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Anna Pietruczuk-Padzik1, Joanna Stefaska1, Katarzyna Semczuk2, Danuta Dzieranowska2, Stefan Tyski1,3

OCENA TWORZENIA BIOFILMU PRZEZ SZCZEPY STAPHYLOCOCCUS AUREUS WYIZOLOWANE Z PLWOCINY PACJENTÓW Z MUKOWISCYDOZ

1 Zaklad Mikrobiologii Farmaceutycznej, Warszawski Uniwersytet Medyczny Kierownik: prof. dr hab. S. Tyski 2Zaklad Mikrobiologii i Immunologii Klinicznej, Instytut Pomnik - Centrum Zdrowia Dziecka w Warszawie Kierownik: prof. dr hab. D. Dzieranowska 3Zaklad Antybiotyków i Mikrobiologii, Narodowy Instytut Leków, Warszawa Kierownik: prof. dr hab. S. Tyski Celem przedstawionych bada byla ocena dwóch metod wykrywania biofilmu bakteryjnego tworzonego przez kliniczne szczepy Staphylococcus aureus wyizolowane z plwociny pacjentów z mukowiscydoz oraz ocena tworzenia biofilmu na powierzchni dolków plytek polistyrenowych w zalenoci od uytego podloa hodowlanego (LB, BHI, TSBglu). Zdolno i intensywno tworzenia biofilmu analizowano metod barwienia z uyciem fioletu krystalicznego (CV) oraz metod oceny redukcji chlorku 2,3,5-trójfenylotetrazoliowego (TTC). Stwierdzono, e obydwie metody barwienia okazaly si przydatne do oceny powstajcego biofilmu gronkowcowego. Za najbardziej optymalne podloe do hodowli biofilmu gronkowcowego uznano podloe LB.

A. P i e t ru c z u k-P a d zi k, S . Tys ki J. S t ef a ska , K. S e mc z u k, D. D z i e r a n o w s ka ,

EVALUATION OF BIOFILM FORMATION BY STAPHYLOCOCCUS AUREUS ISOLATED FROM SPUTUM OF CYSTIC FIBROSIS PATIENTS SUMMARY The purpose of this study was to evaluate two screening methods for detection of biofilm formation by eighty clinical Staphylococcus aureus isolates from patients with cystic fibrosis, and evaluation of biofilm production on the polystyrene 96-well tissue culture plates, depending on media applied. All clinical strains were incubated in three different media: Luria­Bertani broth (LB), tryptic soy broth supplemented with 2% glucose (TSBglu) and brain heart infusion (BHI). Biofilm production was screened by staining with crystal violet (CV) or with 2,3,5-triphenyltetrazolium chloride (TTC). Both CV and TTC assays showed, that all analyzed isolates created biofilm, in all tested media, however with different intensity. In conclusion, the CV method was found to be more sensitive than the TTC method, when we need information about whole mass of biofilm. The most optimal medium for the biofilm culture was LB medium.

Katarzyna Piekarska1, Katarzyna Zacharczuk1, Jolanta Szych1, Elwira Zawidzka2, Elbieta Wilk2, Sebastian Wardak2, Marek 1 Jagielski1, Rafal Gierczyski1

WYSTPOWANIE W JEDNYM Z WARSZAWSKICH SZPITALI PALECZEK KLEBSIELLA PNEUMONIAE WYTWARZAJCYCH KARBAPENEMAZ TYPU KPC

1 Zaklad Bakteriologii Narodowego Instytutu Zdrowia Publicznego ­ Pastwowego Zakladu Higieny Kierownik: prof. dr hab. M. Jagielski 2 Niepubliczny Zaklad Opieki Zdrowotnej, Laboratoria Analiz Lekarskich ­ ALAB. Kierownik: mgr E. Wilk

2

1 2

autor do którego naley kierowa korespondencj badania wspólfinansowane ze rodków na nauk w latach 2008-2009 w ramach projektu badawczego NN 404 165034

Okrelono profile lekowraliwoci oraz podobiestwo profili XbaI-PFGE jedenastu szczepów K. pneumoniae wykazujcych oporno na ertapenem. Szczepy te wyizolowano od dziesiciu pacjentów jednego z warszawskich szpitali i jednego pacjenta hospicyjnego. Analiza fenotypowa (test z kwasem boronowym) i genetyczna (PCR) badanych szczepów wykazala, e wytwarzaj one karbapenemaz typu KPC. Wród badanych szczepów wyróniono 7 genotypów PFGE, przy czym 5 szczepów zaliczono do tego samego genotypu, co moe wskazywa na ich epidemiczne szerzenie si w szpitalu. Pozostale 6 szczepów nalealo do rónych genotypów. Dendrogram genetycznego podobiestwa badanych szczepów skladal si z dwóch galzi obejmujcych 2 i 9 szczepów, o podobiestwie wynoszcym odpowiednio 86,5% i 93,2%. Genetyczne zrónicowanie badanych szczepów na dwie wyranie odrbne grupy moe wynika z horyzontalnego i wertykalnego transferu genu blaKPC u paleczek K. pneumoniae wystpujcych u pacjentów szpitala.

K. P i e ka rs ka , K. Za c h a rc z u k, J . S z yc h , E. Za w i d z ka , E. Wi l k, S . Wa rd a k, M. J a gi e l s ki , R . G i e rc z y s ki

DISSEMINATION OF THE KPC CARBAPENEMASE PRODUCING KLEBSIELLA PNEUMONIAE IN A HOSPITAL IN WARSAW, POLAND SUMMARY Within the last decade, human infections caused by enterobacteria which produce the Klebsiella pneumoniae carbapenemase (KPC) became a serious therapeutic and epidemiological problem worldwide. The KPC producing strains of K. pneumoniae broadly disseminated in the USA then spread to Europe. Recently, the KPC-2 was found in Poland. In the presented study we tested 11 ertapenem resistant isolates of K. pneumoniae. The isolates were obtained from 10 patients of a regular hospital (RH) and from one patient of a palliative care hospital (PH) in Warsaw, Poland. Expression of the KPC was confirmed in all the tested isolates by the positive result of phenotypic test with boronic acid. All the isolates were also shown to harbour the blaKPC gene by PCR with primers targeting the core 372 bp fragment of the gene, and all but two were resistant to imipenem and meropenem as determined by the disc-diffusion method. The DNA sequence analysis of the complete blaKPC gene from representative isolate DM0269 revealed variant 2 of KPC (KPC-2). Tested isolates were subjected to genotyping by the PFGE with XbaI. Dendrogram based on the PFGE profiles was composed of two main branches with 82,3% of similarity. Branch A encompassed 9 isolates (93,2%), including the one from the PH-patient, while the two remaining isolates (86,5%) were located in branch B. Five isolates of the branch A were indistinguishable by the PFGE. The high genetic similarity of the branch A isolates strongly suggests the intra-hospital dissemination of epidemic K. pneumoniae KPC+ sensu stricto strain. Most probably, the strain was also transferred to the palliative care hospital. In contrast, the branch B isolates appear to belong to the distinct sensu stricto strain, that has acquired the blaKPC gene via horizontal transfer. This is the first report on the intra-hospital dissemination of the KPC producing K. pneumoniae in Poland. It is noteworthy, all the tested strains were also resistant to cefotaxime, ceftazidime, aztreonam, ciprofloxacin and sulphonamides, but sensitive to colistin.

Waldemar Rastawicki, Stanislaw Kaluewski, Natalia Rokosz

OCENA PRZYDATNOCI IMMUNOENZYMATYCZNEGO ODCZYNU ELISA DO WYKRYWANIA PRZECIWCIAL DLA LIPOPOLISACHARYDU PALECZEK KLEBSIELLA RHINOSCLEROMATIS U CHORYCH NA TWARDZIEL

Zaklad Bakteriologii NIZP-PZH w Warszawie Kierownik: prof. dr hab. M. Jagielski Odczynem ELISA poszukiwano swoistych przeciwcial dla antygenu 2a paleczek K. rhinoscleromatis w 65 próbkach surowicy uzyskanych od osób z klinicznym podejrzeniem twardzieli. Próbki te przyslano w latach 1970-2009 do rutynowych bada odczynem wizania dopelniacza. Obecno przeciwcial klasy IgA i IgG na poziomie diagnostycznie znamiennym wykryto w 33 (50,8%) a przeciwcial klasy IgM w 28 (43,1%) próbkach surowicy. Przeprowadzone badania wykazaly wysok czulo i swoisto odczynu ELISA z uytym antygenem w poszukiwaniu przeciwcial dla paleczek twardzieli.

W. Rastawicki, S. Kaluewski, N. Rokosz

APPLICATION OF ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) FOR DIAGNOSIS OF ANTIBODIES TO LIPOPOLYSACCHARIDE OF KLEBSIELLA RHINOSCLEROMATIS IN PATIENTS WITH RHINOSCLEROMA

SUMMARY The ELISA were performed on polystyrene microtiter plates (Nunc, MaxiSorp) coated with LPS (2a antigen) at the final concentration of 10 µg/ml. The antigen was extracted from Klebsiella rhinoscleromatis Rh32 by the trichloroacetic acid and separated by ethanol (Boivin method). The antibodies against the LPS were detected by ELISA in serum samples collected from 65 patients suspected in clinical investigation for rhinoscleroma in Poland from 1970 to 2009. Additionally, the specificity of the antigen was tested using serum sample of immunized rabbit and 30 sera of patients from control group, with high level of antibodies to different bacterial pathogens. All serum samples were diluted 1:100. The concentrations of IgA, IgG and IgM antibodies were expressed as optical density (OD) measured at the wavelength of 450 nm. The cut-off limit of serum antibodies was set at mean antibody OD determined in the sera of 30 blood donors exceeded by three standard deviations. The presence of IgA and IgG antibodies were detected by ELISA in 33 (50,8%) and IgM in 28 (43,1%) of patients. Most of the serum samples (75%) with high level of specific antibodies were obtained from patients before 1980. On the other hand antibodies to K. rhinoscleromatis were detected only in 2 (6,7%) patients from the control group and none of blood donors. In conclusion, our home­made ELISA, based on purified LPS of K. rhinoscleromatis showed high specificity and sensitivity in the diagnosis of antibodies to K. rhinoscleromatis in comparison to the complement fixation test. The presence of high level of specific IgA, IgG and IgM antibodies in the sera obtained in different stages of disease may showed that during the rhinoscleroma is permanent stimulation of antibody production.

Grzegorz Madajczak, Jolanta Szych

OCENA PRZYDATNOCI TESTU PREMI®TEST SALMONELLA DO IDENTYFIKACJI PALECZEK SALMONELLA NIETYPUJCYCH SI METODAMI KLASYCZNYMI

Zaklad Bakteriologii Narodowego Instytutu Zdrowia Publicznego -Pastwowego Zakladu Higieny w Warszawie Kierownik: prof. dr hab. M. Jagielski Oceniono przydatno komercyjnego testu Premi®Test Salmonella do identyfikacji nietypujcych si szczepów paleczek Salmonella. Sporód 37 przebadanych szczepów, ocenianym testem w pelni zidentyfikowano 21 (56%) szczepów, identyfikacja 5 (14%) szczepów wymagala dodatkowego potwierdzenia, niepeln identyfikacj uzyskano w przypadku 5 (14%) szczepów, natomiast 6 (16%) szczepów nie udalo si zidentyfikowa. Uzyskane wyniki wiadcz o przydatnoci testu Premi®Test Salmonella do okrelania serotypu paleczek Salmonella, w przypadku gdy jest to niemoliwe tradycyjnymi metodami.

G. Madaj czak, J. Szych

EVALUATION OF PREMI®TEST SALMONELLA KIT FOR IDENTIFICATION OF NOT-TYPABLE BY CONVENTIONAL METHODS SALMONELLA SUMMARY Despite the downward trend, Salmonella is still one of the most important bacterial intestinal infections agents. For example, in 2007 y. in Poland over 14 thousands human salmonelosis cases were notified, in 2008 y. - over 10 thousands. Among all Salmonella isolated from human source, most common (more then 80%) are two serotypes ­ S. Enteritidis and S. Typhimurium, but every year the number of non-typable (because of the lack I or II phase flagellar antigens, autoagglutinative or non-motility properties) Salmonella is growing. This work describes the results of the evaluation of commercially available kit Premi®Test Salmonella (DSM, Netherlands), which uses the microarray hybridization in ArrayTube, for serological identification of non-typable Salmonella strains. All 37 researched strains were submitted to the Department of Bacteriology in 2007-2008 y, were reidentified according to standard operating procedures and serotyped by slide agglutination for somatic and flagellar antigens if it was possible. All strains were tested using the Premi®Test Salmonella Kit, according to manufacturer instructions. In 21 cases (56%) full identification were achieved, in 5 cases (14%) additional tests were required for precise identification (Salmonella Choleraesuis or Paratyphi C, what was detailed using examination of additional biochemical features), 5 strains (14%) achieved an incomplete identification (three of them ­ S. diarizonae were confirmed in National Reference Laboratory for Salmonella) and 6 cases (16% ) were not identified at all. The total number of recognized strains is 30 (81%). The results of present studies show, that Premi®Test Salmonella Kit is useful for nontypable Salmonella identification.

Agata Bialucha, Sylwia Kouszko, Eugenia Gospodarek

SZCZEPY SALMONELLA SP. OPORNE NA LEKI PRZECIWBAKTERYJNE

Katedra i Zaklad Mikrobiologii Collegium Medicum im. L. Rydygiera w Bydgoszczy Uniwersytetu Mikolaja Kopernika w Toruniu Kierownik: dr hab. n. med. E. Gospodarek, prof. UMK Badaniami objto 338 szczepów Salmonella sp. izolowanych z próbek materialu od chorych Wojewódzkiego Szpitala Obserwacyjno-Zakanego w Bydgoszczy oraz Szpitala Uniwersyteckiego nr 1 w Bydgoszczy w latach 2006-2009. Stwierdzono, e wród 90 izolatów paleczek Salmonella sp. opornych na leki przeciwbakteryjne najwyszy odsetek (56,7%) stanowily paleczki S. enterica serowar Enteritidis. Odnotowano wzrost szczepów opornych na ciprofloksacyn (0,0-26,7%) i na kwas nalidyksowy (57,1-97,3%).

A . B i a l u ch a , S. Ko u s z ko, E. G os po d a re k

SALMONELLA SPP. STRAINS RESISTANT TO DRUGS SUMMARY The aim of the study was retrospective analysis of Salmonella spp. strains isolated from patients of State Infectious Diseases Observatory Hospital of T. Browicz in Bydgoszcz (SZAK) and University of dr. A. Jurasz in Bydgoszcz (SU CM UMK) in 2006-2009. The percentages of Salmonella spp. strains resistant to at least one drug were: 19,0% in 2006, 12,5% in 2007, 50,6% in 2008 and 43,8% in the first half of 2009 year. The highest number of Salmonella spp. strains resistant to drugs were isolated from stool (96,7%) and from patients of SZAK (83,3%). Among all isolated Salmonella spp. strains resistant to drugs the highest percentage were S. enterica serovar Enteritidis (56,7%). Among S. enterica bacilli predominated resitant phenotypes to ampicillin, amoxicillin, chloramphenicol and nalidixic acid. The increasing number of strains resistant to ciprofloxacin (0,0 ­ 26,7%) and high percentage of strains resistant to nalidixic acid (97,3%) were noted. Decreasing resistance to chloramphenicol was observed in our study (54,5 ­ 14,3%).

Patrycja Zalas-Wicek, Eugenia Gospodarek, Katarzyna Piecyk

LEKOWRALIWO I WLACIWOCI ADHEZYJNE SZCZEPÓW ESCHERICHIA COLI IZOLOWANYCH Z MOCZU I Z KRWI

Katedra i Zaklad Mikrobiologii Collegium Medicum im. Ludwika Rydygiera w Bydgoszczy Uniwersytetu Mikolaja Kopernika w Toruniu Kierownik: dr hab. n. med. E. Gospodarek, prof. UMK Stwierdzono, e paleczki E. coli izolowane z moczu istotnie czciej ni izolowane z krwi wytwarzaly betalaktamazy typu ESBL i byly wielolekooporne. Z kolei szczepy E. coli wyosobnione z krwi w wyszym odsetku adherowaly do cewników lateksowych i cewników z polichlorku winylu.

ANTIMICROBIAL SENSITIVITY AND ADHESIVE PROPERTIES OF ESCHERICHIA COLI STRAINS ISOLATED FROM URINE AND BLOOD SAMPLES SUMMARY The aim of this study was comparison of the antimicrobial sensitivity and adhesive properties of E. coli strains isolated from urine and blood samples. This study included 169 of E. coli strains isolated in the Clinical Microbiology Department of dr. A. Jurasz University Hospital in 2003-2006. E. coli strains isolated from urine samples revealed statistically higher production of Extended Spectrum Beta-Lactamases and multidrug resistance than strains isolated from blood samples. Stains isolated from the blood adhered to latex and PCV catheters more frequently when compared with strains isolated from urine.

Patrycja Zalas-Wicek, Eugenia Gospodarek, Dorota Kamiska

WLACIWOCI MORFOLOGICZNYCH WARIANTÓW PALECZEK ESCHERICHIA COLI

Katedra i Zaklad Mikrobiologii Collegium Medicum im. Ludwika Rydygiera w Bydgoszczy Uniwersytetu Mikolaja Kopernika w Toruniu Kierownik: dr hab. n. med. E. Gospodarek, prof. UMK Porównano lekowraliwo, wlaciwoci adhezyjne oraz wzory chromosomalnego DNA morfologicznych wariantów kolonii paleczek Escherichia coli. U 90,0% z nich stwierdzono rónice we wraliwoci na antybiotyki. Zdolnoci adhezji do cewników moczowych rónilo si 40,0% wariantów. Warianty o morfologii kolonii jasnych w wyszym odsetku ni warianty o morfologii kolonii ciemnych adherowaly do wszystkich objtych badaniem cewników. Metod PFGE u 35% wariantów wykazano 100% podobiestwa genetycznego. Pozostale warianty byly blisko ze sob spokrewnione.

P . Za l a s -Wi c e k, E. G o s po d a re k, D . Ka mi s ka

COMPARISON OF THE PROPERTIES OF THE MORPHOLOGICAL VARIANTS OF E. COLI STRAINS SUMMARY The aim of this study was comparison of the antimicrobial sensitive, adhesive properties and genetic relatedness of the morphological variants of E. coli strains. This study included 10 of E. coli strains isolated in the Clinical Microbiology Department of dr. A. Jurasz University Hospital nr 1 in 2003-2006, which have grown in two morphological variants ­ light and dark variant. Ninety percent of morphological variants of E. coli strains differed among themselves in antibiotic sensitivity. Forty percent of morphological variants differed among themselves in adhesion to catheters. The light-colony variants more often adhered to all used catheters. The PFGE analysis of chromosomal DNA showed that 35,0% of morphological variants (two pairs and one triple) had 100,0% relatedness, and the rest of them were closely related.

Agnieszka Kaczmarek, Anna Budzyska, Eugenia Gospodarek

WYSTPOWANIE PALECZEK ESCHERICHIA COLI Z ANTYGENEM POWIERZCHNIOWYM K1 U KOBIET CIARNYCH I NOWORODKÓW*

Katedra i Zaklad Mikrobiologii, Collegium Medicum im. Ludwika Rydygiera w Bydgoszczy, Uniwersytetu Mikolaja Kopernika w Toruniu Kierownik: dr hab. E. Gospodarek, prof. UMK Oceniono czsto wystpowania polisacharydowego antygenu K1 wród 425 szczepów E. coli izolowanych z wymazu z odbytu i 67 szczepów izolowanych z pochwy kobiet ciarnych oraz wród 40 szczepów E. coli wyosobnionych z przedsionka jamy nosowej noworodków w okresie czerwiec-wrzesie 2008 roku. Wystpowanie antygenu K1 oznaczono testem aglutynacji lateksowej Pastorex Meningitis (Bio-Rad).

A . Ka c z ma re k, A . B u d z y s ka , E. G o sp od a re k

THE OCCURRENCE OF ESCHERICHIA COLI WITH K1 SURFACE ANTIGEN IN PREGNANT WOMEN AND IN NEWBORNS SUMMARY The aim of the study was to determine the frequency of occurrence of K1 surface antigen in Escherichia coli strains isolated from the pregnant women and newborns. A total of 425 of E. coli strains isolated from the faecal samples, 67 strains isolated from the vagina of pregnant women and 40 strains isolated from the newborns' nasal cavity were included into the study. All strains were collected between June and

September of 2008. Identification of isolates was followed by the assessment of presence of K1 surface antigen in E. coli strains. The presence of K1 antigen was found in 17,6% of E. coli strains isolated from the faecal samples, 20,9% of E. coli strains isolated from the vagina of pregnant women and in 17,5% of E. coli strains isolated from the newborns' nasal cavity. Routine screening of E. coli K1 colonization gives an opportunity to identify women with the risk of E. coli K1 transmission to neonates during delivery and thereby with major probability of perinatal infections. Latex agglutination test Pastorex Meningitis (Bio-Rad) provides fast identification of E. coli K1 strains.

Alicja Budak1,2, Paulina Paluchowska1, Dorota Wlodarczyk1, Pawel Nowak1, Malgorzata Wgrzyn, Wojciech Mudyna3

GENETYCZNA CHARAKTERYSTYKA SZCZEPÓW ACINETOBACTER BAUMANNII ORAZ PSEUDOMONAS AERUGINOSA IZOLOWANYCH OD CHORYCH Z ZAPALENIEM PLUC HOSPITALIZOWANYCH W ODDZIALE INTENSYWNEJ TERAPII

1Zaklad Mikrobiologii Farmaceutycznej Wydzial Farmaceutyczny UJ CM, Kraków 2Pracownia Mikrobiologii ZDL, Wojewódzki Szpital Specjalistyczny im. L. Rydygiera, Kraków Kierownik: prof. dr hab. A. Budak 3Oddzial Anestezjologii i Intensywnej Terapii WSS im. L. Rydygiera, Kraków Ordynator: dr med. W. Mudyna Przedmiot bada stanowily 33 szczepy Acinetobacter baumannii oraz 31 szczepów Pseudomonas aeruginosa izolowanych z aspiratów tchawicznych, pobranych od 19 chorych, z potwierdzonym mikrobiologicznie szpitalnym zapaleniem pluc, hospitalizowanych w Oddziale Anestezjologii i Intensywnej Terapii Wojewódzkiego Szpitala Specjalistycznego im. L. Rydygiera w Krakowie. Izolaty genotypowano z wykorzystaniem metody RAPD-PCR, przy uyciu primerów 208, 272, ERIC-2 oraz PAL-2. Obserwowano zrónicowanie w podobiestwie genetycznym analizowanych populacji szczepów niefermentujcych paleczek Gram-ujemnych.

A. Budak, P. Paluchowska, D. Wlodarcz yk, P. Nowak, M. Wgrz yn, W. Mudyna

THE GENETIC DESCRIPTION OF ACINETOBACTER BAUMANNII AND PSEUDOMONAS AERUGINOSA STRAINS ISOLATED FROM PATIENTS WITH HOSPITAL ACQUIRED PNEUMONIA HOSPITALIZED IN INTENSIVE CARE UNIT SUMMARY The aim of study was to recognize the epidemiology of hospital acquired pneumonia on the base of genotypes of Acinetobacer baumannii and Pseudomonas aeruginosa strains. Gram negative- non fermenting bacilli are the most frequent aetiologic agent of hospital acquired pneumonia among patients from Anesthesiology and Intensive Care Unit in the Rydygiers hospital in Cracow . In the following research RAPDPCR method was applied and there 272, 208, ERIC- 2 and PAL- 2 primers were used. The investigation was conducted among 33 Acinetobacter baumannii and 31 Pseudomonas aeruginosa strains which were isolated from endotracheal aspirates (ETA). ETA were taken from 19 patients with microbiologically confirmed pneumonia. The result of our study shows that most A. baumannii strains came from hospital habitat. In the investigated group of A. baumannii strains, 31 belonged to 3 clonal groups and probably came from two bacteria subpopulations which were present in ICU from a long time. There were only two isolates which had got the posthospital origin. Moreover, the assay of genetic similarity between A. baumannii strains, isolated from individual patients, showed that only in two patients were observed strains with different genetic profiles. Isolates which came from 8 patients had got the same pattern of bands. There were many genetic differences between investigated P. aeruginosa strains. In the group of 31 isolates, which were investigated by using 208, 272, ERIC- 2 and PAL- 2 primers, recognized respectively 10, 10, 13 and 8 of genetic profiles. All isolates of P. aeruginosa probably came from a few subpopulations of hospital strain which has undergone evolutionary divergence phenomenon in time as a result of changing condition of hospital environment and application of antibiotics. The assay of genotype of P. aeruginosa strains which were repeatedly isolated from individual patients showed that only from two patients strains with 100% genetic homology were isolated.

Dorota Wultaska1, Piotr Obuch-Woszczatyski1, Aleksandra Banaszkiewicz2, Andrzej Radzikowski2, Hanna Pituch1, Grayna Mlynarczyk1.

WYSTPOWANIE CLOSTRIDIUM DIFFICILE W PRZEWODZIE POKARMOWYM HOSPITALIZOWANYCH DZIECI DO DRUGIEGO ROKU YCIA

1Katedra i Zaklad Mikrobiologii Lekarskiej WUM w Warszawie Kierownik: prof. dr hab. G. Mlynarczyk 2Klinika Gastroenterologii i ywienia Dzieci WUM w Warszawie Kierownik: prof. dr hab. A. Radzikowski Badano wystpowanie C. difficile w przewodzie pokarmowym dzieci do drugiego roku ycia oraz porównano fenotypowe i genotypowe wlaciwoci wyhodowanych szczepów. Sto siedemdziesit osiem próbek kalu pobranych od dzieci w wieku od 2 miesicy do 2 lat, hospitalizowanych w latach 2003-2006, zbadano na obecno toksyn A/B C. difficile. Toksynotwórczo szczepów potwierdzono metod PCR. Lekowraliwo szczepów oznaczono stosujc metod E-test. Odsetek zakaonych dzieci laseczk C. difficile wyniósl 68,6%. Szczepy o profilu toksynotwórczoci A+B+CDT- stanowily 35%, o profilu toksynotwórczoci A-B+CDT- 10%, natomiast 5% stanowily szczepy A+B+CDT+. 50% wyhodowanych szczepów bylo nietoksynotwórczych. Odsetek szczepów opornych na erytromycyn i klindamycyn wyniósl odpowiednio 52% i 42%. Oporno na cyprofloksacyn byla powszechna i dotyczyla 98% badanych szczepów, za na moksyfloksacyn i gatyfloksacyn wyniosla 8%. Odsetek szczepów opornych na imipenem wyniósl 50%. Wszystkie badane szczepy byly wraliwe na metronidazol i wankomycyn.

D. Wultaska, P. Obuch-Woszczatyski, A. Banaszkiewicz, A. Radzikowski, H. Pituch, G. Mlynarcz yk

PREVALENCE OF CLOSTRIDIUM DIFFICILE IN THE GASTROINTESTINAL TRACT OF HOSPITALIZED CHILDREN UNDER TWO YEARS OF AGE SUMMARY The aim of this study was to determine prevalence of C. difficile in the gastrointestinal tract of hospitalized children under two years of age and a comparison of phenotypic and genotypic features. Hundred and seventy-eight samples collected from the faecal samples of children aged 2 months to 2 years, hospitalized in 2003-2006 were examined for the presence of toxin A/B of C. difficile. Toxigenicity of strains was confirmed using PCR. Susceptibility to antimicrobials was determined using E-test. The percentage of children infected with C. difficile was 68.6%. Toxigenic of C. difficile strains A+B+CDT- accounted for 35%, A-B+CDT- 10%, and 5% were strains of A+B+CDT +. 50% of the cultivated strains were non-toxigenic. The percentage of strains resistant to erythromycin and clindamycin was respectively 52% and 42%. Resistance to ciprofloxacin was widespread concern 98% of strains, and the moxifloxacin and gatifloxacin was 8%. The percentage of resistant strains to imipenem was 50%. All tested strains were susceptible to metronidazole and vancomycin.

Anna Midak-Siewirska1, Karolina Karabin2, Emilia Chudzik2, Tomasz Dziecitkowski1, Maciej Przybylski1, Anna Majewska1, Miroslaw Luczak1, Grayna Mlynarczyk1

ZASTOSOWANIE TECHNIKI REAL-TIME PCR DO WYKRYWANIA DNA LUDZKIEGO WIRUSA OPRYSZCZKI TYPU 1

1Katedra i Zaklad Mikrobiologii Lekarskiej, Warszawski Uniwersytet Medyczny Kierownik: prof. dr hab. G. Mlynarczyk 2Wydzial Rolnictwa i Biologii, Szkola Glówna Gospodarstwa Wiejskiego Kierownik: dr hab. G. Garbaczewska Celem pracy bylo zaprojektowanie i zbadanie uytecznoci metody real-time PCR do diagnostyki zakae ludzkim wirusem opryszczki typu 1, zwlaszcza u pacjentów poddanych immunosupresji oraz z zakaeniami orodkowego ukladu nerwowego.

A . Mi d a k-S i e w i rs ka , K. K a ra b i n , E.C hu d z i k, T. D z i e c i t ko w s ki , M. P rz yb yl s ki , A . Ma j e w s ka , M. Lu c z a k, G . Ml yn a rc z yk

APPLICATION OF REAL-TIME PCR ASSAY FOR INVESTIGATING THE PRESENCE OF HERPES SIMPLEX VIRUS TYPE 1 DNA SUMMARY Herpes simplex virus type 1 is a member of the Alphaherpesviridae subfamily, as it can infect both skin and nerves and develop latent infection within the dorsal root and trigeminal ganglia. Infection with this virus is common and causes a wide range of clinical syndromes. Although HSV-1 infect healthy children and adults, disease is more severe and extensive in the immunocompromised individuals. The goal of the study was development of real-time PCR assay for detection of herpes simplex virus type 1 DNA in clinical samples, using primers targeting a conserved region of the viral DNA glycoproteine G gene and a specific TaqMan hydrolyzing probe. The analytical sensitivity of assay was tested using serial dilutions of HSV-1 DNA in range between 100 and 10-6 (4,35x105 - 4,00x101 copies/ml). Fifteen cell line isolates and twenty plasma samples taken from a group of adult recipients of allogeneic HSCT were tested for the presence of HSV-1 DNA in the LightCycler® system. For comparison commercial quantitative HSV-1/2 LC PCR Kit (Artus/Qiagen) was used, according to the manufacturer's instructions. Both LightCycler® assays, including in-house real-time PCR, detected HSV-1 DNA in 23 specimens. The conclusion is that developed TaqManbased probe real-time PCR test is very reliable and valuable for detection of HSV-1 viremia in different kind of samples. The high level of sensitivity and accuracy provided by the LightCycler® instrument is favorable for the use of this method in the detection of herpes simplex virus 1 DNA also in clinical specimens.

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zestawienie prac za numer 1.2010

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