheorghe Cantacuzino Institute, Splaiul Independentei 103, Bucharest, R-70.100 Romania The objective was to survey the circulation and the macrolide resistance of L-hemolytic streptococci in our country. The streptococcal strains were collected by the National Reference Laboratory for Streptococci between 1999 ^ 2002. The identi¢cation of streptococcal strains was performed by slide agglutination, using the co-agglutination reagents for A, B, C, G serogrouping and antisera for T serotyping ^ prepared by the Cantacuzino Institute. The susceptibility to macrolides and other antibiotics was tested by the agar di¡usion method and MIC determination by agar dilution method. Results: 55.14% of strains were group A streptococci; 23.62% group G; 5.79% group C; 5.17% group B and 10.28% were non-groupable. 46.41% of group A were isolated from respiratory infections (acute tonsilitis and scarlet fever), 7.87% from skin infections, 41.71% from healthy carriers. The origin of non-group A and non-groupable streptococci was: respiratory tract (47.20%), carriers (46.69%), other infections (3.40%). The incidence of pattern T 5/11/12/27 was predominant (56.77%), followed by the pattern 2/4/6/28 (10.50%). The resistance to erythromycin, clindamycin, tetracycline and chloramphenicol was found. 12.32% of

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group A streptococci were erythromycin resistant. The MLS resistance phenotypes (M, iMLS, cMLS) were determined by erythromycin-clindamycin double disc test. Conclusions: The group A streptococci infection still presents a health public problem in our country as all over the world. The pattern T 5/11/12/27 is predominant. The rate of erythromycin resistance is increasing. P6^13 PHENOTYPIC AND GENOTYPIC DETERMINATION OF ANTIBIOTIC RESISTANCE OF CAMPYLOBACTER JEJUNI AND C. COLI FROM HUMANS AND POULTRY ¤ T. Zorman(1), L. Herman(2), I. Berce(3), S. Uzunovic¤ (4), A. Klancnik(1), S. Smole Mozina(1) > > Kamberovic (1) University of Ljubljana, Biotechnical Faculty, Department for Food Science and Technology, Ljubljana, Slovenia; (2) Center for Agricultural Research ^ Ghent, Department for Animal Product Quality and Transformation Technology, Melle, Belgium ; (3) Institute of Public Health Nova Gorica, Nova Gorica, Slovenia; (4) Cantonal Center for Public Health in Zenica, Zenica, Bosnia and Herzegovina Macrolides and £uoroquinolones are regarded as the drugs of choice for the treatment of human Campylobacter infections. The use of antimicrobials for the treatment of infections and as growth promoters has induced antimicrobial resistance of Campylobacter spp. Since poultry is a potential source of human infections the use of antibiotics in animal production can a¡ect the lifetime of therapeutics for human use. In Slovenia, antibiotic resistance of Campylobacter spp. is still not monitored at the national level. There are some data available for clinical but not for poultry isolates. More than 150 poultry and human campylobacters were isolated in the years 2000-03, some of them were tested for susceptibility to eight antimicrobials. The resistance was studied by disc di¡usion method and MICs for cipro£oxacin and erythromycin were determined by E-tests. Higher portion of isolates was resistant to cipro£oxacin than to erythromycin. The occurrence of resistance was higher at C. coli than at C. jejuni as well as at poultry compared to human isolates. This is noteworthy as the extent of C. coli contamination of poultry in Balkan region is signi¢cant, so transfer of resistance genes to human is relieved. Susceptibility tests are not internationally standardised. To con¢rm the accuracy of our test results we used a mismatch ampli¢cation mutation assay (MAMA) PCR to detect the gyrA mutation in 30 quinolone resistant C. jejuni isolates from clinical and food environment. There was 100% correlation between results of both techniques. The PCR procedure to detect the resistance genes in C. coli is still under investigation.

P6^14 CORRELATION BETWEEN BACTERIAL VAGINOSIS AND pH VALUE OF VAGINAL SECRETION ¤ V. Vuksanovic and M. Bujko Institute of Health, Faculty of Medicine, University of Montenegro, Podgorica, Montenegro Bacterial vaginosis (BV) is disbalance of vaginal ecosystem which is caused by changing of dominant lactobacillus with association of bacteria Gardnerella vaginalis, anaerobic bacteria and Mycoplasma hominis. Normal pH value of vaginal secretion is 3.8-4.2 Pathogenic bacteria grow rapidly when pH value is pH s 5. The aim is to examine whether there is correlation between present BV and pH value of vaginal secretion and to determinate whether pH value of vagina has the practical signi¢cance for estimation of the status of vaginal ecosystem. The presence of BV in vaginal secretion was examined in 642 women by using Nugent method. The pH value of vaginal secretion was paralelly tested by using pH indicator strips. Diagnosis of Trichomonas vaginitis was made by native preparation, and Candida vaginitis was diagnosed by stained preparation methylene blue. Diagnosis of other bacterial infections was made by using appropriate base. BV was diagnosed in 120 (18.7%) women, and intermediate £ora in 17 (2.6%) women. Analysis of pH value of vaginal secretion in women with BV showed that patients with BV had increased pH values pH s 4,5 of vaginal medium and this condition was found in 118 (98.3%) patients. This correlation is statistically signi¢cant for level p 6 0.001. Trichomonas vaginitis was found in 51 (7.9%) patients, and 45 (88.2%) of them had pH s 4.5. The number of women whose microbiological test was normal was 152 (23.7%) and 123 (80.9%) of them had normal pH value ^ pH 6 4.5. Statistical signi¢cance was not determined for Candida vaginitis (pH 6 4.5 was found in 76 ^ 58.9% women). In conclusion : There is signi¢cant correlation between BV/intermediate condition and increased pH value of vaginal secretion. Since the method of determination of disbalance of vaginal ecosystem is simple, rapid, economic and e¡ective, the routine following of pH value of vaginal secretion as screening method that can suggest need for further microbiological and biochemical analysing is recomanded.

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P6^15 MULTIPLE BACTERIAL CHRONIC WOUNDS SPECIES COLONIZE

K. Gjodsbol(1), T. Karlsmark(2), B. Jorgensen(2) and K. A. Krogfelt(1) (1) Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark; (2) Bispebjerg University Hospital, Bispebjerg Bakke 23, DK-2400 Copenhagen NV, Denmark The aim of the study was to investigate the bacterial pro¢le of chronic venous leg ulcers. Forty-six patients were included in the study. All had persisting ulcers for more than three months. Of the patients 29 were female and 17 were male, age 29-91 years (median 78 years). The healing of the ulcers was followed for 8 weeks or until the ulcer healed. Every second week swaps; biopsies and ¢lter paper pads were collected. Specimens were plated on 5 % blood agar plates and anaerobic agar plates and incubated at 37C both aerobic and anaerobic. Primary isolation plates were examined after 24 hours and then incubated for at least 4 days. Bacteria were isolated and identi¢ed at species level by standard methods. More than one bacteria species were detected in close to all the samples (94,4 %). The most common species found in the ulcers were Staphylococcus aureus (isolated at least once during the study period in 97,8 % of the patients), E. faecalis (71,7 %), Pseudomonas species (58,7 %, mainly P. aeruginosa), coagulase-negative staphylococci (45,7 %), Proteus species (41,3 %), anaerobic bacteria (39,1 %), E. cloacae (37,0 %), E. coli (32,6 %) and corynebacteria (30,4 %). Identi¢cation of the bacteria species is essential for supplying the proper treatment, since colonisation may be a key parameter in sustaining chronicity in venous leg ulcers. Further investigation of the importance of the bacterial pro¢le of the chronic wounds will be performed, by correlating the bacterial pro¢le to the course of the ulcer healing. P6^16 INFECTIVE AGENTS PNEUMONIA IN EARLY CHILDHOOD

were selected in order to clear clinical, radiological and laboratory characteristics. Results: It has been proved that infective agents as causes of pneumonia were ¢nd out in 59 children (36,6%). Viruses were the most frequent cause (37 / 23.0%). Bacteria was determined in 16 (9.9%) patients and mixed (viral-bacterial) infection in 6(3.7%). In the group of the patients with viral infection there was absolute domination of adenoviruses with 70.3%, than combination of AD and In£uenzae A (16.2%) and In£uenzae A with 13.5%. Among the patients with bacterial cause of pneumonia, Staphylococcus was with the greatest percentage (87.5%), while E. coli and Staphylococcus + E. coli were represented with 6.2%. It was evident that mixed infection (combination of AD + Staphylococcus / 50.0%) were presented in the great number of children. The other agents were more rare (16.7%). Conclusion : The small percentage of con¢rmed agents was based on delayed admission in hospital, previous antibiotic treatment, lack of fast, exact and sophisticated methods of discharge. Con¢rmation of the exact cause is the base of successful treatment and determinated prognosis. P6^17 ETIOLOGY OF BACTERIAL MENINGITIS IN CHILDREN DURING 5 YEARS (1997-2002) IN MINSK O. M. Morozova, L. P. Titov, A. A. Astapov, N. L. Klyuyko, L. A. Grak Byelorussian Research Institute Epidemiology and Microbiology (BelRIEM), Minsk, Belarus. The aim of the study was to assess the etiology of bacterial meningitis in children during 5 years (1997-2002). Material for research (CSF, blood and nasopharyngeal swabs) was immediately inoculated on medium and incubated at 35370C for 24 h under CO2 (5-10%) atmosphere. Typing of strains was carried out by api BioMerieux and latex agglutination (KIT5 BioMerieux). 142 bacterial cultures from children with bacterial meningitis were isolated. 85 ^ N. meningitides (group B 69 strains, group C -12 and group A -4), 16 ^ Haemophilus in£uenzae type b, 15 ^ Streptococcus pneumoniae, 9 ^ Streprococcus spp, 7 ^ Listeria spp, 3- Staph.aureus, 3 ^ Candida, 2 ^ Enterobacter, 1 ^ Pseudomonas aeruginoza, 1 ^ Salmonella enteritidis. 22 strains of N. meningitides (25,9 %) was isolated from CSF and blood, 28 (32,9 %) ^ from CSF, 20 (23,5 %) ^ only from blood, 16 (17,7 %) ^ only from nasopharynx. 6 strains of H. in£uenzae type b (37,5 %) was isolated from CSF and blood, from CSF ^ 9 (56,25 %) and only from blood ^ 1 strain. (6,25%). 5 strains of Streptococcus pneumoniae (33,3 %) was isolated from CSF and blood, 8 (53,3 %) from CSF and 2 (13,4 %) ^ from blood. In conclusion : In etiological structure of bacterial meningitis in children in Minsk dominate N. meningitides (59,9 %) with

M. Maneva, Z. Sarovska and L. Stojanovska Institute for Respiratory Diseases in Children, Skopje, Macedonia Object: Retrograde study involved evaluation of clinic histories of patients that were treated at the Department for patients under age of three years. Material: In the period of three years 1.204 children were hospitalized for di¡erent respiratory diseases. Pneumonia was diagnosed and treated in 321 patients (26.66%). From them, 161 patients

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prevalence N. meningitides group B (81,2 %). Second of the importance agents was H. in£uenzae type b (11,3 %), third ^ Streptococcus pneumoniae (10,6 %). P6^18 EVALUATION OF TWO CASES OF MALARIA IN í NIS REGION-SERBIA IN THE LAST FIVE YEARS ¤ ¤ ¤ S. Tasic(1), N. Miladinovic(1), A. Tasic(2), A. Ni¤ (2), Z. Velickovic(1) and S. Pesic(1) > ¤ >¤ kolic > (1) Medical Faculty, Nis, Serbia; (2) Institute of Public >, Serbia Health, Nis In the ¢ve last years in laboratory of parasitology in In> stitute of Public health in Nis only two cases of malaria were diagnosed. The aim of this study was to evaluate these two cases. In both of cases malaria was diagnosed by parasitical investigation of blood smears stained by Giemsa. First case was the man who returned from Zaire. Patient was hospitalized in Clinic of In¡ective disease with typical symptoms of malaria (fever, anemia, weakness), and he did not take the chemioprophylactic drugs for malaria even before or after his stay in Zaire. Malaria vivax was diagnosed based on morphologically characteristics of evolution forms of plasmodium. Second case was the man from group of our workers in Zambia. Also, in this case patient was hospitalized with typical symptoms of malaria. In this case mixed infection with Plasmodium vivax and Plasmodium falciparum was found by parasitically investigation of blood. Patient did not receive drugs for chemioprophylaxis before stay, but he started with Artemisinin immediately after the ¢rst sings of malaria appearance. The singes of illness were less pronounced than in the ¢rst case. Other workers from the same group received drug during the stay and no one obtained disease. In conclusions, only two cases of malaria were diagnosed in the > ¢ve last year in region of Nis-Serbia. In both of cases patients did not receive drugs before stay in endemic area, but one of them started Artemisinin after appearance of singes and illness was less pronounced.

P6^19 FREQUENCY OF UREAPLASMA UREALITYCUM AND MYCOPLASMA HOMINIS IN FEMALE'S GENITAL TRACT AND THEIR SUSCEPTIBILITY TO ANTIMICROBIAL AGENTS S. Stojanova, M. Panovska, Z. Bozinova and S. Ivic-Kolevska Republic Institute for Health Protection, 50 Divizija br.6 1000 Skopje, Macedonia There are 6 mycoplasmas in the female genital tract, out of which the most common are Ureaplasma urealyticum (U. urealyticum) and Mycoplasma hominis (M. hominis). The role of U. urealyticum and M. hominis in pathogenesis of certain genital infections has been proved. The aim of this investigation was to determine the incidence of the genital U. urealyticum and M. hominis alone and in combination with other microorganisms and to assess their susceptibility towards antimicrobial agents. During our work 180 patients were examined. Three cervical swabs of each patient were used: the ¢rst for examination of the quantitative frequency of U. urealyticum and M. hominis detected by Mycoplasma IST kit; the second was used for isolation of cultivable pathogenic and opportunistic pathogenic microorganisms by standard microbiological techniques; the third for detection of antigens of Chlamydia trachomatis using the ELFA-technique on the VIDAS-analyzer. From 180 patients, in 63 patients(34.99%) U. urealyticum and/or M. hominis were isolated. In 13 patients U. urealyticum and M. hominis were both isolated, from which 4 patients associated with Candida albicans. In 6 patients only M. hominis was isolated from which 2 patients associated with E. coli. In 44 patients U. urealyticum was isolated, from which 7 patients associated with Candida albicans. During our work was examined the susceptibility of U. urealyticum and M. hominis to 8 antimicrobial agents. The highest susceptibility was found towards doxycycline, josamycin and tetracycline. The resistance was found towards erythromycin and cipro£oxacin. There is a high presence of U. urealyticum and M. hominis alone or in association with other microorganisms in patients with genital infections, which is showing their role in these infections.

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P6^20 SEROSITIS AS A COMPLICATION OF SEPTICAEMIA, WITHOUT MENINGITIS, CAUSED BY N. MENINGITIDIS: A CASE REPORT E. Trikka-Graphakos, Z. Salem, N. Charalambakis, M. Nepka, Ch. Pappas, F. Ioannidou Department of Clinical Microbiology, `` Thriassio'' General Hospital, Athens, Greece Complications after meningococcal septicaemia have become extremely rare since antibiotics have been used. Serositis caused by Neisseria menigitidis, especially group C, have been described mainly in patients with underlying disease (diabetes mellitus, complement component de¢ciency). We describe a case of a diabetic patient with pericarditis and pleuritis caused by N. meningitidis group C. A 68-year-old diabetic patient was admitted to the emergency department with high fever, chills and sore throat. During physical examination there were not meningeal signs or other abnormal ¢ndings. On admission, white blood cell count was increased with a shift to the left while CRP was also elevated. The laboratory and immunological ¢ndings were negative. The chest X- ray revealed opacity in the lower lobe of the right lung. N. meningitidis group C (2a:P1.2, 5) was isolated from 2 sets of blood cultures. According to the MIC (E- test), the microorganism was sensitive to penicillin, 2nd and 3rd generation cephalosporins sulfonamides, tetracycline, erythromycin, rifampicin, chloramphenicol, and quinolones. The patient was treated with a 3rd generation cephalosporin, 2 g i.v., every 12 h, and his clinical condition improved rapidly. On the 12th day of hospitalization, the patient's clinical condition was deteriorated with fever, dyspnoea and chest pain while during physical examination paroxysmal pulse rate and hypotention were present. From the cardiac U.S pericardial £uid collection with ¢ndings of pericardial tamponate and from the chest X- ray, pleural e¡usion were present. The pleural £uid was exudate while the culture was negative. Dramatic improvement of patient's clinical condition followed after immediate periocardiocentesis and administration of prednisolone (1 mg/kg). In conclusion, Neisseria meningitidis group C, can cause serositis, with negative culture, in patients with underlying disease.

P6^21 EMERGENCE OF METALLO-L-LACTAMASE PRODUCING PSEUDOMONAS AERUGINOSA AS NOSOCOMIAL PATHOGEN E. Edalucci, C. Lagatolla, E. Medessi, L. Dolzani, F. Gionechetti, F. Gombac, C. Monti-Bragadin and E. A. Tonin Department of Biomedical Sciences, Microbiology Section, University of Trieste, via Fleming 22, I-34127 Trieste, Italy In the last three years, 165 imipenem resistant (MIC =16 Wg/ml) Pseudomonas aeruginosa clinical isolates were collected at the Laboratory for Clinical Microbiology of the ``Cattinara'' Hospital (Trieste-Italy). Total DNA was extracted from all strains and the presence of the blaVIM determinant was investigated by dot blot. Among the 165 strains, 122 (about 74%) were blaVIM-positive. 110 of them carried the allelic form blaVIM1 (90%) and 12 (10%) the blaVIM2. This is the ¢rst report of high-level endemicity of metallo-L-lactamase producing P. aeruginosa. Typing of the isolates, performed by two di¡erent methods (RAPD and AFLP analysis), revealed that most of the isolates (78%) might be considered related. In fact, both methods identi¢ed two clusters (indicated as cluster A and B) which included strains with a Dice similarity coe/cient higher than 88%. 4 blaVIM-positive isolates were unrelated both with either clusters and among each other, so they were considered sporadic. Cluster A included 119 isolates : 109 of them (92%) carried the blaVIM1 determinant, 10 were blaVIM-negative. They were widely distributed in the hospital and were also found in 11 outpatients. Cluster B included 10 isolates, 9 of them carried the blaVIM2 determinant, 1 was blaVIM-negative. They were collected from 4 wards. 28 of the 122 blaVIM-positive isolates have been typed by macrorestriction analysis (PFGE) after digestion of total DNA with SpeI. This technique con¢rmed substantially the previous results but, being more discriminant, allowed the identi¢cation of di¡erent subtypes inside the previously identi¢ed clusters. P6^22 STENOTROPHOMONAS MALTOPHILIA. AN EMERGING PATHOGEN IN A LARGE GREEK GENERAL HOSPITAL S. Kanavaki, E. Galani, S. Karambela, M. Makarona, E. Alexandraki, A. Pefanis, H. Giamarellou Sotiria General Hospital, Athens, Greece During the ¢ve year period 1998-2002 we observed a signi¢cant increase in the incidence of Stenotrophomonas maltophilia (SM) isolation from clinical specimens from hos-

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pitalized patients. From a total of 31298 isolated microbial strains, 238 strains were characterised as SM. The incidence was increased by sixfold from the beginning to the end of the ¢ve year period (from 0,2 to 1,8%, respectively). One hundred and seventy nine (75%) strains were isolated between 1/1/2001 and 30/9/2002. Eighty six (48%) of the strains were isolated from sputum, 48 (26,8%) from blood, 22 (12,3%) from bronchial secretions, 12 (6,7%) from pus, 5 (2,8%) from urine, 5 from pleural £uid and one from the tip of a central vein catheter. Some of these isolations probably represent colonisation rather than true infection. However, the fact that a signi¢cant percent of SM strains were isolated from the blood underscores the signi¢cance of SM as an emerging nosocomial pathogen. Regarding the antibiotic susceptibility, 97,2% of the strains were susceptible to cotrimoxazole, 90% were susceptible to cipro£oxacin, and 82,7% were susceptible to ticarcillin/clavulanic acid, while 98,4% of the strains were resistant to imipenem/cilastatin. A variety of antibiotic susceptibility phenotypes were observed, suggesting that there were no SM epidemics in the hospital. These ¢ndings were con¢rmed in part by applying molecular techniques (PFGE) in a small (n=30) number of strains isolated from blood cultures. In conclusion, Stenotrophomonas maltophilia represents a signi¢cant emerging nosocomial pathogen in our hospital. P6^23 INVESTIGATION OF AN OUTBREAK OF VANCOMYCIN RESISTANT ENTEROCOCCI J. Papaparaskevas(1), D. Katsarelias(2), D. Voros(2), P. T. Tassios(1), E. Kouskouni(1,3), N. J. Legakis(1) and L. Zerva(1,3) (1) Department of Microbiology; (2) Department of Surgery; (3) Laboratory of Microbiology, Areteion Hospital, Medical School, University of Athens, Athens, Greece The objective of this study was to investigate an outbreak of vancomycin resistant enterococci (VRE) in a Greek University Hospital. During the period 05/2001-04/2002, 13 VRE isolates were studied. Species identi¢cation and susceptibility to ampicillin, erythromycin, cipro£oxacin, rifampicin, tetracycline, gentamicin, streptomycin, vancomycin and teicoplanin were performed by the VITEK and the ATB systems (bioMerieux), while susceptibility to linezolide and quinupristine-dalfopristine by disk di¡usion. Genes vanA, vanB, vanC, ddlE. faecalis and ddlE. faecium were detected by multiplex PCR. Pulsed Field Gel Electrophoresis (PFGE) was used for DNA ¢ngerprinting. Five VRE strains were isolated from patients' clinical samples in ICU or a clinical ward, 5 from ICU environmental cultures and 3 from patients' surveillance cultures in two clinical wards, all harbouring the vanA gene. Twelve iso-

lates were identi¢ed as Enterococcus faecium and 1 as Enterococcus faecalis. All E. faecium isolates were resistant to ampicillin, erythromycin and cipro£oxacin and susceptible to linezolid and dalfopristine+quinupristine, whilst the E. faecalis strain was susceptible only to ampicillin and linezolide. PFGE allocated 8 isolates (all environmental, 1 clinical and 2 colonizing strains) into 1 cluster (A), 4 clinical strains into 2 clusters (B and D) and 1 strain into cluster C. A thorough cleaning in ICU resulted in outbreak eradication. This constitutes the second report of a VRE nosocomial outbreak in Greece. The majority of VRE were multiresistant E. faecium. A link was established between environmental, colonizing and clinical strains. The outbreak started in ICU and involved two clinical wards. Infection control measures e¡ectively eradicated the outbreak. P6^24 BACTERIA CARRIERS IN SOME PROFESSIONAL GROUPS IN THE MUNICIPALITY OF OHRID WITHIN THE PERIOD FROM 2000 TO 2002 J. Sturlakova-Korovesovska and S. Dimovska Republic Sanitary and Health Inspectorate (RSHI), Regional Unit Ohrid, 16 Lazo Trposki, 6000 Ohrid, Republic of Macedonia Objective: To show the most frequent bacteria carriers found in some professional groups during periodical health examinations. Material and Methods : The data are taken from the records of the RSHI, Regional Unit ^ Ohrid, and are obtained during the microbiological examinations of throat and nose smear, feces, and cellophane smear for parasite identi¢cation of some professional groups within the period 2000-2002. Standard statistical data processing method was used. Results : Within the given period, a total number of 5240 individuals were examined, of which 902 education employees; 1338 health-care employees ; 62 pharmaceutical employees and 2938 employees involved in food processing. The percentage of positive nose smear ¢ndings ranged from 1.9% in health-care and pharmaceutical employees, 2.7% in education employees and 5.4% in employees engaged in food production and tra/c. The most frequent cause of bacteria carriers in all cases was Staphylococcus aureus. The presence of positive throat smear ¢ndings was the following: 0.2% in pharmaceutical employees, 1.1% in education employees, 0.8% in health-care employees and 0.3% in employees engaged in food processing. The most frequently isolated bacteria was Streptococcus pyogenes. No bacteria carrier was detected in the copraculture examinations. Conclusion: In evaluating the results obtained, it has been stated that Staphylococcus aureus dominates as a bacteria carrier. Nose was found as the most frequent

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location, while the frequency of incidence was the greatest in employees engaged in food processing and tra/c. Due to the increased consumption of creams and ice creams in summer, systematic examinations should be more frequent. P6^25 E. COLI O6 NOSOCOMIAL OUTBREAK IN NEONATAL INTENSIVE CARE UNIT I. Tomova(1), E. Georgieva(1), T. Kantardjiev(1), S. Staneva(2), M. Christova(1), P. Vajarova(2) (1) National Center of Infectious and Parasitic DiseasesSo¢a, Bulgaria ; (2) Hygienic Epidemiological Inspectorate-Varna, Bulgaria The nosocomial outbreak concerns a neonatal infection of the type endotoxic shock (septicemia) in the background of immaturity and preterm birth. A thorough laboratory control has been initiated establishing contamination of some solutions and equipment for infusion therapy. All isolates from clinical materials and parenteral solutions perfained to Escherichia coli and were identi¢ed as O6 serotype of the enterotoxigenic group (ETEC). The strains were tested by complex of bacteriological and genetic methods, demonstrating their identity. Dissemination of infection was realized by the contact route, the hands of personnel and special apparatuses being proved to be of primary importance among factors for distribution of infection. P6^26 USE OF PFGE AND FLAA-RFLP FOR TYPING OF CAMPYLOBACTER JEJUNI AND C. COLI FROM POULTRY AND CLINICAL ENVIRONMENTS ¤ T. Zorman(1), M. Heyndrickx(2), S. Uzunovic-Kamber¤ (3), S. Smole Mozina(1) > ovic (1) University of Ljubljana, Biotechnical Faculty, Department for Food Science and Technology, Ljubljana, Slovenia; (2) Center for Agricultural Research Ghent, Department for Animal Product Quality and Transformation Technology, Melle, Belgium; (3) Cantonal Center for Public Health in Zenica, Zenica, Bosnia and Herzegovina. Thermotolerant Campylobacter species C. jejuni and C. coli are recognised as one of the major causes of acute bacterial enteritis in many countries as well as in Slovenia and Bosnia. Our previous study indicated that high degree of poultry meat on Slovenian and Bosnian retail market is contaminated with campylobacters. In order to indicate the animals and meat as reservoirs of campylobacters for

causing the disease in the community two high resolution genotyping techniques were applied. Pulsed-¢eld gel electrophoresis (PFGE) and £aA typing were used to investigate the genetic diversity among isolates from chickens in farm environment, chicken meat from retail market and sporadic cases of campylobacteriosis in the same geographical area. Macrorestriction pro¢les were analysed using the GelCompar software. Among 124 analysed isolates we observed 78 di¡erent SmaI macrorestriction pro¢les. FlaA typing by using CfoI distributed the strains into 10 £aA types, so PFGE technique was much more discriminative. Di¡erent C. jejuni and C. coli clones were isolated from the same meat product. On the other side, the same macrorestriction pro¢les of human isolates in the same geographical area show on their clonal origin. Very similar or identical genotypes indicate the transmission of these pathogens from chickens in farm environment to poultry meat on retail market, as well as from meat to humans. There is an evidence for subsequent contamination of poultry meat with Campylobacter strains present in the market environment. The results of this study suggest a link between campylobacters in farm environment with those causing disease in the community. P6^27 PLASMID AND DIGESTION OF CHROMOSOMAL DNA OF L. MONOCYTOGENES J. Nowroozi, M. Hakemi Iran University of Medical Science, Tehran, Iran L. monocytogenes is a Gram positive, facultative anaerobic and intracellular bacteria. Epidemiological studies suggested that contaminated foods such as pasteurized milk, soft cheese, raw vegetables and etc, are probable vehicle of infection to human. The purpose of this study was to compare plasmid DNA pro¢les and chromosomal DNA after digestion with Hpa-1 in L. monocytogenes isolated from dairy and clinical specimens. Plasmid DNA was prepared by alkali lysis and chromosomal DNA was obtained by using lysozyme, pronase and extracted by phenol / chloroform. Concentration of DNA was measured by using a spectophotometer. Cleavage reaction with Hpa-1 restriction enzyme was done for three hours. In clinical specimens, 2 bands of plasmid DNA, and in dairy specimens, one band of plasmid DNA were detected. After digestion, 4 bands of chromosomal DNA in clinical specimens and 2 bands in dairy specimens were found. All L. monocytogenes isolated from di¡erent sources had one band of similar of plasmid DNA and also, after digestion, 2 bands of similar chromosomal DNA in clinical and dairy specimens, but another one band of plasmid DNA and two bands of chromosomal DNA were detected in

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clinical specimens. This probably indicated di¡erent virulent strains of Listeria monocytogenes. P6^28 TOXOPLASMA INFECTION AT EYE LEVEL D. Steriu(1), M. Pop(2), C. Cedru(1) (1) Cantacuzino Institute, Splaiul Independentei 103, Bucharest, R-70100, Romania; (2) Hospital of Ophthalmology, Bucharest, Romania 595 serum samples from patients with di¡erent eye diseases, with a possible toxoplasmic etiology, were investigated by ELISA. 341 positive sera were detected. Starting from the idea that toxoplasmosis is a systemic infection frequently encountered in the healthy population, only those cases in which the titers pointed to an exacerbation of the infection were taken into consideration. Thus, 131 such cases were reported. Most of them presented initial or recurrent chorioretinitis (77 cases) and uveitis (34 cases), most of them showing a posterior localization. It is worth mentioning that no positive IgM samples were identi¢ed, therefore no possibly toxoplasmic primary infection could be detected. At the same time, the lesions that occurred, even the recurrent ones, do not have a history that can be traced back to the early childhood. Of particular interest was the 0 ^ 5 years age group that included 27 children, of whom 12 had positive titers for toxoplasmosis. In ten of these children, the positive titres were indicative of an exacerbation of infection. The observation that in all the cases recurrent chorioretinitis was incriminated enables us to presume that even in the absence of investigations performed at birth, these can be considered to be congenital toxoplasmic infection. P6^29 ENTEROBACTER SAKAZAKII IN MILK POWDER A. E. Heuvelink, M. Ahmed, F. D. Kodde, J. T. M. Zwartkruis-Nahuis, H. van der Zee and E. de Boer Inspectorate for Health Protection and Veterinary Public Health, P.O. Box 202, 7200 AE Zutphen, The Netherlands Enterobacter sakazakii is a rare but important cause of life-threatening neonatal sepsis and meningitis. We examined the occurrence of this bacterium in di¡erent milk powders. A portion of 25 g of milk powder was carefully added to 225 ml bu¡ered pepton water (BPW), dissolved by slowly swinging and incubated for 18 to 24 h at 37Cfollowed by inoculating1 Wl onto violet red bile lactose (VRBL) agar (18 to 24 h at 37C ) and 10 ml was transferred to 90 ml of Enterobacteriaceae enrichment

(EE) broth (18 to 24 h at 37C). Then, also 1 Wl of EE culture was inoculated onto VRBL agar (18 to 24 h at 37C). Typical colonies (Enterobacteriaceae) on VRBL were tested for the formation of a yellow pigment and aglucosidase activity on tryptone soy agar (TSA) and nutrient agar supplemented with 4-methylumbelliferyl-a-Dglucopyranoside (NA-MUG), respectively. Yellow-pigmented colonies (2 d at 25C) which produced £uorescent colonies on NA-MUG agar (366 nm) were also tested for a-glucosidase activity with the 4-paranitrofenol (PNP) test. Colonies from TSA were suspended in a tube with 2 ml 0.85% NaCl. After the addition of 2 ml PNP-a-glucosidase solution (5 g/l, 1.15 M phosphate bu¡er, pH 7.0) the tubes were incubated at 37C for 4 h. The formation of yellowcoloured PNP was examined by measuring the absorbance at 405 nm. Isolates that showed a-glucosidase activity (94 uur) were streaked onto sorbitol-MacConkey agar (18 to 24 h at 37C) and identi¢ed as E. sakazakii by using an API 20E biochemical test strip. Additionally, isolates were tested with a tDNA-PCR and subtyped with PFGE. A total of 211 samples of milk powder were examined, including 40 samples of powdered-infant formula. Strains of E. sakazakii were isolated from 8 samples : 1 sample of infant formula and 7 samples collected from cargos and bulk goods of milk powder. Among the 8 isolates, 7 distinct restriction patterns could be discriminated. During a follow-up study, 102 samples of powdered-infant formula were examined. E. sakazakii strains were isolated from 2 of these samples. P6^30 SURFACE BIOPOLYMERS OF THE MELIOIDOSIS AGENT AS POSSIBLE COMPONENTS OF CHEMICAL VACCINE S. I. Zhukova, V. S. Rybkin, N. N. Piven, I. V. Avrorova, N. G. Plekhanova, D. V. Viktorov and O. B. Proshina Antiplague Research Institute, Volgograd, Russia The problem of creating good specimens of prophylaxis and treatment of melioidosis infection has been actual and important recently. In the present work there have been studied characteristics of some antigen complexes of the melioidosis agent including exoprotease, biopolymers of exterior and interior membranes of a microbe cell and surfactant antigen complexes contained the antigen 8, one of the pathogenic factors. Experiments were made on white mice and white rats. Animals were immunized twice with antigen specimens in the mixture (1:1) with the Freind full adjuvant in doses of 30 mkg(for mice) and 100 mkg(for rats). Animals were infected with the virulent culture of the melioidosis agent C-141(for mice) and 100 (for rats) 14 days after the second immunization. There have been shown exoprotease increased the quantity of alive

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mice in 2,5-4 times more, compared with the control group, but the median duration of life of dead animals increased on 36-66 %. The specimen of cytoplasmatic membranes protected 30% of mice from 30 LD50 melioidosis agent. Mice were also protected with surfactant antigen fractions C, C1 and B. Common using of these fractions with immunomodulators(IM) (bromantan and glutaxim) increases the survival rate of mice on 30-40%. The specimen of exterior membranes without immunomodulators didn't have protective characteristics. Surfactant antigen fractions C, C1, D,H were tested in experiments with rats. C and D fractions were mostly protective defending 50-58% of rats from 18-180 LD50 strain 100 of the melioidosis agent. So, surfactant fractions of the melioidosis agent used in the coplex with IM should be the most perspective for creating chemical vaccine. We have tested a lot of IM and chosed adaptogens of adamantanic group(bromantan) and specimens from tiopoetins type(glutoxim) as the most e¡ective. P6^31 MULTIPLEX PCR FOR IDENTIFICATION OF ESCHERICHIA COLI O157 :H7, AS APPLIED TO MONITORING OF HEMORRHGIC COLITIS CAUSATIVE AGENT IN RUSSIA V. A. Bannov , N. V. Volozhantsev , E. A. Svetoch, V. V. Verevkin State Research Center for Applied Microbiology, 142279, Obolensk, Moscow region, Russia A multiplex polymerase chain reaction (PCR) assay was designed to simplify detection of Escherichia coli O157:H7 and to identify the H serogroup and the type of Shiga toxin produced by this bacterium. The system contains four pairs of primers complementary to nucleotide sequence of cytotoxin genes (stx1 and stx2), and intimin protein responsible for the microbe adhesion (eae A), and gene rfb responsible for antigen 0157 synthesis. Besides, primers for a plasmid-encoded hemolysin gene (hly933), or chromosomal £agellar (£iC h7; £agellar structural gene of H7 serogroup) gene were used in a multiplex PCR for coampli¢cation of the corresponding DNA sequences from enterohemorrhagic E. coli (EHEC) O157:H7. The PCR system was tested by using 17 reference strains of E. coli O157:H7 isolated from hemorrhagic colitis patients in Japan and USA, 12 strains of E.coli of other serotypes and 26 strains of other species of bacteria. Samples of clinical materials isolated in 1999-2002 from patients su¡ered from diarrhea, including patients with HUS ; from samples of calf and pigs fecal mass have been analysed by the developed PCR system. PCR gene typing revealed the availability of rfb, eae, stx, ehx and £iC genes. Samples of epizootic material from poultry

farms of 8 regions of Russia were analysed. PCR with chicken samples produced only rfb amplicons, and was not produce eae speci¢c amplicon. In samples from dairy plant ¢lters were isolated strains bearing eae, rfb, stxII and £iC(h7) genes. P6^32 EVOLUTION OF ANALYTICAL METHODS FOR CRYPTOSPORIDIUM PARVUM AND GIARDIA SPP IN SURFACE WATER: RESULTS OF A TIME EXTENDED MONITORING IN THE NORTH WEST OF ITALY E. Carraro(1), S. Bonetta(1), S. Bonetta(1), P. Secco(2), E. Fea(3), C. Pignata(3), G. Gilli(3) (1) Dept. of Science and Advanced Technology, University of Piemonte Orientale A. Avogadro, Alessandria, Italy; (2) Dept. of Medical Sciences, University of Piemonte Orientale A. Avogadro, Novara, Italy ; (3) Dept. of Public Health and Microbiology, University of Turin, Torino, Italy Our research group started a study in 1992 to assess microbiological risk related to the presence of Cryptosporidium parvum and Giardia spp. in a surface source for drinking water in the North West of Italy. Sampling, concentration and determination of the protozoa were performed according to standard methods (APHA-AWWAWEF, 1995; US-EPA, 1999). The overall analysis of the results underlines the occurrence of the protozoa in the raw water examined (Giardia cysts 0,002 ^ 67/L , and Cryptosporidium oocysts 0 ^ 36,86/L) pointing out an association between protozoa contamination levels and runo¡. Nevertheless, cysts and oocysts were never found in drinking water, showing the ability of the water multistage treatment to remove the contamination. Since standard methods for a routine detection of Cryptosporidium and Giardia in water samples only provide presumptive information about cyst/oocysts species identity and viability, RT-PCR and PCR-based assays have been developed to increase detection sensitivity and speci¢city. The recovery e/ciency of IMS-RT-PCR assay for detection of viable Cryptosporidium parvum and Giardia spp. in spiked water samples was evaluated. The IMS showed a recovery of 9099% for Cryptosporidium and 88-100% for Giardia and a sensitivity of 10 cysts/oocysts was obtained with IMS/RTPCR method. The occurrence of Cryptosporidium oocysts and Giardia cysts in surface raw water points out the importance of routinary control of water for human consumption. Considering how complex the analytical methods are, the setting up references laboratories for Giardia and Cryptosporidium control in the waters is of paramount importance.

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P6^33 PREVALENCE AND PROPERTIES OF VEROCYTOTOXIN-PRODUCING ESCHERICHIA COLI ISOLATED FROM SHEEP IN SERBIA í >¤ ¤ ¤ D. Opacic, M. Cobeljic, B. Dimic Military Medical Academy, Veterinary Centre `Belgrade', Belgrade, Serbia The aim of this study was to elucidate the importance of sheep as reservoir of verocytotoxin-producing E. coli (VTEC) in Serbia. In the surroundings of Belgrade, from March through June 2002, fecal samples were collected from lambs and mutton sheep and cultured on Mac Conkey agar plates. Isolated E. coli strains were tested for verocytotoxin production on Vero tissue culture cells. Detected VTEC were examined for phenotypic traits that are frequently exhibited by VTEC associated with illness in humans (lack of sorbitol fermentation, enterohemolysin production, expression of characteristic O antigen, localized adherence to epithelial tissue culture cells). Of 202 examined lambs and mutton sheep, VTEC were isolated from feces of 135 (66.8%).Of them, 16 (11.8%) were sorbitol-negative and 6 were hemolytic (all produced alfa hemolysin). Agglutination with antisera for typical human VTEC serogroups (O26, O55, O111, O128, and O157) revealed that only 7 strains belonged to O128, and 1 to O55 serogroup. By using HEp-2 cells adherence test it was found that 17 (12.6%) strains adhered to these cells (14 displayed aggregative, 2 di¡use, and 1 localized pattern of adherence). 29 (21.5%) VTEC strains showed resistance to antimicrobial drugs: 28 were resistant to cephalexin only, and 1 strain displayed multiple resistance to 4 of 11 tested antibiotics. Although sheep are frequent carrier of VTEC, their importance as a reservoir of these pathogenic agents should be further investigated since most of the isolated strains in this study lacked properties that are commonly encountered among human VTEC. P6^34 COLD, HEAT SHOCK AND STARVATION INFLUENCE VIABILITY, CULTURABILITY AND MORPHOLOGY OF CAMPYLOBACTER JEJUNI CELLS > > > A. Klancnik, P. Skocir, T. Zorman, S. Smole Mozina University of Ljubljana, Biotechnical Faculty, Department for Food Science and Technology, Ljubljana, Slovenia Campylobacter jejuni is a leading cause of acute bacterial foodborne gastroenteritis worldwide. Its physiology is in many senses unusual. It seems to lack many of adaptive responses to environmental stresses known in other food-

borne pathogens. However, under stress conditions campylobacters transit from a spiral-shaped to the more resistant coccoid form, usually accompanied by a transformance into a viable but nonculturable state (VBNC). More research is needed to elucidate the importance of this phenomenon for survival of pathogenic campylobacters during food production chain. This was the reason for our investigation of transition of C. jejuni actively growing and stationary phase cells into a VBNC and coccoid form following their viability, culturability and cellular morphology after cold shock at 25C and heat shock at 55C. Acridine orange direct count (AODC), growth on Karmali agar as well as £uorescent and electron microscopy were methods used, respectively. In addition, the e¡ect of starvation before cold as well as before heat shock and cold shock before heat shock was investigated. In some experiments, chloramphenicol was added as protein synthesis inhibitor after 1, 2, 3, 4, 5 and 24 h of starvation before heat shock. We con¢rmed that low temperatures enhanced survival of C. jejuni cells for cca 20%, viability and culturability were preserved at least 30 days in rich medium (Preston) and more than 40 days in starved cells. High temperature provoked quick transformation from culturable spiral-shaped to nonculturable coccoid cells. It is remarkable that starved C. jejuni cells increased cold and heat resistance. However, the latter was more pronounced, especially in exponentially growing cells after 5 hours of starvation before heat treatment (25% increase of survival). The e¡ect of chloramphenicol treatment was more obvious, when added as earlier after start of starvation. The results indicate the absence of phenotypic stationary phase response of Campylobacter cells. P6^35 PCR DETECTION OF CRYPTOSPORIDIUM SP. IN HUMAN AND ANIMAL FAECES IN SLOVENIA B. Soba(1), M. Petrovec(1), A. Vergles Rataj(2), J. Logar(1) (1) Institute of Microbiology and Immunology, Medical Faculty, University of Ljubljana, Ljubljana, Slovenia; (2) Institute of Microbiology and Parasitology, Veterinary Faculty, University of Ljubljana, Ljubljana, Slovenia Cryptosporidium is a pathogenic protozoon of humans and animals. It can cause acute or chronic diarrhoea or even life-threatening disease in immunocompromised host. An infection, cryptosporidiosis, can be transmitted by contaminated food or water. In recent years, molecular biology-based methods have provided insight into the transmission dynamics of cryptosporidiosis. The aim of this study was to develop a method based on polymerase chain reaction ^ PCR for detection of Cryptosporidium in faecal samples. We extracted DNA from four human and ¢ve

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calf faecal samples in which Cryptosporidium oocysts were previously detected by modi¢ed Ziehl-Neelsen stain. A fragment of Cryptosporidium 18S rRNA gene encompassing the hypervariable region was ampli¢ed by polymerase chain reaction. In all nine faecal samples a fragment of this gene was sucessfully ampli¢ed. In the future it is suggested that detection of Cryptosporidium sp. by polymerase chain reaction and by its genotyping could provide insights into the transmission dynamics, epidemiology and possible zoonotic origin of this parasite in Slovenia. P6^36 IDENTIFICATION OF HELICOBACTER SPECIES IN DIFFERENT MOUSE STRAINS BY PCR-DGGE AND DNA SEQUENCING î W. A. Al-Soud, H.-O. Nilsson, A. Ljungh, T. Wadstrom « Department of Medical Microbiology, Dermatology and Infection, Lund University, Lund, Sweden The genus Helicobacter now comprise at least 24 formally named species, which are widely distributed in the gastrointestinal tract of mammals, birds and other animals. The mouse models are widely used in research. Recently, the complete mouse genome has been published, which will have a huge impact on the use of mouse models. The aim of this study was to address the problem of Helicobacter gut colonisation and infections of di¡erent mouse strains. A PCR-denaturing gradient gel electrophoresis (PCR-DGGE) technique was optimised for the detection and identi¢cation of mice Helicobacter species. Eight mice strains were included in the study. They were obtained from four di¡erent laboratory animal facilities C57Bl/6 (n=12), SPF-SCID (n=5), conventional SCID (n=2), B6sJl (n=3), Balb/cA (n=5), C3H/HEJ (n=4), and Apo-E (n=5), and IL-10 de¢cient C57Bl/6 (n=6). Mice were sacri¢ced and blood, liver, stomach, the small intestine, and faecal samples were collected. DNA from faeces was extracted by the Qiagen DNA Stool Kit (Qiagen), and from all other samples using the Qiagen DNA Kit. Helicobacter DNA was detected by genus-speci¢c semi-nested PCR assay, in all mice strains tested except the IL-10 de¢cient C57Bl/6 mice. Using this assay 85 of 204 (42%) samples were positive. Approximately 6% of the mice were found co-infected by v 2 Helicobacter species. The identi¢cation results of PCR-DGGE were con¢rmed by DNA sequencing and H. ganmani speci¢c PCR designed in our laboratory, which allowed identi¢cation of H. typhlonius, H. rodentium, H. bilis, H. ganmani, H. hepaticus, and H. trogontum. We showed that a number of Helicobacter species are colonising the gastrointestinal tract of di¡erent mouse strains. In addition, di¡erences in colonisation among di¡erent mouse strains were found, which may be related to the level of immune de¢ciency and to di¡er-

ences among breeding houses. The PCR-DGGE technique, which was optimised in this study, was found e/cient and simple for determination of the Helicobacter status in laboratory mice. P6^37 EPIDEMIOLOGY OF HELICOBACTER SP. INFECTION OF DOGS IN SLOVENIA í > > M. Gombac, M. Cerne, M. Pogacnik, I. Gruntar, J. Mehle, B. Krt, M. Ocepek Institute of Pathology, Administrative and Forensic Veteri> nary Medicine, Veterinary Faculty, Gerbiceva 60, Ljubljana, Slovenia Our research included 213 randomly chosen dogs of 44 di¡erent breeds from all parts of Slovenia (6 regions), which died or had been euthanised. Both genders were included in the age from 9 days to 15 years. They lived either indoor or outdoor and were fed either with commercially prepared food or home made food or were still suckling. Using toluidine-blue staining method on necropsy taken stomach tissue we detected Helicobacter in 92,4 % of dogs in all examined parts of stomach i.e. fundus, corpus and antrum. We determined a mild infection in 17,3% of dogs, medium in 48,1 % and a very strong infection in 27 % of dogs. Helicobacters were located in gastric mucus, gastric pits and in gastric glands' lumen. For the determination of e¡ects of epidemiological parameters (gender, age, breed, nutrition, region and housing conditions) on infection and the infection rate with helicobacters we calculated the prevalence of infection and statistically evaluated results.We determined that age and feeding regimen a¡ect the infection and the degree of infection, whereas gender, breed, location and indoor/outdoor living conditions do not. The rate of infection was 33,3 % in dogs up to 2 months of age, still living with the bitch and in dogs older than 2 months 94,4 %. The infection rate of dogs fed with mixed (home prepared) food was 95,9 % and for dogs fed with commercially prepared dog food 91,5 %. All suckling puppies were uninfected. Di¡erences in the prevalence of infection between the regions were small, with the highest infection rate detected in Primorje region.

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P6^38 NEW APPROACHES IN ISOLATION OF HELICOBACTER SPP. FROM GASTRIC MUCOSA IN DOGS I. Gruntar, J. Mehle, B. Krt, M. Ocepek, M. Gombac, M. Cerne, M. Pogacnik Veterinary Faculty, Ljubljana University, Gerbiceva 60, 1000 Ljubljana, Slovenia Helicobacters are spiral shaped Gram-negative bacteria, frequently present in stomach and intestine of humans and several animal species. Clinical importance of these bacteria is unclear, with an exception of H. pylori, a causative agent of chronic gastritis in humans. One of the reasons for such situation arises from the fact that research on Helicobacter sp. is seriously hampered by considerable di/culties in isolation of these bacteria. In our study we tried to increase the isolation e/ciency of Helicobacter spp. by introducing some new approaches in culturing these bacteria from gastric mucosa from freshly euthanised dogs. Suitability of di¡erent isolation media was evaluated, as well as an e¡ectiveness of a variety of antibiotics added to these media. Diverse physical and chemical methods of sample preparation and sample decontamination were evaluated in order to get rid of contaminants that frequently obstructed successful isolation of helicobacters despite great numbers of spiral bacteria present in gastric mucosa. Brain Heart Infusion (BHI) agar supplemented with 10% de¢brinated horse blood, 7% horse serum, Vancomycin, Polymyxin B, Trimetoprim and Amphotericin B was found to be the best medium for the recovery of Helicobacter spp. Among the standard media, chocolate agar gave the best results. We noticed that isolation rate was considerably better when gastric mucus was lique¢ed using cysteine solution before plating the samples. The initial growth of helicobacters was enhanced by use of few drops of BHI broth, supplemented with foetal calf serum and freshly prepared yeast extract. A combination of these methods resulted in successful isolation of Helicobacter spp. from virtually every sample with a positive preliminary urease test. Moreover, using our techniques, viable helicobacters could be recovered even from correctly frozen gastric mucosa samples.

P6^39 PECULIARITIES OF PATHOGENESIS AND ADAPTATION MECHANISMS OF ANTIGENIC VARIANTS OF BURKHOLDERIA PSEUDOMALLEI BY EXPERIMENTAL MELIOIDOSIS V. V Alekseev, T. N. Vjazmina, N. G. Plekhanova, S. V. Zamaraeva, V. I. Kapliev and T. Y. Kivokurtseva Antiplague Research Institute, Volgograd, Russia The present study examines the pathogenesis of melioidosis, induced by typical and defected in the synthesis of capsule antigen and protease strains, and the enzyme activity of the microbe on the di¡erent stages of the disease. Tests with subcutaneous infection of the animals with strains and their antigenic variants showed that Ag8 is the main factor, indicating pathogenicity of the microbe, and proteases played a minor role. Some di¡erences in pathogenic action of the defected strains were determined by the level of the resistance of macroorganism to the infection. By aerogenic infection the antigen 8 of the microbe had a relative meaning, as the strain displayed a pathogenic action, inducing death of 50% of animals in comparisom with the subcutaneous infection. The respiratory melioidosis developed atypical course including macro- and micromorphological changes, dissemination of the microbe and its antigens, interaction with phagocytes, clinical manifestations. Long-duration bacterium carrying by the infection with Australian serovar of the agent was noted. The strains, not producing hydrolase, were avirulent for the animals. This fact con¢rms that hydrolyse is considered to be the main factor of pathogenicity by aerogenic infection. It was determined that realization of the pathogenic properties of strains B.pseudomallei free of a capsule occurred by means of signi¢cant activation of hydrolytic enzymes. It is considered as the manifestation of the adaptation mechanism of the agent. The necessity of the correction of the tactics of laboratory diagnostics by respiratory melioidosis, induced by atypical strains, and means of production of diagnostic preparations have been shown. P6^40 THE IMMUNE RESPONCE IN MICE AND RATS BY B. PSEUDOMALLEI ANTIGENS VACCINATION I. V. Avrorova, V. S. Rybkin, N. N. Piven, S. I. Zhukova, N. P. Khrapova, D. V. Viktorov and N. M. Drefs Antiplague Research Institute, Volgograd, Russia. Improving the methods of prophylaxis and treatment of melioidosis infection is impossible without studying its

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pathogenesis and immunogenesis. Therefore the dynamics of making of the immune responce in animals with di¡erent susceptibility to melioidosis has been interesting. The purpose of this work is studying peculiarities of the vaccinative process caused with protective antigens of the melioidosis agent in mice and rats. Surfactant antigen complexes C, C1, D and H of the meliodosis agent were used for the immunization of animals. Mice and rats were de¢ned the titer of speci¢c ILISA antibodies, the delayedtype hypersensitivity level and phagocytic activity of peritoneal macrophages using the chemiluminescentic method. The protective characteristics of melioidosis antigens were assessed on the mortality rates: the percentage of dead animals and the median life duration were calculated. There have been shown similar antigens of melioidosis agent in host of mice and rats have di¡erent immune activity. Most studied specimens inoculated to mice didn't in£uence cellular or humoral immunity or highly depressed it. The immunization of mice with these antigens didn't prevent animals from infecting with the virulent strain of the melioidosis agent.However inoculating these specimens to rats su/ciently stimulated the cellular immunity which increased the protective activity by the highly virulent strain B.pseudomallei 100. So, the inoculation of studied antigens to rats (not to mice) made the adequate cellular immune responce which was the most su/cient part in the protection of melioidosis infection. All experienced fractions decreased mortality rates in rats. Antigens C and D had mostly expressed protective activity in rats by melioidosis. P6^41 ADAPTATION MECHANISMS OF YERSINIA PSEUDOTUBERCULOSIS AND LISTERIA MONOCYTOGENES TO ABIOTIC FACTORS OF ENVIRONMENT L. S. Buzolyova(1,2), G. P. Somov(1) (1) Institute of Epidemiology and Microbiology of Siberian Branch Russian Academy of Medical Sciences; (2) The Far East National University, Russia Yersinia pseudotuberculosis and Listeria monocytogenes can be adapted to di¡erent conditions of ecological niches, in animals as well as in environmental objects with regularly changing abiotic factors. Adaption of biochemical mechanisms explain the wide ecological plasticity of Yersinia and Listeria to changing environmental conditions. Our experiments showed that during long habitation in soil and water, these bacteria change their biological properties. But these changes have an adverse e¡ect. We arranged that Y. pseudotuberculosis and L. monocytogenes can pass to gasotrophy in the conditions of limited carbon and nitrogen. In starvation period, they can use the autolysis products of dead bacteria. Y. pseudotuberculosis and

L. monocytogenes can store nourishing substances. L. monocytogenes changed their morphological properties. We also arranged that Yersinia in low temperature conditions supports the necessarily vital level of metabolism, using conformational and enzymolysis strategy. We found that Yersinia have a cholinesterase enzyme (HE) with high catalytic activity. A fall in temperature to 4 0C accelerates hydrolysis of acetylcholinesterase (ATH) and propionilcholinesterase (PTH) by 20-50 % (for butyrylcholinesterase (BTH) the speed didn't change). When Yersinia was at 4 0 C, Km of HE increased (with hydrolysis ATH and BTH) 2-fold; PTH 6-fold. So, the temperature e¡ect was paradoxical: reducing the cultivation temperature resulted in increasing enzymolysis of substrates. To reveal changes in enzymolysis structure with changing temperature, we used phosphororganic combinations which speci¢cally block the hydroxyl group of serine of HE active center. We found a distinction in the quantity of inhibition of enzymolysis activity between low and high temperature, showing that the conformation of active surface structure of Yersinia HE depends on temperature of habitation. Evidently, the adaptation temperature of Yersinia is accompanied by conformational changes in the proteins of molecular enzymolysis. P6^42 EFFECTS OF SUBINHIBITORY CONCENTRATIONS OF ANTIBIOTICS ON ADHESION TO AND INVASION OF HEP-2 CELLS BY SOME BACTERIAL STRAINS WITH DIFFERENT SOURCES OF ISOLATION V. Lazar(1), C. Balotescu(1), R. Cernat(1), A. M. Israil(2), L. Petrache(1), C. Dinu(2) (1) Dep. Microbiology-Immunology, Faculty of Biology, University of Bucharest, Aleea Portocalelor 1-3, Sect.5, Bucharest 77206, Romania ; (2) Cantacuzino Intitute for Research and Development in Microbiology and Immunology, Spaiul Independentei 23-26, Sect.5, Bucharest, Romania Adherence of microorganisms to epithelial cells is a pivotal event in the pathogenesis of infectious diseases. The anti-adhesion e¡ects of antibiotics were intensively studied and con¢rmed for abiotic surfaces (polymers, metals and cerams), but studies on the e¡ect of antibiotics on bacterial adhesion to cellular substrate are very scant and controversial. The aim of this study was to evaluate the e¡ects of subinhibitory concentrations of PEN, AMP, AMC, CAZ, VAN, CHL, KAN, ERY, STR, NOR on the adhesion and invasion capacity of some Aeromonas hydrophila, Citrobacter freundii, Enterobacter cloacae, Escherichia coli and Listeria monocytogenes strains isolated from water, food, acute diarrhea, urine and stool cultures. The pro/ anti adhesion and invasion e¡ects were evaluated quanti-

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tatively by Cravioto's modi¢ed method (gentamycin protection assay). As concerning the Gram-negative bacterial strains, all antibiotics exhibited overall inhibitory e¡ect on the adherence/invasion capacity, with no respect of species or source of isolation. As concerning the Gram-positive strains, it is to be noticed that AMP, AMX and VAN at subtherapeutic concentrations promoted the adherence and invasion capacity of Listeria monocytogenes strains. These antibiotics have also changed the adherence pattern of these strains, shifting from di¡use to localized adherence, with the occurrence of speci¢c bacterial clusters, probably by changing the external electric charge of bacterial wall. Our results demonstrated that some antibiotics could speci¢cally stimulate the synthesis of adhesion molecules in certain bacterial strains. The pro-adhesion e¡ects demonstrated by the cell wall acting antibiotics on Grampositive strains might have important implications in the selection of the appropriate antibiotic treatment of patients with systemic infections. P6^43 THE ROLE OF TOXINS IN CLOSTRIDIUM DIFFICILE-ASSOCIATED DISEASE B. Geric, M. Grabnar, and M. Rupnik Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia Clostridium di/cile is a Gram positive rod causing antibiotic-associated diarrhea and potentially lethal pseudomembranous colitis and is the most common infectious cause of nosocomial diarrhea. Two toxins were up to now recognized as main virulence factors responsible for development of disease. Recently, some C. di/cile strains have been reported to produce an additional toxin, binary toxin CDT. Among C. di/cile strains we can therefore di¡erentiate six toxin production types according to the combination of one, two or all three toxins produced. Toxin A (TcdA), primarily an enterotoxin, and toxin B (TcdB), a cytotoxin, are GTP- ribosyltransferases which cause depolymerisation of actin micro¢laments and destroy the cytoskeleton of cells. This, as well as some other systemic e¡ects of both toxins, cause extensive tissue damage and £uid response in the gut. Binary toxin is an ADPribosyltransferase which most probably works synergisticaly with TcdA and TcdB in the destruction of actin micro¢laments of the cytoskeleton. The production of binary toxin is less than 1% of extracelulary proteins, but the concentrated binary toxin was shown to be cytotoxic in cell cultures. The prevalence of binary toxin producing strains is estimated to be from 1.6 to 6 %. Most of the binary toxin producing strains also produce TcdA and/or TcdB, but we have also found a few strains that produce the binary toxin alone. Since the role of binary toxin in the

development of disease is not understood, we used these latter strains to study the role of binary toxin in the development of disease in di¡erent animal models. P6^44 A NEW TWO-COMPONENT SIGNAL TRANSDUCTION SYSTEM IN BARTONELLA HENSELAE K. Hercik and P. Branny Institute of Microbiology, Czech Academy of Sciences, Videnska 1083, Prague 4, 142 20, Czech Republic A major mechanism of signal transduction, widespread in bacteria, is the so-called two-component system. These systems are structured around two conserved proteins: a histidine kinase and a response regulator protein that are phosphorylated at His and Asp residues, respectively. Pathogenic bacteria often use two-component system to regulate expression of the virulence factors. We have succeeded to found both members of a new two-component signal transduction system in Bartonella henselae, a major causative agent of ``cat scratch disease''. Based on their sequence similarities, the genes belong to the family of NtrY/NtrX systems. Deduced amino acid sequences of both genes contain all conserved domains typical for two-component systems. The corresponding genes were cloned and overexpressed in E. coli. Autophosphorylation activity of isolated histidine kinase, on its conserved His residue, was veri¢ed by kinase reaction in vitro. The function of identi¢ed system is not known. P6^45 ADAPTIVE RESISTANCE TO BIOCIDES AND CROSS-RESISTANCE TO ANTIMICROBIAL AGENTS IN SALMONELLA ENTERICA AND ESCHERICHIA COLI M. Braoudaki and A. C. Hilton Aston University, Birmingham, UK The inappropriate use of domestic disinfectants has raised concern about promoting microbial resistance and potential cross-resistance to therapeutic antibiotics. The aim of this study was to investigate the potential for adaptive resistance in Salmonella and Escherichia coli O157 to commonly used biocides, to identify mechanisms underlying resistance and any cross-resistance to antibiotics. Salmonella and E. coli were serially exposed in sub-inhibitory concentrations to erythromycin, benzalkonium chloride, chlorohexidine and triclosan. Following each passage any adaptive resistance, or cross-resistance in a panel of antibacterials was recorded. Adaptive resistance was ob-

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served in all strains investigated. Erythromycin-resistant S. enteritidis expressed cross-resistance to chloramphenicol, whereas in erythromycin- resistant S. typhimurium crossresistance to chlorohexidine was detected. Benzalkonium chloride resistant S. virchow showed an elevated resistance to chlorohexidine, however chlorohexidine resistant S. virchow did not demonstrate cross-resistance to benzalkonium chloride suggesting speci¢c rather than general mechanisms. Triclosan-resistant E. coli O157 strains repeatedly exerted decreased susceptibility to various antimicrobials, including chloramphenicol, erythromycin, imipenem, tetracycline and trimethoprim, as well as to a number of biocides. Conversely, E. coli O55 when adapted to triclosan did not show any cross-resistance to any of the antimicrobial agents tested. The adaptive mechanisms underlying resistance were stable for up to 30 days when strains were passaged in antibiotic/biocide free media. It appears possible that the domestic kitchen may provide a selective environment for adaptive resistance to biocides. This may eventually lead to the undesirable situation of resident strains becoming resistant to disinfection and cross-resistant to other antimicrobials. P6^46 VACCINE POTENTIAL OF S. PNEUMONIAE (PNC) SURFACE PROTEINS Y. Mizrachi-Nebenzahl(1), G. Feldman(1), M. Portnoi(1), R. Dagan(1), K. Overweg(2), J. Wells(2), E. Ling(1) (1) Ben Gurion University, Beer Sheva, Israel; (2) Institute of Food Research, Norwich, UK Pnc is an important respiratory pathogen that induces a wide range of diseases, varying from asymptomatic carriage to lethal meningitis and sepsis. Children are particularly susceptible to Pnc. The current polysaccharidebased vaccines are only partially e¡ective in children. Pnc surface proteins are highly immunogenic and the antibody response to these proteins is enhanced with the age. We hypothesize that immunization with Pnc surface proteins that are common to virulent and non virulent strains and are immunogenic later in life can elicit protective immune response. Pnc cell wall (CW) proteins were isolated and subjected to 2-D PAGE. Proteins selected according to the above mentioned criteria were sequenced and cloned. Mice were immunized with CW, recombinant Aldolase and GAPDH proteins or with aldolase cDNA and challenged intranasally with 108 cfu of Pnc serotype 3 (WU2). Immunization with CW and aldolase cDNA induced antibody response, determined by Western blot. 79.1% of CW, 43% of rAldolase, 33% with pVAC Aldolase, 36% of rGAPDH and 40% with pVAC GAPDH immunized mice and 0% of control mice survived challenge. The non-survivals remained alive for 48 hours lon-

ger then controls. The ability of these proteins to circumvent the immune evasion of Pnc WU2, previously observed in our laboratory, is currently studied. P6^47 OBSERVATION OF LISTERIA MONOCYTOGENES IN HELA CELL CULTURE J. Nowroozi(1), J. V. Youse¢(2), R. K. Moakhar(2), M. A. Jebelli(1) (1) Iran University of Medical Sciences and (2) Institute of Vaccines and Serum Hesarak Razi, Tehran, Iran Listeria monocytogenes can grow inter and intracellular. Immunosuppressed individuals, elderly people and pregnant woman are at speci¢c risk with this bacteria. So, the aim of this study was to determine the incidence of this bacteria in the local cheese, observation them in Hela cell culture by light microscopy. In this experimental study, selective enrichment medium with yeast extract, Palkam and listeria selective agar with cold enrichment was used. Then, after isolation of bacteria, di¡erent concentration of L. monocytogenes obtained from local cheese in qum city were inoculated in Hela cell culture. Then, samples were taken intervally (12, 24, 36, 48 hrs), stained with Gimsa, detected by light microscopy and photographed. It was found that 3.5% of local cheese in qum city was contaminated by L. monocytogenes. Pictures were taken by microscope, showed that this bacteria in concentration more than 5x10 5 after 24 hours can enter into cells and after 36 hours lysed the host cells.The results showed that L. monocytogenes exist in contaminated local cheese. Contamination of dairy products by this bacteria can occur during preparation, transport and distribution. So, because of the high consumption of these products in our country, it is necessary to surveillance the production and distribution to prevent of incidence of listeriosis in susceptible people. P6^48 INVESTIGATION OF CTPA AMONG LISTERIA MONOCYTOGENES AND ITS TRANSFER INTO E. COLI J. Nowroozi Iran University of Medical Sciences, Tehran, Iran Listeria monocytogenes is a Gram positive bacteria, frequently found in the environment and is responsible for serious food-borne diseases such as perinatal infections, septicaemia and meningoencephalitis in humans and animals. For this reason, distribution of the ctpA (copper

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transport protein A) among L. monocytogenes isolated from clinical, environment, dairy, and poultry samples were investigated. Then, ctpA gene was transferred into E. coli DH5-a. This investigation was carried out in two steps (ctpA was found in L. monocytogenes isolated from di¡erent sources, then ctpA gene was transferred into E. coli DH5-a). CtpA DNA from L. monocytogenes was ampli¢ed by PCR, identi¢ed on agarose gel, puri¢ed by phenol, and ligated into pGEM-T vector. Then transferred on X-gal plate containing ampicillin. The sequencing of ctpA DNA was determined by using dye terminator kit and sequencing machine. Using PCR to identify the homologous DNA in 69 isolates, 38% of isolates tested contained the ctpA deteminant. Our results showed that 90% of clinical and dairy isolates, 85% of environmental isolates and 7% of poultry isolates of L. monocytogenes contained ctpA in chromosome DNA. Fortunately, transformation of ctpA from L. monocytogenes into E. coli DH5-a was successful. Since, the existance of ctpA in clinical, dairy and environmental samples was 90% and in poultry was 7%, so, the virulence of all strains of this bacteria are not the same. By introducing of such clone (ctpA gene) into suitable carrier strains, could be expected to produce a good oral immunogen against L. monocytogenes. P6^49 INFLUENCE OF CAPSULE FORMATION IN MELIOIDOSIS AGENT ON ITS PERSISTENCE IN HOST ORGANISM S. F. Popov, V. V. Alekseev and V. Y. Kurilov Antiplague Research Institute, Volgograd, Russia The melioidosis agent of Burkholderia pseudomallei is highly pathogenic for humans and animals. Mechanisms of its survival in vivo haven't been studied thoroughly. The present study evaluates the in£uence of capsule formation in melioidosis agent on its interaction with host cells. The typical strains of B. pseudomallei were used: C-141, 57576, 60806. To reproduce a subacute form of the disease per 6 guinea-pigs were infected subcutaneously with 24-hours cultures with the doses of 103, 104, 105 microbe cells per each animal respectively. The animals were killed with ether at the 22-26 day post inoculation. By autopsy the infection process had a generalized character with multiple necrosis in lungs, liver and spleen. Electron microscopic examination of ultrathin sections of the given organs showed absence of direct damage of parenchymatosous cells by bacteria. Nevertheless, distrophic changes and common intoxication were observed. The other peculiarity of the melioidosis infection was the presence of the agents predominantly in cells of mononuclear phagocytes. One can see bacteria, parasitizing intracellular and having normal ultrastructure, to obtain a capsule. The capsule

was presented by a wide electron transparent zones, evenly surrounding a procaryote body. These zones moderately ¢lled electron solid granulous capsule substance. The part of intact bacteria was in phagolysosomes of macrophages. In these cases the capsule prevented contact of lysosomal contents and the cell wall of microbes. The other part of undamaged incupsulated bacteria was free arranged in cytoplasma. The bacteria, obtaining properties of destruction were revealed, as a rule, in phagolysosomes and hadn't a capsule. Thus, the ability of capsule formation in B. pseudomallei may be considered as a factor of pathogenicity, in£uencing persistance of an agent in a macroorganism. P6^50 CLINICAL AND ENVIRONMENTAL STENOTROPHOMONAS STRAINS : DIVERSITY AND DIFFERENTIATION K. Ribbeck, A. Roder, M. Hagemann and G. Berg University of Rostock, Institute for Molecular Physiology and Biotechnology, Albert-Einstein-Str. 3, D-18051 Rostock, Germany In recent years, the importance of the Gram-negative bacterium Stenotrophomonas maltophilia in biotechnology and as a nosocomial pathogen has increased, giving rise to a need for new information about its diversity and interactions with other organisms. Stenotrophomonas isolates from clinical and environmental sources including strains of biotechnological interest were characterized regarding the production of antifungal metabolites and enzymes, osmoprotective substances and plant growth promoting substances as well as by in vitro interactions with fungi and plants. The 16S rDNA sequence analysis supported the high intraspeci¢c diversity of the isolates found for all parameters and, together with phenotypic properties led to the proposal of a new plant-associated species: Stenotrophomonas rhizophila. The production of osmoprotective substances was di¡erent in clinical and environmental strains while the former ones produced only trehalose additionally glucosylglycerol was found in the latter one. The genes encoding for the biosynthetic enzymes, trehalosephosphate synthase (Tps) or glucosylglycerol-phosphate synthase (GgpS), were partly cloned and sequenced from S. rhizophila strain DSM 14405. Using Southern-blot experiments and PCR applying universal primers designed to amplify speci¢cally partial tps and ggpS fragments from many isolates a clear correlation between the exclusive accumulation of trehalose and the only detection of tps in S. maltophilia strains was found, while in the glucosylglycerol and trehalose accumulating S. rhizophila strain both genes were detectable. The absence or presence of glucosylglycerol and the ggpS gene could be used as a

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speci¢c indicator to distinguish between potential humanpathogenic S. maltophilia strains and environmental strains of the species S. rhizophila. P6^51

P6^52 SEROLOGICAL DIFFICULTIES IN NEUROBORRELIOSIS DIAGNOSTICS ¤ ¤ O. Dakovic Rode, T. Maretic

COMPARISON OF THE FATTY ACID PROFILES AND WHOLE-CELL PROTEINS OF BORRELIA BURGDORFERI SENSU LATO L. Cechova(1), M. Nemec(1), E. Durnova(2), J. Halouzka(3) (1) Department of Microbiology, Faculty of Science, Masaryk University Brno, Tvrdeho 14, 602 00 Brno, Czech Republic; (2) Regional Institute of Hygiene Ostrava, Partyzanske nam. 7, 728 92 Ostrava, Czech Republic ; (3) Institute of Vertebrate Biology AVCR Brno, Department of Medical Zoology, Klasterni 2, 691 42 Valtice, Czech Republic Ten strains of Borrelia burgdorferi sensu lato were analysed in this study. There are represented strains that are undoubtedly associated with Lyme disease (B. burgdorferi sensu stricto, B. garinii, B. afzelii), next B. valaisiana, strains (nearer unappointed) isolated from arthropods and rodents and one non-Borrelia strain. Analysis of the fatty acid methyl esters (FAMEs) of bacteria is a commonly used chemotaxonomic technique. Application of this method to spirochaetes associated with Lyme borreliosis is important for more detailed identi¢cation of bacteria Borrelia burgdorferi sensu lato. Fatty acids are of interest because of their role in pathogenesis of several bacterial diseases. Fatty acid metabolism is not under plasmid control and the presence of certain fatty acids has been shown to correlate with taxonomic conventions. Analysis was performed using a gas liquid chromatography column in conjunction with Microbial Identi¢cation System software (MIS). In B. burgdorferi s. l. were recognized fatty acids with number of carbon atoms from 12 to 18. The major fatty acid components of the Borrelia species used in this study are hexadecanoate (C16:0), cis-octadec-9-enoate [C18:1(9c)] and octadecanoate (C18:0).The cellular debris obtained after lysis of Borrelia were solubilized by sodium dodecylsulfate and studied by polyacrylamide electrophoresis, comparing the molecular weight and relative concentration of the protein bands. Cluster analysis of the PAGE protein pro¢le grouped Borrelia strains into a single cluster at r=0.7. A higher number of all bands in the area between 18 and 60 kDa were observed in the pro¢le of Borrelia strains. In conclusion, the FAME analysis and analysis of whole-cell protein pro¢les by SDS-PAGE proved to be useful to contribute to more detailed study of bacteria Borrelia burgdorferi sensu lato.

University Hospital for Infectious Diseases HDr. Fran Mi¤ haljevicg, Mirogojska 8, 10000 Zagreb, Croatia Neuroborreliosis includes a variety of neurological disorders caused by Borrelia burgdorferi sensu lato. Clinical manifestation of neuroborreliosis is not pathognomonic. The etiological diagnosis is based mainly on serological tests and determination of speci¢c anti-B. burgdorferi antibodies in CSF. Speci¢c antibodies response is in£uenced by phenotypic di¡erences among B. burgdorferi species, di¡erent antigenic structure, their di¡erent geographic spreading, and patient's possibility to react to infection. In some patients with neuroborreliosis antibodies could be produced only intrathecally. Therefore, it is always necessary to test serum and CSF simultaneously and determine the CSF/serum index. In our study serological results of 28 patients with clinical diagnosis of neuroborreliosis were analyzed. Serum and CSF anti-B. burgdorferi IgM and IgG antibodies were determined by recombinant indirect ELISA (Biomedica, Wien, Austria) and capture ELISA (IDEIA Lyme Neuroborreliosis, Dako, Denmark). All sera positive results were con¢rmed by Western blot (Mikrogen, Germany or DPC Biermann GmbH, Germany). The speci¢c antibody index in capture ELISA was calculated as de¢ned by manufacturer. 11/28 (39,3%) patients had proved intrathecal antibodies to B .burgdorferi by capture ELISA antibody index. 21/28 (75,0%) patients had signi¢cant CSF antibody titre by indirect ELISA. 19/28 (67,9%) patients had con¢rmed EIA results by IgM and/or IgG WB. One patient with capture positive ELISA antibodies had no con¢rmation by WB. While serologic assays for anti-B. burgdorferi antibodies have not been standardized yet, all serologic results should be interpreted according to epidemiological and clinical data and used to con¢rm the clinical diagnosis.

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P6^53 THE MOLECULAR EVIDENCE OF BABESIA MICROTI INFECTION IN SMALL MAMMALS COLLECTED IN SLOVENIA D. Duh(1), M. Petrovec(1), T. Trilar(2), K. Prosenc(1) and T. Avsic-Zupanc(1) (1) Institute of Microbiology and Immunology, Medical Faculty, Ljubljana, Slovenia; (2) Slovenian Museum of Natural History, Ljubljana, Slovenia Human babesiosis is an important emerging tick-borne disease. Life cycle of Babesia microti, the main causative agent of this zoonosis in the United States of America, has been well described. In Europe, the zoonotic cycle of B. microti awaits to be determined. Eventhough, B. microti was found in a variety of small mammals in di¡erent countries across Europe, it was only recently detected in Ixodes ricinus ticks in Slovenia by using molecular methods. In order to investigate the mammalian hosts of B. microti in Slovenia we collected 261 small mammals representing 11 species. They were tested for the presence of babesial parasites with a PCR assay based on the nuclear small subunit rRNA gene (nss-rDNA). Only two species, the bank vole (Clethrionomys glareolus) and yellow-necked mouse (Apodemus £avicollis), were infected with B. microti. The prevalence rate was 15.9 % for C. glareolus and 11.8 % for A. £avicollis. Nucleotide sequences of ampli¢ed portions of B. microti nss-rDNA from C. glareolus and A. £avicollis were indistinguishable from each other and identical with those previously described in I. ricinus ticks collected in Slovenia. The results of this study represent the ¢rst molecular evidence of B. microti in small mammals in Europe. Furtheron, the molecular characterisation of B. microti in small mammals as well as in ticks in Slovenia clearly indicates the wide distribution of this parasite in nature. P6^54 IN VITRO EFFECT OF SALIVARY GLAND AND MIDGUT EXTRACTS FROM IXODID TICKS ON THE GROWTH OF BORRELIA GARINII I. Rudolf and Z. Hubalek Medical Zoology Laboratory, Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic, Klasterni 2, CZ-69142, Valtice, Czech Republic The e¡ect of salivary gland extract (SGE) and midgut extract (MGE) from Ixodes ricinus and Dermacentor reticulatus was tested in vitro on the growth, motility and morphology of Borrelia garinii. Concentration of motile

spirochetes (the number of motile cells/ml) was determined at speci¢c intervals using dark¢eld microscopy. In I. ricinus, the concentration of motile spirochetes increased signi¢cantly between days 2 and 11 post inoculation (p.i.) with both SGE and MGE compared to control (C). In D. reticulatus, a signi¢cant increase in concentration of motile spirochetes was only detected with SGE (compared to control) on day 5 p.i., while a marked decrease in concentration of motile spirochetes was observed on day 9 p.i. due to MGE e¡ect, and on day 12 p.i. due to the e¡ect of both organ extracts compared to control. The e¡ect of SGE and MGE on the growth of B. garinii in vitro differed between the two tick species tested : while the extracts derived from I. ricinus (a competent vector for Lyme borreliosis) stimulated signi¢cantly the growth, those from D. reticulatus (a non-competent species) did not a¡ect the growth of borreliae markedly or even inhibited it on days 9 (MGE) and 12 p.i. (MGE and SGE). The results indicate that the tick organ extracts surprisingly need not be inhibitory for pathogen survival in the body of even a non-competent tick species like D. reticulatus in a short-term exposure. P6^55 IMMUNITY IN RABBITS IMMUNIZED CHALMYDOPHILA PSITTACI W. DeptuTa and M. Pawlikowska Chair of Microbiology and Immunology, Faculty of Natural Sciences, University of Szczecin, ul. Felczaka 3a, 71-412 Szczecin, Poland Present study aimed at determining parameters of nonspeci¢c cell-mediated and humoral immunities in rabbits experimentally immunized with a killed antigen of Chlamydophila (Chl.) psittaci strain 6BC (the earlier avian strain of Chlamydia psittaci). The studies were performed on 20 rabbits, 10 received Chl. psittaci strain 6BC antigen, and 10 rabbits served as a control. Blood for the tests was sampled from the marginal ear vein on day 1, 7, 14, 21, 28, 35, 42, 49 and 56 of the experiment. In the blood, the capacity of neutrophilic granulocytes to adhere (ZA) and to ingest standard bacterial strains (expressed by index of phagocytosis, IP, and by the percentage of phagocytes, %kp), reduce nitrotetrazolium blue (NBT) in the spectrophotometric, spontaneous and stimulated NBT test, coef¢cient of granulocyte metabolic activity (WAMG), established in spontaneous and stimulated NBT tests, stimulation index (IS), activity of myeloperoxidase (MPO), concentration and activity of lysozyme (LZM) were estimated in serum. Titre of speci¢c serum antibodies anti-Chlamydia was estimated by complement-¢xation (CF) test. Analysis of the results demonstrated that four out of 12 examined parameters demonstrated augmented WITH

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values (concentration and activity of LZM, activity of MPO, IP), 6 parameters manifested augmented values (ZA, spectrophotometric NBT reduction test, spontraneous and stimulated, spontaneous and stimulated WAMG) while two parameters showed no alterations (%kp, IS). The alterations began on day 7-14 and persisted to days 49-56 of the experiment. Speci¢c anti-Chlamydia antibodies could not be detected by CF test until the day 42 of the experiment. P6^56 VIRULENCE CHARACTERS OF RABBIT ENTEROPATHOGENIC ESCHERICHIA COLI (REPEC) IN HUNGARY AND CZECH REPUBLIC ¤ M. A. Dow(1), I. Toth(1), P. Zs. Fekete(1), P. Alexa(2), E. Oswald(3) and B. Nagy(1) (1) Veterinary Medical Research Institute of the Hungarian Academy of Sciences, Budapest, Hungary ; (2) Veterinary Research Institute, Brno, Czech Republic; (3) UMR960 INRA de Microbiologie Moleculaire, Ecole Nationale Veterinaire de Toulouse, France In a collection 70 E. coli strains, isolated from cases of post weaning diarrhoea of rabbits in Hungary and Czech Republic, PCR investigations were performed for virulence genes. As many as 47 E. coli strains proved to have the eae gene (encoding for intimin) and all of them proved to be of Þ- type of intimin. All eae+ strains had the LEE (locus of enterocyte e¡acement) indicated by the presence of espD gene. Neither the enteropathogen E. coli speci¢c EAF (EPEC Adhesion factor) plasmid nor bfp (bundle forming pili) gene (characteristic for human EPEC) were detected. None of the strains had genes of stx (Shiga-like toxins: stx1 or stx2), lt (thermolable enterotoxin), or sta or stb (thermostable enterotoxin) genes. However, the sequence of porcine attachment associated (paa) gene was detected in 20 strains. The eae+ strains belonged mainly to serogroups O15, O55, O49, O103, O132, O145, O153, and O157. By PCR, the ¢rst described rabbit adhesive factor (AF/R1) sequence was detected in two strains (both of O153 serogroup). Adhesive factor/rabbit2 (AF/ R2) sequence was detected in eleven strains (all of O103 serogroup), and the REPEC adherence locus (ralG) gene was detected in eighteen strains representing: O145, O153, O49, O15, O55, O157 and O132 serogroups. The ral speci¢city was veri¢ed with colony hybridization. The tested eae+ strains strongly adhered to HeLa cells in culture assays in a di¡use way. The type or intensity of HeLa adhesion did not seem to relate to the ¢mbrial type carried by the strains investigated.

P6^57 INFECTIONS OF EXOTIC FISH IN SLOVENIA V. Jencic, M. Ocepek, I. Zdovc, P. Hostnik University of Ljubljana, Veterinary Faculty, Gerbiceva 60, Ljubljana, Slovenia Aquarium and other exotic ¢sh breeding is a popular and economically important amateurish activity. Although ¢sh kept in the closed systems causative agents of diseases can endanger populations in open waters and in the intensive ¢sh farming. Some ¢sh Mycobacteria can be harmful for ¢sh breeders as well. Developed and organised diagnostics of exotic ¢sh diseases can help to reduce the current ¢sh health problems. From epizootic, environmental and ethical point of view, education the owners of exotic ¢sh is extremely important, as well. Diverse ¢sh health problems could be established through systematic clinical examinations. High incidence of bacterial infection caused by Mycobacterium (TBC) was found and control methods were applied. The acidoresistent bacteria have been identi¢ed in clinically changed ¢sh using direct Ziel Nielson staining method. The majority of clinically examined suspicious aquarium ¢sh were positive on Mycobacteriae using special media for bacteriological examinations. Isolated bacteria were M. fortuitum, M. chelonae and M. gordonae. Stained and native bacteriological samples we examined and super¢cial bacteria from the family Myxobacteriae were established. When bacterial nutrient media were employed most often bacteria from the family Aeromonas and species hydrophila/caviae and A. sobria were determined. Carp pox ^ herpes virus was established using pathohistological method in clinically suspicious coi carp (Cyprinus carpio) All clinically suspicious ¢sh were found negative when virologicaly examined, using di¡erent cell cultures. P6^58 MOLECULAR EVIDENCE OF BACTERIA FROM THE GENUS BARTONELLA SP. IN SMALL MAMMALS COLLECTED IN SLOVENIA N. Knap, M. Petrovec, D. Duh and T. Avsic-Zupanc Institute of Microbiology and Immunology, Medical Faculty, Ljubljana, Slovenia Bacteria of the genus Bartonella are emerging human pathogens causing an increasing variety of diseases, most of which are thought to be zoonoses. Therefore, it is important to determine di¡erent vectors and mammalian reservoir hosts of bartonellae. The aim of our study was to investigate the prevalence and the genetic diversity of bar-

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tonellae in small mammals in Slovenia. We collected 146 animals representing 4 species, yellow-necked mouse (Apodemus £avicollis), wood mouse (Apodemus sylvaticus), striped ¢eld mouse (Apodemus agrarius) and bank vole (Clethrionomys glareolus), in 3 di¡erent zoogeographical regions of Slovenia. Animals were tested for the presence of bartonellae with PCR assay based on the ITS regions of Bartonella genome. On overall, 48.6 % (71 of the 146) of small mammals were infected with bartonellae. The prevalence rate di¡ered among 4 species of small mammals tested: 71.8 % (23 of the 32) for A. sylvaticus, 62.7 % (27 of the 43) for A. £avicollis, 31.7 % (13 of the 41) for C. glareolus and 26.6 % (8 of the 30) for A. agrarius. In addition, the evident di¡erence in the prevalence rate of infection was noticed regarding the zoogeographical regions in which the animals were trapped. The PCR ampli¢cation yielded products of di¡erent lengths implying genetic diversity of bartonellae detected in small mammals. To characterise and identify the species of bartonellae the representative amplicons were sequenced. P6^59 IDENTIFICATION AND ANALYSIS OF VIBRIO VULNIFICUS BIOTYPE 2-SPECIFIC DNA SEQUENCES C.-T. Li(1), E. Sanjuan(2), C. Amaro(2) and L.-I. Hor(1) (1) Department of Microbiology and Immunology, National Cheng Kung University, Tainan, Taiwan, R.O.C ; (2) Department of Microbiology and Ecology, University of Valencia, Spain Vibrio vulni¢cus is a bacterial species from warm brackish water with pathogenic potential for humans and several species of aquatic animals. In humans, this pathogen causes wound infections and, occasionally, septicemia. In eels and other aquatic animals, it causes an emergent disease whose symptoms are similar to the vibriosis due to V. anguillarum. This disease was ¢rst registered in Europe in 1989, and since then is the main cause of economic losses in anguilliculture. The eel-pathogenic strains are classi¢ed in the biotype 2 of the species. These strains share highMw plasmids and belong to di¡erent serovars, being the serovar E the most virulent one. The main objective of our work is to determine the genetic basis of eel virulence in V. vulni¢cus. This objective will be developed in di¡erent phases, and we present here the results obtained in the ¢rst one. Suppression subtractive hybridization was used to select biotype 2-speci¢c DNA fragments. The obtained fragments were cloned and analyzed by Southern hybridization, DNA sequence determination and searching of homologous sequences. The selected sequences and the primer pairs derived from them were tested for speci¢city with more than eighty strains, including V. vulni¢cus and

non-V. vulni¢cus isolates. The results obtained in the screening showed that three sequences were speci¢c for serogroup E, and two sequences for eel-virulence. The last sequences were located on one of the common plasmids. The entire nucleotide sequence of this plasmid is now under determination. P6^60 ATTACHING EFFACING ESCHERICHIA (AEEC) BACTERIA IN WEANED PIGS ¤ A. Malik(1), I. Toth(1), L. Beutin(2), B. Nagy(1) (1) Veterinary Medical Research Institute of the Hungarian Academy of Sciences, Budapest, Hungary ; (2) Robert Koch Institute, Berlin, Germany The aim of these studies was to examine the occurrence and the characteristics of the AEEC in postweaning diarrhoea of piglets. Therefore we collected mucosal membranes from the ileum and colon of weaned pigs that died as a result of postweaning diarrhoea. Besides, we have collected rectal swabs from healthy and diarrhoeal weaned piglets. The eae+ isolates were identi¢ed by PCR and further investigated for additional virulence genes (tir, EAF, bfp, stx1, stx2, espD, paa). From 133 piglets that died as a result of postweaning diarrhoea 37 eae+ of 1702 strains were detected (representing 9 % of the pigs). Further 45 eae+ strains were identi¢ed among 535 strains, (from rectal swabs of 57 healthy and 31 diarrhoeal live pigs). The eae-positivity in non-diarrhoeal animals was 14 % and in diarrhoeal animals was 29 %. The main O types of eae+ strains were O45, O49, O28 and O108. The dominant group was O4/O123 carrying both antigens. None of the strains had stx1, stx2, EAF or bfp genes, but 91% was espD+ indicating the presence of the LEE pathogenecity island in almost all strains. The eae genes were typed by PCR: 72 % of the intestinal strains were of L-type, while only 31% of the rectal swab strains were Ltype, the rest having untypable eae gene, suggesting the existence of new variant(s) of eae gene in this collection. Our results indicate that the AEEC bacteria in weaned pigs are relatively frequent and they may have some new types of EPEC virulence traits. COLI

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P6^61 EVALUATION OF IMMUNOPROFILACTIC EFFECTIVENESS OF LEUKOTOXIN INACTIVATED WITH VARIOUS METHODS P. Mikucki, A. Wernicki, A. Puchalski, R. Urban-Chmiel and M. FaTkowska-Podstawka Department of General Prophylaxis and Avian Diseases, Faculty of Veterinary Medicine, Agricultural University Lublin, ul. Akademicka 12, 20-033 Lublin, Poland Mannheimia haemolytica serotype 2 and especially leukotoxin (Lkt) produced by it, is known to be as one of the essential virulence agents participating in etiopathognesis of pulmonary diseases in sheep. Subunit vaccines used so far contain chromatographically puri¢ed or biologically inactivated Lkt. The purpose of the study was to compare protection e¡ectiveness of antibodies induced by Lkt either native or inactivated with formaldehyde or glutaraldehyde. The experiment was carried out on sheep, immunized twice at a two-week interval with various Lkt forms and with commercial vaccine including Lkt. The control animals received only an adjuvant. The immunogenic properties of antigens used, was estimated based on absorbance values of sera antibodies (ELISA). Antibodies protective e¡ect, after intratracheal challenge (1x108 cfu/ml M. haemolytica serotype 2) was estimated on the basis of the extent of lung lesions. Immunized sheep lungs, in comparison with the control group, showed lower extent of in£ammatory process. However, no signi¢cant di¡erences in lung lesions were observed in all experimental groups. Statistically signi¢cant higher absorbance values were observed in all immunized sheep. The relationship between intensity of lung lesion extent, and absorbance values in experimental sera, suggest correlation between antibodies level and their protective e¡ect. The results obtained in our study suggest no signi¢cant in£uence of aldehydes used to Lkt inactivation on immunogenicity and thus on protective e¡ect of antibodies produced. This asssumption, however, needs con¢rmation in further experiments. P6^62 DEVELOPMENT OF A PROTOCOL FOR THE SPECIFIC ISOLATION OF THE PATHOGEN VIBRIO VULNIFICUS BIOTYPE 2 ¤ E. Sanjuan and C. Amaro ¤ ¤ Dpto. Microbiolog|a y Ecolog|a, Universidad de Valencia, Valencia, Spain The haemorrhagic septicemia due to Vibrio vulni¢cus biotype 2 is the main cause of economic loss in eel culture.

This disease emerged in Japan in 1975, arrived to Spain in 1989 and spread to Northern Europe in 1995. Since then, the number of human infections associated with ¢sh manipulation has increased. Some of these cases have been caused by V. vulni¢cus biotype 2, which even though it has pathogenic potential for humans, has been epidemiologically underestimated. This biotype, or more precisely pathovar, comprises strains of at least three di¡erent serovars, and serovar E is the most virulent of them. The recommended protocols for the isolation of V. vulni¢cus from water and seafood samples involve an enrichment step in alkaline peptone water, supplemented or not with polymyxin B, followed by seeding on selective media containing colistin and polymyxin B as selective compounds, and cellobiose as the di¡erential compound. In this work we tested the e/cacy of the recovery of biotype 2 strains from environmental samples of these classical procedures and of a new protocol based on the exclusive capacity of biotype 2 to grow in eel blood. The results obtained in the laboratory and the ¢eld demonstrate that this new protocol is more e¡ective, suggesting that its use for the isolation of the biotype 2 of this species from environmental samples could clarify its epidemiological importance in human and eel health. P6^63 THE CHICKEN HUMORAL IMMUNE RESPONSE IS INVOLVED IN PROTECTION AGAINST ORNITHOBACTERIUM RHINOTRACHEALE INFECTION D. F. Schuij¡el(1), P. J. M. Nuijten(1), A. M. M. A. Pennings(1), J. van Putten(2), P. C. M. van Empel(1) (1) Intervet International BV, Bacteriology R&D, Wim de Korverstraat 35, 5830 AA Boxmeer, The Netherlands; (2) « Utrecht University, Faculty of Animal Health, Department of Bacteriology, Yalelaan 1, 3584 CL Utrecht, The Netherlands Over the last decade, Ornithobacterium rhinotracheale has emerged as an important pathogen associated with respiratory tract infections in poultry. To study the chicken immune response after O. rhinotracheale infection, the role of antibody-mediated immunity was investigated by using two di¡erent strategies. First, young broiler chickens were treated with immunosuppressive cyclophosphamide, thereby depleting antibody-producing B-lymphocytes, followed by a challenge with O. rhinotracheale serotype A. At post-mortem, organ lesion-scores were compared to those of challenged birds with a normal immune system. Immune-suppressed birds su¡ered from signi¢cant higher lesion-scores than the control animals. In the second strategy, B-lymphocyte depleted broiler chickens received sera from protected animals prior to a homologous challenge with O. rhinotracheale serotype A. Lesion-scores from im-

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munized and non-immunized birds were compared. The immune-suppressed birds that received a dose of anti-serotype A antibodies showed lower lesion scores after homologous challenge. Passive immunization with non-speci¢c antiserum prior to challenge did not reduce organ lesions. The results of both studies point out the importance of speci¢c O. rhinotracheale circulating antibodies in protection against infection. Furthermore, administration of serotype A antiserum prior to a heterologous challenge with O. rhinotracheale serotype G signi¢cantly reduced organ lesions, indicating an important role for the humoral immune response in cross-protection against infection with other O. rhinotracheale serotypes. The next step is to identify cross-protective antigens of O. rhinotracheale that induce humoral immunity in order to develop a suitable cross-protective vaccine. P6^64 SOME LESS FREQUENT POTENTIAL ZOONOTIC ANIMAL PARASITES IN SLOVENIA > > A. Vergles Rataj, A. Dovc, J. Posedi, J. Racnik > Veterinary Faculty, Gerbiceva 60, Ljubljana Some less frequent potential zoonotic animal parasites were found in Slovenia in the past few years. Pentastomids were found in 8.3% of examined geckos, 12.2 % of examined monitor lizards and in 16.1% of examined snakes. Acarine ectoparasites ^ Ophionyssus natricis was found on boa skin. Dermanyssus gallinae were often detected on poultry and other birds. It can infestate humans, causing serious problems. Among free-living birds, Argas sp. was noted. Potential zoonotic parasites were detected and determined in pet animals : Tryxacarus caviae in guinea pigs, Cheyletiella sp. in rabbits, Notoedres cati and Otodectes cynotis in cats, Sarcoptes scabiei in dogs. Hymenolepis nana was found in one mouse. Strongyloides sp. was found in monkey. In this article, adult parasites and some of their developing forms and eggs are described and presented. P6^65 FOOT-AND-MOUTH DISEASE EMERGENCE IN U.P. (NORTH INDIA) S. K. Yadav Dept. of Epidemiology and Preventive Medicine, College Of Veterinary Science & Animal Husbandry Mathura U.P., Veterinary University & Cow Research Institute, India The continents like North America, Australia and Antarctica are currently reported free from FMD. But the virus is

still endemic in Asia, Africa, the Middle East and Japan. The other regions supposed to be free are sporadically a¡ected as reported by World organization for Animal Health. It has also been observed that FMD occurred in Italy in 1993, in the eastern part of Greece close to the Turkish border in 1994, 1996 and 2000 and the UK, Ireland, France and the Netherlands in 2001, with the cessation of vaccination against FMD. A systematic epidemiological study on Foot-and-mouth disease was conducted to Asses geographical distribution and seasonal occurrence of di¡erent types and subtypes of the virus associated with this disease and the factors associated with the maintenance and spread of infection in selected areas of North India. For FMD virus isolation and typing work, a total of 21 specimens of vesicular epithelium were collected during this year from di¡erent outbreaks of FMD. All of these were collected from bu¡aloes, the main species involved during the various outbreaks. While 11 of the strains were typed O FMD virus. A bu¡alo trembled and died instantly; autopsy revealed presence of a single small vesicular lesion on the dorsum linguae and pinpoint haemorrhages on the myocardium.The piece of myocardium yielded type O virus. Reports are also available from Mexico and Brazil recording occurrence of malignant form of FMD. The zoonotic impotence of the disease was also studied. P6^66 THE POST FLOOD LEPTOSPIROSIS IN CZECH REPUBLIC K. Zitek and C. Benes Institute for Public Health, Centre for Epidemiology and Microbiology, Prague, Czech Republic Leptospirosis is a typical zoonosis with natural nidality and its occurrence in the climatic conditions of the Czech Republic is sporadic and the incidence is normally about 0,3 cases per 100.000 inhabitants. In 1997 and 2002, however, the incidence of leptospirosis has been in£uenced by the actual natural phenomenon ^ catastrophic £oods, which increased the numbers of serologically diagnosed and registered cases three times, i.c. to 0,9/100.000 in comparison with previous years. In 1997 there have been examined for a leptospirosis a total of 7,156 subjects in the Czech Republic, and the disease was diagnosed and registered in 94 of them (and in 2002 in 92 patients respectively). Two-third of these cases came from inundate areas, half of them in direct relation with the £oods. Four of registered cases (1997) of Weil disease have died in the Czech Republic. The di¡erence between the actual and reported morbidity is critically discussed. The poster contains graphs, maps and tables which described over mentioned.

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P7^1 IDENTIFICATION OF HELICOBACTER PYLORI: COMPARISON OF STAINING METHODS T. Babic(1), H. Basic(2), M. Otasevic(1)

P7^2 TOXOPLASMA GONDII ANTIBODIES IN WOMEN DURING REPRODUCTIVE PERIOD ^ OUR EXPERIENCES D. Cvetkovic, T. Grdanoska, N. Panovski, M. Petrovska

(1) Institute for Public Health, Dept. of Microbiology, Brace Taskovic 50, 18000 Nis, Yugoslavia; (2) Clinical Centre Nis, Institute of Pathology, Brace Taskovic 48, 18000 Nis, Yugoslavia Aim : To compare the sensitivity of detecting H. pylori in gastric biopsy and resection specimens using modi¢ed Giemsa stain and immunohistochemistry using a commercially available anti-H. pylori antibody (Dako, Denmark). Methods: Gastric antral biopsy specimens showing chronic gastritis (27 cases) together with tissue blocks from gastrectomy specimens for duodenal ulcer were histology reviewed. The para/n sections were stained with modi¢ed Giemsa and immunoenzymatic by alcaline phosphatase anti-alkaline phosphatase (APAAP) method. Results : The modi¢ed Giemsa and immunoenzymatic treated sections were carefully examined for the presence of H. pylori. Using a modi¢ed Giemsa stain, the spiral shaped bacteria of H. pylori stained blue, were attached to the brush border of the gastric foveolar epithelial cells and inside gastric pits. In some cases masked bacteria hidden within mucous were obvious only in immunostained preparations (red deposits). Similarly, in modi¢ed Giemsa treated sections, coccoid forms, which were particularly seen in sections from resection specimens, caused some uncertainty. These coccoid H. pylori were obvious in immunostained preparations. Immunoenzymatic staining can be performed on cryostat and para/n tissue sections, but reaction was more intense and di¡use in a cryostatic sections. H. pylori was identi¢ed in 75.9% sections stained with modi¢ed Giemsa, but it could be identi¢ed with greater frequency in sections stained with APAAP (93.1%). In all cases the bacteria were more prominent and easier to detect in the immunostained sections than in sections stained tinctorially. Conclusion : immunohistochemical identi¢cation of H. pylori by the APAAP procedure is a highly sensitive and easy to use method for detecting this organism.

Institute of Microbiology and Parasitology, Medical Faculty, Vodnjanska 17, 1000 Skopje, Macedonia The aim of this study was to analyse the seroprevalence of anti T. gondii antibodies in women during their reproductive period in our country. A total of 400 women were examined: 200 women with pathologic pregnancy (103 with spontaneous abortion, 19 habitual abortion, 39 sterility and 39 with still-born) and 200 who have never been pregnant or had normal pregnancy, as a control group. The sera from women with pathologic pregnancy were collected at the Institute of Microbiology, and the sera from the control group were received from the Republic Center for Blood Transfusion. Following tests were used for detection of speci¢c IgG antibodies : Indirect immuno£uorescent assay (IIF) ( Toxo-Spot IF bioMerieux), France; direct agglutination test (DA) (Toxo-Screen DA bioMerieux), France; enzyme immunosorbent assay (EIA) EIA DIALAB, Wien. The tests for detection of speci¢c IgM antibodies: EIA EIA DIALAB, Wien and immunosorbent agglutination test (ISAGA) (toxo Isaga bioMerieux), France. Obtained results, according to IgG antibodies, are the following: 74.5% of the female patients in reproductive period had no and 25.5% had contact with Toxoplasma gondii. Acute and chronic infection was more frequent in the women with pathology during the pregnancy versus control group. Acute infection was serodiagnosed with presence of IgM antibodies in 10 women from the examined group and one from the control group (p 6 0.01). Acute and chronic infection was more frequently detected in women with diagnosis of habitual abortion. P7^3 AN ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) FOR THE DETECTION OF THE FISH PATHOGEN MORITELLA VISCOSA K. J. Heidarsdottir and E. Benediktsdottir Institute of Biology, Microbiology Labaratory, University L ¤ ¤ of Iceland, Armuli 1A, 108 Reykjav|k, Iceland Moritella viscosa is a psychrotrophic bacteria causing winter ulcers in salmonid ¢sh reared in saline water at temperatures below 10C. The clinical signs of the disease are skin lesions and sometimes haemorrhages in internal or-

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gans. The bacteria has been isolated from winter ulcers in Iceland, Norway and Scotland. The occurrence of the disease might have been underestimated, because M. viscosa is slow growing and possible overgrown by other marine bacteria. Identi¢cation of M. viscosa is also impeded by rather fastidious growth and weak reactivity in most biochemical tests. Therefore an immunosorbent assay (ELISA) with horse radish-peroxidase system was developed. Research have shown that the main antigens of M.viscosa are lipooligosaccharides and a 17-19 kDa antigen in the wall. For coating, two di¡erent antibodies were tested, polyclonal antibodies raised against the cell wall produced in mice, and puri¢ed IgG from rabbits that were raised against whole cell. Puri¢ed and cross-adsorbed polyclonal antibodies from sheep (Microtek) were used for catching. Di¡erent treatment of kidney samples spiked with bacterial cells were conducted to ¢nd the optimal treatment. No cross reaction was observed against non pathogenic strains of M. viscosa or other marine bacterial species tested. P7^4 INCIDENCE OF HEMOLYSIN AND GELATINASE AMONG UROISOLATED ENTEROCOCCI G. Jankoska, M. Petrovska, G. Mirchevska, B. TrajkovskaCurchic, T. Spasenovski Institute of Microbiology and Parasitology, Medical Faculty, Vodnjanska 17, 1000 Skopje, R. Macedonia Hemolisyn, aggregation substance and enterococcal protease, commonly called gelatinase, are some phenotypic markers that have been proposed as possible virulence factors of enterococci. The aim of this study was to determine the production of hemolysin and gelatinase among di¡erent species of enterococci isolated from urine samples. Total of 45 strains of Enterococcus spp. isolated from urine samples were examined. CPS ID2 agar (bio¤ Merieux) was used for isolation and identi¢cation of the strains as Enterococcus spp. VITEK automated system (GPI card) was included in identi¢cation of di¡erent species. Hemolisyn production was detected on Columbia CNA agar as clear zone of L-hemolysis around the streak. Production of gelatinase was determined by using of tripticase soy agar supplemented with 1.5% skim milk. A clear halo around the colonies after overnight incubation at 37C was considered positive for gelatinase production. The strains were identi¢ed as: Enterococcus faecalis (39), Enterococcus faecium (6 isolates). Only 3 strains (6.67% of all) were negative for both hemolysin and gelatinase production. In 14 (31.12% isolates of all) there were present both factors. The rest of isolates (22-48.89% in total) were positive for only one factor, hemolysin or gelatinase (hemolysin in 10, gelatinase in 12 strains). There weren't found any signi¢cant di¡erence in the presence of hemo-

lysin and gelatinase within the di¡erent species. These two markers of virulence, either together or separated, were present in 80% of uroisolated enterococci. P7^5 CARBON SOURCE UTILIZATION FOR DIFFERENTIATION OF THE TEMPE FUNGUS RHIZOPUS OLIGOSPORUS AND RELATED SPECIES J. Jennessen(1), A. R. B. Eriksson(1), J. Olsson(2) and J. Schnurer(1) « (1) Department of Microbiology, Swedish University of Agricultural Sciences, P.O. Box 7025, SE-750 07 Uppsala, Sweden ; (2) Centre for Clinical Trials of Foodstu¡s, Uppsala University, P. O. Box 609, SE-751 25 Uppsala, Sweden The zygomycete Rhizopus oligosporus is used in the production of tempe, a fermented food traditionally made from soybeans, but which also can be made from cereal grains. Di¡erent strains of R. oligosporus di¡er in properties such as mycelium growth, metabolite production, spore production, and optimal growth conditions. The substrate also in£uences the growth and metabolism of R. oligosporus. This makes knowledge from soybean production not directly transferable to production of cereal grain tempe. Increased understanding of basic features of R. oligosporus, e.g. growth on di¡erent substrates, is needed in order to produce tempe of good quality. R. oligosporus and R. microsporus are morphologically very similar. Some strains of R. microsporus have, as opposed to R. oligosporus, been found to produce toxic metabolites. There is thus a need for new methods able to distinguish R. oligosporus from closely related species, as well as to di¡erentiate between R. oligosporus strains. A range of factors in£uences the biology of the tempe fungus. One important factor is the composition of carbon sources of the substrate. Rhizopus species and R. oligosporus strains have been characterised according to their carbon source utilisation patterns during growth in microtiter plates. This presentation will discuss the importance of this new tool for elucidating Rhizopus taxonomy, as well as its use in strain di¡erentiation.

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P7^6 IMPROVED SERODIAGNOSIS OF LYME BORRELIOSIS BY BORRELIA AFZELII IMMUNOBLOT V. L. J. Jovicic(1), E. M. Grego(2), B. L. Lako(3), B. M. Ristovic(3), Z. A. Lepsanovic(3), N. T. Stajkovic(3) (1) Institute of Public Health of Serbia ``Dr Milan Jovanovic, Department for Microbiology; (2) Institute of Molecular Genetics and Genetic Engineering; (3) Institute for Microbiology Military Medical Academy, Belgrade, Yugoslavia To improve serodiagnosis of Lyme borreliosis (LB) the performances of two tests were evalueted.An indirect immuno£uorescent assay (IFA) based on Borrelia burgdorferi sensu stricto, and immunoblot (IB) based on local isolate of Borrelia afzelii were prepared. The serum panels contained 199 serum samples: control group (n=70), and patients at di¡erent stages of LB (n=129). The speci¢city of IB was 96%, and 89% of IFA. In early LB the sensitivity of the IFA was 55% and 92% of IB. In late stage of LB sensitivity was: 70% of IFA and 93% of IB. IgM and IgG antibodies from sera of patients with early and late LB, the most frequently demonstrated reactivity to the OspC. The other signi¢cant proteins in early LB were: p39, p41 in IgM IB, and p83/100, p39, Osp17 in IgG IB; in late LB: p39, p41 in IgM IB, and p83/100, Osp17, p43, p30, p14, and p21. In our study OspC was the most important protein in early and late disease and we postulate that these results are the outcome of performance of IB based on local Borrelia afzelii strain abundantly expressing OspC. The using IB as a con¢rmatory test improves serodiagnosis in our geographical region. P7^7 SUSCEPTIBILITY OF EXTENDED SPECTRUM LLACTAMASES (ESBL) PRODUCING STRAINS OF KLEBSIELLA SPP. AND E. COLI TO ANTIMICROBIAL AGENTS A. Kaftandzieva, V. Kotevska, K. Popovska-Jovanovska, N. Panovski Institute of Microbiology and Parasitology, Medical Faculty, ul. Vodnjanska br. 17, 1000 Skopje, Macedonia ESBL enzymes confer resistance to oxyimino-cephalosporins and monobactams. These enzymes occur predominantly in Klebsiella spp. and E. coli. ESBL producing organisms contain multi-resistant plasmids. As a result, they are often resistant to other classes of antibiotics. We aimed to detect ESBL producing strains of Klebsiella spp. and E. coli and to determine their susceptibility to di¡erent

classes of antimicrobial agents. All strains of Klebsiella spp. and E. coli, isolated from 8 di¡erent clinics from Clinical center in Skopje in a 10-month period, were tested by the disc di¡usion method for antibiotic susceptibility (NCCLS). Intermediate susceptible or resistant to ceftriaxone (CRO) were 144 strains. They were tested for ESBL production by the disc di¡usion test (DDT) and combination disc (CD) method. Identical results were obtained using DDT and CD. In the group of 144 strains, ESBL positive were 86 (59.7%): Klebsiella spp. 56/110 and E. coli 30/34. The DDT method is useful and necessary since ESBLs producers often have a low level resistance to third generation cephalosporins. Moreover, imipenem, meropenem, amikacin and cipro£oxacin showed good in vitro activity against ESBL producing strains of Klebsiella spp. and E. coli. P7^8 THE POTENTIAL OF MABS FOR DETECTION OF BURKHOLDERIA PSEUDOMALLEI N. P. Khrapova, S. N. Tikhonov, E. V. Prokhvatilova and M. Y. Kulakov Antiplague Research Institute, Volgograd, Russia Burkholderia pseudomallei is widely distributed in tropical and subtropical areas. The removal of infection is possible o¡ the endemic areas with persons who temporarily stayed on these territories. Intensive tourism, business trips, migratory movement promote original export of B. pseudomallei. Risk of spreading of melioidosis exists constantly. The actuality of rapid and reliable detection of B. pseudomallei in di¡erent samples increases. That is why national programms of preventing and control the spread of dangerous diseases include the section providing improvememnt of e¡ectiveness of immunodiagnostic means and methods for express detection and identi¢cation of B. pseudomallei. We have created the collection of di¡erent Mabs against conserved termostable epitopes of exopolysaccharide and LPS of B. pseudomallei. On the basis of some of evaluated Mabs preparations for immuno£uorescent test, indirect hemagglutination test and ELISA were prepared. These preparations allow to detect the B. pseudomallei and/or B. mallei and di¡erentiate these two members of the genus. They don't bind to the epitopes located on the antigens of microorganisms of other genera. The qualitive parameters of these preparations make the detection of 80% Burkholderia strains available. The e¡ectiveness of using of these preparations on the basis of evaluated Mabs prevails the e¡ectiveness of using of monoclonal analogues, makes the researches of various specimens of the environment and clinical material more reliable.

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P7^9 MICROBIOLOGICAL MONITORING IN PROPHYLAXIS OF POSTOPERATIVE COMPLICATIONS AT RECONSTRUCTION OPERATIONS OF CHILDREN'S MAXILLOFACIAL SURGEON R. Kipshakbayev Kazakh National Medical University, Department of Children's Stomatology, Tole bi st. 88, Almaty, Kazakhstan, 480090 Studying the micro£ora of an oral cavity both in the preoperative and postoperative periods is of great importance in prophylaxis of post-operative complications following maxillofacial surgery. This research studied the micro£ora of the oral cavity, its sensitivity dynamics in children with congenital clefts of a lip and palate, the development of an optimum algorithm of sampling in an oral cavity and the upper respiratory ways, an optimum set of nutrient media for these micro£ora, and an algorithm of the circuit for e¡ective antibacterial prophylaxis of postoperative complications. We surveyed 240 children from 6 months to 6 years of age with congenital clefts of a lip (118) and palate (122). Material was taken from: 1) border of skin and a mucous upper lip in the ¢eld of a cleft, 2) area of nose on the defect side, 3) edge of palate cleft, 4) back wall of larynx, 5) pharynx. Identi¢cation of speci¢c structure was made by the developed original technique (scienti¢c know-how No 537, 20.12.1999). The research designed an optimum algorithm of 5-days sampling, an optimum set of nutrient media, and tests for identi¢cation of microorganisms in conditions of limited resources. Microbiological monitoring for contamination of postoperative wounds under in£uence of antiseptics and antibiotics was made. We o¡ered a method of unitary introduction of the antibiotic ceftriaxone before operation and processing of a postoperative wound by a 0,01% miramistine (scienti¢c know-how No 518, 2.06.1999). Thus, on the basis of complex qualitative and quantitative comparison of micro£ora of an oral cavity and para-oral areas of healthy children and children with congenital clefts of a lip and the palate, the development of dysbiotic changes for the ¢rst time is revealed at congenital clefts of a lip and the palate which are the important risk factor of development of postoperative complications. Introduction in clinic of the new circuit of postoperative microbiological monitoring have lowered postoperative complications of in£ammatory character and have raised results of surgical treatment by 20% and 12.5% accordingly.

P7^10 ALTERNATIVE METHOD FOR EFFICIENT CONCENTRATION OF PLANT VIRUSES USING MONOLITHIC CHROMATOGRAPHIC SUPPORTS í > P. Kramberger(1), N. Petrovic(2), A. Strancar(1) and M. Ravnikar(2) (1) BIA Separations, d.o.o., Teslova 30, SI-1000 Ljubljana, > Slovenia; (2) National Institute of Biology, Vecna pot 111, POB141, SI-1001 Ljubljana, Slovenia It was shown that plants can acquire a number of plant viruses through the roots. In these cases irrigation water can play a vector role. To detect plant viruses, which are highly diluted in water, a concentration step prior to the detection procedure is essential. Conventional procedures for virus concentration are di/cult and time-consuming. We have applied new chromatographic media, named CIM Convective Interaction Media0 disk monolithic columns for virus concentration. We have tested the usefulness of these columns on two model single stranded RNA plant viruses: rod-shaped Tomato mosaic virus (ToMV) and isometric Cucumber mosaic virus (CMV). We successfully concentrated ToMV on a strong anion exchanger (QA disk monolithic column) and CMV on a weak anion exchanger (DEAE disk monolithic column). The elution of concentrated virus from the monolith was performed by high salt concentration. As shown by ELISA, a few orders of magnitude higher virus titter has occurred in the eluent. Described procedure enables more e/cient and, compared to other methods, much faster concentration of plant viruses. The procedure could be conveniently used in monitoring plant viruses in irrigation waters, and applied to other laboratory manipulations of plant viruses. P7^11 EVALUATION OF FOUR DIFFERENT MEDIA FOR ISOLATION AND IDENTIFICATION METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS FROM REGIONAL HOSPITAL DERIVED SAMPLES í í í P. Kuralt, T. Zohar-Cretnik, A. Storman Institute of Public Health Celje, Department of Medical >> Microbiology, Gregorciceva 5, 3000 Celje, Slovenia Rapid isolation and identi¢cation of Methicillin resistant Staphylococcus aureus from samples, especially surveillance cultures is critical step in preventing spread of bacteria inside the hospital environment. 1089 swabs from patient in the regional hospital were collected from October to December 2002 and 117 were MRSA positive. The purpose of our work was to compare three di¡erent media

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recommended by NCCLS and Phenyl Red Mannitol Broth with aztreonam and ceftizoxime (PRMB) recently described in the literature. All the clinical samples were submerged in Todd Hewitt Broth with supplement (THBS), and PRMB and then streaked onto Manitol Salt Agar with oxacillin plates (MSA) and 5% bovine blood agar plates (KA). The frequency and speed of isolation of MRSA were recorded for each medium separately. The limit of detection was determined with ¢ve control ATCC MRSA strains. In 47% of positive samples MRSA was isolated from all four media, in 9.4% from both broths, in 9.4% only from THBS and in 2.6 % only from PRMB. As the PPV for PRMB after 24 hours was only 19.7%, the combination of solid and liquid media for rapid detection of MRSA is still needed. Interestingly growth of ¢ve control MRSA strains was supported to a very di¡erent extent by each medium.

P7^12 INCIDENCE OF ACTIVE TOXOPLASMOSIS IN REGION OF NISH, SERBIA N. Miladinovic-Tasic(1), S. Tasic(1), A. Tasic(2), B. Petrovic(1), D. Zdravkovic(2) (1) Medical Faculty, Nis; (2) Institute of Public Health, Nis, Serbia Toxoplasmosis, world-spread antroposoonosis, is great problem in many countries today. The aim of the study: The occurrences of active toxoplasmosis on Nis region, in the period from 1998 to 2002, have been surveyed. Samples and methods : the study used the data obtained from the Parasitology Department of the Institute of Public Health, Nis and has examined patients who were permanent citizens of Nis region in the period from 1998 to 2002. Active toxoplasmosis incidence rates in this region have been determined of the sample of 100,000 citizens according to the census from 1991 (396.043). The diagnosis was made on the bases of clinical report and positive immune test by ¢nding speci¢c IgM antibodies. Speci¢c IgM and IgG antibodies have beed detected by indirect immunoenzyme assay. Results : In last ¢ve years in Parasitology Department 1467 patients were examined for speci¢c antibodies to toxoplasmosis. Prevalence of chronic toxoplasmosis was 17,85%, and active toxoplasmosis was diagnosed in 2,93% of patients. The cumulative rate of active toxoplasmosis in ¢ve year period is 10,85, while the average cumulative incidence per a year is 2,17. During the studied period the values of incidence varied from 1,51 in 1998 to 3,02 in 2002. The highest incidence was registered in 2002 (3,02). The incidence of active toxoplasmosis in other years was 2,01 (1999, 2001) and 2,27(2000). Conclusion : In the city of Nis region the incidence of active toxoplasmosis was not high in the last ¢ve years and therefore does not represent a great clinical or epidemiological problem. P7^13 CHLAMYFAST AND MYCOFAST TEST IN DIAGNOSIS OF GENITAL TRACT INFECTIONS IN MEN A. Vasic(1), S. Tasic(2) and N. Miladinovic-Tasic(2) (1) Institute of Public Health, Nis; (2) Medical Faculty, Nis 18000, Yugoslavia Infections with Chlamydia trachomatis (Ch. trachomatis), Mycoplasma hominis (M. hominis) and Ureaplasma urealyticum (U. urealyticim) have been recognised as being common sexually transmitted disiase in industrial countries, and all there suspected to have a pathogenic role in human

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reproductive failure.The prevalence of Ch. trachomatis, M. hominis and U. urealyticum infections in male patients were evaluated. In this study 400 men with symptoms of urethritis were analyzed in the Bacteriology Section of Institute of Public Health in Nis, Serbia. Infection with Ch. trachomatis was ditected with Chlamyfast (International, Mycoplasma ^ France). Mycofast (International, Mycoplasma, France) was used for identi¢cation of M. hominis and U. urealyticum. Ch. trachomatis infection was detected in 35,55% of male patients.In signi¢cant lower percents of patients M. hominis (2,32%) and U. urealyticum (6,20%) infections were found. Mixed infections were detected in only 8 cases.In one case mixed infection was with Ch. trachomatis and M. hominis, and in second case mixed infetion with Ch. trachomatis and U. urealytica was found. M. hominis and U. urealyticum were identi¢ed in material from 6 patients. P7^14 A SCANNER READING ASSISTED MIC (COLORIMETRIC) METHOD FOR SUSCEPTIBILITY TESTING OF ENVIRONMENTAL GRAM NEGATIVE FERMENTATIVE BACTERIA M. Rahman(1), I. Kuhn(1), M. Rahman(2) and R. Moll« « by(1) (1) Microbiology and Tumor Biology Center (MTC), Karolinska Institutet, SE-171 77 Stockholm, Sweden; (2) International Center for Diarrhoeal Disease Research, GPO Box 128, Dhaka 1000, Bangladesh The ScanMIC method is a scanner reading assisted colorimetric MIC determination method, designed for susceptibility testing of environmental gram-negative fermentative bacteria. The method is a slight modi¢cation of the NCCLS recommended broth micro dilution method, using a tetrazolium salt (TTC) as indicator to estimate the endpoint of growth in a microtiter plate (i.e. growth inhibition) and a £atbed scanner to capture the image of the microtiter plate. The choice of TTC as indicator was based upon toxicity values, ease to read by scanner and relation to bacterial growth. A simple in-house software was developed to transform the microtiter plate scan image into numerical values of the pellet sizes and automatically to generate the MIC values. We compared the ScanMIC method for 119 separate coliform strains against seven antimicrobial agents to the NCCLS recommended agar dilution method and another 98 separate coliform strains to the NCCLS recommended broth microdilution method. ``Absolute categorical agreement'' was obtained in 91 %. Agreement for MIC di¡erences (within þ 1 log2 dilution) was obtained in 94 % for ScanMIC versus agar dilution and 98.5 % for ScanMIC versus broth micro dilution. This ScanMIC method was found to be labour saving and

needed only a cheap scanner device for reading, and it may thus be useful for epidemiological surveys of susceptibility pattern in large numbers of environmental isolates. P7^15 X-RAY MICROANALYSIS ^ A POWERFUL METHOD FOR DETECTION OF MICROBIAL CELLS IN THE ENVIRONMENT V. Sorokin, A. Mulyukin, G. El'-Registan and V. Galchenko Institute of Microbiology of RAS, Prospekt 60-let Oktjabrja, 7/2, 117312 Moscow, Russia Standard microbiological culture methods make it possible to reveal only a small fraction of the microorganisms actually present in the environment and gives limited information on the physiological slate of microbial cells in situ. It is often di/cult to distinguish microbial cells and abiotic particles in environmental samples by using only the electron microscopy. The content of S, P, Ca, and K and the Ca/K and P/S ratios considerably di¡er in cells with di¡erent physiological state (vegetative cells, resting cells, endospores, dead cells) and abiotic particles. X-ray microanalysis allowed us to reveal the remarkable and statistically signi¢cant di¡erences of these parameters in various bacterial cells. Also, we used these parameters as markers for the direct detection of microbial cells in natural habitats, such as permafrost. X-ray microanalysis was successfully used for the detection of microbial cells among celllike particles immediately in the samples of ancient Antarctic permafrost deposits (170 thousand years old). The X-ray spectra of cell-like particles were compared with the data obtained for microbial cells of various physiological state. Thus, the absence of P and S peaks in X-ray spectra in the most of cell-like particles allowed us to regard them as non-living objects. Among some other particles that were preliminary recognized as most probable microbial cells by a set of characteristics of the element composition, we were able to ¢nd the resting forms of microorganisms. It had the increased intracellular level of Ca, high Ca/K ratio and low P/S ratio. The distinguishing of cells with di¡erent physiological statuses is important for ecological monitoring of the environment. X-ray microanalysis can be a regarded as a promising tool for a primary detection of microbial cells in situ and to predict their physiological state.

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P7^16 COMPARISON OF TWO METHODS FOR DETERMINATION OF GIARDIA LAMBLIA CYSTS IN FECES N. Svent-Kucina, I. Grmek-Kosnik, M. Ravnik, B. Virnik, M. Petrevcic, M. Erzar Institute of Public Health Kranj, Gosposvetska ulica 12, 4001 Kranj, Slovenia We made comparison of two methods for determination Giardia lamblia cysts in feces in laboratory for gastrointestinal diseases in department of medical microbiology of Institute of public health in Kranj. First method was examination of native samples with Lugol under light microscope, made by two independent examinators. Second method was ELISA test for determination Giardia lamblia cysts from feces. We examined 322 samples from patients and from people who prepare food (preventive examination). 318 samples were negative by both methods. Two samples were positive by both methods. Two samples were negative by ¢st Lugol and positive by ELISA method. However, by second Lugol examination, those two samples turned out to be positive. We conclude that ELISA method is more sensitive for determination of Giardia lamblia cysts from feces. P7^17 EVALUATION OF FLUORESCENT POLARIZATION TEST IN DIAGNOSIS OF HUMAN BRUCELLOSIS V. Taleski Institute of Preventive Medicine, Military Hospital, Department of Microbiology, Skopje, Macedonia Human brucellosis is a zoonosis that remains a worldwide veterinary, medical, and economical problem. Last 8 years, approximately 500-600 new cases of human brucellosis are registered yearly in Macedonia. Tests ranging from culture to serodiagnostic tests to the recent molecular techniques are available. Mostly used are conventional serologic techniques such as RBT (Rose Bengal, Slide Agglutination Test), Wright (Tube Agglutination Test) and Coombs (Antihuman Globulin Test), than ELISA and competitive-ELISA. Fluorescence Polarization Assay (FPA) has been validated for number of species including cattle, swine, bison (Nielsen and Gall). Objectives: Evaluation of Fluorescence Polarization Assay (FPA) in comparison with ELISA and the Conventional serologic techniques in diagnosis of human brucellosis. Patient samples were collected at 5 regional hospitals in Macedonia. Many of the patients were on treatment when blood samples

were collected. Samples were held frozen at -200C until they were processed. A total of 217 sera samples were tested. Initial diagnoses were made by serology: RBT, Wright and Coombs tests. To detect IgM and IgG antibrucella antibodies NOVUM Diagnostica micro plates, coated with Brucella LPS antigen and reader ELISA TECAN-Classic, were used. In FPA, O-polysaccharide prepared from B. abortus strain 1119-3 conjugated with FITC (Fluorescein isothiocyanate) and Fluorescence Polarization Analyzer FPM-1, were used. Results and conclusions: Sensitivity of Wright, Coombs, ELISA and FPA were 82%, 89%, 98% and 86% respectively. Speci¢city of Wright, Coombs, ELISA and FPA were 98%, 100%, 100% and 92% respectively. FPA is a very promising tool in diagnosis of human brucellosis beside diagnosis in animals, further studies concerning sensitivity and speci¢city are needed. ELISA remains a reference method in serologic diagnosis of human brucellosis. P7^18 MORPHOLOGICAL AND MORPHOMETRIC CHARACTERISTICS OF DIROFILARIA SPECIES A. Tasic(1), S. Tasic(2), N. Miladinovic(2), D. Zdrav' kovic(1) and N. Bakrae(3) (1) Institute of Public Health Nis; (2) Medical Faculty Nis; (3) Veterinary Faculty Beograd, Yugoslavia A large number of publications and scienti¢c studies from Europe and other parts of the world show that ¢lariasis of dogs have an important in£uence on the health of dogs and on human health. The aim of this study is the di¡erentiation of ¢lariosis caused by Diro¢laria immitis and Diro¢laria repens in Zrenjanin, the territory known as a district of mosquitoes. In this study there were included peripherical blood smears of 32 dogs. Nostandardized Knott's test with modi¢cation and DIFIL test (evsco, buena, nj, usa) were used for the detection of micro¢lariae. Micro¢laria identi¢cation and determination were based on their morphologic and morfometrical characteristics. Softver Lucia 32G, version 4.11 was used for the determination of morphological and morphometric characteristics of diro¢laria species. Micro¢lariae of Diro¢laria repens were found in all investigated dogs (32), but in two dogs mixed infection with Diro¢laria immitis end Diro¢laria repens was found.

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P7^19 IMMUNOLOGICAL INVESTIGATION IN VAGINAL MYCOSES S. Tasic(1), N. Miladinovic(1), A. Tasic(2) and D. Zdravkovic(2) (1) Medical Faculty and (2) Institute of Public Health, Nis, Yugoslavia Despite numerous researches about primary recurrent genital candidiasis which a¥icts 10% of female population in the world, the cause of it is still unknown. The aim of this study was to investigate whether the disregulation of system immune response, that is to say, the domination TH-2 immune response, could be the cause of recurrent vaginal candidiasis in women. The examination of the parameters of cellular immunity included 20 women with RGC (group A), who were in the remission phase at the moment of examination and 20 women of the control group (group B), who had never had a diagnosis of genital candidiasis. The production of IFN-gamma and IL-4 by mononuclear cells of peripheral blood was stimulated by joined activity of standard mitogen Concavalin-A and speci¢c antigen HKB (heat killed blastospores of Candida albicans). The quantity of produced interferon gamma and IL-4 was detected by immunoenzyme test Quantikine (R/D system USA). The production of cytokine by a joint stimulation of the mononuclear cells of the women's peripheral blood did not di¡erentiate in the examined groups (A-IFN-gamma-average value (AV)1115pg/ml; IL-4-AV-59 pg/ml i BIFN-gamma-AV-1371 pg/ml; IL-4-AV-48pg/ml). The relationship between two ``key'' cytokines was important for the detection of the relationship between Th-2 i Th-1 immune response. In the relation expressed as a quotient of IL-4 cytokine values and IFN-gamma values there was not any statistically signi¢cant di¡erence between the examined groups, or in other words, in all the women there was proved the domination of TH-1 immune response. A- IL-4/IFN-gamma= 55x10-3 ; B- IL-4/IFN-gamma = 51x10-3, p 6 0.05. P7^20 DETECTION OF SERUM SPECIFIC IgG AND IgA ANTIBODIES TO CANDIDA ALBICANS IN WOMEN WITH RECURRENT GENITAL CANDIDIASIS S. Tasic(1), N. Miladinovic(1), A. Tasic(2), P. Vlahovic(3), B. Petrovic(1) and D. Zdravkovic(2) (1) Medical Faculty, (2) Institute of Public Health and (3) Clinical Centre, Nis, Yugoslavia

There have been only a few studies so far which attempted to examine the role of speci¢c serum and secretolytic immunoglobulin in women with recurrent genital candidiasis (RGC). The aim of this study was to prove a possible cause-e¡ect relation between speci¢c immunoglobulin ¢ndings and the ¢ndings of Candida sorts in women's genital tract by the detection of anti-Candida IgG and IgA antibodies in the blood of examined women. The examined test group for serological analysis included 60 women with RGC who had positive ¢ndings of Candida albicans in vaginal mucosa. The control group included 60 women without Candida sp. infection/colonization of genital tract. The speci¢c anti-Candida IgG and IgA antibodies in the blood of the examined women were found by a non-standardized indirect imuno£uorescent test. The speci¢c antiCandida IgG antibodies were proved in 90% of the women with RGC and in 45% of the women in the control group. IgA speci¢c immunoglobulin were found in 12 women of the test group and in only one women in the control group. Lower titer of IgG antibodies was proved in almost the same number of the women in both examined groups. Higher titer of IgG antibodies was proved in a signi¢cantly greater number of the women with a chronic fungal genital infection (34) in comparison to the women in the control group (9). P7^21 DETECTION OF SPECIFIC ANTIBODIES FOR ACTINOBACILLUS PLEUROPNEUMONIAE IN SWINE BLOOD SERA B. Vidic, Z. Grgic, S. Savic-Jevdjenic Scienti¢c Veterinary Institute ``Novi Sad'', Rumenacki put 6, 21 000 Novi Sad, Yugoslavia Actinobacillus pleuropneumoniae, also known as Haemophilus pleuropneumoniae, causes pleuropneumonia in swine, a disease that presents a signi¢cant health and economical problem in intensive swine production. The disease mostly appears in fattened swine, at the age of 2-6 months, but spreads to older and younger animals in the herd. In Europe, mostly serotype 2, 8 and 9 of A. pleuropneumoniae are present. Serologic methods are very important for diagnostics of pleuropneumonia in swine, but also for the determination of infection prevalence in swineherds. Few methods are yet described for diagnosis : agglutination, indirect haemagglutination, complement ¢xation, ELISA, hemolisin-neutralisation test and cytotoxinneutralisation test. The goal of our research was to apply several serologic methods to analyze for the ¢rst time the presence of A. pleuropneumoniae in swineherds in this country. Analysis of 764 swine sera was done, from boars, sows and piglets of 4 swineherds. The samples were taken by free choice, from those that came for regular yearly

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examination for brucellosis and leptospirosis. For detection of speci¢c antibodies to A. pleuropneumoniae, the following methods were used: microagglutination, 2-ME-agglutination and complement ¢xation (CF). Antigens were prepared from a reference strain of A. pleuropneumoniae serotype 2. Antibodies speci¢c for A. pleuropneumoniae were found in all analyzed swineherds. By microagglutination, 27,5-66,6% of animals were positive, with the antibody titre from 1:8 to 1:128. 2-ME-agglutination detected a lower percentage of positive swine and also a lower antibody titre (GMT 16). By modi¢ed CF reaction, 21,147,2% of positive swine were found with the titre of complement ¢xation antibodies from 1:4 to 1:64. Our results con¢rm the presence of A. pleuropneumoniae infection in swineherds and show that there is a need for wider episootiologic research in our region. P7^22 A PCR MICROARRAY BASED APPROACH FOR IDENTIFICATION OF ROTAVIRUS STRAINS C. Booth(1), N. Boonham(1), R. Stones(1), M. Iturriza¤ Gomara(2), J. Gray(2) and N. Cook(1) (1) Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK; (2) Enteric Virus Unit, Enteric, Respiratory and Neurological Virus Laboratory, Central Public Health Laboratory, London NW5 9HT Microarrays, containing immobilised gene-speci¢c DNA or oligonucleotide probes, allow monitoring and analysis of a large number of genetic features in a single hybridisation test. This technology has been used for gene expression analysis, bacterial phylogenetic classi¢cation, gene mapping, and polymorphism analysis. It can be used to identify variants of speci¢c genes, and thus to determine the strain type of a microorganism. In this study, the potential of a PCR microarray-based approach for the detection and identi¢cation of rotavirus strains was demonstrated. The array was designed to identify ¢ve rotavirus genotypes found in the UK. Seven oligonucleotide probes, speci¢c for the VP7 and VP4 genotypes, were spotted onto the glass slides. Rotavirus particles were extracted from faeces and infected cell cultures, using a silica microcolumn. Nucleic acid was extracted and single tube RTPCR was performed using conserved primers, allowing the incorporation of amino-allyl dNTPs. Amplicons were then labelled with £uorescent dyes (Cy3 and Cy5), and hybridised to the microarray. The array was scanned and the identity of the isolate determined by the hybridisation pattern. All rotavirus isolates were correctly identi¢ed by the microarray. The method described is capable of performing all the steps in the analysis from sample treatment to strain identi¢cation, and holds the potential

for expanding the tools available to the public health scientist and molecular epidemiologist. P7^23 PCR EVALUATION OF ESCHERICHIA STRAINS PATHOGENICITY COLI

M. Damian(1), C. R. Usein(1), C. Capusa(2), R. Fagaras(2), M. Cosman(1), D. Tatu-Chitoiu(1), M. Nica(3), M. Florea(1) and G. Mircescu(2) (1) Cantacuzino Institute, Splaiul Independentei 103, Bucharest, R-70.100, Romania; (2) ``Dr. C. Davila'' Clinical Hospital of Nephrology, Bucharest, Romania; (3)''Victor Babes'' Infectious Diseases Hospital, Bucharest, Romania Escherichia coli is a heterogeneous species consisting of both enteric commensal and pathogenic strains. Di¡erent types of E. coli cause various diseases in a range of hosts including intestinal and extra-intestinal infections. Classical bacteriological methods are not able to discriminate among the pathogenic and non-pathogenic E. coli strains. PCR technique was used to detect virulence encoding genes in a collection of isolates from patients with di¡erent types of urinary tract infections. In order to establish the pathogenic power of the strains, the genes coding for adherence (pap, sfa/foc, afa) and for toxins (hly, cnf) was targeted. Urinary and fecal isolates from patients were compared with fecal isolates from controls.

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P7^24 MOLECULAR IDENTIFICATION BY ITS-PCR OF STAPHYLOCOCCAL ISOLATES FROM OVINE SUB-CLINICAL MASTITIS S. F. F. Pereira(1), I. Couto(1,2), C. Queiroga(3), A. Marinho(3), C. L. Vilela(4), I. Santos-Sanches(1,5) and H. de Lencastre(1,6) ¤ ¤ (1) Laboratorio Genetica Molecular, Instituto de Tecnolo¤ ¤ gia Qu|mica e Biologica (ITQB/UNL), Rua da Quinta Grande, 6, Apartado 127, 2780-156 Oeiras, Portugal; (2) ¤ " Centro de Recursos Microbiologicos, Faculdade de Ciencias e Tecnologia (FCT/UNL), Quinta da Torre, 2829-516 Monte da Caparica, Portugal; (3) Departamento de SaniL dade Animal e Vegetal, Universidade de Evora, Apartado L 94, 7002-554, Evora, Portugal; (4) Centro Interdisciplinar de Investigacao em Sanidade Animal, Faculdade de Medic°< ¤ ¤ ina Veterinaria, Polo Universitario Alto da Ajuda, 1300-477 " Lisboa, Portugal; (5) Faculdade de Ciencias e Tecnologia da Universidade Nova de Lisboa (FCT/UNL), Quinta da Torre, 2829-516 Monte da Caparica, Portugal; (6) Laboratory of Microbiology, The Rockefeller University, New York, NY 10021, USA Infectious ovine mastitis is responsible for serious economic losses in sheep farms, as a result of the decreased milk production, decline in milk quality and increased demand for use of drugs and veterinary care. Coagulase-negative Staphylococci species, in particular S. epidermidis, are the most frequent causative agents of ovine intramammary infections. The use of molecular methods for the precise identi¢cation of the staphylococcal species is of enormous importance for the recognition of the etiology of infection. In this study, Internally Transcribed Spacer PCR (ITSPCR) was used to identify a collection of 97 staphylococcal isolates collected from milk samples of 88 ewes with L sub-clinical mastitis in 19 herds from Evora, Portugal. Eighty-three isolates were identi¢ed : the majority (47) as S. epidermidis and the other isolates as S. xylosus (12), S. warneri (8), S. chromogenes (7), S. simulans (4), S. haemolyticus (3), S. lentus (3) and S. hyicus (1). The remaining 14 isolates could not be identi¢ed by ITS-PCR because their ampli¢cation patterns di¡ered from those of the staphylococcal control strains used. The ITS-PCR results were compared with the ones previously obtained by the com¤ mercial kit API STAPH (BioMerieux) and 11 isolates were found to be misidenti¢ed by the kit. ITS-PCR proved to be a simple method that met our needs for the rapid and more reliable identi¢cation of large numbers of isolates. Further characterisation of the S. epidermidis isolates by molecular typing methods should identify di¡erences in strain populations between sheep herds.

P7^25 EFFECTIVENESS OF DIFFERENT DNA EXTRACTION METHOD FOR SALMONELLA ENTERITIDIS DETECTION IN MEAT BY SYBR-GREEN REAL TIME PCR E. Delibato, S. Di Pasquale, D. De Medici, L. Croci and L. Toti Food department, Italian National Institute of Health, Viale Regina Elena 299, 00161 Rome, Italy The objective of this study was to develop a rapid, reproducible and robust method for detecting Salmonella Enteritidis in poultry samples, ¢rst comparing four methods of DNA extraction and puri¢cation from the pre-enrichment culture (i.e., boiling, alkaline lysis, nucleospin and Dynabeads DNA Direct System I) and then combining the most e¡ective method with a Real-Time PCR method based on double-stranded DNA binding dye SYBR Green I using the ABI Prism1 7700 system. The speci¢city of the reaction was determined by the melting temperature (Tm) of the amplicon obtained. The experiments were conducted both on samples of chicken experimentally contaminated with S. Enteritidis and on commercially available poultry samples, which were also used for comparisons with the standard cultural method (i.e., ISO 6579/2001). The comparison between the four DNA extraction methods showed signi¢cant di¡erences for all comparisons, except when comparing boiling and nucleospin (i.e., the two methods that produced the lowest threshold cycle). Boiling was chosen because of its simplicity and rapidity and was combined with SYBR Green I Real-Time PCR, using primers SEFA-1 and SEFA-2. The speci¢city of the reaction was con¢rmed by the Tm, which was consistently speci¢c for the amplicon obtained; the mean peak Tm obtained with curves speci¢c for S. Enteritidis was 82.56þ0.22. The standard curve constructed using the mean threshold cycle and various concentrations of S. Enteritidis (ranging from 103 to 108 cfu/ml) showed a good linearity (R2=0.9767) and a sensitivity limit of less than 103 cfu/ml. The results of this study demonstrate that this SYBR Green I Real-Time PCR constitutes an e¡ective and easy-to-perform method for detecting S. Enteritidis in poultry samples.

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P7^26 UREAPLASMA SPP. DETERMINATION IN WOMEN ATTENDING A FAMILY PLANNING CLINIC IN L GUINE-BISSAU, USING POLYMERASE CHAIN REACTION OF THE MULTIPLE-BANDED ANTIGEN GENE ¤ D. Domingues(1), L. Tavora Tavira(1), A. Duarte(2), A. Sanca(3), E. Prieto(1), F. Exposto(1) (1) Unidade de DST/IHMT, Universidade Nova de Lisboa, Lisboa, Portugal; (2) Departamento de Microbiologia/Fac¤ uldade de Farmacia/Universidade de Lisboa, Lisboa, Portugal; (3) Centro de Medicina Tropical de Bissau/IHMT, ¤ Bissau, Guine-Bissau Although commonly found in the genital tract of assymptomatic women, it has been suggested that only certain subgroups of Ureaplasma spp. are disease associated. Vaginal specimens were collected for identi¢cation of Ureaplasma spp. and to estimate a possible association of this microorganisms with age, absence of lactobacilli, and tetracycline resistance. From the 94 women studied, 40 (43%) carried Ureaplasmas spp. and therefore were biotyped by PCR. Twenty-nine (73%) strains belonged to Ureaplasma parvum, 10 (25%) were Ureaplasma urealyticum and one patient (2.5%) carried both species. Ureaplasma parvum was the most frequently found in all age groups and seems to be more frequent in young women in the 20 to 25 years age group (41%), while Ureaplasma urealyticum was common in women who had between 30-35 years of age (22%). In this study, Ureaplasma spp. was not associated with changes in the vaginal £ora, although the inverse seemed to be true for Mycoplasma hominis. However, Ureaplasma urealyticum was more associated with the loss of lactobacilli than Ureaplasma parvum. The number of Ureaplasma urealyticum strains that presented tetracycline (40%) and multiple resistance (100%) was higher than that presented by Ureaplasma parvum strains (27% and 69%, respectively). P7^27 THE IMPORTANCE OF MOLECULAR TESTING OF PARVOVIRUS B19 INFECTION IN IMMUNOCOMPETENT INDIVIDUALS D. Duh, M. Petrovec and T. Avsic-Zupanc Institute of Microbiology and Immunology, Medical Faculty, Ljubljana, Slovenia Human parvovirus B19 is associated with a growing spectrum of disease manifestations. Infection with parvovirus B19 is usually a mild self-limiting disease. However, im-

munosuppressed and immunocompromised individuals as well as pregnant women and their fetuses are at great risk for serious consequences from parvovirus B19 infection. Thus, the diagnostic method for detection of parvovirus B19 must be chosen carefully considering the type of pathology and the type of patient. While molecular testing should be the test of choice when diagnosing parvovirus B19 infections in patients at great risk, serological testing should be performed in immunocompetent individuals. In some cases, viral DNA may persist at low level in the blood despite the presence of circulating IgG antibody and therefore virus can not be detected by serological methods. In our study, 11 immunocompetent patients with an acute infection were tested by serological and molecular methods. In consecutive serum samples speci¢c IgM antibodies were detected up to 3 months. The results of serologic testing agreed with the results of molecular methods used, nested and real-time PCR. No viral DNA was detected after 3 months, when IgM antibodies decreased to the undetectable levels. The sensitivity of both molecular methods used was the same. Real-time PCR allowed us to detect minimum of 3 copies of viral genome. Among 11 patients there was 1 individual in which viral DNA persisted for more than a year yet IgM antibodies were no longer detectable. P7^28 CHARACTERISATION OF ANTIBIOTIC RESISTANT SALMONELLA TYPHIMURIUM STRAINS ISOLATED IN THE CZECH REPUBLIC BETWEEN 1984 AND 2002 M. Faldynova(1), M. Pravcova(1), F. Sisak(1), H. Havlickova(1), I. Kolackova(2), A. Cizek(3), R. Karpiskova(2) and I. Rychlik(1) (1) Veterinary Research Institute, Hudcova 70, 621 32 Brno, Czech Republic; (2) National Institute of Public Health, Center for Food Chain Hygiene, Palackeho 1-3, 612 42 Brno, Czech Republic; (3) University of Veterinary and Pharmaceutical Sciences, Palackeho 1-3, 612 42 Brno, Czech Republic In 1985, a new clone of pentadrug resistant Salmonella enterica serovar Typhimurium appeared. Since that time it has spread throughout the world. In this study we focused on the introduction of this clone in the Czech Republic and in the evolution of the resistance to antibiotics in general. In a collection of 66 strains isolated between 1984 and 2002, genes coding for the antibiotic resistance were determined using speci¢c PCRs and DNA sequencing. We found that the pentadrug resistant clone ¢rst appeared in the Czech Republic in 1990. The genetic basis of antibiotic resistance in strains isolated before this date was considerably di¡erent from that observed in current

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strains. Strains isolated in 1984-85 were typical by the presence of strA, tetB, tetC, cat, TEM and sul2 genes. Most of these strains were free of integrons. Rarely isolated integron-positive strains from this period were always of di¡erent genetic composition from recent isolates i.e. they never coded for aadA2, £oR, tetG and PSE1. Such genes are on the other hand found in 80% of present antibiotic resistant strains. In a single strain isolated in 1991 we found a new variant of the aadA gene designated aadA21, the 5' end of which was identical with aadA2 and the 3' end of which was identical with aadA1. This ¢nding further con¢rms introduction and mixing of a new bacterial population in the beginning of the 1990's in the Czech Republic. P7^29 THE IDENTIFICATION AND DISCRIMINATION BETWEEN EUROPEAN AND AMERICAN PATHOGENIC STRAINS OF YERSINIA ENTEROCOLITICA BY PCR-MSSCP TECHNIQUE R. Gierczynski, M. Jagielski, W. Rastawicki National Institute of Hygiene, 00-791 Warsaw, Chocimska 24, Poland The species Yersinia enterocolitica includes human-pathogenes as well as strains considered non-pathogenic. Hence, strains of Y. enerocolitica are divided to two phylogenetic groups called European and American according to regions of their predomination. The European strains are considered less virulent than American but they often show the higher resistance to L-lactams. Pathogenic strains of both groups possessing gene ail, encoding an adhesine Ail. The nucleotide sequences of ail from European and American isolates show some stable di¡erences. In this report we show the simple, sensitive and highly reliable method for identi¢cation of pathogenic strains of Y. enterocolitica and determination of its phylogenetic group by the multitemperature single strand conformation polymorphism (MSSCP) technique. We compared the single strand conformation pro¢les of 425 bp fragment of ail gene from 6 strains of Y. entrocolitica O :8 (American) and 32 European strains representing serogroups O:3 (28 strains), O:9 (3), O:5,27 (1). All tested DNA fragments of European strains revealed identical pattern that was easily distinguishable from pattern observed for American strains. The comparison of the nucleotide sequences of analysed amplicons of O:8 and O :3 strains indicated the presence of 15 point mutations. All the test procedure including PCR, MSSCP and silver staining was ¢nished within 6 hours. If comparing with classic RFLP, the PCR-MSSCP appears to be more reliable and sensitive but less time and funds consuming when the large numbers of samples are analysed.

P7^30 EPIDEMIOLOGICAL STUDIES OF A SALMONELLA ENTERICA SEROVAR TYPHIMURIUM OUTBREAK IN HUMANS AND ANIMALS IN ICELAND E. Gunnarsson(1), S. Gudmundsdottir(2), H. Hardardottir(3), S. Bjarnadottir(1) (1) Institute for Experimental Pathology, University of Iceland, Keldur, 112 Reykjavik ; (2) Icelandic Fisheries Labo¤ ¤ ratories, Skulagata 4, 101 Reykjav|k; (3) Department of Microbiology, Landspitali University Hospital, 101 Reykjavik, Iceland The present study deals with the use of Pulsed-Field Gel Electrophoresis (PFGE) and phage typing (PT) as an epidemiological tool in a study of an outbreak of Salmonella enterica Serovar Typhimurium (S. Typhimurium) in Iceland. S. Typhimurium was diagnosed as a cause of death of two foals on one farm late in September 1999. Two months later S. Typhimurium was isolated from a dead cow on a big dairy farm in the same district. In a survey of all cattle on the farm S.Typhimurium was found in 40 % of the samples examined. During the next weeks and months this serovar was isolated in connection with disease or deaths of horses on three additional farms. S. Typhimurium was also traced back to one more farm after isolation in a slaughterhouse. In a survey on those farms involved S. Typhimurium was found in cattle, horses, sheep and dogs. On two of the farms some people in the household got sick. During the winter S. Typhimurium was also isolated from a poultry farm, horses and a dog in the Reykjavik area and from horses in the south-western part of the country. Many isolates of Salmonella from humans at this time were identi¢ed as S. Typhimurium but no strains of this serovar were isolated from food in Iceland. It was suspected that the transmission was from animals to humans. The typing revealed that the majority (84%) of the strains belonged to the same PFGE and phage type. This outbreak strain might be endemic in Iceland.

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P7^31 DYNAMIC MODIFICATION OF MICROORGANISMS BY PYRENE-BUTANOATE FOR FLUOROMETRIC DETECTION IN CAPILLARY ELECTROKINETIC TECHNIQUES í ¤ fi> > M. Horka(1), F. Ruzicka(2), K. Slais(1) (1) Institute of Analytical Chemistry, Academy of Sciences >| of the Czech Republic, Vever¤ 97, 611 42 Brno, Czech Republic; (2) Department of Microbiology, Faculty of Medicine, Masaryk University Brno, Czech Republic The capillary electrokinetic techniques such as capillary zone electrophoresis and capillary isoelectric focusing can be used to on-line rapidly separate, identify, quantify or study the mixed bacterial cultures and fungi. The capillary isoelectric focusing of the low-molecular-mass pI markers and mixed cultures of microbial populations of Escherichia coli, Candida albicans, Staphylococcus epidermidis and Enterococcus faecalis with UV detection were recently shown. It was possible to quantify bacterial cells according their peak areas; the minimum detectable number of microbial cells was 5 x 102 ^ 1 x 103. The infectious doses of pathogenic bacteria can be very low in order of tens to hundreds of cells. Therefore, the sensitive and selective £uorescence detection is needed. Pyrenebutanoate and non-ionogenic tenside on the basis of pyrenebutanoate as the £uorescent compounds were used as a bu¡er additives in capillary zone electrophoresis or capillary isoelectric focusing, respectively, for a dynamic modi¢cation of the microbial samples. Using the deuterium lamp UV excitation for the on column £uorometric detection, the minimum detectable amounts of the microorganisms in order of ones to tens of cells sampled on the separation capillary was achieved. P7^32 FTIR MICROSPECTROSCOPY AS A NOVEL METHOD FOR DETECTION OF MALIGNANT CELLS INFECTED WITH RETROVIRUSES AND CELLS INFECTED WITH HERPES VIRUSES M. Huleihel, M. Talyshinsky, Y. Souprun, I. Mukmanov and V. Erukhimovitch. The Institute for Applied Biosciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel In the present study we used microscopic Fourier-Transform Infrared spectroscopy (FTIR) to investigate and to detect malignant cells which were transformed in culture by murine sarcoma virus (MuSV) and cells infected with herpes viruses (herpes simplex 1,2 and varicella viruses).

For this study various primary cells from di¡erent animals were used for transformation with MuSV and Vero cells (cell line) were used for infection with herpes viruses. The advantage of microscopic FTIR spectroscopy over conventional FTIR spectroscopy is that it facilitates inspection of restricted regions of cell culture or the tissue. Our results showed signi¢cant and consistent di¡erences between the various tested normal cells and malignant cells transformed by MuSV and cells infected with herpes viruses. A considerable decrease in carbohydrates and phosphates levels was seen in malignant cells compared to the normal cells. Whereas, a signi¢cant increase in phosphates levels and a decrease in carbohydrates levels were seen in infected cells with herpes viruses compared to uninfected cells. In addition, the peak attributed to the PO2- symmetric stretching mode at 1082 cm-1 in normal cells was shifted signi¢cantly to 1087 in malignant cells, whereas, in herpes infected Vero cells there was no shift in this peak. These results in addition to further di¡erences in the shapes of various bands throughout the spectrum strongly support the possibility of developing the FTIR microscopy as diagnostic method for the detection of malignant and virus infected cells. P7^33 STUDY OF SPECIFIC BINDING OF BACTERIA AND ENTEROVIRUSES ONTO THE AFFINE SUBSRTRATES BY ATOMIC FORCE MICROSCOPY T. E. Ignatyuk(1), I. A. Golutvin(1), S. N. Virjasov(2), S. G. Ignatov(2), N. S. Nasikan(1), I. A. Kabanov(1), A. L. Suvorov(1) (1) State Science Center Institute for Theoretical and Experimental Physics, 25 B. Cheremushkinskaya, Moscow, 117259 Russia ; (2) Science Research Center of Applied Microbiology, Obolensk, Moscow Region 142279, Russia Onrush of nanotechnologies has resulted in a large scale production of scanning probe microscopes (SPM), operating with nanometer resolution, which in its own turn gave rise to the tendency for creation of biosensors that can detect single biological interactions. We used an atomic force microscope (AFM) to study morphological and immune properties of bacteria (Legionella micdadei, Escherichia coli, Micobacterium tuberculosis) and enteroviruses (poliovirus, adenovirus, rotavirus), adsorbed on the surface of gold, silicon nitride, glass, mica and graphite. Both speci¢c and non speci¢c binding to the substrates was studied. In case of bacteria we used Protein A/ antibody complexes to produce a/ne substrates. In case of enteroviruses, amphiphilic polyelectrolites were used to form Langmuir ¢lms of antibodies which were transferred onto the solid substrates. Special image processing software which a¡ords to extensively automatize biological

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objects identi¢cation procedure according to their AFM images was developed. Atomic force microscopy based method can be used for rapid detection and identi¢cation enteroviruses and microbiological objects in water quality assay. P7^34 MOLECULAR TYPING STRAINS IN RUSSIA N. GONORRHOEAE

P7^35 SIMULTANEOUS GROWTH AND DETECTION OF SALMONELLA AND LISTERIA MONOCYTOGENES > B. Jersek Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia Salmonella spp. belong to important food contaminating pathogenic bacteria causing a high number of human infection. Listeria spp. are widely distributed in nature and are frequently isolated from foods. Among Listeria spp. L. monocytogenes has been recognised as the most threatening human pathogen. Traditional culture detection methods take 4 ^ 6 days to obtain results, are time consuming and laborious. The objective of the present study was to detect Salmonella and L. monocytogenes simultaneously. As the best broth for growth of both bacteria universal enrichment broth (UPB) was chosen. It was incubated at least 24 h at 35 C. There was no di¡erence in growth of Salmonella strain when incubated alone or with L. monocytogenes strain, but L. monocytogenes strain grew at slower rate in the presence or absence of Salmonella strain. As simultaneous detection method for Salmonella and L. monocytogenes multiplex PCR was used. Two sets of previously published speci¢c primers were combined in one reaction. DNAs of Salmonella and L. monocytogenes were prepared together. Crude cell bacterial lysate were prepared by mixing bacterial cultures after growth in UPB and lysis bu¡er containing NaOH and SDS and subsequent heating of samples at 94 C for 15 min. The lowest detection level for both bacteria was 104 cfu/ml in the original culture media or 500 bacterial cells in the PCR reaction. The combination of simultaneous growth of Salmonella and L. monocytogenes and simultaneous detection of these bacteria with PCR prove to be sensitive and timee/cient and therefore promising for further development of detection method. P7^36 INFECTION WITH CHLAMYDIA TRACHOMATIS AMONG FEMALES IN GREECE K. Avdeliodi, M. Simou, E. Glynou, and H. Kada Laboratory of Microbiology, Elena Venizelou Hospital, 2 E. Venizelou Sq., Athens 115 21, Greece The objective of this study was to determine the frequency of infection with Chlamydia trachomatis among female outpatients attended at a Greek Hospital during a 6-year period. Materials and methods: During a 6-year period (1/

E. N. Ilina, M. V. Malahova, V. A. Vereschagin, V. M. Govorun, M. M. Zubcov and A. A. Kubanova Research Institute of Physico-Chemical Medicine and Central Research Institute of Dermatology and Venerology, Moscow, Russia The ¢rst typing method for N. gonorrhoeae was developed with monoclonal antibodies in 1984 year. This typing system based on the study of structure of gonococcal major outer membrane protein I (Por I, PI). Peptide mapping analysis divided N. gonorrhoeae strains into two distinct groups designated serovar A (PIA) and serovar B (PIB). It was shown that PIA strains especially associated with disseminating gonococcal infection and PIB strains ^ with antibiotic resistance to wild specter of drugs. Now the molecular biology methods using to typing N. gonorrhoeae strains. During last years gonococcal serovars distributions were studied in several countries. There are no data about gonococcal serovars distribution in Russia. The aim of our study was to use the genetics methods to typing Russian N. gonorrhoeae clinical strains. The N. gonorrhoeae strains were obtained from patients in Moscow and its region. The resistance to quinolones, macrolides, tetracycline and cefatoxime were analyzed by serial agar dilution method using chocolate agar with the selective additions + antibiotics in various concentrations. We report the typing of 41 clinical strains by protein I gene sequencing and Opa gene PCR-RFLP analysis. Genotyping showed there were 11 various groups according Opatyping. Among 41 N. gonorrhoeae clinical strains 34 (83%) believed to PIB serovar and 7 (17%) ^ to PIA serovar. All resistant strains to £uoroquinolones were found to have the PIB type, which is associated to multidrug resistant.

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1/1996 ^ 31/12/2001) specimens from 7.366 consenting outpatients examined at the Department of Obstetrics and Gynaecology were submitted for testing for C. trachomatis. All consecutive specimens were included in the study. Diagnosis of infection was established by a molecular commercial method, the Ligase Chain Reaction (LCR, Abbott) according to the instructions of the manufacturer. A questionnaire was developed for the collection of patient demographical data. Results : Among the total group of patients, there were 6.876 women of Greek nationality (93%) and 488 foreigners (7%). There were 3.315 symptomatic (45%) and 4.051 asymptomatic patients (55%), who were referred for some other reason. Overall, 266 out of 7.366 women were positive by LCR (infection incidence: 3.6%). Among the 266 C. trachomatis infected women 202 were Greek (infection incidence among Greek patients 2.9%) and 64 foreigners (infection incidence among foreigners patients 13.1%) (p 6 0,00001). Only 122 women out of 266 C. trachomatis infected patients were symptomatic (46%). Conclusions : This is the ¢rst report on the incidence of infection with C. trachomatis among patients in Greece as detected by a molecular method. An overall incidence of 3.6% was detected, however infection was more prevalent among foreign patients (13.1%) (p 6 0,00001). P7^37 BACTERIAL IDENTIFICATION AND FINGERPRINTING BASED ON SIMPLE SEQUENCE REPEATS (SSR) Y. Kashi, L. Somer, E. Diamant, R. Gur-Arie, Y. DaninPoleg and Y. Palti. Department of Food Engineering and Biotechnology and Grand Water Research Institute, Technion, Haifa 32000, Israel Analysis of the complete genomic DNA sequences of prokaryotes showed the existence of tens of thousands well distributed Simple Sequence Repeat (SSR) tracts, with core motifs ranging from 1-6 nucleotides. DNA sequences at arbitrarily chosen SSR tracts were compared among strains of E. coli and Listeria. Polymorphisms of SSR copy number were observed at mononucleotide SSR tracts screened, with all polymorphisms occurring in non-coding regions. The identi¢ed SSR polymorphism could prove important as a genome-wide source of variation, both for practical applications (including rapid detection, strain identi¢cation) and for evolutionary adaptation of microbes. SSRs loci in the non-coding portion of microbial genomes are located in areas near gene regulatory elements, supporting hypothesis that SSR polymorphisms may be a part of a ¢ne-tuning mechanism in molecular processes that e¡ect gene regulation. The ¢nding that SSR

tracts are hyper-variable in bacterial genomes, but stable at the strain level, enables the use of SSR for bacterial strains identi¢cation, including discrimination of pathogenic and non-pathogenic strains. The SSR based ¢ngerprinting method identi¢es bacterial strains, assigns an identity number to bacterial strain, and can be developed to a rapid high-throughput typing technology. P7^38 RETROSPECTIVE PCR ANALYSIS OF VIBRIO CHOLERAE EL TOR STRAINS ISOLATED DURING DIFFERENT EPIDEMIC SITUATIONS IN TURKMENISTAN E. A. Kostromitina, N. B. Cheldyshova, N. I. Smirnova and V. V. Kutyrev Russian Anti-Plague Research Institute ``Microbe'', Universitetskaya Str. 46, 410005, Saratov, Russia PCR is important in the diagnosis of dangerous infections including cholera. PCR makes it possible not only to identify the infectious agent early, but also to retrospectively analyse the data enabling the epidemiologic situation to be prognosticated. The aim of this work was to identify genes associated with virulence (ctxA, tcpA, toxR, zot, ace, rtxA, attRS1) in the genome of 123 V. cholerae E l Tor strains isolated in Turkmenistan in the 70-80's from people and the environment using PCR. The complete set of the main virulence genes (ctxA+ tcpA+ toxR+ ) was present only in strains isolated during cholera epidemics from clinical cases or carriers as well as from the environment. 77% of the interepidemic environmental isolates lacked the genes for both the cholera toxin and toxin-coregulated piles. About 43% of vibrio carriers in Turkmenistan were also shown to be infected with non-toxigenic variants that contained the tcpA genes whose product provides for e/cient small-intestinal colonization. Such strains inhabited the surrounding medium, too (18%). Some V. cholerae El Tor strains whose genotype was ctxA- ace+ zot+ were found to carry CTX phi bacteriophage with an incomplete set of virulence genes. Gene rtxA encoding the main subunit of the RTX toxin capable of causing gastroenteritis, was present in the chromosome of 92% of isolates analysed. In fact, all ctxA- tcpA+ strains were also shown to carry a genomic site, attRS1, indicative of their possible infection with CTX phi bacteriophage. Thus, our data may suggest that under the environmental conditions in Turkmenistan in the 70-80's, toxigenic strains could originate from non-toxinogenic ones, because a large portion of the El Tor cholera vibrio population living in vibrio carriers and in open-water reservoirs during the interepidemiologic period contained not only the tcpA genes encoding the receptor for CTX phi bacteriophage, but also a genomic attRS1-site necessary for bacteriophage invasion

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into the chromosome resulting in the formation of stable lysogens. P7^39 A STUDY OF THE POPULATION STRUCTURE OF NONTOXIGENIC VIBRIO CHOLERAE O1 ISOLATED FROM THE IMMUNOCOMPROMISED PATIENT E. A. Kostromitina, N. I. Smirnova Russian Anti-Plague Research Institute ``Microbe'', Universitetskaya Str. 46, 410005, Saratov, Russia A single case of cholera is described in a 69-year old-man, su¡ering from hormonal bronchial asthma and chronic hypoacidic gastritis for a long time. A Vibrio cholerae O1 El Tor Inaba serotype strain with typical morphological, cultural and biochemical properties with low virulence for suckling-rabbits and epidemically safe according to epidemiological observations was isolated from the patient. Despite the typical clinical symptoms (more than 30 liters liquid loss), the chromosome of the isolate was negative by PCR for cholera toxin gene (ctxA), responsible for severe watery diarrhea, and for toxin-coregulated pilus gene (tcpA), encoding critical colonization factor. It was positive for essential regulatory gene toxR and gene of soluble hemagglutinin/protease (hapA). To understand the nature of virulence of this strain we investigated the structure of its population. The study of 2000 clones of V. cholerae El Tor strain revealed the uniformity of its population in production of some pathogenic enzymes : all the clones expressed hemolysin, phospholipase and hemagglutinin / protease activity. PCR assay of 30 random clones demonstrated the absence of ctxA (that was con¢rmed by DNA-hybridization with CT-probe) and of tcpA, as well as of zot and ace, encoding additional toxins and situated on CTX-genetic element along with genes ctxAB, and additional regulatory gene toxT. All clones were positive for rtxA, forming a part of a RTX-genetic element which encodes biosynthesis of some enterotoxin able to cause weak diarrhea, and were negative for the NAG-speci¢c st gene, encoding heart-stable toxin. We came to the following conclusions : (i) the examined population of V. cholerae El Tor is homogeneous in structure and does not contain ctxA and tcpA genes con¢rming its epidemiological safety; (ii) the severe gastroenteritis in the patient was probably caused by other toxins (RTX-toxin, hemolysin, as well as Sanyal toxin) on the background of the obvious decrease of host antibacterial resistance resulting from hypoacidity and immunosuppression, caused by steroid hormones given to the patient over a long period.

P7^40 A THREE-DAYS PCR-BASED METHOD FOR THE DETECTION OF LISTERIA MONOCYTOGENES IN FOOD ¤ ¤ ¤ E. Kacl|kova(1), D. Pangallo(1), H. Drahovska(2), K. ¤ (1), T. Kuchta(1) Oravcova (1) Department of Microbiology and Chemistry, Food Re¤ search Institute, Priemyselna 4, P.O. Box 25, SK-82475 Bratislava 26, Slovakia ; (2) Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, ¤ Mlynska dolina B-2, SK-84215 Bratislava, Slovakia A three-days PCR-based method is presented for the detection of Listeria monocytogenes in food. The method is equivalent to EN ISO 11290-1 or ISO 10560 in terms of the same detection limit of 100 cfu per 25 g or 10 g and 100 % relative accuracy. The version alternative to EN ISO 11290-1 begins with a primary enrichment in half Fraser broth (24 h), secondary enrichment in Fraser broth (24 h) and post-enrichment in Brain heart infusion broth (5 h). The version alternative to ISO 10560 begins with enrichment in Listeria enrichment broth (48 h) and postenrichment in Tryptone soya broth (5 h). These steps are followed by bacterial cell lysis by boiling, PCR oriented to inlB gene using a mimic internal control, and agarose gel electrophoresis. At the evaluation of the method on model food samples arti¢cially contaminated with decimal dilutions of a L. monocytogenes culture (cheese, smoked ¢sh, ready-to-eat meat products; 21 samples altogether), a detection limit of 100 cfu per 25 g or 10 g was determined. Dead L. monocytogenes cells did not cause false positivity, as determined using model food samples arti¢cially contaminated with decimal dilutions of dead L. monocytogenes cells. At the evaluation of the method on naturally contaminated food samples (same as above, 140 samples altogether) identical results (8 positives) as with the reference method were obtained. P7^41 MULTIPLEX PCR ANALYSIS FOR SIMULTANEOUS DETECTION OF GENERA CARNOBACTERIUM AND LEUCONOSTOC M. C. Macian(1,2), E. Chenoll(2), R. Aznar(1,2) (1) Department of Microbiology and Ecology, University of Valencia ; (2) Institute of Agrochemistry and Food Technology (IATA), Spanish Council for Scienti¢c Research (CSIC), P. O. Box 73, Burjassot E-46100, Valencia, Spain Members of genera Carnobacterium and Leuconostoc have been reported during last years as potential food spoilers,

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especially of vacuum-packed meat products. Several authors focused their interest on developing rapid and reliable methods for detection of both LAB genera, and subsequently several genus-speci¢c primers for PCR detection are available in the literature. However, as a result of taxonomic rearrangements new species have been described in both genera. In the present work we have evaluated the speci¢city of the reported PCR primers and have designed new genus- , group- and species-speci¢c primers in order to accommodate speci¢city to the new taxonomic situation. As a result a Multiplex-PCR assay has been developed that allow simultaneous detection of members of both genera as well as members of the non-motile Carnobacterium species: C. divergens, C. maltaromicum and C. gallinarum, that are the most frequently isolated as potential food spoilers. Species-speci¢c identi¢cation is also possible by applying another Multiplex-PCR reaction. Both PCR procedures have been tested using reference strains as template and have proved useful for colony identi¢cation of isolates (grown on MRS or Blood-Agar plates). In addition, they could be successfully applied for PCR detection of genus Carnobacterium and Leuconostoc, and non-motile Carnobacterium species, in arti¢cially inoculated as well as naturally contaminated vacuum-packed meat products. P7^42 CORRELATION BETWEEN THE PLASMID PRESENCE AND THE RESISTANCE TOWARDS BETALACTAMS IN KLINICAL ISOLATES OF KLEBSIELLA SPECIES I. H.-P. Meloska, G. Jankoska, G. Mircevska, M. Petrovska Institute for Microbiology and Parasitology, Medical Faculty, Vodnjanska 17, Skopje, Macedonia The increasing use of extended spectrum beta-lactams in the last decade has been associated with the emergence of plasmid spread of resistant bacterial strains. The aim of the present study was to determine, if the resistance of our hospital isolates of Klebsiella species towards beta lactams is correlated with their plasmid content. This study included a total of 60 (45 K pneumoniae and 15 K oxytoca) strains, originating from in-patients in Surgical Clinics in Skopje and isolated from respiratory samples, wound swabs and drains. The susceptibility testing was preformed with disc di¡usion method and VITEK (bioMerieux, France) automated technique. The plasmid content was determined by thermal-alkal lyses method, as described by Kado and Liu followed by agarose gel electrophoresis. The resistance of Klebsiella spp was the following: amoxicillin clavulanic acid (68.3%), piperacillin (100.0%), piperacillin tazobactam (78.3 %), ceftazidime (83.3%), ceftriax-

one (90.0%), cefotaxime (85.0%) and ce¢pime (41,7%). Plasmids sized : 55kb, 65kb, 75kb, (50 & 80kb) and (55 & 85kb), were isolated from 53.3% of examined Klebsiella spp strains. The presence of plasmids was signi¢cantly higher (84.4% and 96.9%) in strains resistant to penicillins and third generation cefalosporins (p 6 0.0000). However, out of 25 strains resistant to ce¢pime, 18 (72.0%) had plasmids. The plasmids analyses showed that all Klebsiella spp strains with one 65kb plasmid were resistant to penicillins and to cephalosporins. Only two strains without plasmids were resistant to all four examined cephalosporines. The predominant isolation of plasmids from strains resistant to beta-lactam antibiotics suggests the plasmid origin of this resistance. P7^43 ARE THE ONCOGENIC HUMAN PAPILLOMA VIRUS TYPES INCLUDED IN CERVICAL HPV INFECTION OF WOMEN IN MONTENEGRO? G. Mijovic(1,2), N. Jokmanovic(3), Z. Zekovic(1), M. Bujko(1,2) (1) Institute of Public Health, (2) School of Medicine, University of Montenegro, and (3) Clinical center of Montenegro, Podgorica, Montenegro Human papillomaviruses (HPV) are small DNA viruses associated with epithelial lesions ranging from certain benign proliferative lesions to invasive carcinomas. More than 100 HPV types were identi¢ed, but some of them have certain oncogenic potential, and are associated with cervical neoplasia. The aim of this study was to investigate if the oncogenic HPV types are included in cervical HPV infection of women in Montenegro. HPV infection was diagnosed by using the method of hybridization in situ, and 115 cervical swabs of women (aged 17- 62 years) who visited Gynecologic Ambulance of Clinical Center in Podgorica from April 2000 to November 2001 were examined. Cervical samples of 85 women with clinical signs of cervicitis and 30 cervical swabs of women without any clinical signs of cervical disorder were tested. Screening test was positive in 51 cases (44,3 %). HPV typing was done in 41 samples and the results were positive for: HPV type 6/11 in 18 (35,3%), type 16/18 in 21 (51,2%), types 31/ 33/35/51 in 23 (45,1%). 30% of women without clinical signs of cervical disorder were HPV positive. The results of this investigation showed that among women with cervical HPV infection, oncogenic HPV 16/18, and HPV 31/ 33/51 types were present in 51% and 45% cases, respectively. These results indicated that HPV infection, especially caused by oncogenic types, represent a signi¢cant public health concern in Montenegro.

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P7^44 COMPARISON OF THREE PCR-BASED METHODS FOR THE DETECTION OF LISTERIA MONOCYTOGENES IN FOOD ¤ ¤ ¤ E. Kacl|kova, K. Oravcova, T. Kuchta Department of Microbiology and Chemistry, Food Research ¤ Institute, Priemyselna 4, P.O. Box 25, SK-82475 Bratislava 26, Slovakia Following PCR-based methods for the detection of Listeria monocytogenes in food were compared: 1. BAX for Screening L. monocytogenes (Cat. No. 1771 0605 ; Qualicon, Wilmington, DE, USA), 2. Probelia L. monocytogenes (Ampli¢cation Kit Cat. No. 357 8004 and Detection Kit Cat. No. 357 8005; Bio-Rad, Marnes-la-Coquette, ¤ ¤ France), 3. three-days PCR-based method of Kacl|kova et al. presented elsewhere in this Congress. Method 1 features all PCR reagents supplied in a tablet, the ampli¢cation product is detected by gel electrophoresis and a 2days enrichment is required prior to PCR. Method 2 features PCR reagents in a stabilized everything-containing mastermix, the ampli¢cation product is detected by hybridization and subsequent ELISA and a 2-days (1-day for non-meat samples) enrichment is required prior to PCR. Method 3 is a cheap protocol requiring pipetting all reagents, the ampli¢cation product is detected by gel electrophoresis and a 2-days enrichment is required prior to PCR. All the three methods employ an internal ampli¢cation control. The methods were evaluated at the analysis of model food samples arti¢cially contaminated with L. monocytogenes (4 types of cheese, 3 types of meat products, 1 type of smoked ¢sh and 1 type of frozen vegetable salad) along with the standard method for the detection of L. monocytogenes in food, EN ISO 11290-1. All the three PCR-based methods had the same detection limit as the standard method, 100 cfu / sample. However, methods 1 and 2 produced false positive results for samples contaminated with dead L. monocytogenes cells.

P7^45 DIAGNOSTICS OF DIARRHEAGENIC ESCHERICHIA COLI BY USE OF MULTIPLEX-PCR DIRECTED TOWARDS CHARACTERISTIC VIRULENCE GENES S. Persson, K. E. Olsen, F. Scheutz, J. Sonne-Hansen, K. A. Krogfelt, V. Fussing and P. Gerner-Smidt Dept. of Gastrointestinal Infections, Statens Serum Institut, Artillerivej 5, 2300S Copenhagen, Denmark Diagnostics of diarrheagenic E. coli groups have traditionally been based on culturing faecal samples, followed by biochemical characterization, DNA hybridisation, serotyping and/or detection of toxins. Many reports have all ready shown the potential of PCR as a diagnostic tool, by directing primer towards the virulence genes that characterise these pathogens. However, most of these methods, are not taking the routine diagnostic problems of good sensitivity limits and proper positive controls into account. The present method relies on multiplex-PCR on a simple DNA preparation from plate grown cells, followed by PCR product detection by gel electrophoresis. This was done by designing one multiplex-PCR containing 6 genes necessary for classifying the E. coli groups: ETEC, VTEC, EPEC and EIEC, where each amplicon had a distinguishable size. The assay was also designed to contain a primerset directed towards a universal 16S DNA sequence, serving as a positive control for the PCR. By this assay we were able to detect as little as 1 pathogenic colony in a bacterial mixture of 104 commensal colonies on the plate. Compared to a probe-hybridisation method previously used in our laboratory, the PCR assay showed superior sensitivity, when both methods were applied on the same 425 clinical samples. Compared to culturing the faecal samples, PCR on template DNA puri¢ed directly from faeces is an attractive strategy. This would result in a faster analysis, which is not a¡ected by the selectivity of in vitro growth, and should be able to detect dead cells. The performance of a number of commercial DNA puri¢cation kits will be compared.

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P7^46 KINETIC PARAMETERS OF IMMOBILIZED DEOXYRIBONUCLEASE I ON CIM0 MONOLITHIC SUPPORT í > > N. Berginc(1), K. Bencina(2), M. Bencina(1), A. Strancar(2), A. Podgornik(2) (1) Laboratory of Biotechnology, National Institute of Chemistry, Hajdrihova 19, P.O.B. 600, SI-1001 Ljubljana, Slovenia; (2) Bia separations d.o.o., Teslova 30, SI-1001 Ljubljana, Slovenia Traces of DNA in RNA samples present impurities that could a¡ect results of mRNA quanti¢cation and cDNA synthesis. In most cases, the DNA impurities in RNA samples are removed using enzyme deoxyribonuclease (DNase), which speci¢cally breaks down DNA impurities. In order to avoid the addition of DNase into the analyzing sample, the use of immobilized DNase on solid support is recommended. Because of the size of DNA however, very few supports available to the matrix enable e/cient interaction between immobilized enzyme and DNA. Furthermore, since DNA mobility inside the closed pores of the supports is extremely slow the entire removal of DNA is extremely long and consequently very ine/cient. In recent years a new group of support named monoliths was introduced. They consist of a single piece of highly porous material. Pores are interconnected forming a network of channels through which the liquid is pumped. Because of enhanced exchange between mobile and stationary phase separation and bioconversion processes are signi¢cantly accelerated. Therefore also the e/ciency of DNA removal might be competitive to the degradation with free enzyme. In this work we immobilized DNase on CIM monoliths. The kinetic parameters of immobilized DNase were determined compared with kinetic parameters of free DNase. The use of immobilized DNase in real samples was tested.

P7^47 NUCLEIC ACID SEQUENCE-BASED AMPLIFICATION (NASBA) DETECTS DNA IN MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS D. Rodriguez-Lazaro(1), J. Lloyd(2), M. Pla(1), I. Ikonomopoulos(3) and N. Cook(2) (1) Institute of Food and Agricultural Technology (INTEA). University of Girona, Campus Montilivi s/n. E17071 Girona, Spain; (2) Central Science Laboratory (CSL), Sand Hutton, York, YO41 1LZ, UK; (3) Department of Histology-Embryology, School of Medicine, University of Athens, Athens, Greece Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease in ruminants, and has been implicated as the agent of Crohn's in humans. Three nucleic acid sequence-based ampli¢cation (NASBA) assays were developed for MAP, based on di¡erent target sequences. NASBA has been previously shown to selectively mediate the detection of RNA in microbial cells. Nucleic acids were extracted from MAP and Salmonella enterica serotype Typhimurium, and subjected to four enzymatic treatments prior to ampli¢cation, to determine the nature of the template nucleic acid which was ampli¢ed. These enzymatic treatments were DNase, RNase, S1 nuclease, and RNase / S1 nuclease. With each assay, the latter three treatments had no e¡ect on the MAP NASBA signal, whereas DNase treatment abolished it. As expected, the converse was observed with the S. Typhimurium signal. Thus, whereas NASBA selectively ampli¢ed RNA in S. Typhimurium, it ampli¢ed DNA in Mycobacterium avium subsp. paratuberculosis. P7^48 DETECTION OF ENDOTOXIN USING GAS-LIQUID CHROMATOGRAPHY CONNECTED WITH MASS SPECTROMETRY (GLC/MS) J. Rybka, A. Gamian Institute of Immunology and Experimental Therapy, Weigla 12, 53-114 Wroclaw, Poland LPS (endotoxin) is a major component of the outer membrane of Gram-negative bacterial cell. The LPS molecule consists of three structurally di¡erent regions: lipid A, core oligosaccharide and O-antigenic polysaccharide. LPS possess immunostimulatory and toxic properties to human organism even in trace amounts. Detection and quantitation of endotoxin in biological £uids, environment or biotechnological products is a matter of great importance for medicine as well as for the industry. Nowadays

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used endotoxin quantitation methods: biological rabbit pyrogen test and Limulus Amebocyte Lysate (LAL) test are indirect and expensive methods with several limitations. There is no valid diagnostic method for direct detection of endotoxin in body £uids for example in blood or blood serum so far. Sepsis ^ development of bacterial infection in blood ^ and septic (endotoxic) shock are ones of the most important medical problems leading frequently to the death of the patient. Despite intensive investigations there is no reliable procedure of diagnosis and monitoring of septic shock. Elaboration of a simple and reliable method of chemical endotoxin detection and its quantitation would be very helpful in sepsis and septic shock treatment. In the present work we propose a detection of endotoxin by one of its integral component : 2-keto-3-deoxyoctulosonic acid (Kdo). Kdo is one of the components, inherent of the inner part of the LPS core region; up to three Kdo residues form the Kdo region of the lipopolisaccharide molecule. Kdo, after chemical derivatization, is detected as a chemical marker of endotoxin using gas-liquid chromatography/mass spectrometry analysis (GLC/MS). The detected Kdo derivative is acetylated methyl ester of Kdo methyl glycoside. P7^49 APPLICATION OF MOLECULAR METHODS TO THE EPIDEMIOLOGY OF DRUG-RESISTANT TUBERCULOSIS IN POLAND A. Sajduda(1), A. Dela(2), J. Dziadek(2), E. Augustyno¤ wicz-Kopec(3), and F. Portaels(4) ¤ ¤ ¤ ¤ (1) University of Jodz, Jodz, Poland ; (2) Centre for Mi¤ ¤ crobiology and Virology, Polish Academy of Sciences, Jodz, Poland; (3) National Research Institute of Tuberculosis and Lung Diseases, Warsaw, Poland ; (4) Institute of Tropical Medicine, Antwerp, Belgium The aim of this study was to investigate drug-resistant M. tuberculosis isolates from tuberculosis patients in Poland by molecular epidemiological methods. A total of 268 drug-resistant M. tuberculosis isolates from 251 patients were analyzed by IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping. The generated IS6110 ¢ngerprint patterns were highly variable. The number of IS6110 copies per isolate varied from 5 to 20. 203 of the 251 strains (81%) contained 6 to 11 IS6110 copies, with a mean of 10 bands. 203 distinct IS6110 ¢ngerprint patterns were observed in the 251 isolates analysed, of which 179 patterns were observed only once each, whereas 24 were shared by two or more isolates. Some strains with identical IS6110 patterns were identi¢ed in patients of the same family or patients living in the same area, indicating active transmission. 2 clusters, consisting of 2 and 3 identical strains, respectively, were isolated both from Polish

and foreign-born patients. The 3 members of this latter cluster exhibited an IS6110 RFLP pattern characteristic for Beijing genotype family. Altogether, 7 strains (2.8%) of Beijing genotype were found. All the isolates were subject to spoligotyping analysis and the results compared with those obtained by IS6110-associated RFLP. A total of 96 spoligotypes were identi¢ed in the 251 isolates. There were 20 spoligotypes identi¢ed among 72 isolates assigned to 24 IS6110 clusters. In addition, among 179 isolates with unique IS6110 pro¢les, 88 spoligotypes were identi¢ed. Twelve spoligotypes were shared by clustered and unique isolates. By spoligotype alone, 183 isolates were divided into 33 spoligotypes, whereas 63 isolates showed unique spoligopatterns. The results obtained in this study indicate that transmission of drug-resistant M. tuberculosis strains contributes to the emergence of drug-resistant tuberculosis in Poland, including members of the Beijing genotype family. This work was supported by grant from the State Committee for Scienti¢c Research (KBN, contract no. PBZ03015). A. S. was supported by FEMS Fellowship in the Institute of Tropical Medicine, Antwerp, Belgium P7^50 CLONALITY OF GROUP A STREPTOCOCCI FROM CARRIAGE I. Santos-Sanches(1,2), G. Ribeiro(1,2), and D. Rolo(1,2) ¤ (1) Centro de Recursos Microbiologicos, Faculdade de " ncias e Tecnologia (FCT/UNL), Quinta da Torre, Cie ¤ 2829-516 Monte da Caparica, Portugal; (2) Laboratorio ¤ nese Microbiana, Instituto de Tecnologia Qu|mica ¤ de Patoge ¤ e Biologica Rua da Quinta Grande, 6, Apartado 127, 2780156 Oeiras, Portugal Group A streptococci (GAS) are responsible for a multiplicity of human diseases. In many countries, macrolides are extensively used in the empirical treatment of pharyngitis and other upper respiratory track infections, contributing to the decreased susceptibility against these antimicrobials. There is no evidence of the role of asymptomatic carriers in the dissemination of drug-resistant GAS in Portugal. The aim of this study was the recognition of drugresistant GAS clones capable of colonizing unrelated populations from di¡erent settings in the district of Lisbon. Antimicrobial resistance was determined by disc di¡usion. Macrolides resistance phenotypes: M and MLSB types were de¢ned by a triple-disc test with erythromycin, clindamycin and josamycin. Clonality of drug-resistant strains was assessed by pulsed-¢eld gel electrophoresis (PFGE) using SmaI and S¢I restriction enzymes. In 2000-2001, 2,066 oropharyngeal swabs were taken from children at day care centers, elementary schools students, personnel and family members. A total of 218 GAS were isolated

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and 32 were resistant to antimicrobials (15%). Resistance to macrolides was 66% (21/32) and M-type was prevalent (16/21). Resistance to tetracycline (Te) was 34% (11/32). The M-type isolates were uncut by SmaI but clustered into four S¢-I PFGE patterns with one comprising 13 isolates. MLSB-type strains were genotipically unrelated. Strains resistant to Te were grouped in ¢ve patterns with SmaI and six with S¢I. Two major disseminated clones were recognized, one resistant to macrolides (M-type) and the other resistant to Te. Further comparison with diseasecausing isolates will contribute to a better understanding of the molecular epidemiology of GAS. P7^51 RESTRICTION ANALYSIS OF CONSERVATIVE REGIONS IN THE GENOME OF ACETOBACTER SPP. M. Kretova(1), P. Siekel(2), J. Grones(1) (1) Department of Molecular Biology, Faculty of Natural ¤ Sciences, Comenius University, Mlynska dolina B-2, SK84215 Bratislava, Slovakia; (2) Food Research Institute, ¤ Priemyselna 4, P.O. Box 25, SK-82475 Bratislava 26, Slovakia Acetic bacteria are ubiquitous microorganisms which have found use in food and pharmaceutical technologies because they can be cultured at di/cult conditions, at low pH, and are able to produce organic acids from ethanol, mannose, xylose and other saccharides. Phenotypic identi¢cation of acetic bacteria is di/cult, unprecise and timeconsuming. One of the reasons is a high frequency of spontaneous mutations and the presence of insertion elements plays a role as well. An improvement in identi¢cation of Acetobacter spp. is achieved by the use of DNAbased methods. In this study, regions of 23S rRNA which are conserved within individual genera as well as 16S-23S rRNA spacer regions were ampli¢ed by PCR. Di¡erences in the primary structure of 23S rRNA were observed for Acetobacter pasteurianus 3614, A. pasteurianus 3612, A. pasteurianus 2374 a A. aceti 3620, which cannot be distinguished by microbiological identi¢cation methods. For a more detailed identi¢cation of individual species, a fragment of the 16S-23S rRNA spacer region was cloned in a pBSSK+ plasmid and analysed by restriction using endonucleases HaeII and HpaII. By this method, di¡erent restriction pro¢les were obtained for the tested strains. Primary structures of the 16S-23S rRNA spacer region of selected Acetobacter species were compared with previously published data in DNA databases.

P7^52 PEPTIDE NUCLEIC ACID-MEDIATED PCR CLAMPING AS A MOLECULAR METHOD FOR DIFFERENTIATION OF tox-GENE OF TOXIGENIC FROM THE `tox-GENE' OF NON-TOXIGENIC CORYNEBACTERIUM DIPHTHERIAE N. V. Volozhantsev, V. A. Bannov and K. I. Volkovoi State Research Center for Applied Microbiology, 142279, Obolensk, Moscow Region, Russia In nature, both toxigenic and non-toxigenic strains of Corynebacterium diphtheriae exist. Toxigenic strains bear propage containing tox-gene. This gene expresses diphtheria toxin, which contributes essentially to pathogenesis of diphtheria. Non-toxigenic strains mostly do not contain tox-gene and do not cause clinical diphtheria. During epidemic rise in the 1990s in Russia, non-toxigenic tox-gene bearing (NTTB) C. diphtheriae strains were often isolated. In most of NTTB strains, two mutations in the tox-gene were detected. One of them (one nucleotide deletion in region 52-55) results in the shift of the DNA open reading frame due to which toxin formation is not observed. NTTB strains can hardly play an epidemiologically signi¢cant role in diphtheria. Nevertheless, the probable phage conversion along with DNA recombination suggests the possibility of ``silent'' tox-gene recovery in these strains. Therefore, monitoring of such strains is important. To provide rapid identi¢cation of the NTTB strains and to di¡erentiate them from toxigenic strains, peptide-nucleic acid (PNA)-mediated PCR clamping was used. Oligonucleotide primers for tox-gene fragment ampli¢cation and PNA molecule complementary to the nucleotide sequences located between the PCR primers were constructed. PNA was bound to complementary sequences of native toxgene, thus blocking PCR, and was not bound to the nucleotide sequences of mutant gene. In the latter case, PCR was not blocked, and ampli¢cation products appeared. Simultaneous performance of PCR in the presence and absence of PNA allows for clear di¡erentiation of toxigenic C. diphtheriae strains containing native tox-gene, nontoxigenic strains not containing tox-gene, and non-toxigenic strains bearing ``silent'' tox-gene.

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P7^53 BACTERIAL VIRULENCE AND ACQUISITION OF ERYTHROMYCIN RESISTANCE IN ITALIAN ISOLATES OF STREPTOCOCCUS PYOGENES C. Zampaloni, P. Cappelletti, M. S. Pasquantonio, D. Petrelli, M. Prenna, S. Ripa, L. A. Vitali Department of MCA Biology, University of Camerino, via Camerini 2, 62032 Camerino (MC), Italy The in£uence of macrolides on the expression of bacterial virulence mechanisms has been extensively studied. However, the relationship between bacterial genetic background and acquisition of speci¢c antibacterial resistance has not been yet addressed. The aim of this work was to determine the level of genetic correlation between macrolide resistance and M protein virulence factor distribution in S. pyogenes. 167 erythromycin (ER) resistant S. pyogenes were analyzed for the macrolide resistance phenotype and subsequently screened for the genetic determinants of resistance (erm(A) and mef(A) genes). This set of isolates plus 48 ER sensitive isolates were then analyzed to determine the emm gene type. The following distribution of resistance phenotypes was obtained: 18% cMLSB, 31% iMLSB, 51% M phenotype. All emm2 strains showed the M phenotype, while all strains harboring emm22 gene were cMLSB. More than 85% of the emm89 positive strains was iMLSB and the same proportion of emm4 strains showed the M phenotype. emm3 is recorded only among sensible strains. The distribution of frequencies of the emm gene type was signi¢cantly di¡erent both when compared to the type of resistance and when the resistant versus the sensible population of S. pyogenes under study were considered. Therefore, resistance genes and the corresponding phenotypes are not randomly distributed among the ER resistant S. pyogenes strains, indicating that the virulence potential and its related expression may a¡ect the acquisition of speci¢c resistance traits. Presented data indicate that particular genetic backgrounds render this bacterium less prone to acquire resistance to macrolides (i.e. emm3 strains).

P7^54 CIPROFLOXACIN RESISTANT STRAINS 0F NEISSERIA GONORRHOEAE IN SLOVENIA > >¤ A. Zore(1), M. Petrovec(1), D. Kese(1), E. Ruzic¤ (1), M. Potocnik(2), M. Gubina(1) > Sabljic (1) Institute of Microbiology and Immunology, Medical > Faculty, Zaloska 4, Ljubljana, Slovenia; (2) Dept. of Der> matology, Univ. Medical Centre Ljubljana, Zaloska 4, Ljubljana, Slovenia Sensitivity of Neisseria gonorrhoeae to antibiotics is changing. Oral penicillins and tetracyclines are no longer recommended for therapy. Fluoroquinolones are recommended for therapy of uncomplicated infections (MMWR, 1998). The ¢rst £uoroquinolone resistant strains of N. gonorrhoeae have been diagnosed in Slovenia in 2002. The aim of the study was to determine the susceptibility of N. gonorrhoeae isolates from patients in Slovenia to antibiotics and to determine underlying genetic mechanisms of £uoroquinolone resistance. In a two-year period (20012002), we isolated N. gonorrhoeae from 40 patients (35 male, 5 female) treated at the outpatient clinic of Dept. of Dermatology. Swabs of urethra and cervix were transported to laboratory in transport culture system. GC-Agar plates with 1% de¢ned growth supplement were used for isolation of N. gonorrhoeae and determination of minimal inhibitory concentrations by E-test (AB Biodisc) to penicillin, tetracycline, azithromycin, cefotaxime, ceftriaxone and cipro£oxacin. GC-Agar plates, supplemented by Vitox (Oxoid) were cultured at 5% CO2 , and 35C for 1-2 days. L-lactamase was determined by nitrocephine test. For quality control, we used N. gonorrhoeae strain ATCC 49226 (NCCLS standards: M 100-S12, M7-A5, 2002). Genetic heterogeneity of £uoroquinolone resistant isolates was determined for gyrA and parC genes by sequencing PCR products. Nine of 40 (22.5%) N. gonorrhoeae strains were resistant to cipro£oxacin. No strain was resistant to other antibiotics or produced (-lactamase. All cipro£oxacin resistant strains had point mutations in gyrA (on codon Ser-91 and Asp-95) or parC genes (on codon Asp-86 and Leu-131). Because all resistant strains were found in 2002, the actual £uoroqinolone resistance of N. gonorrhoeae in Slovenia might be even higher than the 22.5% we found. Double alterations at Ser-91 and Asp-95 in GyrA plus a single or double ParC alteration probably play an important role in the development of high-level £uoroquinolone resistance in N. gonorrhoeae.

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P8^1 GENES INVOLVED IN BIOSYNTHESIS OF LINCOMYCIN AND RELATED ANTIBIOTICS ^ REGULATION OF EXPRESSION í ¤ ¤ ¤ ¤ ¤ M. Albrechtova, M. Jel|nkova, J. Kopecky, L. Cermak, J. ¤zek > Janata and J. Sp| Institute of Microbiology, Academy of Sciences of the ¤ > ¤ Czech Republic, V|denska 1083, 142 20 Prague-4, Czech Republic Lincomycin, produced by Streptomyces lincolnensis, and its derivatives (e.g. clindamycin) are important, clinically used antibiotics. Twenty-seven putative open reading frames with biosynthetic or regulatory functions (lmb genes) and three resistance genes (lmrA, lmrB, lmrC) have been identi¢ed. A distinct order of gene transcription initiation was observed. The aim of this experiment was to ¢nd possible regulatory genes. S. lincolnensis was cultivated in a semi-synthetic production media and samples of culture were collected from 18h of cultivation. Total RNA was isolated and cDNA from random primers was prepared. By using RT PCR the presence of particular transcripts was demonstrated during cultivation. It was found that transcripts of lmbU, lmbR, lmbQ, and lmbV genes were present in 18h of cultivation. All genes of the lincomycin gene cluster were transcribed starting from 20h of cultivation. cDNA from speci¢c primers was further prepared from 28h total RNA. An arrangement of particular genes in transcriptional units was ascertained using RT PCR. A set of prepared probes was also used to search for homologous genes in Streptomyces caelestis producing a structurally related antibiotic celesticetin. Homologues of lmbQ and lmbF were detected. Chromosomal DNA of producer of anthramycin, an antibiotic structurally di¡erent, but biosynthetically related to lincomycin is screened for implied analogues of lmb genes coding for the shared biosynthetic pathway. In order to test the regulatory relationships several constructs for a directed inactivation were prepared (e.g. construct for lmbJ inactivation with respect to the expected interconnection between lmbJ and lmbIH expression).

P8^2 THE INFLUENCE OF URINE ON THE IN VITRO ANTIMICROBIAL ACTIVITY OF VARIOUS ANTIBIOTICS AGAINST DIFFERENT ESCHERICHIA COLI PHENOTYPES M. Ang-Kucuker(1), N. Gonullu(2), O. Buyukbaba-Boral(1), O. Kucukbasmaci(2), O. Ang(1) (1) Department of Microbiology and Clinical Microbiology, Istanbul Faculty of Medicine, University of Istanbul, 34390 Capa, Istanbul, Turkey; (2) Institute for Experimental Medical Research, University of Istanbul, Istanbul, Turkey The purpose of this study was to assess the in£uence of urine on the in vitro activities of various antibiotics used in the therapy of this clinical infections. Thirty Escherichia coli strains isolated from patients with urinary infections were used in this study : the ¢rst group was susceptible to ampicillin. The second group was resistant to ampicillin and ampicillin + sulbactam with an inhibitor resistant beta-lactamases (IRT) producer phenotype. The third group was an extended spectrum beta-lactamases (ESBL) producing E. coli group detected by the double disk synergia between ceftazidime and amoxicillin/clavulanic acid. MICs were determined by the microbroth dilution method. The media were Mueller-Hinton broth (Oxoid, Istanbul, Turkey) and human, pooled antibiotic-free urine with a pH of 6.4 and sterilized by passage through a 0.2 Wm ¢lter. MICs of approximately all antibiotics were higher in the urine media. We obtained an equal MIC90 for cephalothin in broth and urine media in ampicillin susceptible and IRT producer groups. The most evident di¡erence of MIC90 (128 fold higher in urine) was obtained for amikacin and cipro£oxacin in the E. coli susceptible group. The lowest di¡erence resulted for ampicillin + sulbactam (two fold higher in urine) in IRT and ESBL producer groups. In conclusion, we assessed the activity of di¡erent antibiotics in broth and urine media and we found a lower activity of these antibiotics in urine media with the exception of cephalothin.

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P8^3 THE EVALUATION OF MUMMY'S INHIBITORY EFFECT ON SOME BACTERIAL AGENTS OF WOUND INFECTION Sh. Assar, M. Khaksari, M. Allahtavakoli and A. R. Rezaee Rafsanjan School of Medicine, Rafsanjan University of Medical Sciences, Rafsanjan 77179, Iran One of the most important goals of therapeutic medicine is to amend wounds in a short time and safe position as possible. Since natural drugs have these characters and a variety of e¡ective compounds, medical communities and World Health Organization paid more attention to them. The aim of this study was to evaluate the inhibitory e¡ect of mummy on some agents of wound infection. We collected Mummy, a natural, semisolid, black or brown substance, from ¢ssures of some mountain caves. Then, the inhibitory e¡ect of the mummy in concentration of 30, 50, 80 and 100 percent was measured on growth of Pseudomonas aeroginosa, Staphylococcus aureus, Escherichia coli by kirby-bauer as an antibiogram method. The mummy in above concentrations did not show any inhibitory e¡ect on Staphylococcus aureus or E-coli's growth, but Pseudomonas aeroginosa was a¡ected in a upgrading manner. This natural drug had an inhibitory e¡ect on the most important agent of wound infection, Psedomonas aeroginosa. Because of its routine application among some people, we suggest it for pharmacological investigations and con¢rmation of usage in concentration about 18 gm/dl. P8^4 THE ACTIVITY OF L-LACTAMASES AGAINST NEW CEPHALOSPORIN ANTIBIOTICS IN KLEBSIELLA PNEUMONIAE ISOLATES J. Blahova, K. Kralikova, V. Krcmery Institute of Preventive and Clinical Medicine, Limbova 14, 83301 Bratislava, Slovakia Cefoperazone is one of the most promising third-generation cephalosporins, active also against strains of Pseudomonas aeruginosa and/or Stenotrophomonas maltophilia. Favorable e¡ects of cefoperazone or cefoperazone/sulbactam against nosocomial bacteria resistant to a palette of other antibiotics leads the clinicians to use it extensively mainly in hospitals and their intensive care units (ICU). However, this habit might produce an unwanted and increased selection pressure in direction of emergence of cefoperazone-resistant strains. Among 22 isolates of Klebsiella pneumoniae, two strains phenotypicaly expressed the production of extended-spectrum L-lactamases (ESBL) hy-

drolyzing cefoperazone, ceftazidime, cefotaxime and cefepime and transferred the genes of the resistance to these antibiotics to susceptible recipient strains indicating that the gene(s) for ESBL production are localized on a plasmid. Hydrolytic activity of lactamases in cell lysate was determined also by macroidometric method and con¢rmed active hydrolysis of cefoperazone, cefotaxime and ceftazidime in original strains and also in transconjugant clone. Clavulanate added to the cell lysate together with antibiotics, actively inhibited hydrolytic activity of lactamases (decreasing almost to 25 %) in both, original strain and transconjugant. Similar activity was shown also when sulbactam was used as a L-lactamase inhibitor. ESBL producing strains have a strongly negative epidemiological signi¢cance being coded by transmissible plasmids. The appearance of transmissible genes coding for resistance to these drugs might further deteriorate the outlook of successful therapy of a series of infectious diseases caused by these problematic bacteria. It seems reasonable to perform at least phenotypic tests to detect strains producing ESBLs in all clinical relevant Gram negative bacteria. P8^5 A SEARCH FOR SUBUNITS OF N-DEMETHYLLINCOMYCIN SYNTHETASE ¤ ¤ >| D. Byrtusova, J. Novotna, S. Kadlc¤k, J. Janata, J. Ko¤ , J. Sp|zek ¤> pecky Institute of Microbiology, Academy of Science of the Czech ¤ > ¤ Republic, V|denska 1083, Prague, Czech Republic N-demethyllincomycin (NDL)-synthetase is one of the enzymes involved in biosynthesis of the antibiotic lincomycin in Streptomyces lincolnensis. This enzyme, consisting of several nonidentical subunits, catalyzes formation of the amide bond between propylproline and methylthiolincosamide. To identify its still unknown subunits we used two approaches. First, we searched for proteins with similar function in databases and selected proteins LmbC, LmbD, LmbE and LmbF. Protein LmbC has a conserved domain, which is responsible for amino acid activation. An analog of LmbE catalyzes cleavage of the amide bond in the detoxi¢cation pathway in Mycobacterium. Selection of proteins LmbD and LmbF was based on the localization of their coding sequences in the neighborhood of the gene lmbE. Second, we looked for interactions among proteins involved in lincomycin biosynthesis. We assume that the detection of these interactions should help us to reveal subunits of di¡erent enzyme complexes and of some regulatory proteins. At present, we test in vivo interactions using the yeast two-hybrid system. We prepared soluble proteins LmbC, LmbD, LmbE, LmbF by heterologous production in E. coli BL21(DE3) and developed the protocol for the puri¢cation and storage of these proteins.

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The ability of the puri¢ed proteins to catalyze the formation of NDL from supplied substrates is now tested. Recombinant proteins can also be used to detect the interactions in vitro. P8^6 GLYCOPEPTIDE RESISTANCE IN THE TEICOPLANIN PRODUCER STRAIN ACTINOPLANES TEICHOMYCETICUS ATCC 31121 AND IN THE A40926 PRODUCER STRAIN NONOMURAEA SP. ATCC39727 A. Consolandi, B. Fabrizio, F. Bagatin, R. Rossi, F. Marinelli Biosearch Italia S.p.A., Via R. Lepetit, 34, 21040 Gerenzano (VA), Italy Glycopeptide antibiotics are active against several grampositive bacteria due to their ability to bind the D-alanylD-alanine terminal dipeptide of cell wall intermediates. Two di¡erent mechanisms of resistance are reported for pathogens. In enterococci the van genes encode for a resistance apparatus that determines the synthesis of altered peptidoglycan by replacing terminal D-alanyl-D-alanine dipeptide with D-alanyl-D-lactate. In staphylococci, resistance originates from the accumulation of several di¡erent mutations determining the increase in cell wall thickness. The presence of van-like genes has been reported in the glycopetide producer strains Streptomyces toyocaensis, Amycolatopsis orientalis and Actinoplanes teichomyceticus ATCC 31121. However, genetic data have not been so far supported by microbiological and physiological studies. In order to characterize resistance mechanisms in the producer strains A. teichomyceticus, and Nonomuraea sp. we developed systems for the determination of Minimal Inhibitory and Bactericidal concentrations in these myceliar microorganism. A. teichomyceticus was resistant to high levels of vancomycin and teicoplanin showing a typical VanA phenotype of resistance. On the other side, Nonomuraea sp. showed the typical response to glycopeptides observed for staphylococci. This was also consistent with the genetic studies already reported for A. teichomyceticus. So far, typical van glycopetide resistance genes in Nonomuraea sp. have not been evidenced.

P8^7 SEROTYPES AND ANTIMICROBIAL SUSCEPTIBILITY OF SALMONELLA SPP. ISOLATED FROM PATIENTS WITH ACUTE DIARRHEA IN BELGRADE, SERBIA I. Dakic(1), I. Cirkovic(1), M. Vivoda(1), S. Stepanovic(1), B. Stosovic(2), M. Svabic Vlahovic(1) (1) Institute of Microbiology and Immunology, School of Medicine, Belgrade, Serbia; (2) Institute of Infectious and Tropical Diseases ``Dr Kosta Todorovic'', Belgrade, Serbia The inappropriate use of antibiotics in the clinical practice as well as in the agriculture, resulted in increased antibiotic resistance in Salmonella spp. The objective of the present study was to determine the serotype distribution and antimicrobial susceptibility of Salmonella spp. isolated from patients with acute diarrhea in Belgrade. A total of 150 Salmonella strains were isolated from patients with acute diarrhea at the Institute of Infectious and Tropical Diseases in Belgrade, between August 2001 and July 2002. The strains were identi¢ed and serotyped by conventional methods. Antimicrobial susceptibility tests for 11 antibiotics were performed by disk-di¡usion method according to NCCLS recommendations. Extended-spectrum beta-lactamases (ESBL) were screened by double disk synergy method using amoxicillin-clavulanate, ceftazidime and cefotaxime. S. enteritidis (127 isolates; 84.7%) was the predominant serotype, followed by S. typhimurium (13; 8.7%) and S. infantis (4; 2.6%), while the remaining 6 isolates (4%) were identi¢ed as members of other serogroups. Susceptibility to all antibiotics was established in 78 (52%) Salmonella isolates. All the isolates were sensitive or intermediary susceptible to cefuroxime and cefotaxime, and fully susceptible to gentamicin and cipro£oxacin. However, 35 (23.3%) isolates were resistant to nalidixic acid, 18 (12%) to tetracycline, 14 (9.3%) to ampicillin and amoxicillin-clavulanate, 8 (5.3%) to trimethoprim-sulfamethoxazole, 5 (3.3%) to chloramphenicol and 5 (3.3%) to kanamycin. Nine (6%) isolates showed multipleresistance, while ESBL production was observed in 3 (2%) isolates. The presence of 48% strains resistant to one or more antibiotics suggests the need for continuous surveillance of changes in Salmonella spp. antimicrobial susceptibility patterns.

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P8^8 ISOLATION AND CHARACTERIZATION OF A NEW MICROCIN PRODUCED BY ESCHERICHIA COLI: MICROCIN F S. Dandrieux, S. Sable, C. Barthelemy, G. Cottenceau, A. M. Pons ¤ ¤ " Laboratoire de Genie Proteique et Cellulaire, Batiment ¤ de La Rochelle, Avenue Michel Marie Curie, Universite ¤ Crepeau, 17042 La Rochelle CEDEX 01, France Numerous members of the Enterobacteriaceae produce substances that inhibit phylogenetically related species. Microcins (Mcc) form a family of inhibitory substances that can be distinguished from the colicins by their much smaller size ( 6 10 kDa), and the fact that their synthesis non lethal for the producing strains, is not inducible by DNA-damaging SOS agents. The production of microcins depends predominantly on plasmids. Microcins exhibit di¡erent mechanisms of action: inhibition of DNA replication, inhibition of protein synthesis or abolition of membrane potential. Usually, their synthesis is optimal in minimal medium, early in the stationary phase. Up to date, nine microcins have been identi¢ed according to the cross resistance of the mono-producers strains, as well as the biochemical or genetic properties: MccB17, C7, D93, E492, H47, J25, L, N24 and V. Here, we report the isolation and the characterization of a new microcin produced by an Escherichia coli strain: MccF. The wild type strain also produces MccV that has been previously characterized. MccF is an original compound produced both in minimal and rich media, at the beginning of the stationary phase. This molecule presents a molecular weight ranging from 12 to 20 kDa, as revealed by ultra¢ltration and tricine-SDS-Page electrophoresis. Microcin activity is not modi¢ed by pH variation from 4 to 12 values. MccF loses activity upon heat treatment (70C, 30min). The genetic determinants for MccF production are located on plasmid. P8^9 Withdrawn.

P8^10 RESISTANCE OF GENITAL MYCOPLASMAS M. Dimitrova, L. Puzderliska, and L. Karakerezova Department Of Preventive Health, Stip, Macedonia In our laboratory the susceptibility of genital mycoplasmas was followed in the period of three years i.e. 2000'02. For this purpose Mycoplasma JST were used. We examined 471 endocervical swabs, from which 256 (54%) were positive to mycoplasma. Ureaplasma urealyticum were 209 (82%), Mycoplasma hominis were 13 (5%) and 34 (13%) were combined isolates of both species. U. urealyticum shows intermediary susceptibility to O£oxacin in 49%, almost identical moderate resistance is shown to Erythromycin 7%, Tetracyclin 11%, O£oxacin 6% and to Doxycyclin 3%. M. hominis is resistant to Erythromycin 100%, to Tetracyclin 46% and it shows intermediary susceptibility to O£oxacin in 15%. Both combined isolates of U. urealyticum and M. hominis were resistant to Erythromycin in 82%, Tetracyclin 20%, intermediary susceptibility to O£oxacin in 70%. In the current year, there is increasing isolation of M. hominis (in relation) compared with the same one in the last few years. There are also more often combined isolates of both species. Because of resistance occurrence of great importance is the examination of mycoplasmas susceptibility in the most frequently used antibiotics. All examined mycoplasmas do not have resistance to Josamycin and Pristinamycin.

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P8^11 EFFECT OF ESSENTIAL OIL OF SAGE-BRUSH ON RESISTANCE OF STAPHYLOCOCCUS TO ANTIBIOTICS T. S. Ermakova, L. P. Titov, E. F. Panshina Research Institute for Epidemiology and Microbiology, Minsk, Republic of Belarus Proceeding increase of microbial resistance to antibiotics becomes very acute problem. The purpose of present research was study of in£uence of essential oil of sage-brush bolchan (Artemisia balchanorum Krasch) (EOS) in subbactericidal doses on resistance of Staphylococcus spp. to antibiotics. The kinetics of microbial growth in liquid medium at presence of various concentration of oil was recorded by spectrophotometer. Sensitivity of cultures to antibiotics was investigated after 2, 4 and 8 passages by disc-di¡usion method. At 0,05% of the oil solution in medium the lagphase of bacterial growth was extended up to 5h (in the control 3h), and in a log-phase bacterial growth does not reach a control level. The oil concentration in thin agar layer 0,05, 0,02 and 0,01% brakes the formation of microcolonies. The EOS inhibiting e¡ect ampli¢es with increasing of their contents in medium. At study 99 cultures of staphylococcus isolated from carriers, changes of their sensitivity to antibiotics under in£uence EOS were marked. In result of passages with bacteriostatic concentration of EOS (0,01%) increase quantity of cultures sensitive to tested preparations (more often Neomycin, Tetracycline and Erythromycin). After 8 passages 80-90% of investigated cultures became sensitive to one and more antibiotics. Spectrum and level of resistance in control (passages without oil) did not change. Thus, e¡ect of sage-brush essential oil components there was a loss of staphylococcus resistance to many antibiotics. It is interesting to investigate biochemical and genetic basis of this phenomenon, equal as possibility of practical application of this property of sage-brush essential oil. P8^12 PHENOTYPIC CHARACTERIZATION OF MACROLIDE RESISTANCE IN PHARYNGEAL ISOLATES OF GROUP A STREPTOCOCCUS Sp. Fokas, St. Fokas, F. Markatou and M. Dionysopoulou Clinical Microbiology Department, General Hospital of Sparta, Sparta, Lakonia 23100, Greece Resistance to macrolides within Group A Streptococcus (S. pyogenes) is an increasing problem worldwide making necessary the prudent use of this class of antibiotics. The

aim of our study was to record the current trend regarding macrolide resistance in S. pyogenes in our region (Lakonia, Southern Greece) and determine the phenotypes of resistance. We investigated 231 non duplicated S. pyogenes strains isolated in 2 years period (2000-2001) from patients with the clinical diagnosis of pharyngitis. The identi¢cation to the species level was achieved by colony morphology, beta hemolysis on blood agar, and agglutination technique. The pattern of susceptibility to erythromycin, clindamycin and penicillin was examined for all the strains performing the disk di¡usion method according to the criteria of NCCLS (1999). All the isolates of S. pyogenes tested were susceptible to penicillin while the resistance to macrolides was signi¢cant (20,78%, 48 strains). The expression (%) of the macrolide resistance phenotypes among the resistant strains as they were evaluated by the erythromycin-clindamycin double disk test were : M-phenotype 69%, inducible iMLS(B) phenotype 29% and the constitutive cMLS(B) phenotype 2%. The overall resistance rate to clindamycin was 6,5%. Macrolide resistance within S. pyogenes is relatively frequent and responsible for many cases of treatment failure in patients treated with macrolides. Continued epidemiological surveillance of the resistance is required. However, in our region treatment of S. pyogenes infections with clindamycin in patients allergic to penicillin is considered to be adequate alternative option due to the high prevalence of the Mphenotype. P8^13 PURIFICATION, CLONING AND CHARACTERIZATION OF BACTERIOCIN PRODUCED BY PROPIONIBACTERIA THEONII P-127 N. Gollop, V. Zakin and G. Ben-Shushan Dept of Food Science, ARO, P.O. Box 6, Bet-Degan, 50250 Israel The bacteriocin GZB-1 with molecular weight of 3000 Dalton, was puri¢ed from the growth media of Propionibacteria theonii P-127, a strain which is know to produce, under speci¢c growth conditions, the bcateriocin PLG-1 with molecular weight of 9,500. The N-terminal of GZB1 was microsequenced, the gene was cloned and the DNA sequence was determined. GZB-1 is highly homologous to PAMP (Faye et al, J. Bacteriol. (2002) 184, 3649-3656), but in contrast to PAMP it was puri¢ed in its active form and no protease digestion was needed. The survival curve of indicator bacteria Lactobacillus delbrukii with 100 units per ml of GZB-1 showed two phases. The fast phase of 20 minutes was followed by a slow phase. During the fast phase bacterial survival was reduced by two logs and during the slow phase bacterial survivals was reduced by 3 additional logs in 200 minutes. GZB-1 activity is a¡ected

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by magnesium and is activity is completely abolished at 50 mM magnesium chloride. Other divalent cations had no e¡ect on GZB-1 activity. This is the ¢rst report, to our knowledge that bacteria may produce two di¡erent bacteriocins under di¡erent growth condition. P8^14 RESISTANCE TO ANTIBIOTICS STRAINS OF S. AUREUS ISOLATED AT DIFFERENT INFECTIONS IN REPUBLIC OF BELARUS V. A. Gorbunov, L. P. Titov, T. S. Ermakova, K. H. Blyha Research Institute for Epidemiology and Microbiology, Minsk, Belarus S. aureus is an important causative agent of nosocomial infections. Monitoring antimicrobial resistance of S. aureus forms the basis of measures to prevent the spread of multiresistant strains, the development of e¡ective prophylaxis and treatment of infections caused by them. Identi¢cation and de¢nition of susceptibility to antibiotics was carried out by ATB expression (BioMerieux, France). The frequency of isolation S. aureus from urological infections was 2,99% (n=1440), intestinal 15,35% (n=1590), respiratory 5,88% (n=204), infections of skin and soft tissues 10,0% (n=110), sepsis 13,97% (n=136). Percentage of resistant strains S. aureus in 1996-1999 was: urinary infections to penicillin 92,3%, oxacillin 38,5%, cefazolin 2,6%, gentamicin 20,7%, tetracycline 75,0%, erythromycin 48,7%, lincomycin 84,6%, cipro£oxacin 15,4%, co-trimoxazole 17,1%, vancomycin 10,3%; from respiratory infections to penicillin 92,3%, oxacillin 53,8%, cefazolin 0,0%, gentamicin 12,5%, tetracycline 53,8%, erythromycin 30,8%, lincomycin 53,8%, cipro£oxacin 25,0%, co-trimoxazole 0,0%, vancomycin 0,0%; from intestinal infections to penicillin 100,0%, oxacillin 100,0%, cefazolin 9,7%, gentamicin 9,7%, tetracycline 19,4%, erythromycin 33,3%, lincomycin 22,6%, cipro£oxacin 3,2%, co-trimoxazole 12,9%, vancomycin 9,7%; from skin infections to penicillin 92,5%, oxacillin 25,5%, cefazolin 3,0%, gentamicin 12,1%, tetracycline 89,0%, erythromycin 53,5%, lincomycin 82,0%, cipro£oxacin 2,1%, co-trimoxazole 13,0%, vancomycin 9,0%; from blood infections to penicillin 100,0%, oxacillin 62,5%, cefazolin 12,5%, gentamicin 50,0%, tetracycline 56,3%, erythromycin 56,3%, lincomycin 87,5%, cipro£oxacin 16,7%, co-trimoxazole 0,0%, vancomycin 0,0%. In conclusion, the frequency of isolation of resistant strains of S. aureus varies from 25,5 to 100% and depends on the form of infection and such as a drug. Methicillin-resistant staphylococci are more often isolated from intestinal infections and sepsis (62,5-100%). They are found much less often at urological and skin infections. The high frequency of cipro£oxacin-resistant staphylococci isolated from respiratory tract and blood (16,7 and 25,0%, repectively), and

also the increase in resistance to vancomycin (up to 10%) demands attention. Monitoring MRSA is crucial in Belarus. P8^15 RESISTANCE TO ANTIBIOTICS OF STRAINS E. COLI ISOLATED FROM PATIENTS WITH UROLOGICAL AND INTESTINAL INFECTIONS IN REPUBLIC OF BELARUS V. A. Gorbunov, L. P. Titov, T. S. Ermakova, K. H. Blyha Research Institute for Epidemiology and Microbiology, Minsk, Belarus Monitoring of resistance clinically signi¢cant microorganisms to antibiotics is a part of strategy of containment their spread in clinical terms. Increasing resistance of E. coli emphasizes the necessity to monitor antibacterial susceptibility of this pathogen. Identi¢cation and de¢nition of susceptibility to antibiotics was carried out on the ATB expression (BioMerieux, France) Results: E. coli is one of the main causative agent of urological and intestinal infections. The frequency of these isolation at urological infections was 39,0% (n=1440), intestinal-12,1% (n=1590). Strains E. coli isolated from patients with urinary infections in 1996-1999 were resistant to amoxicillin in 59,2-60,4%, cefotaxime-13,1-16,6%, aztreonam-21,327,3%, imipenem-1,1-4,4%, gentamicin-15,3-26,2%, amikacin-29,2-42,3%, chloramphenicol ^ 66,7-68,7%, tetracycline-86,5-92,3%, o£oxacin-15,2-24,4%, cipro£oxacin10,2-16,3%. Strains E. coli isolated from patients with intestinal infections in 1996-1999 were resistant to amoxicillin in 58,21-70,2%, cefotaxime-4,1-18,2%, aztreonam ^ 0,054,5%, imipenem ^ 0,0-54,5%, gentamicin ^ 17,2-52,6%, amikacin ^ 22,1-42,1%, chloramphenicol ^ 47,3-63,6%, tetracycline ^ 61,4-54,5%, o£oxacin ^ 0,0%, cipro£oxacin ^ 0,0-54,5%. In conclusion: Strains E. coli are highly resistent to amoxicillin, chloramphenicol, tetracycline. The reliable increase of resistance E.coli to aminoglycozides is observed at urinary infections, to aztreonam, imipenem and cipro£oxacin at intestinal infections. Probably, it is connected to high frequency of using these drugs in the investigated period of time.

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P8^16 NaCl IN THE SELECTIVE SALT ENRICHMENT BROTH SUPRESS GROWTH OF MRSA I. Grmek-Kosnik, U. Dermota, B. Krivic, Z. Jokovic, A. Kodran, J. Smrekar, N. Meglic and T. Blazic Institute of Public Health Kranj, Gosposvetska ulica 12, 4001 Kranj, Slovenia Study of the growth kinetics in the selective salt (NaCl) enrichment broth showed that high concentration of salt in the selective enrichment broth inhibits the growth of MRSA (2). It is thus essential to test the salt tolerance of an epidemic MRSA isolate before undertaking widespread and costly screening programmes. We compared the growth of 10 isolates (2 ATCC strains and 8 domestic strains) MRSA after 24 hours incubation in the di¡erent concentrations of NaCl salt enrichment broth. Growth kinetics demonstrated a logarithmic inhibition of growth of MRSA with increase of NaCl concentration. Growth of MRSA in salt enrichment broth was signi¢cantly suppressed even with NaCl concentration of 0.5% (p 6 0.05, Student's t-test) but growth was detected in all tubes even at NaCl concentration of 10%. We con¢rmed, that concentration of NaCl salt enrichment broth suppresses the growth of MRSA. Our ¢ndings and ¢ndings in literature (2) suggest that the sensitivity of the screening method might be diminished for at least some isolates of MRSA at the recommended concentration of NaCl (7%). Thus, a policy of screening to control an outbreak could be seriously undermined by a high level of false negative results. P8^17 EFFECT OF pH VARIATIONS ON MEASUREMENTS OF INHIBITION ZONE AROUND ANTIBIOTIC-IMPREGNATED DISKS Z. Jokovic, U. Dermota, I. Grmek Kosnik, M. Ceh, C. Stropnik Institute of Public Health Kranj, Gosposvetska ulica 12, 4001 Kranj, Slovenia We studied the pH variations in Mueller-Hinton medium on reference strain susceptible to antibiotics. NCCLS standardizes antibiotic susceptibility testing by the diskdi¡usion (Kirby-Bauer) method with agar medium pH 7.2 to 7.4. We compared zones of inhibition around penicillin, vancomycin, gentamycin and erythromycin disks with reference strain Staphylococcus aureus ATCC 25923 on Mueller-Hinton medium contained di¡erent pH value (from 6.2 to 8.4 with intervals of 0.2). Accuracy pH measurements were obtained with a pH meter. We measured

inhibition zone diameter. Using a standard table of antibiotics susceptibilities, we determined if the strain was resistant, intermediate or susceptible. We also looked, if the control limits for reference strain were appropriate. Zones of inhibition around penicillin deviated at pH 8.2 and 8.4, around vancomycin at pH 8.0, 8.2 and 8.4, around gentamycin at pH 7.6, 7.8, 8.0, 8.2 and 8.4, around erythromycin at pH 8.0, 8.2 and 8.4. All measures the diameter of the zones of growth inhibition around penicillin, vancomycin and gentamycin were within the limits of susceptibility, for erythromycin the zones were out of the expected ranges. Inhibition zones around erythromycin at pH 6.2 and 6.4 were within the limits of intermediate, at pH from 6.6 to 8.4 was within the limits of susceptibility. Our ¢ndings are, if the pH is not between 7.2 and 7.4, the rate of the di¡erent antimicrobial agents or the activity of the drugs may be a¡ected. Zones of inhibition with reference strain are out the limit, if the pH is from 7.6 to 8.4. P8^18 DETECTION OF EXTENDED-SPECTRUM L-LACTAMASES : COMPARISON OF THE MAST DD TEST, THE DOUBLE DISK, E-TEST ESBL AND VITEK 2 B. Jutersek, T. Zohar Cretnik, A. Storman, V. Bozanic Institute of Public Health Celje, Department of Microbiology, Gregorciceva 5, 3000 Celje Extended-spectrum L-lactamase (ESBL) type of resistance is an important problem in the treatment of nosocomial infections. Detection of ESBL expression has proven to be di/cult with routine susceptibility test, because in-vitro tests may not determine resistance to cephalosporins at the NCCLS interpretative breakpoint for susceptibility. In our laboratory, double disk di¡usion test is used routinely for ESBL detection for all K. pneumoniae isolates. 298 isolates of ESBL producing K. pneumoniae were collected from patients in the regional hospital in last 5 years and 40 of them were randomly selected for the study. The aim of the study was to evaluate and compare the ability of four di¡erent methods to detect ESBL production : double disk di¡usion test (DD), ESBL E-test method, MAST disk test and VITEK 2. Detection of genes coding for SHV family L-lactamases was carried out by PCR with SHV speci¢c primers. The sensitivity of di¡erent methods were as follows : DD 100%, Mast disks 90%, E-tests 100% and VITEK 2 70%. DD is reliable, easy to and relatively cheap. E-tests and Mast disks are also practically useful for detection of ESBL. Problem of VITEK 2 is a limited database for recognition of ESBL producers. Co-operation with manufacturer and upgrading of their data is necessary.

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P8^19 MYCOPLASMA IDENTIFICATION AND SENSITIVITY TEST IN FEMALE GENITAL TRACT : A PROSPECTIVE STUDY M. Simou, S. Tzortzatou, T. Lazaraki, E. Thomou, E. Kada Laboratory of Microbiology, Elena Venizelou Hospital, Athens, Greece This is a prospective study to assess the prevalence of Mycoplasma hominis and Ureaplasma urealyticum in cervical smears and their susceptibility to antimicrobial agents. Methods: 430 cervical smears of women in reproductive age who attended the Outpatient Department of our Hospital were tested for the presence of Mycoplasma hominis and Ureaplasma urealyticum. The identi¢cation of mycoplasma and the sensitivity test were performed using the Mycofast Screening Evolution 2, kit (International Microbio, France). Results : Out of 430 specimens 145 (33.7%) were positive for mycoplasma. 134 specimens (31.16%) were positive for Ureaplasma urealyticum. From 134 U. urealyticum positive specimens 20 gave positive results in 1/1000 dilution, 35 in 1/10.000 dilution and 79 in 1/100.000 dilution. 11 specimens (2.5%) were positive for M.hominis in s 1/10.000 dilution. In 9 cases (2.0 %) both Ureaplasma urealyticum and Mycoplasma hominis were identi¢ed. Resistance to antimicrobial agents was 9.6 % to Roxithromycine (14 strains were resistant : 8 M. homiiniss and U. urealyticum , 4 U. urealyticum and 2 M. hominis), 6.2 % to O£oxacine (9 strains : 4 M. hominis and U. urealyticum and 5 U. urealyticum) and 0.6 % to Doxycycline (1 strain of U. urealyticum). In the total 24 (16.55 %) strains of mycoplasma were resistant to antibiotics. Conclusions : The incidence (33.7 %) of mycoplasma in genital tract of women in reproductive age is relatively high. The percentage 16.5 % of resistance to antimicrobials could be a therapeutic problem in£uencing conception and leading to infertility.

P8^20 IN VITRO ACTIVITY OF TELITHROMYCIN COMPARED TO MACROLIDES AND FLUOROQUINOLONES AGAINST STREPTOCOCCUS PNEUMONIAE, HAEMOPHILUS INFLUENZAE AND MORAXELLA CATARRHALIS º O. Kucukbasmaci(1), N. Gonullu(1), Z. Aktas°(2), D. «°« « « « Gurol(2), R. Berkiten(2) « (1) Istanbul University, Institute of Experimental Medical Research, 34390 Capa, Istanbul, Turkey ; (2) Istanbul University, Istanbul Faculty of Medicine, Department of Microbiology, 34390 Capa, Istanbul, Turkey This study has the objective of comparing the in vitro activity of the ketolide telithromycin with those of macrolides and £uoroquinolones against major respiratory pathogens recently isolated in a Turkish university hospital. A total of 336 strains including 83 S. pneumoniae, 168 H. in£uenzae and 85 M. catarrhalis isolated from outpatients with CA-RTIs were examined. MICs were determined by the agar dilution method. Of the 83 S. pneumoniae isolates, 25.3% (21/83) were intermediately resistant and 10.8% (9/83) were highly resistant to penicillin G. Telithromycin demonstrated 4-8 fold higher potent activity against S.pneumoniae than all the macrolide antibiotics. In fact, telithromycin (MIC90, 0.008 Wg/ml) was the most active compound against S. pneumoniae among all the antibiotics tested. Both penicillin G resistant and penicillin G susceptible S. pneumoniae strains were highly susceptible to telithromycin, moxi£oxacin and gemi£oxacin. MIC90 of gemi£oxacin was two fold lower than that of moxi£oxacin and 32 fold lower than those of both cipro£oxacin and levo£oxacin. Of the 168 H. in£uenzae isolates, seven (4%) were L-lactamase positive. Telithromycin (MIC90, 4 Wg/ml) was more active than erythromycin (MIC90, 8 Wg/ml) or clarythromycin (MIC90, 16 Wg/ml) and was as active as azithromycin (MIC90, 4 Wg/ml). L-lactamase status did not seem to a¡ect the activity of telithromycin, macrolides or £uoroquinolones. All four £uoroquinolones were highly active against H. in£uenzae and the most active compound was gemi£oxacin with a MIC90 of 0.015 Wg/ml. The MIC90 values for M. catarrhalis of macrolides and telithromycin ranged from 0.015 to 0.06 Wg/ml. Against M. atarrhalis, the activities of the four £uoroquinolones were similar and their MIC90 were within two dilution steps of each other while gemi£oxacin was superior to others with a mode MIC of 0.015 Wg/ml.

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P8^21 COMPARISON OF INTRAVENOUS AMINOGLYCOSIDE THERAPY WITH SWITCH THERAPY TO CEFIXIME IN URINARY TRACT INFECTIONS A. R. Lari(1), S. Noorbakhsh(2), F. Masjediyan(1), M. Habibi(1), R. Alaghehbandan(1) and H. Mostafavi(3) (1) Department of Microbiology, Iran University of Medical Sciences, P.O. Box 14515-717, Tehran, Iran; (2) Department of Pediatric, Iran University of Medical Sciences, P.O. Box 14515-717, Tehran, Iran; (3) Exir Pharmaceutical Company, Tehran, Iran Urinary tract infections (UTIs) are one of the most important causes of hospitalization among children in Iran. As intravenous antibiotic therapy is associated with side e¡ects, toxicity, high cost, and long hospitalization period in treatment of UTIs, ``switch'' therapy (intravenous-tooral antibiotic) has been considered. The aim of this study was to compare the e/cacy of intravenous aminoglycoside (amikacin or gentamicin) therapy with intravenous ceftriaxone and switch therapy to ce¢xime in children with UTIs. In this single-blind randomized clinical trial, 54 children aged 9 10 years with complicated urinary tract infections, who were determined by positive urine analysis/ culture, were enrolled and divided in two groups A and B. Children in group A (n = 30) treated with intravenous amikacin (15 mg/kg daily) or gentamicin (3 mg/kg daily) with ampicillin (100 mg/kg daily) for 7-10 days. Patients in group B (n = 24) treated with intravenous ceftriaxone (50 mg/kg daily) for the ¢rst 2 days and then switch to ce¢xime (8 mg/kg daily) orally for 8 days. One week after treatment, patients were evaluated clinically/microbiologically. Rate of response (clinically/microbiologically) to intravenous aminoglycoside therapy in patients of group A was 80% (24/30). Children of group B, who received ceftriaxone with switch to ce¢xime, had 88% (21/24) response rate. However, there was not statistical signi¢cant di¡erence between rate of response in two groups (P (2) = 0.82). Switch therapy with ce¢xime in children with UTIs increases e¡ectiveness and convenience. Switch therapy shortens duration of hospitalization, and of course, decreases costs and risk of nosocomial infections. Also, ce¢xime could be considered as switch therapy in children with UTIs.

P8^22 ANTIMICROBIAL ACTIVITY OF ESSENTIAL OIL OF SAGE (SALVIA OFFICINALIS L.) AND DIFFERENT TERPENOID FRACTIONS I. Bratic, A. Bratic, D. Mitic, J. Knezevic-Vukcevic, B. Vukovic-Gacic, D. Simic Chair of Microbiology, Faculty of Biology, University of Belgrade, Serbia Herbs, spices and fruits have been used through the ages as a source of £avour in food and parfume preparation. Many essential oils and their ingredients have been shown to exhibit a range of biological activities, including antibacterial and antifungal activity. This study was performed to investigate the antimicrobial activity of essential oil of Salvia o/cinalis L., and its fractions distinguished by di¡erent content of mono- and sesqui- terpenoids. For this purpose we used:disk di¡usion test for pre-screening of antimicrobial potential of test agents; minimum inhibitory concetration (MIC) tests to identifying the amount of antimicrobial agent required for visible antimicrobial e¡ect and time-kill assay to measure the time required for antimicrobial e¡ect.The essential oil and their fractions demonstrated a signi¢cant antimicrobial e¡ect on Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 10707 in disk di¡usion test. Moreover, B. subtilis was more sensitive than S. aureus. The MIC concentration for S. aureus was 1.25Wl/ml for essential oil and all tested fractions except F2 fraction (2.5 Wl/ml). B. subtilis was more sensitive than S. aureus and the MIC concetration was in the range 0.1-2.5 Wl/ml. The time-kill test showed that essential oil and its fractions have bactericidal e¡ect on S. aureus, while the e¡ect on B. subtilis is bacteriostatic. P8^23 MECHANISMS OF RESISTANCE TO MACROLIDES AND LINCOSAMIDES IN METHICILLIN RESISTANT STAPHYLOCOCCI ¤ ¤> ¤ ¤> G. Novotna, K. L|ckova, J. Janata, and J. Sp|zek Institute of Microbiology, Academy of Science of the Czech ¤ > ¤ Republic, V|denska 1083, Prague 14220, Czech Republic We identi¢ed di¡erent types of resistance mechanisms to macrolides and lincosamides in a group of methicillin resistant coagulase negative staphylococci. One-hundred-¢ve clinical isolates in vitro resistant to one of erythromycin (macrolide), lincomycin or clindamycin (lincosamide) were tested by southern blot analysis for the presence of the genes ermA, ermC (resistance by target site alteration) msrA (e¥ux resistance) and linA (resistance by antibiotic

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inactivation). The resistance was mainly due to the presence of msrA gene, which was detected in 57 strains [54%]. Genes ermC and ermA were detected in 48 strains [46%] and the linA was detected in 22 strains [21%]. The dissemination of resistance types di¡ers strongly from the published data. While in other countries cross-resistance to macrolides and lincosamides conferred by ermA and ermC predominates, in the Czech Republic the gene msrA is the most frequent genetic determinant conferring resistance only to macrolides, while sensitivity to lincosamides is preserved. We observed a relationship between the type of resistance and the level of resistance to erythromycin: whereas MICERY for the strains bearing msrA only was 16 to 128 mg/l, for the strains with erm it was MICERY 512 to 1024 mg/l. In 14 strains we detected no resistance genes although the strains were resistant to lincomycin and clindamycin. The fact that lincosamides are not inactivated indicates that a new type of resistance di¡erent from inactivation is involved. P8^24 EFFECTIVENESS OF TREATMENT OF RESPIRATORY FORM OF ACUTE MELIOIDOSIS USING LIPOSOMAL CEFAZOLIN IN EXPERIMENT K. A. Rotov, V. V. Alekseev, V. I. Kapliev and S. N. Tikhonov Antiplague Research Institute, Volgograd, Russia The choice of antibiotics is very important due to a wide spread of Pseudomonas pseudomalei. To solve this problem, one may use liposomal forms of antibacterial agents. These forms improve the penetration into the cells and provide an increased time of drug elimination with minimized toxicity, immunologic reaction. In this study we have determined the e¡ectivity in treatment of respiratory form of melioidosis using cefazolin in free and liposomal forms in mice. Liposomal cefazolin was prepared by reverse phase evaporation and was sterilized by Q-radiation in doses of 500 krad. Experimentaly respiratory form of acute melioidosis was induced by aspiration of P. pseudomalei 100. E¡ectivity of treatment was determined by percent of defence, ED50 of drugs, morphology during autopsy, bacteriology of organs. Usage of cefazolin made it possible to raise the percent of defence from 50% for free antibiotic to 75% for immobilized form. It was found out that ED50 of the drug was 3.59 mg in liposomal form and 9.77 mg in free antibiotic. Thus, inclusion of cefazolin in liposomes helped to raise e¡ectiveness in treatment of respiratory form of acute melioidosis in mice.

P8^25 DISTRIBUTION OF NIM AND CFI A GENES IN BACTEROIDES SPP. IN BELARUS V. Slizen(1), L. P. Titov(1), J. S. Brazier(2), M. Gal(2) (1) Belarussian State Medical University, Department of Microbiology, Immunology, Virology, Minsk, Belarus; (2) PHLS Anaerobe Reference Unit, Cardii¡, UK Despite the widespread use of metronidazole and imipenem, bacteroides resistance to these antibiotics remains low (about 1%). A rise in resistance to 3-5% was recorded. Nim (A,B,C,D,E) and c¢A genes confer resistance to these drugs, respectively, though these genes frequently are not expressed. Low incidence of c¢A and nim genes is indicative of recent acquisition of these resistance determinants. We investigated the distribution of c¢A and nim genes among Bacteroides spp. to evaluate the risk of emergence of phenotypic resistance via gene activation. 40 isolates of Bacteroides spp from patients with perirectal abscesses were screened for the presence of c¢A and nim (A, B, C, D, E) genes by PCR technique with the universal primers. RFLP analysis (HpaII and Taq I restriction) of ampli¢cation products from nim gene PCR produced pro¢les that enabled identi¢cation of nim (A,B,C,D,E) genes. Agar disk di¡usion method was used to test phenotypic resistance to metronidazole (5 Wg) and imipenem (10 Wg). All isolates were susceptible to metronidazole and did not give positive reaction in detection of nim (A,B,C,D) by PCR. All tested bacteroides were susceptible to imipenem in routine disk testing and lacked c¢A gene (absence of PCR products after ampli¢cation with the speci¢c primers). The data support the concept of low distribution of nim and c¢A genes in bacteroides. Imipenem and metronidazole remain to be highly potent drugs against bacteroides in Belarus. PCR-RFLP technique will permit improved identi¢cation of Bacteroides spp. and facilitate the recognition of nitroimidazole resistance determinants.

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303

P8^26 MONITORING OF ANTIMICROBIAL RESISTANCE IN REPUBLIC OF BELARUS L. P. Titov(1), V. A. Gorbunov(1), T. S. Ermakova(1), K. H. Blyha(1), E. F. Panshina(1), N. I. Tochko(2), E. A. Selesneva(3), N. A. Matveeva(4) (1) Research Institute for Epidemiology and Microbiology, Minsk, Belarus; (2) City Center of Hygiene and Epidemiology, Minsk, Belarus ; (3) Region Center of Hygiene and Epidemiology, Grodno, Belarus ; (4) Region Center of Hygiene and Epidemiology, Gomel, Belarus Microbiological monitoring allows increased e/ciency of microbiological diagnostics, rational antimicrobial chemotherapy and improvement in their control. Creation of a really functioning national surveillance system for containment of antimicrobial resistance ^ an important task of public health services. Material for research : sputum, pleural liquid, tracheal aspirations, blood, urine, faeces etc. The monitoring was carried out using ATB-expression (BioMerieux) in three Belarus regions (Minsk, Gomel, Grodno) in 1996-1999. Results I. 6584 strains of microorganisms were isolated. 218 species of bacteria and fungi are identi¢ed. Most frequent microorganisms were: Staphylococcus spp.- 28,8%; Escherichia spp. ^ 17,7%; Streptococcus spp. ^ 6,3%; Candida spp. ^ 5,8%; Enterobacter spp. ^ 4,0%; Pseudomonas spp. ^ 3,9%; Citrobacter spp. ^ 3,5%; Klebsiella spp. ^ 3,3%. II. Increasing resistance of strains S. aureus to ampicillin (39%), oxacillin (73%), cefuroxime (14%) was observed. III. Signi¢cant increase of resistance E. coli in 1996-1999 was observed to moxalactam (19%), aztreonam (16%), cipro£oxacin (16%), ticarcillin/clav.acid (15%), amikacin (14%), gentamicin (13%), imipenem (11%), netilmicin (10%). IV. The share of resistant strains Candida spp. in 1996-97 were 2-5% depending on a drug, whereas in 1998-99 these indexes have increased in 2 and more time (9-14%). V. Analysis of dynamics antimicrobial resistance P. aeruginosa for the period with 1996-97 on 1998-99 has revealed increase of resistant forms to ticarcillin / clav.acid (55%), imipenem (30%), aztreonam (68%), gentamicin (77%), netilmicin (86%), cipro£oxacin (51%). Conclusion: Increase of antimicrobial resistance of microorganisms from 1996 to 1999 bears witness to the necessity of carrying out constant monitoring of multidrug resistance, including introduction of hospital formularies for the practical doctors, control of antibiotics usage in hospitals and other measures, whose ultimate goal is containment of antimicrobial resistance.

P8^27 ANTIBIOTIC RESISTANCE IN BANK (CLETHRIONOMYS GLAREOLUS) AND MICE (APODEMUS SYLVATICUS) VOLES WOOD

N. J. Williams(1), T. R. Jones(1), D. Hunt(1), N. P. French(1), M. Begon(2), M. Bennett(1) and C. A. Hart(3) (1) University of Liverpool Veterinary School, Leahurst, Chester High Road, Neston, South Wirral, CH64 7TE; (2) School of Biological Sciences, Nicholson Building, University of Liverpool, L69 3GP; (3) Department of Medical Microbiology, Duncan Building, University of Liverpool, L69 3GA A previous survey of wild bank voles and wood mice in a woodland site, demonstrated antibiotic resistant organisms in their faecal £ora, even though they had no obvious contact with antibiotics. These results raised a number of questions: such as the source of the antibiotic resistance genes; their transferability; and whether a selective pressure was required for their maintenance. This report describes attempts to address some of these questions, through a longitudinal survey of the antibiotic susceptibility of E. coli and carriage of vancomycin-resistant enterococci (VRE) in wild rodents. Rodents have been livetrapped monthly since June 2001, using 200 traps placed in pairs at 10m intervals over a permanently marked grid of 100m2 of woodland. Faecal samples have been collected from traps and associated with individual animals identi¢ed by transponder chips. E. coli is isolated from faeces using media with and without antibiotics. Isolates are screened for resistance against six commonly used antibiotics. The prevalence of antibiotic resistance in faecal E. coli and faecal carriage of VRE have both been found to vary with season. Furthermore, bank voles appear to be half as likely to excrete an ampicillin (OR 0.49, P=0.03) or trimethoprim (OR 0.44, P=0.07) resistant E. coli than wood mice and one-third less likely to excrete a chloramphenicol (OR 0.28, P=0.005) or tetracycline (OR 0.33, P= 6 0.001) resistant E. coli. Overall a higher prevalence of VRE was also found in wood mice (7%) than in bank voles (0.5%). The genes responsible for resistance are currently being investigated.

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P9^1 ANALYSIS OF THE BILE STRESS RESPONSE IN LISTERIA MONOCYTOGENES LO28 M. Begley, C. G. M. Gahan and C. Hill Department of Microbiology, National University of Ireland, Cork, Ireland Bile is one of the many barriers that pathogens must overcome in the human gastrointestinal tract in order to infect and cause disease. We have demonstrated that Listeria monocytogenes LO28 can tolerate the average concentration of bile/bile acids encountered in vivo. Data indicates that prior adaptation of the bacterium to sublethal levels of bile or other stresses encountered during food processing and sanitizing treatments (acid, heat, salt, SDS) may alter cellular physiology to enhance bile resistance. Present studies are focusing on the molecular mechanisms underlying bile tolerance. Three genes possibly involved in the degradation / transformation of bile salts were identi¢ed following analysis of the recently published L. monocytogenes EGDe genome. These genes are absent from the genome of the non-pathogenic strain L. innocua. Initial physiological analyses of deletion mutants suggest that all three genes contribute to listerial bile tolerance. In order to identify other genetic loci involved in bile stress, transposon and plasmid integration banks were screened for bile sensitive mutants. This revealed a role for a number of genes including btlA, gadE and zurR in bile resistance. Interestingly, all loci identi¢ed so far play putative roles in the maintenance of the cell envelope or in stress responses. P9^2 ACTIVITY OF ASCORBIC ACID AGAINST HELICOBACTER PYLORI E. T. Dokic, M. Petrovska, N. Panovski Institute of Microbiology, Medical faculty, Skopje, Republic of Macedonia Introduction : The most successful treatment for the eradication of H. pylori includes combination of proton pump inhibitors with antibiotics such as amoxicillin, clarithromycin, tetracycline and metronidazole. Toxic e¡ects of ascorbic acid in the presence of metal ions and oxygen were reported for a number of viruses and bacteria. The role of vitamin C, both in vitro and in vivo, in H. pylori infection hasn't been studied enough so far. The aim of this study is to determine the in vitro activity of vitamin C against 30 H. pylori clinical isolates. Materials and methods 30 H. pylori clinical isolates cultured from gastric bi-

opsies taken at routine endoscopy were studied. Ascorbic acid was diluted and 2-fold dilution prepared to obtain a ¢nal concentration range of 4000 to 62,5 mg/L. An agar dilution method using Columbia agar supplemented with yeast extract and sheep blood was used. A high inoculum (approximately 106 CFU/ml) was applied using a replicator and plates were incubated in a microaerophilic atmosphere at temperature of 370 for 5 days. Minimum inhibitory concentration (MIC) was recorded as the lowest concentration that inhibited visible growth. Results The number of strains that were inhibited at each concentration of ascorbic acid were as follows : 3 strains with MIC=125 mg/L, 8 with MIC= 250 mg/L, 11 with MIC=500 mg/L, 6 with MIC=1000 mg/L and 2 with MIC=2000 mg/L. Conclusions Ascorbic acid shows good in vitro activity against H. pylori clinical isolates and could explain some of the bene¢cial properties of vitamin C. P9^3 MEASUREMENT OF GLUTATHIONE AS STRESS INDICATOR IN ACTIVATED SLUDGES M.-A. Dziurla(1), P. Leroy(2), G. Strunkmann(3), M. « Salhi(4), P. Camacho(5), V. Heinz(6), J. Muller(3), E. « Paul(4), Ph. Ginestet(5) and J. C. Block(1) (1) Laboratoire de Chimie, Physique et Microbiologie pour ¤ l'Environnement ^ LCPME, Unite Mixte de Recherche ^ ¤ ¤ UMR 7564, CNRS ^ Universite Henri Poincare, Nancy 1, ¤ de Pharmacie ^ Pole de l'eau, 15, avenue du Char" Faculte mois ^ 54500 Vandoeuvre-les-Nancy, France; (2) Equipe ¤ Thiols et Fonctions Cellulaires, Faculte de Pharmacie, Uni¤ Henri Poincare Nancy 1, 30 rue Lionnois, 54000 ¤ versite Nancy, France; (3) Institute of Mechanical Process Engineering, Technical University of Braunschweig, Volkmaroder Str. 4/5, 38104 Braunschweig, Germany ; (4) Institut ¤ National des Sciences Appliquees de Toulouse (INSA-Toulouse), Complexe Scienti¢que de Rangueil, 31077 Toulouse Cedex 04, France ; (5) CIRSEE-ONDEO Services, 38, rue ¤ du President Wilson, 78230 Le Pecq, France ; (6) Institut fur Lebensmitteltechnologie und Prozesstechnik, Technische « Universitat Berlin, Konigin Luise Strasse 22, 14195 Berlin, « « Germany Excess biomass produced during the biodegradation of waste water by activated sludges requires costly disposal. Production of excess sludge can be reduced by submitting sludges to mechanical, electrical, thermal, or oxidative stress. These treatments induce biological stress responses allowing the restoration of cellular structures and metabolic pathways. The adaptation and the resistance of the sludge microbial ecosystem to stress conditions is a major question as it may de¢nitively limit the e¡ect of stress treatments. Defense mechanisms developed, in particular in response to oxidative stress involve various antioxidant

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activities and compounds such as glutathione. In order to determine the redox status of activated sludges before and after stress, an HPLC method was developed for measuring reduced and total glutathione (GSH and GSHt) in perchloric acid sludge extracts. The method was sensitive, highly speci¢c and validated for linearity, precision and recovery. To our knowledge, this is the ¢rst report on GSH measurement in activated sludges. Extraction yield for GSH was estimated to be 84 %. Considering an oxidation of 30 % of GSH during extracts storage at -80C, the concentration of GSH measured was estimated to represent 60 % of the e¡ective GSH content in activated sludges. Total glutathione ranged from 0.32 to 3.34 Wmol per g volatile solids. In ``normal'' activated sludges, GSH and GSHt concentrations varied without knowing the reason for the variations. In sludges submitted to thermal and mechanical stress, decrease in glutathione and oxygen uptake rate occured indicating that glutathione is a stress indicator. P9^4 GENERATION OF REACTIVE OXYGEN SPECIES (ROS) IN YEAST CANDIDA INTERMEDIA AS RESULT OF COPPER AND ZINC EXPOSURE í ¤ S. Fujs(1), Z. Gazdag(2), M. Pesti(2), J. Belagyi(3), P. > (1) Raspor(1) and M. Batic (1) Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia ; (2) Department of General and Environmental Microbiology, Faculty of Sciences, University of Pecs, P.O. Box 266, H-7601 Pecs, Hungary ; (3) Central Research Laboratory, University of Pecs, P. O. Box 266, H-7601 Pecs, Hungary Reactive oxygen species (ROS) are generated during normal cellular metabolism and can damage cellular components by oxidizing lipids, proteins and nucleic acids. The redox active metal copper can be toxic at high concentration, due to generation of reactive hydroxyl radical (OHà) via a Fenton-type reaction and other reactive oxygen species, including H2O2 and superoxide radical (O2à). The less redox active zinc has important role in antioxidant protection of DNA. Zinc displaces bound iron from macromolecules thereby reduces iron catalysed Fenton reaction of OHà generation. The objective of this study was estimation of ROS generation in yeast Candida intermedia to 5 mg/l of copper and 50 mg/l of zinc exposure. To estimate intracellular peroxide and superoxide levels, £uorescence indicators dihydrorhodamine 123 (DHR) and dihydroethidium were used, respectively. The formation of rhodamine and ethidium (ET) was quanti¢ed spectro£uorimetrically. Hydroxyl radical formation induced by Cu and Zn was measured by electron spin resonance spectroscopy in cell

extract of C. intermedia by using the spin trap N-tert-butyl-a-phenyl nitrone (PBN). The cellular copper and zinc concentrations in biomass were determined by £ame atomic absorption spectrometry (FAAS). O2à-dependent ET £uorescence in yeast C. intermedia to copper and zinc exposure was increased by 111% and 18%, respectively. Opposite, H2O2-dependent DHR oxidation was decreased compared to control sample. Hydroxyl radical formation in yeast extract exposed to 2 mM copper and zinc was not detected by ESR. High concentrations of copper and zinc stimulated superoxide radical generation in yeast C. intermedia. Moreover, copper exposure showed more deleterious ROS generation compared to zinc exposure. We would like to acknowledge the Ministry of Agriculture, Forestry and Food and Ministry of Education, Science and Sport of the Republic Slovenia for support project No. V4-0402-00. P9^5 CELL CYCLOPROPANE FATTY ACID CHANGES AS A KEY FACTOR IN STRESS TOLERANCE OF SALMONELLA M. E. Guerzoni and L. Vannini Dipartimento di Protezione e Valorizzazione Agroalimen' ' tare, Facolta di Agraria, Universita degli Studi di Bologna Via Fanin 46, 40127 Bologna, Italy To date most of the work on stress response of pathogenic species as well as the relationship between stress exposure and virulence has been focused on de novo protein synthesis while the potential role of phospholipid or fatty acid modi¢cation has been virtually ignored. In this work fatty acid adaptation, and particularly cyclopropane fatty acid (CFA) changes, following oxidative and thermal stresses in 12 strains of Salmonella was analysed. The 12 strains, that were characterised by di¡erent antibiotic resistances, plasmid pro¢les and ribotype patterns, showed di¡erent viability responses and CFA changes when exposed to various H2O2 concentrations and cold and heat stresses. For some of the strains a relevant increase in CFA percentage with a consequent decrease in unsaturated fatty acid content was observed following the stress exposure; on the other hand negligible changes in fatty acid composition was evidenced with other ones. The relationship between stress tolerance, virulence factors and fatty acid modulation patterns will be discussed.

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P9^6 CILIATE Cd-METALLOTHIONEIN GENES: A HIGHLY CONSERVED MICROBIAL RESPONSE TO HEAVY METAL STRESS ¤ ¤ ¤ ¤ ¤ J. C. Gutierrez, S. D|az, L. Ben|tez and A. Mart|n-Gonzalez ¤ ¤ ¤ Dpto. Microbiolog|a-III, Facultad de Biolog|a, C/. Jose Antonio Novais 2, Universidad Complutense (UCM), 28040 Madrid, Spain Metallothioneins (MTs) are cysteine (Cys)-rich heavy metal-binding proteins, which are present in animals, plants as well as in cyanobacteria and eukaryotic microorganisms. MTs are induced by heavy metals (Cd, Zn and Cu), and they are responsible for the bioaccumulation and inactivation of these metals in the cell. We report three di¡erent macronuclear gene isoforms (MTA, MTB and MTC) encoding Cd-MTs isolated from very di¡erent ciliates. All these MTs show a high average identity among them; the identity among MTAs is about 74%, for MTB isoforms is 97-100% and for MTCs is about 89%. A lower average identity is showed among the three isoforms (MTA ^ MTB : 32%, MTA-MTC : 35%, MTB ^ MTC : 55%). Ciliate MTs present unique features with regard the rest of known MTs: a)- They show Cys motifs composed by three Cys residues (-CCC-) very conserved among ciliates MTs, but not reported in other MTs. b)The total number of Cys residues is higher than that in other MTs [ MTA (22 ^ 31), MTB (33 ^ 34), MTC (48) ]. c)- MTB and MTC isoforms have aromatic amino acids (Tyr, Phe) and His, which are not present in any MT. d)Their molecular weights are higher (11-16 KDa) than those ( 6 7 ^ 10 KDa) for typical MTs. e)- They are over-expressed after heavy metal exposure. The singular features of ciliate MTs make possible that they can be used as biological tools to design molecular biosensors to detect environmental pollution by heavy metals. P9^7 CHAOTROPICITY: A NEW CONCEPT IN WATER STRESS J. E. Hallsworth(1), S. Heim(2) and K. N. Timmis(1,2) (1) Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, UK; (2) Division of Microbiology, GBF ^ National Research Centre for Biotechnology, Braunschweig, D-38124, Germany Low water availability is the most ubiquitous cause of stress for terrestrial plants, animals and microorganisms, and has a major impact on ecosystem function and agricultural productivity. Studies of water stress have largely

focused on conditions that a¡ect cell turgor, ie osmotic stress. We show that chaotropic chemicals that do not a¡ect turgor reduce water activity, perturb macromolecule-water interactions and thereby destabilize cellular macromolecules, inhibit growth, and are powerful mediators of water stress in a typical soil bacterium, Pseudomonas putida. Such bacteria mineralise organic wastes, including chaotropic xenobiotics, play a critical role in the carbon cycle, and detoxify polluted habitats by natural attenuation or bioremediation. Chaotropic solute-induced water stress resulted mostly in the up-regulation of proteins involved in stabilization of biological macromolecules and membrane structure. We suggest that some chemicals currently considered to be toxic are primarily chaotropic agents of water stress, and that associated changes in entropy represent a common factor in di¡erent sources of stress and the corresponding cellular stress responses. P9^8 MEASURING VITALITY AND GENETIC STRESS RESPONSE OF BACILLUS LICHENIFORMIS FOR OPTIMIZATION OF PROPAGATION METHODS T. HornbAk(1,2), A. Nielsen(1), M. Jakobsen(2) and J. Dynesen(1) (1) Novozymes A/S, SmÖrmosevej 25, DK-2880 BagsvArd, Denmark; (2) KVL, Dairy and Food Science, Food Microbiology, Rolighedsvej 30 4th, DK-1958 Frederiksberg, Denmark Bacillus licheniformis is a microorganism widely used by the enzyme industry in fermentations for the production of enzymes. Prior to fermentation, the strain undergoes different propagation steps potentially a¡ecting the physiology of the strain i.e. the inoculum used for the production of enzyme. In order to minimize variations in inoculum quality it is a prerequisite to know how the culture is a¡ected by di¡erent propagation conditions. Methods applied for this purpose include the use of 5(6)-carboxy£uorescein diacetate succinimidyl ester in combination with £ow cytometry or £uorescence ratio imaging microscopy for determination of cell vitality. Using DNA arrays, examinations of the genetic stress response at changing propagation conditions are carried out. Ongoing work that examines the e¡ect on the vitality and genetic stress response of B. licheniformis found when substituting an agar substrate with a liquid growth medium in the propagation process will be described. The vitality of the culture is examined after growth on the solid agar surface respectively in the liquid medium and the genetic stress response is investigated shortly after inoculation into the subsequently used seed tank medium.

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P9^9 TRANSPOSITION OF F-FACTOR LEADS TO SUPPRESSION OF lon MUTATIONS IN ESCHERICHIA COLI CA-154 HfrH H. G. Hovhannisyan S. S. Hovhannisyan Institute of Microbiology, NAS RA, Abovyan City, 378510, Armenia The Lon ATP-dependent protease of E. coli participates in the regulation of heat-shock and SOS stress responses and responsible for the degradation of a number of regulatory proteins and of many abnormal proteins. E. coli K-12 strains lacking Lon display two characteristic phenotypes, increased sensitivity to UV and overproduction of capsular polysaccharides. These phenotypes result from failure to degrade the speci¢c Lon protease substrates, SulA ^ cell division inhibitor whose expression is induced as part of the SOS response and RcsA ^ positive regulator of expression of the colanic acid synthesis genes. For selection nonmucoid revertants of lon mutant E.coli K-12 CA154 HfrH we have used the virulent bacteriophage M59 speci¢cally lysates the capsulated cells overproducing colanic acid. Among more than two hundred UV sensitive revertants one was UV-resistant, but still carrying mutation in lon gene. Interrupted conjugation experiments showed that in the UV-resistant revertant called NM144 have changed of genes transfer gradient and polarity to the F- recipient strains. Chromosome transfer was started from point of origin 60 min in counterclockwise direction. Transposition of F-factor back to former at zero point region abolishes the lon suppression. The ability to suppress two independent lon phenotypes was most easily explained as expression of an unknown protease activity which can substitute for Lon-protease. In the new region of F-factor insertion located ssrA gene which acts as a negative regulator of alternative Lon-protease (Alp) synthesis (Gottesman, 1996). Thus it is possible that insertion of F-factor inactivated ssrA and induced Alp activity. P9^10 THE USE OF MICROBES AS INDICATOR FOR BIOAVAILABILITY OF MERCURY IN SOIL P. Jaeckel and M. Schloter GSF-National Research Center for Environmental and Health, Institute of Soil Ecology, Ingolstadter Landstr.1, « D-85764 Neuherberg, Germany Due to industrial activities in South East Germany until the late 60th of the last Century, soils and sediments close to the industrial sites were highly contaminated with heavy

metals, especially with mercury. As most of the Hg2+ is either bound in soil minerals or adsorbed on solid surfaces, the bioavailability of Hg is low. In the present study the in£uence of farming on the bioavailability of mercury was investigated. As bacteria are only a¡ected by bioavailable substances and have direct contact to the soil solution, they can be used as sensitive bioindicators for pollutants. Therefore the structural diversity, the microbial adaptation (Pollution Induced Community Tolerance, PICT) and gene response could be a sensitive way to measure the bioavailability of mercury in the environment. The structural diversity was studied by bacterial community ¢ngerprints (ARDRA) and PICT by community level physiological pro¢les (CLPP) with and without mercury. Due to the Hg-stress a horizontal gene transfer of resistance plasmids containing the mer-gene may occur. Thus a correlation between mer-gene transfer and bioavailability of mercury was investigated. P9^11 Hsp104 INDUCTION IN THE YEAST CANDIDA INTERMEDIA EXPOSED TO Cr(VI) P. Jamnik and P. Raspor Food Science and Technology Department, Chair of Biotechnology, Biotechnical faculty, Jamnikarjeva 101, 1000 Ljubljana, Slovenia The survival of yeast cells is dependent on their ability to sense alternations in the environment and to appropriately respond to the new situation. One of the most conserved mechanisms of cellular protection is the expression of a polypeptide family named heat shock or stress proteins. Among them Hsp104 has drawn a great attention, because it promotes survival under extreme stresses such as high temperatures, severe oxidative damage and high concentrations of ethanol. These stresses cause protein aggregation and Hsp104 promotes survival by facilitating the resolubilization of protein aggregates. The aim of our work was to investigate whether Hsp104 is induced in the yeast Candida intermedia exposed to Cr(VI). Cr(VI) compounds are widely used in many industrial processes and cause environmental toxicity. Cr(VI) belongs to redox active metals, which play an important role in the generation of reactive oxygen species in the cell. In our previous study we have already reported that Cr(VI), speci¢cally in concentration of 100 WM causes elevated intracellular oxidant level. Herein, we demonstrate that Cr(VI) stress result in variation in protein synthesis, which indicate that Cr(VI) might regulate the expression of some genes. At 100 WM Cr(VI) induction of Hsp104 was observed, which is connected to formation of protein aggregates. Hsp104 assists their resolubilization and so contributes to cell survival. Therefore, we showed that Hsp104 plays an important

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role in defence system of yeast Candida intermedia to Cr(VI). We are grateful to the Ministry of Education and Sport of the Republic of Slovenia for grant (no.: 35/8) and the Ministry of Science and Technology of the Republic of Slovenia (Project no.: J4-7454-490) for ¢nancial support. P9^12 ENDOGENOUS ADP-RIBOSYLATION OF THE G PROTEIN L SUBUNIT MODULATES GLQ ACTIVITY IN FUNGI N. Jeraj, H. Lenasi and K. Breskvar University of Ljubljana, Faculty of Medicine, Institute of Biochemistry, Vrazov trg 2, 1000 Ljubljana, Slovenia Progesterone is toxic for ¢lamentous fungus Rhizopus nigricans and at higher concentrations leads to inhibition of fungal growth. The fungus responds to toxic e¡ects of progesterone by detoxi¢cation of this steroid via P450 11K-hydroxylation. One of progesterone signaling routes includes membrane progesterone receptors coupled to G proteins. We demonstrate here some of the subsequent steps of this signaling route. A reduction of cAMP levels was observed after stimulation with progesterone, but was not blocked by pertussis (PTX) or cholera toxin (CTX). This ¢nding suggests that the K subunits of classical Gi or Gs proteins are not involved in the inhibition of fungal adenylyl cyclase. These studies prompted us to explore the possibility that G protein LQ subunits (GLQ) might play an important role in the response to progesterone. Using a combination of (i) ADP-ribosylation assay ^ labelling with [32P]NAD; (ii) ADP-ribosylation in the presence of cholera toxin and pertussis toxin; and (iii) Western blotting with a panel of anti-G-protein antibodies, we demonstrated that R. nigricans expresses homologues of the L like subunits (35kDa) and K subunits of the Gi1 subfamily (45kDa) found in mammals. The activity of GLQ can be modulated by mono-ADP-ribosylation in a similar way to the regulation of some G protein K subunits. Here we present the ¢rst report of the endogenous mono-ADP-ribosylation of the G protein L subunit in an organism from the kingdom of fungi.

P9^13 INFLUENCE OF EXOGENOUS STRESS FACTORS ON THE PRODUCTION OF CAROTENOIDS BY RED YEAST R. Koci, I. Marova, J. Pokorna, O. Koutny Department of Food Chemistry and Biotechnology, Faculty of Chemistry, Technical University of Brno, Czech Republic Carotenoids are the most widespread natural lipid-soluble pigments with many important biological activities and industrial applications. The aim of this study is to compare the composition and content of carotenoids produced by several strains of carotenogenic yeasts in the presence of exogenous stress factors. Three strains of red yeasts (Rhodotorula glutinis, Sporidiobolus salmonicolor, Pha/a rhodozyma) were grown aerobically on glucose medium. Oxidative stress was induced by 2-5 mM H2O2, osmotic stress by 2-5% NaCl during 40- and/or 80-hours exposition. In£uence of aeration, temperature and heavy metals was studied too. Growth parameters, oxygen consumption, concentration of carotenoids (HPLC/MS) and other metabolites (e.g. glycerol) were analyzed. Stress factors resulted in di¡erent responses according likely to original environment. P. rhodozyma was more sensitive to all types of stress than the other strains. Under salt stress only R. glutinis produced higher amount of all carotenoids. Oxidative stress led in S. salmonicolor and R. glutinis to an induction of both growth and production of carotenoids (4-8x higher concentration of L-carotene, 3-4x lycopene, 23x phytoene ; 2-4x K-carotene). Aeration and temperature led to an increase of L-carotene production in limited range. Pretreatment of R. glutinis with low concentration of NaCl and/or H2O2 led to further induction of L-carotene (2.3x), lycopene (3.9x) and phytoene (5.7x) and to higher tolerance to additional oxidative stress. Higher production of carotenoids in yeast cells under stress conditions can act as an adaptive mechanism. Because yeast cells exposed cross-protection against di¡erent stress agents, carotenoids probably take part in general stress response of red yeast.

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P9^14 COLONY AND ULTRASTRUCTURAL DIFFERENCES AND SIMILARITIES IN TWO TRIMMATOSTROMA SPP. ISOLATED FROM DIFFERENT WATER-STRESS ENVIRONMENTS T. Kogej(1), A. A. Gorbushina(2), A. Schulte(2) and N. Gunde-Cimerman(1) (1) University of Ljubljana, Biotech. Faculty, Dept. of Biology, Vecna pot 111, SI-1000 Ljubljana, Slovenia; (2) Geomicrobiology, ICBM, Carl von Ossietzky Universitat « Oldenburg, POB 2503, D-26111 Oldenburg, Germany Black yeasts are rare known extremophilic eukaryotic microorganisms. The morphology of black yeasts is important for their survival in extreme environments, and includes characteristics on a colony level: polymorphism, meristematic growth, endoconidiation, and on a cell level: melanization, thick cell walls. Common features of the black-yeast genus Trimmatostroma are melanized thickwalled muriform cells that develop by conversion from undi¡erentiated hyphae. Genus Trimmatostroma is classi¢ed in ascomycetous order Dothideales and contains 15 species, which are inhabitants of phylloplane (conifer needles), wood, rock surfaces, and hypersaline water. All of the mentioned environments are osmotically stressful due to a relatively low water activity. We have chosen to study the colony- and cell-level adaptations to saline environment of two di¡erent species of the genus Trimmatostroma, which were previously isolated from two di¡erent environments with comparably low water activity. A halophilic species Trimmatostroma salinum was isolated from hypersaline water of a saltern. A xerophilic species Trimmatostroma sp. was isolated from a glass-window surface of a church. Several di¡erent non-saline and saline growth conditions were chosen according to their ability of growth in the presence of NaCl. Both species were then studied microscopically. Their colony structure was studied histologically, and their cell-wall structure was examined by transmission electron microscopy. The growth patterns of both species were identi¢ed and compared on a colony level. Cell-wall structures with emphasis on melanization patterns were also compared in both species as their response to osmotic conditions.

P9^15 CHANGES IN LIPID COMPOSITION AS ADAPTIVE RESPONSE OF ROCK INHABITING ASCOMYCETOUS BLACK YEASTS TO DEHYDRATION AND REHYDRATION E. R. Kotlova(1), A. A. Gorbushuna(2), A. Schulte(2) (1) Komarov Botanical Institute, Prof. Popov Str. 2, St. Petersburg, Russia ; (2) Carl von Ossietzky University, P. O. Box 2503, 26111 Oldenburg, Germany The representatives of rock dwelling ascomycetous black yeasts are highly adapted to the existence in stressed conditions such as prolonged dehydration. Among the most principal features responsible for their tolerance the following could be mentioned: slow growth rate, protective structures surrounding cells, internal and external compounds (polysaccharides, proteins) binding water. In the present work we attended to e¡ects of de- and rehydration on colonial organization, ultrastructure and lipid composition. For this purpose one strain of black yeasts isolated from a marble substrate and identi¢ed as the Phaeococcomyces sp. was used. Developed colonies were subjected to slow or fast desiccation followed by rehydratation. Under desiccation, distinctive intracolonial strati¢cations were detected, which were visible due to the discernible distribution of di¡erent cell types. Three individual cell types were described, including round yeast-like cells, dividing meristematic cell clusters and cylindrical hyphae. In accordance with our previous ¢ndings lipid droplets inside the cells were noticed as well. Among substances which attribute to the reserve lipid particles, triacylglycerols, sterol esters and free fatty acids formed the most part. The content of each was practically doubled during fast desiccation and reverted to the initial one after rewetting. Slow dehydration induced the considerable decrease of their amount which was also reverted after rehydration. On the contrary, the amount of membrane lipids (phosphatidylcholines, phosphatidylethanolamines, sterols) remained unchangeable during dehydration. Such stability of membrane components might be due to the reserved lipids which are not only material for energy production and water supply but also for membrane lipid synthesis.

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P9^17 SPHERICAL NANOSTRUCTURES BY PSEUDOMONAS AERUGINOSA DURING PHOSPHATE STARVATION : COMPLEX VESICLES OR NANOCELLS ? ' ' S. La Porta, A. Gio¡re, M. Nicolo, S. Guglielmino Dept. of Microbiological, Genetic and Molecular Sciences, University of Messina, sal. Sperone, 31, 98166 Messina, Italy Pseudomonas aeruginosa, like many Gram negative bacteria such as Vibrio spp., Escherichia coli and other pseudomonads, is able to survive in starvation conditions thanks to the activation of complex regulatory systems leading to physiological and structural adaptation, including variation of cell shape. In 0.2 Wm-¢ltered culture supernatant after ¢ve days of phosphate starvation, spherical nanostructures (NSs) of about 200 nm diameter were visualised by scanning electron microscopy. NSs structures were investigated by SDS-PAGE, enzyme activities and nucleic acids content. Electrophoretic analysis showed a complex protein pro¢le with more than 30 bands. Enzymatic activity analyses revealed the presence of malate dehydrogenases and cytochrome-oxidases. DNA and RNA have also been detected into NSs; further analysis of DNA was performed and particularly REP-PCR showed a pro¢le similar to generating-cells, suggesting that NSs contain large amounts of the genome. These data and further SEM observations on phosphate-starved cells address to either asymmetric septation or cell debris release due to structural alterations. P9^18 EFFECT OF HEAVY CARBON ISOTOPE 13C ON THE CYTOLOGY OF RHODOSPIRILLUM RUBRUM N. N. Ladyguina Pushchino State University, Pushchino, Moscow region, 142290, Russia Photosynthesis of bacteria is accompanied with of carbon isotope fractionation. Usually the isotope ratio of 12C and 13 C of natural substrate (CO2) is evaluated 99:1. The paper presents study on e¡ect of initial isotopic composition of the CO2 carbon on cytology of the photosynthetic purple bacteria Rhodospirillum rubrum VKM B-1621 (type strain). Initial content of the carbonate carbon in experiments was 20 mM NaHCO3, however, enrichment with 13 C in di¡erent experiments varied from 10 to 57 %. The control (blank) variant contained 1 % 13C, i.e. natural composition. Morphology of bacterial population in control variant was presented with long chains of spirillas.

The number of single cells increased with the increase of the 13C concentration in medium. At the highest concentrations of 13C (45 and 57 %), the size of single cells decreased and was about 90 % of natural size in the blank variant. Thus, concentration of heavy carbon in NaHCO3 a¡ects cytology of the photosynthetic purple bacteria. This e¡ect is realable at high concentration of 13C and reveals in two ways: increases the single cells number and diminishes their sizes. The data provide evidence that 13C concentration in substrate can be an a¡ecting agent for bacteria. Author thanks Drs. N.E.Suzina, A.L.Mazanov and M.B.Vainshtein for their kind support. P9^19 RESPONSE OF SULFATE-REDUCING BACTERIA DESULFOVIBRIO DESULFURICANS TO MERCURY A. Lapanje(1), D. Drobne(1), B. Beaumont(2), R. J. M. van Spanning(2), M. Rupnik(1) > (1) Biotechnical Faculty, Department of Biology, Vecna pot 111, 1000 Ljubljana, Slovenia; (2) Vrije Universiteit, NL1081 HV Amsterdam, The Netherlands Presence of mercury is unfavourable for growth of sulfatereducing bacteria (SRB) for at least two reasons: mercury toxicity and mercury derived higher redox potential. SRB detoxify mercury by sul¢de precipitation into HgS. Also, SRB transform mercury into more toxic methylmercury. Methylmercury is mostly toxic to multicellular animals because it is lipid soluble compound which di¡uses through the membrane. Methyl mercury is then transformed into ionic form where persists and make severe e¡ects. For single cell organism the methylation of mercury can be the mechanism for detoxi¢cation. The SRB biochemically transform the ionic form of mercury, which enters the cell, into lipid soluble form after the methylation process. This produced methylmercury goes out from the cell with di¡usion. Besides this the methylation of mercury is known to be enzymatic process and SRB can actively transform mercury into methyl mercury. These two mechanisms can serve SRB to survive high concentrations of mercury. Because of that we tried to ¢nd physiological e¡ects of mercury on Desulfovibrio desulfuricans. This could help us to understand detoxifying mechanisms. We added di¡erent amounts of mercury into the media for fermentative and respiratory growth. We inspected growth curves as well as protein synthesis at the beginning of the stationary phase. We investigated whole concentration of proteins as well as patterning proteins on SDS PAGE gels. The changes in expression were observed in soluble although we investigate both phases ^ lipid soluble as well as water soluble proteins. We also found that the

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growth curve shape was changed and showed biphasic growth. P9^20 REDUCTION OF EXOGENOUS FERRIC IRON BY A CELL SURFACE-ASSOCIATED REDUCTASE OF ENTEROCOCCUS SPP. P. Lisiecki, P. Wysocki Department of Pharmaceutical Microbiology, Medical Uni¤ ¤ ¤ ¤ versity of Jodz, 90-235 Jodz, Pomorska 137, Poland Over the past few years enterococci have become one of the most important noscomial and community acquired pathogens. For several bacteria, a correlation between mechanisms of iron assimilation and virulence has been demonstrated. Enterococci as facultative anaerobes have an obligate iron requirement. For this reason, there is an increasing interest in strategies of iron acquisition in enterococci. We showed that enterococci produced hydroxamate class siderophore. In this study we have tried to estimate the ability of enterococci to reduction of exogenous Fe3+ by means of cell surface-associated reductases. A total of 122 strains genus Enterococcus belonging to ten species and isolated from clinical and environmental sources were used. Ten iron compounds were tested: ferric ammonium sulphate, ferric ammonium citrate, ferric versenate, ferric nitrate, ferric pyruvate, ferrioxamine B, horses ferritin, human transferrin, lactoferrin and haemoglobin. The reduction of Fe3+ was assayed with ferrozine test. The ability of enterococci to reduce of Fe3+ was detected with agar plate technique and reductase activity was estimated in liquid medium. All strain of enterococci used 3+ in this study were able to reduce Fe of all tested iron sources. The iron in form of ferric ammonium citrate, lactoferrin and ferrioxamine B were the best iron sources for enterococcal reductases. We found that reduction of iron was slightly stimulated in NADH presence but not in NADPH, and was enhanced by addition of FMN. Addition of Mg2+ to the reaction mixture had no in£uence on reduction of exogenous ferric iron. All the strains showed no di¡erences in reductases activities when they grew under iron-poor or iron rich conditions. Haemin or haemoglobin in the culture medium did not increased reductases activities. The cell surface-associated ferric reductases may be one of the important way of iron-acquisition in enterococci.

P9^21 IN VIVO ANALYSIS OF CHAPERONES-DEPENDENT REFOLDING OF BACTERIAL LUCIFERASES DIFFERING IN THE THERMOSTABILITY AND LUMINENCENCE DECAY RATE I. V. Manukhov, M. M. Mazhul, V. U. Kotova, G. B. Zavilgelsky State Scienti¢c Center of the Russian Federation, GosNIIgenetika, 1_yi Dorozhnyi Proezd 1, 117545 Moscow, Russia The role of chaperones Hsp70 and Hsp100 in refolding invivo of thermoinactivated luciferases from marine bacterium Vibrio ¢scheri, Vibrio harveyi, Photobacterium leiognathi and the terrestrial bacteria Photorhabdus luminescence in Escherichia coli cells has been studied. These luciferases are homologous but di¡er greatly in the rate of thermal inactivation and in the rate luminescence decay. It was shown that refolding of thermoinactivated luciferases is completely determinate by the DnaKJ system. However these luciferases markedly di¡er in rate and degree of refolding. The degree of refolding of thermolabile, ``fast'' V. ¢sheri luciferases reaches 80% of the initial level over several minutes, whereas renaturation of thermostable, ``slow'' Ph. luminencence luciferases proceeds substantially slower. Intermediate parameters of refolding are characterized for V. harvei and Ph. leoignathi luciferases. The measurement of the rate of thermo inactivation of luciferases in E. coli wild strain and strains containing mutations in clpA, clpB and clpX genes showed that Ph. luminencence luciferase revealed reduced thermostability in mutant strains E. coli clpA or clpB. In E. coli clpA this e¡ect is not connected with DnaK-dependent refolding, in E. coli clpB this e¡ect is determinate by DnaK-dependent refolding and connected with the ability of chaperone ClpB to provide disaggregation of the proteins. The addition of uncoupler of oxidative phosphorylation, carbonylcyanide-3-chlorophenylhydratione (CCCP) results in a sharp decrease in thermostability of luciferase to the level typical of the enzyme in vitro. We suggest that thermostable luciferases have enhanced a/nity with respect to chaperone ClpA in comparision with DnaK, whereas thermolabile luciferases are characterized by enhanced a/nity with respect to chaperone DnaK.

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P9^22 GENES INVOLVED IN STRESS RESPONSE AND RESISTANCE OF ESCHERICHIA COLI TO THE LACTOPEROXIDASE SYSTEM J. Sermon, P. Despiegeleer, K. Vanoirbeek, A. Aertsen, and C. W. Michiels Department of Food and Microbial Technology, Katholieke Universiteit Leuven, Leuven, Belgium Haem peroxidases such as lactoperoxidase (LP) catalyze the oxidation by hydrogen peroxide of a wide range of substrates into oxidizing reaction products with antimicrobial properties, and play a role in host defence against infection. While the e¡ects of, and bacterial response to, other oxidative stress agents such as hydrogen peroxide and superoxide has received considerable attention in recent years, little is known about oxidative stress caused by these peroxidase enzymes. We report here the initial results of a study into the stress response and resistance factors of Escherichia coli MG1655 to the LP-thiocyanate system. In a ¢rst approach, we have selected random transposon insertion mutants with an increased resistance to the LP system and identi¢ed the location of the insert. In a second approach, we used a gfp reporter gene assay to identify promotors that are speci¢cally induced by the LP / thiocyanate system but not by hydrogen peroxide alone. These approaches resulted in the identi¢cation of three classes of genes : (i) genes which have been previously implicated in (oxidative) stress response, such as cysJ and lrp; (ii) genes with a known function, but unrelated to stress response such as rfaQ, rfaB, sgaT; (iii) genes with no assigned function to date. Together, these results suggest that response and resistance to the LP system involve a unique set of genes in E. coli. Further analysis will provide insight in the cellular targets of the LP system and in the relation of these genes to other forms of oxidative stress. P9^23 FUNCTIONAL ANALYSIS OF THE LYSOZYME INHIBITOR PROTEIN IVY IN E. COLI D. Deckers(1), A. Aertsen(1), M. Atanassova(1), B. Masschalck(1), C. Abergel(2), and C. W. Michiels(1) (1) Department of Food and Microbial Technology, Katholieke Universiteit Leuven, Leuven, Belgium ; (2) CNRS Institute of Structural Biology & Microbiology, 13402 Marseille Cedex 20, France The product of the E. coli ORF ykfE was recently shown to be a potent inhibitor of C-type lysozymes. This inhib-

itor, designated as Ivy, is the ¢rst lysozyme-inhibiting protein to be reported, and the objective of this work was to study its potential function in E. coli. First we studied the role of Ivy in E. coli sensitivity to hen egg white lysozyme. These experiments were conducted under high pressure to permeabilize the outer membrane for lysozyme. Compared to the wild-type strain MG1655, lysozyme sensitivity was increased in an ivy knock-out mutant, and reduced in the same mutant overexpressing Ivy from a plasmid. Further, we found that lysates of the knock-out strain displayed lytic activity on a Micrococcus lysodeikticus cell suspension. Lysates from the wild-type and overexpression strain did not exhibit such activity but inhibited the lytic activity of the lysate obtained from the knock-out strain. Finally, we observed under some conditions an increased resistance of the ivy knock-out mutant to ampicillin, a beta lactam antibiotic that interferes with cell wall synthesis and that can induce autolysin activity. Together, these results suggest a possible role of Ivy in (i) protecting pathogenic or commensal E. coli against lysozyme produced by its host; and (ii) regulating peptidoglycan metabolism. P9^24 INFLUENCE OF THE ENVIRONMENTAL CONDITIONS ON THE GROWTH OF AEROPYRUM PERNIX ¤ > I. Milek, B. Cigic, L. Pogacnik, M. Skrt, N. Poklar Ulrih University of Ljubljana, Biotechnical Faculty, Chair of Chemistry, Jamnikarjeva 101, 1000 Ljubljana, Slovenija Aeropyrum pernix is the ¢rst obligately aerobic and neutrophilic hyperthermophilic archaeon to be isolated. It belongs to Desulforococcales order in Crenarchaeota kingdom. The fact that the majority of hyperthemophilic Archaeas are anaerobic makes A. pernix very interesting for laboratory experiments. Its optimum growth temperature is between 90 and 95C and the optimum pH for growth around 7. With the purpose to obtain high cell densities in liquid culture, we systematically studied the e¡ect of the medium composition and culture conditions on growth of A. pernix. For cultivation 300 mL Nephelo culture £asks were used. We have found out that the highest cell density was achieved by using 50 mL of marine medium (Bacto Marine broth 2216, Difco) at pH 7.0 in reciprocating water bath at 92C. We discuss di¡erent types of cultivation (liquid medium, solid medium) and the in£uence of the environmental conditions including oxygen availability and pH on growth curve.

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P9^25 BEYOND THE STATIONARY-PHASE : LONG-TERM SURVIVAL OF PSEUDOMONAS AERUGINOSA ' M. Nicolo, S. Carnazza, M. Capone, S. Guglielmino Dept. of Microbiological, Genetic and Molecular Sciences, University of Messina, sal. Sperone, 31, 98166 Messina, Italy In the last decades, much attention has been focused on survival of bacterial populations under nutrient limitation, such as nutrient starvation and prolonged stationary phase. Bacterial response to starvation is generally homeostatic, but in some cases, such as phosphate starvation, the emergence of ¢tter mutants, called PSOM, that take over the culture has been described for Pseudomonas putida. On the other hand, studies on prolonged stationary phase revealed that, in Escherichia coli, mutants with higher ¢tness, so-called ``GASP'' phenotypes, appear weekly. These mutants, showing mutations in rpoS, are unable to enter stationary phase and the continuous emergence of ¢tter mutants seems to be the strategy which ensures the survival of the population. It should also be remarked that the terms ``starvation'' and ``stationary phase'' have been used as synonyms, by the aprioristic assumption that nutrient starvation should be one of the limiting conditions in prolonged stationary phase, but little is known about the true role of nutrient starvation in a prolonged stationary phase. In this work, the survival mechanisms of Pseudomonas aeruginosa have been investigated. The study has been conducted in two parallel ways. First, as holistic criterion, the phenomenon has been observed in a batch culture, incubated for 24 months. Second, from a cartesian point of view, the incidence of single variables has been analysed. The data suggest that, di¡erently from the increase of ¢tness in E. coli, P. aeruginosa survival is based on the modulation of cell lysis which, in turn, releases nutrients recycled by remaining cells. P9^26 HEAT SHOCK PROTEIN EXPRESSION IN BALB/C (H-2d) MICE AT COURSE OF MOUSEPOX J. Cymerys and M. G. Niemialtowski Immunology Laboratory, Department of Preclinical Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, ul. Ciszewskiego 8, 02-786 Warszawa, Poland Ectromelia virus (ECTV) can develop a mousepox. It is well known from studies with some other viruses that expression of heat shock proteins (hsp) may be modulated during viral infections. To assess the role of hsp at di¡er-

ent clinical phases of mousepox (up to 20 days p.i.), liver, spleen, brain and conjunctivae were collected and the levels of hsp27, hsp70, and hsp90 were detected in studied tissues of BALB/c mice infected with Moscow strain of ECTV (ECTV-MOS). Results. In all examined organs hsp27, hsp70, and hsp90 were expressed not only during primary viraemia, but also during the peak (10-20 days p.i.) of disease. In liver highest level of hsp27 and hsp90 was found at 1 day p.i,, whereas at 15 day p.i hsp70 predominated ( s 60% and s 80% positive cells, respectively). In spleen hsp90 reached maximal expression at 5 day p.i., hsp27 at 10 day p.i., and hsp70 at 15 day p.i. ( s 80%, 65% and s 80% positive cells, respectively). Our results con¢rmed that primary viraemia in parenchymal organs during the course of mousepox is strongly associated with hsp expression. In the brain level of hsp90 was signi¢cantly elevated between 1 and 5 d.p.i., then diminished to 20% positive cells. Similarly, hsp27 and hsp70 levels in brain cells were highest at 5 day p.i. In conjunctivae expression of hsp accompanied the onset of clinical sings of mousepox. Hsp27 expression reached maximum value at 15 d.p.i., hsp70 signi¢cantly increased between 10 and 15 d.p.i., whereas hsp90 level between 1 and 15 d.p.i. ( s 80%). Conclusion. Our studies demonstates that ECTV-MOS can modulate hsp levels at course of mousepox. This work was supported by the Foundation for Polish Science [grant no 10/2000]. P9^27 CHARACTERIZATION OF STREPTOCOCCUS PNEUMONIAE SERINE / THREONINE PROTEIN KINASE StkP AND PROTEIN PHOSPHATASE PhpP AND THEIR PHYSIOLOGICAL FUNCTION L. Novakova, L. Prenosilova, J. Echenique, M.-C. Trombe and P. Branny Institute of Microbiology, Czech Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic Transmission and ampli¢cation of a signal by a network of Ser/Thr/Tyr protein kinases plays a principal role in di¡erentiation and cell-to-cell communication in eukaryotes. During the last several years, however, a considerable number of eukaryotic-type Ser/Thr protein kinases (STKPs) were identi¢ed in prokaryotes. Being absent in E. coli and present only in a few examples in many bacteria they are represented by a multigene families in Streptomyces, Myxococcus, and Mycobacterium. All these bacteria display developmental characteristics comparable to multicellular eukaryotes. Alternatively, STPKs have been shown to be required for the full virulence of some pathogens in mouse models. Searching in the genome sequence

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of S. pneumoniae revealed presence of a single eukaryoticlike Ser/Thr protein kinase gene associated with a gene encoding protein phosphatase. The genes were named stkP and phpP, respetively. We expressed, puri¢ed and characterized PhpP, StkP proteins. In vitro kinase assays demonstrated that StkP is a functional kinase that is dependent on either magnesium or manganese ions. A twodimensional phosphoamino acid analysis revealed strong phosphorylation at a threonine residues. The PhpP phosphatase is an Mn2+-, but not a Ca2+- or a Mg2+, dependent phosphatase. Its activity is inhibited NaF, but not by okadaic acid, and is similar to that of PP2C phosphatase. In S. pneumoniae both proteins are likely functionally coupled as PhpP e/ciently dephosphorylates autophosphorylated form of StkP. Analysis of S. pneumoniae stkP deletion mutant showed that protein kinase is likely involved in the control of cell wall biosynthesis. P9^28 OXIDATION STRESS AND SYSTEMS OF ANTIOXIDANT PROTECTION IN HETEROTROPHIC COLORLESS SULFUR BACTERIA D. A. Podkopaeva(1), M. Yu. Grabovich(1), G. A. Dubinina(2) (1) Voronezh State University, University square 1,394006, Voronezh, Russia ; (2) Institute of Microbiology,Prospect 60-let Oktyabrya 7/2, 117811, Moscow, Russia The in£uence of the oxygen regime of Spirillum winogradskii strain D-427, DSMZ 12756 cultivation on biosynthesis processes, enzyme activity and role of di¡erent systems of antioxidant cell protection from oxidation stress, are determined. Under aerobic conditions (21% O2 in gas phase) in contrast to microaerobic ones (2% O2 in gas phase), bacteria growth is accompanied by changes in metabolism ^ lower activity of constructive metabolism processes and more intense synthesis of exopolysaccharides as a way of external cell protection from excess O2. This brings to twofold reduction of economic growth factor and cellular harvest. In spite of the fact that low activity of catalase is compensated by a many-fold increase in the activity of other cytoplasmic enzymes protecting from toxic O2 forms, mass lysis of cells begins in the middle of exponential phase growth and brings to the destruction of culture in the stationary phase because of the accumulation of H2O2 in periplasm up to 10 Wm (mg of protein)-1. No cytochrome-c peroxidase, which is the main periplasmic enzyme removing H2O2, is detected. The addition of thiosulfate to the medium results in cessation of cell lysis, 3 ^ fold increase of Qmax, and stabilization of culture as a result of removing H2O2 in aerobic conditions. During bacteria growth with thiosulfate, the activity of sulfur metabolism enzymes of dissimilatory type is not observed,

which is indicative of microorganisms inability to lithoheterotrophic growth. Thus, we suggest that the main reason of oxidation stress in cells is the spatial separation of active systems of antioxidant protection from O2 localized in cytoplasm and accumulation of H2O2 in periplasm because of cytochrome-c peroxidase absence. This work was supported by the Russian Foundation of Basic Research, grant no. 02-04-49185. P9^29 CHANGES OF METABOLIC ACTIVITY OF ERWINIA SP. GROWN UNDER SOME TYPES OF EXTERNAL STRESS J. Pokorna, I. Marova, R. Koci, M. Drabkova, M. Knoppova Department of Food Chemistry and Biotechnology Faculty of Chemistry, Technical University of Brno, Czech Republic The genus Erwinia represents nonphotosynthetic bacteria pathogenic mostly for higher plants. The strains E. herbicola and E. carotovora, respectively, are of increasing interest as industrial microbes producing pectinolytic, cellulolytic and proteolytic enzymes and antileukaemic asparaginase. Cultures of Erwinia are producents of carotenoids, which caused yellow-orange coloured phenotype. The aim of this work was to study in£uence of exogenous stress factors on genome integrity, metabolic activity and carotenoid production by E. herbicola and E. carotovora. Bacteria were grown aerobically (28C, permanent lighting). Exogenous stress was induced by addition of 3% NaCl (osmotic stress), 5 mM NiCl2 and/or 5 mM H2O2 (oxidative stress). During the experiment cell morphology, amount and composition of carotenoids (HPLC/MS), production of neutral lipids, glycerol and extracellular pectinases and proteases were followed. Chromosomal DNA changes were followed by PFGE using macrorestriction enzymes SmaI and NotI. Bacteria were more sensitive to osmotic stress, a signi¢cant decrease of biomass (2.2x), lipids (2.3x), oe- and oe-carotene (1.8x), lutein (1.8x) and extracellular enzymes (2-3x) was observed. Osmotic stress led to some changes in set of macrorestriction fragments too. On the other hand, addition of H2O2 led to increased production of lipids (1.4x), enzymes (1.1x), carotenes (1.3x) and lutein (1.6x). Production of glycerol was 1.8x higher under salt stress, but 1.6x lower under oxidative stress when compared with control cultivation. Carotenoids produced by Erwinia sp. could play a role in protection of cells from both oxidative stress in nature and reactive oxygen species (superoxide, H2O2) produced as a ¢rst response by plant-pathogen interaction.

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P9^30 ROLE OF CCPA IN REGULATION OF CENTRAL METABOLISM AND IN RESPONSE TO STRESS CONDITION IN LACTOBACILLUS PLANTARUM C. Castaldo(1), R. Marasco(2), L. Muscariello(1) and M. Sacco(1) (1) Dipartimento di Scienze Ambientali, Seconda Universi' ta di Napoli, Caserta, Italy ; (2) Dipartimento di Scienze ' Biologiche ed Ambientali, Universita del Sannio, Benevento, Italy In Gram-positive bacteria of low G+C content, CcpA (catabolite control protein A) is a master regulator which can function either as a repressor or as an activator of transcription. We characterised the ccpA gene of Lactobacillus plantarum LM3 and isolated the LM3-2 strain carrying a ccpA null mutation (ccpA1). We analysed proteins from membrane and cell wall fractions of the L. plantarum LM3 and LM3-2 strains. Proteins were extracted from cells grown on glucose or ribose up to exponential phase. The electrophoretic pattern of membrane proteins extracted from exponential cells grown on glucose showed a di¡erential protein expression in the two strains. The presence of a 49 kDa protein only in the wild type strain suggested a CcpA-mediated activation in the expression of this protein. The expression of 5 proteins, ranging from 44 to 67 kDa, was detected only in the mutant strain while the expression of two other proteins of 41 and 47 kDa resulted increased in the mutant strain, suggesting a CcpA-dependent negative control of their expression. Different electrophoretic patterns were also shown in the cell wall fractions, where we found a 45 kDa protein whose expression resulted CcpA repressed in the wild type strain. We also analyzed the protein expression in the wild-type and mutant strains grown in salt stress condition, ¢nding at least two proteins whose expression is repressed by CcpA. We are now identifying the di¡erentially expressed proteins to study the CcpA-mediated regulation of the corresponding genes. We are also investigating the role of the CcpA protein in response to starvation and environmental changes in temperature and carbon source in Lactobacillus plantarum.

P9^31 RIBOFLAVIN ENHANCES YEAST RESISTANCE TO CHROMIUM D. Fedorovych(1), H. Ksheminska(1), L. Babyak(1), P. Kaszycki(2), H. Koloczek(2), A. Jaglarz(2), and A. A. Sibirny(1,3) (1) Institute of Cell Biology, Drahomanov Street 14/16, 79005 Lviv, Ukraine; (2) University of Agriculture, Bio¤ chemistry Department, al. 29 Listopada 54, 31-425 Krakow, Poland ; (3) Institute of Biotechnology, Rzeszow University, ul. Rejtana 16C, 35-310 Rzeszow, Poland Some yeast strains, for example, Pichia guilliermondii, responded to Cr(VI) by stimulation of ribo£avin (RF) biosynthesis (Fedorovych et al., 2001). The P. guilliermondii strain UKD 1453 incapable of RF oversynthesis possessed higher sensitivity to Cr(VI). We suggested that extensive £avinogenesis is a response to Cr(VI) treatment and a mechanism leading to higher yeast survival. The e¡ect of Cr(VI) on growth of P. guilliermondii mutants defective in the regulation of RF biosynthesis and possessing di¡erent levels of £avinogenesis was studied. The sensitivity of yeast strains to Cr(VI) did not correlate with the level of £avinogenic activity. However, when Cr(VI) was added to the cultures which actively synthesized RF, growth inhibition was diminished. Exogenous RF decreased the toxicity of Cr(III) and Cr(YI). In the presence of Cr(III) or Cr(VI) the growth of £avinogenic strain UKD 66 of P. guilliermondii and strain UKD 1453, unable to RF oversynthesis, was characterized by a prolonged lag phase. Culture medium supplementing with exogenous RF (200 Wg/ml) led to a decrease in lag phase. Diminished toxicity of Cr(III), but not of Cr(VI), was caused by reduced chromium uptake. These data suggest the existence of interaction between RF metabolism and chromium toxicity. It was shown earlier that RF can decrease the nephrotoxic e¡ect of chromate in young and adult rats (Appenroth et al., 1996). Our data on RF protection of chromium sensitivity in P. guilliermondii strains provide evidence for RF crucial role in chromium detoxi¢cation.

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P9^32 STRESS RESPONSE OF YEAST CANDIDA INTERMEDIA UPON CR(III) SUPPLE