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Salmonella:

Pathogenesis, Epidemiology, and Vaccine Development

TABLE OF CONTENTS

General Information Map Scientific Program Abstracts for Oral Presentations Abstracts for Poster Sessions Index

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GENERAL INFORMATION GENERAL INFORMATION SCIENTIFIC PROGRAM ORGANIZERS Salvatore Rubino, M.D. University of Sassari, Italy Andreas Baumler, Ph.D. Texas A&M University, USA LOCAL HOST COMMITTEE Guido Leori, D.V.M. Elena Muresu, Ph.D. Sergio Uzzau, M.D. IN COOPERATION WITH The University of Sassari, Center for Biotechnology and Biodiversity Research and Department of Biomedical Sciences ACKNOWLEDGMENTS

GENERAL

The American Society for Microbiology would like to acknowledge Azko Nobel Banco di Sardegna Banca di Sassari Camera di Commercio di Sassari Comune di Alghero Consgilio Regionale della Sardegna ERSU ESIT Fondazione Banco di Sardegna Lohmann Animal Health GmbH & Co. KG Merial Select Pilgrim's Pride Regione Autonoma della Sardegna Sardaleasing Statens Serum Institut United Egg Producers US Department of Agriculture (USDA) for their generous contributions to this meeting.

Additional thanks to Mrs. Iolanda La Gatta Professor Bruno Masala Dominico Delogu Valentina Camboni and Across Sardinia for their excellent assistance.

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ASM Conference on Salmonella

GENERAL INFORMATION GENERAL INFORMATION REGISTRATION During the scientific sessions, the conference registration desk will be located in the area of the auditorium of the Porte Conte Ricerche. GENERAL SESSIONS

All general (oral presentations) sessions will be held in the auditorium. A name badge is reL INFORMATION for entry into all sessions. In consideration of other participants, no children are perquired mitted in the sessions. POSTER PRESENTATIONS Poster boards are located in the main building of the Porto Conte Ricerche. Presenters should mount posters the morning of their assigned poster session (A, B, or C noted next to your poster in the program) and should remove them immediately following the session. Please check your assigned number in the abstract index and mount your poster on the board space bearing that number. Official poster sessions will be held Sunday through Tuesday, as noted within the program. Please present your poster during the session noted next to your poster number in the program. CONFERENCE MEALS Breakfasts are provided at the hotel in which each participant is staying. Lunches for conference participants are offered in the dining room at the Porto Conte Ricerche. SOCIAL PROGRAM Saturday 20 th 2003: Sunday 21st 2003 : Monday 22 nd 2003: Tuesday 23 rd 2003: 6:30 pm Opening of the Congress 8 pm Welcome Sardinia party at Porto Conte Ricerche Visit to Alghero and free evening (dinner on own) Agritourism ­ Typical Sardinian dinner at the Porticciolo Gala Dinner ­ At the "Casale 117"

TRANSPORTATION Shuttle buses will be provided between official conference hotels and the Porto Conte Richere, as well as to official evening functions. STUDENT TRAVEL GRANTS ASM encourages the participation of graduate students and new postdocs at ASM Conferences. To support the cost of attending the conference, ASM has awarded travel grants of $500 to each of the following:

Anne Beeston Maria Braoudaki Geovana Brenner Michael Susan Bueno Kaman Chan Angela Cordone Sigrid De Keersmaecker Caleb Dorsey Helen Garmory Monica Giacomodonato John Geddes Deanna Gibson Daniela Gregorova Mellisa Hamilton Andrea Haraga Andrea Humphries A. Maryke Keestra Arlene Kelly Charles Kim Wook Kim Paivi Kyllonen Abigail Lazzerine Sebastien Lemire Vanessa Lopes Jitka Matiasovicova Erin Mitchell Marsibil Morgensen Chloe Mortimer Gerald Murray Ana I. Prieto Marquez Anna Reinicke Gary Rowley Kathleen Sonck Akamol Suvarnapunya Connie Kwai Ping Tam Kwai Lin Thong Eric Weening

Pathogenesis, Epidemiology, and Vaccine Development

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MAP OF MEETING SPACE MAP

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ASM Conference on Salmonella

SCIENTIFIC PROGRAM SCIENTIFIC PROGRAM SATURDAY, SEPTEMBER 20

6:30 pm Welcome Address

Chairs: Andreas Baumler, USA, and Salvatore Rubino, Italy

Dignitaries from the following organizations will provide welcoming remarks:

The Town of Alghero The Ministry of General Affair of the Sardinian Regional Government The Italian Society for Microbiology The University of Sassari

7:30 pm 8:30 pm 8:45 pm

Keynote Lecture: Salmonella Enterica Serovar Typhi

Gordon Dougan, England

Sardinian Science Award Welcome Reception

SUNDAY, SEPTEMBER 21

Session 1: Molecular Epidemiology

Chair: John Threlfall, England

9:00 am

John Threlfall, United Kingdom

9:30 am

WHO Initiatives on Global Foodborne Salmonellosis Surveillance

Awa Aidara-Kane, Switzerland

10:00 am

Surveillance Of Salmonellosis In Developing Countries: A Model Of Cooperation

Salvatore Rubino, Italy

10:30 am 10:45 am 11:15 am

Coffee Break Plasmids of S. Typhi

John Wain, England

Amoxycassia And Other Plant Derived Antimicrobial Substances For The Treatment Of Multi-drug Resistant Salmonella Infections in Karachi- Pakistan

Shahana Kazmi, Pakistan

11:30 am

Molecular Properties of Salmonella enterica Serotype Paratyphi B Distinguish Between its Systemic vs. Enteric Pathovars

Rita Prager, Germany

Pathogenesis, Epidemiology, and Vaccine Development

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SCIENTIFIC PROGRAM SCIENTIFIC PROGRAM

11:45 am Acquisition and Transfer of Resistance to ExtendedSpectrum Cephalosporins to Salmonella in the Intestinal Tract

Cornelius Poppe, Canada

12-1 pm 1 - 2 pm

Lunch Poster Session A Session 2: Genomics

Chair: Brett Finlay, Canada

2:00 pm 2:30 pm 3:00 pm 3:30 pm 3:45 pm

Genomics and the Host Cell Response to Salmonella

Brett Finlay, Canada

Evolutionary Genomics of the Salmonella

Michael McClelland, USA

Comparative Sequencing of Salmonella Strains

Mark Roberts, Scotland

Coffee Break

Robert Edwards, USA

4:15 pm

SlyA Regulates SPI2 Expression in Salmonella enterica serovar Typhimurium

Steve Libby, USA

4:30 pm

Use of a Salmonella enterica sv. Typhimurium DNA Microarray to Identify Transposon Mutants Compromised in Their Ability to Survive in vitro and in vivo

Kaman Chan, USA

4:45 pm

Intracellular Salmonella enterica Has Distinct Gene Expression Profiles in Macrophages and Epithelial Cells

Jay C. Hinton, United Kingdom

5pm-6pm 8pm-11pm

Poster Session B Visit to Alghero, a beautiful coast town in Sardinia.

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ASM Conference on Salmonella

SCIENTIFIC PROGRAM SCIENTIFIC PROGRAM MONDAY, SEPTEMBER 22

Session 3: Pathogenesis 1

Chair: Eduardo Groisman, USA

9:00 am

Eduardo Groisman, USA

9:30 am

A Bacterial Protein Adhesin Mimics a Host Polyscharide to Bind Extracellular Matrix Receptors

Robert Kingsley, USA

10:00 am

The Interaction of Salmonellae with Bile and Gallstone Surfaces

John Gunn, USA

10:30 am 10:45 am

Coffee Break The SpvB Protein Delivers a Fatal Blow to the Host Cell Cytoskeleton

Don Guiney, USA

11:15 am

virK, somA, and rcsC are Important for Systemic Salmonella Enterica Serovar Typhimurium Infection and Cationic Peptide Resistance

Corrie Detweiler, USA

11:30 am

Salmonella Rapidly Kill Dendritic Cells Via a Caspase-1-Dependent Mechanism

Ando van der Velden, USA

11:45 am

Cellular Immune Responses During Salmonella Infection

Brad Cookson, USA

12-1pm 1pm-2pm

Lunch Poster Session C Session 4: Pathogenesis 2

Chair: Ferric Fang, USA

2:00 pm 2:30 pm

Mice, Metals & Macrophages

Ferric Fang, USA

Salmonella-Host Cell Interactions in the Fibroblast Infection Model

Francisco Garcia-del Portillo, Spain

3:00 pm

The Molecular Pathogenesis of Salmonella typhimurium-Induced Enterids

Beth McCormick, USA

3:30 pm

Coffee Break

Pathogenesis, Epidemiology, and Vaccine Development

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SCIENTIFIC PROGRAM SCIENTIFIC PROGRAM

3:45 pm Intracellular Activities of the SPI-2 Type III Secretion System

David Holden, England

4:15 pm

Down Regulation of PMCA Induced By Salmonella enterica Typhimurium In Intestinal Epithelial Cells Increases Cytosolic Levels of Calcium

Garry Adams, USA

4:30 pm

Coupling Gene Expression to Flagellar Assembly in Salmonella

Kelly Hughes, USA

4:45 pm

Regulation of Conjugal Transfer in the Salmonella Virulence Plasmid: Roles of Dam methylation and the Leucine-Responsive Regulatory Protein

Josep Casadesus, Spain

5pm-6pm 8pm

Poster Session Dinner

TUESDAY, SEPTEMBER 23

Session 5: Host Adaptation

Chair: Tim Wallis, England

9:00 am

New Insights into the Molecular Basis of SalmonellaSerotype Host Specificity

Tim Wallis, England

9:30 am 10:00 am 10:30 am 10:45 am 11:15 am

How Does the Host Contribute to Host Adaptation?

Andreas Baumler, USA

Does Genetic Load Determine Host-Specificity?

Stanley Maloy, USA

Coffee Break Adaptation to the Alimentary Tract in Salmonella

Paul Barrow, England

Early Immune Responses In Newborn Chickens Infected With Salmonella Typhimurium

Gamini Withanage, United Kingdom

11:30 am

Assessment of the Intracellular Activities of Salmonella Effector Protein SopA

Abigail Blakely, United Kingdom

11:45 am

Biology of Salmonella Enterica Serovar Gallinarum Host-Specificity in the Avian Host

John Olsen, Denmark

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ASM Conference on Salmonella

SCIENTIFIC PROGRAM

12-1pm 1pm-2pm

Lunch Poster Session Session 6: Bacteriophages

Chair: Lionello Bossi, France

2:00 pm 2:30 pm

Why Lysogeny Prevails...and Spreads

Lionello Bossi, France

Contribution of a Lambdoid Prophage and an Intergeneric IS Element to the Virulence of Salmonella Enterica Serovar Abortusovis

Sergio Uzzau, Italy

3:00 pm

Phage Mediated Transfer of the SPI-7 Effector Protein Gene SopE

Wolf-Dietrich Hardt, Switzerland

3:30 pm 3:45 pm

Coffee Break A Genetic Process that Speeds Adaptation Without Affecting Mutation Rate: Effects of Growth Under Strong Selection

John Roth, USA

4:15 pm 4:30 pm

Salmonella Hijacks Phagocytes

Micah Worley, USA

A Salmonella Enterica Serovar Typhimurium Translocated Leucine-rich Repeat Effector Protein Inhibits NF-êB-Dependent Gene Expression

Andrea Haraga, USA

4:45 pm

Retrospective Analysis of Salmonella Enterica Serovar Enteritidis by the New Phage Typing Scheme (Ward) in Eastern Germany Before 1989 Reveals a Heterogeneity of Clones in Laying Flocks

Wolfgang Rabsch, Germany

5pm-6pm 8pm

Poster Session Banquet

Pathogenesis, Epidemiology, and Vaccine Development

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SCIENTIFIC PROGRAM WEDNESDAY, SEPTEMBER 24TH, 2003

Session 7: Vaccine Development

Chair: Roy Curtiss III, USA

9:00 am

Recombinant Attenuated Salmonella Vaccines for Induction of Cross-Protective Immunity and Antigen Delivery

Roy Curtiss III, USA

9:30 am

Attenuated Salmonella Spp. as Carriers for Protein and DNA-Based Vaccines

Carlos Guzman, Germany

10:00 am 10:30 am 10:45 am

Salmonella Typhi Live Vector Vaccines

Marcella Pasetti, USA

Coffee Break The Use of Salmonella's TTSS for Vaccine Development

Holger Russmann, Germany

11:15 am

Vaccination of Chickens Against S. Enteritidis with the S. Gallinarum 9R Strain

Maarten Witvliet, Netherlands

11:30 am

Protection Against Murine Listeriosis by Oral Vaccination with Recombinant Salmonella Expressing Protective Listerial Epitopes Within a Surface-Exposed Loop of the TolC Protein

Simone Spreng, Switzerland

11:45 am 12-1pm

Live Vaccines for Salmonella Control in Poultry

Heike Scharr, Germany

Lunch

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ASM Conference on Salmonella

SPEAKER ABSTRACTS

(in alphabetical order)

WHO INITIATIVES FACING THE GLOBAL THREAT OF FOODBORNE SALMONELLOSIS

A. Aidara-Kane, Strategy Development of Monitoring of Zoonoses, Foodborne Diseases and Kinetoplastidae/ Control, Prevention, Eradication/ Communicable Diseases/World Health Organization , Geneva, Switzerland Salmonella is a leading cause of foodborne diseases worldwide. The human illness burden of salmonellosis is particularly high in developing countries where many causes of salmonellosis are painfully obvious : ubiquitous contamination of the environment by human and animal waste, poor water supplies, unhygienic food handling practices, unadequate cooking and other processing steps, etc...More than one million people are traversing international bounderies daily, and with the globalisation of food production manufacturing and marketing, the risk of importing and exporting Salmonella strains is substancial. Salmonella Enteritidis appeared simultaneously around the world in the 1980s and has since undergone a 20-fold increase in Europe and North America. Infections with this bacteria are often associated with contaminated poultry and eggs. The continuing emergence of Salmonella strains that are resistant to antimicrobials is also a cause of increasing concern. Following the discovery that antibiotics promote growth as well as prevent diseases in farm animals, the use of antibiotics in farms increased considerably. Continuous and often low-level dosing of antibiotics provides ideal conditions for the development of drug-resistant strains of Salmonella normally present in the intestinal flora of amimals. In many cases multiresistant bacteria infecting humans have been directly linked to resistant organisms in animals. Salmonella Typhimurium DT104, which is resistant to five commonly prescribed antibiotics has recently spread throughout many countries., in the USA, multidrug-resistant strains of Salmonella Newport have increased by more than 30% in less than a decade, in 1999 a new multiresistant Salmonella serotype, Salmonella Keurmassar, has emerged simultaneously in poultry and human in Senegal. The consequences for human medicine include increased incidence of human infections caused by resistant pathogens and more frequently therapeutic failure. To reduce the public Health burden of foodborne salmonellosis requires an effective and comprehensive public Health surveillance system .The overall gaol of WHO programme on Surveillance of foodborne diseases is to provide countries with the necessary data to reduce the foodborne disease burden by providing information which allows the food safety system to be improved. In order to address these issues, the WHO programme on Surveillance of foodborne Diseases has initiated the following activities: · -Global Salmonella Surveillance The Global Salmonella Surveillance (GSS) network is coordinated by the World Health Organization (WHO)and is a partnership between the Danish Veterinary Institute in Denmark (DVI) , the Centres for Disease Control and Prevention in Atlanta (CDC), Institut Pasteur in Paris and Health Canada. Global Salm Surv was conceived as a strategy for improving global surveillance of Salmonella while also Pathogenesis, Epidemiology, and Vaccine Development

strengthening laboratory capacity, particularly for monitoring of drug resistance in developing countries. Its main activities are developing and maintaining a website, a listserve and a country databank of Salmonella serotypes, managing a laboratory quality assurance program and developing and organizing regional training courses. · -WHO Network of Networks on foodborne Diseases surveillance To facilitate communication, sharing intelligence, experience and surveillance approaches between existing surveillance networks, to strengthen surveillance in networks, regions and countries, especially developing countries and to facilitate sharing of appropriate urgent information (Early Warning System). · -WHO sentinel sites programme As a part of the broader objective of developing a global strategy to reduce the burden of salmonellosis and other foodborne diseases, WHO has considered establishing sentinel sites to measure the burden of illness in order to provide important information for more accurate selection of strategies for diseases control.

DOWN REGULATION OF PMCA INDUCED BY SALMONELLA ENTERICA TYPHIMURIUM IN INTESTINAL EPITHELIAL CELLS INCREASES CYTOSOLIC LEVELS OF CALCIUM

J. Figueiredo, R. B. Mouneimne, S. Khare, S. Zhang, R. C. Burghardt, A. J. Bäumler, L. G. Adams; Texas A&M University, College Station, TX. Salmonella enterica Typhimurium causes severe enterocolitis and fatal diarrhea in calves, which is characterized by an acute profound infiltration of polymorphonuclear neutophilic leukocytes (PMN) in the intestinal mucosa. Down regulation of plasma membrane calcium transporting ATPase (PMCA) gene has been previously demonstrated by differential display to be associated with S. typhimurium infection in calves. Our hypothesis is that down regulation of PMCA in intestinal epithelial cells results in increased cytosolic levels of calcium essential for IL-8 expression and PMN chemotaxis. Caco-2 cells were infected in vitro with S. typhimurium strain IR715 (MOI 1:100), and the level of PMCA expression was evaluated by Real Time PCR (RTPCR) at 1, 2.5, and 5 hours post infection. Infected Caco-2 cells had a 29.69% decrease of PMCA expression at 2.5 hours post infection when compared to the uninfected control cells. At the same time points, we assessed the cytosolic concentration of calcium by confocal microscopy. We detected a statistically significant increase of 30.84% in cytosolic calcium in infected Caco-2 cells at 2.5 hours post infection as compared to the uninfected controls. These findings indicate that down regulation of PMCA may result in an increase in cytosolic levels of calcium in Salmonella infected intestinal epithelial cells. Using RTPCR, we evaluated the in vivo expression of PMCA in tissues from bovine ligated ileal loops inoculated with S. typhimurium at 30 min, 1, 2, 4 and 8 hours post infection. At 2 hours post infection, we measured a 30% down regulation of PMCA expression in Salmonella infected bovine tissues when compared to the uninfected control. In summary, our in 13

SPEAKER ABSTRACTS

vitro and in vivo observations suggest that Salmonella typhimurium induced down regulation of PMCA in intestinal epithelium results in increased cytosolic calcium which may be one of the factors responsible for enhanced IL-8 expression, chemotaxis of PMNs, degranulation and subsequent mucosal injury, leading to clinical diarrhea. type III secretion system (TTSS-1). However, a TTSS-1 dependent induction of CXC chemokine gene expression by epithelial cells was only elicited in the bovine mucosa. These data suggest that different disease outcomes between mice and calves may be partly due to differences in how the S. Typhimurium sipA, sopA, sopB, sopD, and sopE2 gene products affect CXC chemokine gene expression in epithelial cells.

ADAPTATION TO THE ALIMENTARY TRACT IN SALMONELLA

P. A. Barrow, UNITED KINGDOM From the point of view of infection biology Salmonella can be divided into two groups, (1) a small group of serotypes that characteristically produce typhoid in a host-specific manner and colonise the gut poorly (2) the remaining serotypes which colonise the gut of many animal species. Members of both groups apparently have the capacity to induce gastro-enteritis in mammalian species although the extent to which they do this is unclear. Amongst the typhoid serotypes the basis of host specificity is unclear although it is likely that the major effect is expressed during infection of mononuclear cells. The molecular basis is unknown. It seems likely that a common mechanism of this group is the avoidance of induction of pro-inflammatory cytokine production during infection of intestinal epithelial cells and the down regulation of other inflammatory responses in mononuclear cells mediated by TTSS-related mechanisms. The basis of intestinal colonization is also poorly understood. The nature of the interaction with the mucosa is unclear and, although there is little extensive attachment to the mucosa, a number of mutational screens have identified involvement of fimbria and TTSS. Colonization of the chicken intestine by S. Typhimurium involves responses to stresses, including heat shock and amino acid starvation. Metabolism is largely anaerobic and genome sequence analysis can pin-point key colonization determinants, such as cobalamin biosynthesis, which gives some indication of important pathways of energy generation.

ASSESSMENT OF THE INTRACELLULAR ACTIVITIES OF SALMONELLA EFFECTOR PROTEIN SOPA

A. N. Blakey1, C. D. Rapier 1, P. J. Brown 1, R. W. Carter2, E. E. Galyov1; 1Institute for Animal Health, Compton, UNITED KINGDOM, 2Edward Jenner Institute for Vaccine Research, Compton, UNITED KINGDOM. Salmonella secretes and translocates a range of proteins, known as effectors, into host cells via the use of two type III secretion systems. These effector molecules elicit a variety of responses inside the host cell including cytoskeletal rearrangements and induction of proinflammatory cytokines. The responses aid the uptake of Salmonella into host cells and allow it to harness the host cell machinery for its own benefit and for the induction of enteropathogenesis. As part of the on-going work in our group on the molecular and functional characterisation of Salmonella effector molecules, we created human embryonic kidney cells (HEK-293) which stably expressed individual Salmonella outer protein (Sop) effectors under the regulation of a tetracycline inducible promoter. These cells were used in a variety of assays to elucidate the function of the Sop effectors. Flow cytometric analysis of surface marker expression on the cells showed that ICAM-1, a cell adhesion molecule, was up-regulated on cells expressing SopA. In addition human monocytes demonstrated increased adherence to the SopA expressing cells compared to untransfected cells. Thus it appeared that SopA acts to increase expression of ICAM-1 and possibly other receptors on host cells, which allows stronger binding of monocytes during the inflammatory response. To confirm the data in an infection situation, an in vitro infection model was used. Semi-confluent T84 cell monolayers were infected with wild-type Salmonella Dublin or a Salmonella Dublin sopA mutant and ICAM-1 expression was analysed by flow cytometry. ICAM-1 was up-regulated on cells infected with wild-type Salmonella compared to uninfected cells agreeing with data in the literature. Surprisingly cells infected with the sopA mutant strain also showed increased expression of ICAM-1 and there was no significant difference statistically between the mutant and wild-type strains. Thus this data indicates that in an infection situation, factors in addition to SopA contribute to the up-regulation of ICAM-1 on infected host cells. Alternatively in polarised cells SopA may play an initial role followed by other factors later in the infection. The cells in this infection study were not polarised, so this may provide a feasible explanation for the data. Work is continuing in this area to identify the other factors that may be involved in up-regulating ICAM-1 expression on Salmonella infected cells.

HOW DOES THE HOST CONTRIBUTE TO HOST ADAPTATION?

A. J. Bäumler; Texas A&M University SHSC, College Station, TX. Salmonella enterica serotype Typhimurium causes a systemic infection without diarrhea in mice while infection of calves results in a localized infection characterized by diarrhea. To understand how host genetic differences lead to different disease outcomes, we have performed a comparative analysis of S. Typhimurium infection mice and calves. Perhaps the most striking difference between host responses of mice and calves was the type of infiltrate elicited in the intestine. S. Typhimurium infections of mice were characterized by a slowly developing infiltrate composed predominantly of mononuclear inflammatory cells. In contrast, infection in calves resulted in a rapidly developing infiltrate containing neutrophils as the predominant cell type. Both responses were dependent on the presence of effector proteins (SipA, SopA, SopB, SopD, and SopE2) of the invasion-associated

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ASM Conference on Salmonella

SPEAKER ABSTRACTS

WHY LYSOGENY PREVAILS...AND SPREADS

L. Bossi; CNRS, Gif-sur-Yvette, FRANCE. Most Salmonella enterica strains contain multiple prophages integrated in their genomes. This prophage complement accounts for most of the genomic diversity between members of a given serogroup. Some prophages express functions that improve Salmonella interactions with host cells. These loci may contribute to retention of phage DNA in the bacterial chromosome in spite of undoubted pressure to economize. Still, the forces responsible for the conservation of other portions of prophage genomes, such as those connected with the viral lifestyle (e.g., replication and morphogenesis genes) have remained elusive. Our recent work reveals that selective value of lysogeny becomes apparent under mixed culture conditions. Co-cultures of Salmonella strains carrying or lacking specific prophages evolve into an all-lysogen population as a result of phagemediated killing of sensitive bacteria and lysogenization of survivors. Apparently, spontaneous prophage induction in a few lysogenic cells ignites massive viral replication at the expenses of the non-immune population. With strains from separate lineages (i.e., carrying distinct prophage repertoires) the co-culture conditions lead to antagonistic viral bursts resulting in the strain with the more vigorous prophage arsenal outgrowing the other. Therefore, the prophage complement of a strain can boost its fitness in a competition. Spontaneous phage release during unchallenged bacterial growth sets a selection regime that forces maintenance and spread of the lysogenic condition in bacterial genomes. This could be critical for how bacterial populations evolve in their natural environment and might contribute to the evolutionary success of certain phylogenic lineages.

the regulatory gene traJ. Lrp activates traJ transcription from an upstream-activating sequence (UAS) that contains an UP element and two sites related to the consensus Lrp binding site (LRP-1 and LRP-2). The LRP-1 site, which is bound by Lrp with high affinity, is absolutely needed for traJ promoter activity. The LRP-2 site overlaps with the UP element, and both share a GATC site. The UP element is more active if unmethylated. Altogether, these data support a model in which Lrp plays a dual role in transcriptional activation of traJ: (i) Lrp switches on the traJ promoter by binding the LRP-1 site; (ii) Lrp activates the UP element by protecting the GATC site of LRP-2 from Dam methylase.

USE OF A SALMONELLA ENTERICA SV. TYPHIMURIUM DNA MICROARRAY TO IDENTIFY TRANSPOSON MUTANTS COMPROMISED IN THEIR ABILITY TO SURVIVE IN VITRO AND IN VIVO

K. Chan, S. Falkow; Stanford University, Stanford, CA. The development of spotted DNA microarrays has provided an opportunity to combine the principles of signaturetagged mutagenesis (STM) with microarray technology in order to identify potentially important bacterial virulence genes.The scope of the DNA arrays provide for screening on a much larger scale than possible by STM alone. Moreover, the combined approach utilizes a transposon library without the need to synthesize individual signature molecules. We have adapted a transposon-based microarray technique, TraSH (transposon site hybridization), for use with a Typhimurium DNA microarray in order to identify genes that are important for survival and replication in RAW mouse macrophage cells or in the spleens of BALB/cJ mice. A 10X transposon library of Typhimurium strain SL1344 was opsonized for macrophage infection or used to intraperitoneally infect mice. Bacteria were either harvested from RAW macrophages and re-passaged in cells or harvested from mouse spleens 2-days post-infection. In each case, the genomic was DNA isolated and processed for analysis on the microarray. Of the genes that are absent in the output pools of each selection condition, a significant proportion are components of the SPI-2 pathogenicity island which is known to be critical for intracellular survival and replication and systemic disease in the mouse. Additional genes, including many putative ones, not previously known to have a virulence function have also been identified in the output pool and are currently under study.

REGULATION OF CONJUGAL TRANSFER IN THE SALMONELLA VIRULENCE PLASMID: ROLES OF DAM METHYLATION AND THE LEUCINE-RESPONSIVE REGULATORY PROTEIN

J. Casadesus, E. M. Camacho, A. Serna; Universidad de Sevilla, Seville, SPAIN. A genome-wide search for genes regulated by Dam methylation in Salmonella enterica serovar Typhimurium identified the transfer operon of the virulence plasmid as a Damrepressed locus (Torreblanca and Casadesus, Genetics 144: 15-26, 1996). Dam methylation represses tra operon expression by sustaining high levels of FinP RNA synthesis (Torreblanca et al., Genetics 151: 31-15, 1999). The effect of Dam methylation on finP gene transcription is indirect, and involves an hypothetical Dam-dependent regulator, still to be identified. However, genetic trials for this elusive regulator have been useful to find other regulators of tra operon expression. For instance, mutations that prevented tra operon derepression in a Dam- strain identified the leucineresponsive regulatory protein (Lrp) as an activator of conjugal plasmid transfer (Camacho and Casadesus, Mol. Microbiol. 44: 1589-1598, 2002). The target of Lrp control is Pathogenesis, Epidemiology, and Vaccine Development

CELLULAR IMMUNE RESPONSES DURING SALMONELLA INFECTION

B. T. Cookson, L. Cummings, M. Bergman, W. Wilkerson, S. Rassoulian-Barrett, I. Fellnerova; University of Washington, Seattle, WA. Infection with attenuated Salmonella provides long lasting protection against subsequent challenge with virulent strains for both mice and humans. To better understand the mechanisms by which T cells contribute to immunity, we 15

SPEAKER ABSTRACTS

characterized the diversity and regulation of antigens recognized by CD4 + T cells isolated from protectively immunized mice. We used a combination of mutant Salmonella strains, wild type bacteria grown in media simulating the phagosomal environment inside macrophages, as well as infected splenocyte APC directly ex vivo to thoroughly examine the antigens recognized by T cells from immune mice. Clonal analysis of T cell specificity reveals the host preferentially recognizes conserved pathogen surface organelles, with two important consequences: triggering of the initial innate response and directing Ags with particular attributes (association with Tlr ligands) for adaptive immune recognition. Because regulation of Ag expression is genetically coupled to the induction of bacterial resistance inside phagocytes, Salmonella has evolved coordinated mechanisms for escaping both innate and adaptive immune responses. Regulation of antigen expression, which we demonstrate can occur at both transcriptional and post-transcriptional levels and is subject to both temporal and spatial regulation during infection in vivo, effectively makes antigen significantly less bio-available for host surveillance. This can be overcome by inducing antigen expression from a heterologous promoter, refuting the notion that during systemic infection Salmonella resides in cellular niches apart from immune T cells. We present a model in which bacterial regulation of antigen expression during the priming of, and subsequent detection by, the host immune response is an important component of Salmonella pathogenesis. wall because of an inability to synthesize muramic acid and diaminopimelic acid. The strains possess additional mutations to uncouple any requirement of protein synthesis and growth from display of the cell wall-less death phenotype. Other mutations ensure complete biological containment to the vaccine constructs to ensure their ability to lyse completely in vivo to release recombinant protective antigens from diverse pathogens or DNA vaccine vectors encoding protective antigens to be synthesized within the immunized animal host.

VIRK, SOMA, AND RCSC ARE IMPORTANT FOR SYSTEMIC SALMONELLA ENTERICA SEROVAR TYPHIMURIUM INFECTION AND CATIONIC PEPTIDE RESISTANCE

C. S. Detweiler; University of Colorado, Boulder, CO. Salmonella must express and deploy a Type III secretion system located in Salmonella Pathogenicity Island 2 (SPI-2) in order to survive in host phagocytic vacuoles and to cause systemic infection in mouse models of typhoid fever. A genome-wide approach to screen for Salmonella genes that are transcriptionally co-regulated in vitro with SPI-2 genes was used to identify bacterial loci that might function in a mouse model of systemic disease. Strains with mutations in three SPI-2 co-expressed genes were constructed and tested for their ability to cause disease in mice. We found that virK, a homolog of a Shigella virulence determinant, and rcsC, a sensor kinase, are important at late stages of infection. A second Salmonella gene that has VirK homology, somA, is also important for systemic infection in mice. We have shown that expression of both virK and somA requires the transcription factor PhoP, whereas rcsC does not. Additionally, rcsC expression does not require the transcription factor OmpR, but expression of one of the known targets of RcsC, the yojN rcsB putative operon does require OmpR. virK, somA, and rcsC are expressed in tissue culture macrophages and confer Salmonella resistance to the cationic peptide polymyxin B. We conclude that virK, somA, and rcsC are important for late stages of Salmonella enteric fever, and that they likely contribute to the remodeling of the bacterial outer membrane in response to the host environment.

RECOMBINANT ATTENUATED SALMONELLA VACCINES FOR INDUCTION OF CROSS-PROTECTIVE IMMUNITY AND ANTIGEN DELIVERY

R. Curtiss III, X. Zhang, W. Kong, S.-Y. Wanda, W. Bollen; Washington University, St. Louis, MO We have designed strains of Salmonella typhimurium that optimize expression of wild-type attributes that facilitate successful colonization and invasion of the GALT and other lymphoid effector organs before undergoing changes that cause their attenuation. Modifications have also been included to minimize expression of serotype-specific antigens and cause antigens that display immunological relatedness among diverse serotypes of S. enterica and closely related E. coli pathovars to be over expressed. These strains possess a Dpmi mutation for a mannose dependent synthesis of O-antigen side chains that ceases to occur in vivo, a D(gmd-fcl) mutation to block conversion of GDP-Mannose to GDP-Fucose to block colanic acid synthesis, a DPfur::TTaraC PBAD fur deletion-insertion mutation that causes constitutive high-level expression of all gene products needed for iron acquisition, and DfliC and DfljB mutations to abolish synthesis of variable flagellar antigen epitopes. In other vaccine constructions designed for delivery of antigens or DNA vaccine vectors, we engineered strains to exhibit a regulated delayed cell lysis in vivo. By combination of chromosomal deletion-insertion mutations and vector construction gene sequences, we designed strains that are arabinose-dependent and after 5 to 10 generations of growth in vivo cease to synthesize the rigid layer of the bacterial cell

TYPHOID:HOW DOES THE GENOME SEQUENCE HELP STUDIES ON HOST/ PATHOGEN INTERACTIONS ?

G. Dougan; Imperial College London, UNITED KINGDOM. Typhoid is still a serious problem in many tropical areas of the world. Further, the emergence of multiple antibiotic resistance in the etiological agent, Salmonella enterica serovar Typhi (S. Typhi) is now complicating treatment of the disease. Despite these problems, the clinical disease in humans has not been extensively studied, in part because S. Typhi exhibits human host restriction and many scientists prefer to use the murine salmonellosis (S. Typhimurium) model for genetic and immunological investigations. Work ASM Conference on Salmonella

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on S. Typhi (and other human restricted pathogens) can potentially be facilitated by whole genome sequencing programme such as that recently completed at the Sanger Centre on a Vietnamese multi-drug resistant S. Typhi strain. Other DNA sequencing centres have also established important sequencing programmes on several S. enterica serovars, stimulating possible programmes on comparative genomics. We and others have investigated aspects of the epidemiology, immunology and human susceptibility to typhoid in a programme designed to look at multiple aspects of host pathogen interactions. Microarray studies can be used to define genome variation in S. Typhi and facilitate new approaches for defining the position of the serovar S. Typhi within S. enterica Work has also been initiated to define human typhoid susceptibility genes as a step towards exploiting the human genome. These studies provide a platform for future investigations on typhoid and other diseases. Opportunities may emerge for improved vaccine design and for the exploration of new therapeutic approaches. Details of some of these studies will be presented.

intravacuolar environment that is experienced by Salmonella. This approach promises to become a useful tool for unravelling the biology of infection by Salmonella and other bacteria.

GENOMICS AND THE HOST CELL RESPONSE TO SALMONELLA

B. Finlay; University of British Columbia, Vancouver, BC, CANADA. Salmonella encounters several host cells as it progresses through infections. These cells include epithelial, dendritic, macrophage, and neutrophils. With the advent of arrays, it is now possible to examine the entire transcriptional response to Salmonella of isolated host cells. The predominant reponse is that mediated by innate immunity, especially in response to LPS. However, other responses occur, and there is considerable interest in determining specific responses to bacterial virulence factors. In addition, following up on array results yields several interesting biological events, while posing even more questions. The host response to Salmonella will be discussed, as will some pathways that have been pursued following initial array results.

INTRACELLULAR SALMONELLA ENTERICA HAS DISTINCT GENE EXPRESSION PROFILES IN MACROPHAGES AND EPITHELIAL CELLS

J. C. Hinton1, S. Eriksson2, I. Hautefort1, S. Lucchini1, A. Thompson 1, M. Rhen 2; 1Institute of Food Research, Norwich, UNITED KINGDOM, 2MTC, Karolinska Institute, Stockholm, SWEDEN. The success of Salmonella enterica as a pathogen is partly explained by its ability to adapt to the intracellular environment of the phagocytic cell. However, we have understood little about this process of adaptation because of the difficulty in measuring bacterial gene expression during infection. In the past, reporter gene fusions were used to monitor changes in expression of certain genes during the transition from the extracellular to the intravacuolar environment, but until now it has not been possible to visualise gene expression at the RNA level. We have now developed a method for stabilising and purifying Salmonella RNA from infected mammalian cells, and have used DNA microarrays to determine the complete transcriptional profile of intracellular S. enterica sv. Typhimurium following infection of macrophage and other cell lines. During replication in murine macrophage-like J774-A.1 cells, 919 of 4451 S. Typhimurium genes showed significant changes in transcription. The expression profile identified alterations in numerous virulence genes, and revealed unexpected findings concerning the biology of the Salmonella/macrophage interaction. Almost half of the in vivo-regulated genes were of unknown function, suggesting that intracellular growth involves novel bacterial properties. Our latest experiments have revealed that Salmonella has a distinct expression profile during infection of human HeLa epithelial cells. Comparison with the data from macrophages showed differential expression of many Salmonella gene clusters including SPI1. We are now generating expression profiles for S. Typhimurium grown under different environmental conditions in vitro, and are making new deductions about the Pathogenesis, Epidemiology, and Vaccine Development

SALMONELLA-HOST CELL INTERACTIONS IN THE FIBROBLAST INFECTION MODEL

F. Garcia-del Portillo; Centro Nacional de Biotecnologia-CSIC, Madrid, SPAIN. A common trait of all Salmonella enterica infections is the residence of this pathogen within membrane-bound compartments (vacuoles) of the host cell. Studies performed in cultured eucaryotic cells and in vivo infection models have shown that the lifestyle of S. enterica in this specialized vacuole differs substantially depending on the host cell type. The in vitro infection models pointed out epithelial cells as prototype for permissiveness of intracellular bacterial growth. However, S. enterica intracellular proliferation in vivo occurs preferentially within macrophage cells. No evidence for bacterial growth within non-phagocytic cells has been found in vivo. We are pursuing the fibroblast infection model with the aim of dissecting the S. enterica lifestyle within vacuoles of non-phagocytic cells. S. enterica does not grow within fibroblast cells and remain within the vacuole in a latent state. Certain bacterial functions are responsible for this `intracellular growth attenuation' response, including the two-component system PhoP-PhoQ, other transcriptional regulators as SlyA, SvpR and RpoS, and a novel protein termed IgaA, for intracellular growth attenuator. Loss of function of any of these functions results in intracellular overgrowth phenotypes. Due to the novelty of the IgaA protein, we have been interested in its function. IgaA is a membrane protein that acts as a negative regulator of the RcsC-YojN-RcsB regulatory system. IgaA is also required for virulence solely due to its function as repressor of the RcsCYojN-RcsB system. Thus, IgaA is fully dispensable for virulence if any of the components of the RcsC-YojN-RcsB is inactivated. Other bacterial functions have been identified

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as playing a role in sustaining intracellular survival of the pathogen within fibroblasts. They include the sigma factor RpoE, and the Salmonella-pathogenicity island 2. SPI-2 effectors such as SseF, SseG are required for that intracellular survival phenotype whereas other such as SifA and SifB do not play any apparent role in maintaining bacterial survival. Infection of rat fibroblasts is not followed by Sif formation and sifA mutants remain enclosed in vacuoles, phenotypes that contrast to what has been reported in epithelial cells. Another striking difference found in fibroblasts is that bacterial entry can proceed in the absence of a functional SPI-1-encoded type III secretion system. Taken together, these observations highlight the diversity of strategies used by Salmonella to engage host cellular functions. What it is remarkable is that bacterial functions described in macrophage or epithelial cells as necessary for intracellular survival (PhoP-PhoQ) or alteration of vesicular trafficking (SifA), have either totally opposite roles (attenuators of growth as PhoP-PhoQ) or no apparent role (SifA) when S. enterica invades fibroblasts. cell cytoplasm may be independent of the SseB/SseC translocon components. These observations suggest a mechanism by which SpvB activity in the cytoplasm can be modulated independently of the delivery of the SPI2 effectors that are transported through the translocon structure. We propose that SpvB is transported across the phagosome membrane by the action of the N-terminal domain.

THE INTERACTION OF SALMONELLAE WITH BILE AND GALLSTONE SURFACES

J. S. Gunn; The Ohio State University, Columbus, OH. Bile, which is stored in the gallbladder, contains bile acids that act as detergents to solubilize lipids and are therefore potent antimicrobials. Salmonella spp. have been shown to be resistant to high concentrations of bile and can sense and respond to bile by altering mechanisms of resistance and virulence properties. Salmonellae can exist in an asymptomatic carrier state in the human gallbladder. Individuals with gallstones are more likely to become typhoid carriers, and antibiotic treatments are often ineffectual against S. typhi carriers with gallstones. Therefore, we hypothesize that Salmonella spp. form biofilms on the surface of gallstones, where the bacteria are protected from high concentrations of bile and antibiotics. We have utilized a number of methods to look at biofilm formation on human gallstones in vitro, including confocal microscopy and scanning electron microscopy. Salmonellae form a full biofilm on the surface of gallstones and excrete an exopolysaccharide that binds the bacteria to the surface and to each other. Efficient biofilm formation on gallstones is dependent upon the presence of bile, as a biofilm does not form on gallstones in Luria Broth alone. Numerous factors have been investigated for a role in gallstone biofilm formation, including fimbriae, flagella, LPS, RpoS, PhoPQ, and exopolysaccharide. In addition, we have examined the interaction of salmonellae with bile by 2-D gel electrophoresis and DNA microarray. Using these techniques, the Salmonella typhimurium marRAB and acrAB operons, flagellum-related genes, and invasion-related genes were identified as bile-regulated. Transcriptional and functional assays have confirmed this regulation. These data further demonstrate that bile is an important environmental signal sensed by Salmonella spp. that plays a role in regulating gene expression in multiple virulence-associated pathways. Furthermore, these studies will help gain a better understanding of the Salmonella carrier state, an important but under-researched area of typhoid fever pathogenesis. By understanding the basis of carrier development, it may be possible to identify effective strategies to prevent/treat this chronic infection.

THE SPVB PROTEIN DELIVERS A FATAL BLOW TO THE HOST CELL CYTOSKELETON

D. G. Guiney, S. Browne; UCSD School of Medicine, La Jolla, CA. Salmonella produces several effector proteins that induce cytoskeletal rearrangements during the course of intracellular infection. Effectors translocated by the SPI1 type three secretion system (TTSS) induce localized actin polymerization resulting in bacterial uptake. Actin condensation around the Salmonella-containing vacuole (SCV) has been described and depends on genes in SPI2. The Salmonella SpvB protein possesses ADP-ribosylating activity in the C-terminal domain that targets actin monomers in the host cell and prevents actin polymerization. The net effect of SpvB activity in the cytoplasm is profound loss of actin filaments. This cytopathology is seen in cells transfected with an SpvBexpressing vector and also in human macrophages and epithelial cells infected with spvB-containing Salmonella strains. These observations raise the issue of how SpvB translocation into the cytoplasm is regulated so as not to interfer prematurely with the actin polymerization required for invasion and formation of the SCV. In addition, human epithelial cells and macrophages infected with Salmonella undergo delayed apoptosis that is dependent on SpvB and is characterized by caspase 3 activation and DNA fragmentation. The SpvB effect on actin depolymerization also requires certain components of the SPI2 (TTSS), including the core proteins SsaJ and SsaV. In addition, the SpvB effect requires the SpiC protein. SpiC has been shown to be essential for the secretion of the putative translocon components SseB and SseC. SpiC itself also appears to have effector activity. However, sseB and sseC mutants still show residual SpvB-mediated actin depolymerization during macrophage infection. As anticipated, a mutation in the effector protein SifA has no effect on SpvB activity in the cytoplasm. These results suggest that SpvB is secreted by the core components of the SPI2 TTSS including SpiC. However, delivery of SpvB from the vacuole into the host 18

ASM Conference on Salmonella

SPEAKER ABSTRACTS

ATTENUATED SALMONELLA SPP. AS CARRIERS FOR PROTEIN- AND DNABASED VACCINES

C. A. Guzmán; GBF-German Research Centre for Biotechnology, Braunschweig , GERMANY. Most microbial infections are either restricted to the mucosal membranes or the etiologic agents need to transit through the mucosa. Thus, it is desirable to stimulate a local mucosal response to block both infection and disease development after vaccination. However, antigens are usually poorly immunogenic when administered by the mucosal route. The use of live attenuated bacterial carriers as antigen delivery system is a proven strategy to overcome this problem. Bacterial carriers can be also used as a delivery system for nucleic acid vaccines. This new approach adds to the multiple advantages of bacterial carriers and DNA vaccination, the possibility to specifically target antigen presenting cells, thereby promoting their activation and maturation by the delivery of a potent danger signal. Carrier-based vaccine prototypes stimulate efficient immune responses at both systemic and mucosal levels, which are able to protect not only against infections, but also against non-infectious diseases (e.g. cancer). However, the delivery of antigens to the immune system is not sufficient per se to engender a protective response following immunization. A successful vaccination requires the elicitation of an adequate type of immune response (e.g. antibodies versus cell-mediated immunity, Th1 versus Th2 cells). Thus, the development of a carrier-based vaccination strategy demands selection of the optimal host and expression strategy. This will ensure carrier fitness, promoting also optimal immune responses. Strains carrying mutations affecting the specific course of the infection can be exploited to modify the quality of the immune responses elicited. The topology and form of antigen display may also have a significant impact in the overall efficacy. The use of selected promoters to drive antigen expression further facilitates the optimization of the elicited response. Finally, the co-delivery of immune stimulatory molecules allows fine-tuning, according to specific applications.

invasion of intestinal epithelial cells. The Salmonella translocated effector proteins SspH1 and SptP participate in this process. SspH1 is a member of the bacterial LPX repeat protein family that localizes to the mammalian nucleus and inhibits NF-êB-dependent gene expression. A Shigella flexneri translocated effector, IpaH9.8, which has similar structure and subcellular localization in mammalian cells, also inhibits NF-êB-dependent gene expression. This suggests that inhibition of NF-êB may be a common property of nuclear LPX repeat proteins. We propose that suppression of inflammatory responses by intracellular S. enterica serovar Typhimurium, and perhaps Shigella flexneri, contributes to bacterial colonization of host tissues and pathogenesis.

PHAGE MEDIATED TRANSFER OF THE SPI-1 EFFECTOR PROTEIN GENE SOPE

W. Hardt, K. Ehrbar, C. Pelludat, S. Mirold; Institute for Microbiology, 8092 Zürich, SWITZERLAND. Salmonella spp. are enteropathogenic Gram-negative bacteria which use a large array of virulence factors to colonize the host, manipulate host cells and resist the host's defense mechanisms. Even closely related Salmonella strains have different repertoires of virulence factors. Bacteriophages contribute substantially to this diversity. There is increasing evidence indicating that the reassortment of virulence factor repertoires by converting phages might represent an important mechanism in the adaptation of Salmonella spp. to specific hosts and to the emergence of new epidemic strains. Here, we report on SopEÖ, a P2-like phage from S. Typhimurium DT204 which encodes the SPI-1 Type III effector protein SopE. We describe the attachment site of SopEÖ (ssrA) and the sequence analysis of the "captured" SopEÖ-prophage. Interestingly, SopEÖ encodes SopE, but no secretion chaperone (a type of small protein required for stabilization/recognition of effector proteins by TTSS). We found that SopE uses the SPI-1 encoded chaperone InvB. This provides a model how the phage encoded effector protein SopE can function properly after lysogenic conversion of "naïve" Salmonella strains.

A SALMONELLA ENTERICA SEROVAR TYPHIMURIUM TRANSLOCATED LEUCINE-RICH REPEAT EFFECTOR PROTEIN INHIBITS NF-ÊB-DEPENDENT GENE EXPRESSION

A. Haraga, S. I. Miller; University of Washington, Seattle, WA. Nontyphoidal salmonellae are enteric pathogens that cause acute gastroenteritis and colonize the intestinal tract for prolonged periods. In the intestinal epithelia these bacteria induce secretion of proinflammatory cytokines, such as interleukin-8 (IL-8), which leads to a profound inflammatory response through recruitment of polymorphonuclear leukocytes. Production of IL-8 induced by Salmonella is due to the activation of the transcription factors NF-êB and AP1. This work demonstrates that Salmonella enterica serovar Typhimurium can downmodulate IL-8 production after Pathogenesis, Epidemiology, and Vaccine Development

INTRACELLULAR ACTIVITIES OF THE SPI-2 TYPE III SECRETION SYSTEM

D. Holden, Imperial College London, UNITED KINGDOM. The type III secretion system (TTSS) encoded by the Salmonella SPI-2 pathogenicity island is activated after bacteria are internalised by host cells, and is required for intravacuolar growth of Salmonella in epithelial cells and macrophages. It transfers effector proteins from the bacterial cell into the phagosomal membrane and the host cell cytosol. A variety of functions have been attributed to the SPI-2 TTSS. These include the inhibition of various aspects of endocytic trafficking, an avoidance of NADPH oxidasedependent killing, the induction of a delayed apoptosis-like host cell death, the control of membrane dynamics of the Salmonella-containing vacuole (SCV), the assembly of a meshwork of F-actin around the SCV, an accumulation of cholesterol around the SCV, and interference with the localization of inducible nitric oxide synthase to the SCV. 19

SPEAKER ABSTRACTS

Several effector proteins that are translocated in a SPI-2dependent manner have now been identified. These are encoded both within and outside SPI-2. The characteristics of some of these effectors, and their relationship to the physiological functions listed above, will be the subject of this talk.

AMOXYCASSIA AND OTHER PLANT DERIVED ANTIMICROBIAL SUBSTANCES FOR THE TREATMENT OF MULTI-DRUG RESISTANT SALMONELLA INFECTIONS IN KARACHI- PAKISTAN

S. U. Kazmi; Department of Microbiology ,University of Karachi, Karachi, PAKISTAN. Although the wide spread use of antibiotics in recent years has revolutionized the therapy and has substantially reduced mortality from infectious diseases, misuse of antibiotics , poor patient compliance and easy over the counter availability of prescription drugs in Pakistan and some other countries has unfortunately helped in the emergence of Multiple Drug Resistant strains of Salmonella , M. tuberculosis , Staph aureus (MRSA) and other potential human pathogens. The emerging antibiotic resistance is a serious health problem worldwide. Medicinal plants have long been used as a source of potential anti- microbial substances. We are currently screening large number of endogenous plant derived substances for their antimicrobial and immunomodulating properties. Recently , we developed a synergistic compound by the name of Amoxycassia (patent approved) which is highly effective against MDR Salmonella strains. This novel compound consists of amoxicillin and water /organic extract of Cassia fistula fruit. The therapeutic value of Cassia fistula( Amaltas ) commonly found in Pakistan has been recognized in several systems of medicine . A total of 2708 Salmonella isolated from blood samples during a two year surveillance study were characterized and screened for antibacterial susceptibility against antibiotics known to provide in-vivo clinical cures in addition to our newly developed preparation. All first line drugs exhibited significantly lower activity as compared to Fluoroquinolones. Cotrimoxazole ,in particular covered the least number of isolates ( 35.2%). MDR Salmonella isolates were found to be quite susceptible to aqueous / alcoholic extract of fruit of C.fistula alone as well as in combination with Amoxcillin . MIC of C. fistula when tested alone was found to be in the range of 390-3120 ug/ml however the combination of Amoxcillin and fruit extract of C. fistula , MIC of C.fistula lowered it to 390-1560 ug /ml and there was a four-fold decrease in MIC of Amoxcillin for MDR Salmonella typhi . Amoxycassia , showed synergism for 63%of MDR Salmonella typhi tested by checker board method ( FIC < 0.5 ). Both Amoxicillin and C.fistula have been used for several years for the treatment of various ailments in humans and there are no reports of their toxicity , as such our new formulation Amoxycassia can be developed as a potent drug for the treatment of MDR Salmonella typhi infections.

COUPLING GENE EXPRESSION TO FLAGELLAR ASSEMBLY IN SALMONELLA

K. T. Hughes; University of Washington, Seattle, WA. Salmonella enterica is peritrichously flagellated possessing 6 ­ 10 individual flagella per cell. As the cell divides and continues to grow new flagella structures are initiated. Thus, during steady state growth different flagella will be at different stages of development. We present evidence for a mechanism that allows the cell to regulate the assembly of individual flagella that are at different stages in the assembly process. This mechanism involves an RNA signal at the 5' end of flagellar mRNA sequences that acts to regulate translation of specific flagellar genes in a manner to target these mRNA sequences for localized translation at the base of individual flagella. We have recently shown that the FljA protein inhibits both the transcription and translation of fliC. Analysis of potential mRNA secondary structures in the wild-type fliC 5'UTR sequence revealed two stem-loop structures followed by two mutually exclusive stem-loop structures of equal energies. Mutants in the 5'-UTR were isolated that allowed fliC transcription and translation in the presence of FljA. These mutants were located to one of the competing stem-loop structures. One mutant, a 5 base insertion, exhibited a dominant-negative phenotype in that it prevented the expression of the second filament gene fljB. This suggested that the 5'UTR might play a role in flagellin assembly. Replacement of the chromosomal promoter for the flagellar filament gene, fliC, with the tetA promoter and structural gene, results in a P tetA-tetA-fliC operon fusion. With only 2 or 10 bases between tetA and fliC, transcription and translation of fliC occurred. However, the efficient assembly of translated FliC protein into the external filament structure required the 5'-untranslated region (5'-UTR) of fliC mRNA to be included between the tetA and fliC structural genes. Localized mutagenesis of the fliC 5'-UTR was used to isolate base changes that were defective in transcription only, translation only and FliC assembly only in the absence of FljA. Transcription-defective mutants were in the fliC promoter sequences, translation-specific mutants were in the ATG start site, and assembly mutants were located in two stem-loop structures at the very 5' end of the fliC UTR. These results suggest that FljA binds the fliC 5'UTR to stabilize an attenuator stem-loop structure and inhibit translation of transcripts that are not attenuated. In the absence of FljA, the fliC mRNA is targeted for translation and secretion by the very 5' end of the UTR.

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ASM Conference on Salmonella

SPEAKER ABSTRACTS

A MOLECULAR MECHANISM FOR SALMONELLA PERSISTENT INTESTINAL CARRIAGE

R. A. Kingsley; Texas A&M University SHSC, College Station, TX. The primary risk factor for the introduction of non-typhoid serotypes of Salmonella into meat and meat products is their presence in the intestine of apparently healthy animals at slaughter and subsequent fecal contamination during processing. A critical control point for improvement of food safety is therefore intervention strategies aimed at a reduction in the prevalence of Salmonella in livestock and poultry at preharvest. The development of such strategies requires elucidation of the molecular mechanisms of intestinal persistent carriage for Salmonella in natural hosts. To this end we characterized ShdA, the first Salmonella-specific determinant to be identified that is involved in persistent intestinal carriage in the murine and bovine hosts. This protein is a member of the autotransporter family of outer surface located proteins and binds to the extracellular matrix protein fibronectin. Localization of the binding site in fibronectin and inhibition studies revealed that the mechanism of ShdA binding involved molecular mimicry of the host polysaccharide ligand heparin. We are currently testing the hypothesis that ShdA binding to fibronectin, or other heparin-binding proteins, is required for persistent intestinal carriage using a murine persistence model.

promoter regions. Whether OmpR and SlyA bind cooperatively or competitively remains to be determined. Regulation of SPI2 by SlyA may help to account for the inability of slyA mutant Salmonella to replicate in host phagocytes. The involvement of dual independent transcriptional regulators in the activation of SPI2 expression may provide a mechanism for the integration of diverse signals in virulence gene regulation.

DOES GENETIC LOAD DETERMINE HOST-SPECIFICITY?

A. Thierauf1, R. Helm 2, S. Tsuji3, S. Maloy3; 1University of Illinois, Urbana, IL, 2Cancer Research Center, Columbia, MO, 3 Center for Microbial Sciences, San Diego, CA. Host-specific Salmonella serovars have a much greater genetic load than serovars with a broad host-specificity. It is possible that the host-specific serovars evolved by sequential accumulation of compromising mutations which caused a stepwise limitation in the ability to thrive in multiple hosts. Alternatively, the limited host range may have resulted abruptly via acquisition or loss of a single island or a small number of changes, allowing the accumulation of neutral mutations in loci no longer needed in the narrower niche. Determining how the mutation load affects host-specificity may provide insight into how host-specificity evolved. Comparative genomic analysis indicates that host-specific Salmonella serovars have substantially more pseudogenes than serovars with broad host specificity. Analysis of genetic hybrids indicate that many of the pseudogenes have no effect on host-range, but a subset of mutations in loci distributed around the chromosome modulate the host range. In addition to point mutations, chromosome rearrangements are much more frequent in host-specific Salmonella. Although serovars with broad host specificity maintain an extremely stable chromosome order, natural isolates of Salmonella serovars that are host-specific exhibit multiple inversions and levitations of large chromosomal regions. Comparison of the inversion frequency in generalist vs host-specific serovars indicates that the differences in chromosomal rearrangements are due to the exclusive niche of host-specific serovars. Taken together, the results support the hypothesis that evolution of host-specificity occurred via a stepwise ratchet.

SLYA REGULATES SPI2 EXPRESSION IN SALMONELLA ENTERICA SEROVAR TYPHIMURIUM

D. Walthers1, S. Linehan2, T. Halsey 3, W. Navarre4, J. Potter3, l. J. Kenney 1, F. C. Fang4, D. Holden2, S. J. Libby3; 1Oregon Health Sciences University, Portland, OR, 2Imperial College, London, UNITED KINGDOM, 3NC State University, Raleigh, NC, 4 University of Washington, Seattle, WA. The ability to survive and replicate within host phagocytic cells is crucial for the ability of Salmonella to cause disease. Mutants that are unable to survive in host phagocytes are severely attenuated for virulence in animal models. The pathogenicity island SPI2 promotes Salmonella survival in activated macrophages by diverting the NADPH phagocyte oxidase away from Salmonella-containing vacuoles. The regulation of SPI2 gene expression is complex: maximal expression of SPI2 effector proteins occurs at low pH and low magnesium, conditions believed to mimic the intravacuolar environment. Previous studies have demonstrated that the OmpR protein regulates SPI2 transcription. Recently, we have found that the transcriptional regulator SlyA, already known to be required for Salmonella virulence, oxidative stress resistance, and survival in phagocytes, also controls the expression of SPI2 genes. Utilizing DNA microarray analysis, real-time PCR, DNA footprinting, electrophoretic mobility shift assays, beta-galactosidase fusions, and competitive infections assays, we have characterized the role of SlyA in SPI2 expression. Interestingly, SlyA and OmpR protect similar regions of the ssrA and ssrB Pathogenesis, Epidemiology, and Vaccine Development

UNDERSTANDING THE MOLECULAR PATHOLOGY OF SALMONELLA TYPHIMURIUM-INDUCED ENTERITIS

B. A. McCormick; Massachusetts General Hospital and Harvard Medical School, Charlestown, MA. The pathphysiology of localized enteritis caused by nontyphoidal Salmonella is characterized by movement of electrolytes and water as well as polymorphonuclear neutrophils (PMN) into the intestinal mucosa and lumen from the underlying vasculature. We have previously used an in vitro model of S. typhimurium infection to demonstrate that the intestinal epithelium is not merely a barrier to PMN movement but rather, in response to this enteric pathogen,

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intestinal epithelial cells direct PMN movement via the polarized secretion of chemokines. Specifically, S. typhimurium activates the transcription factor NF-kB resulting in the epithelial synthesis and basolateral release of a potent PMN chemokine, interleukin-8 (IL-8). Such basolateral secretion of IL-8 recruits PMNs through the matrix (lamina propria) to the subepithelial space but is not involved in PMN migration across epithelial tight junctions. Instead, this final step of PMN movement across the epithelium has been shown to be directed by the NF-kBindependent release of a soluble bioactivity from the apical surface of the epithelium - previously termed PEEC (pathogen-elicited epithelial chemoattractant). This presentation will highlight recent advances we have made toward understanding the molecular mechanisms by which S. typhimurium-epithelial interactions elicit the production of inflammatory regulators that promote the movement of PMN across the intestinal epithelium. at 41°C, indicating that temperature regulation of virulence expression does not play a role in host specificity. Sgal did not show an increased ability to invade or survive inside HD11 (chicken macrophage-like cell line) but tended to be less cytotoxic than other serotypes investigated. This may indicate a stealth-like type of infection, which may be important for the development of the fowl typhoid. Sgal did not cause a significantly higher oxidative burst in the HD-11 cell line, nor in primary peritoneal macrophages from hens, and Sgal did not show increased ability to survive in serum from chicken compared to other serotypes. In summary, none of the investigated parameters have been shown to be decisive for the outcome of the host specific infection of Sgal in the avian host.

SALMONELLA ENTERICA SEROVAR TYPHI LIVE VECTOR VACCINES

M. F. Pasetti, M. M. Levine; Center for Vaccine Development, University of Maryland, Baltimore, MD. At the Center for Vaccine Development (CVD) we have concentrated our efforts in the development of attenuated S. Typhi strains that can be used as oral typhoid vaccines and as live vectors expressing foreign antigens or delivering DNA vaccines. CVD 908htrA has been tested in Phase 1 and 2 clinical trials, and has proven to be safe and highly immunogenic. This strain was adapted to express foreign antigens such as tetanus toxin Fragment C, and to carry DNA plasmids encoding the same antigens. In a mouse model of intranasal immunization strong mucosal and systemic immune responses including antibodies, antibody secreting cells and cell-mediated immunity (CMI) against both foreign and bacterial antigens were demonstrated. CVD 908htrA expressing Frag C combined with tetanus toxoid in a mucosal prime-parenteral boost approach induced stronger serum antibody responses and IFN-ã production than 2 doses of either the recombinant strain or parenteral tetanus toxoid. CVD 908htrA given intranasally to rhesus macaques induced IgG anti-Frag C responses despite the presence of prior immunity to the vector. Successful mucosal prime with CVD 908htrA expressing malaria CSP antigen and parenteral boost with CSP-DNA vaccine was also demonstrated. CVD 908htA was recently used to mucosally deliver DNA measles vaccines in cotton rats, inducing neutralizing antibodies and CMI that conferred protection upon viral challenge. This strategy aims to prime the immune system in infants, for whom the traditional live measles attenuated vaccine is ineffective due to the blocking effect of maternal antibodies. Another typhoid vaccine candidate, CVD 915 -a guaBA mutant, has also shown great promise as live vector for antigen expression and DNA delivery. CVD 915 is being tested in a tumor murine model and it was found to efficiently infect tumoral cells, favoring tumor remission. A novel strain, CVD 909, which constitutively expresses S. Typhi Vi capsular polysaccharide, was recently evaluated in Phase 1 studies. IgA secreting cells against LPS, flagella and Vi were observed in vaccinated volunteers. In a novel approach, we have successfully used attenuated S. Typhi and S. Typhimurium strains as live vectors to mucosally prime immune responses in neonatal mice. Bacteria-induced Th1 ASM Conference on Salmonella

BIOLOGY OF SALMONELLA ENTERICA SEROVAR GALLINARUM HOST-SPECIFICITY IN THE AVIAN HOST

J. E. Olsen1, M. S. Chadfield1, S. Aabo2, D. J. Brown3, J. P. Christensen 1; 1Royal Veterinary and Agricultural University, Frederiksberg C, DENMARK, 2Danish Veterinary and Food Administration, Søborg, DENMARK, 3Scottish Salmonella Reference Laboratory, Glasgow, UNITED KINGDOM. Salmonella enterica serovar Gallinarum (Sgal) causes systemic salmonellosis known as fowl typhoid in the avian host. This poster reports the results of an investigation into the biology of the host specific infection. The host specific infection was characterized by a selective ability of Sgal to propagate to high numbers at systemic sites in day old as well as one-week-old chickens compared with other serovars of Salmonella. Sgal was less invasive in jejunal loop assays in 10-12 week old hens than S. Typhimurium (Stm) and equally as invasive as S. Dublin (Sdu) and S. Choleraesuis. S. Abortusovis was significantly less invasive than the other serovars. The significant difference between Sgal and Stm in invasion was also seen in the caecal tonsils and in the bursa of Fabricius. Thus the invasion per se does not seem to be a decisive factor in the host specific infection. In contrast to previous reports for biovar Pullorum, Sgal did not show a predilection for invasion via the bursa, as this site showed the lowest number of invading Sgal (and Stm). Sgal is unique among salmonellae in its lack of flagella. However, a flagella knock out mutant of Sdu remained avirulent in oral challenge of chicken while it was still as invasive as the wild type Sdu strain in the loop assay. This indicates that the presence of flagella is not the reason why Sdu is avirulent in chickens. Displacing the virulence plasmid of Sgal with the virulence plasmid of Stm did not alter pathogenicity, nor did it influence the avirulence of Sgal in the mouse model. This suggests that none of the genes harboured on the virulence plasmid determine the difference between the Sgal infection and the infection caused by other serotypes in the avian host. No significant differences were detected in the expression of the plasmid virulence genes in Stm or Sgal at either 37°C or

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cytokines are expected to increase maturation of neonatal DC, leading to the induction of cell mediated immunity, otherwise absent in early life stages and critical to clear infection by intracellular pathogens. In this model S. Typhi invades the nasal lymphoid tissue and Peyer's patches. S. Typhi-delivered DNA vaccine plasmids persisted up to 7 days in murine lymphoid tissues. Expression of mRNA encoding measles antigen was documented in the nasal tissue of vaccinated animals, which we believe is a major site involved in the priming of immune responses. The progress of these studies will be discussed in the presentation.

ACQUISITION AND TRANSFER OF RESISTANCE TO EXTENDED-SPECTRUM CEPHALOSPORINS TO SALMONELLA IN THE INTESTINAL TRACT

C. Poppe 1, L. Martin1, C. Gyles2, K. Rekker1, R. Reid-Smith1, P. Boerlin2, S. McEwen3, J. Prescott 2, K. Forward4; 1Health Canada, Laboratory for Foodborne Zoonoses, Guelph, ON, CANADA, 2 University of Guelph, Department of Pathobiology, Guelph, ON, CANADA, 3University of Guelph, Department of Population Medicine, Guelph, ON, CANADA, 4Dalhousie University and Queen Elisabeth II HSC, Halifax, NS, CANADA. Concerns: Resistance of Salmonella Newport, other Salmonella serovars and Escherichia coli to extended-spectrum cephalosporins (ESCs) is being reported with increasing frequency. Ceftiofur, a veterinary ESC, may be used increasingly for the treatment of bacterial infections in animals. In humans, infections with ESC resistant Salmonella threaten the efficacy of ceftriaxone, the drug of choice for treating septicaemic salmonellosis in children. Methods: 12 turkey poults were placed in each of four pens in the animal isolation unit at the University of Guelph. The treatment of the poults was as follows: pen 1 - poults were dosed orally with an antimicrobial susceptible (AMS) S. Newport on day 1 and inoculated subcutaneously (s.c.) with 0.17 mg ceftiofur on day 2; pen 2 - poults were dosed orally with an AMS S. Newport on day 1 and inoculated s.c. with 0.2 mL saline on day 2; pen 3 - poults were dosed orally with an AMS S. Newport and an E. coli encoding resistance to the ESCs and other antimicrobials on a self-transmissible 72 Mda plasmid, but were not treated with antimicrobials; pen 4 - poults received no treatment. Environmental and cloacal samples were taken from poults on days 2 and 4, and cecal contents on day 10. E. coli and Salmonella were isolated and antimicrobial susceptibility (AMS) testing was performed on all isolates. Salmonella isolates resistant to the ESCs were further characterized by PCR, plasmid profiles and hybridization studies. The trial was repeated once. Results: AMS testing of isolates from poults in pen 3 revealed that Salmonella Newport isolates showed the same antimicrobial resistances including resistance to the ESCs as the E. coli donor strain and had acquired a plasmid of the same size as the 72 Mda plasmid in the E. coli donor strain. PCR amplification resulted in a product consistent with that of the cmy-2 gene. One E. coli isolate in pen 1 became weakly resistant to cephalothin, a first-generation cephalosporin. When bacteriophages isolated from the cecal contents and environment of poults in pens 1 and 3 were propagated on Pathogenesis, Epidemiology, and Vaccine Development

multi-resistant E. coli and S. Newport, they were able to transduce antimicrobial resistance determinants, including resistance to ESCs, to an AMS S. Newport. Conclusions: Resistance to the ESCs encoded by the cmy-2 gene is transferable from E. coli to an AMS S. Newport in the intestinal tract. The transfer may occur by conjugation and transduction of the AMS S. Newport. Implications: ESC resistant E. coli and Salmonella isolated from animals, the animal environment and foods of animal origin are encountered with increasing frequency. Resistance to the ESCs can be transferred to Salmonella in the intestinal tract, which will limit treatment options when humans experience foodborne salmonellosis and become infected with such highly drug-resistant strains.

MOLECULAR PROPERTIES OF SALMONELLA ENTERICA SEROTYPE PARATYPHI B DISTINGUISH BETWEEN ITS SYSTEMIC VS. ENTERIC PATHOVARS

R. Prager, W. Rabsch, W. Streckel, W. Voigt, E. Tietze, H. Tschäpe; Robert Koch Institute, Wernigerode, GERMANY. Salmonella enterica serotype O1,4,5,12:Hb:1,2, designated according to the current Kauffmann-White scheme as S. Paratyphi B, is a very divers serotype with respect to its clinical and microbiological properties. PCR and blot techniques which enable to identify the presence, polymorphism, and expression of various effector protein genes, help to distinguish between strains with more systemic vs. enteric outcome of diseases. All serotype Paratyphi B strains from systemic infections have been found rather genetically related with respect to the pattern of their virulence genes sopB, sopD, sopE1, avrA, and sptP as well as other molecular properties (MLEE, PFGE, ribotype, IS200). They have been classified as members of the systemic pathovar (SPV). All these SPV strains possess a new, sopE1 gene carrying bacteriophage (designated FSopE309 ) with a high SopE1 protein expression but they miss the commonly occurring avrA determinant. They reveal normal SopB protein expression but fail in their SopD protein production. In contrast strains from enteric infections classified as enteric pathovar (EPV), possess various combinations of the respective virulence genes, PFGE pattern, and ribotypes. In order to identify both pathovars of serotype S. Paratyphi B we propose as a diagnostic tool the PCR technique for testing the presence/absence of the virulence genes sopE1 and avrA. This will be of great public health importance since strains of serotype Paratyphi B have been re-emerged recently worldwide.

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SPEAKER ABSTRACTS RETROSPECTIVE ANALYSIS OF SALMONELLA ENTERICA SEROVAR ENTERITIDIS BY THE NEW PHAGE TYPING SCHEME (WARD) IN EASTERN GERMANY BEFORE 1989 REVEALS A HETEROGENEITY OF CLONES IN LAYING FLOCKS

W. Rabsch1, I. Schröder 2, U. Methner3, R. Prager1, H. Tschäpe1; 1 Robert Koch Institute, Wernigerode, GERMANY, 2Lohmann Animal Health GmbH & Co. KG, Cuxhaven, GERMANY, 3 Bundesforschungsanstalt für Viruskrankheiten der Tiere, Jena, GERMANY. In the 1980s, public health laboratories in Europe and the Americas reported a dramatic increase in the number of human S. Enteritidis infections. In the former German Democratic Republic (GDR), S. Enteritidis became the Salmonella serovar most frequently isolated from humans in 1986 and the highest incidence of human cases was reported in Germany in 1992. After breeder lines for laying flocks had been imported from Canada in 1966 the egg industry in the GDR was completely isolated from that in Western Germany or other Western European countries until the reunification of Germany in 1989. Because of is isolation from other countries, we reasoned that an analyzed of the clonal diversity of isolates from GDR chicken flocks cultured during the 1980s would provide a unique opportunity to obtain new insight into factors that may have triggered the S. Enteritidis epidemic. While isolates had previously been typed by the phage typing scheme of Lalko and Laszlo we applied for the first time the phage typing scheme by Ward for retrospective analysis of these strains. Although overall the Ward phage type (PT) 4 dominated between 1986 and 1989, other phage types dominated in the early 80s and still comprised a considerable fraction of isolates until 1989. For instance, PT 8 was isolated from the collective (Kombinat Industrielle Mast) Gutenberg, district Halle, from July 1988 until December 1989. In the collective Roggosen, district Cottbus, PT8 was isolated between 1988 and 1989. PT4 was isolated from neither of these two collectives. Since collectives harboring either PT4 or PT8 obtained laying hens from the same sources (Breeder lines in Deersheim and Spreenhagen) it is likely that S. Enteritidis infection was aquired from the environment in each individual collective. The presence of different phage types in different collectives suggests that in each case S. Enteritidis strains present in the environment (i.e. in rodent populations) were able to enter chicken flocks. This scenario is consistent with the idea that the S. Enteritidis epidemic was caused by eradication of a competitor (S. enterica serovar Gallinarum), thereby providing an opportunity for strains from the environment to enter chicken flocks.

COMPARATIVE SEQUENCING OF SALMONELLA STRAINS.

M. Roberts1, D. Maskell2, P. Barrow3, T. Humphrey4, N. Thomson5, J. Parkhill5; 1University of Glasgow, Glasgow G61 1QH UK; 2, University of Cambridge, Cambridge, UK; 3Institute for Animal Health, Berks, UK; 4University of Bristol, Bristol, UK; 5 The Sanger Institute, Cambridge, UK. The Wellcome Trust has provided funding for the comparative genome sequencing of 5 salmonella strains. One strain of Salmonella bongori and four strains belonging to Salmonella enterica subspecies I (enterica) were selected for sequencing. The Salmonella enterica subspecies I strains are: two strains of serotype Typhimurium, SL1344 and a phage type DT104 strain; a serotype of Enteritidis phage type 4; a serotype Gallinarum strain. S.bongori strains are usually only isolated from cold-blooded hosts but have occasionally been reported as a cause of gastroenteritis in humans. S.Typhimurium SL1344 is a virulent, widely used laboratory strain. S.Typhimurium DT104 strains cause infection in humans and animals and are multiply antibiotic resistant due to chromosomally encoded resistance genes. S. Enteritidis is the most common cause of human salmonellosis in many countries and S. Enteritidis PT4 strains are responsible for a pandemic of S.Enteritidis infection. Infection is mainly associated with ingestion of improperly prepared poultry products, particularly eggs. S.Gallinarum causes fowl typhoid and unlike the other Salmonella enterica subspecies I being sequenced has a very narrow host-range. The presentation will discuss the selection of the strains for sequencing, the sequencing strategy, sequencing progress and preliminary analysis of the genome sequences.

A GENETIC PROCESS THAT SPEEDS ADAPTATION WITHOUT AFFECTING MUTATION RATE -- EFFECTS OF GROWTH UNDER STRONG SELECTION.

J. R. Roth1, D. I. Andersson2, E. S. Slechta1, E. Kugelberg2, K. L. Bunny1, E. Kofoid 1; 1University of California, Davis, CA, 2Swedish Institute for Infectious Disease Control, Solna, Sweden. All organisms adapt to stress by means of mutation and natural selection, which together dictate the genetic composition of a population. A 100-year-old question is whether selection affects mutation rate and specificity or only the relative growth rate of genetically distinct organisms. To avoid the effects of selection on mutant frequency, mutation is usually studied under non-selective conditions. Some surprising things happen when one observes the mutation process while selection is imposed. -- A unique experimental system developed by Cairns and Foster seems to indicate that selective stress directs mutations to useful sites or induces generalized mutability in non-growing cells. We suggest that the behavior of this system is fully explained by effects of natural selection on growing cells, i. e. with no change in mutation rate. Cells destined to adapt arise prior to selection (as concluded by Luria and Delbruck and by Lederberg). These cells carry a duplication of the growthASM Conference on Salmonella

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SPEAKER ABSTRACTS

limiting gene. Under selection, these cells grow and give rise to descendents with higher amplification. Each added gene copy improves growth and adds a mutational target to the clone until the number of target copies (cell number x copy per cell) is sufficient to allow a mutation to arise by normal processes. The final mutation is made more probably by an increase in target size under selection, not an increase in mutation rate or a change in specificity. The increase in mutant number appears to reflect directed mutation because only the target gene amplifies during growth under selection and only the selected revertant allele remains in the final strain following segregation. -- A distraction in understanding this process is an increase in general mutation rate experienced by a few revertants. The dinB gene for an errorprone polymerase happens to lie near the gene under selection and is included in the amplified unit in about 10 to 20% of the developing clones. This results in a low-level mutagenesis that is neither necessary nor sufficient to explain the effect of selection on mutant yield. ---- Thus selection does increase the frequency of mutant organisms in the population and can appear to direct mutations to useful targets, but only if that target amplifies during growth under selection. The genetic events underlying this process duplication, amplification, mutation, segregation - are likely to occur in all biological systems. We suggest that this process may help explain how pathogens adapt to a host, how cells in a metazoan become malignant and how new genes evolve from old ones. In these process genetic adaptation is accelerated because it occurs during growth under selection.

and tested for antibiotic resistances. This last issue is important to aid the countries in the political of drug supplies. Molecular epidemiology and identification of virulence genes were also performed. Few examples of these activities will reported and discussed. In Zimbabwe, the establishment of a National Reference Center for Enteropathogens, created by international cooperation donors, allowed to monitor the diffusion of serovars Typhimurium and Enteritidis in human and uncovered the dissemination of multidrug resistant (MDR) strains of the, otherwise, rarely found serovar Decatur. In Brazil (Rio Grande do Sul) we investigated the capability to trace the spread of serovar Typhimurium strains in swine by using IS200 fingerprint. Four distinct profiles of IS200 were detected in swine isolates, with a prevalent pattern in almost all strains characterized. The IS200 profile of serovar Typhimurium seems to be highly conserved and there is no correlation with the most prominent pattern of antibiotic resistance identified (Te-Su-Tsu-Cm). In Pakistan and in the nearby Iran, we demonstrate two serovar Typhi IS200 profiles, with a high degree of relatedness, only one present in both countries. However, we identified a common MDR pattern (Ap, Cm, Pip, Tsu, and Te) only in Pakistanian isolates. None of the Iranian isolates was MDR, suggesting that MDR strains of serovar Typhi have not yet spread throughout Iran. Furthermore we analyzed the presence of virulence factors in S. keurmassar, a new MRD serovar recently isolated in Senegal. In conclusion the capability to establish surveillance systems is a good model to characterize and control Salmonella in DC.

SURVEILLANCE OF SALMONELLOSIS IN DEVELOPING COUNTRIES: A MODEL OF COOPERATION

S. Rubino; Dept. Biomedical Science, Center for biotechnology development and biodiversity research,University of Sassari, Sassari, ITALY. Salmonellosis is a worldwide health problem, with a rilevant impact in developing countries (DC). This is due to inadequate measures of surveillance. Furthermore, Salmonella infections are often not recorded by public health institutions.The situation is becoming dramatic with the spread of HIV in Africa and Asian countries. Since early 90', our group sponsored by the Ministry of Foreign Affair and by Sardinian Regional Government, is working on surveillance programs for Salmonella. Our aim is to strengthen the capabilities of DC to create reference centers that may assess Salmonella serovars prevalence, drugs resistance, and carry molecular epidemiology studies to trace the routes of Salmonella diffusion. The research projects are implemented with intensive training on job for scientific personnel qualification. So far, the countries involved are in South America (Brazil, Colombia, Perù, Venezuela), Asia (Jordan, Pakistan, Vietnam), Africa (Angola, Egypt, Libya, Maroc, Mozambic, Senegal, Tanzania, Zimbabwe), and Albania. The projects activity includes the collection of strains of human, food and animal origin by Universities and PH laboratories. All strains were characterized biochemically and serologically Pathogenesis, Epidemiology, and Vaccine Development

INVERTED PATHOGENICITY: HOW TO USE SALMONELLA´S TTSS FOR VACCINATION PURPOSES

H. Rüssmann; Max von Pettenkofer-Institute for Hygiene and Medical Microbiology, Munich, GERMANY. The term "inverted pathogenicity" stands for the exploitation of microbial virulence factors and mechanisms for preventive or therapeutic purposes. It will be demonstrated that Salmonella´s major pathogenicity concept can be used to develop a new vaccination strategy. Upon infection, many gram-negative animal and plant pathogens evade the host´s immune response by utilizing a specialized protein secretion machinery, known as type III secretion system (TTSS), for the export of bacterial virulence factors delivered directly into the cytosol of target cells. This unique translocation mechanism can be used by attenuated Salmonella carrier vaccines for the delivery of large protein fragments derived from immunodominant viral and bacterial heterologous antigens to the MHC class I-restricted antigen processing pathway. Among different bacterial species, many components of TTSSs reveal functional conservation probably due to the fact that shared type III genes were recruited by horizontal transfer during evolution. One of the best studied type III effector proteins is the 25-kDa Yersinia outer protein E (YopE). During the interaction of Yersinia species with professional phagocytes, YopE translocation disturbs eukaryotic cytoskeleton dynamics and inhibits phagocytosis. YopE is a GTPase-activating protein that is active towards G

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SPEAKER ABSTRACTS

proteins from the Rho family. It has been demonstrated by our laboratory that the fusion of the N-terminal 138 amino acids of YopE comprising the translocation domain of the type III molecule to listerial antigens results in hybrid proteins that are engaged and translocated by both Salmonella´s and Yersinia´s TTSS. In mice orally immunized with attenuated Salmonella vaccine strains expressing translocated chimeric YopE, this novel vaccination strategy results in the induction of pronounced peptide-specific cytotoxic CD8 T cell responses that confer protective immunity.

CONTRIBUTION OF A LAMBDOID PROPHAGE AND AN INTERGENERIC IS ELEMENT TO THE VIRULENCE OF SALMONELLA ENTERICA SEROVAR ABORTUSOVIS

S. Uzzau; Dept Biomedical Sciences, Center for Biotechnology Development and Biodiversity Research,University of Sassari, Sassari, ITALY. Horizontal transfer of virulence genes via mobile genetic elements has been a major driving force for the evolution of Salmonella pathogenicity. Temperate phages appear to play a pivotal role in this process by sampling virulence genes, allowing their transfer between phage genomes, and promoting reassortment of virulence effectors in Salmonella by lysogeny. Salmonella enterica serovar Typhimurium strains are consistently lysogenic for two lambdoid phages that encode virulence effectors, Gifsy-1 and Gifsy-2. Other serovars, including serovars Dublin, Gallinarum, Enteritidis, and Hadar, harbor distinct prophages with similarity to the Gifsy phages. Serovar Abortusovis, a pathogen exclusively associated to ovine infection, carries a cryptic prophage (Gifsy-2AO) related to Gifsy-2. Gene mapping and sequence analysis of Gifsy-2AO showed that the prophage "b" region is highly homologous to equivalent regions in serovars Typhimurium, Enteritidis, and Dublin. The Gifsy-2AO element contributes to serovar Abortusovis systemic disease, while this element does not influence intestinal colonization. In addition, extensive analysis of Gifsy-2AO in epidemic strains isolated in Europe (Italy, France, and Albania) and in Asia (Iran), uncovered the presence of an insertion sequence, IS1414, in all European strains tested. IS1414, previously identified in pathogenic Escherichia coli strains, encodes a heat-stable toxin, EAST1 (astA), structurally related to ETEC stable toxin ST and to a group of mammalian heat-stable peptides. To our knowledge, this is the first evidence for intergeneric transfer of virulence genes via IS elements in Salmonella. Serovar Abortusovis harbors both sodC1 and gtgE genes, the major virulence determinants encoded by serovar Typhimurium Gifsy-2. However, insertion of IS1414 within Gifsy-2AO, caused the `adjacent deletion' of a large portion of the `b' region, encompassing gtgB (sseI, srfH), gtgC, part of gtgD, and tail fiber protein genes. Consistently, all serovar Abortusovis European strains tested (41) were negative for gtgB. These data suggest that GtgB might be dispensable during serovar Abortusovis infection in ovine. EAST1-positive serovar Abortusovis strains showed a high number of IS1414 copies (i.e., more than 20). In addition to the IS1414 insertion within Gifsy-2AO, copies of this element were mapped within other pathogenic loci, including SPI1 (between hilAiagB and sptP genes) and the SCI genomic island (within trascriptional regulator sinR). The wide distribution of IS1414-positive epidemic strains suggest that horizontal acquisition of EAST1 and IS1414 frequent transposition within serovar Abortusovis genome might have improved the fitness of serovar Abortusovis within its narrow ecological niche.

LIVE VACCINES FOR SALMONELLA CONTROL IN POULTRY

I. Schröder, H. Scharr, M. Iburg; Lohmann Animal Health, Cuxhaven, GERMANY. Increasing awareness of food borne diseases in humans have prompted stricter requirements for food production and have given way to the introduction of new preventive methods in poultry husbandry, including intensive vaccination programmes. TAD Salmonella vac E (Salmonella Enteritidis) and TAD Salmonella vac T (Salmonella Typhimurium) are live vaccines from Lohmann Animal Health, two efficacious tools to further improve Salmonella control programmes in poultry production, to increase food safety and consumer protection by continuously reducing Salmonella burden in poultry products. Sensitivity to Salmonella infections is known to vary widely between different species, i.e. mouse, calf, chicken. The concept of selecting naturally occurring Salmonella Drift Mutants and establishing a correlation between growth characteristics and virulence offers the unique opportunity to design live vaccines that match the species specific sensitivity to Salmonella bacteria. The concept of vaccinating via the drinking water mimics the natural oral route of infection and triggers rapid and appropriate immune reactions to combat potential field challenge with Salmonella Enteritidis and/or Salmonella Typhimurium. Studies demonstrate the increased IgA synthesis post vaccination, which is considered to be most relevant to initially prevent colonization at gut level. In either case, the 3-dose vaccination regime starts at day one, minimising the risk of an early Salmonella infection. The two following vaccinations during rearing induce solid protection throughout the laying period. Efficacy has been proven in a series of experimental challenge studies. This is the accepted standard approach to evaluate protection since currently there are no reliable methods of correlating antibody response to a protection index against this bacteria. Safety is a pre-requisite for today's live vaccines. The strains are designed for reduced persistence in the target species and in the environment. Marker technology allows reliable identification of the vaccine strains if required. Since the alterations are located in the chromosome, high genetic stability is given. Studies have also shown absence of egg transmission of the drinking water vaccine strains. In conclusion, TAD Salmonella vac E and TAD Salmonella vac T combine reliable efficacy with ease of use, safety and animal welfare.

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ASM Conference on Salmonella

SPEAKER ABSTRACTS

SALMONELLA RAPIDLY KILL DENDRITIC CELLS VIA A CASPASE-1DEPENDENT MECHANISM

A. W. van der Velden, M. Velasquez, M. N. Starnbach; Harvard Medical School, Boston, MA. Dendritic cells provide a critical link between innate and acquired immunity. In this study, we demonstrate that the bacterial pathogen Salmonella enterica serovar Typhimurium can efficiently kill these professional phagocytes via a mechanism that is dependent on sipB and the Salmonella pathogenicity island 1 (SPI1)-encoded type III protein secretion system. Phosphatidylserine redistribution, caspase activation, and rapid loss of plasma membrane integrity were characteristic of dendritic cells infected with wildtype Salmonella, but not sipB mutant bacteria. Caspase-1 was particularly important in this process since Salmonellainduced dendritic cell death was dramatically reduced in the presence of a caspase-1-specific inhibitor. Furthermore, dendritic cells obtained from caspase-1-deficient mice, but not heterozygous littermate control mice, were resistant to Salmonella-induced cytotoxicity. We hypothesize that Salmonella have evolved the ability to selectively kill professional antigen presenting cells in order to combat, exploit, or evade immune defense mechanisms and discuss the significance of our findings in the context of both Salmonella pathogenesis and host response to infection

regions appear to be hot spots for DNA acquisition in incHI1 plasmids. Analysis of a global collection of S. Typhi resistance plasmids using a gene specific microarray designed from the pHCM1 sequence has shown that the core region of the plasmids is conserved and that variation is confined to the same five variable regions that difere between R27 and pHCM1. There is clear acquisition and loss of genes over time which has allowed the re-construction of the genetic process in the molecular evolution of the MDR plasmids of S. Typhi.

NEW INSIGHTS INTO THE MOLECULAR BASIS OF SALMONELLA-SEROTYPE HOST SPECIFICITY

T. S. Wallis; Institue For Animal Health, Compton, Berkshire, UNITED KINGDOM. Our current understanding of Salmonella virulence mechanisms is largely based on studying the behaviour of S. typhimurium in genetically highly susceptible mice. Recently progress has been made in identifying factors that influence enteropathogenesis using larger animal models of infection. However the factors that influence Salmonella serotype hostspecificity and that determine if Salmonella remain localised in the intestines or become disseminated to cause systemic forms of disease, remain poorly characterised. Previous studies have correlated Salmonella persistence within macrophage and intestinal invasiveness with host specificity. Using a range of Salmonella serovars of defined virulence in cattle, pigs, poultry and mice we assessed intestinal invasion, Salmonella replication and killing rates in vivo, persistence within macrophages in vitro and dissemination from mesenteric lymph nodes (mln) in the context of hostspecificity. No correlation between host-specificity and the magnitude or route of intestinal invasion was found. Salmonella persistence within macrophages was both serotype- and host-specific, but did not correlate with systemic virulence. Rapid replication rates in vivo correlated with enteropathogenicity but not the ability to cause systemic disease. Passage through the mln was serotype-specific and correlated with systemic virulence and was dependent on a functional TTSS-1 but not TTSS-2. Bacteria that had traversed the mln were predominantly free in the lymph and were not host-cell associated. These observations indicate that the nature of host/pathogen interaction in the mln are potentially pivotal in influencing the nature and severity of disease and that Salmonella exploit both intra- and extracellular niches during pathogenesis. S.typhimurium has a very broad host range and the role of virulence genes in different animal species is not clear. The repertoire of virulence gene usage by S.typhimurium in cattle and chickens was compared using the technique of signature-tagged mutagenesis. This analysis has led to the identification of several new pathogenicity islands and demonstrates that S.typhimurium uses different sets of virulence genes in different animal species. These observations have implications for the design of effective vaccines for controlling Salmonella infections in different animal species.

PLASMIDS OF S. TYPHI

J. R. Wain; Imperial College, London, UNITED KINGDOM Resistance to a drug of treatment for typhoid fever, chloramphenicol, was first reported in Salmonella Typhi in 1950 but it was not until 22 years later that the first outbreaks of chloramphenicol resistant typhoid fever occurred. Today multi-drug resistant (MDR) Salmonella Typhi has a world-wide distribution causing typhoid fever which does not respond to any of the first line drugs; chloramphenicol, co-trimoxazole or ampicillin. Typhoid fever in Vietnam has followed this global pattern. The first chloramphenicol resistant outbreak was reported from 1972, the first anecdotal reports of MDR typhoid fever in the late 1980's, and the first outbreak in 1993. Genome analysis of Salmonella Typhi strain CT18, an MDR isolate from a patient admitted to The centre for Tropical Diseases, Ho Chi Minh City, Vietnam, in December 1993 has revealed that the MDR plasmid pHCM1 is very closely related to plasmid R27, a Salmonella plasmid first seen in 1961 in the UK. There is a core region shared by the two plasmids with five regions of variation. Two of these regions contain the genes encoding resistance. The largest region is 34.954Kbp in length and is bordered by two almost identical IS10 left elements associated with a truncated transposon Tn10. The second, smaller region is 14.75Kbp and encodes a trimethoprim resistance gene, dfr14A associated with a class one integrase. Restriction enzyme analysis has shown that the variation in MDR plasmids collected during the emergence of MDR Salmonella Typhi in Vietnam, map to five variable regions. These Pathogenesis, Epidemiology, and Vaccine Development

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SPEAKER ABSTRACTS EARLY IMMUNE RESPONSES IN NEWBORN CHICKENS INFECTED WITH SALMONELLA TYPHIMURIUM.

G. S. Withanage1, P. Wigley2, P. Kaiser 2, H. J. Brooks1, P. Mastroeni1, I. McConnell1, D. J. Maskell1; 1University of Cambridge, Cambridge, UNITED KINGDOM, 2Institute for Animal Health, Compton, UNITED KINGDOM. Poultry meat and eggs contaminated with Salmonella enteritidis and S.typhimurium are the most common source of acute gastro-enteritis in humans. When chickens are infected with Salmonella, some of them may harbour the bacteria and excreat to the environment intermittently over a long time. Much research has therefore recently been focused on investigating the host immune response to S. enteritidis and S.typhimurium . Infiltration of immune effector T lymphocytes, heterophils and macrophages into the intestinal tract shortly after the primary infection has been reported. However, the exact nature of the protective immune mechanisms against Salmonella infection in chickens is yet to be dissected. The bacterial colonization was investigated in the liver, ileum and cecal contents in newly hatched chickens 6, 12, 24 and 48 hrs after S. typhimurium oral infection. The development of pathological lesions as well as cytokine and chemokine expression were investigated by light microscopy and by quantitative real time RT-PCR respectively in the liver, spleen, cecal tonsils, jejunum and ileum after the same time points. Very high bacterial counts were found in the ileum and cecal contents throughout the experiment whereas Salmonella starts to appear in the liver from 24 hrs after the infection. The initiation of inflammation was evident only in the liver 48hrs after the infection. Proinflammatory cytokines and most of the chemokines were significantly increased in all the organs examined. In the liver, there was a several thousand-fold increase in chemokine expression, which correlated with the presence of inflammation only in the liver. before the onset of lay against an oral S. enteritidis infection was tested. One group of 30 chickens was vaccinated at 6 and 18 weeks of age and another group of 30 chickens served as unvaccinated controls. Following oral challenge infection at 23 weeks of age, cloaca swabs were taken regularly and the animals were necropsied at 26 weeks of age to culture internal organs. Furthermore, all eggs laid during the three week period after challenge were cultured for the presence of the challenge strain. In the control group, 87% of the birds and 5.6% of the eggs were found to be positive for S. enteritidis, compared to a 29% infection rate and 1.0% positive eggs in the vaccine group. These differences were highly statistically significant. In addition, the efficacy of different vaccination schedules was compared. Laying-type hens were vaccinated subcutaneously or by drinking water at 6 weeks of age. At 18 weeks of age, the animals received a subcutaneous or intramuscular booster vaccination, followed by a S. enteritidis challenge infection at 23 or 57 weeks of age. No significant differences in efficacy were observed between the various vaccination schemes tested.

SALMONELLA HIJACKS PHAGOCYTES

M. J. Worley; Oregon Health and Science University, Portland, OR. In many tissues a single layer of epithelial cells is the only physical barrier separating a host from the external environment, and thus is augmented with an array of defences against the systemic spread of microbes. Regardless, numerous pathogens quickly traverse epithelial barriers, access deeper tissue, and cause life-threatening systemic illnesses. Salmonella enterica arrives in the bloodstream inside phagocytes within minutes following ingestion and thereby rapidly disseminates beyond the gastrointestinal (GI) tract to internal organs. Here we provide a molecular explanation for this phenomenon. We demonstrate that Salmonella injects proteins into the cytosol of infected phagocytes via a type III secretion system that radically alter the kinetics and migratory patterns of these cells. We show that one of these effectors, SrfH, binds the host protein TRIP6, a member of the zyxin family of proteins that abrogates motility. Thus, Salmonella surmounts host barriers to the systemic spread of microbes by forcing infected phagocytes to traverse the GI epithelium and enter the bloodstream. The active subversion of phagocytes as vehicles for dissemination into deeper tissue is a new paradigm in host-pathogen interplay.

VACCINATION OF CHICKENS AGAINST S. ENTERITIDIS WITH THE S. GALLINARUM 9R STRAIN

M. H. Witvliet, T. G. Mols, L. T. Wijnhoven; Intervet International, Boxmeer, NETHERLANDS. The S. gallinarum 9R live vaccine strain, which has been developed by Williams Smith in the 1950s, has been used safely and effectively for the prevention of fowl typhoid for many years. Previous work has shown that intramuscular injection of chickens with the 9R strain will induce crossprotection against S. enteritidis under laboratory conditions (Barrow et al. 1991. Avian Path 20: 681-692). This finding was confirmed in a large field study in 80 commercial layer flocks with a high risk for S. enteritidis infection in The Netherlands (Feberwee et al. 2001. Avian Dis. 45: 83-91; Feberwee et al. 2001. Avian Dis. 45: 1024-1029). In the same study, safety and spread of the vaccine strain were evaluated. No safety problems or spread of the vaccine strain were observed under field conditions. In the present study, the efficacy of two subcutaneous vaccinations with the 9R strain

28

ASM Conference on Salmonella

POSTER ABSTRACTS

1(A)

ANTIMICROBIAL RESISTANCE PATTERN OF SALMONELLA'S STRAINS GAINED IN VLADIVOSTOK (RUSSIA) IN 2001-2002 YEARS

V. B. Turcutyuicov, A. V. Martynova, N. A. Syrzova; the State Medical University of Vladivostok, Vladivostok, RUSSIAN FEDERATION. Background: Salmonellosis is the leading cause of acute intestinal infections throughout all of the world. Our region ( Vladivostok/Far East of Russia) is not an exception. Serological pattern and antimicrobial agents resistance of the main pathogens of this disease complicate treatment of this infection. Methods: We had isolated and investigated strains of salmonellas according to common procedures and NCCLS standards. Results: In 2001-2002 years there were identified microorganisms of different serological groups. We had revealed that 86,7% of cases salmonellosis in 2001 year were caused by group B salmonellas (S.typhimurium, S.derbi, S.java), in 0,7% - salmonellas of serogroup C1(S.infantis), and in 5,7% of all cases the main ethiological pathogens were strains of group B (S.typhimurium, S.derbi, S.java). In 2002 year in 1,5% of all salmonellosis were isolated salmonellas of group B (S.typhimurium, S.derby), in 2,2 % -salmonellas of group C1 (S.infantis). S.duesseldorf, S.tspiongwe (serogroup C2) were isolated in 2,2 % of cases, and salmonellas of serogroup B (S.enteritidis) were isolated in 86,9% of all cases in 2002 year. Antimicrobial agents resistance of strains gained in 2001-2002 years had the tendency to increasing, e.g. there were 7,5% of resistance strains to polymixin in all S.enteritidis's strains in 2001 year, and 34,5% of these microorganisms were resistant to the polymixin in 2002 year. Resistant strains to canamycin were isolated in 51,7% in 2001 year and in 78,9% in 2002 year. Resistance strains of S.enteritidis to gentamycin had increased from 1% in 2001 year to 16.6% in 2002 year. In addition resistance this microorganism had increased from 25,5% in 2001 year to 62% in 2002, and accordingly tetracyclin resistant strains were observed in 90% and 100% of all cases. There is increasing of S.enteritidis' s strains resistant to ciprofloxacin (11%) and to cefuroxim (35%). Another strains of salmonellas were resistant to canamycin (S.infantis) and to tetracycline (S.derby) in 100%. Conclusion: The intensive antimicrobial agents therapy may lead to increasing of resistance in these microorganisms.

tional mutants, termed genome scanning mutagenesis (GSM), and (ii) a high-throughput cell culture infection assay for the selection of attenuated mutants. Short chromosomal fragments were cloned into a temperature-sensitive vector, and 9,125 individual mutant clones were created by insertional-duplication mutagenesis and selected at non-permissive temperature. Large-scale screening for replication defects in mouse macrophages revealed 438 attenuated mutants that were further characterized by PCR amplification and sequencing of the insertional fragments. The majority of attenuating mutations was found to be accumulated within the two chromosomal segments 1,100 to 2,331 kb and 2,730 to 3,050 kb. These regions not only comprise the pathogenicity islands SPI-5, SPI-2 and SPI-1, but another 16 non-collinear islands carrying up to 10 GSM mutations that led to attenuated phenotypes of serovar Typhimurium mutants in macrophages.

3(C)

GENOME SCANNING MUTAGENESIS (GSM): A POWERFUL TOOL FOR THE IDENTIFICATION OF NOVEL ANTIMICROBIAL TARGETS

T. M. Fuchs; Institut für Mikrobiologie, Freising, GERMANY. As a novel genetic strategy for the discovery of prokaryotic targets for antimicrobial agents, we have developed the Genome Scanning Mutagenesis (GSM) technology. Key features of GSM are i) random knockout-mutagenesis of an entire bacterial genome, ii) high throughput screening (HTS) of mutants under defined conditions followed by iii) PCR based identification of essential genes. In the first example for GSM, we screened the whole genome of Salmonella enterica serovar Typhimurium for genes that are indispensable for bacterial growth in rich medium. We not only detected the majority of genes involved in essential cellular mechanisms against which the traditional antibiotics are directed, but also discovered numerous novel antibacterial targets. By homology search based on the genome sequence data of Staphylococcus, Streptococcus, Mycobacterium and other clinically relevant bacteria, we derived a map of GSM targets that are highly conserved in a defined range of pathogens. Since GSM was successfully applied to Gram negative and Gram positive bacteria, it is a powerful tool to provide access to nontraditional targets that could give rise to new classes of wide-range as well as species-specific antibacterial agents.

2(B)

SCREENING THE SALMONELLA GENOME FOR NOVEL GENES CONTRIBUTING TO INTRACELLULAR SURVIVAL AND GROWTH

T. M. Fuchs; Institut für Mikrobiologie, Freising, GERMANY. A novel screening approach was applied to Salmonella enterica serovar Typhimurium in order to identify genes required for intracellular proliferation. This approach comprises (i) a genetic system based on homologous recombination for the genome-wide generation of inserPathogenesis, Epidemiology, and Vaccine Development

29

POSTER ABSTRACTS POSTER ABSTRACTS

4(A)

SOPD2 IS A NOVEL TYPE III SECRETED EFFECTOR OF SALMONELLA TYPHIMURIUM THAT TARGETS LATE ENDOCYTIC COMPARTMENTS UPON DELIVERY INTO HOST CELLS

J. H. Brumell1, S. Kujat-Choy 2, N. F. Brown2, B. A. Vallance2, L. A. Knodler2, B. B. Finlay 2; 1Hospital for Sick Children, Toronto, ON, CANADA, 2University of British Columbia, Vancouver, BC, CANADA. Salmonella Typhimurium is a facultative intracellular pathogen that utilizes two type III secretion systems to deliver virulence proteins into host cells. These proteins, termed effectors, alter host cell function to allow invasion into and intracellular survival/replication within a vacuolar compartment. Here we describe SopD2, a novel member of the Salmonella Translocated Effector (STE) family, which share a conserved N-terminal type III secretion signal. Disruption of the sopD2 gene prolonged the survival of mice infected with a lethal dose of Salmonella Typhimurium, demonstrating a significant role for this effector in pathogenesis. Expression of sopD2 was induced inside host cells and was dependent on functional ssrA/B and phoP/Q two component regulatory systems. HA-tagged SopD2 was delivered into HeLa cells in a SPI-2-dependent manner and associated with both the Salmonella-containing vacuole and with swollen endosomes elsewhere in the cell. Subcellular fractionation confirmed that SopD2 was membrane associated in host cells while the closely related effector SopD was localized to the cytosol. A SopD2 fusion to GFP associated with small tubular structures and large vesicles containing late endocytic markers, including Rab7. Surprisingly, expression of N-terminal amino acids 1-150 of SopD2 fused to GFP was sufficient to mediate both binding to late endosomes/lysosomes and swelling of these compartments. These findings demonstrate that the N-terminus of SopD2 is a bifunctional domain required for both type III secretion out of Salmonella as well as late endosome/lysosome targeting following translocation into host cells. We are currently examining the role of SopD2 in Salmonella pathogenesis.

rDNA sequence have been developed for Salmonella detection, in this report, we tried to design the ITS region based PCR primers for the detection of Salmonella spp. Sequences of the ITS regions of 51 Salmonella serovar. were determined by PCR-sequencing technique using the primers designed from the conserved sequences of 23S rDNA. By comparison of the ITS sequences of these Salmonella spp. and other nonSalmonella spp. available in Gene bank database, two PCR primers were designed. When Salmonella strains with various serotypes were PCR assayed, all the Salmonella strains generated PCR products with molecular weight equal to 300 bp. On the other hand, 53 non-Salmonella isolates including strains of Enterobacteriaceae and strains of other food pathogens generated the negative results. Detection limits of this PCR method was N×10 0 (N=1~9) cfu per assay. As these PCR primers were used for the detection of Salmonella cells artificially contaminated in foods, such as chicken and whole milk, the detection limit was 103 cfu per assay. When an 8h pre-enrichment step was performed prior to the PCR assay, the detection limit became N×100 cfu per gram of sample.

6(C)

ASSIGNMENT OF SALMONELLA PATHOGENICITY ISLAND ASSOCIATED SEQUENCES TO XBAI-RESTRICTION FRAGMENTS OF SALMONELLAE

B. Guerra1, S. M. Soto2, R. Helmuth1, M. C. Mendoza2; 1Federal Institute for Risk Assessment (BfR), Berlin, GERMANY, 2Dpto. Biología Funcional. Universidad de Oviedo, Oviedo, SPAIN. The Salmonella pathogenicity islands (SPI1-5) carry important genes for expressing the full pathogenicity potential of Salmonellae. Their homology to other genetic elements suggests that they have been acquired by horizontal transfer from phage or plasmids of unknown origin. However, little information has been gained on the prevalence and chromosomal location of the SPIs in field isolates of different Salmonella serotypes. The aim of this study was to detect and locate the five SPIs on the genomic XbaI-restriction maps of the epidemiologically important Salmonella serotypes Enteritidis (E), Typhimurium (T) and Paratyphi B d-tartrate+ (PdT+). DNA-gene-sequences representing SPI1-5 were analyzed by the following three-step procedure on forty six strains (20 E, 20 T and 6 PdT+) collected from food samples from animal origin in Germany. First, SPI DNA-sequences were detected by PCR using primer-pairs designed to target each SPI (1: invE/A, 2: ttrC, 3: mgtC, 4: spi4R, and 5: sopB). Results showed that all the strains were positive for the five SPI-sequences. Second, pulsed-field gel electrophoresis (PFGE) of the whole DNA by restriction with XbaI. The strains were discriminated into 29 PFGEprofiles (7 in E, 16 in T, and 6 in PdT+) which were serotype-characteristic. Third, probes for the selected SPI DNA-sequences were hybridized with the PFGE restriction fragments. It was found that: the SPI2-probe mapped on an identical fragment of about 250-260 kb in all restriction patterns, whereas the other probes mapped on different fragments. So, the SPI1-probe hybridized with fragments of approximately 662 kb in E, [649, 367, 480, and 506] kb in T, ASM Conference on Salmonella

5(B)

DESIGNING OF PCR PRIMERS FROM THE INTERNAL TRANSCRIBED SPACER (ITS) REGION OF 16S-23S RRNA GENE FOR THE SPECIFIC DETECTION OF SALMONELLA SPP

H. Y. Tsen, T. H. Chiu, T. R. Chen, C. L. Hung; National chung Hsing University, Taichung, Taiwan, ROC, Taichung, TAIWAN REPUBLIC OF CHINA. Salmonella is one of the most important pathogens that may cause human salmonellosis and animal infections. Rapid detection of this bacteria spp. in food, feed, and clinical samples is important. Since 16S rDNA and the 16S-23S rDNA spacer region were the most popular target for bacteria taxonomy and some PCR primers based on the 16S 30

POSTER ABSTRACTS POSTER ABSTRACTS

and [380, and 341] in PdT+ patterns; SPI3-probe with fragments of 185 kb in E, [70, and 80] kb in T, and 63 kb in PdT+ patterns; the SPI4-probe, with fragments of 296 kb in E, [226, 367, 261, and 199] kb in T, and 306 kb in PdT+patterns; and the SPI5-probe, in 820 kb in E, [289, 506, 517, 725, and 302] kb in T, and 840 kb in PdT+ patterns. These data suggest that SPI2 is inserted in one conserved XbaI-fragment in all three Salmonella serotypes investigated. Each of the other four SPIs in contrast, appear to be located in four different conserved XbaI segments in serotype Enteritidis, and four different non-conserved fragments in serotype Typhimurium. In Paratyphy B (dT+) strains, apparently, SPI3, SPI4 and SPI5 are located in three serotype-conserved XbaI-fragments. the potential for adaptive resistance in Salmonella enterica serovar Enteritidis, Typhimurium, and Virchow to commonly used antibacterial agents and to identify mechanisms underlying any resistance obtained. Salmonella strains were serially exposed to sub-inhibitory concentrations of erythromycin (ERY), benzalkonium chloride (BKC), chlorohexidine (CHX) and triclosan (TLN). Following each passage the MIC of the antibacterial and any adaptive resistance was recorded. Adaptive resistance was promoted in all strains investigated. Permeability changes in the outer membrane, including LPS, cell surface charge and hydrophobicity and the presence of an active efflux were investigated as possible resistance strategies. The outer membrane and LPS bands were analysed by SDS-PAGE and visualised by Coomassie blue and silver staining. The cell surface charge and hydrophobicity were investigated employing microelectrophoresis and microbial adhesion to hydrocarbons (MATH assay), respectively. Efflux activity was examined by comparing the level of resistance in pre- and post-adapted strains in the presence of the efflux inhibitor, reserpine. Examination of the outer membrane LPS did not reveal any significant changes between pre- and post-adapted strains. There was no correlation between cell surface charge and hydrophobicity, however most of the parent strains were not significantly hydrophobic, whereas those that were resistant to the presence of antibacterials were. An efflux system was the most likely mechanism of BKC and CHX resistance in all strains investigated. The adaptive resistance to erythromycin, BKC, CHX and TLN in cells of Salmonella was stable for over 30 passages in biocide-free media. In summary, increased cell surface hydrophobicity and the presence of an active efflux pump could facilitate the acquisition of antibacterial resistance in Salmonella enterica, providing crossresistance to a range of antibiotics/biocides.

7(A)

CHARACTERISATION OF THE S. TYPHIMURIUM RPOE REGULON

G. Rowley1, S. Humphreys1, M. Fookes 2, A. Thompson3, A. Stevenson 1, A. Ivens2, J. Hinton3, J. Kormanec4, M. Roberts1; 1 University of Glasgow, Glasgow, UNITED KINGDOM, 2The Sanger Institute, Cambridge, UNITED KINGDOM, 3The Institute of Food Research, Norwich, UNITED KINGDOM, 4Institute of Molecular Biology, Bratislava, SLOVAKIA. The extracytoplasmic stress response (ESR) is controlled by at least two partially overlapping pathways in Salmonella, the alternative sigma factor óE (rpoE) and the two-component regulator, cpxAR. These pathways regulate the expression of various genes in response to different environmental stimuli. We are currently investigating the potential roles of ESR regulated genes in the Salmonella stress response and virulence, and have previously demonstrated that rpoE is critically important for virulence of S. typhimurium in a mouse model. In order to establish putative members of the Salmonella rpoE regulon we have utilised a variety of techniques. These include microarray analysis, a 2 plasmid system, S1 mapping, RT-PCR and reporter studies. Each of these methods has their own limitations, but through using them in combination we aim to draw a definitive consensus of rpoE regulated genes. From these preliminary investigations, as well as identifying genes reported to be rpoE regulated in other organisms, we have also discovered a number of other genes that thus far have not been reported as rpoE regulated. For some of the putatively rpoE regulated genes identified we have tried to ascertain their roles in the ESR and virulence, using a variety of in vitro assays as well as the well recognised competition index in vivo.

9(C)

MIMOTOPES OF VI ANTIGEN OF SALMONELLA ENTERICA SEROVAR TYPHI IDENTIFIED FROM PHAGE DISPLAY PEPTIDE LIBRARY

K. Thong1, S. Tang1, W. Tan2, S. Devi1, T. Pang3, L. Wang4; 1 University of Malaya, Kuala Lumpur, MALAYSIA, 2University Putra Malaysia, Kuala Lumpur, MALAYSIA, 3WHO, Geneva, SWITZERLAND, 4CSIRO, Victoria, AUSTRALIA. The capsular polysaccharide Vi antigen (ViCPS) is an essential virulence factor and also a protective antigen of Salmonella enterica serovar Typhi. A random 12-mers phage displayed peptide library was used to identify mimotopes (epitope analogues) of this antigen by panning against a ViCPS-specific monoclonal antibody, mAb ATVi. Two consensus sequences were identified after 4 rounds of biopanning. These two sequences were also obtained in a similar panning process using pooled sera from patients with a confirmed diagnosis of typhoid fever, suggesting they mimic immunodominant epitopes of ViCPS antigens. Binding of mAb ATVi to the mimotopes were specifically blocked by ViCPS, indicating that they interact with the same binding site (paratope) of the mAb. Data and reagents generated in this study have important implications for the

8 (B)

MECHANISMS OF ADAPTIVE RESISTANCE IN SALMONELLA ENTERICA

M. Braoudaki, A. C. Hilton; Aston University, Birmingham, UNITED KINGDOM. Bacterial resistance to antibiotics and biocides is a widespread problem, which may be exacerbated by the commonplace and often unnecessary addition of biocides into household products. The aim of this study was to investigate Pathogenesis, Epidemiology, and Vaccine Development

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POSTER ABSTRACTS POSTER ABSTRACTS

development of peptide-base diagnostic test and may also provide a better understanding of the pathogenesis of typhoid fever. methods and is considered a `a gold standard' for subtyping of Salmonella spp. However, the current protocol is time consuming and can hamper rapid, real time results in cases of outbreaks. Thus, a simple PFGE protocol that is rapid, simple but at the same time retain the quality of results, needs to be adopted. We have modified our previous PFGE protocol (a 3- day method) to a one-day method based on the PULSENET protocol. We applied this revised protocol to subtype more than 200 strains of S. Typhi to construct a `genotype' database of Malaysian isolates for purpose of surveillance and identification of new clusters of outbreaks. Considerable genetic diversity exists among Malaysian S. Typhi isolates associated with sporadic cases of typhoid fever but outbreak isolates were more clonal in nature,supporting our previous findings (JCM 32:1135-1141). The revised rapid protocol was very useful in providing realtime data in identifying a new cluster of S.Typhi in a recent outbreak of typhoid fever in Malaysia.

10(A)

MOLECULAR EPIDEMIOLOGY OF SALMONELLA ENTERICA SEROVAR PARATYPHI B IN MALAYSIA

K. Thong1, Y. Goh 1, S. Puthucheary1, R. Yasin 2; 1University of Malaya, Kuala Lumpur, MALAYSIA, 2Institute of Medical Research, Kuala Lumpur, MALAYSIA. In Malaysia,Salmonella enterica serovar Paratyphi B (1,4,[5],12: b: 1,2) is relatively important as it is the fourth most common Salmonella serovar isolated. Eighty-six human strains of S.Paratyphi B isolated in Malaysia between 19821983, 1992, and 1996-2002 were analyzed by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility tests. Sixty-four strains were d-tartrate-negative (dT-) while 22 strains were d-tartrate-positive (dT+). Thirty-seven percent of the S. Paratyphi B strains were resistant to one or more antimicrobial agents. PFGE analysis clearly distinguished the dT- and dT+ strains into two clusters based on the unweighted pair group average method (UPGMA). Twentytwo XbaI-pulsotypes and 17 pulsotypes were observed among the dT- and dT+ strains respectively. The present study showed that PFGE was very discriminative with 33.7% of the strains yielding distinct fingerprints. Paratyphoid fever in Malaysia is probably caused by one predominant, endemic clone of S. Paratyphi B dT- with various subtypes. The data also showed that there was no distinct correlation between the molecular subtypes and the different localities in Malaysia, implying mobility and movement of strains in the country. Moreover, there was no association between the pulsotypes with the severity of the disease indicating that the disease outcomes are probably multifactorial. This is the first report on the application of PFGE on a large collection of S. Paratyphi B in Malaysia.

12(C)

ANTIBIOGRAMS FOR THE HUMAN AND ANIMAL ISOLATES OF SALMONELLA TYPHIMURIUM IN TAIWAN AND TRACING THE CLONAL RELATIONSHIP BY PULSED FIELD GEL ELECTROPHORESIS ANALYSIS

T. Hau Yang, L. Jer-shan, H. Hsien-Yee; National Chung Hsing University, Taichung, Taiwan, ROC, Taichung, TAIWAN REPUBLIC OF CHINA. Studies on the antibiograms for 45 human isolates and 87 animal isolates of Salmonella Typhimurium collected from 1990 to 1996 in Taiwan using tetracycline (Tc), sulfisoxazole (G), ampicillin (Am), chloramphnicol (C), streptomycin (S), trimethoprim-sulfamethoxazole (Sxt), kanamycin (K), gentamycin (Gm), norfloxacin (Nor) and cefoperazone (cfp) revealed that the antibiograms for human and animal isolates are quite similar. The major resistant pattern for these Salmonella strains is TcGAmSC. Both the human and animal isolates are highly resistant to front line antibiotics, such as tetracycline, sulfisoxazole, ampicillin, streptomycin and chloramphenicol, but are sensitive to fluoroquinolone antibiotics, such as norfloxazin and cefoperazone. 58.6% of the animal isolates and 68.9% of the human isolates are multidrug- resistant. When pulsed field gel electrophoresis (PFGE) patterns obtained from the digestion of chromosomal DNA with XbaI and SpeI for those isolates were compared, results showed that the major subtypes for isolates from both origins were the same. They showed X5S4 (or XgSf) pattern. Strains in this major PFGE pattern are multi-drug resistant, i.e., they are chloramphenicol, streptomycin, sulfonamide and tetracycline (ACSSuT) resistant. As the sizes of the integron genes were analyzed using PCR primers 5'CS/3'CS (specific for class I integron), strains from those major subtype, i.e., X5S4, for both the animal and human isolates showed identical molecular sizes, i.e., 1.0 and 1.2 kb. Thus, certain PFGE patterns are the major subtypes and the similarity of antibiotic resistant pattern for S. typhimurium strains may be due to the recirculation of those strains between human and animal hosts. ASM Conference on Salmonella

11(B)

MACRORESTRICTION ANALYSIS OF MALAYSIAN ENTERICA SEROVAR TYPHI STRAINS ISOLATED FROM YEARS 1989 THROUGH 2003

K. Thong1, K. Lai1, S. Puthucheary1, A. Ismail2, K. Kam3, R. Yasin 4; 1University of Malaya, Kuala Lumpur, MALAYSIA, 2 University Science Malaysia, Kelatan, MALAYSIA, 3Public Health Laboratory Centre, Hong Kong, HONG KONG SPECIAL ADMINISTRATIVE REGION OF CHINA, 4Institute for Medical Research, Kuala Lumpur, MALAYSIA. Typhoid fever is a systemic prolonged febrile illness caused by S. Typhi. It continues to be a worldwide health problem, especially in developing countries with poor sanitation, and poor standards of personal hygiene. Effective epidemiological surveillance requires rapid, discriminative, reproducible tools to differentiate closely related strains in outbreak situations. Pulsed field gel electrophoresis (PFGE) is recognized to be superior to most commonly used molecular

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POSTER ABSTRACTS POSTER ABSTRACTS

13(A)

GENE EXPRESSION PROFILING OF SALMONELLA TYPHIMURIUM DT104 DURING ACID ADAPTATION

A. Hermans 1, P. Berk 2, M. Mols1, T. Abee 3, M. Zwietering3, H. Aarts 1; 1RIKILT Institute of Food Safety, Wageningen, NETHERLANDS, 2Microbiological Laboratory for Health Protection, RIVM, Bilthoven, NETHERLANDS, 3Laboratory of Food Microbiology, Wageningen University, Wageningen, NETHERLANDS. During the last decades, the incidence of foodborne diseases has increased in many parts of the world and new foodborne pathogens, like Salmonella Typhimurium DT104, have been identified. Foodborne pathogens have the potential to adapt to a wide variety of food preservation related stress conditions, i.e. starvation, temperature extremes and (weak) acids, which can result in an increased survival in the GItract. The objective of our research is to analyze gene expression profiles of acid tolerant and acid sensitive Salmonella Typhimurium DT104 isolates to gain detailed insight in the molecular pathways involved in low pH survival. The expression of stress response and virulence related genes during acid adaptation and differences in gene expression between acid tolerant and acid sensitive Salmonella Typhimurium DT104 isolates are of our interest. Acid tolerant and acid sensitive variants were identified from a number of food and human Salmonella Typhimurium DT104 isolates, by exposure to pH=2.5 after adaptation at pH=5.0. A small thematic pilot microarray was constructed containing 60mer oligo's homologous to 70 Salmonella Typhimurium LT2 stress response and virulence related genes and Salmonella Typhimurium DT104 specific antibiotic resistance. Cells of acid tolerant and acid sensitive Salmonella Typhimurium DT104 food and human isolates were harvested at four time points in the late exponential, and stationary growth phase during the acid adaptation at pH=5.0. The obtained RNA samples were hybridized to the thematic microarray. Furthermore the acid survival at pH=2.5 was measured at the different growth phases. Microarray data analysis resulted in many differential expressed genes between growth phases at pH=5.0. Furthermore a comparison of the acid tolerant and acid sensitive isolates resulted in a limited number of differential expressed genes. Nevertheless some interesting acid survival related genes were up regulated in the acid tolerant isolate. Subsequently, a larger thematic microarray was constructed. This microarray contains 50mer oligo's homologous to approximately 450 Salmonella Typhimurium LT2 stress response and virulence related genes, housekeeping genes, LPS related genes, many putative genesand Salmonella Typhimurium DT104 specific antibiotic resistance genes. New results obtained from the larger microarray, will be compared with the results from the smaller microarray. Furthermore the differential expression of the additional genes will be studied between acid tolerant and acid sensitive Salmonella Typhimurium DT104 food and human isolates. The thematic microarray provides indeed a tool to gain detailed insight in the molecular pathways involved in low pH survival. Pathogenesis, Epidemiology, and Vaccine Development

14(B)

INVESTIGATION OF THE DISTRIBUTION AND CONTROL OF SALMONELLA ENTERICA SEROVAR ENTERITIDIS PT6 IN A LAYER BREEDING AND LINKED COMMERCIAL LAYING OPERATION

R. H. DAVIES, E. Liebana, M. Breslin; Veterinary Laboratories Agency, Addlestone, surrey, UNITED KINGDOM. Investigations were carried out in a layer breeder hatchery, a layer parent rearing farm, a layer parent farm and in a commercial pullet rearing and cage layer farm where S. Enteritidis PT6 had become established in birds originating from an infected breeding company. PT6 was initially found to be persisting in focal points in the hatchery, such as hatcher ventilation ducting, tray wash areas and waste areas, but improvements to the tray washer and metering of disinfectant was followed by a rapid disappearance of contamination. Several different phage types of S. Enteritidis were found in the hatchery but most of these proved to be genotypically identical with PT6 by PFGE, Pst1/Sph1 ribotyping and plasmid profile analysis. Investigations of contaminated layer breeder and rearing sites showed that the terminal disinfection programmes in place were largely effective in that no carry-over of infection occurred, despite a low level of contamination persisting after cleaning and disinfection, and the organism was rapidly eliminated from the organisation. Infection with PT6 originating from chicks from the hatchery was investigated on a commercial pullet rearing farm. After several rounds of treatment with a fluoroquinolone antibiotic and competitive exclusion infection was cleared from 5 of 6 houses. In one house no Salmonella was found in faeces or cloacal swabs but was present in dust in one of six houses. Sampling carried out after cleaning and disinfection and repopulation confirmed clearance of the organism from the site, but infection did become established in a large commercial cage laying house receiving the birds. This infection has persisted for over 2 years to infect new flocks placed in the laying house, and has spread to other flocks on site despite prior vaccination of birds with killed and live vaccines. In common with many other cage layer sites the difficulties of effective cleaning and disinfection and pest control have led to levels environmental contamination challenge which overcome vaccine protection. Molecular fingerprinting results demonstrated both persistence and diversification of clones originally found in the breeding company. The implications of these findings for monitoring and control of S.Enteritidis in poultry breeding and production and the results of ongoing interventions using competitive exclusion combined with vaccination will be discussed in the presentation.

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15(C)

SALMONELLA CONTAMINATION OF EGGS FROM VACCINATED AND NON-VACCINATED COMMERCIALLY LAYING FLOCKS

R. H. Davies, M. Breslin; Veterinary Laboratories Agency, Addlestone, surrey, UNITED KINGDOM. Eggs were collected monthly from 12 cage-layer flocks on 4 farms where Salmonella Enteritidis was present in vaccinated flocks despite vaccination with an S.Enteritidis bacterin. Where possible hens were also taken for culture at the end of the laying period and faecal and environmental samples were taken from the laying houses before and after cleaning and disinfection. 24 batches of 6 egg shells from the 13,652 tested (0.18% [0.11-0.26 CI95] single egg equivalent) were positive for S.Enteritidis and 54 (0.40% [0.30-0.52 CI95] single egg equivalent) for other serovars. 6 batches of 13,640 (0.04% [0.02-0.10 CI 95] single egg equivalent) egg contents, bulked in 6 egg pools, contained S.Enteritidis and 3 batches contained other serovars. In addition 3 further batches contained S.Enteritidis in both contents and shells and 2 other batches contained other serovars in both. The total level of contamination by S.Enteritidis of both contents and shells found in vaccinated flocks was therefore 33 batches/ 13,682 eggs (0.24% [0.17-0.34 CI95] single egg equivalent). The total of contamination for any Salmonella serovar was 92 batches/13,682 eggs (0.68% [0.55-0.84 CI95] single egg equivalent). These results contrasted significantly with the findings of testing of eggs from 3 unvaccinated flocks prior to this study where 21 batches of egg shells from a total of 2,101 eggs (1.0% [0.63-1.56 CI95] single egg equivalent) and 6 batches of contents from 2,051 eggs (0.29% [0.11-0.64 CI 95] single egg equivalent) were contaminated with S.Enteritidis. S.Enteritidis was found in 67/699 (9.6%) of vaccinated spent hens and 64/562 (11.4%) of bulked fresh faecal samples taken from laying houses. Serotypes other than S.Enteritidis were found in 0.4% of spent hens and 14.4% of bulked faeces samples, but the presence of Salmonella in the flocks was most readily detected by testing environmental samples such as spillage from egg belts, beneath cage stacks and dust, of which 25.6% and 19.0% of 961 samples contained S.Enteritidis or other serovars respectively. Failure to adequately clean and disinfect laying houses and to control mice appeared to be a common feature on the farms.

acid, and ciprofloxacin) was demonstrated for mutants of Salmonella enterica serovar Enteritidis induced following exposure to the sanitizer chlorine (25 ppm) or to the preservatives sodium benzoate (1.0% w/v) and acetic acid (0.05% w/v). Chlorine was tested since it is a common sanitizing agent used on the farm and in food processing facilities. Extent of decreased sensitivity to antibiotics varied between mutants selected on a given agent. Mutants induced on chlorine or acetic acid exhibited cross-resistance to all antibiotics, however, sodium benzoate mutants exhibited cross-resistance to only tetracycline and ciprofloxacin. Antibiotic susceptibility of selected mutants was restored to levels similar to wildtype strains following complementation experiments with a functional marR, suggesting that mar mutation was responsible for resistance. The multiple antibiotic resistance (mar) operon is a global regulator controlling intrinsic resistance towards structurally and functionally unrelated antibiotics and other noxious agents. Mutants (n = 10) serial subcultured (10 passages) in media void (TSB) of the inducing agent maintained decreased antibiotic sensitivity. Surprisingly, no mutation was found upon sequence analysis of the mar operon of the chlorine induced mutants. Expression of marA, tolC, soxS, and acrB in mutants is being determined using Northern blotting. No naturally occurring mar mutants were found among Salmonella isolates (n = 20) obtained from produce or irrigation water. Isolates collected from farm (animal environments) and food processing environments have yet to be screened. Results highlight the importance of monitoring the use of antimicrobial agents to ensure that concentrations capable of inactivating target pathogens are used.

17(B)

PRECISE EXCISION OF THE LARGE PATHOGENICITY ISLAND OF SALMONELLA ENTERICA SEROVAR TYPHI

S. M. Bueno1, J. A. Fuentes2, A. N. Trombert 2, A. A. Murillo2, P. Youderian 3, G. C. Mora2; 1Universidad de Chile, Santiago, CHILE, 2Universidad Católica, Santiago, CHILE, 3Texas A&M University, College Station, TX. The large pathogenicity island (LPI, also known as SPI-7) is a 133.606 bp segment closely linked to the phenylalanine tRNA (tRNApheU) coding gene, pheU, in the Salmonella enterica sv. Typhi genome. The LPI contains at least 151 open reading frames, including the virulence factor SopE and genes involved in the synthesis of the Vi antigen and type IV pili. The present work describes the precise excision of the entire pathogenicity island, occurring spontaneously in clinical isolates of S. Typhi as well as in the laboratory strain Ty2. Strains that have lost this region acquire altered colony morphology and fail to produce Vi antigen. As a consequence these strains are no longer aggluttinable by anti-Vi antibodies and they become resistant to the litic phage Vi-II. Furthermore, strains that have lost the LPI are almost ten-fold more invasive in vitro in the human epithelial cell line HEp-2 than wild type strains. Analysis of S. Typhi CT18 sequenced genome allowed us to determine that LPI is flanked by two 52-bp direct repeats, one of them overlapping the tRNApheU coding gene. PCR and sequence ASM Conference on Salmonella

16(A)

DECREASED SUSCEPTIBILITY OF SALMONELLA ENTERITIDIS TO ANTIBIOTICS FOLLOWING EXPOSURE TO CHLORINE OR FOOD PRESERVATIVES

K. Matthews, M. Gandhi; Rutgers University, New Brunswick, NJ. The ability of bacteria to persist on the farm or during food processing may be linked to acquired or inherent resistance to antimicrobial agents. This has a direct impact on food safety and ultimately human illness. Decreased sensitivity to multiple antibiotics (tetracycline, chloramphenicol, nalidixic 34

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analyses showed that the mayority of the LPI minus strains have lost the entire island by recombination between the 52bp direct repeats and that the precise excision event produces an intact copy of pheU gene at its novel join point. In addition, we have also detected the partial excision of LPI, occurring beetwen one of the 52-bp repeats mentioned above and a related 16-bp sequence present downstream of the viaB locus. The excision frecuency of the entire LPI was estimated to be 5x10-6 per cell in the wild type strain, and occurs by a recombination event dependent on several related prophage integrases encoded in the S. Typhi genome. Taken together our results suggest that LPI, as other pathogenicity islands described in the literature, is a mobile genomic element, perhaps in the process of becoming stably inserted in the S. Typhi genome.

This work was funded by grants FONDECYT 1020485 to G.C.M and Senior International Fellowship TW05645 to P.Y. from the NIH Fogarty Center. S. M. B. is supported by fellowships from CONICYT-Chile.

19(A)

THE INFLUENCE OF SALMONELLA ENTERICA SV. TYPHIMURIUM FIMBRIAE ON COLONIZATION AND PERSISTENCE OF THE INTESTINE AND ORGANS OF MICE

E. H. Weening, R. A. Kingsley, A. D. Humphries, R. M. Tsolis, A. J. Baumler; Texas A&M HSC, College Station, TX. Fimbriae are involved in host pathogen interactions by mediating attachment and by serving as targets for the host immune response. Whole genome sequencing reveals that S.Typhimurium contains 11 putative fimbrial operons. Of these fimbriae, 10 are assembled by the usher-chaperone pathway. We are studying the role of these fimbriae in virulence and persistence in mice. We constructed a collection of strains, each carrying a deletion of a different fimbrial operon. Virulence of the fimbrial deletion strain collection was compared with that of the wild type in competitive infection experiments using the susceptible mouse lineage Balb/c. The ability of mutants to persist long term in the intestine was compared to the wild type using a resistant mouse lineage (CBA/J). Our results suggest that the main function of fimbriae is to mediate intestinal persistence while deletion of individual fimbrial operons has little effect on the ability to cause lethal morbidity in mice.

18(C)

VIRULENCE OF SALMONELLA TYPHIMURIUM DT104 IN VITRO AND IN VIVO

P. A. Berk, R. de Jonge; National Institute for Public Health and the Environment, Bilthoven, NETHERLANDS. During the last decades, the incidence of foodborne diseases has increased in many parts of the world and new foodborne pathogens have been identified. Some findings indicate that these emerging bacteria, in particular Escherichia coli O157:H7 and Salmonella Typhimurium DT104, are more virulent in humans than other E.coli's and Salmonellas. In patients infected with S. Typhimurium DT104 a higher mortality and morbidity are found and E. coli O157:H7 strains are found to be more acid resistance, particularly at low temperature, than other E. coli's. Since the stomach with its acidic pH is recognised as the first line of defence against foodborne pathogens, acid resistance may be an indicator for virulence. We selected 4 strains from S. Typhimurim DT104: two strains isolated from food products and two from patients, in both cases one acid resistant and one acid sensitive strain. These selected strains will be tested for virulence in in vitro cell-lines and in vivo in rats. The human intestinal cell line, Caco-2, and a rat intestinal cell line, IEC-18 will be used to determine invasion and adhesion of the pathogens into these cell lines. Also the IL-8 and MIP-2 production by respectively the Caco-2 and the IEC-18 cells after infection with the selected bacteria will be determined. Preliminary results show no correlation in vitro in Caco-2 cells between acid resistance and invasion, adhesion or IL-8 response, nor between isolation source and invasion, adhesion or IL-8 response. In vivo, several microbiological (e.g. cell counts in spleen) and heamatological (e.g. white bloodcell concentrations) parameters in the rat will be determined after infection with the selected S. Typhimurim DT104 strains. These data will also be used to do a parallellogramapproach to predict the human situation in vivo using the in vitro data from the human- (Caco-2) and rat- (IEC-18) cell-lines and the in vivo data from the experiments with rat.

20(B)

REPRODUCTIVE TRACT COLONISATION BY SALMONELLA ENTERITIDIS IN LAYING HENS

J. M. De Buck, F. M. Van Immerseel, F. Haesebrouck, R. Ducatelle; Ghent University, Merelbeke, BELGIUM. The contamination of table eggs with Salmonella is the most important source of Salmonella food poisoning in humans. Infected laying hens may intermittently lay infected eggs for a long time. How the infection of the oviduct is established and maintained hitherto was largely unknown in laying hens. The infected hens may harbour the bacteria in the ovary or in the oviduct. The purpose of the present study was to characterize the pathogenesis of oviduct infection in laying hens. In a first series of experiments, primary cell cultures of the isthmal and magnal tubular gland cells were used to detect differences in invasion and proliferation between S. Enteritidis isolates. Internalisation in the glandular cells was demonstrated by confocal scanning microscopy and corroborated with a gentamicin protection assay. High invasive and low invasive isolates were found, with mostly more invasion in the isthmus than in the magnum. In a second series of experiments in vivo oviduct loops were created for the investigation of the invasion of the oviductal tissue from the lumen of the oviduct. Loops in the isthmus and magnum were injected with S. Enteritidis. The infection ratio of the loops was significantly higher for the isthmus (1.3 x 10-3) than for the magnum (5.3 x 10-5). In a last experiment, laying hens were intravenously inoculated with 5.10 7 cfu of S. Enteritidis. Four days post infection the bacteria were found in the tubulary glands by immunohis35

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tochemistry. Isolation of the tubulary gland cells of the isthmus and magnum under gentamicin pressure proved the S. Enteritidis bacteria to be located intracellularly in these glands. The ratio of S. Enteritidis bacteria per isolated tubular gland cell was 2.1 x 10 -4 and 4.8 x 10 -5 for isthmus and magnum. In conclusion : S. Enteritidis can colonise the oviductal tissue, with a preference for the isthmus by invading the tubular glands intracellularly. Different isolates of S. Enteritidis invade oviductal glandular epithelial cell at a different rate. The bacteria may reach these cells from the oviductal lumen as well as from the blood.

22(A)

LIVE SALMONELLA VACCINES BY GRANULOCYTE ADAPTATION

T. T. Kramer; Iowa State University (emeritus), Fort Collins, CO. Live vaccines were developed against septicemic salmonellosis of pigs caused by Salmonella cholerae-suis (SCS), against gut infection and egg transmission by Salmonella Enterica var. enteritidis (SE) of chickens, and against septicemia of chickens caused by Salmonella Enterica var. gallinarum and pullorum (SGP) . Virulence attenuation of these strains was achieved by sequential (repeated) adaptations of Salmonella to granulocytes of pigs and chickens respectively. Virulence attenuation has not affected immunogenicity. All vaccinations and challenges were by oral gavage. I. Virulence attenuation by neutrophil (granulocyte) adaptation of SCS occurred stepwise. Each passage through neutrophils decreased virulence for mice and pigs vis a vis the previous passage. The safety and efficacity of the SCS vaccine was tested satisfactorily in numerous field trials. II. A virulent avian strain of SE was made avirulent for hens by eleventimes repeated exposure to heterophils (avian granulocytes). Fecal excretion of the vaccine strain, and subsequently of challenge strains, ceased one week after gavage of hens with 108 colony forming units (CFUs) of the vaccine or challenge strains respectively. Seventy five percent of control hens remained infected after challenge for 40 days or longer. None of 1,019 eggs and egg shells collected after vaccination and challenge of vaccinated hens was infected with Salmonella. An average of 6 percent of control hens laid infected eggs for 70 days or longer after challenge. The SE vaccine strain was given to 60 one-day old chicks at a dose of 10 6,107 or 108 CFUs. None became ill, none died and their six-week weight gains were statistically comparable to uninfected controls. Vaccine SE persisted in their ceca up to 4 or 5 weeks, but not in intestines and internal organs. The SE vaccine was eliminated from ceca in a dose-dependent fashion. III. SGP vaccine was given to 25 day-old chicks at a dose of 107 and 10 8 CFUs. It did not cause illness or death and persisted on the 9th day post infection (p.i.) in only 1 of 25 chicks. All 25 control chicks given the source strain died before the 9th day p.i., and SGP was isolated from internal organs of all. The cellular and molecular basis of virulence attenuation by granulocyte adaptation is under investigation. Granulocyte adapted Salmonellae are phagocytosed with greater avidity than wildtype strains. Granulocyte adapted vaccine strains may be spontaneous DNA-adeninemethyltransferase (Dam-) mutants.

21(C)

PROPHAGE PROFILING OF SALMONELLA TYPHIMURIUM DT104 ISOLATES

A. Hermans 1, A. Beuling 1, T. Abee2, M. Zwietering2, H. Aarts 1; 1 RIKILT Institute of Food Safety, Wageningen, NETHERLANDS, 2Laboratory of Food Microbiology, Wageningen University, Wageningen, NETHERLANDS. During the last decades, the incidence of foodborne diseases has increased in many parts of the world and new foodborne pathogens, like Salmonella Typhimurium DT104, have been identified. An important reason of the emergence of these new foodborne pathogens,in our case Salmonella Typhimurium DT104, could be the acquisition of horizontal transferable genetic elements. The laboratory Salmonella Typhimurium LT2 strain, of which the genome was sequenced, contained already many of those islands and prophages, for example Gifsy and Fels prophages, and Salmonella pathogenicity islands (SPI1-5), which all play a major role in infection and virulence. Furthermore five antibiotic resistance genes of Salmonella Typhimurium DT104 are localized on the Salmonella genomic island I (SGI-I). A genomic subtraction was performed between a Salmonella Typhimurium DT104 and LT2 strain to identify genomic differences between both strains. The resulting Salmonella Typhimurium DT104 specific DNA fragments had many similarities with prophages, SGI-I and also a new lipopolysaccharide (LPS) related gene, which is associated with virulence, was found. These Salmonella Typhimurium DT104 specific DNA fragments were aligned to the recently available public database of the unfinished Salmonella Typhimurium DT104 genome. These sequence data were produced by the Salmonella spp. Sequencing Group at the Sanger Institute and can be obtained from ftp:// ftp.sanger.ac.uk/pub/pathogens/Salmonella. The alignments indicated the presence of three prophages in the Salmonella Typhimurium DT104 genome, which were not present in the Salmonella Typhimurium LT2 genome. One prophage contained the new LPS related gene. Twenty-one food and human Salmonella Typhimurium DT104 isolates were characterized for the presence of the borders and an internal prophage part of the three prophages by PCR. Differences between the Salmonella Typhimurium DT104 isolates were found. Two groups were identified: one group contained all 3 prophages and the LPS related gene, while the other group only contained 1 prophage and not the novel LPS related gene. These results can be useful for additional characterization within the group of a Salmonella Typhimurium DT104.

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ASM Conference on Salmonella

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23(B)

THE USE OF FLOW CYTOMETRY TO DETECT EXPRESSION OF SUBUNITS ENCODED BY 11 SALMONELLA ENTERICA SEROTYPE TYPHIMURIUM FIMBRIAL OPERONS

A. D. Humphries 1, M. Raffatellu1, S. Winter1, E. Weening1, R. A. Kingsley 1, R. Droleskey2, S. Zhang3, J. Figueiredo3, S. Khare3, J. Nunes3, L. Adams3, R. M. Tsolis1, A. J. Baumler1; 1Texas A&M University HSC, College Station, TX, 2USDA, College Station, TX, 3College of Veterinary Medicine, TAMU, College Station, TX. The Salmonella enterica serotype Typhimurium (S. Typhimurium) genome contains 13 putative fimbrial operons termed agf (csg), fim, pef, lpf, bcf, saf, stb, stc, std, stf, sth, sti and stj. Evidence for in vitro expression of fimbrial proteins encoded by these operons is currently only available for agf, fim and pef. We raised antisera against putative major fimbrial subunits of S. Typhimurium, including AgfA, FimA, PefA, LpfA, BcfA, StbA, StcA, StdA, StfA, SthA and StiA. Elaboration of StcA on the bacterial surface could be detected by flow cytometry and immuno electron microscopy after expression of the cloned stcABCD operon from a heterologous T7 promoter in Escherichia coli. To study expression of fimbrial antigens in S. Typhimurium by flow cytometry we constructed strains carrying deletions of either agfAB, pefBACDI, lpfABCDE, bcfABCDEFG, stbABCDE, stcABCD, stdABC, stfACDEFG, sthABCDE, or stiABCDE. Using these deletion mutants for gating, expression of fimbrial antigens was measured by flow cytometry in cultures grown in vitro or in samples recovered 8 hours after infection of bovine ligated ileal loops with S. Typhimurium. FimA was the only fimbrial antigen expressed by S. Typhimurium after static growth in Luria Bertani (LB) broth. Injection of static LB broth cultures of S. Typhimurium into bovine ligated ileal loops resulted in expression of BcfA, FimA, LpfA, PefA, StbA, StcA, StdA, StfA, and StiA. These data show that in vivo growth conditions drastically alter the repertoire of fimbrial antigens expressed in S. Typhimurium.

strains varied from 0.125 ­ 0.5 ìg/ml and 0.002- 0.125 ìg/ml for Nalidixic Acid Sensitive Salmonella typhi (NASST). The common clinical features besides fever were vomiting 38%, diarrhoea 19% and abdominal pain 38%. Forty three percent patients developed complications. Complication rates were significantly higher in NARST strains. Twenty four percent patients with NARST showed failure to Ciprofloxacin, requiring alternative therapy. The median time to fever clearance was 158 hrs (range 96-264hrs) for NARST and 96 hrs (72- 144hrs) in NASST.Salmonella with decreased susceptibility to Ciprofloxacin are on the rise. In view of the increased complications, high treatment failure rates and poor response, quinolones should not be used for managing infections with NARST. Nalidixic acid can be used by clinical laboratories as a indicator drug, as resistance to the drug always confers reduced susceptibility to ciprofloxacin.

25(A)

A GENOMIC ANALYSIS ESTABLISHES THE ROLE OF GUANOSINE TETRAPHOSPHATE (PPGPP) IN THE ENVIRONMENTAL REGULATION OF SALMONELLA VIRULENCE GENE EXPRESSION

A. Thompson1, K. Tedin2, M. Rolfe1, J. Hinton1, S. Lucchini1; 1 Inst. of Food Research, Norwich, UNITED KINGDOM, 2Institut fur Mikrobiologie, Freie Universitat Berlin, GERMANY. Salmonella causes infection of mammalian hosts by coordinating the expression of a number of key virulence genes. In this work we used in-house constructed microarrays to define the ppGpp regulon in Salmonella enterica sv. Typhimurium using a strain deleted for the relA and spoT genes. We investigated the expression of several pathogenicity islands and virulence related genes because it had previously been shown that the mutant strain is severely attenuated in BALB/c mice, and that the strain exhibited reduced expression of both invF and hilA, the major transcriptional activators required for Salmonella Pathogenicity Island (SPI1) expression (K. Tedin, pers. comm.) The expression profile of Salmonella was defined in both the mutant and wild-type strains grown under conditions which are relevant to the gastrointestinal environment. We discovered that ppGpp plays a central and specific role in virulence gene expression and that the expression of all of the known regulators of SPI1, except hilACD and invF are either unchanged or elevated in the mutant strain relative to the wild-type strain. Based on these observations, we propose a model to explain how ppGpp regulates virulence gene expression in Salmonella.

24(C)

QUINOLONE RESISTANT SALMONELLA TYPHI: NEED OF A NEWSCREENING TEST AND CLINICAL RESPONSE TO TREATMENT

R. Gaind, M. Walia, R. Mehta, P. Paul, P. Aggarwal; Vardhman Mahavir Medical College and Aasoc Safdarjang Hospital, Delhi, INDIA. Eighty eight patients admitted with bacteriologically confirmed enteric fever from April 2001- May 2003 were studied for clinical profile and complications in relation to antibiotic susceptibility pattern of S.typhi isolates. Forty three percent of the isolates were resistant to Chloramphenicol, Co-trimoxazole and Ampicillin (MDR). All strains were sensitive to Ciprofloacin as per NCCLS guidelines (MIC<1 ìg/ml). Seventy four percent of the isolates were resistant to nalidixic acid (NARST). Ciprofloxacin MIC of the NARST

Pathogenesis, Epidemiology, and Vaccine Development

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26(B)

SALMONELLA SURFACE-EXPRESSING YERSINIA PESTIS F1 ANTIGEN PROTECTS MICE AGAINST PLAGUE

M. Morton1, H. S. Garmory1, S. D. Perkins1, A. M. O'Dowd 2, K. F. Griffin1, A. K. Turner 2, A. M. Bennett 1, R. W. Titball1; 1 Defence Science and Technology Laboratory, Salisbury, UNITED KINGDOM, 2Acambis, Cambridge, UNITED KINGDOM. The F1 antigen of Yersinia pestis is a major protective antigen against plague. A sub-unit vaccine including the F1 antigen has been shown to protect mice in models of both bubonic and pneumonic plague and is currently in clinical development. However, since the sub-unit vaccine is delivered by injection, there is a requirement to develop an orally delivered plague vaccine. Salmonella-based vaccines potentially offer the advantage of being orally delivered to stimulate long-lived cellular and humoral immune responses following one or two doses. Thus, Salmonella expressing F1 antigen is being considered as a candidate orally delivered plague vaccine. In a previous study, Salmonella enterica serovar Typhimurium expressing the caf operon resulted in high-level expression of F1 antigen on the cell surface (Titball et al., 1997). This Salmonella-based vaccine was shown to afford protection against plague in the murine model of infection following oral administration. In order to further develop the Salmonella-based plague vaccine towards clinical trials, we have investigated the use of attenuated S. enterica serovar Typhi strain BRD1116 (aroA aroC htrA) for the surface-expression of F1 antigen. A recombinant strain of BRD1116 surface-expressing F1 antigen was generated by transforming BRD1116 with plasmid pAH34L encoding the caf operon, which includes the gene encoding F1 antigen and other genes involved in the export and surface assembly of F1 antigen in Y. pestis. Plasmid pAH34L was stably inherited in strain BRD1116 both during in vitro growth and during colonization of murine lungs following intranasal administration. In mice, an immunization regimen of two doses of 1x10(8) cfu of BRD1116/pAH34L given intranasally 7 days apart induced the strongest F1 antigen-specific antibody responses compared to other regimens and protected 13 out of 20 mice from lethal challenge with Y. pestis. The results demonstrate that attenuated strains of S. enterica serovar Typhi expressing F1 antigen may provide an oral vaccine against plague suitable for use in humans.

Salmonella strains from 27 serovars and 15 non-Salmonella strains from 8 different genera were used in this study. PCR with all the Salmonella strains produced a 784 bp DNA fragment, which was absent in all the non-Salmonella strains tested. The detection limit of the PCR was 100 pg with genomic DNA and 3 x 10 4 CFU/ml with serial dilutions of bacterial culture. An enrichment-PCR method was further developed to test the sensitivity of the hilA primers for the detection of Salmonella in faecal samples spiked with different concentrations of Salmonella typhimurium. The method described allowed the detection of S. typhimurium in faecal samples at a concentration of 3 x 102 CFU/ml. In conclusion, the hilA primers are specific for Salmonella species and the PCR method presented may be suitable for the detection of Salmonella in feces.

28(A)

MICROBIOLOGICAL AND CLINICAL CHARACTERIZATION OF EXTRAINTESTINAL SALMONELLA INFECTION

A. V. Rakov1, F. N. Shubin1, V. A. Ivanis 2; 1Institute of Epidemiology and Microbiology SB RAMS, Vladivostok, RUSSIAN FEDERATION, 2Vladivostok State Medical University, Vladivostok, RUSSIAN FEDERATION. Salmonella infection usually proceeds as gastroenteritis, having a brief duration, not demanding treatment of antibiotics, and has low mortality. Much less frequently Salmonella infection proceeds as extraintestinal salmonellosis appeared bacteremia-septicemia and focal infections. It is characterized by high severity and a plenty of lethal outcomes. The aim of this study was an evaluation of focal forms of extraintestinal salmonellosis cases in Primorye Region and the comparative analysis of plasmid characteristics of Salmonella enterica subsp. enterica serovar Enteritidis strains isolated from patients with extraintestinal salmonellosis and Salmonella gastroenteritis. Studying of Salmonella infection in Primorye Region in 1995-2002 has shown that the basic form of an infection was gastroenteritis caused by S. Enteritidis. Extraintestinal salmonellosis was revealed in two patients. At the first patient, 65 years, disease proceeded as Salmonella pleural empyema and has ended with recovery. From feces, pleural pus and blood of the patient it has been isolated S. Enteritidis with plasmid profile 57:3.5 kb. Strains of similar plasmid profile for corresponding half-year have been isolated at 9.6% of gastroenteritis patients, caused by S. Enteritidis, that is they did not dominate over the given period of time, and took only the third place in Salmonella gastroenteritis morbidity. The second patient, a child of 5 months has developed severe Salmonella sepsis and the purulent meningitis, ended with the death of the patient after 8 day of illness. From the patient's cerebrospinal fluid, blood and feces demonstrate isolated S. Enteritidis with plasmid profile 57:2.1 kb. Strains with same plasmid profile in the corresponding half-year had first place in Salmonella gastroenteritis morbidity and have made 49.4 %. The relativity of pathogen strains isolated from patients of extraintestinal salmonellosis and gastroenteritis proves to be true in their uniformity of antibiotic pattern. S. Enteritidis ASM Conference on Salmonella

27(C)

RAPID DETECTION OF SALMONELLA STRAINS BY DIRECT PCR-AMPLIFICATION OF THE HILA GENE

K. Thong1, S. Pathmanathan1, N. Cardona-Castro 2, M. CorreaOchoa3, S. Puthucheary1; 1University of Malaya, Kuala Lumpur, MALAYSIA, 2Instituto Colombiano de Medicina Tropical, Sabaneta, COLOMBIA, 3Universidad de Antioquia, Medellin, COLOMBIA. This study was performed to evaluate the suitability of a PCR procedure using a pair of primers targeting the hilA gene as a means of detecting Salmonella species. A total of 33

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strains of both plasmid profiles isolated in both forms of infection were sensitive to all tested antibiotics. Hence, the conducted studies have shown that extraintestinal salmonellosis was caused by S. Enteritidis demonstrating the leading importance in etiology of infections. At the same time, according to the received data, it is possible to conclude that extraintestinal salmonellosis can be caused by S. Enteritidis of different plasmid profiles, both dominating, and not dominating in a population of the pathogen. Our data agrees with the results received in Spain by Ì. Rodriguez et al. (1998). Extraintestinal salmonellosis was caused as by the dominating genomic group of S. Enteritidis, having the basic etiological importance in development of gastroenteritis, and the others genomic groups of the pathogen which is recognized at Salmonella gastroenteritis.

30(C)

ETIOLOGICAL VALUE RARELY DETECTED SALMONELLA SEROVARS

N. I. Kovalchuk, F. N. Shubin; Research Institute of Epidemiology and Microbiology, Vladivostok, RUSSIAN FEDERATION. The two serovars dominating in the etiology of salmonellosis in Primorsky region are S. Enteritidis and S. Typhimurium. The share of other serovars, which we called rarely detected, is 8.36 %. However, our attraction was drawn by variety of rarely detected Salmonella serovars. 349 strains belonging to 94 serovars were isolated on Primorsky region territory (19 cities and districts) in from 1995 to 2002. All the Salmonella strains were divided into three groups: serovars isolated from patients and bacteria carriers (group A), strains of serovars isolated from food products and objects of an environment (group B) and serovars isolated from people and food products at the same time (group C). Group A includes 69 strains belonging to 37 serovars. One strain represents 23 serovars. 23 strains of 18 serovars (33.33%) had no plasmids. Group B consist of 61 strains differentiated into 28 serovars. Nine strains without plasmids belonged to 8 serovars. Strains of 5 serovars were of two types - with and without plasmids. The most numerous group C includes 219 strains belonging to 29 serovars. For seven years from 2 to 27 strains of each serovar have been isolated. In 25.14% of cases strains did not contain plasmids (more strains without plasmids were isolated from people than from food products-15.06 % and 10.04 % respectively). The link between human disease incidence and food products was detected in 5 cases. Two strains S. Branderburg isolated from minced turkey contained the same set of plasmids as the strain isolated from bacteria circulator two months after. Two strains S. Glostrup isolated from minced turkey in Vladivostok in 1997 and 1998 also had plasmid profile similar to strain S. Glostrup isolated from patient in Terney in 1999. Plasmid profile 55 : 2.6 MDa combined three strains S. Heilerberg isolated from minced turkey and strain isolated from patient a month after that. Two plasmidovars S. Saint-paul were common for people and food stuffs. Strains with plasmid profile 34 : 2.3 : 1.4 MDa were isolated from patient and chicken ham. The second plasmidovar ­ 37 : 2.3 : 1.4 MDa combined strains isolated from minced turkey and from patient. Thus, the obtained results prove the efficiency of plasmid analysis for interspecies typing of rarely detected Salmonella serovars and execution of continuous coordinated salmonella monitoring in the region.

29(B)

MAIN PLASMID VARIANTS SALMONELLA ENTERITIDIS AND THEIR ETIOLOGICAL VALUE

N. I. Kovalchuk, F. N. Shubin; Research Institute of Epidemiology and Microbiology, Vladivostok, RUSSIAN FEDERATION. Salmomella serovar dominating in salmonellosis etiology in Primorsky region is S. Enteritidis, which makes up more than 80 % of all cases. Investigation of plasmid profiles of strains S. Enteritidis isolated on the territory in 1992-2001 identified 76 plasmid variants (plasmidovars) of the bacterium. 5 of which, isolated annually, combined 87.9 % isolates. They were determined as the main ones by frequency criterion with plasmid profiles 38 MDa; 38 : 1.4 MDa; 38 : 2.3 MDa; 38 : 2.6 MDa; 38 : 2.8 MDa. Etiological value of certain plasmidovars varied. The value of 38 MDa decreased from 81.5 % down to 11.2 % from 1992 till 1999. on the contrary, the value strains of plasmidovar 38 : 1.4 MDa was growing constantly during this period and by 1999 it has reached 55.6 %. The same tendency was observed in 2000. The three remaining plasmidovars played less important role in salmonellosis etiology. Plasmidovar 38 : 2.3 MDa was the most valuable in 2000, when it caused 21.6 % of the cases. Since 1999 plasmidovar 38 : 2.8 MDa has no longer been isolated from people, totally it amounted 2 % of all cases. On the other hand in 1998 a new of plasmidovar 38 : 2.6 MDa appeared which caused an outbreak and continues to give low rate of sporadic disease incidence. Its etiological value made up 1.2 %. The investigation allow the following generalization: S. Enteritidis population is characterized by explicit heterogeneity in plasmid profile: different plasmidovars S. Enteritidis are not equal in their value for the infection etiology, etiological value of certain plasmidovars is subject to changing.

Pathogenesis, Epidemiology, and Vaccine Development

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31(A)

NON-TYPHOIDAL SALMONELLA BACTERAEMIA AMONG HIV-INFECTED MALAWIAN ADULTS: HIGH MORTALITY AND FREQUENT RECRUDESCENCE

M. A. Gordon1, H. T. Banda2, A. L. Walsh1, C. F. Gilks 3, C. A. Hart 4, M. E. Molyneux1; 1Malawi-Liverpool-Wellcome Trust Clinical Research Programme, College of Medicine, Blantyre, MALAWI, 2University of Malawi College of Medicine, Blantyre, MALAWI, 3Imperial College, London, UNITED KINGDOM, 4 University of Liverpool, Liverpool, UNITED KINGDOM. Objectives: Non-typhoidal salmonella (NTS) bacteraemia is a common, severe, recurrent illness in HIV-infected African adults. We aimed to describe the presentation and outcome of NTS bacteraemia, the pattern of recurrence, and to determine whether recurrence results from re-infection or recrudescence. Methods: One hundred consecutive adult in-patients with NTS bacteraemia were treated with chloramphenicol for 14 days (all isolates sensitive). Survivors were prospectively followed at monthly intervals to detect recurrence of bacteraemia, or death. Index and recurrent blood culture isolates were typed by antibiogram, pulsed-field gel electrophoresis and plasmid analysis, to distinguish recrudescence from re-infection. Results: Isolates: Index event blood culture isolates comprised 75 Salmonella enteritidis serovar Typhimurium, 19 serovar Enteritidis, 5 other serovars, 1 dual growth Typhimurium and Enteritidis. Susceptibility testing showed 79% resistance to ampicillin and 73% resistance to co-trimoxazole. In-patient survival: In-patient mortality was 47%. 77/78 subjects tested were HIV positive. Median CD4 count was 101 cells/micl. Median haemoglobin was 6.8 g/dl. Anaemia was strongly associated with inpatient death (OR 1.4 (CI 1.11.8) per fall in Hb by 1g/dl, p=0.0014). Out-patient survival: Follow-up was for up to 609 days (median 214). Overall 1-year mortality was 77%. Several features of AIDS (oral thrush, pruritic rash, previous TB, low CD4 count) were associated with poor outpatient survival. Recurrence: Among survivors discharged from hospital, 43% (19/44) had at least one recurrence of NTS bacteraemia. The first episodes of recurrence occurred at 23186 days. 26% (5/19) of these subjects later developed multiple recurrences up to 245 days. Recurrence was not associated with any clinical or laboratory feature of presentation. Focal or suppurative infections were not found at presentation, and were seen only twice at recurrence. Index and recurrent pairs/series of isolates were identical by phenotyping and genotyping, consistent with recrudescence rather than re-infection. Conclusions: NTS bacteraemia has a high mortality (47%) and recurrence rate (43%) in HIV-infected African adults. Anaemia is strongly associated with in-patient mortality. Recurrence is not predicted by any feature of presentation, and is caused by recrudescence with the same organism, rather than re-

infection. As focal or suppurative infections were rarely found, recrudescence may be a consequence of intracellular tissue sequestration. There is an urgent need for improved primary treatment and secondary prophylaxis in Africa.

32(B)

INACTIVATION OF THE SALMONELLA ENTERICA SHUFFLON CORRELATES WITH DECREASED VIRULENCE FOR HUMANS

C. Tam, J. Hackett, C. Morris; Hong Kong University of Science and Technology, Kowloon, HONG KONG SPECIAL ADMINISTRATIVE REGION OF CHINA. Salmonella enterica serovar Typhi uses PilV-free Type IVB pili to facilitate bacterial self-association under conditions (such as low oxygen tension in the gut) favoring DNA supercoiling, which affects Rci-mediated inversion activity of the shufflon. Comparison of the pil operon sequences of serovars Typhi and Paratyphi C, a Salmonella enterica serovar less virulent for humans, identified a remarkable difference located in the shufflon region. In serovar Typhi, the Rci recombinase acts upon two 19 bp inverted repeats to invert the terminal region of the pilV gene, thereby disrupting PilV synthesis and permitting bacterial self-association. In serovar Paratyphi C, however, the shufflon is inactivated by a single basepair insertion in each of the Rci 19 bp substrates, and thus is locked in one orientation such that the PilV2 protein is synthesized and bacterial self-association does not occur. Nevertheless, serovar Paratyphi C also synthesizes and exports the PilS structural pilus protein identical to that of serovar Typhi. This protein, polymerized into pili, may presumably use CFTR as a eukaryotic cell receptor. Taken together, the data strongly suggest that bacterial selfassociation mediated by the serovar Typhi pil operon in the anaerobic human intestine is a key step in the pathogenesis of epidemic enteric fever.

33(C)

AVTIVATION OF HUMAN MONOCYTE-DERIVED MACROPHAGES WITH INTERFERON-GAMMA PRIOR TO INFECTION WITH SALMONELLA TYPHIMURIUM RESULTS IN INCREASED CELL DEATH AT 2 HOURS, AND REDUCED INTRACELLULAR BACTERIAL REPLICATION OVER 24 HOURS.

J. E. Folb, J. D. Edgeworth, G. E. Griffin; St George's Hospital Medical School, London, UNITED KINGDOM. There is a large body of data from clinical, in vitro and in vivo sources, to suggest a crucial protective role for interferon-gamma (IFN-gamma) in the primary immune response to Salmonella infection. In particular, studies in humans genetically deficient in IFN-gamma signaling suggest that IFN-gamma may limit systemic spread of nontyphoidal Salmonella strains. Since Salmonella disseminates within phagocytic cells, it is likely that IFN-g acts at least in ASM Conference on Salmonella

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part by directly modifying phagocytic cell handling of Salmonella. We therefore investigated the effect of preactivation of human monocyte-derived macrophages (MDM) with IFN-gamma, on their subsequent interaction with Salmonella typhimurium. In particular, the effect on early macrophage cytotoxicity and bacterial intracellular replication, was examined. Overnight activation of macrophages with 300U/mL IFN-gamma resulted in increased cell death at 2 hours, as measured by release of lactate dehydrogenase, upon subsequent infection with S. typhimurium. This effect was dependent upon bacterial uptake. However, although the increase in cytotoxicity was partially blocked by inhibition of caspase-1, it did not require intact SPI1 or SPI2. Increased cytotoxicity was also observed after infection of activated macrophages with E. coli and heat-killed S. typhimurium. Macrophage activation with IFN-gamma resulted in a decrease in intracellular bacterial replication over a 24 hour period following infection. CONCLUSIONS: Together these experiments suggest two mechanisms whereby IFN-gamma might serve to limit dissemination of non-typhoidal Salmonella strains: by increasing macrophage cell death after phagocytosis and restricting the subsequent proliferation of Salmonella in surviving macrophages. We are currently investigating whether IFN-gamma has a similar effect on human dendritic cells infected with S. typhiumurium. toxoid fragment C. The group inoculated with the pTETnirB-containing strain showed increased persistence, with Salmonella recovered from more birds and in higher numbers than the 9R strain alone. This preliminary experiment shows that the 9R vaccine strain may be a useful carrier for poultry vaccines. This may be particularly useful as 9R protects both against fowl typhoid and against colonisation by Salmonella serovar Enteritidis. However considerable further characterisation is needed due to the higher levels and longer persistence of the pTETnirB-containing strain.

35(B)

COEXISTENCE OF ANTIMICROBIAL RESISTANT SALMONELLA AND E. COLI IN FOOD

F. Hilbert, S. Mayrhofer, P. Paulsen, F. J. Smulders; Institute of Meat Hygiene, Vienna, AUSTRIA. Horizontal gene transfer in bacteria for acquiring virulence factors or antimicrobial resistance determinants in the host environment has been studied extensively. Foods of animal origin constitute an important environment for the coexistence of pathogens and commensals. We analyzed chicken meat from retail stores for Salmonella and coexisting E. coli and determined the antimicrobial resistance pattern. When Salmonella showed resistance to tetracycline in 91% the E. coli isolate of the same food was also resistant to tetracycline. For ampicillin the rate was slightly lower, i.e. 83%. In contrast, coexisting chloramphenicol resistance occurred in only 11%. For resistance to streptomycin and the quinolones nalidixic acid and ciprofloxcin (most likely not acquired by horizontal gene transfer) the rate was 45%, 50% and 60%, respectively. These results indicate that horizontal gene transfer of antimicrobial resistance determinants in related microbes such as Salmonella and E. coli occurs often and coexistence in food is rather the rule than the exception.

34(A)

EXPRESSION OF A HETEROLOGOUS ANTIGEN IN THE SALMONELLA ENTERICA SEROVAR GALLINARUM 9R VACCINE STRAIN PRODUCES A STRONG HUMORAL IMMUNE RESPONSE IN THE CHICKEN

P. Wigley, R. K. Beal, C. M. Powers, A. L. Smith, P. A. Barrow; IAH, Newbury, UNITED KINGDOM. The use of attenuated strains of both Salmonella enterica serovar Typhimurium and serovar Typhi as vaccine carriers in man and biomedical animal models has been extensively investigated. In contrast, the potential of Salmonella carriers of veterinary vaccines is largely unknown. In this study we investigated the ability of the Salmonella serovar Gallinarum 9R vaccine strain to be used to a deliver a heterologous antigen to chickens. A plasmid, pTETnir15, directing expression of fragment C of tetanus toxoid under control of the nirB promoter, was transformed into the Salmonella serovar Gallinarum 9R vaccine strain by electroporation. The plasmid was checked for stability in the vaccine strain, prior to oral inoculation of 108 cfu into 3-week old SPF Rhode Island Red chickens. A control group was inoculated with the 9R strain without the plasmid. At 11, 18, 26 and 33 days post infection, five birds from each group were killed for post mortem analysis. Blood was taken to determine serum antibody responses to Salmonella and to tetanus toxoid fragment C and liver and spleen samples were taken for bacteriological analysis. Both groups produced a strong antibody (IgG) response to Salmonella, whilst the group inoculated with the 9R strain containing the pTETnir15 also produced a strong antibody response against the tetanus Pathogenesis, Epidemiology, and Vaccine Development

36(C)

SALMONELLA TOXOID VACCINE FOR POULTRY

S. K. Chauhan, V. D. Sharma; G B Pant University, Pantnagar, INDIA. Salmonellosis is havoc to poultry industry and hazard to human health as well. Poultry and poultry products including eggs have a major role as vehicles of transmission in human cases of salmonellosis. Despite of huge amount of work done on immunoprophylaxis against salmonellosis in poultry, much has not been achieved. Till date no single vaccine is available which can protect against ever increasing serovars of Salmonella. The present investigation was under taken to develop a potent, safe and cost effective vaccine against salmonellosis in poultry. Crude toxins extract of Salmonella Weltevreden was isolated. Enterotoxins were then partially purified by fractional salt precipitation, dialysis and Sephadex gel filtration, and evaluated for toxicity on Vero cell line, ilial loop test and mouse paw edema test, which revealed cytotonic and cytotoxic enterotoxin. Two types of saponin adjuvanted toxoid vaccines were prepared by treating crude 41

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toxins and purified enterotoxins (pooled cytotonic and cytotoxic enterotoxins) with formalin. The efficacy of toxoids was tested by vaccinating 21-day-old White Leghorn chicks, subcutaneously. Serum antibody titres by ELISA and lymphocyte stimulation index were examined weekly. At 21day-post vaccination, birds of different groups were challenged with 1x109 C.F.U. of S. Enteritidis, S. Gallinarum and S. Weltevreden intraperitonealy. Both crude and purified toxoids were found to be equally effective and vaccinated birds showed more than 90% protection, measured in terms of mortality, clinical signs, shedding of Salmonella in droppings, postmortem lesions and recovery of Salmonella from liver, spleen and caecum. In addition, 19 Salmonella serotypes tested for agglutination with the serum of crude toxoid vaccinated birds, showed positive results. The dose of crude toxoid was optimized at 100ìg of toxoid per bird, subcutaneously. A booster dose given at 10 weeks after first dose conferred the immunity for one year. Our findings suggest that both purified and crude toxoid vaccines are equally protective and safe. However, crude toxoid vaccine is more cost effective than purified toxoid and can be used efficiently in poultry to prevent salmonellosis. invasion up to 8 hours post infection. After 5 hours post infection both strains were detected only in the lamina propria, indicating that epithelial transmigration requires up to 4 hours to complete in vivo. Ultrastructurally both strains had the same ability to invade intestinal epithelial cells. No differences were detected in the tissue distribution of the sopB mutant and the wild type organism. At 3 hours post infection, when a strong chemokine and cytokine host response occurs, the same profile of CXC chemokine and pro-inflammatory cytokine expression was induced by both strains. In conclusion, our results indicate that the attenuation of the sopB mutant is associated with pathogenic mechanisms other than invasion and distribution in host intestinal tissues.

38(B)

REGULATORY MECHANISM OF SALMONELLA PATHOGENICITY ISLAND 1 (SPI1) GENE EXPRESSION BY LON PROTEASE

A. Takaya1, Y. Kubota 1, T. Tomoyasu 1, E. Isogai2, T. Yamamoto1; 1 Department of Microbiology and Molecular Genetics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, JAPAN, 2Department of Preventive Dentistry, School of Dentistry, Health Sciences University of Hokkaido, Hokkaido, JAPAN. Salmonella invasion of intestinal epithelium requires the expression of the Salmonella Pathogenicity Island 1 (SPI1) genes which are controlled in a complex manner. The expression of hilA gene, which encodes a protein to play a central role in the regulatory hierarchy for SPI1 genes expression, is modulated by several environmental signals and transcriptional regulators. Results from our laboratory have revealed that Lon protease, which is one of stressinduced ATP-dependent proteases, functions as a negative regulator for controlling the hilA expression (Takaya et al., 2002 J. Bacteriol. 184:224-232). To determine how the Lon protease regulates the SPI1 expression, we first examined the transcription of invF from its HilA-dependent and independent promoters by assessment of the amount of SipC protein in the wild type or ÄHilA background. The results have showed that the lon mutation stimulates the expression from both the HilA-dependent and -independent promoters, suggesting that the Lon protease is involved in the degradation of the regulator(s) to activate the both promoters for invF transcription and for hilA transcription. It is known that HilC and HilD proteins activate both promoters. To determine the possibility that the Lon protease interacts with HilC and HilD proteins on SPI1 regulation, we constructed lon and hilC or hilD null mutants and measured the transcriptional activities of SPI1 genes in each mutant. The increased SPI1 expression by lon mutation disappeared by addition of hilC or hilD mutation, suggesting that the both HilC and HilD proteins may be related to the SPI1 regulation by Lon protease. To examine this possibility, we constructed a lon, hilC and hilD triple mutant and examined the amounts of SipC protein in the mutant by the immunoblotting with antiserum. The result revealed that the accumulation of SipC protein by lon mutation disappeared in the lon, hilC and hilD triple mutant. On the otherhand, ASM Conference on Salmonella

37(A)

THE ATTENUATED SOPB MUTANT OF SALMONELLA ENTERICA SEROVAR TYPHIMURIUM HAS THE SAME TISSUE DISTRIBUTION AND HOST CHEMOKINE RESPONSE AS THE WILD TYPE IN BOVINE PEYER'S PATCHES

B. P. Reis1, S. Zhang2, R. M. Tsolis2, A. J. Bäumler 2, L. Adams 2, R. L. Santos1; 1Universidade Federal de Minas Gerais, Belo Horizonte, BRAZIL, 2Texas A&M University, College Station, TX. Salmonella enterica serovar Typhimurium is an important cause of enteric infections in farm animals and it is one of the most frequent food borne infections worldwide. Serovar Typhimurium lacking the sopB gene is attenuated for induction of host inflammatory response and fluid accumulation into the intestinal lumen, which correlates with clinical diarrhea. SopB is an inositol phosphate phosphatase, but its exact role in the pathogenesis of salmonellosis is still unclear. We employed the bovine ileal ligated loop model to compare the tissue distribution of a sopB mutant and wild type serovar Typhimurium. Sections of the Peyer's patches were histologically processed and immuno-stained for detection of serovar Typhimurium using a monoclonal antibody and a peroxidase-based detection system. In addition, samples were processed for transmission electron microscopy, and the profile of expression of host chemokine and cytokine responses was assessed. At 5 min after the inoculation, both sopB mutant and wild type attached to the apical surface of the domed villus epithelium. Attachment to the apical surface of the epithelium at the tips of the absorptive villi was detected at 15 min after inoculation. Both the mutant and wild type were located predominantly at the apical side of the epithelium up to 30 minutes post infection. A significant number of organisms was detected in the lamina propria at 1 hour post infection with progressive

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expressions of both hilC and hilD genes are not affected by the lon mutation. Taken together, it is suggested that Lon protease regulates the expression of SPI1 genes through the modulation of HilC and HilD proteins. was found to be 139/141 published by Rahn et al., 1992, which targets the invA gene. An extended determination of the selectivity using 364 strains showed that the inclusivity was 99.6% and the exclusivity 100% for the invA primer set. For the indication of possible PCR inhibitors derived from the sample DNA, an internal amplification control (IAC) was constructed, which is co-amplified with the invA target gene. In the presence of 300 DNA copies of the IAC, the detection probability for primer set 139/141 was found to be 100%, when using a 10^4 CFU/ml cell suspension as template in the PCR reaction (50 CFU per reaction). The primer set was further validated in an international collaborative study participating 16 laboratories. Analysing 28 coded "blind" DNA samples resulted in an analytical accuracy of 98%. The performance of a PCR method for the detection of Salmonella on artificially inoculated chicken-rinse and pork swab samples was conducted in a second ring-trial participating 15 laboratories. The diagnostic specificity, sensitivity, accordance (repeatability) and concordance (reproducibility) was 100% for the pork samples. For the chicken-rinse samples, the sensitivity, accordance and concordance was also 100% and the specificity of 87.2%. Thus, a simple PCR method specific for Salmonella spp., which amplifies a chromosomal DNA fragment detected by gel electrophoresis, is established through extensive validation and is currently proposed as international standard document. The work done in this study can contribute to meet the increasing demand of quality-assured laboratories for standard diagnostic methods and to facilitate international comparison and exchange of epidemiological data\

39(C)

THE BURDEN OF INVASIVE NTS DISEASE IN MALAWIAN CHILDREN: 1996-2002

S. M. Graham; Wellcome Trust Research Laboratories, Blantyre, MALAWI. The Wellcome Trust Research Laboratory has provided a microbiology service to the paediatric wards of the Queen Elizabeth Central Hospital, Blantyre, Malawi, since 1996. The results of CSF isolates is representative of the burden of bacterial meningitis from 1996 until 2002, but the number of blood cultures received by the laboratory has increased over time as capacity has expanded. Over the 7-year period, there have been 1,735 cases of proven bacterial meningitis in children between 0 and 14 years. Non-typhoidal Salmonella (NTS) was the cause in 226 cases (13%) being the third commonest after Streptococcus pneumoniae (31%) and Haemophilus influenzae type b (23%) and more common than Neisseria meningitidis (5%). The case-fatality rate for NTS meningitis was 58%, significantly higher than for other causes. Over the same period, there were 4,457 cases of paediatric bacteraemia and the commonest cause was non-typhoidal Salmonella (1,873 cases or 42%) followed by S.pneumoniae (13%). The common species were Salmonella typhimurium (1,204 cases) and Salmonella enteritidis (546 cases). In contrast, Salmonella typhi was uncommon (34 cases or <1%). NTS bacteraemia was more common during the rainy season, was associated with malaria and anaemia compared to other causes of bacteraemia, and case-fatality rate was 24%. Chloramphenicol is the first-line antibiotic for suspected septicaemia on the paediatric wards. However, the recent marked increase in multi-drug resistant NTS isolates including resistance to chloramphenicol (in 1999 for S.enteritidis and in 2001 for S.typhimurium) poses a major management dilemma in a resource-poor country such as Malawi where alternative antibiotics are usually not available or too expensive.

41(B)

NTS BACTERAEMIA IS COMMON IN MALAWIAN CHILDREN WITH SEVERE MALARIAL ANAEMIA

S. M. Graham 1, J. Mwenechanya1, M. Tembo1, A. L. Walsh1, M. E. Molyneux1, T. E. Taylor2; 1 Wellcome Trust Research Laboratories, Blantyre, MALAWI, 2 Blantyre Malaria Project, Blantyre, MALAWI. Previous studies, including many studies that pre-date the HIV epidemic, have found that non-typhoidal Salmonella is the commonest cause of bacteraemia/septicaemia in children living in tropical Africa and that mortality is significant. A number of these studies have noted retrospectively that nontyphoidal Salmonella bacteraemia is associated with malaria and with anaemia compared to other common causes of childhood bacteraemia. We determined the prevalence and pattern of bacteraemia in Malawian children with severe malaria and the impact of bacteraemia on case-fatality rate. A prospective study was undertaken involving Malawian children admitted consecutively to the Malaria Research Project ward, Blantyre, between February 1996 and June 1999. Blood cultures were routinely performed on admission. Independent associations with bacteraemia and mortality were determined by logistic regression. Of 701 children with a final diagnosis of severe malaria, 36 (5.1%) had bacteraemia. A wide range of bacteria was isolated and the commonest was non-typhoidal Salmonella (n=18). The rate of bacteraemia was significantly higher in children with

40(A)

MULTI-CENTER VALIDATION OF SALMONELLA PCR: TOWARDS AN INTERNATIONAL STANDARD

B. Malorny 1, J. Hoorfar2, N. Cook3, R. Helmuth1; 1Federal Institute for Risk Assessment, Berlin, GERMANY, 2Danish Veterinary Institute, Copenhagen, DENMARK, 3Central Science Laboratory, York, UNITED KINGDOM. As part of a major international project for the validation and standardization of PCR for detection of five major foodborne pathogens, four Salmonella-specific primer sets were evaluated in-house with respect to their analytical accuracy (selectivity and detection limit) on 43 Salmonella and 47 non-Salmonella strains. The most selective primer set Pathogenesis, Epidemiology, and Vaccine Development

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severe malarial anaemia without coma (11.2%) than in children with cerebral malaria without anaemia (3.2%). This difference was wholly due to the significant association of non-typhoidal Salmonella bacteraemia with severe malarial anaemia (8.2%) compared to cerebral malaria without anaemia (0%) (p<0.001). The overall case-fatality rate was 15% and was higher in children with bacteraemia (22%) than in those without (15%), but this difference was not significant. Independent risk factors for mortality included severe anaemia, low coma score and hypoglycaemia but not bacteraemia. This is the first prospective study of African children to show that NTS bacteraemia is a common complication of severe malarial anaemia. Enteritidis. This serotype has the utmost adaptibility to parasitism among all animal species in which intestines can be sustained for a long time and easily transmitted from one unit to another. The appearance of «uncommon» serotypes and significantly changed appearance of each serotype over 27 years is probably the result of increased consumption of imported food and international travel. Antibiotic resistance in general is low and varies among serotypes.

43(A)

STUDIES ON INTESTINAL COLONISATION INHIBITION AND VIRULENCE OF SALMONELLA PHOP, RPOS AND OMPC DELETION MUTANTS IN CHICKENS

U. Methner1, P. A. Barrow2, D. Gregorova 3, I. Rychlik 3; 1Federal Research Centre for Virus Diseases of Animals, Jena Branch, Jena, GERMANY, 2Institute for Animal Health, Compton, UNITED KINGDOM, 3Veterinary Research Institute, Brno, CZECH REPUBLIC. Immunisation with live and inactivated Salmonella vaccines represents one of the most important methods to increase the resistance of both very young and adult chickens against Salmonella infection. In addition to the development of an adaptive immune response, oral administration of nonattenuated live Salmonella strains to day-old chicks provides protection against infection within a matter of hours by colonisation inhibition. Although currently available, commercial , live attenuated Salmonella vaccines induce protection by adaptive immunity, however, none of them are able to induce protection against Salmonella organisms by colonisation-inhibition and, therefore, are unable to protect newly-hatched birds immediately after oral vaccination. Despite the fact that the degree of colonisation-inhibition is most profound between Salmonella organisms of the same serovar its exploitation by oral administration of an inhibitory live attenuated Salmonella vaccine strain could increase the efficacy of vaccination in the control of the most important serovars of Salmonella enterica. In this study mutants of Salmonella Typhimurium and Enteritidis with deletions in phoP and rpoS, either alone or in combination with ompC, were characterised and tested for their level of attenuation and their ability to inhibit the intestinal colonisation of the isogenic parent strains in chickens. Mutants with deletions in rpoS only demonstrated an unaffected potential to inhibit the intestinal colonisation of the challenge strain but were still fully virulent for the chickens. Mutants with deletions in phoP, either alone or in combination with rpoS, resulted in a high level of attenuation, unimpaired ability to colonise the gut and a nearly unaffected potential to inhibit the challenge strain from caecal colonisation. Mutants with an additional deletion in ompC revealed a reduced capacity of intestinal colonisationinhibition when compared to the control strains and both the single rpoS and the phoP deletion mutants. Mutations in phoP or phoP-regulated genes may therefore be used for the development of live attenuated Salmonella vaccines possessing these novel characteristics.

42(C)

SURVEILLANCE DATA OF SALMONELLA INFECTION IN SPLIT AND DALMATIA COUNTY

A. Babic-Erceg; Public Health Institute, Split, CROATIA. Objectives: To study and determine isolated serotypes of Salmonella spp. strains during 1998-2002, their susceptibility to antibiotics and to compare them to those from 1975 and to International Surveillance Network for Salmonella. Methods: Strains of Salmonella were isolated from stool samples according standard protocols. All isolates were serotyped according to Kauffmann-White scheme. Antibiotic resistance was assesed with Kirby-Bauer disc-diffusion method. Results: Over a 5-year period, 34345 stool samples were examined in Public Health Institute of Split and Dalmatia County. Out of 6665 (19,4%) positive results, 5478 (82,19% ) were Salmonella spp. The average number of isolates over 5 years was 1095 per year. 30 different Salmonella serotypes were determined. The most common serotypes were: S.enterica serotype Enteritidis (84,81%), S.enterica serotype Typhimurium (5,48%) and S.enterica serotype Heidelberg (2,17%). Presence of other serotypes like Leith, Zaiman, Stanleyville, Akanji, Bareilly and other, for our area rare serotypes were observed. Results of serotyping are in concordance with those of International Surveillance Network for Salmonella. Resistance to ampicillin and sulfamethoxazole/trimethoprim occured in 2,26% and 0,24%, respectively. Resistant to nalidixic acid was 13% of strains. No strains resistant to ciprofloxacin, ceftriaxone and ceftazidim were detected. S.Enterica serotype Typhimurium was proved to be the most resistant. Over the same period in microbiological laboratory of the Clinical Hospital Split there were 20 salmonella positive blood cultures, 12 urines, 4 punctates, 2 pus and 1 wound. S.enterica serotype Enteritidis was the most frequent serotype, followed by Typhimurium, Derby and Typhi. In comparison with data from 1975, the increase of salmonella isolates from feces and rectal swabs and decrease from blood cultures, liquor and urine was observed. According to serotypes, in 1975 the most frequent was S.Wien (53,33%), then S.Abony and S.Saint paul, whereas currently most frequent S.Enteritidis was on fourth place with frequency of 5,25%. Conclusion: The most common Salmonella serotype in our region as well as in other countries is S.enterica serotype

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ASM Conference on Salmonella

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44(B)

SALMONELLA PATHOGENICITY ISLAND 10, ADJACENT TO TRNALEUX, IS A HYPER-VARIABLE REGION OF THE SALMONELLA GENOME

A. L. Bishop1, S. Jenks1, S. Baker1, M. Fookes 2, P. O'Gaora1, N. Thomson2, C. Kidgel1, D. Pickard1, A. Ivens2, G. Dougan1; 1Centre for Molecular Microbiology and Infection, Department of Biological Sciences, Imperial College London, London, UNITED KINGDOM, 2The Sanger Centre, Wellcome Trust Genome Campus, Cambridge, UNITED KINGDOM. Background:Salmonella enterica subspecies I contains serotypes that commonly infect warm-blooded animals, whereas members of the other five Salmonella enterica subspecies, and Salmonella bongori, are predominantly isolated from cold-blooded animals. Subspecies I serotypes are highly variable in pathogenicity and host restriction. For example, Salmonella enterica serovar Typhi (S. Typhi) is restricted to human or primate hosts, and causes the systemic disease of Typhoid fever, whereas S. Typhimurium has a broad host range, causing gastroenteritis in hosts such as humans and a Typhoid-like disease restricted to rodents. Salmonellae share a genomic backbone with E. coli. Comparison of different Salmonella and E. coli genomes helps us to understand the evolution of the Salmonella genus and to identify genes that may contribute to pathogencity and host restriction. Ten large genomic regions have been identified, termed Salmonella Pathogenicity Islands 1 to 10 (SPI's 1 to 10), which vary between non-pathogenic E. coli K12 and S. Typhi CT18. Here we present our investigation of one of these islands, SPI-10, which is adjacent to tRNAleuX. Aims: To investigate the SPI-10 region across the Salmonella genus, with a focus on genes that differ between serovars, as these genes could contribute to differences in pathogenicity and host restriction. Methods: Bioinformatics, microarrays, Southern blotting and PCR. Results: E. coli K12, E. coli O157:H7, S. Typhi and S. Typhimurium all contain different tRNAleuXadjacent genes. In contrast, the SPI-10 regions of S. Paratyphi A and S. Enteritidis share a group of fimbriaerelated genes with S. Typhi, but are otherwise divergent from S. Typhi and from each other. Thus, there are at least four different tRNAleuX-adjacent gene organizations within the Salmonella genus. The organization of SPI-10 genes is largely, but not entirely, conserved between different strains within a serovar. However, the majority of Salmonellae do not appear to share the four known SPI-10 gene organizations, suggesting that further arrangements have yet to be identified. Many P4 phage and IS element-related genes, or gene fragments, are found adjacent to tRNAleuX in both Salmonellae and E. coli, suggesting that targeting of P4 phages to tRNAleuX and insertion of IS elements may drive the observed variability in this region. Conclusions: SPI-10 is a hyper-variable region of the Salmonella genome, a hot spot for horizontal gene transfer, which contains different genes in different Salmonellae. In contrast, most of the other SPI's, with the clear exception of SPI-7, are similar between S. Typhi and S. Typhimurium.

45(C)

TRANSCRIPTIONAL PROFILING REVEALS THE ROLE OF THE H-NS OLIGOMERISATION DOMAIN IN CONTROLLING GENE EXPRESSION IN SALMONELLA ENTERICA SEROVAR TYPHIMURIUM

M. D. Goldberg, S. Lucchini, J. C. Hinton; Institute of Food Research, Norwich, UNITED KINGDOM. The bacterial nucleoid of Gram-negative bacteria comprises a circular chromosome and several DNA-binding proteins whose role is to direct folding and packaging of the DNA. The nucleoid-associated proteins act to modulate gene expression at the global level in response to environmental and physiological stimuli. H-NS is a 15.5kDa nucleoidassociated protein which regulates a diverse range of genes in response to factors such as temperature, pH, osmolarity and aerobicity, functioning as a repressor of transcription. The protein comprises two functional domains: a C-terminal DNA-binding domain and an N-terminal oligomerisation domain. The role of the H-NS oligomerisation domain in the regulation of gene expression is poorly understood. Other proteins have been identified that can interact with HNS via the oligomerisation domain to form hetero-oligomers, including StpA (58% identical to H-NS) and Hha. To investigate the function of the H-NS oligomerisation domain, we constructed an hns deletion mutation in S. Typhimurium LT2a and found that it had a severe growth defect compared with the truncated hns mutants that have been previously described for Salmonella, which all express functional oligomerisation domains. Transcriptional profiling showed that approximately 12% and 25% of the genes from the truncated hns mutant CH1838 and the hns null mutant JH4000, respectively, showed differential expression relative to LT2a. We identified many sets of genes that were only deregulated in JH4000. Particularly noteworthy was the 50 to 100-fold derepression of many SPI2 genes in JH4000, caused by deregulation of the SPI2 regulator SsrAB. We also discovered a differential effect on the flagellar and chemotaxis regulons in the two hns mutants. By deleting stpA from CH1838, which still expressed the H-NS oligomerisation domain, we were able to recreate many of the transcriptional changes displayed in JH4000 indicating that the H-NS oligomerisation domain interacts with StpA to control the expression of many genes. We therefore provide conclusive evidence that the intimate relationship between H-NS and StpA is responsible for the direct or indirect control of expression of ~600 S. Typhimurium genes.

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46(A)

ANTIBIOTIC RESISTANCE OF FOODBORNE SALMONELLA SPP.IN CANADA:1999-2002

J. Y. D'Aoust; Health Canada, Ottawa, ON, CANADA. Salmonella spp. continue as the leading cause of human foodborne bacterial diseases in many countries. Public health risk concerns are further heightened by the emergence of salmonellae that are resistant to a wide range of antibiotics notably therapeutic agents used for the treatment of human systemic bacterial infections. In this study (1996-2002), a total of 1853 Salmonella strains were isolated from raw and processed foods and from farm materials during monitoring and compliance activities of Canadian government agencies. NCCLS disk diffusion analysis of strains using a test panel of 14 antibiotics showed that 32% of 1707 isolates (19961999) and 21.2% of 146 isolates (2000-2002) were resistant to ?2 antibiotics. The incidence of resistant salmonellae in raw poultry and raw pork (?50.0%) greatly exceeded that encountered in raw beef, fish, shellfish, fresh produce, egg products, spices and animal feeds. The detection of 34.4 % ( 1996-1999) and 28.7% (2000-2002) salmonellae that are resistant to one or more antibiotic underlines the importance of animal feeds as reservoirs of resistant microorganisms. Highest levels of resistance were associated with streptomycin, tetracycline and sulfonamide which are widely used in animal husbandry at subtherapeutic doses for prophylaxis or growth promotion. Levels of resistance to â-lactams (ampicillin > piperacillin > cephalothin) and aminoglycosides (streptomycin >> gentamicin > kanamycin) ranged from 3.0 to 9.8% and from 2.7 to 41.2%, respectively. Low levels (3.0%) of chloramphenicol- resistant salmonellae were also detected whereas all isolates were found to be sensitive to ciprofloxacin, enrofloxacin and norfloxacine. Although strains harboring resistance determinants to ?4 antibiotics were not uncommon in raw meats, several isolates from raw poultry and minced beef were resistant to nine antibiotics. Comparison of resistance data from the current and a 19861989 survey of foods and agricultural products in Canada showed a 13% increase in the incidence of both resistant and multi-resistant salmonellae in the Canadian food chain within the last decade.

other species. The infection in itys mice is therefore atypical. Salmonellosis in the rat resembles self-limiting gastroenteritis. S. enteritidis colonises the gut, invades and spreads in moderate numbers to the liver and spleen. However, little or no proliferation occurs in systemic tissues and bacterial numbers decline between 6 and 12 days. Infiltration of inflammatory cells and disruption of the gut are evident. Resistant (ityr) mice, such as C3H/HeN, develop a persistent gastrointestinal infection when exposed orally to high numbers of S. enteritidis. Moderate numbers are found in the liver and spleen but only limited proliferation occurs in these tissues. There is infiltration of inflammatory cells and disruption of the gut. The infection is not self-limiting, at least over a 12 day period. The rat and resistant (ityr) mouse could be useful in evaluating the roles of host and dietary factors in modulating salmonellosis.

48(C)

SALMONELLA-ASSOCIATED CHILDREN DIARRHOEA IN ZLITEN

M. Ben Ali1, K. S. Ghenghesh2, R. Ben Aissa3, A. Abuhelfaia1, M. Dufani2; ; 1Faculty of Arts and Sciences, El-Ghomes, LIBYAN ARAB JAMAHIRIYA, 2Faculty of Medicine, Tripoli, LIBYAN ARAB JAMAHIRIYA, 3Institute Pasteur, Tunis, TUNISIA. Objectives: In Libya, very few studies were carried out in the past regarding the role of Salmonella in children diarrhea and such studies were mainly in the two major cities (Tripoli and Benghazi) of the country. The Aims of the present study were to determine the prevalence, serotypes, and antimicrobial resistance of Salmonella isolated from children with diarrhea in a small semi-urban city (Zliten) in Libya. Methods: Included in the study 169 children aged few days to 12 years with acute diarrhea admitted to Al-Shafa Clinic and the Central Hospital in Zliten from April 2000 to Mars 2001. Liquid stool samples from patients were examined for Salmonella and other agents using standard microbiological procedures. Results: Salmonella was isolated from 23 (14%) children and more than 80% of affected children had fever, vomiting, and dehydration. Serotyping resulted in 21 being Salm. Heidelberg and 3 Salm. Enteritidis. Two different serotypes were isolated from on patient. More than 78% of isolates were resistant to multiple antibiotics including amoxicillinclavulanic acid, cefoxitin, chloramphenicol, doxycycline and gentamicin. All (100%) isolates were susceptible to norfloxacin. Salmonellae were significantly isolated from children < 12 months (P<0.01), acquired in hospital than in community (P< 0.03), and isolated in autumn and winter than in Summer (P< 0.005, P< 0.003 respectively). Sex, type of feeding and water used for drinking, and antibiotic use had no significant effect on the prevalence of salmonellae. Conclusions: Salmonella is an important cause of children diarrhoea in Zliten. Very high rates of resistance to antibiotics among isolated salmonellae warrants the need for prudent use of such drugs in clinical and veterinary practice.

47(B)

THE RAT AND RESISTANT (ITYR) MOUSE IN EVALUATING THE ROLES OF HOST AND DIETARY FACTORS IN MODULATING SALMONELLOSIS

P. J. Naughton1, G. Grant2; 1University of Ulster, Co. Londonderry, UNITED KINGDOM, 2Rowett Research Institute, Aberdeen, UNITED KINGDOM. S. typhimurium causes a lethal infection in susceptible (itys) mouse strains. This is widely used a model of human typhoid-like disease. However, lethality in itys mice is primarily linked to the rapid systemic proliferation of pathogen, whereas S. typhimurium generally causes a selflimiting gastroenteritis-type infection in humans and most

46

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49(A)

USE OF MIXED INFECTIONS TO STUDY INVASION AND INTRACELLULAR SURVIVAL AND PROLIFERATION IN EUKARYOTIC CELL LINES

F. Ramos-Morales, I. Segura, J. Casadesús; Universidad de Sevilla, Sevilla, SPAIN. The standard test used to measure invasion and proliferation of Salmonella in cell cultures is the gentamicin protection assay. In this communication we propose a combination between the gentamicin protection assay and the calculation of a competitive index (CI), in order to quantify invasion or proliferation defects in mutants of Salmonella enterica serovar Typhimurium. The main advantage of the method is that, in a mixed infection, both strains (mutant and wild type) are exposed to the same conditions throughout the process. This decreases the variability between independent trials and allows a direct measurement of the impairment of the mutant in invasion or intracellular proliferation. The method has been tested in HeLa epithelial cells and J774.A1 macrophages. The assays have involved Salmonella mutants with defects in invasion (invH, invA) or in intracellular proliferation (recB, phoP). The distinction between the mutant and the wild-type strains has been carried out using antibiotic resistance markers. As an alternative, we have used plasmids encoding the Red Fluorescent Protein (RFP) and the Green Fluorescent Protein (GFP). The plasmids used have proven to be remarkably stable upon infection of eukaryotic cells. Because of their distinct colors, colonies from each strain can be distinguished (and counted) on the same plate.

response in S. enterica. On these grounds, we propose that exposure of S. enterica to bile causes DNA lesions or replication errors that are eliminated by Dam-dependent mismatch repair. In the absence of hemimethylated DNA, the MutHLS system fails to deal properly with such lesions or errors, and generates DNA strand breaks. However, this model does not explain why inactivation of the MutHLS system also relieves bile sensitivity in a Dam methylase overproducer. Mutations in mutH, mutL or mutS do not cause bile sensitivity on their own, suggesting that the cell can overcome bile-induced DNA damage by mechanisms other than methyl-directed mismatch repair.

51(C)

THE DNAK/DNAJ CHAPERONE MACHINERY IS ESSENTIALLY REQUIRED FOR INVASION OF EPITHELIAL CELLS AND SURVIVAL WITHIN MACROPHAGE CELLS OF SALMONELLA ENTERICA SEROVAR TYPHIMURIUM

T. Yamamoto1, A. Takaya1, T. Tomoyasu1, H. Matsui2; 1 Department of Microbiology and Molecular Genetics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, JAPAN, 2Department of Infection Control and Immunology, Kitasato Institute for Life Sciences, Kitasato University, Tokyo, JAPAN. Salmonella enterica serovar Typhimurium, similar to various facultative intracellular pathogens, has been shown to respond to the hostile conditions inside macrophagephagosomes of the host organism by induction of stress proteins such as DnaK. The DnaK is known to form a chaperone machinery with co-chaperones, DnaJ and GrpE and to be involved in many cellular processes such as DNA replication, RNA synthesis, protein transport and cell division. Furthermore, this chaperone machinery nonspecifically interacts with many unfolded and misfolded proteins and therefore assists proper folding, helps refolding, prevents aggregation, and mediates degradation of misfolded proteins. To elucidate a potential role of the DnaK/DnaJ chaperone machinery in pathogenesis of S. enterica serovar Typhimurium, we initially constructed an insertional mutation in the dnaK-dnaJ operon of the pathogenic strain ÷3306. The DnaK/DnaJ-depleted mutant was temperature-sensitive for growth, that is, non-viable above 39°C. We then isolated a spontaneously occurring mutant to suppress the growth defect of DnaK/DnaJ-depleted mutant at 39°C and then used it for infection of BALB/c mice by oral or subcutaneous route. The mutant (ÄdnaK/dnaJ, Ts+ suppressor) lost the ability to cause a lethal systemic disease of mice. The impaired ability for virulence was restored by providing a functional copy of dnaK-dnaJ operon, suggesting that the DnaK/DnaJ chaperone machinery is essentially required for the systemic Salmonella infection of mice. This result also indicates that the cellular requirements for DnaK/ DnaJ chaperone machinery to grow at high temperature are not identical to the cellular requirements for the machinery on the pathogenesis of Salmonella. Macrophage-survival assays revealed that the DnaK/DnaJ-depleted mutant could not survive or proliferate at all within macrophages, 47

50(B)

INACTIVATION OF DAM-DIRECTED MISMATCH REPAIR SUPPRESSES BILE SENSITIVITY IN DNA ADENINE METHYLASE MUTANTS OF SALMONELLA ENTERICA

A. I. Prieto, F. Ramos-Morales, J. Casadesús; Universidad de Sevilla, Sevilla, SPAIN. Lack and overproduction of DNA adenine methylase cause bile sensitivity in Salmonella enterica serovar Typhimurium. Mutations that suppressed bile sensitivity in a Dam- strain were sought after MudJ mutagenesis. Sequencing of MudJ boundaries identified the loci whose inactivation had resulted in suppression. One such locus was the mutS gene, which encodes one component of the Dam-directed mismatch repair system, MutHLS. Mutations in any of the mutH, mutL and mutS genes suppress bile sensitivity, both in a Damstrain and in a Dam methylase overproducer. These results suggest that the bile sensitivity phenotype of Dam- mutants is caused by an active MutHLS system. An interpretation is that, in the absence of Dam methylation, lack of DNA strain discrimination results in MutH-induced DNA breaks or other kinds of DNA damage. This view is supported by an additional finding: using lacZ fusions in genes of the SOS regulon, we observed that bile is able to induce the SOS Pathogenesis, Epidemiology, and Vaccine Development

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suggesting that the loss of virulence of the mutant could be due to its inability to survive bactericidal mechanisms of macrophages. Of further interests are the findings that the mutant could not invade the cultured epithelial cells or secreted any of invasion proteins encoded by Salmonella Pathogenicity Island-1. This is the first evidence that the DnaK/DnaJ chaperone machinery is critically involved in bacterial invasion of epithelial cells.

53(B)

IMMUNOMICS AND VACCINE DESIGN AGAINST SALMONELLA

M. M. Mogensen, M. P. Gallagher, C. J. Inchley; University of Edinburgh, Edinburgh, UNITED KINGDOM. Salmonella enterica is a Gram negative, facultative, intracellular pathogen of clinical importance to both humans and animals. The widespread and inappropriate use of antibiotics has led to the emergence of antibiotic resistant strains, which creates a major problem for infection control and therapy. However, vaccination provides a possible solution. Currently, there are a number of live-attenuated vaccines available for Salmonella, but the efficacy and safety aspects of these are not optimal for all applications. A sub-unit vaccine provides an alternative but firstly, it is necessary to identify suitable antigens. By combining 2D-gel electrophoresis with immunoblotting, we have pin-pointed immunodominant antigens using hyper-immune serum raised in mice by sequential infection with S. typhimurium - hyperimmune serum is not only likely to enrich for antibodies against immunodominant antigens but also, against in vivo expressed antigens. Using mass spectrometry, we have determined the identities of approximately 30 of the prominent antigens. Among those identified are known outer membrane proteins (OMPs) or surface antigens, such as OmpA and phase 1 flagellin, as well as previously unrecognised OMPs. We have also identified several periplasmic protein antigens of known or unknown function. These include components with roles in sugar or ion binding and chemotaxis. Amongst the cytosolic proteins identified, we have found previously recognised immunodominant antigens such as Hsp60, as well as a Salmonella-specific heat shock protein. Other antigens include proteins which play roles in `house-keeping' functions, DNA binding or protein synthesis. Importantly, several of the identified antigens show little homology to mammalian proteins, and as such, may be of particular value as vaccine components. We have successfully cloned and expressed a number of the identified antigens and ongoing work includes immunological studies and analysis of their antigenicity. The evidence for this work and the implication of these findings for Salmonella virulence and vaccine development will be addressed within the poster.

52(A)

PRIMING OF HUMAN MACROPHAGES WITH RECOMBINANT INTERFERON-GAMMA ENHANCES PHAGOCYTOSIS AND KILLING OF SALMONELLA ENTERICA SEROVAR TYPHIMURIUM, AND MODIFIES CYTOKINE RESPONSES

M. A. Gordon1, R. C. Read2; 1Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Blantyre, MALAWI, 2Divison of Genomic Medicine, University of Sheffield Medical School, UNITED KINGDOM. We investigated the effects of priming human monocytederived macrophages (MDM) with recombinant interferon gamma on bacterial internalisation, intracellular killing and cytokine release, after infection with S. enterica serovar Typhimurium. 11-day-old human MDM, primed for 72 hours with rIFN (100ng/ml) and exposed to S. enterica serovar Typhimurium (NCTC12023) at MOI 50 for 15 minutes, exhibited an increased proportion of MDM with cellassociated bacteria (31% primed vs 26% unprimed), and an increase in both the number of cell-associated bacteria and the number of internalised bacteria per cell by 67%, compared to unprimed cells. 90% of cell-associated bacteria were internalised in both groups. Intracellular killing was assessed using a gentamicin exclusion method, over a 24h period. Overall, intracellular killing was increased in 72h primed cells compared to unprimed cells by 3.5-fold (CI 2.4 - 5.2, n=8) over 24 h. This difference in killing was maximal in the earliest phase 0.5h after internalisation (4.8-fold, CI 3.1 - 7.4) , but was also observed at 24h (3.4-fold CI 2.3 4.9) By contrast, overall killing of bacteria in cells that had only been primed for 24 hours was not significantly increased, compared to unprimed cells (2.3 fold, CI 0.9 - 5.4) There was no difference in cell density counts at 24h between groups, but infection with S. enterica serovar Typhimurium resulted in a minor increase in cell death (typan blue uptake) in both primed and unprimed cells. IL-10 and IL-12 (p40/70) were measured by ELISA in conditioned 24h media. IL-10 levels were reduced in primed compared to unprimed cells, and IL-12 levels were increased in primed compared to unprimed cells. Uninfected control cells did not release detectable IL-10 or IL-12. We conclude that priming with rIFN-gamma increases the efficiency of phagocytic killing of S. enterica serovar Typhimurium by human MDM, and modifies subsequent cytokine release.

54(C)

THE CYTOKINE RESPONSE TO SALMONELLA INFECTION IN AN IN VITRO AVIAN MODEL

A. N. Lazzerine, P. Barrow, P. Kaiser; Institute for Animal Health, Newbury, UNITED KINGDOM. We recently used a chicken primary cell culture model (chick kidney cells ­ CKC) to show that invasion of the broad host range serotype S. Typhimurium caused an increase in production of the pro-inflammatory cytokine IL-6, whilst invasion by the host-specific serotype S. Gallinarum caused no increase. This finding correlated with the pathogenesis of Salmonella in poultry. S. Typhimurium invasion produces a strong, pro-inflammatory response that may limit the spread ASM Conference on Salmonella

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of Salmonella largely to the gut, whilst S. Gallinarum does not induce an inflammatory response and may not be limited by the immune system, leading to the severe systemic disease: fowl typhoid. Further investigation with defined invasion mutants of S. Typhimurium, S. Gallinarum and another host-specific serotype, S. Pullorum, suggested that SPI-1 proteins may modulate host pro-inflammatory immune responses (IL-1â , IL-6 and IL-8) during a S. Pullorum infection. This again correlates with the pathogenesis of S. Pullorum infection in poultry, suggesting evasion of host immune responses and action as a stealth killer.

56(B)

REGULATION OF SALMONELLA TYPHIMURIUM VIRULENCE GENE EXPRESSION BY CATIONIC ANTIMICROBIAL PEPTIDES

M. W. Bader, S. I. Miller; University of Washington, Seattle, WA. Cationic antimicrobial peptides (CAMP) represent a conserved and highly effective component of innate immunity. During infection, the Gram-negative pathogen Salmonella typhimurium induces different mechanisms of CAMP resistance that promote pathogenesis in animals. This study shows that exposure of S. typhimurium to sublethal concentrations of CAMP activates the PhoP/PhoQ and RpoS virulence regulons, while repressing the transcription of genes required for flagella synthesis and the invasionassociated type III secretion system. We further demonstrate that growth of S. typhimurium in low doses of the á-helical peptide C18G induces resistance to CAMP of different structural classes. Inducible resistance depends on the presence of PhoP, indicating that the PhoP/PhoQ system can sense sublethal concentrations of cationic antimicrobial peptides. Growth of S. typhimurium in the presence of CAMP also leads to RpoS-dependent protection against hydrogen peroxide. Since bacterial resistance to oxidative stress and CAMP are induced during infection of animals, CAMP may be an important signal recognized by bacteria on colonization of animal tissues.

55(A)

A PHOSPHOTRANSACETYLASE (PTA) MUTANT OF S. TYPHIMURIUM 14028 IS IMPAIRED IN NRAMP1 WILD TYPE BUT NOT NRAMP1 MUTANT MICE

J. Fierer1, Y. Kim 1, J. C. Escalante-Semerena 2; 1UC San Diego and VA Healthcare, San Diego, CA, 2Univ. Wisconsin, Madison, WI. In Salmonella typhimurium, phosphotransacetylase enzyme activity is required for the efficient metabolism of short chain fatty acids such as acetate and propionate. Virulence of a pta derivative of S. typhimurium 14028 (pta::MudJ[kan+]) was tested in BALB/c and BALB/c.D2 congenic mice. The former carries a point mutation in the Nramp1 gene that makes it susceptible to S. typhimurium infections. The latter have a wild-type Nramp1 gene from DBA/2, and these mice are about 1,000 times more resistant to S. typhimurium infections. We injected 7x10 3 cells of pta+ and pta- mixed together by the i.p. route. Mice were sacrificed 5 and 10 days after infection, and the ratio of pta+/pta bacteria was determined in livers and spleens. In repeat experiments the ratio in livers and spleens was 20-50:1 on both days, indicating that the pta mutation significantly reduced the ability of S. typhimurium to grow in resistant mice, even though it grew normally in LB broth. When we injected 50100 pta mutant bacteria i.p. into BALB/c mice they died, though more slowly than the mice infected with 14028. Quantitative cultures of livers and spleens done 3 and 6 days after infection with the pta mutant showed that the mutant grew more slowly than the pta+ strain, but by day 6 it reached near lethal concentrations. We then injected congenic mice with iron dextran 24 hours before they were infected with mixtures of the two strains of S. typhimurium. The growth of 14028 was enhanced >200 fold, while the number pta mutants was increased only 40 fold. We conclude that the pta mutation compromises the ability of S. typhimurium to obtain iron in vivo when the host has a normal Nramp1 gene. Phosphotransacetylase is less important in mice with a mutant Nramp1. This implies that Nramp1 might be involved in withholding iron from the pathogen, and that Salmonella may utilize a mechanism that requires pta to scavenge iron in vivo.

57(C)

CHARACTERIZATION OF A NOVEL DNA VACCINE VECTOR WITHOUT ANTIBIOTIC RESISTANCE GENE AND ITS TRANSPORTING SYSTEM BASED ON ATTENUATED SALMONELLA

X. Jiao, X. Zhang, Z. Pan, J. Huang, X. Liu; Yangzhou University, Yangzhou, CHINA. DNA vaccine is regarded as the third generation vaccine. It can elicit both cellular and humoral immunity and has several advantages, such as easy to produce, transport and store, easy to construct multivalent vaccines and easy to incorporate several adjuvant molecules. But two major factors restrict its development. One is the issue of safety, and the existence of antibiotic resistance gene within DNA vaccine is always a concern. The other factor is the immune efficacy is not so satisfactory. In this study both safety and efficacy issue of DNA vaccine were dealt with. By introducing asd gene from Salmonella typhimurium into DNA vaccine vector pVAX1 and destroying kanamycin resistance gene at the same time, a new DNA vaccine vector asd-pVAX1 which contained no antibiotic resistance gene was constructed. Then enhanced green fluorescence protein (EGFP) gene was inserted into its multiple cloning site (MCS). This new recombinant plasmid asd-pVAX1-EGFP was harvested fromÄasd E. coli X6212 in vitro and strong green fluorescence was observed in transfected mouse mastocytoma P815 cells. asd-pVAX1EGFP was transformed into Äasd S. typhimurium X4550 49

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and in vitro and in vivo tests showed asd-pVAX1-EGFP can be stably maintained within X4550. When X4550(asdpVAX1-EGFP) was used to infect COS-7 cells, EGFP positive COS-7 cells can be observed 24 hours postinfection. asd-pVAX1-EGFP was used to immunize BALB/c mice intramuscularly and specific serum anti-EGFP antibody was elicited. The titer of anti-EGFP antibody was no lower than the antibody induced by pVAX1-EGFP. Prokaryotic promoter Ptrc and multiple cloning site (MCS) was inserted into the downstream of eukaryotic expression promoter Pcmv in DNA vaccine vector pVAX1. Endonucleases digestion and nucleotide sequencing confirmed the rightness of this ligation. This novel double expression plasmid vector was named as pVAXD and was 3115 bp in length. To verify this novel vector EGFP gene was placed into the downstream of both promoters and consequently transformed into Salmonella typhimurium X4550 and transfected into COS-7 cell. The EGFP expression in X4550(pVAXD-EGFP)was detected by flow cytometer, visible spectrum scanning and SDS-PAGE and its expression in COS-7 cell was monitored by fluorescence microscope. EGFP was expressed both in X4550 and COS-7 cell and its expression level was comparable with corresponding recombinant S. typhimurium X4550(pYA3334-EGFP)and EGFP eukaryotic expression plasmid pVAX1-EGFP. A prokaryotic-eukaryotic double expression plasmid was successfully constructed and it has potential applications in designing new recombinant Salmonella vaccines. contamination of the respective lot of chocolate. Phage typing and PFGE have been found as rapid and reliable tools applied successfully for surveillance of S. enterica serovar Oranienburg.

59(B)

CIRCULATING GAMMA/ DELTA T CELLS IN CHICKENS IMMUNISED AND INFECTED WITH DIFFERENT SALMONELLA STRAINS

A. Berndt, U. Methner; Federal Research Centre for Virus Diseases of Animals, Jena Branch, Jena, GERMANY. For successful development of vaccines against Salmonella infections in poultry, comprehensive knowledge on immunological defence mechanisms of chickens against Salmonella exposure is very important. Circulating and tissue gamma/ delta T cells seem to play a crucial role in Salmonella infections of young chicks. For phenotypic and functional characterisation of these cells, day-old chicks were immunised either orally with an attenuated S. Enteritidis live vaccine and a S. Enteritidis wild-type strain or parenterally with a S. Enteritidis inactivated vaccine. Additionally, all animals were infected orally with the S. Enteritidis wild-type strain on day 44 of life. The peripheral blood lymphocytes of the treated birds and appropriate control animals were examined flow-cytofluometrically for the occurence of TcR1+ cell subpopulations between days 3 and 71 of life. Three different CD8alpha+/TcR1+ cell subpopulations were identified in non-treated animals. These three populations could be phenotypically discriminated by their varying antigen expression (CD8alpha+high/TcR1+ cells; CD8alpha+dim/TcR1+ cells; CD8alpha-/TcR1+ cells) and additionally specified by the detection of different further surface antigens. It could be shown that the CD8alpha+high/TcR1+ cells express the CD8alpha beta heterodimer and the CD8alpha+dim/TcR1+ cells the CD8alpha alpha homodimer. After immunisation and infection of chickens using the different Salmonella strains, defined reactions of the three TcR1+ cell subpopulations could be observed. In particular, the CD8alpha-/TcR1+ cell population revealed characteristic changes depending on the treatment and seems to be of special significance for Salmonella immunity of chickens. It can be summarised that the group of TcR1+ cells in avian peripheral blood represents a heterogeneous population. The divergent expression of special antigens on TcR1+ cells also indicates different functions of these cell populations. The examination of the emergence of these cell subpopulations in avian blood after Salmonella infection or immunisation may serve as a suitable parameter to characterise the efficacy of potential Salmonella vaccine candidates in future.

58(A)

CLONAL DIVERSITY OF SALMONELLA ENTERICA SEROVAR ORANIENBURG AND IDENTIFICATION OF AN INTERNATIONAL OUTBREAK TRACING BACK TO CHOCOLATE

H. Tschäpe1, R. Prager1, W. Rabsch 1, D. Werber2, B. Gericke1, A. Ammon 2; 1Robert Koch Institute, Wernigerode, GERMANY, 2 Robert Koch Institute, Berlin, GERMANY. Clinical cases S. enterica serovar Oranienburg have been observed very rarely in Germany. However, beginning with October 2001 reported cases of S. Oranienburg infections increased 100 fold ranking up to 350 cases in two month. Using phage typing and molecular fingerprinting (PFGEpattern, plasmid profiles and ribotype) all respective isolates have been identified as clonal identically as well as isolates from a distinct lot of chocolate suggesting that its consumption was the source of the epidemic outbreak. This lot of chocolate was exported to other European and oversee countries and consequently infections due to S. Oranienburg were reported in Denmark, Sweden, Netherlands, Belgium, Austria, Canada, and Czech Republic also after consumption of chocolate. The same epidemic clone could be identified among S. Oranienburg isolates from these countries. In contrast, S. Oranienburg strains isolated from other geographical or temporal sources revealed a great molecular diversity underlining their sporadic nature. Interesting to note is the identification of the epidemic clone also, but rarely, among S. Oranienburg strains isolated before the epidemic outbreak and from other sources indicating a cross-

50

ASM Conference on Salmonella

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60(C)

HOST ADAPTATION OF PIGEON ISOLATES OF SALMONELLA ENTERICA SUBSP. ENTERICA SEROVAR TYPHIMURIUM VAR. COPENHAGEN PT 99 IS ASSOCIATED WITH ENHANCED MACROPHAGE CYTOTOXICITY

F. R. Pasmans 1, F. Van Immerseel 1, M. Heyndrickx2, A. Martel1, C. Godard 3, C. Wildemauwe3, R. Ducatelle1, F. Haesebrouck1; 1 Ghent University, Merelbeke, BELGIUM, 2Center for Agricultural Research, Melle, BELGIUM, 3Pasteur Institute, Brussels, BELGIUM. Phage type 99 (PT 99) of Salmonella enterica subsp. enterica serovar Typhimurium var. Copenhagen (ser. Typhimurium var. Copenhagen) is commonly isolated from pigeons. In this study, Salmonella PT 99 strains isolated from pigeons were examined for the presence of genotypic and phenotypic characteristics which would confirm the hypothesis that these strains are adapted to pigeons. Genotypic characterization was performed by comparing the pulsed field gel electrophoresis (PFGE) patterns obtained with XbaI and BlnI from 38 strains of pigeon ser. Typhimurium var. Copenhagen PT 99 with those obtained from 89 porcine, poultry and human strains of ser. Typhimurium var. Copenhagen. Identical patterns with XbaI and 4 closely related patterns with BlnI were obtained in the pigeon strains, whereas 16 XbaI patterns were found in the poultry, porcine and human strains. The XbaI patterns of the pigeon strains showed a low genetic similarity to the patterns of the porcine, poultry and human strains and invariably showed a low molecular weight band that was absent in the majority of the other strains. The virulence genes shdA, spvR, pefA, sopE and spvB were uniformly present in 6 pigeon isolates representing the genetic diversity found with BlnI. These strains as well as 3 porcine isolates were used in in vitro and in vivo studies to determine their virulence for pigeons. Intracellular survival and macrophage cytotoxicity were determined in pigeon peritoneal macrophages using 6 pigeon and 3 porcine strains. The pigeon derived strains were highly cytotoxic for the macrophages compared to the porcine strains. After experimental infection of pigeons with a pigeon strain, clinical symptoms, faecal shedding and colonisation of internal organs were much more pronounced compared to those after infection with a porcine strain. These data suggest that the PT 99 strains used in this study are highly adapted to pigeons and should be classified as a host-restricted lineage of the serovar Typhimurium.

61(A)

ADAPTIVE RESISTANCE TO BIOCIDES AND CROSS-RESISTANCE TO ANTIMICROBIAL AGENTS IN SALMONELLA ENTERICA

M. Braoudaki, A. C. Hilton; Aston University, Birmingham, UNITED KINGDOM.

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 enterica to commonly employed biocides, to identify mechanisms underlying resistance and any crossresistance to antibiotics. Salmonella enterica was serially exposed in sub-inhibitory concentrations to erythromycin, benzalkonium chloride, chlorohexidine and triclosan. Following each passage any adaptive resistance or crossresistance in a panel of antibacterials was recorded. Adaptive resistance was observed in all strains investigated. Erythromycin-resistant Salm. Enteritidis expressed cross-resistance to chloramphenicol, whereas in erythromycin- resistant Salm. Typhimurium cross-resistance to chlorohexidine was detected. Benzalkonium chloride resistant Salm. Virchow showed an elevated resistance to chlorohexidine, however chlorohexidine resistant Salm. Virchow did not demonstrate cross-resistance to benzalkonium chloride suggesting specific rather than general mechanisms. 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 crossresistant to other antimicrobials.

62(B)

THE SURVEILLANCE OF ANTIMICROBIAL RESISTANCE OF SALMONELLA PULLORUM/GALLINARUM

X. Jiao, Z. Pan, W. Liu, S. Gao, X. Liu; Yangzhou University, Yangzhou, CHINA. 346 Salmonella pullorum/gallinarum strains were collected and preserved from some regions in China from 1962 to 1999. All strains were studied for antimicrobial susceptibility testing by means of Kirby- Bauer method. The results showed that almost all of 346 isolates had the capacity of antimicrobial resistance. The resistance rates of Salmonella pullorum/gallinarum isolates to antimicrobials were increasing in the last 40years. The level of resistance to a set of antimicrobials, including Ampicillin, Spectinomycin, Sulfarmethoxazole/Trimethoprim, Sulfafurazole, Trimethoprim, Carbeni-cillin, Tetracycline, Streptomycin and Penicillin Gincreased is significant increased (P < 0.01) . In contrast their resistance to other antimicrobials remained low level. The percentages of isolates were 55.5% with resistance 51

Pathogenesis, Epidemiology, and Vaccine Development

POSTER ABSTRACTS POSTER ABSTRACTS

to 2 or 3 antimicrobials in 1960s, 60.5% with resistance to 4 or 5 antimicrobials in1970s, 80.2% with resistance to 5,6 or 7 antimicrobials in 1980s, 83.7% with resistance to more than 7 antimicrobials in 1990s. The later the isolates were derived from the field, the broader their resistance spectrum expanded during the period from 1962-1999. According to epidemiological investigation, the isolates had significantly higher resistance rates to some antimicrobials which were prescribed repeatedly and used for long time in veterinary clinics ,such as Penicillin G, Streptomycin, Tetracycline and Sulfanilamides. The results suggested that there was a close correlation between the antimicrobial resistance and the frequency of agents prescribed in veterinary clinics. Changing trends in antimicrobial resistance of these isolates would be very useful formulating guide lines for the medication in the control of bacterial infections. of serovar Typhimurium, down-regulation of, IL-1â expression. In both lines there was little expression of IL-6 mRNA at 20 minutes, but 5 to 20-fold increases were found at 1 hour. Salmonella serovar Typhimurium infected macrophages showed no increase or down-regulation of expression at 4 hours. In contrast high expression of IL-6 mRNA was seen in Salmonella serovar Gallinarum infected macrophages. These initial studies suggest that resistant line chicken macrophages are more rapidly activated than susceptible line cells corresponding to their phenotype of more rapidly clearing Salmonella and that Salmonella serovar Gallinarum infected macrophages express pro-inflammatory cytokines over a more prolonged period than serovar Typhimurium infected cells. If replicated in vivo this could contribute to the substantial pathology and inflammation seen in Salmonella serovar Gallinarum infections of chickens.

63(C)

CYTOKINE RESPONSES OF MACROPHAGES FROM SALMONELLARESISTANT AND SUSCEPTIBLE INBRED LINE CHICKENS

P. Wigley, S. D. Hulme, P. Kaiser, L. Rothwell, P. A. Barrow; IAH, Newbury, UNITED KINGDOM. Studies using inbred lines of chickens have indicated that a single genetic locus, SAL1, is the major factor in resistance to systemic salmonellosis in the chicken, with Nramp1 and Tlr4 having minor influences. Macrophages from resistant birds have a phenotype of increased clearance and oxidative burst activity compared with macrophages from susceptible birds, a phenotype displayed most strongly during Salmonella serovar Gallinarum infection. We have begun to determine the expression of cytokines by resistant and susceptible chicken macrophages following in vitro infection by use of quantitative RT-PCR (TaqMan). Monocyte-derived macrophages from Salmonella-resistant W1 line and Salmonellasusceptible Line 72 chickens were challenged with Salmonella enterica serovar Gallinarum and serovar Typhimurium at a MOI of 10, or were left uninfected as controls. Invasion and survival of intracellular Salmonella was determined by gentamicin protection assay at 20 minutes, 1 hour and 4 hours post-infection. At the same time points RNA was isolated from cells using a Qiagen RNeasy mini-kit. Expression levels of pro-inflammatory chicken cytokines IL-1â and IL-6 in comparison to control cells were determined through TaqMan analysis using 28S RNA gene expression as a `housekeeping' gene to correct total RNA values. Both Salmonella serovars invaded resistant and susceptible macrophages to a similar level but by 4 hours post infection levels of Salmonella serovar Gallinarum and to a lesser degree serovar Typhimurium were significantly reduced in the resistant (W1) but not susceptible (72), macrophages as expected. At 20 minutes after infection up to 20-fold increases in IL-1â mRNA expression were found in the resistant line macrophages. In contrast there were only minimal changes in IL-1â expression in susceptible line macrophages at this time point. By 1 hour post infection both lines showed around 20-fold increases of expression. At four hours post infection there was little, or in the case

64(A)

PHYLOGENETIC ANALYSIS OF PROTEINS HOMOLOGOUS TO THE SALMONELLA MGTC VIRULENCE FACTOR

A. Blanc-Potard, B. Lafay; UMR CNRS-IRD 9926, Montpellier, FRANCE. MgtC is a virulence factor in Salmonella typhimurium, being required for intramacrophage survival and growth in low Mg2+ medium. The mgtC gene is carried by the SPI-3 pathogenicity island, that is likely to have been acquired through horizontal gene transfer. The acquisition of mgtC occurred probably prior to the Salmonella genus diversification because mgtC sequences were detected by Southern experiments in all eight subspecies of S. enterica. In contrast, Southern experiments indicated a narrow and sporadic distribution of the mgtC gene in 10 enterobacteria. A MgtC homologue, that is also required for intramacrophage survival and growth in low Mg2+, is found in Mycobacterium tuberculosis, a pathogen not phylogenetically related to Salmonella. We have investigated further the presence of mgtC-like sequences in eubacterial genomes. Analyses of MgtC-like proteins phylogeny and mgtC-like chromosomal context support the hypothesis that mgtC has been acquired by horizontal gene transfer repeatedly throughout bacterial evolution. In addition, the phylogenetic analysis revealed a subgroup of proteins, that includes the S. typhimurium and M. tuberculosis MgtC proteins, as well as MgtC-related proteins from other pathogens that are able to survive in macrophages, B. melitensis and Y. pestis. We propose that MgtC has a similar function in all these distantly related pathogens, most likely providing the ability to grow in a low Mg2+ environment.

52

ASM Conference on Salmonella

POSTER ABSTRACTS POSTER ABSTRACTS

65(B)

A SEQUENCE-BASED APPROACH TO MOLECULAR SEROTYPING OF SALMONELLA ENTERICA.

C. K. Mortimer, J. Logan, T. Peters, C. Arnold; HPA, London, UNITED KINGDOM. In outbreak situations, typing of bacteria can help deduce the mode of transmission and extent of the outbreak. For meaningful epidemiological investigation, it is vitally important that bacteria are accurately identified and assigned correct nomenclature. Reference laboratories identify Salmonella by the reactions of flagellar and somatic antigens with specific antisera, according to the Kauffman-White scheme, established over 70 years ago. Salmonella serotyping methods are stable, reproducible and have high typeability. Nevertheless, there are several drawbacks of the conventional serotyping system not least the ethics, cost and quality control measures necessary to maintain the supply of antisera upon which the system relies. The fliC gene codes for the H1 flagellin protein, which confers part of the Salmonella serotype. There are 50 H1 determinants recognised by specific antisera, a single flagellin protein may carry a number of different determinants. Sequence data has been collected from strains of various H1-types. By in silico analysis alone, the amino acids that may contribute to a discontinuous epitope cannot be identified, but through comparison of data from representative strains it may be possible to determine the molecular basis for the differences in H1 antigens. In this study preliminary data indicating the feasibility of a sequence based molecular serotyping for Salmonella are presented.

suggesting that signal production is a reflection of the metabolic state of the cell rather than cell density. Since regulation of AI-2 production occurs at the level of pfs transcription, we have examined the control of pfs expression. Due to the dependence on certain carbohydrates for AI-2 production we have examined the regulatory role of the cAMP receptor protein (CRP) on AI-2 signal generation. By utilizing crp and cya mutant strain backgrounds for these studies we have demonstrated dramatic increases in pfs expression and AI-2 production in the absence of a functional CRP or adenylate cyclase. These results suggest that CRP functions (directly or indirectly) as a negative regulator of pfs transcription and AI-2 production in Salmonella. A random Tn10dCm mutagenesis approach was also used to isolate transcriptional regulatory mutants that alter the pfs transcriptional profile. pfs promoter activity of approximately 10 000 stable transposon mutants was analyzed under conditions that result in maximal (LB+glucose) and minimal (LB media alone) pfs expression in the wildtype strain. Mutants showed pfs expression differences ranging from ten fold decreases to five fold increases. Recovery of these transposon insertion sites by arbitrary-primed PCR and subsequent sequencing has revealed multiple mutations in transcriptional regulatory proteins including leuO, hha, yifA, and yojN. These results suggest that pfs transcription is under multiple regulatory controls. The conditions that result in maximal pfs expression and AI-2 production reflect those that Salmonella encounters in the host intestine. These observations contribute to our understanding of the regulation of AI-2 production by Salmonella and potentially elucidate the role of AI-2 signaling in the host during colonization and infection.

66(C)

MOLECULAR BASIS OF THE REGULATION OF AUTOINDUCER-2 PRODUCTION IN SALMONELLA ENTERICA SEROVAR TYPHIMURIUM

A. L. Beeston, M. G. Surette; University of Calgary, Calgary, AB, CANADA. Bacterial intercellular communication provides a mechanism for signal-dependent regulation of gene expression to promote coordinated population behaviour. Salmonella enterica serovar Typhimurium (S. typhimurium) produces an autoinducer-2 (AI-2) signaling molecule in exponential phase growth in the presence of preferred carbohydrates. The luxS and pfs gene products and the Pfs substrate Sadenosylhomocysteine (SAH) are sufficient to mediate the production of AI-2 in vitro. Pfs converts SAH to Sribosylhomocysteine that is converted to AI-2 by LuxS. To further investigate the nature of AI-2 production, pfs and luxS promoter fusions to a luxCDABE reporter were used to examine transcriptional-level regulation. We have reported that transcription of pfs (unlike luxS) is correlated to AI-2 production in S. typhimurium, where several conditions that result in maximal AI-2 production also result in maximal pfs transcription. The results of this work indicate that AI-2dependent signaling may be regulated by substrate availability Pathogenesis, Epidemiology, and Vaccine Development

67(A)

COMPARISON BETWEEN PROMOTERS REGULATED BY PHOP/Q BY EXPRESSION OF GFPMUT3 (* ): CONTRUCTION OF A PLASMIDIAL SYSTEM FOR STABLE EXPRESSION OF HETEROLOGOUS ANTIGENS AND BY ATTENUATED SALMONELLA ENTERICA TYPHIMURIUM STRAINS

L. G. Brandão1, A. V. Dias1, W. D. Silveira 2, M. Brocchi 1; 1 University of São Paulo - USP, Ribeirão Preto - SP, BRAZIL, 2 University of Campinas - UNICAMP, Campinas - SP, BRAZIL. Mutants of Salmonella enterica Typhimurium vaccine strains attenuated for virulence are able to cause transient infections and to elicit a powerful immunological response against the foreign as well as to self antigens. Factors as level of expression and stability may modulate the immune responses to antigens when expressed by these live attenuated organisms and could therefore modify the imunogenicity of the vaccine candidates. The construction of a plasmidial regulated system using the balanced lethal asd system and in vivo induced promoters to guarantee stability and to ameliorates immunogenicity was proposed. The ability of the pagC, pagD, mgtC and phoN promoter genes, inserted in pYA3137 and pYA3074 asd+ plasmids, to drive the expression of a reporter gene in S. enterica Typhimurium ÷3987ÄcyaÄcrpÄasd 53

POSTER ABSTRACTS POSTER ABSTRACTS

e H683ÄaroAÄasd vaccine strains was investigated. All four promoters are regulated by the system PhoP/Q and they are induced when the bacteria are in the intracelular environment, particularly in macrophage cells. The PhoQ membrane kinase protein is activated in low Mg2+ concentration (ìM concentration) and phosphorilates PhoP that activates the expression of pagC, pagD, mgtC and phoN. The promoters of those genes were PCR amplified and fused with gfpmut3 (* ) that was obtained by site direct mutagenesis. The highcopy number plasmid pYA3137 and the low copy number plasmid pYA3074 were PCR modified in order to clone the genic fusions between the reporter gene and the promoters. The plasmids were introduced in the S. enterica Typhimurium ÷3987 and H683 trains. The expression of gfpmut3(*) were evaluated under different growth conditions, as different Mg 2+ concentrations and in the intracellular environment of BALB/c peritoneal macrophages cells by Florescent Activated Cell Sort (FACSort). The level of expression was compared with that obtained with the constitutive promoter (trc). Results indicate that expression of GFPmut3(*) protein from pYA3137pagC is the strongest between all inducible promoters, but is generally lower than that obtained with the constitutive promoter trc. The constructions containing the promoters under the control of the PhoP/Q system, were associated with higher levels of GFPmut3(*) expression in the absence of Mg2+, differently from the construction using the constitutive promoter trc. The results with the two different S. enterica strains indicated that the genetic background of the strain influences the level of gfpmut3(*) expression as ÷3987ÄcyaÄcrp Äasd expressed higher levels of fluorescence in comparison toH683 ÄaroAÄasd. Supported by: FAPESP, CAPES and CNPq. during invasion of epithelial cell lines and interaction with macrophages is at present under investigation. The cheA and cheB mutants of Sdu and the cheR mutant of Stm were all attenuated in virulence after oral challenge in mice. No attenuation with either the chemotactic or the flagella mutants was demonstrated in oral infections in the chicken and in the intestinal loop invasion. The fliC mutant of Sdu and the double mutation of fliCfljB in Stm demonstrated significantly reduced virulence in murine macrophages. Mutation in Stm of fljB alone demonstrated no attenuation. Results indicate that the chemotaxis system of Sdu appears to be important for virulence in mice but limited for virulence of Stm. Expression of flagella were linked to systemic virulence in mice (involving intracellular survival in macrophages and colonisation) for both Stm and Sdu but not for triggering the oxidative response in macrophage cell lines. Furthermore, hyper-flagellation was seen to induce increased cytotoxicity in macrophages.

69(C)

MOLECULAR DIFFERENTIATION OF SALMONELLA ENTERICA SUBSP. ENTERICA SEROVAR AGONA ISOLATES OBTAINED FROM SWINE IN SOUTHERN BRAZIL

G. Brenner Michael1, M. Cado Bessa2, S. M. Ferraz Castagna2, P. Schwarz 2, M. Cardoso 2, S. Schwarz1; 1Institute for Animal Science (FAL), Neustadt-Mariensee, GERMANY, 2Universidade Federal do Rio Grande do Sul, Porto Alegre, BRAZIL. Introduction: Salmonella enterica subsp. enterica (S.) strains were isolated at three swine slaughterhouses in three different regions in Southern Brazil between 1999 and 2001. Mesenteric and head lymph nodes, tonsils, intestinal contents and meat products were sampled. S. Agona was one of the most frequently isolated Salmonella serovars. The aim of the present study was to determine the relatedness between 45 S. Agona isolates. Methods: All strains were identified as S. Agona by biochemical and serological tests. Antimicrobial resistance patterns were determined by disk diffusion. Moreover, plasmid profiles as well as XbaI- and BlnImacrorestriction patterns were determined and compared. These two restriction endonucleases, XbaI and BlnI, have proved in previous studies to be suitable for macrorestriction analysis of S. Agona and other Salmonella serovars. Results: Five different plasmid profiles consisting of either one or two plasmids were detected among the six plasmid-bearing strains. Four of these strains harboured plasmids larger than 90 kb. Macrorestriction analysis with XbaI yielded ten different patterns. Common patterns were seen in 32 and five strains, respectively, and unique patterns were detected in the remaining eight strains. This corresponded to a low discriminatory index of D = 0.489. BlnI digestion yielded eight patterns. Only four strains showed unique patterns while common patterns were detectable in three to 30 strains respectively. The D value of the BlnI patterns was slightly higher at 0.544. Comparative analysis of plasmid profiles and macrorestriction patterns revealed that 30 of the 45 S. Agona strains were indistinguishable by their XbaI- and BlnIpatterns and also did not carry plasmids. However, nine ASM Conference on Salmonella

68(B)

THE ROLE OF FLAGELLA AND CHEMOTAXIS ON VIRULENCE OF SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND DUBLIN

M. S. Chadfield, K. Hobolt Høegh-Andersen, S. Aabo, J. P. Christensen, J. E. Olsen; KVL, Copenhagen, DENMARK. The aim of this study was to investigate the role of the flagella and chemotaxis systems in Salmonella infection. To determine the effect of mono-phasic and bi-phasic expression of flagella, isogenic flagella and chemotaxis mutants of the host adapted single flagella phased Salmonella enterica serovar Dublin (Sdu) and the ubiquitous broad host spectrum bi-phasic flagella phased Salmonella enterica serovar Typhimurium (Stm), were constructed and used to determine the effects on virulence mechanisms. Expression of specific adhesins may be important for organ colonisation and/or host specificity of a strain, therefore two different hosts (mice and chicken) were included in the investigation. Strains were assayed for virulence in vivo by oral and intraperitoneal infections and intestinal loop assays in chickens. In vitro assays included intracellular survival, cytotoxicity and production of reactive oxygen species through the respiratory burst in macrophage cell lines HD11 and J774A.1. Additionally, cytokine expression induced

54

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different antimicrobial resistance patterns were detected among these strains with 15 strains being resistant exclusively to sulfonamides. Single strains showed either resistance to fluoroquinolones or exhibited extended resistance patterns including resistances to sulfonamides, trimethoprim, tetracycline, amikacin, tobramycin, gentamicin, neomycin and streptomycin. Conclusion: Comparative analysis of the S. Agona isolates from porcine sources showed that a major clone is obviously prevalent among swine in Southern Brazil with members of this clone being found during 1999 and 2000 in all three regions in Southern Brazil. did the wild type SL1344 strain. sdiA, controlled by iron concentration and pH, therefore contributes to the virulence regulation of S. typhimurium in the initial stages of the infection in mice.

71(B)

EPIDEMIOLOGY OF SALMONELLOSIS IN SARDINIA, ITALY: 1995-2001

A. Azara, A. Piana, I. Maida, B. Are, G. Sotgiu, E. Muresu; University of Sassari, Sassari, ITALY. Background: Changes of food-producing and fooddistributing techniques and increase of international travels explain the rising incidence of foodborne illnesses, mainly Salmonella infections of humans and animals. It represents a problem for clinicians and for responsibles of public health. Objectives: Aim of the study is to describe the epidemiology of salmonellosis in Sardinia since 1995 to 2001, evaluating geographic distribution, incidence of occurrence and antimicrobial drug resistance in vitro. Material and methods: Hygiene and Preventive Medicine Institute of University of Sassari, Italy, receives human, animal, environmental Salmonella strains from numerous laboratories, because it is the reference centre, in Sardinia, of a European surveillance network, named Enter-Net. Results: 2,785 strains were isolated: 51% in the province of Cagliari, 30% Sassari, 14% Nuoro, 5% Oristano. Main serovars were (55%): S.enteritidis (33%) and S.typhimurium (23%). S.typhimurium was more frequently recovered during 1996, 1998, 2001; at the same time S.enteritidis had a lower frequence. Ceftriaxone, cefotaxime, ciprofloxacin showed good activity in vitro (> 98% susceptibility), Lower susceptibility rates were observed for ceftazidime, gentamycin, amykacin, trimethoprim/sulfamethoxazole, tobramycin (9098%). Conclusions: These data, linked to those obtained from molecular techniques (R.A.P.D., P.F.G.E., IS200, automated ribotyping) and compared to national and international informations, will show the complexity of epidemiological chain of salmonellosis, emphasising critic targets, like bacterial vehicles and sources of transmission, whose knowledge is condition for decreasing human infections.

70(A)

SDIA CONTRIBUTES TO VIRULENCE REGULATION IN SALMONELLA ENTERICA SEROVAR TYPHIMURIUM

J. Volf 1, M. Sevcik1, H. Havlickova1, F. Sisak1, J. Damborsky2, I. Rychlik 1; 1Veterinary Research Institute, Brno, CZECH REPUBLIC, 2National Centre for Biomolecular Research, Masaryk University, Brno, CZECH REPUBLIC. sdiA in Salmonella enterica serovar Typhimurium (S. typhimurium) encodes a protein belonging to the LuxR family of transcriptional regulators which are frequently linked with the quorum sensing systems in bacteria. Computer analysis of S. typhimurium sdiA gene revealed the presence of a fur box 19 bp upstream of the start codon of the sdiA gene and a helix-turn-helix motif typical for transcriptional regulators in the carboxy-terminal part of the SdiA protein. Using transcriptional fusions we observed that the deletion of the fur box in S. typhimurium F98 resulted in twofold increase of sdiA transcription. Addition of dipyridyl, an iron chelator, to culture media increased sdiA transcription to the level observed in the fur box mutant, confirming that sdiA is suppressed in the presence of iron. sdiA was also stimulated by the decrease in pH of LB broth. Next, in frame deletion mutants lacking the helix-turn-helix domain of SdiA were constructed in S. typhimurium F98 and S. typhimurium SL1344 strains. The former strain is free of the virulence plasmid and is attenuated for mice after per oral infection. The latter strain causes typical typhoid disease in mice. Mutants in both the strains were used for per oral infection of Balb/C mice with LD50 of each of the strains. Mortality was monitored for 3 weeks and after this period, surviving mice were sacrificed and Salmonella counts were determined. When compared with the group of mice infected with the wild type F98 strain, minor increase in mortality was observed in mice infected with the sdiA mutant. No such difference was observed in SL1344. In sacrificed mice, higher counts of Salmonella were observed in caecum, liver and spleen of mice infected with sdiA mutants than in mice infected with the wild type strains in both the F98 and SL1344. Finally, mice were infected either per orally or intraperitoneally with the sdiA mutant and the wild type SL1344 strain, and in one day intervals, 3 mice were taken from both groups and analysed for the presence of Salmonella. There was no difference in counts and distribution of the wild type strain and sdiA mutant in mice after intraperitoneal mode of infection. However, when the mice were infected per orally, sdiA mutant reached maximal counts in tissues 2 days later than Pathogenesis, Epidemiology, and Vaccine Development

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72(C)

IS200 FINGERPRINT OF SALMONELLA TYPHIMURIUM SWINE STRAINS ISOLATED IN RIO GRANDE DO SUL BRAZIL

M. C. Bessa , S. Castagna , G. B. Michael , N. Canu , M. Cardoso1, S. Rubino2; 1Universidade Federal do Rio Grande do Sul, Porto Alegre - RS, BRAZIL, 2Dept of Biomedical Sciences, University of Sassari, Sassari, ITALY.

1 1 1 2

73(A)

CHARACTERIZATION OF SALMONELLA ISOLATES FROM CAPTIVE LIZARDS

F. R. Pasmans1, A. Martel1, F. Boyen1, I. Wybo2, F. Van Immerseel1, M. Heyndrickx3, J. Collard 2, R. Ducatelle1, F. Haesebrouck1; 1Ghent University, Merelbeke, BELGIUM, 2Scientific Institute of Public Health, Brussels, BELGIUM, 3Center for Agricultural Research, Melle, BELGIUM. Reptile-associated salmonellosis in humans is an increasing public health issue. This study aims at characterizing Salmonella isolates from captive lizards and to compare them to human isolates. The presence of Salmonella in 112 faecal and cloacal samples from lizards was determined. The strains were serotyped and the antimicrobial susceptibility pattern was examined using disk diffusion tests. For comparison, 17 human Salmonella isolates belonging to 17 different serotypes of subspecies I were included. Invasion assays in Caco-2 cells were performed with 40 saurian isolates of subspecies I, 14 isolates of subspecies II, 4 strains of subspecies IIIb, 6 subspecies IV isolates and the human isolates. In these strains, the presence of the virulence genes agfA, shdA, spvR, pefA and sopE was determined using PCR. Salmonella was isolated from 75.8% of the cloacal and 60% of the faecal samples. The strains belonged to subspecies I, II, IIIb and IV (44 serotypes in total). Only two saurian strains, one of serotype Enteritidis and one of serotype Amsterdam, were resistant to nitrofurantoin. In the human strains, resistance to nitrofurantoin was detected in the serotype Enteritidis strain, resistance to sulphonamides in a serotype Urbana isolate and resistance to tetracycline, ampicillin, spectinomycin and sulphonamides in the serotype Shubra strain. Serotypes belonging to subspecies I invaded the Caco-2 cells to a higher extent than those from the other subspecies. Within the same serotype, the human isolates invaded the Caco-2 cells to a lesser degree compared to their saurian counterparts. Whereas agfA was detected in all strains, pefA was only detected in one saurian and in the human serotype Enteritidis strains. The spvR gene was detected in the same human and saurian serotype Enteritidis strains and in 33% of the subspecies IV strains. The shdA gene was present in all the human isolates and in 87% of subspecies I saurian isolates. SopE was found in 17% of the human isolates, in 22% of the saurian subspecies I strains and in all of the subspecies IV strains. In conclusion, the only indications that Salmonella subspecies I isolates from humans might represent a subpopulation different from poikilothermic isolates, are their lower intestinal invasion ability and decreased antibiotic susceptibility.

The importance of pork as a source of human salmonellosis has gained increasing attention in recent years. In studies, conducted in Southern Brazil, Salmonella Typhymurium was the most frequent serovar isolated from pigs and pork products. Thus, the aim of this study was to characterize these S. Typhimurium strains, using IS200 fingerprint, antibiotic resistance profile and spvR gene PCR amplification. A collection of 55 S. Typhimurium strains isolated in Southern Brazil during 1999/2000 was used in this study. Strains were isolated from mesenteric lymph nodes and intestinal content of healthy pigs slaughtered in three abattoirs located in different regions of Rio Grande do Sul (Brazil). Furthermore, another 20 S. Typhimurium strains isolated from intestinal content, tonsils, mesenteric or submandibular lymph nodes from pigs slaughtered during 2001 were also characterized. Finally, one strain isolated from an outbreak of gastroenteritis in a swine farm, and one strain isolated from meat implicated in an human outbreak in Rio Grande do Sul in the same period were included. Strains were submitted to hybridization with IS200 probes and antibiotic susceptibility test, using the gel diffusion method. The PCR assay for the spv region was performed using oligonucleotides sequences corresponding to the spvR gene. Using the Dice coefficient and the UPGMA clustering method, a dendrogram was constructed for IS200 fingerprinting patterns. All strains contained from 6 to 9 copies of the IS200 element, located in restriction fragments with sizes ranging from ~2,03 to 23,13 Kb. Four distinct profiles of IS200 were detected in swine strains. One pattern (A) included 47 strains isolated in 1999/2000 and all strains isolated in 2001. Each of the other three patterns (B, C and D) was represented for only one strain isolated in 1999/ 2000. Another pattern (E) was represented for strains isolated from human and swine outbreaks, which showed a common band profile. Pattern A was the most distantly related group with homology ranging from 47 to 71% to the other patterns. Furthermore, pattern E showed a homology of about 75% with pattern D. The spvR region was detected in only one strain of pattern A and in all strains belonging to pattern B, D and E. The antibiotic resistance profile showed a high diversity even in the same IS200 pattern. The pattern resistance to tetracycline-sulphonamide-sulphamethoxazole/ trimethoprim-chloramphenicol was the most common among the strains. It was concluded that the IS200 profile of S. Typhimurium swine strains may be highly conserved in Southern Brazil.

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74(B)

ASSESSMENT OF VIRULENCE OF PIGEON ISOLATES OF SALMONELLA ENTERICA SUBSPECIES ENTERICA SEROVAR TYPHIMURIUM VARIETAS COPENHAGEN

F. R. Pasmans 1, F. Van Immerseel 1, K. Hermans1, M. Heyndrickx2, R. Ducatelle1, F. Haesebrouck 1; 1Ghent University, Merelbeke, BELGIUM, 2Center for Agricultural Research, Melle, BELGIUM. Pigeon isolates of Salmonella enterica subspecies enterica serovar Typhimurium varietas Copenhagen (var. Copenhagen) are a host adapted lineage of the serovar Typhimurium. In this study, the virulence of these strains was determined. Upon necropsy, var. Copenhagen was isolated from 5 of 152 (3,3%) wild roaming pigeons from the city of Ghent, Belgium. Pooled faecal samples from 114 pigeon lofts yielded 26 positive results (22,8%). Seven of these pigeon isolates belonging to phage type (PT) 99 were further compared in vitro to five human var. Copenhagen isolates belonging to PT 208 (2x), 120, U302 and non typeable. No differences in invasiveness in human intestinal epithelial Caco-2 cells were found. The human strains, however, were able to multiply significantly more inside human THP-1 macrophages compared to the pigeon strains. Using a WST-1 cell proliferation test, no cytotoxic effects of the pigeon and the human strains on the THP-1 cells were noticed. Finally, 5 mice were inoculated intragastrally with 10 exp 4 CFU of a pigeon PT 99 strain. At 5 days after inoculation (PI), all the mice showed severe clinical symptoms including a rugged appearance, anorexia and apathia. Four of the five animals were moribund at day 7 PI and were euthanized. The following numbers of Salmonella bacteria were excreted in the faeces during the 7-day period (mean log (10) CFU / g faeces): 2,3 (day 1 PI); 1,3 (day 2 PI); 1 (day 3 PI); 1,1 (day 4 PI); 2,1 (day 5 PI); 4,3 (day 6 PI) and 4,5 (day 7 PI). Splenomegaly and granulomatous lesions were noticed at necropsy. High numbers of Salmonella were found in the livers (mean log (10) CFU / g: 6,6), spleens (mean log (10) CFU / g: 6,2) and caeca (mean log (10) CFU / g: 5,6). In conclusion, the less pronounced ability of the pigeon var. Copenhagen strains to multiply inside human macrophages compared to human strains is in line with the hypothesis of adaptation of these strains to pigeons. The virulence for mice of the tested strain and the relatively high prevalence in the pigeon population might suggest that pigeon adapted strains might occasionally cause clinical salmonellosis in humans. The rareness of this event in humans is reflected by the lack of isolation of these strains from humans in Belgium in the period 2000 - 2002.

75(C)

ENDOCARDITIS DUE TO AMPICILLINRESISTANT SALMONELLA ENTERITIDIS

G. Cartolano, C. Leclerc, C. Halphen, Y. Welker; CHI Poissy-St Germain en Laye, St Germain en Laye, FRANCE. Usually infections with non-S. typhi Salmonella strains are self limiting, but bacteremia occurs in 5% of patients. The risk of developing endovascular infection after salmonellosis has been estimated to be 25% in patients older than 50 years of age. In addition infective endocarditis is a rare manifestation of Salmonella infection mostly observed in patients with predisposing heart conditions. In the present case, mitral valve endocarditis was due to ampicillin-resistant Salmonella enteritidis, and antibiotherapy alone was successful to obtain a favourable outcome with a normalization of the echocardiography after 6 weeks of treatment.

76(A)

PARENT AS A VECTOR FOR NOSOCOMIAL INFECTION DUE TO SALMONELLA BRANDENBURG IN NEONATAL INTENSIVE CARE UNIT (NICU)

G. Cartolano, M. Moulies, J. Séguier, A. Boisivon; CHI PoissySt Germain en Laye, St Germain en Laye, FRANCE. During the 1970's, nosocomial infections due to Salmonella species were frequently reported. Since the introduction of general infection control measures, these nosocomial infections disappeared. We report the case of a septic shock due to Salmonella brandenburg observed in the Neonatal Intensive Care Unit of St Germain en Laye Hospital (France). DL was a newborn boy admitted for the management of his prematurity (34 weeks and 4 days of gestation). He had a twin sister, AL, hospitalized in the same two-bed room. A septic shock appeared on day-12, and lead to a brief cardiac arrest. Salmonella brandenburg was isolated from the faeces and from the gastric aspirate of DL, and from the faeces of his mother. Intravenous antibiotherapy was conducted during 8 days and followed with amoxicillin per os for 3 weeks. Clinical and biological evolution was favourable. Hand washing and general infection control measures were reinforced and nosocomial Salmonella gastroenteritis remained limited to DL. Recent publications have recommended that stool specimens from patients hospitalized for more than 3 or 4 days should be processed only for viruses and Clostridium difficile toxin. We think that applicability of proposed rejection criteria and exception rules must be evaluated in each hospital, and certainly in each care unit

Pathogenesis, Epidemiology, and Vaccine Development

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77(B)

CHARACTERISATION OF A NOVEL PRENYLATION MOTIF AT THE C-TERMINUS OF THE SALMONELLA TYPHIMURIUM EFFECTOR PROTEIN, SIFA

A. T. Reinicke, A. P. Kelly, J. Trowsdale; Cambridge University, Cambridge, UNITED KINGDOM. The Salmonella typhimurium virulence factor, SifA is an effector protein secreted by the SPI-2 encoded type III secretion system into the host cell. SifA has been shown to induce aggregation of host endosomal compartments into Lgp positive tubules called sifs in HeLa cells and is thought to function in maintaining the vacuolar membrane around the bacteria. In this study we have characterised a prenylation motif present at the C-terminus of SifA. Transient expression of N-terminal and C-terminal GFP fusion constructs of SifA showed differences in subcellular localisation of the protein within the melanoma cell line, Mel JuSo. A sequence comparison of the SifA C-terminal region with the Ctermini of members of the small G protein family containing mammalian C-terminal prenylation motifs showed that SifA's motif most closely resembles a type I prenylation signal and predicted that SifA be modified by a geranylgeranyl transferase. Analysis of SifA's prenylation motif by mutation of the last 6 amino acids at the Cterminus of SifA showed alteration in the protein's subcellular localisation. Each of three cysteine residues within this motif are possible targets of isoprenoid addition, a processing step which allows a protein to attach to membrane. Mutagenesis studies showed that when mutated to serine individually or in combination each cysteine contributes towards the localisation of the protein. Prenylation of proteins is step I of several steps in post translational processing and is accompanied by increases in SDS gel mobility and hydrophobicity. Futher mutational analysis of HA tagged SifA showed that the protein is accompanied by such an increase relative to its triple cysteine mutant. Recent work has showed that SifA requires its C-terminal hexapeptide to attach to membrane and that this motif is important for the biological function of the protein (Boucrot, E. et al., J. Biol. Chem., 2003). Work is in progress to demonstrate post translational processing by SifA to further understanding of the molecular function of SifA.

Systemic salmonellosis depends on the presence of the big virulent plasmid. The products of the genes located in the plasmid can influence the survival and multiplication of bacteria in host macrophages. Among other genes the spv gene group was detected. This group is highly conserved fragment of DNA, consisting of five open reading frames: R, A, B, C, D. It seems that the most important gene of this group is spvR which encodes the promouer for other spv genes. The other gene, which function was determined is spvB which encodes protein-mono(ADP-ribosyl)transferase. Functions of the of the rest of spv genes are still poorly understand. Present studies were focused on determining the frequency of plasmid-encoded spv genes occurring among S. Enteritidis strains isolated from poultry. In our studies we determined that spv operon can be detected in isolated strains in complete (all R, A, B, C, D genes) or incomplete form. The main goal of thes studies is to find the correlation between detecting complete or incomplete form of spv genes and the S. Enteritidis ability to survive in chicken macrophages and its pathogenicity for chicken. Spv genes in isolated strains were detected using PCR, then the intracellular survival and multiplication of bacteria was ... in MQNCSU macrophage like cell line using CFU counting from lised by 0,1% Triton X-100 macrophages after 1, 4, 12 , 24 and 48 hours after chalange. Preliminary data of donned experiments show that exist a few variants of spv operon (with or without one or more spv genes). It allows to conclude that there is different in intracellular survival beetwen S. Enteritidis strains with spvB gene in operon and other variants of this bacteria without spvB gene. There is no diferenties between strains with spvB gene but with miscellaneous occurrence of others spv genes.

79(A)

CHARACTERIZATION BY ANTIMICROBIAL RESISTANCE, PLASMID PROFILE, PCR-RIBOTYPING AND PFGE OF SALMONELLA ENTERITIDIS ISOLATED FROM FOODS INVOLVED IN FOODBORNE OUTBREAKS OCCURRED IN SOUTH OF BRAZIL, 19992000

M. P. Geimba1, E. C. Tondo2, C. D. Paula2, K. S. Heckler2, E. R. Caron2, F. A. Oliveira2, A. Brandelli2, C. D. Paula 2; 1Pontifícia Universidade Católica do Rio Grande do Sul - PUC/RS, Porto Alegre, BRAZIL, 2Universidade Federal do Rio Grande do Sul, Porto Alegre, BRAZIL. Salmonella sp. was identified as the principal microorganism responsible for reported foodborne diseases in the last 9 years in Rio Grande do Sul State (RS), South of Brazil. The present work aimed to characterize Salmonella Enteritidis isolated from foods involved in foodborne outbreaks occurred during the period of 1999-2000 in RS. Salmonella Enteritidis strains (n=76) were studied by antibiotic resistance, plasmid profile, PCR-ribotyping and Pulsed-Field Gel Electrophoresis (PFGE). The highest susceptibility percentages were detected to chloramphenicol (99%), ampicillin (97%), sulfamethoxazole/trimethoprim (97%), sulfazotrim (96%), kanamycin (93%), and nalidixic acid ASM Conference on Salmonella

78(C)

INFLUENCE OF PLASMID-ENCODED SPV GENE GROUP ON SURIVAL AND MULTIPLICATION OF SALMONELLA ENTERITIDIS IN CHICKEN MACROPHAGES

G. Madajczak; Warsaw Agriculture University, Veterinary Medicine Faculty, Warsaw, POLAND. Salmonellosis in chicken, caused by Salmonella enterica subspecies enterica serovar Enteritidis may cause hard systemic infection with numerous lethal outcomes, or enteritis with diarrhoea and cycling sowing of pathogens. 58

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(84%). Strains were also sensitive to gentamycin (72%), streptomycin (63%), neomycin (59%), and tetracycline (43%). Resistance percentages were demonstrated only to streptomycin (36%), gentamycin (13%), and nalidixic acid (13%). Antimicrobial analysis generated 36 profiles, demonstrating variation in antimicrobial resistance patterns. The most frequent pattern was represented by profile 22, which have grouped 24 strains. Four different types of plasmids were identified with approximately sizes of 15, 1.5, 0.8, and 0.2 Kb. Six different plasmid profiles were identified (A to F), being that profiles were very similar. Profiles A and B presented plasmid profiles with only a plasmid of difference and, together, them have grouped 77% of the strains. PCRribotyping analysis generated 3 profiles, with DNA banding patterns close related. Partial PFGE results have confirmed the genetic similarity of the strains. Results suggested the involvement of the same strain of S. Enteritidis in different foodborne outbreaks in Rio Grande do Sul. metabolic physiological state. The down-regulated swarm cell proteins included several outer membrane proteins (OMPs), suggesting that the OM of swarm cells may be relatively less permeable. In addition to the up-regulation of a putative glutathione-S-transferase, decreased OM permeability may represent a general mechanism of increased antibiotic resistance in swarm cells for relieving intracellular toxicity while decreasing the influx of toxic compounds. It was previously shown that SlyA up-regulates the expression of a similar subset of OMPs that were identified in our study. SlyA and several heat shock proteins (i.e. GroEL) that are members of the SlyA-regulon were down-regulated in the swarm cells, suggesting that down-regulation of the SlyAregulon may be a characteristic signature of the swarm state. Thus, a swarming population of serovar Typhimurium represents a unique physiological state that is well equipped to withstand high levels of toxic compounds and tailored for rapid population expansion and migration within a nutrientrich environment.

80(B)

PROTEOMIC ANALYSIS OF SWARMING SALMONELLA: A UNIQUE PHYSIOLOGICAL STATE ASSOCIATED WITH ELEVATED RESISTANCE TO A BROAD SPECTRUM OF ANTIMICROBIAL COMPOUNDS

W. Kim, M. G. Surette; University of Calgary, Calgary, AB, CANADA. Salmonella enterica serovar Typhimurium is capable of swarming over semi-solid surfaces. This form of motility is distinct from the swimming motility, as it is strictly preceded by a morphological differentiation of short swim cells into elongated, hyperflagellated, and polyploid swarm cells. In addition to the relative solidity of the growth medium, specific nutrient availability and cell-density are key factors that trigger such differentiation. We have recently shown that swarm cells exhibit elevated resistance to a wide variety of structurally and functionally distinct classes of antibiotics, including cationic peptides, penicillins, aminoglycosides, and quinolones. As serovar Typhimurium differentiate into swarm cells, the pmr operon is up-regulated, resulting in a more positively charged LPS core. This is one potential mechanism which confers swarm cells increased resistance to antibiotics such as cationic antimicrobial peptides present in the human gastrointestinal tract. However, additional mechanisms are likely associated with the cells in the swarm state that confer elevated resistance to such a broad spectrum of antimicrobial agents. With an aim to gain insight into the other potential mechanisms of antibiotic resistance and to further characterize this unique physiological state, we utilized a proteomic approach based on 2DGE and MALDITOF mass spectrometry. More than 80 differentially regulated proteins were identified by comparing the whole cell-extracts of swim and swarm cells. As anticipated, FliC (flagellin) was up-regulated, reflecting the hyperflagellated swarm cells. The majority of the up-regulated proteins were those involved in pathways of carbon and nitrogen metabolism, nucleic acid biosynthesis, and energy production, suggesting that swarm cell differentiation results in a highly Pathogenesis, Epidemiology, and Vaccine Development

81(C)

PROTEOMIC ANALYSIS OF DRY, WRINKLED COLONIES OF SALMONELLA ENTERICA SEROVAR TYPHIMURIUM

A. P. White1, W. Kim1, D. Gibson2, P. Banser2, W. W. Kay2, M. G. Surette1; 1University of Calgary, Calgary, AB, CANADA, 2 University of Victoria, Victoria, BC, CANADA. Prolonged growth of many Salmonella enterica serovars at temperatures below 30 °C on standard laboratory agar (i.e., LB) results in the formation of dry, wrinkled colonies.This colony "state" is associated with the production of several polymeric substances that mediate cell-cell attachment, namely cellulose, extracellular polysaccharides (EPS), and thin, aggregative fimbriae (Tafi; curli). Disruption of cellulose or Tafi production results in cells that are unable to form this dry, wrinkled phenotype. S. typhimurium 14028s was compared with isogenic ÄbcsA (cellulose-) and ÄagfA (Tafi-) strains to identify proteins up-regulated as a result of cellcell aggregation and micro colony formation. From these, a small subset of proteins present on or near the cell surface was isolated by washing colonies in low ionic strength buffers and > 40 proteins were identified using twodimensional gel electrophoresis followed by MALDI-TOF mass spectrometry. The majority of the proteins identified were found to be common to all three strains, but some "state"-specific proteins were identified. Stress proteins, glycolytic enzymes, sugar transporters and binding proteins and several proteins known to be regulated by RpoS were identified. Production of Tafi and cellulose are normally regulated in an RpoS-dependent manner and the dry, wrinkled "state" takes at least 48 hours to develop at 28 °C. By shifting the regulation of Tafi and cellulose production to be RpoD-dependent, we were able to induce similar colony conditions and protein up-regulation within 24 hours at 37 °C. Cellulose, Tafi and EPS have all been previously implicated in biofilm formation and environmental persistence. Therefore, these results enhance our understanding of the physiological dynamics occurring within individual

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bacterial colonies. Our most significant findings are discussed in relation to current hypotheses.

83(B)

BIOCHEMICAL ANALYSIS OF THE INVH-PEPTIDOGLYCAN ASSOCIATION

M. Pucciarelli, F. Garcia-Del Portillo; Centro Nacional De Biotecnologia-CSIC, Madrid, SPAIN. The Salmonella enterica InvH lipoprotein was identified as a factor required for invasion of eucaryotic cells. Functions assigned to InvH include outer membrane targeting of the secretin InvG, efficient assembly of the needle complex and elicitation of enteropathogenesis. To get insights on how these functions are accomplished, we have examined the precise association of InvH to envelope components. InvH was found among a set of proteins bound to macromolecular peptidoglycan (PG) upon boiling of envelope material in 4% SDS. Association of InvH to PG occurs independently of components of the base (PrgH, PrgK or InvG), the needle (PrgJ, PrgI), the export apparatus (InvA), or needle length regulators (InvJ). However, the interaction of InvH with peptidoglycan was abolished by alteration of peptidoglycan structure introduced by D-aminoacids in live bacteria. Under these conditions, neither the level of membrane-associated InvH nor the outer membrane targeting of the secretin InvG was affected. D-aminoacidtreated bacteria had no InvH associated to peptidoglycan and were impaired for secretion of type III effectors, invasion of cultured epithelial cells and correct assembly of the needle complex. Our current interest is focused in the mapping of the InvH domains mediating association to PG. The analysis of the primary structure of InvH revealed the presence of a region at residues 61-76 with features similar to the PGbinding motif (PBM) described for other PG-associated proteins such as OmpA and Pal. A series of truncated InvH variants are being engineered and tested in in vitro binding assays to purified peptidoglycan material. Our preliminary data show that full-length InvH associates specifically to peptidoglycan isolated from Salmonella enterica but less efficiently to that of Escherichia coli. This observation suggests that yet undefined structural determinants of the S. enterica peptidoglycan are responsible for mediating InvH association. Unexpectedly, the truncated variant devoid of the putative PBM domain associated to PG as the full length form, which indicates that either this region is not critical for association to PG or that InvH might associate by more than one region separated in the primary sequence. To test this second hypothesis, we are performing in vitro competition experiments with peptides that cover distinct regions of the InvH sequence.

82(A)

PHASE VARIATION IN SALMONELLA ENTERICA SEROVAR TYPHIMURIUM

R. E. Isaacson1, S. K. Patterson, II2; 1University of Minnesota, St. Paul, MN, 2University of Illinois, Urbana, IL. Salmonella enterica serovar Typhimurium strain 798 is able to cause persistent asymptomatic infections of swine. This strain has been found to undergo a phenotypic phase variation that correlates with the coordinate expression of a set of genes associated with the ability of the strain to colonize the intestines of animals and to cause disease. Detection of phase variants of strain 798 is readily accomplished by plating cells on Evans Blue-Uranine (EBU) agar. Cells in the "virulent phase" result in white colonies while cells in the "avirulent phase" result in blue colonies. Compared to other virulent S. Typhimurium strains, strain 798 has a very high LD50 in BalbC mice (~109). To determine if other strains of S. Typhimurium are capable of undergoing this phenotypic phase variation we screened two highly virulent strains (SL1344 and 14028) and an attenuated strain (LT2) on EBU agar. When plated on EBU agar all three strains generated white colonies indicating that they were in the "virulent phase". However, using an ampicillin enrichment technique, blue "avirulent phase" colonies of SL1344 and 14028 were detected. Blue colonies of strain LT2 never were observed. When blue colonies from SL1344 or 14028 were picked and re-streaked on EBU agar, all the resulting colonies blue and white sectored colonies. Very few completely blue colonies were observed. Rates of phase variation from the "virulent phase" to the "avirulent phase" were estimated to be 3-4x10-6 per cell per generation for SL1344 and 14028. This is very similar to the rate of phase variation for strain 798. However, the rates of phase variation from the "avirulent phase" to the "virulent phase" for SL1344 and 14028 were significantly higher compared to strain 798: 13X10-2 per cell per generation for SL1344 and 14028 compared to 1-2X10-4 per cell per generation for strain 798. Consequently, while strain 798 can be maintained in the "avirulent phase", strains SL1344 and 14028 can not. A comparison of the abilities of strains 798 and 14028 to invade Henle 407 cells was performed. Unexpectedly, the "virulent phase" 798 cells were much more invasive than 14028 cells. However, "avirulent phase" cells of strain 798 were roughly as invasive as strain 14028. Currently we are using a DNA microarray to detect the differences in gene expression between the two phases of strain 798, SL1344 and 14028.

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ASM Conference on Salmonella

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84(C)

SALMONELLA FROM FISH AND WATER OF THE 23 RD JULY LAKE IN BENGHAZI

H. A. Nashnoush 1, R. S. Tobgi2, K. S. Ghenghesh3; 1Faculty of Science, Benghazi, LIBYAN ARAB JAMAHIRIYA, 2Tuberculosis Center, Benghazi, LIBYAN ARAB JAMAHIRIYA, 3Faculty of Medicine, Tripoli, LIBYAN ARAB JAMAHIRIYA. Objectives: The 23rd of July Lake is an important recreational site in Benghazi. Although, it is polluted by untreated sewage, the lake is used by the locals for fishing and other activities. The aims of the present work was to determine whether salmonellae are present in the fish and water of the lake and if so what are the serotypes that are prevalent and their susceptibility to antibiotics. Methods: Included in the study 106 fish and 24 water samples from the lake and, for comparison, 41 sea fish and 8 seawater samples from the coastal adjacent coastal area. Isolation, identification, serotyping and susceptibility to antibiotics of salmonellae were carried out using standard bacteriological procedures. Results: Salmonella were isolated from 5 (4.7%) and 3 (12.5%) of lake fish and water samples respectively. No salmonellae were isolated from sea fish and seawater samples. Serotyping of salmonellae from lake fish resulted in of 3 different serotypes (2 S. Enteritidis, 1 S. Typhimurium and 2 S. Arizonae). From lake water one each of 7 different serotypes (S. Amesterdam, S. Braemderup, S. Enteritidis, S. Heidelberg, S. Livingstone, S. Mbendaka and S. Wien). All (100) isolates were susceptible to ampicillin, cephalexin, chloramphenicol, ciprofloxacin, gentamicin, tetracycline and trimethoprim-suphamethoxazole. Conclusions: Salmonellae are not uncommon in fish and water of 23rd July Lake and their presence is a serious health problem for the community. The health and environmental authorities in Benghazi City should take proper measures to clean up the lake.

importance of O PS modal length regulation by Wzz is poorly defined. We have reported that S. Typhimurium LPS O PS chains have two distinct modal lengths. Long O PS chains with 16-35 repeat units are determined by wzzST, while wzzfepE (a homologue of E. coli fepE) determines a second modal length (>100 repeat units). Mutation of both wzz genes led to unregulated O PS length, an increase in susceptibility to complement and reduced virulence in the mouse model of infection. This indicates that O PS modal length regulation is required for virulence in the mouse, and that Wzz proteins form the molecular basis for the long O PS chains previously identified as a major factor in complement resistance. Complementation of a wzz double mutant with wzz genes from different bacterial species has resulted in a unique panel of isogenic LPS O PS length derivatives with modal lengths ranging from very short (2-6 repeats) to very long (>100 repeats). When used in various assays, complex relationships between O PS modal length, protection from the bactericidal activity of complement and complement activation were identified. These results highlight the importance of wzz genes as determinants of the structure of LPS, particularly through interaction with a component of the innate immune system. Such interactions are likely to be important in determining the outcome of Salmonella infections.

86(B)

IDENTIFICATION OF NOVEL GENES RESPONSIBLE FOR BIOFILM FORMATION IN SALMONELLA ENTERICA SEROVAR TYPHIMURIUM USING DNA MICROARRAYS

M. S. Hamilton1, R. J. Bongaerts2, S. Lucchini 2, J. D. Porter1, J. C. Hinton2, H. M. Lappin-Scott1; 1University of Exeter, Exeter, UNITED KINGDOM, 2Institute of Food Research, Norwich, UNITED KINGDOM. Opportunistic pathogens, such as Salmonella spp., are of particular importance for the medical and food industries as they cause diseases ranging from gastroenteritis to typhoid fever. The success of Salmonella enterica serovar Typhimurium to survive and overcome environmental stress is apparent from the frequent isolation of these bacteria from a large number of sources, ranging from soils to kitchen surfaces, and in human infection. It has been shown that survival and persistence is enhanced when Salmonella is attached to a surface as a biofilm, allowing bacteria to overcome limited nutrient availability, low pH and the low temperatures used in food storage. The formation of highly structured biofilms on surfaces during food processing allows S. Typhimurium to cause contamination while resisting antimicrobial treatments. In this study we used whole genome DNA microarrays to discover which genes are responsible for biofilm formation and whether they are differentially expressed in comparison to free living (planktonic) cells. Analysis of the microarray data revealed that many genes involved in chemotaxis, motility and amino acid metabolism were affected. We observed the upregulation of genes known to encode cell surface structures that have previously been implicated in biofilm formation, such as fimbriae. Many 61

85(A)

THE ROLE OF WZZ GENES IN SALMONELLA ENTERICA SEROVAR TYPHIMURIUM VIRULENCE AND INTERACTION WITH COMPLEMENT

G. L. Murray, S. R. Attridge, R. Morona; The University of Adelaide and The Pest Animal Control Cooperative Research Centre, Adelaide, AUSTRALIA. Salmonella enterica serovar Typhimurium is responsible for gastroenteritis in humans and a systemic infection in mice that has similarities with typhoid fever. Lipopolysaccharide (LPS) is an important virulence determinant of Salmonella, conferring protection against the innate host defences including complement. LPS is located in the outer leaflet of the outer membrane and consists of lipid A, an oligosaccharide core region, and a polymer of O polysaccharide (O PS) repeat-units. The number of O PS units polymerised into LPS is not random, but determined by Wzz proteins, which impose a modal length typical of each species. While the requirement for O PS for virulence is well established, the Pathogenesis, Epidemiology, and Vaccine Development

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genes located in Salmonella pathogenicity islands were significantly repressed. Interestingly, the expression of many genes of unknown function also showed significant variation in expression in biofilms. Deletion mutants were constructed for selected genes identified by the microarray analysis to investigate their role in biofilm formation. This led to the identification of novel genes that are required for biofilm formation in both static and flowing systems.

88(A)

THE PHYSICAL-CHEMICAL NATURE OF THE DIET AND ITS EFFECTS ON ASSOCIATION OF SALMONELLA IN THE PIG GUT

L. L. Mikkelsen1, M. S. Hedemann1, P. J. Naughton2, B. B. Jensen 1; 1Research Centre Foulum, Danish Institute of Agricultural Sciences, DK-8830, DENMARK, 2University of Ulster (at Coleraine), Co. Londonderry, BT52 1SA, UNITED KINGDOM. Previous studies in chicken suggest that the physicalchemical nature of the diet may have an influence on the incidence of Salmonella. Therefore, we investigated the effect of the physical-chemical nature of the diet on the association of Salmonella in the gastrointestinal tract of pigs. Four groups of 6 Danish Landrace x Yorkshire pigs (approx. 25kg) were fed either a coarse non-pelleted (C-NP), coarse pelleted (C-P) fine non-pelleted (F-NP) or fine pelleted (F-P) standard pig diet according to a 2x2 factorial design. Post mortems were performed and tissue segments were taken from the distal small intestine. The intestinal segments were inoculated with 9.5E7 of Salmonella typhimurium DT12 and the intestinal segments were incubated at 37oC for 1 hour. The sections were washed vigorously with PBS to remove the non-adherent bacteria, the tissue was homogenised and the bacterial numbers calculated per gram wet weight. The numbers of Salmonella isolated from the different treatment groups were log transformed and statistically analysed. The present study demonstrated that the nature of the feed reduced the association of Salmonella typhimurium DT12 in the porcine intestine organ culture model. There was a significant difference between the number of Salmonella isolated when comparing the pelleted with the non-pelleted diet (P<0.05). The data from the present study suggests that the physical-chemical nature of the diet may alter the epithelial tissue and perhaps the expression of receptors on the pig gut surface. The changes in the morphological characteristics of the pig small intestine as a result of different dietary treatments will be discussed.

87(C)

THE IMPORTANCE OF LIPID-A MEDIATED MACROPHAGE ACTIVATION FOR THE CONTROL OF PRIMARY SALMONELLA INFECTIONS

O. L. Wijburg1, D. J. Maskell2, N. Van Rooijen 3, R. A. Strugnell1; 1The University of Melbourne, Melbourne, AUSTRALIA, 2The University of Cambridge, Cambridge, UNITED KINGDOM, 3Vrije Universiteit, Amsterdam, NETHERLANDS. Salmonella spp. are facultative, intracellular bacteria which are thought to reside and replicate inside macrophages. In previous studies, we showed that this Salmonella-macrophage interaction is the cause of the mortality associated with primary infections. Results of recent studies have suggested that lipid A of LPS is an important factor in lethal murine salmonellosis. We investigated the significance of lipid Amediated activation of macrophages in the immunobiology of S.typhimurium infections using a Delta-waaN mutant of S.typhimurium C5, which synthesises a lipid A molecule lacking one fatty acid chain. This DwaaN mutant elicits reduced levels of TNFa, IL1b or NO, grows at the same rate as wild-type S.typhimurium C5 but, in contrast to the wildtype parent, is rapidly cleared at around 10-14 days post infection. Mice depleted of macrophages by toxic liposomes were used to study inflammation in DwaaN-infected mice. These studies showed that unlike wild-type strains, DwaaN S.typhimurium failed to attract inflammatory cells in the absence of macrophages, and although still able to replicate in vivo, was not lethal. We also studied the role of T lymphocytes in control of DwaaN S.typhimurium infections in mice. Flow cytometric analysis of spleen cell suspensions demonstrated that clearance of bacteria coincided with a large influx of lymphocytes which lacked any T or B cell markers, and current studies are focussing on the identification of these cells. Further experiments analysed the production of T cell cytokines by intracellular staining and flow cytometry. The importance of our results for the current understanding of the immunobiology of Salmonella infections will be discussed.

89(C)

MULTIPLE COPIES OF EAST1-ENCODING IS1414 (ASTA) ARE ASSOCIATED WITH STRAINS OF SALMONELLA ENTERICA SEROVAR ABORTUSOVIS.

D. Bacciu, D. Chessa, G. Falchi, S. Rubino, S. Uzzau; Dip Scienze Biomediche, University of Sassari, Sassari, ITALY. Sequence analysis of Gifsy-2AO prophage in Salmonella enterica serovar Abortusovis uncovered the presence of IS1414, an insertion sequence previously identified in epidemic strains of enterotoxigenic Escherichia coli (ETEC). IS1414 encodes for a heat-stable toxin, EAST1 (astA), structurally related to ETEC stable toxin ST and to a group of mammalian heat-stable peptides (i.e., guanylin and uroguanylin) that regulate electrolyte and water transport in intestine by means of the cGMP signaling. To our knowledge, this is the first evidence for intergeneric transfer of virulence genes via IS elements. IS1414 exists in multiple ASM Conference on Salmonella

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genomic copies in ETEC epidemic strains. These data have prompted us to determine whether serovar Abortusovis epidemic strains also carry other copies of astA ­ and of the IS1414 element ­ in addition to the one encoded within the Gifsy-2AO prophage. We examined a collection of 27 strains isolated from ovine in various geographic areas. 81 % of the serovar Abortusovis strains tested were found positive for the astA gene. These strains showed a very high number of restriction fragments hybridizing with the astA/IS1414 probe (i.e., more than 20). The variety of astA/IS1414 profiles, even between serovar Abortusovis strains that are epidemically related, suggests that IS1414 can transpose actively. Sequence analysis of the DNA regions flanking IS1414 showed a large adjacent deletion, possibly resulting by replicative transposition. The presence of a high number of IS1414 copies within serovar Abortusovis chromosome prompted us to evaluate whether its frequent transposition is associated to deletion and/or rearrangements within the bacterial DNA. To date, we cloned and sequenced 12 insertion loci of IS1414 within the genome of serovar Abortusovis strain SS44. We found that IS1414 insertion occurred within pathogenic loci already described in other Salmonella serovars (including SPI1) and within regions without homology in bacterial genomes databases, but encoding putative virulence genes (including fimbrial genes). The wide distribution of epidemic strains carrying IS1414 (EAST1) suggests that horizontal acquisition of EAST1 toxin and the frequent transposition of IS1414 within virulence associated genetic loci might have improved the pathogenic fitness of serovar Abortusovis strains in ovine. results confirmed that the Salmonella virulent plasmid (spvplasmid), usually present in the invasive serotypes Enteritidis, Typhimurium, Dublin and Cholerasuis, is absent in 80 isolates of serotype Virchow. Since serotype Virchow has a known potential to cause invasive disease, our results favor the assumption that chromosomal virulence genes, rather than the virulence plasmid, are the most important determinants for the virulence phenotype in humans; (ii) Virchow has increased invasive potential in very young children (blood invasiveness ratio was 4.4), and is the most common cause of NTS-associated bacteremia during early childhood, causing 31% of cases; (iii) Virchow shows high rates of resistance to multiple antibiotic agents, which have dramatically increased during recent years. Thirty-one percent of the strains were resistant to one antibiotic agent, and 57% to multiple antibiotics. Furthermore: there was a dramatic increase in the resistance to multiple antibiotics over time as follows: chloramphenicol - from 13 to 43% of the isolates, nalidixic acid - from 60 to 80%, ampicillin - from 13 to 30%, and trimethoprim-sulfamethoazole - from 27 to 45%. To study the molecular basis for the resistance strategies, all the resistant isolates were analyzed for the presence of Class 1 integrons and plasmids. The role of the AcrAB efflux pump was studied by means of a panel of reporter plasmids, in which GFP is under the control of acrAB promoter. Answering key questions regarding the spread of Virchow in Israel may help to reverse the trend and design better preventive and control policies. This may intercept not just the spread of Virchow, but also the emergence of other salmonella serotypes and the development of multi-drug resistance.

90(C)

THE EMERGENCE OF SALMONELLA ENTERICA SEROTYPE VIRCHOW AS PREDOMINANT SALMONELLA SEROTYPE IN ISRAEL: MOLECULAR EPIDEMIOLODY AND CHARACTERIZATION OF VIRULENT PROPERTIES

H. Solnik1, M. Weinberger2, S. Yaron1; 1Technion - Israel Institute of Technology, Haifa, ISRAEL, 2Rabin Medical Center, Petach Tikva, ISRAEL. In most developed countries a few Non-typhoid Salmonella enterica (NTS) serotypes, mostly Enteritidis and Typhimurium, are responsible for the vast majority of NTS infections. In Israel, a third serotype, serotype Virchow, has demonstrated a rapid increase in prevalence, and has even become the leading serotype in the year 2000, accounting for 22.5% of reported NTS infections. This worrying distribution is very different from the rest of the world, where serotype Virchow usually accounts for 0-5% of the Salmonella serotypes. The present research was undertaken to study the virulence, epidemiology and resistance properties of serotype Virchow. Results present unfavorable characteristics of serotype Virchow: (i) it is capable of invading the blood stream and causing systemic disease, particularly bacteremia and meningitis. Blood invasiveness ratio (the ratio between the number of blood and blood plus stool isolates) was 2.9, while average of the invasiveness of the most common Israeli serotypes between 1995-1999 was 2.2. PCR Pathogenesis, Epidemiology, and Vaccine Development

91(A)

IMMUNE RESPONSES TO LIVE SALMONELLA VACCINE IN MICE

S. Cho1, J. Park1, S. Seok1, H. Lee1, D. Kim 1, S. Lee2, S. Hur2, S. Ban2, Y. Lee2, H. Won2, J. Park 1; 1Seoul National University, Seoul, REPUBLIC OF KOREA, 2Biological Evaluation Department, Korean Food and Drug Administration, Seoul, REPUBLIC OF KOREA. This study was performed to provide the guideline for evaluating efficacy of recombinant Salmonella live vaccine. We examined the immunologic efficacy of a candidate typhoid vaccine strain (recombinant salmonella typhimurium LF 878) and Salmonella typhi Ty2. Ten mouse was orally administered with 10 5 and 106 CFU of LF878, 107 and 109 of S. typhi Ty2 and control with sterile PBS. Five mice of each group were necropsied at 5 and 12 days after infection, respectively. On ELISA and RT-PCR using serum and spleen tissue, expression of immunoglobulins and cytokines was higher in mice immunized with LF878 and S. typhi Ty2 than in control. When treated with T- and B-cell mitogens, spleen cells of mice immunized with LF878 and S. typhi Ty2 was more proliferating than that of control. And expression of CD4+ T lymphocyte in spleens of mice with LF878 and S. typhi Ty2 also increased more than control. Test methods used in this study could be useful as an evaluating system for efficacy of recombinant attenuated live salmonella vaccine.

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92(B)

VI ANTIGEN AFFECTS THE EARLY INTERACTIONS OF S. TYPHI WITH HUMAN MONOCYTE DERIVED MACROPHAGES BUT IS RAPIDLY RELEASED UPON PHAGOCYTOSIS

D. L. House, K. Unsworth, D. Holden, G. Dougan; Imperial College, London, London, UNITED KINGDOM. Studies of S. Typhimurium infection in the mouse have shown that the interaction between Salmonella and host macrophages is a key event in the pathogenesis of systemic salmonellosis. Unlike S. Typhimurium, the human systemic pathogen S. Typhi can express a capsule, known as the Vi antigen. There is evidence to suggest that the Vi capsule is anti-opsonophagocytic and that it may protect intracellular bacteria from oxidative killing. Studies with other encapsulated species suggest that capsules can also affect the trafficking of intra-cellular bacteria. Using isogenic Vi+ and Vi- strains, we investigated the effect of the capsule on the uptake and intracellular fate of S. Typhi Ty2 within human monocyte derived macrophages (MDMs). We found that both in the presence or absence of opsonisation with nonimmune serum, the Vi- mutant was more readily phagocytosed by MDMs than the Vi+ parental strain. The observed differences in uptake were due to differences in adherence, the rate of phagocytosis being the same in the presence and absence of the capsule. Similarly, in the presence of immune sera from patients with typhoid fever the level of uptake was enhanced with Vi- but not Vi+ S. Typhi. These results indicate that the capsule interferes with phagocytosis both directly and indirectly, by inhibiting and/or blocking the deposition of opsonins. We found no evidence to suggest that the capsule enhanced the subsequent survival of intracellular bacteria over a 6-8 hour infection period. In fact, the proportion of wild type bacteria positive for Vi surface expression declined dramatically following uptake. Concurrent with this decrease in expression, aggregates of the Vi antigen were observed within the cytoplasm of the infected MDMs. These results suggest that the Vi antigen affects only the early interaction between S. Typhi and human MDMs. Further studies are in progress to determine the nature of the Vi aggregates and the potential interaction between the antigen and host-cell antigen-presenting molecules, and to characterise the effect of the capsule on the maturation of the S. Typhi containing vacuole.

93(C)

CHARACTERISATION OF S. TYPHIMURIUM SPI1 TYPE THREE SECRETION SYSTEM (TTSS) EFFECTOR GENES IN A NOVEL MURINE COLITIS MODEL VIA GREEN FLUORESCENT PROTEIN (GFP) REPORTER CONSTRUCTS

B. E. Stecher, W. Hardt; ETH Zurich, Zurich, SWITZERLAND. Salmonella infections are an important health concern both in developed and industrialized countries affecting millions of people each year. Molecular mechanisms and virulence factors involved in gastroenteritis provoked by S. Typhimurium are poorly understood until today. Recently, we have developed a mouse colitis model that allows us to study Salmonella virulence factors involved in intestinal inflammation. The SPI1 TTSS was shown to play a major role in the outcome of disease. However, on the molecular level SPI1 mediated intestinal inflammation remains to be elucidated. We used GFP as a reporter to monitor expression of SPI1 TTSS effector proteins. Promoter-GFP fusions of several S. Typhimurium SPI1 TTSS effector genes were constructed and the GFP-expression levels under different in vitro culture conditions were defined by FACS analysis and compared to the respective levels in vivo. To localize the site and percentage of promoter activation we here also performed in situ immunofluorescence of infected gut tissues.

94(A)

DIFFERENTIAL INVASIVE CAPACITY BETWEEN SALMONELLA ENTERICA SEROVAR TYPHI, CHOLERAESUIS, PULLORUM, ENTERITIDIS AND TYPHIMURIUM TO HEP-2 CELL CULTURES

N. M. Cardona-Castro 1, M. M. Sanchez-Jimenez2; 1Instituto Colombiano de Medicina Tropical, Instituto de Ciencias de la Salud CES, Medellin, COLOMBIA, 2Universidad de Antioquia, Medellin, COLOMBIA. Host specificity of Salmonella spp suggests differences in the early steps of pathogenesis and different expression of invasive genes. Invasion to Hep-2 cells was tested to detect different capacity of invasion between Salmonella serotypes Typhimurium EE 138, Choleraesuis EE86, Enteritidis EE459, Typhi EE8 and Pullorum EE 456. Also, detection of hilA gene (hyperinvasive locus A, a regulator gene of the pathogenicity island 1) by polymerase chain reaction (PCR) was carried out with the aim to detect the sequences of this gene in these serotypes. Invasion was done with the gentamicin protection assay. The Hep-2 cell cultures were inoculated with Salmonella spp isolates obtained from different osmolarity of Luria Berthani broth (1% and 5% NaCl for low and high osmolarity respectively). The percentage of recovering of intracellular Salmonella cells was determined through dilution cultures of the intracellular bacteria ASM Conference on Salmonella

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recovered. The percentage of recover of intracellular Salmonella spp was compare between serotypes to determine its invasive capacity to Hep-2 cells. The percentage of CFU/ ml was compared between isolates from low and high osmolarity medium. The serotype Typhimurium has the higher percentage of invasion to Hep-2 cells with low and high osmolarity isolate inoculum respect to other serotypes. The serotypes Enteritidis, Choleraesuis and Typhi have similar invasion levels and the Pullorum serotype has low invasion index in Hep-2 cells with inuculum obtained from low and high osmolarity. hilA gene was detected in all the serotypes tested.

96(C)

CONTRIBUTION OF INDIVIDUAL SPI-1 EFFECTOR PROTEINS TO S. TYPHIMURIUM COLITIS IN STREPTOMYCIN-PRETREATED MICE

S. Hapfelmeier, B. E. Stecher, K. Ehrbar, W. D. Hardt; Institut of Microbiology ETH, Zurich, SWITZERLAND. Salmonella enterica subspecies 1 serovar Typhimurium (S. Typhimurium) induces gastroenteritis in man and cattle. In these species, infection with S. Typhimurium typically remains non-systemic and localized to intestinal tissues. Current knowledge of the molecular mechanisms of enteric salmonellosis emerged mainly from studies in bovine infection models which revealed the Salmonella pathogenicity island 1 (SPI-1) effector proteins SopA, SopB, SopD, SopE, SopE2 and SipA as the principal elicitors of intestinal inflammation. Orally infected mice develop a systemic disease without overt signs of intestinal inflammation. Nonetheless, we recently showed that orally streptomycinpretreated mice provide a model for S. Typhimurium colitis. We further characterized and validated this model as a sensitive tool to evaluate the contribution of individual virulence factors to intestinal inflammation. Using this murine colitis model we have analyzed the involvement of the SPI-1 type III secretion system (TTSS) and different SPI-1 effector proteins in intestinal inflammation. Results concerning the role of individual SPI-1 effector proteins and their relation to findings in the bovine infection model will be presented.

95(B)

THE LRP GENE OF THE MOUSE PATHOGEN CITROBACTER RODENTIUM

A. Cordone, E. Mauriello, M. De Felice, E. Ricca; University Federico II, Napoli, ITALY. The gram-negative enterobacterium Citrobacter rodentium is the causative agent of transmissible murine colonic hyperplasia and belongs to the family of enteric pathogens, which includes enteropathogenic and enterohemorrhagic strains of Escherichia coli, Shigella and Salmonella strains. This group of enterobacteria show several similarities with regard to their initial interactions with epithelial cells, mediated by protruding structures (pili) or adhesion molecules, and to the use of the type III secretion system to deliver molecules to the host cells. Soon after the attachment of the bacteria to the host plasma membrane, these enteric pathogens cause similar localized changes in the epithelial surface, rearrangement of host actin and related cytoskeletal components, and activation of host signal transduction pathways. C. rodentium is a mouse pathogen that in infected mice induces mild diarrhea, coat ruffling, retarded growth and, in extreme cases, rectal prolapse with moderate to high rates of mortality, depending on the mouse strains. The mechanisms of Citrobacter infection have been recently the object of several studies aimed at using the mouse pathogen as a model for studying human pathogens. We report the identification and characterization of the lrp gene of C. rodentium. lrp encodes a global transcriptional regulator (Lrp, Leucine- responsive Regulatory Protein), initially identified in E. coli but present in various enterobacteria. Lrp controls either positively or negatively the expression of a number of genes including some encoding virulence factors. Lrp of C. rodentium appeared highly homologous to the protein of E. coli and S. typhimurium and was able to complement an lrp null mutant of E. coli. Translational fusion to the E. coli lacZ gene, RT-PCR and primer extention experiments were used to characterize the expression of lrp in C. rodentium. Our results represents a first study of a transcriptional regulator of C. rodentium and provide some tools to analyze its effects on the expression of virulence factors.

97(A)

CREATION OF A HYPER INVASIVE ATTENUATED GENE TRANSFER VEHICLE

L. Marsh, T. Chakraborty; Justus-Liebig Univeristät, Giessen, GERMANY. Salmonella typhimurium is a promising candidate for the in-vivo treatment of cancer.This treatment can be achieved via prodrug converting enzymes, Salmonella expressed Immunoantigens, or delivery of host expressed plasmids. Attenuation must be balanced to avoid overt effects of uncontrolled systemic infection and efficacious expression of antigens. Attenuation of wild- type isolates of Salmonella typhimurium strains S1016, S1017 and the laboratory strain SL7207 was achieved by extensive use of the Lambda Red based recombination system. Deletions were created by replacing between 70-100% of the ORF with an antibiotic resistance marker, which was subsequently removed by recombination leaving a single FRT scar. Genomic deletions were created in aroA, phoP, asd, invA, pnp, and hilA to creating single and multiple mutant strains. Phenotypic characterisation of the mutants were performed to select for strains that are highly invasive and can be controlled for growth in tissue culture and in animal assays. Data will be presented on the use of these vehicles in tumour therapy in animal models.

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98(B)

MOLECULAR EPIDEMIOLOGY OF THREE OUTBREAKS OF SALMONELLOSIS IN GEORGIA IN 2002

M. G. Kekelidze, S. A. Tsanava, L. P. Tevzadze, M. G. Lashkarashvili, N. V. Tarkhashvili, E. G. Adeishvili, E. G. Zhgenti, L. G. Bakanidze, P. G. Imnadze; National Center for Disease Control of Georgia, Tbilisi, GEORGIA. Summer ­ beginning of fall 2002 was "rich" with salmonellosis outbreaks in Georgia. Three of these outbreaks were the biggest ­ totally more than 450 people were infected in different parts of Georgia, quite far away from each other (Adigeni in south of Georgia, Tianeti in the north and Abasha ­ in western part). About 65 % of them were hospitalized. The source of infection in two outbreaks was beef (in Georgian cuisine very often middle-prepared meat is consumed), in the third ­ eggs and chicken. Odds ratio of food products was estimated: undercooked meat was the cause of the disease among 55 attendees of one of the parties (OR 8.80, p - value 0.003), at another party eggs and chicken were strongly associated with the infection (OR 30.43. p-value =0.005). It must be mentioned, that too big ritual parties are very popular in Georgia - patients of all three outbreaks were infected at funeral or wedding parties with the average number of invitees for each was 200. Conditions of food keeping were not satisfactory ­ due to lack of electricity refrigerators are not often used. For all three outbreaks age of patients was 2 - 88 years, incubation period varied from 1 to 4 days (median 2 days). Common symptoms included: diarrhea: 82%, nausea 79%, vomiting 39%, fever 82%, headache 70%, abdominal pain 60%, malaise 80%, dizziness 30%, hypotony 30% Stool specimens from patients, beef, eggs and chicken samples were investigated with bacteriological methods. Different species of Salmonella were isolated - Salmonella enteritidis, and Salmonella isangi. Molecular biological investigation of obtained isolates was carried out by Pulse Field Gel Electrophoresis (PFGE) using methodology worked out for E.coli 0157H7 (Gautom RK., J.Clin.Microbiol., 1997,11). Clinical and food samples were investigated. It was established, that Salmonella enteritidis strains, isolated from samples of Tianeti outbreak were of the same origin. The same can be said for Salmonella enteritidis strains, isolated from samples of Adigeni outbreak. PFGE patterns shown by these two groups differ significantly, that indicates different sources of infection for these two outbreaks. Salmonella isangi strains from Abasha outbreak gave two clonal groups ­ source of one of them is chicken, and the source of another clone could not be identified. As a result of obtained data we can conclude that different strains of Salmonella circulate in Georgia, and what was most interesting ­ for the first time in Georgia Salmonella isangi was isolated.

99(C)

RETRON REVERSE TRANSCRIPTASE RRTT IS UBIQUITOUS IN STRAINS OF SALMONELLA ENTERICA SEROVAR TYPHIMURIUM

J. Matiasovicova1, M. Faldynova1, M. Pravcova 1, R. Karpiskova 2, I. Kolackova2, J. Damborsky3, I. Rychlik 1; 1Veterinary Research Institute, Brno, CZECH REPUBLIC, 2National Institute of Public Health, Brno, CZECH REPUBLIC, 3National Centre for Biomolecular Research, Brno, CZECH REPUBLIC. Certain bacterial species encode specific type of reverse transcriptases called retron reverse transcriptases (RRT). These have been frequently found in Myxococcus sp. while in Escherichia coli they are found in about 10% of field strains only. In other bacteria, RRTs have been reported very rarely. In Salmonella, RRT was detected in 4 out of 70 strains of the SARB reference collection and recently RRT (rrtE, retron reverse transcriptase Enteritidis) encoded by low molecular weight plasmid of S. Enteritidis was described. Bacterial RRT are unusual enzymes which utilise the same RNA molecule as a template and also as a primer for initiation of the reverse transcription. A typical end product of this process is single stranded cDNA (msDNA ­ multicopy single stranded DNA) which accumulates in bacterial cell cytoplasm. Interestingly, when an integronencoded multidrug resistance genomic island was sequenced in Salmonella enterica serovar Typhimurium (S.Typhimurium), immediately downstream from it, the rrtT (retron reverse transcriptase Typhimurium) gene was detected on a genetic locus resembling a phage structure. Using standard BLAST analysis with the rrtT gene sequence encoded by the multidrug resistant S. Typhimurium we found this gene also in the genome of the non-resistant completely sequenced S. Typhimurium LT2. This suggested that the rrtT may not be a rare gene in Salmonella. We have therefore collected field strains of S. Typhimurium and other serovars and detected the presence of rrtT by PCR. This gene was present in 158 out of 175 tested S. Typhimurium strains. Quite frequently it was found also among strains belonging to the serovar Saintpaul. Single positive strain was found in serovar Schwarzengrund. rrtT was absent in all investigated strains belonging to serovars Agona, Heidelberg, Enteritidis, Hadar, Derby, Indiana, Stanley, Dublin and Gallinarum. In 35 rrtT+ strains of S. Typhimurium, presence of msDNA was confirmed. In 8 rrtT- S. Typhimurium strains, no production of msDNA was observed. The presence of the gene in the genome of individual strains therefore correlated with the production of msDNA. Detailed sequence analysis finally allowed us to predict the sequence of msDNA, to propose that the final msDNA is free of any RNA and to classify the rrtT, together with retrons of Vibrio cholerae, Vibrio parahaemolyticus, and E. coli Ec78 and Ec83, among a specific subclass of retron reverse transcriptases.

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100(A)

SALMONELLA PROMOTERS WITH HIGH ACTIVITY DURING INFECTION ARE LARGELY ASSOCIATED WITH VIRULENCE GENES

C. Rollenhagen, M. Soerensen, D. Bumann; Max-Planck-Institute for Infection Biology, Berlin, GERMANY. Salmonella displays diverse adaptations during infection. To determine the relative importance in terms of gene expression, we used a novel quantitative approach to identify 58 Salmonella promoters that are highly active in a murine typhoid fever model. The in vivo activities of these promoters are dramatically different from those in standard broth culture, but are partially reproduced in acidic minimal medium with low magnesium concentration providing quantitative support for low pH and low magnesium as crucial stimuli of the Salmonella microenvironment in infected animals. Most of the identified promoters drive the expression of virulence-associated, AT-rich genes that are absent in closely related, non-pathogenic Escherichia coli. In contrast, metabolic adaptation and general stress responses account for minor shares in gene expression. In conclusion, this study reveals a predominance of horizontally acquired virulence factors among highly in vivo expressed genes suggesting that fighting against the host is the main Salmonella activity in infected animals.

remaining four down-regulated genes were found to be contiguous on the chromosome. Deletion of those genes resulted in a 70% decrease in MDCK epithelial cell invasion. Only three genes were found to be up-regulated in the stpA::mTn5 mutant, including stpA itself. Previous work and microarray data obtained in our laboratory with a stpA deletion mutant show that StpA affects the expression of many environmentally-controlled genes. We suggest that StpA plays a role in the integration of environmental signals for the optimal control of SPI1 gene expression.

102(C)

COMPARATIVE ANALYSIS OF SIPA, SOPA, SOPB, SOPD, AND SOPE2 EXPRESSION PRODUCTS OF SALMONELLA ENTERICA SEROVARS TYPHIMURIUM AND ABORTUSOVIS IN A TISSUE CULTURE MODEL OF INFECTION

D. Bacciu 1, G. Falchi1, A. Spazziani1, G. Leori2, S. Uzzau1; 1 Dip Scienze Biomediche, University of Sassari, Sassari, ITALY, 2 Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, ITALY. Salmonella infections are associated with distinct diseases according to the Salmonella serovar and the infected animal species. In certain serovar-host combinations (i.e., serovar Typhi-human, serovar Abortusovis-ovine), disease syndromes are characterized by a prevalence of systemic over gastrointestinal symptoms. Other salmonellosis, viceversa, are associated almost exclusively with enterocolitis (i.e., serovar Typhimurium-human). Thus, it appears that during their evolutionary adaptation to animal hosts, Salmonella serovars have tuned their cross-talk with the host mucosal tissues toward inflammatory diarrhea or toward a "silent" translocation into the animal bloodstream. Five effector proteins of Salmonella serovar Typhimurium (SipA, SopA, SopB, SopD, and SopE2), are responsible for the induction of diarrhea in calves. Interestingly, sopA and sopE2 alleles are pseudogenes in serovar Typhi. In this study we aimed to evaluate whether serovar Abortusovis, an ovinerestricted pathogen that causes systemic infections with scarce or absent enterocolitis, carries the sipA and sopABDE2 genes and to compare the levels of their expression products to those of the serovar Typhimurium alleles. The 5 genes were identified in serovar Abortusovis by PCR analysis. For each gene we obtained the sequence of its 3' end and downstream adjacent regions (100% identities compared to serovar Typhimurium). This allowed us to design oligonucleotides that are appropriate for the construction of epitope[3xFLAG]-tagged strains in both Salmonella serovars. We then compared the level of SipA, SopA, SopB, SopD, and SopE2 tagged proteins in serovar Typhimurium and serovar Abortusovis strains grown in LB broth and in intracellular bacteria recovered from infected epithelial cell (Hep2). Each of the strains used contained a second FLAG tag in a constitutively expressed chromosomal cat gene as an internal standard. Proteins were detected by western blot analysis with anti-FLAG Mab. Chemiluminescence was captured by a digital imager for accurate quantifi67

101(B)

THE H-NS-LIKE PROTEIN STPA CONTROLS EXPRESSION OF INVASION GENES OF SALMONELLA ENTERICA SEROVAR TYPHIMURIUM

S. Lucchini 1, P. McDermott 1, M. Goldberg 1, B. Raupach 2, A. Thompson 1, D. W. Holden3, S. Falkow4, J. C. Hinton1; 1Institute of Food Research, Norwich, UNITED KINGDOM, 2Max-PlanckInstitut fuer Infectionsbiologie, Berlin, GERMANY, 3Imperial college of Science, Technology and Medicine, London, UNITED KINGDOM, 4Stanford University School of Medicine, Stanford, CA. Salmonella enterica serovar Typhimurium must cross the epithelial barrier during infection of mammalian hosts. Most genes required for invasion of the epithelial cells are encoded on the 40 kb SPI1 pathogenicity island (Salmonella pathogenicity island 1). We searched for additional factors required for cell invasion by screening a mTn5 signaturetagged library for S. Typhimurium mutants unable to invade cultured epithelial cells. This resulted in the identification of a mutant (P3A8) with a 7 to 10-fold decrease in invasion. The mTn5 insertion was found to be in the regulatory region of stpA, which is an orthologue of the global regulator HNS. Western blotting with StpA-specific antibodies showed that insertion of the transposon resulted in a 2-fold increase in StpA levels. Microarray experiments were performed to compare the transcription profile of the stpA::mTn5 mutant with the parental strain (S. Typhimurium SL1344). Strikingly, the effects of StpA over-expression was limited to SPI1 or SPI1-associated genes. Of the 37 down-regulated genes, 33 are known to be co-regulated with SPI1. Three of the Pathogenesis, Epidemiology, and Vaccine Development

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cation of protein levels. Our results show a differential accumulation of these effectors in the two Salmonella serovars. In particular, serovar Typhimurium strains grown in LB broth showed higher levels of each of these effectors up to 8 folds (i.e., SopA), as compared to serovar Abortusovis strains. Analysis of intracellularly grown bacteria in a 24 hours gentamicin protection assay revealed an increase of SipA, SopA, SopB, and SopD levels in serovar Typhimurium tagged strains. A similar increase was observed in serovar Abortusovis tagged strains only for SipA and SopB effectors, although to a lower extent. Taken together, these data suggest that a relatively low expression of enterocolitisassociated effectors, rather than the absence of functional genes, might account for the lack of enterocolitis in serovar Abortusovis infected ovine.

104(B)

DPS FROM A STRAIN OF SALMONELLA IS A MANOSE-CONTAINING GLYCOPROTEIN

E. S. Hanna1, M. C. Roque-Barreira1, E. S. Bernardes1, A. Panunto-Caslelo1, M. Valle-Sousa2, I. C. Almeida3, M. Brocchi1; 1 Universidade de São Paulo, Ribeirão Preto - SP, BRAZIL, 2 Universidade de Brasília, Brasília-DF, BRAZIL, 3Instituto de Ciências Biomédicas, São Paulo - SP, BRAZIL. All organisms living in aerobic atmosphere have powerful mechanisms to protect macromolecules from oxygen reactive specimens. Thus, microrganisms developed biomoleculeprotecting systems in response to starvation and/or oxidative stress. DNA biocrystallisation with Dps (DNAbinding protein from starved cells) is one example among these systems. Dps is a protein produced in large amounts when bacterial cell faces harm. An 18 kDa protein was purified from the crude extract of Salmonella enterica Typhimurium and characterised for its jacalin-binding ability. N-terminal sequencing revealed 100% homology with the Dps of S. enterica Typhimurium. Furthermore, alpha-metilgalactose inhibited the binding of Dps to jacalin in enzymelinked lectin assay, suggesting that the Carbohydrate Recognition Domain (CRD) is involved in this interaction. Chemical deglycosylation with TFMS reduced the molecular mass slightly from 18587 to 18138 Daltons (449 Daltons), indicating that a small oligossacharide branch must be associated with its molecule.Finally, results from GC-MS showed that manose is one possible constituent of the Dps glycan. Even though jacalin is known as a galactosidebinding lectin, there are some pieces of evidence that it might bind manose as well. We are now working on structural and functional characterisation of the Dps glycan(s).

103(A)

TRACING THE ORIGINS OF FLAGELLAR MONOPHASIC S. ENTERICA I 4,[5],12:I:- : GENETIC SIMILARITY TO S. ENTERICA TYPHIMURIUM

A. L. Sales1, V. R. Santos 2, S. A. Fernandes3, W. D. Silveira4, R. Martinez2, M. Brocchi 1; 1Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo, Ribeirão Preto - SP, BRAZIL, 2 Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo, Ribeirão Preto - SP, BRAZIL, 3 Instituto Adolfo Lutz, São Paulo - SP, BRAZIL, 4Universidade de Campinas, Campinas - SP, BRAZIL. Non-typhoid Salmonella enterica subsp enterica is an important and emergent pathogen for humans. Most human infections are caused by the S. enterica species, which is also classified by serological reactions depending on the type of polysaccharide content of LPS (O), flagellar (H) and capsular (Vi) antigens. The combination of these antigens defines a serovar. Some serovars such as Typhimurium exhibit the ability to change the type of flagellin protein expressed. The H1 flagellin is the product of the fliC gene, and the H2 flagellin is the product of the fljB gene. The type of flagellin expressed is under the control of a phase variation system. Since the end of the last century, monophasic Salmonella enterica subsp enterica 4,5,12:i:- has been isolated in Brazil, particularly in São Paulo State. This new serotype might have been originated from a biphasic serovar such as Salmonella Typhimurium (4,[5],12:i:1,2), Lagos (4,[5],12:i:1,5) or from a non-motile serovar that acquired the ability to express the flagellum. Recently, this new serotype was also isolated in Spain and in the United States. In the present study, monophasic Salmonella 4,5,12:i:- isolated in Brazil were typed by different molecular techniques such as REP-PCR, Ribotyping, IS200 fingerprint and plasmidial profile, which indicated that this serotype is derived from Salmonella Typhimurium. The nature of the mutation associated with the lack of flagelar phase variation was also investigated. The fljB was not detected by PCR. On the other hand, Southern blot experiments using specific probes detected this gene. Thus, further analysis will be necessary to characterise the nature of the mutations responsible for the origin of 4,5,12:i:- serotype in Brazil.

105(C)

ANTI-APOPTOTIC ACTIVITY OF THE TTSS-1 EFFECTOR SIGD IN CULTURED EPITHELIAL CELLS

O. Steele-Mortimer1, B. B. Finlay2, L. A. Knodler1; 1NIAID, NIH, Hamilton, MT, 2University of British Columbia, Vancouver, BC, CANADA. Salmonella enterica serovar Typhimurium (S. Typhimurium) infects both phagocytic and nonphagocytic cells. Two type III secretion systems (TTSS-1 and TTSS-2) translocate effector proteins into the host cell and are required for virulence. TTSS-1 is essential for invasion of nonphagocytic cells, which involves remodeling of the actin cytoskeleton and activation of signal transduction pathways. The TTSS-1 effector SigD is an inositol phosphate phosphatase that associates with host cell membranes and activates AKT/PKB via an unknown pathway. AKT is a serine/threonine kinase best known as a transducer of prosurvival signals from a variety of stimuli. In this study we have investigated the roles of SigD and AKT in survival of cultured epithelial cells infected with S. Typhimurium for 3-6 hours. Our results show that levels of apoptosis are increased in cells infected with a sigD deletion mutant compared to those infected with ASM Conference on Salmonella

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wild type S. Typhimurium. Furthermore, wild type but not mutant S. Typhimurium blocked the induction of apoptosis by a topoisomerase inhibitor, camptothecin. The SigDdependent anti-apoptotic patway involves Caspase-3 but not Caspase-1, which has previously been shown to mediate cell death in infected macrophages. These results indicate that Salmonella can selectively block apoptosis in infected cells. Moreover, they illustrate that TTSS-1 effectors such as SigD can influence intracellular events much later in the infection process than previously considered. SD1 to generate nitrites in plasma was found 48h after infection, 8.5 + 0.2 ìM compared to 33,4 + 1.89 ìM induced by the wt strain. Also Western blot analysis showed a delayed translocation of NFkB in the ileal loop inoculated with SD1 compared with the wt strain. In summary, our results demonstrate that S. enteritidis SD1 bearing a defective Dam invades and induces histological changes in PP. These findings, absent in mutants lacking Dam, would account for the moderate protection induced by SD1 strain. The lack of O9 antigen in SD1 mutant would explain the failure to achieve higher protection against challenge with wt S. enteritidis.

106(A)

A SALMONELLA ENTERITIDIS DAM MUTANT OF LEAKY PHENOTYPE AS A POTENTIAL VACCINE STRAIN

M. N. Giacomodonato 1, S. H. Sarnacki 1, F. Sisti2, R. Caccuri3, M. C. Cerquetti 1; 1CEFYBO-CONICET, Buenos Aires, ARGENTINA, 2Universidad Nacional de La Plata, Buenos Aires, ARGENTINA, 3Universidad de Buenos Aires, Buenos Aires, ARGENTINA. DNA adenine methylase (Dam) protein is a global regulator of bacterial gene expression. Dysregulation of Dam activity is potentially a general strategy for the generation of vaccines against bacterial pathogens. S. typhimurium strains lacking Dam protein are excellent vaccines. They are innocuous, unable to invade enterocytes or to cause cytotoxicity and they confer protective immunity. Dam mutants of S. enteritidis have been less analyzed; it was shown that S. enteritidis strains lacking Dam protein fail to generate protective immunity. We obtained a Dam mutant of S. enteritidis, named SD1, that bears a defective Dam (ten amino-acid shorter). SD1 mutant shows a leaky phenotype, its DNA has methylated and unmethylated adenines. We studied S. enteritidis SD1 as a potential vaccine strain. Mice were immunized intragastrically and challenged orally. In some experiments mice received the inocula into the ileal loop. We found that SD1 mutant is highly innocuous (oral LD50 >109 cfu). Light and electron microscopy revealed histological changes in Peyer's patches (PP), suggesting that SD1 is able to induce cytotoxicity. Moreover, SD1 was found inside the PP 60 min after inoculation into the ileal loop. In agreement with these results SipA. SipB and SipC proteins were found in culture supernatants. Immunization with SD1 resulted in a more efficient clearance of wild type (wt) S. enteritidis from spleen, compared with control animals. In separate experiments it was found that 40% of the immunized mice survived the challenge with the wt strain. Thus, leaky mutant SD1 induces better protection than S. enteritidis strains lacking Dam. Survival rates found, however, were not as high as those reported for dam mutants of S. typhimurium. The lower efficacy of SD1 to induce protection may be related to a defective LPS. Silver-stained SDS-PAGE revealed that SD1 lacks O9 antigen, the immunodominant epitope involved in protection against S. enteritidis. Also, a defective LPS could affect the expression of IFN-ã and iNOS which play a critical role in host protection against Salmonella. In fact, SD1 induced lower levels (p < 0.05) of intestinal IFN-ã than wt strain (509 + 62 vs 798 + 82 pg/ ìg of protein, respectively) measured by ELISA. A reduced capacity of Pathogenesis, Epidemiology, and Vaccine Development

107(B)

CHARACTERIZATION OF OMPR- AND SSRB-DEPENDENT REGULATION OF SSRA-B

D. Walthers1, X. Feng2, L. J. Kenney1; 1University of Illinois at Chicago, Chicago, IL, 2Oregon Health and Science University, Portland, OR. The Salmonella pathogenicity island 2 (SPI-2) is essential for replication within macrophages and survival during systemic infection in mice. Expression of genes within SPI-2 is controlled by a novel mechanism whereby the response regulator of one two-component system (EnvZ-OmpR) directly activates transcription of the genes for a second two-component system (ssrA-ssrB) encoded within the pathogenicity island. SsrB, in turn, is required for expression of both its own gene and of ssrA from two distinct promoters. Site-directed mutagenesis of ssrB reveals that D56, the predicted site of phosphorylation, is required for SsrBdependent transcription. Deletion analysis of the ssrB coding region indicates that the isolated C-terminus constitutively activates transcription. These results suggest that phosphorylation of the N-terminus relieves an inhibitory effect on the C-terminus. SsrB activates transcription by binding to sites downstream of the ssrA and ssrB transcriptional start sites. Our preliminary results indicate that the binding sites of SsrB and OmpR at ssrA and ssrB partially overlap. It is unclear whether OmpR and SsrB coordinately or competitively bind to these promoters to regulate gene expression. We are clarifying this issue by performing competitive footprinting analysis and by measuring the affinities of OmpR and SsrB for each binding site using fluorescence anisotropy. In addition, the chemical nuclease Fe-BABE will be cross-linked to unqiue cysteine substitutions within the OmpR DNA binding domain. The OmpR-Fe-BABE footprint will then be examined in the presence and absence of SsrB. Unlike competitive footprinting, this approach will enable us to specifically identify the contribution of OmpR to binding when SsrB is present, but without interference of the SsrB footprint. Finally, we are developing an in vitro transcription system to directly measure the contribution of OmpR and SsrB to ssrA and ssrB gene expression. Supported by NIH GIM 58746 and NSF 024358 to LJK and NIH 1F32GM68364-01 to DW.

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108(C)

OCCURRENCE OF SALMONELLA SPP. IN SWEDISH SEWAGE SLUDGE AND THE HYGIENIC RISKS WITH AGRICULTURAL USE

I. Berggren, L. Sahlström; National Veterinary Institute, SVA, Uppsala, SWEDEN. Conventional treatment of sludge, i.e. sedimentation or mesophilic digestion, cause a limited reduction of the content of pathogens. However, these methods do not result in a hygienically safe sludge. Salmonella spp. may therefore still be present in the final sludge products. At the National Veterinary Institute (SVA) in Sweden, some research projects are focused on hygienic aspects of recirculation of organic wastes back to agriculture and food production. In one project, the incidence of Salmonella spp. has been surveyed in sewage sludge from eight treatment plants at different locations in Sweden. Salmonella was present in 55% of the treated sludge samples and 49 different serotypes were detected during the one-year sampling period, confirming the insufficient hygienic outcome of the conventional treatments used. Moreover, identification with pulsed field gel electrophoresis of the Salmonella serotypes found, demonstrated that these serotypes were identical with Salmonella serotypes isolated within the human population in the area served by the sewage treatment plants and that they were able to persist for a long time in the plants. A further treatment that may result in a more hygienic sludge is to store the sludge before further use. However, international studies have revealed that long-term storage is an insufficient method for hygienisation of sludge. Thus, ongoing studies at SVA aim to assess the Salmonella die-off during long-term sewage sludge storage under Swedish climate conditions. The stored sludge, conventionally treated, will be monitored continuously for presence and quantity of Salmonella during one year. At present, the risk for animal and human infections associated with the use of sewage sludge on arable land is not known. To our opinion, an increased recycling of sewage sludge must therefore be hygienically controlled in order to protect human and animal health and the environment in large.

macrophages. The PmrAB system also confers resistance to Fe3+-mediated killing. Recently some novel targets of the PmrAB system have been discovered to which neither a role in Fe3+-resistance nor in increased resistance against cationic polypeptides could be assigned, pointing towards novel roles of the PmrAB system. These observations suggest that, despite the fact that it has been subject of intensive molecular biological studies, the PmrAB-dependent regulon is still not fully characterized. To further unravel the PmrABdependent regulon, we used an in silico approach. Using a motif model of the PmrA binding site, a genome wide screening was performed to detect novel PmrAB-regulated targets (Motif Locator http://www.esat.kuleuven.ac.be/ ~dna/BioI/Software.html). Genome wide screenings have proven to be successful in detecting novel targets. However, deciding based on their score whether detected motifs are biologically valid turns out to be a non-trivial task, which customarily occurs on an arbitrary basis. More reliable predictions on specific pathways have been obtained by incorporating cross species comparisons (phylogenetic footprinting). In this study we combine both approaches: novel putative targets identified by a genome wide screening are, whenever possible, subjected to a phylogenetic footprint approach based on Gibbs sampling (Motif Sampler). For each dataset we aimed at constructing a local multiple alignment based on the positions of high scoring motifs. Such multiple alignments of intergenic sequences show which motifs are conserved among species and allow us to find out whether the putative PmrA motifs retrieved by the genome wide screening were conserved in the alignments. Recovering most of the known PmrAB-dependent targets showed the efficiency of our methodology. Besides the known targets, we could detect several novel targets with yet unknown function. Expression studies univocally confirmed PmrAB-dependent induction of three novel targets.

110(B)

ANTIMICROBIAL SUSCEPTIBILITY AND SEROTYPE DISTRIBUTION OF SALMONELLA SPP. STRAINS ISOLATED FROM CLINICAL SPECIMENS DURING 20002002 IN LAKONIA REGION, GREECE

S. Fokas, M. Kalkani, M. Tsironi, P. Andriopoulos, M. Dionisopoulou; General Hospital of Sparta, Sparta, GREECE. The aim of our study was to determine the trend in the prevalence of Salmonella serotypes, to analyze their distribution and their antibiotic resistance patterns. We studied the serotypes and we investigated the in vitro antibiotic susceptibility patterns of Salmonella spp. isolated from clinical specimens. The isolates were identified by conventional microbiological methods. Serotyping was carried out by the National School of Public Health in the University of Athens. Susceptibility tests to ampicillin (AMP), ciprofloxacin (CIP), trimethoprim/sulfomethoxazole (SXT) and ceftriaxone (CRO) were performed using the disk diffusion method according to NCCLS recommendations. During the three-year study period (2000-2002) a total of 52 Salmonella spp. strains were isolated : 47 from 465 stool ASM Conference on Salmonella

109(A)

IN SILICO IDENTIFICATION AND EXPERIMENTAL VALIDATION OF NOVEL PMRAB TARGETS IN SALMONELLA TYPHIMURIUM BY REGULATORY MOTIF DETECTION

K. Marchal 1, S. De Keersmaecker 2, P. Monsieurs 1, N. van Boxel2, K. Lemmens 1, B. De Moor1, J. Vanderleyden 2; 1SCD/ bioinformatics, Leuven, BELGIUM, 2Centre of Microbial and Plant Genetics, Leuven, BELGIUM. The PmrAB (BasSR) two-component regulatory system is one of the key regulatory components required for Salmonella typhimurium virulence. The PmrAB-mediated modifications of the peptidoglycan layer confer resistance to cationic antibiotic polypeptides allowing bacterial survival within 70

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specimens (10.1% positive for Salmonella), 4 from blood specimens and 1 from urine specimen. Fourteen different Salmonella serotypes were isolated. S.enterica serotype Enteritidis was the predominant isolate (21 strains - 40.4%), followed by S.enterica serotype Typhimurium (7 strains13.4%), S. kottbus (4 strains-7.7%), S.agona (3 strains-5.7%), S. oranienburg (2 strains-3.8%) and other less common serotypes (29%). The majority of the Salmonella strains (65% - 34 strains) were isolated from children (<14 yrs old) and the seasonal distribution revealed an increased number of isolates in the summer months. Thirty nine (75%) of the Salmonella strains were susceptible to all antibiotics tested. Only 8 strains (15.4%) were resistant to ampicillin and 5 strains (9.6%) to trimethoprim/sulfomethoxazole. S. enteritidis continuous to be the most common clinical isolate although its frequency is decreased according to our previous data. In our geographical area (Peloponnese ­ South Greece) the resistance of Salmonella spp. to AMP and SXT remains at relatively low levels and is worth noted that the resistance is also decreased. Susceptibility to fluoroquinolones is still preserved owing in part to the low frequency of using these antibiotics. Though we did not observe any resistant strains in Salmonella to CIP and CRO, the resistance pattern in general is alerting for rational antibiotic use in salmonellosis and continuous surveillance for changes in antibiotic resistance, necessary for determining therapeutic regimens, whenever needed.

112(A)

THE SALMONELLA SURFACE PROTEASE/ADHESIN PGTE INACTIVATES THE PLASMA PROTEASE INHIBITOR ALPHA-2-ANTIPLASMIN

K. Lähteenmäki, L. Partanen, P. Kyllönen, T. K. Korhonen; University of Helsinki, Helsinki, FINLAND. The Salmonella enterica outer membrane protein PgtE proteolytically activates the mammalian plasma proenzyme plasminogen to plasmin and mediates bacterial adhesion to basement membranes. Plasmin is a serine protease that effectively breaks down basement membrane components, such as laminin. Thus, PgtE can generate localized proteolysis on basement membrane and may this way promote migration of Salmonella through tissue barriers. We found that PgtE also cleaves and inactivates the main inhibitor of plasmin, alpha-2-antiplasmin (a2AP). Generation of plasmin by pgtE-expressing Salmonella and recombinant Escherichia coli leads to degradation of immobilized, radiolabelled laminin, and the degradation takes place also in the presence of a2AP under conditions where degradation by preformed plasmin is inhibited by a2AP. PgtE-mediated cleavage of a2AP can thus further increase the efficiency of plasminmediated proteolysis on basement membranes. However, we have found that the functions of PgtE are prominent only in Salmonella isolates with rough lipopolysaccharide (LPS) wheras the long O-side chain of smooth LPS seems to prevent the action of PgtE. As the patient isolates of Salmonella usually express smooth LPS, which is required for full virulence, the importance of PgtEmediated proteolytic activity towards plasma proteins has remained elusive. pgtE has been found to be upregulated and, on the other hand, the composition of LPS is known to be altered during intracellular growth of Salmonella. Therefore, we measured a2AP inactivation with bacteria released from infected murine macrophages. We found that Salmonella enterica 14028 with smooth LPS acquired ability to cleave a2AP during growth inside macrophages, whereas no cleavage was seen with the pgtE deletion derivative 14028-1 from macrophages, or with 14028 grown outside macrophages. The results suggest that Salmonella released from macrophages have the ability to interfere with the control of host proteolysis in circulation.

111(C)

COMPARISON OF THE PULSED FIELD GEL ELECTROPHORESIS (PFGE) AND RANDOMLY AMPLIFIED POLYMORPHIC DNA ANALYSIS (RAPD) PATTERNS FOR SUBTYPING SALMONELLA CHOLERAESUIS ISOLATED FROM SWINE FIELD IN SOUTHERN TAIWAN

T. H. Chiu 1, J. C. Pang1, W. Z. Hwang1, M. H. Liao 2, H. Y. Tsen1; 1National Chung-Hsing University, Taichung, TAIWAN REPUBLIC OF CHINA, 2National Pingtung University of Science and Technology, Pingtung, TAIWAN REPUBLIC OF CHINA. Pulsed field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA analysis (RAPD) were performed for a total ninety-five Salmonella Choleraesuis strains isolated from swine with diarrhea and systemic infection during 2000~2002 in the swine fields of southern Taiwan. For PFGE, XbaI and NotI restriction enzymes were used for chromosomal DNA digestion. The results showed that, for these 95 Salmonella strains, 46 PFGE patterns combinations were found. Of them, pattern X1N1, was the major subtype since 41 strains belong to this pattern. Strains of this pattern may be the most epidemics strains. On the other hand, RAPD analysis generated 74 patterns in 95 isolated strains. Comparison of the PFGE and RAPD patterns, RAPD could subdivide strains within a PFGE type. Thus, RAPD was the most discriminated tool for subtyping of Salmonella Choleraesuis, if suitable primer was selected for typing.

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113(B)

DETECTION OF ANTIBIOGRAMS, CALSS I INTEGRON AND ANTIBIOTIC RESISTANCE GENES FOR SALMONELLA CHOLERAESUIS STRAINS ISOLATED FROM SWINE IN SOUTHERN TAIWAN

J. C. Pang1, T. H. Chiu1, W. Z. Hwang1, M. H. Liao2, H. Y. Tsen1; 1National Chung Hsing University, Taichung, TAIWAN REPUBLIC OF CHINA, 2National Pingtung University of Science and Technology, Pingtung, TAIWAN REPUBLIC OF CHINA. Antimicrobial susceptibility testing was preformed for the 95 Salmonella Choleraesuis strains collected from the swine field in Southern Taiwan. A total of 17 antibiotics were tested for antibiograms. The results showed that 44 antibiograms were obtained and all these strains were multidrug resistant strains. PCR was used to screen the gene encode the resistance to ampicillin (PSE group), streptomycinspectinomycin (aadA), kanamycin (aphA), sulfadiazine (sulI), rimethoprim (dfrA12), chloramphenicol (cat, cmlA) and class I integron. The results showed that 32.6% of the 95 strains of Salmonella Choleraesuis are with the class I integron. The presence of antibiotic resistance genes in integron cassette was further analyzed by PCR mapping. Five types of patterns were detected, including dfrA12-aadA2, aadA2-oxa1, dfrA12-aad2, pse and dfrA12. Our data demonstrated that antibiotic resistance problem in Salmonella Choleraesuis need to be paid with attention.

identified in several prokaryotic and eukaryotic LPS-binding proteins. Substitution of two arginines of this motif thought to bind 4'-phosphate of lipid A abolished proteolytic activity but not membrane insertion of PgtE. The results suggest that PgtE needs rough LPS to be active but its interactions with large-molecular-weight substrates are sterically inhibited by O-antigen. Expression of PgtE is upregulated in murine macrophages where the genes encoding O-antigen, on the other hand, are repressed. We are currently studying the proteolytic activities of PgtE in bacteria from Salmonella containing vacuoles in infected macrophages.

115(A)

THE PREVALENCE OF CU, ZN-SOD IN SALMONELLA CLINICAL ISOLATES

C. Yuo1, C. Chang2, J. Lin3, C. Liu3, S. Huang3; 1Department of Biology, Kaohsiung Medical University, Kaohsiung, TAIWAN REPUBLIC OF CHINA, 2Clinical Laboratory, Yuan's General Hospital, Kaohsiung, TAIWAN REPUBLIC OF CHINA, 3 Department of Medical Technology, Fooyin University, Kaohsiung Hsein, TAIWAN REPUBLIC OF CHINA. Virulent Salmonella contains two periplasmic Cu, Znsuperoxide dismutases, sodCI and sodCII, to protect the bacteria from exogenous oxidative damage. It has been documented that both sodC loci contribute to the virulence of Salmonella in mice. In our study, the clinical correlations of sodCI and sodCII genes in Salmonella were analyzed in 200 clinical isolates. First, the sodCI and sodCII genes were detected with multiplex PCR by using invA gene as the internal control. The result indicates that the prevalence of sodCI and sodCII was 69% and 100%, respectively. Second, the clinical correlation of Salmonella isolates was analyzed with the isolates either with or without sodCI gene. Third, the correlation of the sodCI gene in Salmonella isolate with the resistance to oxidative stress was analyzed. The resistance to the superoxide generators, either methylglyoxal or xanthine with xanthine oxidase, were measured in the Salmonella clinical isolates. The results indicated that not merely sodCI, but sodCII plays a role to protect the bacteria from the oxidative stress.

114(C)

LACK OF O- ANTIGEN IS ESSENTIAL FOR PLASMINOGEN ACTIVATION BY SALMONELLA ENTERICA

P. Kyllönen, M. Suomalainen, M. Kukkonen, K. Lähteenmäki, H. Lång, R. Virkola, T. K. Korhonen; University of Helsinki, Helsinki, FINLAND. PgtE of Salmonella belongs to the omptin family of enterobacterial outer membrane aspartate proteases. Its functions and role in Salmonella pathogenesis have largely remained unknown. PgtE promotes bacterial resistance to alpha-helical cationic antimicrobial peptides and may this way contribute to bacterial resistance to innate immunity. PgtE also activates the plasma zymogen plasminogen (Plg) to plasmin by proteolytic cleavage. Our measurements of endogenous Plg activation of S. enterica revealed that Salmonella isolates with rough LPS activate Plg much more efficiently than smooth isolates. However, immunoblotting of cell envelope proteins with anti- His 6- PgtE antibodies revealed a similar expression level of PgtE in the rough SL1102 and smooth 14028 strains. Site-specific deletion derivates SL1102-1 and 14028-1 were negative for Plg activation and PgtE expression and complementation with pgtE led to efficient Plg activation in the rough backround only. No difference in surface expression of PgtE between the various tested rough and the smooth host strains were detected. The sequence of PgtE contains a putative, 3-dimensional LPS-binding motif 72

116(B)

THE FIS TRANSCRIPTOME OF SALMONELLA ENTERICA SEROVAR TYPHIMURIUM

A. E. Kelly1, M. Goldberg2, V. Danino 2, J. C. Hinton2, C. J. Dorman1; 1Microbiology Dept, Trinity College Dublin, IRELAND, 2 Institute of Food Research, Norwich, UNITED KINGDOM. Fis, the factor for inversion stimulation is a multifunctional protein and acts as a pleiotropic regulator of gene expression in Salmonella enterica serovar Typhimurium. Microarray analysis was carried out to elucidate the Fis regulon in S. typhimurium. We now report the complete Fis transcriptome following growth in LB. The expression profile confirmed several known functions of the Fis protein including its requirement for the expression of the virulence genes in Salmonella Pathogenicity Island 1 (SPI-1). In addition it was ASM Conference on Salmonella

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revealed for the first time that the absence of Fis produced an up-regulation of the bio operon involved in the biotin biosynthetic pathway and an up-regulation of the pdu operon responsible for the utilization of propanediol. A novel role for Fis in motility was also identified. From the microarray analysis Fis was found to be required for wildtype flagellar gene expression, where the absence of Fis resulted in a decrease in flagellar gene expression. S. typhimurium fis mutants were characterized as being defective in motility on motility agar plates. We examined the effect of a fis mutation on transcriptional fusions to flagellar promoters (flhD, fliA, fliE, flgA and fliC). For each promoter a decrease in transcriptional activity was observed in the absence of Fis. The Fis protein was shown to bind to the flhD, fliA and fliC promoter regions by DNA mobility shift assay. Wild-type motility in the fis mutant could be restored by complementation. These results suggest the Fis protein is a positive regulator of genes involved in the biogenesis of flagella. In addition the microarray analysis revealed a S. typhimurium fis mutant displayed reduced lpp expression, where lpp codes for Braun's lipoprotein. This raises the possibility that a fis mutant may exhibit defects in the structural and functional integrity of the cell. A possible link between lipoprotein expression and motility is discussed. These data propose novel functions for the Fis protein of S. typhimurium. mock pigs. However, the phagocytic and clearance abilities of neutrophils and PBMC declined dramatically when pigs prior challenged with 105 TCID 50 of CSF virus. Moreover, the phogocytic abilities against Serovars Choleraesuis and Typhimurium began declining at 3 and 7 days post CSF virus infection (DPI) respectively, and even dropped to 20-30% of mock phagocytosis ratio at 18-21 DPI. In addition, the respiratory burst of peripheral blood phagocytes was decreasing to 70-80% at 7 DPI and slightly recovered at 14 DPI, however it also significantly dropped to 10-30% at 21 DPI. These results suggested that CSF virus not only induces host leukopenia but also impairs the phagocytic and respiratory burst activities of phagocytes against Salmonella infection in swine.

118(A)

CHARACTERIZATION OF A SALMONELLA ENTERICA TRANSPOSON MUTANT STRAIN WITH AN INCREASED RESISTANCE TO POLYMYXIN B

M. Espariz, J. Barchiesi, E. García Véscovi, F. C. Soncini; IBRConicet, Rosario National University, Rosario, ARGENTINA. Cationic antimicrobial peptides are a structurally diverse group of molecules active against a wide variety of pathogens, including Gram-positive and Gram-negative bacteria, fungi, and viruses. A crucial step in their biological activity against Gram-negative bacteria relies on the interaction with the outer membrane. In this regard, it has been shown for Salmonella enterica that structural modifications of the lipopolisaccharide (LPS) greatly affect the bacterial resistance to most of these microbicidal compounds, and that these modifications are coordinately regulated by the twocomponent systems PhoP-PhoQ and PmrA-PmrB. Salmonella resistance to antimicrobial peptides is a necessary step in the infection process, and mutants with increased sensitivity to these compounds are impeded to prosper within the host tissues. Through a screening for novel genes that modulate the resistance to polymyxin B in Salmonella enterica serovar Typhimurium we isolated a transposon insertion mutant that increased the bacterial resistance to the antibiotic. Direct genomic DNA sequencing allowed us to identify a previously uncharacterized gene whose product displays a weak homology to phosphosugar isomerases. The LPS extracted from the insertional mutant showed an electrophoresis pattern similar to the one that characterizes PmrA constitutive (PmrAc) strains. Nevertheless, expression of this gene was independent of either PmrA-PmrB, or PhoP-PhoQ. Besides, and in contrast to the PmrAc strains, this mutant showed increased susceptibility to the antimicrobial peptides protamine and cecropin P1, and it was defective in swarming and in virulence. These results indicate that the identified gene product is involved in a novel pathway that affects the infection process by altering the resistance to antimicrobial peptides.

117(C)

IMPAIRMENT OF SWINE PHAGOCYTES AGAINST SALMONELLA ENTERICA SEROVARS CHOLERAESUIS AND TYPHIMURIUM CAUSED BY CSFV INFECTION

C. Liao, Y. Lin, Z. Chen, W. Lee, C. Lin, M. Chien; Graduate Institute of Veterynary Pathology, Taichung, TAIWAN REPUBLIC OF CHINA. The diarrheagenic disease of Salmonellosis caused by Salmonella enterica Serovars Choleraesuis and Typhimurium is among the most important bacterial diseases affecting swine worldwide that can induce gastroenteritis as well as systemic septicemia. In Taiwan, classical swine fever (CSF) accompanied with Salmonella infection is the most clinical diagnostic cases from the surveillance in pig farms during 1999-2002. Although the mechanism and relationship between two diseases still remain unclear, the leukopenia of CSF virus infected pigs and the ability of Salmonella to survive intracellularily in phagocytic cells may influence the host immunity while both pathogens co-reside in swine. The aim of this study is to compare phaogcytosis and respiratory burst of phagocytes against Salmonella serovars after challenged with CSF virus. The swine alveolar macrophages, neutrophils and peripheral blood mononuclear cells (PBMC) were collected and manipulated with Serovars Choleraesuis and Typhimurium respectively to evaluate the affect on phagocytosis and respiratory burst using flow cytometry analysis, as well as to monitor intracellular bacterial surviving in vitro. The results demonstrated Serovars Choleraesuis and Typhimurium could be eliminated efficiently in neutrophils but survive well in alveolar macrophages that isolated from Pathogenesis, Epidemiology, and Vaccine Development

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119(B)

DETECTION OF ANTIBIOTIC RESISTANCE GENES IN VARIOUS SALMONELLA SEROTYPES BY OLIGONUCLEOTIDE MICROARRAY ANALYSIS

A. van Hoek, I. Scholtens, H. Aarts; RIKILT, Institute of Food Safety, Wageningen, NETHERLANDS. The spread of antibiotic resistance among pathogenic bacteria, like Salmonella, is recognized as a serious problem that eventually can complicate medical treatment of bacterial infections. For the molecular characterization of the resistance patterns of bacteria a microarray was developed. The microarray contains 50- and 60-mer oligonucleotides representing more than 150 different antibiotic resistance genes, belonging to approximately 15 different antibiotic classes, e. g. chloramphenicol, ESBLs, sulfonamide, tetracycline, and trimethoprim. Furthermore, oligonucleotides to identify genetic elements carrying antibiotic resistance genes were also spotted on the microarray . For example oligonucleotides specific for several integrase genes present on the different classes of integrons. The microarray also contains 60-mers to identify Salmonella spp. S. typhimurium, and S. enteritidis. Two specific labeling methods (multiplex PCR and specific primed labeling) and two generic procedures (random primed labeling and nick translation) were tested to optimize the hybridization results. Different Salmonella serotypes from various sources, i.e. livestock, patients and food, were screened with the microarray to determine their antibiotic resistance profile, the results will be discussed.

of infection. Based on the results hypotheses are formulated on the mode of spread and the relevance of horizontal spread for DT 104 infections.

121(A)

DIFFERENTIAL EXPRESSION OF GENES IN CULTURABLE AND NONCULTURABLE FORMS OF SALMONELLA TYPHIMURIUM

Y. M. Romanova, A. S. Tomova, Y. S. Alyapkina, A. L. Gintsburg; The Gamaleya Research Institute, Moscow, RUSSIAN FEDERATION. Salmonella species are important food-borne pathogens that represent an increasingly significant public health issue in industrialized countries. The problem, at list in part, is that these organisms can persist for long periods in the environment in a heavily stressed state known variously, and often contentiously, as viable but non-culturable (VBC or not immediately culturable) They lose the ability to form colonies on non-selective plating media or to grow in nonselective broth media. Non-culturable populations of pathogenic bacteria in the environment (soil, natural water) or associated with bacteriological spoilage may still be capable of causing disease if ingested by a susceptible host. The our research group in the Gamaleya Research Institute in Moscow is engaged in the investigation of ability of pathogenic bacteria to reversible transformation into nonculturable state under unfavorable conditions. Moleculargenetic approaches to deciphering the mechanisms of genetic control of transformation of pathogenic bacteria into non-culturable state are outlined on the model of Salmonella typhimurium. The genetic investigation, concerning to the identification of the genes and their expression during transition and long lasting existence in nonculturable state (NS) was undertaken. We demonstrated here the identification of a few genes which controlled the process of culturable form generation of Salmonella. The method of successively conducted reverse transcription and quantitative PCR technique was applied for evaluation of pqi and stn genes expression in S.typhimurium cells during NS transition. Moreover the differential expression of several genes of Salmonella typhimurium was shown by molecular display method. The six fragments of cDNA differentially expressed genes in depending on culturable or nonculturable state were isolated, cloned and sequenced. The application of those methods allowed us for the first time to demonstrate that some genes continue to be expressed not only during starvation and conversion to an NS but also in NF of bacteria . A low level of expression of some genes in NF of bacteria is likely to be necessary for fast recovery of growth and propagation when favorable environmental conditions supplant a negative situation.

120(C)

OCCURRENCE AND SPREAD OF SALMONELLA TYPHIMURIUM DT 104 IN DANISH PRODUCTION ANIMAL HERDS INVESTIGATED BY THE USE OF DNA TYPING, SPATIO-TEMPORAL ANALYSIS AND GEOGRAPHIC INFORMATION SYSTEM (GIS)

M. N. Skov, J. S. Andersen, D. L. Baggesen; Danish Veterinary Institute, Copenhagen, DENMARK. The present study describes the occurrence and spread of Salmonella (S.) Typhimurium phage type (DT) 104 in Danish production animal herds. The study included all herds infected between July 1996 until January 2003. We applied spatio-temporal analysis and Geographic Information System (GIS) for description of the development and spread of infection among herds. Data included time of infection, geographical location of the infected herd, epidemiological data on trade contacts, and DNA type of DT 104 isolates from each of the infected herds. The DNA typing was performed by the use of pulsed-field gel electrophoresis (PFGE) and plasmid analysis of 174 DT 104 isolates representing one DT 104 isolate per infected herd. The results suggest that the combined use of spatio-temporal analysis and GIS can be used to overcome the problem that clonality of bacteria can rise in relation to studies of spread 74

ASM Conference on Salmonella

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122(B)

PLASMID-MEDIATED TETRACYCLINE AND STREPTOMYCIN RESISTANCE IN ISOLATED FROM ANIMALS IN ITALY

C. Pezzella1, A. Bertini1, L. Villa1, A. Ricci2, E. Digiannatale3, I. Luzzi1, A. Carattoli1; 1Istituto Superiore di Sanità, Rome, ITALY, 2Istituto Zooprofilattico Sperimentale delle Venezie, Padua, ITALY, 3Istituto Zooprofilattico Sperimentale Abruzzo e Molise, Teramo, ITALY. Background. The increasing incidence of resistance to streptomycin (Sm) and tetracyclines (Te) has been worldwide reported in Salmonella spp. Genes conferring Sm and Te resistance have been extensively studied in S. enterica Typhimurium definitive type 104 strains (DT104), but little information is available on the mechanisms responsible of the wide diffusion of these resistances in other serotypes. The aim of this work was to identify common Sm and Te resistance determinants, such as resistance genes, transposons and plasmids, circulating among Salmonella of animal origin isolated in Italy. Fifty-eight multidrug-resistant S. enterica strains belonging to 19 different serotypes were analyzed. The collection did not include the S. Typhimurium DT104 strains. Results. A large percentage of the Te resistant strains of our collection (63%) were positive for the tetA (class A) gene. Most of these genes were located within the Tn1721 transposon. The 84% of the Sm resistant strains carried the strA-strB genes. The 29% of the strains were also positive for the presence of integrons, frequently associated with the Tn21 transposon. Prototypic resistance plasmids were characterized by conjugation, Southern blot hybridization and DNA sequencing. A replicon typing method based on PCR amplification was devised. This assay revealed the presence of common plasmids circulating among different S. enterica serotypes in animals living in Italy. In particular, a repN-positive plasmid, carrying the strA-strB and tetA genes, was diffused in different Salmonella serotypes. The strA-strB genes were frequently identified within the Tn5393 transposon linked to the IS1133 element, in Hadar, Agona, Bredeney, Give, Heidelberg, London and Tshiongwe serotypes. Conclusions. Salmonella strains of different serotypes, geographical origins and animal sources showed the presence of common, predominant, widely distributed resistance determinants. We have found that the tetA and strA-strB genes were very diffused and often located within transposons. In particular, the strA-strB genes were frequently associated with transposon Tn5393, originally described in the apple-trees-pathogen Erwinia amylovora, in the fish pathogen Aeromonas salmonicida, in Campylobacter jejuni, Pseudomonas aeruginosa and Corynebacterium striatum. This is the first time that this resistance determinant is identified in Salmonella isolates. Moreover, we found that the IS1133-variant of the transposon, previously described only in plant-pathogens, was diffused in Salmonella of different serotypes isolated from poultry. Since the use of Te and Sm in animal farming in Europe is quite limited, it should be explored the possibility that the

selection of resistance determinants may occur in plant pathogens, transferred to bacteria circulating in animals and humans by horizontal gene transfer.

123(C)

LPS AND FLAGELLIN FROM SALMONELLA ENTERITIDIS ACTIVATE CHICKEN MACROPHAGES VIA TOLLLIKE RECEPTORS 4 AND 5

A. M. Keestra, I. Kuhar, E. J. Gaasbeek, J. P. van Putten; Utrecht University, Faculty of Veterinary Medicine, Utrecht, NETHERLANDS. Salmonella enteritidis can invade and survive within macrophages, thus triggering a wide range of cellular responses. LPS and flagella are important factors of S. enteritidis that contribute to the virulence of S. enteritidis in poultry. These pathogen-associated molecular patterns (PAMP) may induce these responses by binding to PAMP receptors, including the Toll-like receptors (TLRs). Toll-like receptors are a family of transmembrane receptors that play an important role in the innate immune system by activating the NF-kB signalling pathway. In human, LPS and flagellin activate the innate immune response via TLR4 and TLR5, respectively. In poultry, however, not much is known about the TLR-NF-kB signalling pathway. We investigated the activation of chicken macrophages (HD11) using recombinant His-tagged flagellin. Purified flagellin from S. enteritidis stimulated the production of nitric oxide (NO) in HD11 cells. NO production was also increased when HD11 macrophages were stimulated by the addition of LPS from S. enteritidis. Transfection of HD11 macrophages with a NF-kB-luciferase reporter system showed that both LPS and flagellin activated the NF-kB signalling pathway. RT-PCR on RNA isolated from HD11 macrophages with primers based on the sequence of chTLR4 (accession number AY064697) and on the sequences of chEST clones from the BBSRC database with homology to human TLR5 showed that both TLR4 and TLR5 are expressed in the HD11 cell line. These results suggest that LPS and flagellin from S. enteritidis activate the chicken innate immune system via chTLR4 and chTLR5.

124(A)

CHARACTERIZATION OF SIX LOW MOLECULAR WEIGHT PLASMIDS PRESENT IN SALMONELLA ENTERICA SEROVAR ENTERITIDIS

D. Gregorova1, J. Matiasovicova 1, A. Sebkova1, R. Karpiskova 2, I. Rychlik 1; 1Veterinary research institute, Brno, CZECH REPUBLIC, 2National institute of Public Health, Brno, CZECH REPUBLIC. Salmonella enterica serovar Enteritidis (S. Enteritidis) frequently possesses plasmids of different sizes and roles. Besides the serovar specific virulence plasmid present in most field strains, S. Enteritidis can harbour plasmids of low molecular weight. The fact that the low molecular weight plasmids are present in about 10% of the field strains suggests that there is considerable selection pressure against 75

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them. However, this simultaneously raises a question of what biological function they have to be to allow their prolonged existence in S. Enteritidis. We therefore characterised the most frequent low molecular weight plasmids in S. Enteritidis. Using DNA hybridisation we found that there are at least three distinct groups of plasmids. After sequencing 6 representative plasmids from each group we concluded that they belong to ColE1, ColE2 and rolling circle-like replicating plasmids. Plasmids pI, pC and pK belonged to the most widespread group of ColE1-like replicating plasmids. Plasmid pI (4053 bp) coded for retron reverse transcriptase and cold shock protein. This plasmid apparently influenced phage resistance of the host strain. Plasmid pC (5269 bp) encoded functional restriction modification which explains its nearly ubiquitous presence in phage highly resistant S. Enteritidis PT14b strains. Plasmid pK (4245 bp) coded for ORFs with unknown function. ColE2 type plasmid pP (4301 bp) encoded two proteins essential for its replication and one ORF for which no function could be predicted. Inside Salmonella cell, pP was present predominantly in a single stranded DNA form. The smallest plasmids pJ (2096 bp) and pB (1983 bp) were classified as rolling circle-like replicating plasmids. Both of them encoded only a single protein essential for their own replication.They were capable of hybridization without denaturation, their DNA could be linearized with S1 nuclease, however, even after such treatment the ability to hybridize without denaturation did not disappear. This suggests that both the plasmids pJ and pB must exist in an unusual molecular structure. We therefore concluded that one of the frequent functions of low molecular weight plasmids in S. Enteritidis is the protection against phage infection as observed for plasmids pI and pC. Although in some cases, such as in the plasmid pJ and pB, no obvious function of this plasmids could be predicted. 2002). Methods: SPA blood isolates from all children (age 0-18 years) suffering from enteric fever who had blood cultures submitted to the Aga Khan University laboratory or collection points throughout the country from April 2002 to March 2003 were analyzed. Randomly selected isolates of SPA from 1998 ­ 2001 were also analyzed. Chromosomal DNA from 123 SPA isolates (obtained from Karachi. Lahore, Islamabad, Rawalpindi, Quetta, Peshawar and Hyderabad) was subjected to enzymatic digestion with xba l (5´-TCTAGA-3´), a rare-cutting enzyme which yields large fragments of chromosomal DNA of varying molecular weights. These fragments were then analyzed by PFGE. Results: PFGE analysis of the 123 SPA isolates revealed all strains to be clonal (identical) or closely-related (one to three band differences only). Strains obtained from different years as well as from different cities exhibited PFGE patterns that were almost identical to each other. PFGE could not discriminate between antibiotic sensitive and antibioticresistant strains. Conclusion: This study demonstrates significant genetic homogeneity among circulating strains of SPA in Pakistan, suggesting a recent point-source origin and epidemic spread. These findings are comparable to the clonal origin of multiple-drug-resistant strains of typhoid in Karachi demonstrated in earlier studies.

126(C)

CARACTERIZATION OF SALMONELLA ISOLATES RECOVERED FROM SWINE, POULTRY AND SPORADIC CASES OF SALMONELLOSIS IN HUMANS WITHIN A LIMITED GEOGRAPHICAL AREA

S. Quessy, N. Rheault; Université de Montréal, St-Hyacinthe, PQ , CANADA. Limited data are available on sources of infection for sporadic cases of human salmonellosis. The aim of this study was to compare the genetic profiles and the antimicrobial sensivity patterns of Salmonella isolates from swine and poultry operations to those of isolates of sporadic cases of salmonellosis in human within a limited geographical area and a defined period of time. Over a one year period, a total of 1812 swine and 2175 broiler chickens were sampled at slaughter. Fecal material and carcass samples were collected to detect the presence of Salmonella. A total of 1754 samples were also collected from human with diarrhea in hospitals located within the same geographical area. All samples were processed by conventional enrichment and culture procedures and serotyped. Salmonella was found in, respectively, 20,4%, 13,4% and 3% of swine, poultry and humans samples. Genetic profiles of isolates were obtained by Pulse Field Gel Electrophoresis using XbaI as first discriminatory restriction enzyme and antimicrobial susceptibility profiles were done using the agar diffusion method. High antimicrobial resistance levels were observed in Salmonella Typhimurium isolates from swine and poultry feces although isolates from carcasses had lesser antimicrobial agent resistance. The frequency of antimicrobial agent resistance was lower in human isolates but it was possible in ASM Conference on Salmonella

125(B)

SALMONELLA PARATYPHI A BLOODSTREAM INFECTIONS IN PAKISTANI CHILDREN ­ AN EPIDEMIOLOGIC ANALYSIS BY PULSED-FIELD GEL ELECTROPHORESIS

A. K. Zaidi, Z. Parween, A. Irshad, R. Hasan, Z. Abbas, A. A. Siddiqui, Z. A. Bhutta; Aga Khan University, Karachi, PAKISTAN. Background: Salmonella paratyphi A (SPA) are now responsible for causing 20-30% of all cases of enteric fever in Pakistan. An increase in the number of cases has been observed in Karachi since 1999. Unlike Salmonella typhi infections which are confined mainly to the pediatric age group, SPA infections show similar attack rates in adults and children, suggesting a non-immune adult population and recent introduction of this pathogen in a susceptible population. Outbreaks associated with this organism have also been reported in other regional countries. Objective: The objective of our study was to use pulsedfield gel electrophoresis (PFGE) to epidemiologically characterize isolates of SPA obtained from bloodstream infections in Pakistani children over a 5 year period (1998-

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few cases to demonstrate genetic links between multiresistant isolates from poultry, swine and humans. These data indicate that swine and poultry may be considered as reservoirs of multiresistant Salmonella strains involved in sporadic cases of salmonellosis. antimicrobials such as Gn, To, Ak, Nt, Apra, Nal, SxT and C. Conclusion: The detection of a major clon in swine for human consumption, added to its appearance in poultryderived food, and together with the isolation in human and environmental strains, might indicate a wide dissemination of a pig-related Salmonella serotype Derby strain in Spain.

127(A)

CLONAL SPREAD OF A SINGLE STRAIN OF SALMONELLA ENTERICA SEROTYPE DERBY FROM SWINE-DERIVED FOOD IN SPAIN

S. Valdezate1, A. Vidal2, S. Herrera-León1, J. Pozo 2, P. Rubio2, M. Usera1; 1LNRSSE. C.N.M. Instituto de Salud Carlos III, Madrid, SPAIN, 2Infectious Diseases Unit. Animal Health Departament. Faculty of León, León, SPAIN. Aim: In order to identify epidemiological linkages between Salmonella enterica subsp. enterica serotype Derby strains, antimicrobial susceptibility profile and genetic characterization by PFGE were performed. Swine-related strains and, other animal, human, food and environmental origin strains, isolated from different Spanish locations were studied. Materials and methods: During a three-year period (20002002) one hundred and six Derby isolates were studied. Sixty-three isolates were obtained from 45 slaughter pigs, proceeding of 6 finishing units belonging to 4 swine operations, and 43 isolates from the LNRSSE collection with the following distribution: human (16), food (22), ill pigs (4), and environment (2). Identification and serotyping were performed according to standard methods. Susceptibility to 25 antimicrobials was carried out by disk diffusion method (NCCLS). NCCLS interpretative criteria (M100-S12) was considered, and intermediate isolates were categorized as resistants. Clonal analysis was performed by molecular characterization of all strains by PFGE, with XbaI restriction under Salm-Gene guidelines (pulse times from 2s to 64 s at 6.0V/cm at 14oC during 22 h). Results: The overall rates of resistance for the studied antimicrobials were: higher than 80.0 % for spectinomicin (Sh), streptomycin (S) and tetracycline (Te); 43.7 % for sulfonamide (Su); ranging from 16.7 to 4.5 % for ampicillin (Amp), ticarcillin (Tic), gentamicin (Gn), tobramicin (To), netilmicin (Nt), nalidixic acid (Nal), trimethoprim/ sulphamethoxazol (SxT) and chloramphenicol (C); and low rates (1.8 %) for apramicin (Apra) and amikacin (Ak). No resistances were detected to amoxicillin-clavulanic, cephalosporins, ciprofloxacin and enrofloxacin. PFGE results in Derby serotype showed a high genetic diversity, yielding 26 profiles. The index Simpsom´s of diversity reached was of 0.862 from LNRSSE collection. In this way, in each isolation source, different resistance phenotypes and PFGE profiles were identified, respectively: in slaughter pigs (13/7), in human (9/10), food (12/8), ill pigs (4/4), and environment (2/2). A major clon were detected in 29 slaughter pigs belonged to 5 finishing unit proceeding of 4 swine operations. This predominant profile also appeared in strains from human source (4/16), food (10/23), ill pigs (1/6) and environmental sources (3/4). The main resistance phenotype displayed by this clon was Sh-S-Te-Su (32strains), but in some occasions the resistance was extended to other Pathogenesis, Epidemiology, and Vaccine Development

128(B)

GENETIC INTERACTIONS BETWEEN THE GIFSY-1 AND GIFSY-2 PROPHAGES OF SALMONELLA ENTERICA SEROVAR TYPHIMURIUM

S. Lemire, N. Figueroa-Bossi, L. Bossi; CNRS, Gif-sur-Yvette, FRANCE. A class of suppressors of recBCD mutations in Salmonella enterica ser Typhimurium LT2 map in the Gifsy-1 prophage (at a locus designated sbcE). They result from mutational changes that activate a normally repressed prophage gene (recE) whose product can substitute for the RecBCD enzyme in DNA recombination and repair. All sbcE alleles are small deletions that remove part of a presumptive lysogenic repressor gene (gogR) and put the recE gene (and other genes of in the prophage's main "left" operon) under the control of the gogR promoter. Intriguingly, recE gene activation in the sbcE mutant background is totally dependent on the presence of a second prophage, Gifsy-2, elsewhere in the chromosome. The Gifsy-1 and Gifsy-2 prophages of strain LT2 are strongly related and have virtually identical sequences (> 99% identity) over an uninterrupted 7 Kb segment that includes the immunity region. The present study was aimed at identifying the Gifsy2 determinants responsible for Gifsy-1 trans-activation in sbcE mutants. We have measured expression of a recE-lacZ gene fusion in strains deleted for selected portions of the Gifsy-2 genome. Preliminary results allow us to delimit the region responsible for trans-activation and suggest the involvement of Gifsy-2 putative repressor gene, gtgR, in the activation mechanism. The product of this gene, 100% identical to Gifsy-1's GogR, appears capable of mediating transcriptional activation at the gogR promoter. Conceivably, this response reflects the capacity of GtgR and GogR proteins to autogenously regulate their own expression.

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129(C)

DEVELOPMENT OF AN INACTIVATED, IRON-RESTRICTED VACCINE FOR POULTRY TO AID IN THE CONTROL OF SALMONELLA ENTERITIDIS AND SALMONELLA TYPHIMURIUM, AND THE POTENTIAL FOR BROAD SPECTRUM PROTECTION AGAINST SALMONELLOSIS

J. F. van den Bosch1, F. A. Clifton-Hadley 2, S. W. Cooles2, S. B. Houghton1, M. J. Woodward 2; 1Intervet UK Ltd, Milton Keynes, UNITED KINGDOM, 2Veterinary Laboratories Agency Weybridge, Addlestone, UNITED KINGDOM. We have shown previously that vaccination of poultry with an inactivated, iron-restricted, Salmonella enterica serovar Enteritidis vaccine (Salenvac) significantly reduces S. Enteritidis infection in commercial flocks. The introduction of this vaccine into the UK correlated with a decrease in cases of human salmonellosis.In order to reduce further the risk to human health of salmonellosis arising from poultry and poultry products contaminated with Salmonella spp., a multivalent salmonella vaccine is indicated. Here we report the development of an inactivated, iron-restricted bivalent vaccine for the active immunisation of chickens to reduce excretion and infection of birds with both S. Enteritidis and S. Typhimurium. Significantly this vaccine, Salenvac T, also confers protection in poultry against other Salmonella serovars which are associated with human disease, as shown in experimental challenge studies with S. Heidelberg and S. Agona.

accumulates more extensively than SodC1 in stationary cultures in the laboratory. In the present study, we confirmed that increased SodC1 levels in intracellular bacteria do not result from gene amplification due to prophage induction. To establish if the differential expression patterns reflect transcriptional as opposed as to post-transcriptional regulation, we have measured the levels of epitope-tagged SodC1 and SodC2 proteins in strains in which the DNA sequences encoding these proteins are switched relative to the respective gene promoter regions. Results show that SodC1 and SodC2 accumulation patterns reverse upon this rearrangement, indicating that the differential regulation occurs primarily at the transcriptional level. In order to validate the above observations, we carried out a similar study with Salmonella enterica, serovar Enteritidis, which also has a chromosomal sodC2 gene and a sodC1 gene within a prophage genome. Unlike in serovar Typhimurium, the prophage involved appears extensively defective, is only partially related to the Gifsy-2 prophage and located at a different chromosomal location. In spite of these differences, competitive index measurements in mice infected with sodC1 and sodC2 single and double mutants yielded results closely comparable to those obtained in serovar Typhimurium; namely, sodC2 inactivation had no effect, whereas the sodC1 mutant was significantly attenuated. Again, this difference correlated with the lack of detection of epitope-tagged sodC2 protein in vivo. These results confirm that regulation of sodC1 and sodC2 genes has evolved to respond to the different conditions encountered by broad-host range Salmonella serovars in the environment or in the host.

131(B)

BACTERIAL TRANSLOCATION FROM MESENTERIC LYMPH NODES CORRELATES WITH SYSTEMIC SALMONELLOSIS IN CALVES, AND IS DEPENDENT ON TYPE THREE SECRETION SYSTEM-1 (TTSS-1), BUT NOT TTSS-2

S. M. Paulin, M. P. Stevens, J. Campbell, T. S. Wallis, B. Villarreal-Ramos; Institute for Animal Health, Compton, UNITED KINGDOM. The host and bacterial factors that determine whether Salmonella enterica serovars remain restricted to the gastrointestinal tract or penetrate beyond the mucosa and cause systemic disease in a given host remain largely undefined. In this study, the route and magnitude of bacterial translocation from the gut was assessed for three Salmonella serovars, with differing virulence for calves, using an efferent lymphatic cannulation model. Serovars Dublin, Typhimurium and Gallinarum were compared as, despite being invasive for bovine intestinal mucosa, these serovars cause systemic disease, enteritis and asymptomatic infections respectively following oral inoculation of calves. Serovar Dublin translocated via the efferent lymph in significantly higher numbers than either serovars Gallinarum or Typhimurium. The latter two serovars were contained by the mesenteric lymph nodes (MLN). Bacteriological and microscopic analysis of efferent lymph demonstrated that serovar Dublin ASM Conference on Salmonella

130(A)

COMPARING THE REGULATORY RESPONSES AND THE RELATIVE CONTRIBUTIONS TO PATHOGENICITY OF CU,ZN SUPEROXIDE DISMUTASE SODC1 AND SODC2 GENES IN SALMONELLA ENTERICA SEROVARS TYPHIMURIUM AND ENTERITIDIS

S. Ammendola1, N. Figueroa-Bossi 2, A. Battistoni1, L. Bossi2; 1 University of Rome Tor Vergata, Rome, ITALY, 2Centre de Génétique Moléculaire, CNRS, Gif-sur-Yvette, FRANCE. Periplasmic Cu,Zn superoxide dismutases (Cu,ZnSODs) are thought to contribute to bacterial virulence by protecting Gram-negative pathogens against the oxidative burst of host's phagocytes. Salmonella enterica serovar Typhimurium has two such enzymes, SodC1 and SodC2, encoded by genes located respectively on a prophage (Gifsy-2) and on the chromosome. Recent studies suggest that the two enzymes do not contribute equally to virulence: disruption of the sodC1 impairs significantly the ability of Salmonella to systemically infect mice, whereas no such effects were observed upon deleting the sodC2 gene. This correlates with the findings that SodC1 is the only one of the two proteins synthesized to significant levels in bacteria proliferating intracellularly or in mouse spleens. In sharp contrast, SodC2

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leaves the MLN in an extracellular niche, free in the lymph, despite being intracellular whilst in the intestinal mucosa and MLN. A pool of signature-tagged strains, derived from mutagenesis studies, was assessed for their ability to translocate from the regional MLN. Disruption of TTSS-1, but not TTSS-2, blocked passage through the MLN. These observations suggest that, at least during systemic salmonellosis, trafficking within the intracellular and extracellular milieu, together with the ability to pass through MLN, correlates with host specificity in calves. types with seven subtypes in MDR strains, whereas sensitive strains yielded four types with six subtypes. Ribotyping analysis identified three different patterns in MDR strains and four patterns in sensitive strains. Overall it can be suggested that the incidence of MDR S. typhi has been increasing in Bangladesh with diverse clonality.Therefore, detailed study of MDR S. typhi is necessary for better understanding of the epidemiology of these strains in order to reduce the morbidity and mortality rate as well as the total burden of S. typhi in Bangladesh.

132(C)

MOLECULAR CHARACTERIZATION OF MULTIDRUG-RESISTANT SALMONELLA TYPHI ISOLATED IN BANGLADESH

K. A. Talukder1, A. Safa1, J. Sultana1, D. K. Dutta1, M. A. Islam1, F. Hassan1, M. A. Hossain 1, K. Alam1, D. J. Gomes 2, G. B. Nair1, D. A. Sack 1; 1International Centre for Diarrhoeal Disease research, Bangladesh, Dhaka, BANGLADESH, 2 Department of Microbiology, University of Dhaka, Dhaka, BANGLADESH. Enteric fever is one of the major public health problems in developing countries like Bangladesh. Annual global incidence of typhoid fever has been estimated to be 16.6 million cases of which 7.7 million cases in Asia alone with 0.44 million deaths. The disease is mainly attributed by Salmonella typhi. Resistant of S. typhi to most relevant drugs have emerged and imposed a serious limitation on the treatment of this disease. Resistance to most accessible antibiotics has been increasing in Bangladesh causing impaired treatment of the disease. Five hundred five S. typhi strains isolated from patients attending at the Dhaka treatment center of the ICDDR,B between January 1997 and December 2001were studied. The results showed that the frequency of multidrug resistance increased from 31% in 1997 to 40% in 2001. Of 505, 70 strains were included in this study to determine the present status of the antibiotic resistance pattern and also to determine the clonal relationship among multidrug-resistant (MDR) and sensitive strains. These strains were isolated, identified, and susceptibility to antimicrobial agents determined by standard methods. Strains were characterized genotypically by determining the resistance factor using conjugation, analyzing the plasmid DNA, pulsed-field gel electrophoresis (PFGE) and ribotyping. Incompatibility group plasmid and different genes encoding resistance to â-lactam antibiotics were identified by PCR. Twenty six percent strains were resistant to ampicillin, chloramphenicol, trimethoprimsulfamethoxazole, tetracycline, streptomycin and nalidixic acid but none were resistant to ceftriaxone and ciprofloxacin. Plasmid analysis showed that all MDR strains except 3 harbored 140 MDa and/or 90 MDa plasmid whereas most of the sensitive strains (89.65%) were plasmid less. PCR result showed that the strains harboring 140 MDa plasmid belonged to the incompatibility group IncHI1. PCR analysis confirmed the presence of TEM-1, strAB and tetB of resistance genes in all the MDR strains. Hybridization experiment recognized the TEM-1 gene located in both chromosome and plasmid DNA. PFGE analysis yielded three Pathogenesis, Epidemiology, and Vaccine Development

133(A)

DESCRIPTION OF A NEW EXTENDEDSPECTRUM B-LACTAMASE CTX-M-15 IN SALMONELLA ENTERICA SEROTYPE ENTERITIDIS

S. Herrera-León1, M. J. Pena-López2, S. Valdezate1, M. A. Echeita1, M. A. Usera1; 1LNRSSE. CNM, Majadahonda. Madrid, SPAIN, 2Gran Canaria Dr. Negrín Hospital, Gran Canaria, SPAIN. Objetives and Methods:Salmonella sp. has been found to express a wide variability of extended-spectrum blactamases (ESBLs) types including TEM, SHV, PER and CTX-M enzymes. Three Salmonella enterica serotype Enteritidis phagetype 1 strains were isolated from three under ten months old children in the "Gran Canaria Dr. Negrin" Hospital located in the Canarian Island (Spain) were analysed in this work. The two first strains were yielded from stool samples and the last one was isolated from blood culture. A wide antimicrobial susceptibility profile was carried out by the disk diffusion method (NCCLS) together with doubledisk synergy test (DDS). Isoelectric focusing was performed. The ESBLs presence were checked by PCR, using primers specifically designed for this work. After amplification, sequencing were realized by the Big-Dye terminor method (ABI PRISM 377). Results: The three strains displayed resistance to the following compounds: ampicillin, ticarcillin, piperacillin, oxacillin, cefuroxime, cefotaxime and ceftazidime. Clavulanic acid, restored susceptibility of the strains to penicillins and cephalosporins. Moreover, the strains displayed also resistance which affect to nalidixic acid, while remained susceptible to cefoxitin, imipenem, ciprofloxacin, enrofloxacin, gentamicin, trimethoprim-sulfamethoxazole, sulfamides, tetracycline, cloramphenicol, streptomycin, and kanamicin. A positive DDS indicated ESBL production. The pI of the enzymes were determined focusing at 5.4 and 8.9, suggesting the expression of a blaTEM-1 gene and a blaCTX-M, respectively. Sequencing confirmed the presence of a blaTEM-1 and a blaCTX-M-15. The CTX-M enzymes confer high-level resistance to cefotaxime, ceftriaxone and aztreonam, but have only marginal effects on ceftazidime MICs. However CTX-M-15 has so far also been found to have a significant degree of resistance to ceftazidime. Conclusion: CTX-M enzymes have been relatively frequently found in ESBL-producing Salmonella spp. although, CTX-M15 has never been found. Due to the fact that Salmonella enterica serotype Enteritidis represents the majority serotype in Europe, production of newer cephalosporin-hidrolyzing

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b-lactamases by strains belonging to a predominant phage type of this serotype is a disturbing development. Further dissemination of such strain could reduce therapeutic options for severe Salmonella infections. lated in chicken skin to study their growth kinetics in the presence and absence of other contaminants in chicken meat. Growth kinetics of Salmonella under different temperatures and in the presence of different chemicals that are used for chicken processing will be studied using GFP as a marker.

134(B)

INTERACTION OF S. ENTERICA SEROTYPE TYPHIMURIUM WITH EPITHELIAL CELLS IN VIVO AND IN VITRO

M. Raffatellu 1, A. D. Humphries1, H. Andrews-Polymenis1, J. Figueiredo2, S. Khare2, S. Lawhon2, C. Rossetti2, L. G. Adams2, A. J. Bäumler1; 1Texas A&M University HSC, College Station, TX, 2 Texas A&M University CVM, College Station, TX. Salmonella enterica serotype Typhimurium infection in calves results in signs of disease and pathology that closely mimic the illness caused by this pathogen in humans. The hallmark of the host response elicited by S. Typhimurium is the production by intestinal epithelial cells of PMN chemoattractants (i.e. the CXC chemokines GRO alpha and IL-8) and a subsequent massive infiltration of the mucosa with PMN. The effector genes sipA, sopA, sopB, sopD and sopE2 are required for eliciting the production of CXC chemokines, PMN infiltration and diarrhea in the calf model. We investigated whether host responses observed in the calf model can be modeled by S. Typhimurium interaction with human epithelial cell lines in vitro. Invasiveness of S. Typhimurium mutants for unpolarized cells correlated with the amount of fluid accumulation elicited in bovine ligated ileal loops. However, GRO alpha and IL-8 production measured during infection of unpolarized human epithelial cell lines was independent of the effector genes sipA, sopA, sopB, sopD and sopE2. We are currently investigating responses of polarized epithelial cells to S. Typhimurium infection.

136(A)

ANALYSIS OF SALMONELLA ENTERICA SUBSPECIES I SPECIFIC FUN GENES: STM437 AND STM557 HAVE ROLES IN INTESTINAL PERSISTENCE

H. L. Andrews-Polymenis, L. R. Garza, J. Schouwenberg, A. J. Baumler; Texas A&M Heath Science Center, College Station, TX. S. enterica subspecies I serotypes are responsible for the vast majority of salmonellosis in mammals and birds, yet only a few factors specific to this group that allow them to persist in this niche have been identified. Recently, genes of S. enterica that are subspecies I specific and unique, have been identified through genome sequencing and microarray studies. Identification of these genes allowed us to investigate the roles of several of these genes in the pathogenesis of infection caused by S. Typhimurium in susceptible and resistant murine models of infection. Three subspecies I specific genes, STM437, STM557, STM3478, chosen on the basis of their predicted location and unknown function (FUN), were inactivated and the resulting strains were tested for virulence and persistence in murine models. In balb/c mice, which are susceptible to infection with S. Typimurium, mixed infections with virulent S. Typhimurium 14028 and individual isogenic mutant strains resulted in only minor defects in colonization of the liver, spleen, mesenteric lymph nodes, peyer's patches and cecum. However in CBA mice, which are a model for intestinal persistence of S. Typhimurium, STM437 and STM557 mutants had striking defects in intestinal persistence over 40 days of infection as compared with virulent S. Typhimurium 14028 in mixed infections. STM3478 mutants were able to persist in the intestine of CBA mice at levels similar to virulent S. Typhimurium 14028 in mixed infections. Our current and future studies focus on further investigating the roles of STM437 and STM557 in intestinal persistence by attempting to identify the subcellular location of these gene products, identifying interacting proteins and ultimately determining the molecular mechanism of these genes in intestinal persistence.

135(C)

USE OF GFP AS A MARKER TO STUDY THE GROWTH KINETICS OF SALMONELLA IN CHICKEN MEAT

K. Dulal; University of Maryland Eastern Shore, Princess Anne, MD. Since chicken meat is one of the most common meat sources in the United States, we are interested to find out the growth kinetics of Salmonella in chicken meat. Plasmid pEGFP containing the gene which expresses green fluorescence protein (GFP), from jellyfish Aequora victora was transformed in two different strains of Salmonellae, S. kentucky (S167) and S. hadar (S169). Plasmid pEGFP and plasmid pPROtet.E233 containing chloramphenicol resistance gene and a tetracycline inducible promoter from E. coli were digested by two enzymes, Not I and BamHI. The insert containing the GFP gene and the vector containing the chloramphenicol resistant gene and tetracycline inducible promoter were ligated. The new recombinant plamid was grown in E. coli and harvested. The plasmid was then transferred to the two strains of Salmonella. Salmonella with GFP and without the GFP expressing gene will be inocu-

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137(B)

DRUG RESISTANCE PATTERNS OF TYPHOIDAL AND NON-TYPHOIDAL SALMONELLA SPECIES IN HAMADAN, WEST OF IRAN

R. Yousefi-Mashouf, R. Yousefi-Mashouf, Sr.; Medical Sciences University of Hamadan, Hamadan, IRAN (ISLAMIC REPUBLIC OF). Introduction: The epidemiological studies indicate that the incidence of salmonellosis is increasing throughout the world. The uncontroled and unappropriate usage of antibiotics has been caused multi- drug resistance in these organisms, in recent years. Materials and Methods: In a cross-sectional descriptive study, 204 strains of typhoidal salmonella (T.S) and 114 strains of non- typhoidal salmonella (N.T.S) were examined to determine drug resistance.The strains were collected from patients who referred to cilinical centers in Hamadan during 1997 to 1999. They were serotyped and then tested for their antibiotic resistance patterns, using Kirby- Bauer method for 8 antibiotics. Results: The salmonella isolated were as follows: "S.typhi, S. paratyphi A, B, C. S.typhimurium, S.enteritidis, S. cholerasuis, S. agona, S. arizona, S. infantis, S.havana, S.lexington and S. virchow." A proportion of strains (>60%) were resistance to Carbenicillin and ampicillin. Resistance to Ciprofloxacin and Nalidixic acid was very low (<15%). S.typhimurim (100%), S. typhi (95.7%) paratyphi B (89.2%) and enteitidis (60%) showed multi- drug resistance (MDR). Conclusion: Our results showed that most of salmonella spp. isolated from patients in Hamadan city (west of Iran) were resistant to beta-lactam antibiotics, whereas, most of them were sensitive to fluoroquinolones antibiotics.We suggest that the use of some newer antibiotics such as new fluoroquinolones, ceftazidime, and aztreonam as effective therapy againts salmonella species in this region.

Many pathogens have evolved strategies to evade the immune response by interfering with antigen processing and presentation. Down-regulation of MHC class II on the surface of infected cells has been observed for many bacterial pathogens. Mechanisms for the down-regulation include degradation of host transcription factors necessary for expression of class II, downregulation of the transcription of the class II transcriptional activator CIITA, changes in the stability of class II heterodimers and disruption of membrane traffic in the endocytic pathway. Salmonella reside and replicate intracellularly within membrane-bound vacuoles and interfere with endosomal trafficking in the host cell. We investigated expression of MHC molecules in cultured cells following infection with Salmonella typhimurium. FACS analysis revealed decreases in class II molecules but not class I molecules on the surface of infected MelJuso and THP-1 cells. Western blotting and metabolic labeling have revealed the decrease is not due to reduced protein synthesis, increased protein degradation or alteration in the stability of class II heterodimers. Efforts are ongoing to determine the mechanism of the cell-surface down-regulation.

139(A)

CHARACTERIZATION OF THE EXOPOLYSACCHARIDE ASSOCIATED WITH THIN AGGREGATIVE FIMBRIAE OF SALMONELLA ENTERICA SEROVAR ENTERITIDIS

D. L. Gibson; University of Victoria, Victoria, BC, CANADA. Salmonella enterica serovar Enteritidis, a frequent food-borne enteric human pathogen, has been shown to form recalcitrant biofilms even on Teflon and stainless steel surfaces. It has been well established that extracellular polymeric substances (EPS) play a significant role(s) in the maturation and stabilization of biofilms. Extracellular polymeric material (EPM) was found to be intimately associated with thin aggregative fimbriae (Tafi) of S. enteritidis. This EPM was detected on western blots using immune serum raised to whole, purified Tafi, which does not recognize lipopolysaccharide (LPS). EM examination using this antiserum to strains deficient in Tafi production revealed thin, branched, fibrous material also detected in S. enterica serovar Typhimurium and diarrheagenic E. coli isolates. It was observed that extracellular bacterial cellulose was required to link this material to cells. Preliminary analysis indicated that this material contained the extracellular polysaccharide (EPS), colanic acid. However, an isogenic mutant of the glucosyltransferase gene involved in colanic acid synthesis (wcaJ) still produced the EPM indicating it was not colanic acid. Purified EPM separated by SDS-PAGE did not stain with gelcode and was resistant to Proteinase K indicating the absence of protein; nor did, purified EPS react with the silver stain for LPS, indicative of it's absence. The EPM was positively stained by Alcian blue as a high MW smear indicating the presence of acidic residues and was,similar to the high MW material observed on western blots using immune serum raised to Tafi. A quantitative assay for uronic acid, a component of the repeating unit of acid mucopolysaccharides, indicated that this purified EPM contains 81

138(C)

DECREASED MHC CLASS II EXPRESSION ON SALMONELLA-INFECTED CELLS

E. K. Mitchell, A. Kelly, J. Trowsdale; University of Cambridge, Cambridge, UNITED KINGDOM. Antigen processing and presentation by MHC molecules is an important component of the adaptive immune system. During pathogen infection, MHC molecules bind pathogenderived peptides which are then displayed on the cell surface for surveillance by T cells. Presentation of peptides is mediated by two classes of MHC molecules, class I and class II which sample peptides from distinctly different cellular locations. MHC Class II molecules are sythesized in the ER and transported via the Golgi to the endocytic pathway. Here, they bind peptides generated from the lysosomal degradation of internalised exogenous antigens. In contrast, MHC class I molecules bind peptides in the ER, derived mainly from endogenous antigens generated in the cytosol and transported into the ER by a specialised transporter. Pathogenesis, Epidemiology, and Vaccine Development

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glucuronic acid. Compositional and structural analyses are underway. Since S. enteritidis often persists as biofilms, understanding the underlying factors and biological processes involved in biofilm structural development are fundamentally and practically important when considering the deleterious effects biofilms pose to human and animal heath.

141(C)

PRO-INFLAMMATORY CYTOKINE DIFFERENCES UNDERLYING THE GENETIC PREDISPOSITION TO VARIOUS SALMONELLA SEROVARS

A. N. Lazzerine, P. Barrow, P. Kaiser; Institute for Animal Health, Newbury, UNITED KINGDOM. A range of Salmonella enterica serovars (Typhimurium, Gallinarum and Pullorum) were tested for their ability to induce pro-inflammatory cytokines from chicken kidney cell (CKC) of genetically resistant and susceptible inbred White Leghorn chickens. In terms of Salmonellosis, line 61 birds are considered resistant, while line 72 birds are considered susceptible; both chicken lines are highly inbred (F >> 0.99) and have the same MHC I (B2). IL-6 production in the more susceptible line 7 2 birds was consistently higher than that produced by more resistant line 61 birds at 2 and 4 hpi, but was not significantly different by 8 hpi. Invasion by S. Pullorum, however, produced no significant difference between the lines and induced lower levels of IL-6 from the host cells. The inherent increase in IL-6 production from line 72 birds may impede the immune system in removing Salmonella from the host. The comparatively low induction of IL-6 from cells invaded by S. Pullorum, repeats responses we have reported previously in outbred CKC, again suggesting the ability of S. Pullorum to manipulate pro-inflammatory factors in the host immune response.

140(B)

THE ROLE OF DNA BASE EXCISION REPAIR IN SALMONELLA PATHOGENESIS

A. E. Suvarnapunya, M. A. Stein; University of Vermont, Burlington, VT. Salmonella enterica serovar Typhimurium proliferates within macrophages in the presence of potentially lethal oxidants. The role of the base excision repair (BER) system, dedicated to repair of oxidatively damaged DNA, in serovar Typhimurium pathogenesis was therefore investigated. Salmonella, unlike most other bacteria, has homologs of every microbial BER gene. Null mutants lacking discrete BER enzymes (Dnth, Dnei, Dxth, or Dnfo) and defective in pyrimidine glycosylase- (Dnth/nei) or endonucleasemediated (Dxth/nfo) repair steps were constructed. Dnth/ nei, Dxth, and Dxth/nfo were markedly impaired for survival in RAW 264.7 and C57BL/6-derived macrophages activated by interferon-g. Abrogation of macrophage phagocyte oxidase and inducible nitric oxide synthase (C57BL/6 gp91phox-/-/NOS2-/-) almost fully restored survival of the same mutants. Moderate attenuation was observed in the murine model by Competitive Index: 5- and 12-fold for Dnth/nei and Dxth/nfo, respectively. A triple glycosylase mutant (Dfpg/nth/nei) was constructed, which is unable to initiate BER, to control for toxic, glycosylasegenerated repair intermediates. Dfpg/nth/nei was radiationresistant, but was significantly impaired for survival within macrophages. Thus, macrophage-mediated DNA damage directly accounts for intramacrophage survival defects of the glycosylase mutants. The disparity between the severe intramacrophage defects and moderate virulence attenuation was also addressed; pre-exposure of Dxth/nfo to mild acid stress, recapitulating a key phagosomal condition, fully restores intramacrophage survival. Furthermore, abrogating SPI2 function in Dxth/nfo results in survival defects much greater than observed for SPI2 or Dxth/nfo mutants alone. Collectively, the data suggests that BER is critical for initial interactions within macrophage, while SPI2 or other oxidant defenses, subsequently limits exposure to DNA-damaging oxidants.

142(A)

DIFFERENTIAL PROTEOME ANALYSIS OF SALMONELLA TYPHIMURIUM SL1344 USING 2D-DIGE TECHNOLOGY

K. A. Sonck, S. De Keersmaecker, G. Schoofs, J. Vanderleyden, I. Nagy; Katholieke Universtiteit Leuven, Heverlee, BELGIUM. In response to various signals, bacteria adapt their behavior. High-throughput analysis methods offer the advantage that these changes can be studied at a global molecular level. Such techniques should reveal as much qualitative and quantitative information as possible. Micro-arrays (transcriptomics) and 2D-gelelectrophoresis (proteomics) are two high-throughput techniques that are complementary. Changes on transcriptional level are not necessarily always reflected on translational level. Moreover, for the study of proteins, not only information about expression patterns is interesting, also post-translational modifications, like phosphorylations (phosphoproteomics), are essential components of a regulatory network. Many signal transduction cascades use phosphorylated intermediates to pass on information to the cell. Proteomics is a research domain that has evolved greatly over the last decade, in which the emphasis moves towards quantifying differences in protein expression between multiple samples. A more recently developed proteomic analysis tool is the 2D-DIGE technology (Amersham Biosciences). Two-dimensional differencein-gel-electrophoresis is based on the labeling of protein samples with Cy-dyes. Due to this labeling and the specific ASM Conference on Salmonella

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excitation and emission spectra of the dyes, more than one protein sample can be run on a single 2D-gel. This decreases the number of gels that have to be run, which can be important in large-scale experiments. 2D-DIGE also uses a pooled internal standard that is applied in every gel. Therefore, this standard can be used to distinguish between experimental variation and biological variation. Statistical relevant data can be obtained on biological variation between protein samples. To optimize and adapt this technology to our needs, Salmonella typhimurium SL1344 was grown in two different conditions: oxic growth, and growth conditions that mimic the intestinal environment (micro-oxic, high osmolarity, neutral pH). Protein samples were prepared and respectively labeled with Cy3 and Cy5 (Amersham Biosciences). A pooled internal standard was labeled with Cy2. After 2D-gelelectrophoresis, the gels were scanned and analyzed for differential protein expression, using deCyderTM software (Amersham Biosciences). Results of this analysis will be presented. of a host. Many of the genes required for Salmonella invasion are encoded on Salmonella pathogenicity island 1 (SPI1). The invasive phenotype varies greatly in response to growth under different environmental conditions (e.g. osmolarity, oxygen tension, pH). An intricate regulatory network is responsible for transmitting the environmental signals into appropriate gene expression. HilA, a member of the ToxR/ OmpR-like family of transcriptional regulatory proteins, is a major player in this network. Its expression is dependent upon several environmental and bacterial factors. HilA on its turn activates the transcription of genes encoding components of the SPI1 type III secretion machinery and of InvF, a regulatory protein located downstream in the HilA dependent regulatory cascade. By analysing a set of preliminary microarray experiments, we discovered additional information on the Salmonella regulatory pathway for invasion of epithelial cells. We applied a dedicated glass microarray, containing 384 Salmonella spp. genes. We performed microarray experiments in 7 different conditions. Cluster analysis revealed an interesting cluster, enriched in genes that were functionally related to the Salmonella invasion pathway. This cluster of coexpressed genes was subjected to motif detection using Gibbs Sampling (Motif Sampler). We retrieved a motif previously in literature reported as the HilA box, in the promoter region of prgH and invF (already published) and two other genes, not yet reported. To univocally demonstrate the biological validity of the novel HilA targets, reporter expression studies of wild type and site directed mutated promoter fragments were performed. For both promoter fusions, the point-mutated base pair, predicted by in silico motif detection, has an important role for the expression of these genes. Future work involving DNA binding studies should shed light on the authenticity of a HilA box in these promoters and concomitantly on its contribution to the transcriptional regulation network that controls gene expression during invasion of Salmonella. Our results allowed some reflection on the design of the HilA transcriptional network.

143(B)

SPI-2 IS UPREGULATED IN RESPONSE TO CATION STARVATION

C. Kim, S. Falkow; Stanford University, Stanford, CA. We observed during studies of iron regulation in Salmonella that SPI-2 mRNA abundance increases upon treatment with 2-2' dipyridyl (DP), a cation chelator with relatively high specificity for iron.Eight of the top 256 genes upregulated by DP were SPI‑2-related genes, and a lexical analysis determined that the SPI-2 genes were significantly overrepresented in the upregulated dataset (p < 0.0004). We confirmed promoter activation of SPI-2 upon DP treatment using SPI-2 GFP reporter fusions (ssrA and ssaG) and observed that the response was dose-dependent and envZ-dependent. Pairwise comparison of induction activity of a panel of chelators was used to generate an ion specificity profile for SPI-2 induction, with Ca(II), Mg(II), Co(II), Mn(II), and Ni(II) being the strongest candidates. These results indicate that SPI-2 is regulated by divalent cations through EnvZ.

145(A)

SEROTYPES OFSALMONELLA STRAINS ISOLATED AT THE INSTITUTE FOR INFECTIOUS AND TROPICAL DISEASES IN BELGRADE

M. Jovanovic; Institute for Infectious and Tropical diseases, Belgrade, YUGOSLAVIA. The number of Salmonella strains at the Institute for Infectious and Tropical Diseases in Belgrade over the 35 year period, from 1968-2002 has been investigated. Salmonellae were isolated from stool and urine, but also from blood, CSF, pus and sputa of our patients. The total number of Salmonella per year was variable, but the number of Salmonella enteritidis compared to the total number of the isolates steadily increased, from 9.40% in 1972 to 94,84% in 2002 (linear trend of growth, p<0.01). During the last 15 years, it was the most prevalent strain at our Institute. Salmonella wien occured in 1973, was the most prevalent in 1974 and 1975 (55.65% and 73.19% respectively), but almost disappeared until 1983. Salmonella typhi-murium and

144(C)

CLUSTER ANALYSIS AND MOTIF DETECTION REVEAL POSSIBLE NEW TARGETS OF THE HILA REGULATORY PROTEIN IN SALMONELLA TYPHIMURIUM

S. C. De Keersmaecker 1, K. Marchal2, T. L. Verhoeven1, C. S. Detweiler3, S. Falkow3, J. Vanderleyden1; 1Centre of Microbial and Plant Genetics, University of Leuven, Heverlee, BELGIUM, 2 ESAT SISTA-SCD, University of Leuven, Heverlee, BELGIUM, 3Dep. of Microbiology and Immunology, Stanford University School of Medicine, Palo Alto, CA. The intestinal Gram-negative pathogen Salmonella enterica serovar Typhimurium (S. typhimurium) is a major cause of food-borne diseases worldwide. This gastrointestinal disease is initiated by S. typhimurium crossing the intestinal mucosa Pathogenesis, Epidemiology, and Vaccine Development

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Salmonella infantis were the most prevalent, after Samonella enteritidis, in the last 10 years. A few strains (1-3) of Salmonella virchow or Salmonella bovis morbificans were isolated per year in the last decade. The number of Salmonella varies from year to year, but the number of Salmonella enteritidis increases in the last 15 years. The number of the highly virulent strain, Salmonella wien, although predominant in 1974 and 1975, was probably decreased due to vigorous therapy of the diseases it caused.

147(B)

MULTIDRUG RESISTANCE IN SALMONELLA SPP. IN ZIMBABWE

G. Falchi1, N. A. Canu1, S. Uzzau1, O. Chitsatzo 2, L. Gwanzura2, C. Pietro 1, S. Rubino1; 1Dip Scienze Biomediche, University of Sassari, Sassari, ITALY, 2University of Zimbabwe, Harare, ZIMBABWE. Nontyphoidal salmonellae cause 1 to 5% of gastroenteritis in developing countries, representing the most frequently isolated Gram-negative bacterial pathogen in the African continent. The majority of these infections are not recorded by public health institutions. Among sub-equatorial African country, Zimbabwe is one of the most developed and it is regarded as a model for hospitalization and treatment point by neighboring nations. However, diarrheal diseases represent a major public health problem also in this country. Drug therapy is heavily used to control enteritis, even against diseases that do not require antibiotic treatment because of their self-limiting nature. This approach, and the use of antimicrobials without medical prescription, favor the development of multidrug-resistant bacteria. In addition, a massive use of antibiotics occurs among HIV-positive patients with enteritis, also to prevent collateral diseases. The high rate of HIV infections in Zimbabwe (estimated of being responsible of about 160,000 deaths for AIDS among adults and children in 1999 in a total population of 11,529,000), contributed to the remarkable development and spread of Salmonella (and other enteric pathogens) strains resistant to multiple drugs, including first-line antibiotics such as chloramphenicol, ampicillin and gentamicin. In this study we have analyzed a collection of Salmonella strains, isolated in Zimbabwe by the Zimbabwean National Reference Center for Enteropathogens, created in Zimbabwe thanks to the aid of the international cooperation. Among the most common serotypes Typhimurium and Enteritidis, we also found the uncommon serotype Decatur, firstly identified in Zimbabwean laboratories as generic Group C Salmonella or S. enterica serotype Choleraesuis. Interestingly, while the most part of the strains belonging to serotype Enteritidis are sensitive to all the antimicrobials tested, serotypes Decatur and Typhimurium presented a remarkable multidrug resistance. Furthermore, molecular techniques like ribotyping, IS200 fingerprinting and plasmid profile analysis were performed to identify a possible common clonal line among strains belonging to the same serotype.

146(B)

IN VITRO EXPRESSION OF STC, STB, STF AND PEF FIMBRIAL OPERONS OF SALMONELLA ENTERICA SEROTYPE TYPHIMURIUM

C. W. Dorsey1, M. Raffatellu1, A. D. Humphries 1, E. H. Weening1, R. Droleskey2, R. A. Kingsley1, A. J. Bäumler1; 1Texas A&M HSC, College Station, TX, 2USDA, College Station, TX. Sequence analysis of the Salmonella enterica serotype Typhimurium genome has identified 13 putative fimbrial operons called agf, fim, pef, lpf, bcf, saf, stb, stc, std, stf, sth, sti, and stj. However, only expression of type 1 fimbriae (encoded by fim) can be detected when S. Typhimurium is cultured in static LB broth. To further characterize putative fimbriae in vitro and to initiate functional studies we have begun cloning S. Typhimurium putative fimbrial operons and expressing these in Escherichia coli as a heterologous host. The stc operon was cloned and expressed in a non-fimbriated E. coli strain (ORN172, Dfim E. coli) from the T7 promoter. Expression of StcA was detected by flow cytometry and western blot. No fimbrial filaments were observed by transmission electron microscopy on the surface of an E. coli expressing the stc operon but expression of StcA on the bacterial surface could be detected by immunogold labeling. The stb and stf operons were cloned under control of the PBAD promoter and expressed in E. coli ORN172. Expression of StbA and StfA were detected by western blot and flow cytometry. Introduction of the pef operon into ORN172 resulted in expression of fimbriae that reacted with anti-PefA serum as shown by immuno-gold labeling. A single protein band of approximately 17kDa was detected in fimbrial preparations and identified as PefA by western blot. These results demonstrate that stc, stb, stf and pef operons can be expressed in vitro in E. coli ORN172 therefore facilitating fimbriae purification for future functional studies.

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148(A)

ANTIBIOTIC RESISTANCE AND CHARACTERIZATION OF CLASS I INTEGRONS IN SALMONELLA ENTERICA SEROVAR NEWPORT ISOLATED FROM ANIMALS AND HUMANS IN MINNESOTA

V. C. Lopes, A. M. Kraska, B. T. Velayudhan, D. A. Halvorson, K. V. Nagaraja; University of Minnesota, Saint Paul, MN. There is increasing concern on the development of antimicrobial resistance in zoonotic microorganisms due to its relevance to public health. Recently, multidrug-resistant strains of Salmonella serovar Newport have been reported in the United States, highlighting the emergence of resistance to multiple antibiotics in this organism. The incidence of drug-resistance in organisms and the identification of factors, that promote development of drug resistance, are critical components of a control program. In this study, strains of S. Newport isolated from humans, poultry, bovine and swine in Minnesota were tested for antibiotic susceptibility (ampicillin, ceftiofur, apramycin, chlortetracycline, clindamycin, enrofloxacin, erythromycin, florfenicol, gentamicin, penicillin, neomycin, sulphathiazole, oxytetracycline, tiamulin, sulphadimethoxine, trimethoprin/ sulphamethoxazole and tylosin). Results illustrate resistance of S. Newport to a greater number of antibiotics in bovine and human strains. Screening for the presence of class I integrons, which are natural mobile genetic elements that have been found to contribute to the spread of resistance, showed them to be present in most of the strains, thus demonstrating their possible role in the development of resistance. In addition, antibiotic resistant genes for tetracycline, chloramphenicol, streptomycin and quinolones were tested by PCR.

late endosomes/lysosomes. SifA, an effector translocated by the SPI2 TTSS, is central in the formation of SIFs. It has been shown that sifA- strains do not form SIFs and also do not maintain the vacuolar membrane surrounding intracellular Salmonella. It has further been shown that uninfected cells transfected with a SifA expressing plasmid possess aggregated late endocytic compartments and structures resembling SIFs. These observations indicate that SifA exerts its effect by altering membrane traffic within host cells. The goal of this study is to identify the regions of SifA which are important for various aspects of its function, such as targeting to the SPI2-encoded TTSS and the ability to form SIFs. To achieve this, 16 internal deletions have been constructed in sifA for expression from plasmids. This set of deletions collectively spans the majority of sifA and may allow for the identification of regions which contribute to various characteristics of SifA function. These constructs have been assessed for their ability to complement a strain harbouring a null chromosomal sifA allele in a number of functional assays in comparison to the parental wild type sifA-expressing construct. These assays include secretion, localisation within host cells and the ability to aggregate late endosomes.

150(C)

A FUNCTIONAL SCREEN TO IDENTIFY TYPE III SECRETED PROTEINS IN SALMONELLA TYPHIMURIUM

J. K. Geddes; Oregon Health and Sciences University, Portland, OR. Many parasitic organisms must secrete proteins into host cells in order to manipulate the host cell environment to promote survival. Gram-negative bacterial pathogens employ the use of type three secretion systems (TTSS) to inject proteins into host cells to promote invasion and replication. Although TTSS have been easily identified by sequence homology, the identification of secreted effector proteins has proven difficult. Here we report a Tn5 based transposon screen using the cycler transposon, which we applied to identify proteins secreted by Salmonella typhimurium during infection of the J774 macrophage-like cell line. This transposon allows for relatively easy identification of secreted proteins by measuring cyclic AMP (cAMP) levels in infected cells using a commercially available ELISA kit. We demonstrated the efficacy of this system by isolating the two previously identified type three effectors: slrP, and sptP, as well as several novel effectors. The cylcer transposon can be introduced either in vitro to purified plasmid DNA or in vivo to competent bacteria. The versatility of this system makes it an ideal tool to identify secreted proteins in a broad range of parasitic organisms.

149(B)

STRUCTURE-FUNCTION ANALYSIS OF SIFA, AN EFFECTOR TRANSLOCATED BY THE SPI2-ENCODED TYPE III SECRETION SYSTEM

N. F. Brown, B. K. Coombes, J. H. Brumell, B. Finlay; University of British Columbia, Vancouver, BC, CANADA. Salmonella pathogenicity island 2 (SPI2) is a locus, positioned at centisome 31 of the Salmonella chromosome. It encodes a type III secretion system (TTSS) which is involved in the systemic phase of pathogenesis in the Salmonella Typhimurium murine model of typhoid fever. Expression of the SPI2-encoded TTSS is induced when Salmonella are present within their membrane-bound compartment inside host cells. The SPI2 encoded TTSS translocates a number of effector proteins across the vacuolar membrane into the host cell cytosol where they alter host cell membrane and protein traffic in order to establish an intracellular niche which supports Salmonella replication. One phenomenon mediated by SPI2 which is observed in epithelial cell lines is the formation of Salmonella-induced filaments (SIFs), a novel tubular endocytic compartment which contains markers of Pathogenesis, Epidemiology, and Vaccine Development

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151(A)

MUTATIONS IN THE LPS OF SALMONELLA ENTERICA SEROVAR TYPHIMURIUM DT104 DISRUPT FORMATION OF THE RUGOSE PHENOTYPE

Y. A. Anriany, K. R. Wessells, S. W. Joseph; University of Maryland College Park, College Park, MD. The rugose (wrinkled) colony phenotype in Salmonella enterica serovar Typhimurium DT104 Rv has been associated with cell aggregation and ability to form biofilms, which are important for the survival of the cells in the environment. We are investigating particular factors that could contribute to the expression of the rugose, which is due to the production of a matrix composed of curli and cellulose. Random mutagenesis was performed and two Tn5 insertion mutants of Rv were selected because of the reduced expression of rugose. Single primer RATE PCR protocol and subsequent sequencing reaction were employed to analyze the insertion sites. The insertions were located at ddhC and waaG, which code for CDP-6deoxy-D-xylo-4hexulose-3-dehydrase and glucosyltransferase I, respectively, both of which are involved in the synthesis of LPS. Both mutants produced low molecular weight LPS and resistance to P22 phage, while only waaG mutant exhibited susceptibility to novobiocin. The reduced rugose expression by the two mutants was associated with reduced binding to Congo red and altered cell adherence to polystyrene surfaces. Examination by SEM revealed the presence of reduced amounts of extracellular matrix as compared to the parent Rv strain. The reduction in the matrix was due, at least in part, to decreased levels of curli production, as judged by the results of Western blots probed with anti-SEF17 (curli) antibodies. Although the production of curli and the associated expression of rugose (also called rdar) in Salmonella has been extensively described, the results from this study suggest that, in some manner, LPS may also have a role.

findings frequently seen in paediatric population were splenomegaly 57%, hepatomegaly 63% and anaemia 44%. Complications were more frequently seen in Paediatric population, hepatitis 25%, neurological manifestations 13.2%, bronchopneumonia 14.7%, bleeding disorder 20.6%, hypotension 11.8%, ascitis 7.4% and intestinal perforation 4.4%. Case fatality rate was 4.5%.Although there were only two isolates with resistance to Ciprofloxacin, number of strains resistant to Nalidixic acid increased from 59.3% in 2001 to 73% in 2002 and 90.4% in 2003. Incidence of resistance to Co-trimoxazole, Chloramphenicol and Ampicillin (MDR strains) was 43%. All isolates were uniformly sensitive to 3rd generation cephalosporins. All strains were negative for Extended Spectrum Beta Lactamases (ESBL). Detection of S.typhi in young children and emerging resistance trends call for development of new vaccines to target infants and young children. Treatment protocols need to be further reviewed.

153(C)

DEVELOPMENT OF A PROMOTERTRAP, BACTERIAL EXPRESSION SYSTEM TO IDENTIFY SALMONELLA TYPHIMURIUM DT104 GENES THAT ARE INDUCED SPECIFICALLY DURING INVASION INTO INT407 CELLS

C. P. Giri1, R. B. Raybourne2, R. Thomas1, J. McDaniel 3, D. J. Kopecko3; 1FDA-CFSAN, College Park, MD, 2FDA-CFSAN, Beltsville, MD, 3FDA-CBER, Bethesda, MD. Multi-antibiotic resistant S. typhimurium DT104 strains have gained recent prominence in U.S. foodborne infections, raising the possibility of novel DT104 virulence attributes. The aim of the present study was to identify DT104 genes specifically induced during infection of human intestinal INT407 cells. Sau3A1-restricted fragments (0.4-1.6 kbp) representing the entire DT104 chromosome were ligated into a promoter-trap, bacterial expression vector containing a BamH1 cloning site upstream from GFPmut3.1/CAT reporter genes in tandem orientation. Recombinant plasmids were electroporated into an Amps DT104 strain and the resultant library of ~40,000 AmpR DT104 recombinants were used to infect INT407 cells. Following a 1-3 hr infection period, any remaining extracellular bacteria were killed by the addition of gentamicin and those intracellular bacteria expressing the CAT reporter gene were selected by incubation with chloramphenicol(Cm) for 16 hrs at 37 C. CmR and GFP+ bacterial clones that were induced intracellularly were additionally enriched by FACS analyses. To eliminate constitutively expressed promoters, these GFP+ bacteria were subsequently grown in BHI broth(~16 hrs) and nonfluorescent(GFP-) bacteria were sorted to enrich for promoter clones active only inside host cells. This protocol was repeated serially 3 times. All of 100 clones randomly picked from the enriched library were found to express the reporter genes intracellularly, but not during growth in broth. Two-thirds of these clones exhibited 6-15 fold intracellular induction while the remaining one-third showed 16-100 fold induction in host cells. Ongoing DNA sequence analyses of the inserts have revealed 5 separate promoters ASM Conference on Salmonella

152(B)

EMERGING TRENDS OF TYPHOID FEVER - IN INDIA

R. Gaind, M. Walia, P. Paul, R. Mehta, P. Aggarwal; Vardhman Mahavir Medical College and Aasoc Safdarjang Hospital, Delhi, INDIA. Retrospective analysis of patients with bacteriologically confirmed Typhoid fever attending tertiary care centre was conducted from April 2001 ­ May 2003. In a total of 279 patients, Salmonella typhi was isolated in 84.4% followed by Paratyphi A in 18.6%. Isolation of S.Typhi was highest in monsoon and summer seasons accounting for 73% of infections. Children under 12 yrs were more frequently infected (71.3%). Thirty one percent of the paediatric infections occurred in children 0-5Yrs. The multisystem nature of the disease caused delayed diagnosis in 11.5% cases. Twenty nine percent of the adult isolates were Paratyphi A as compared to 14.6% in Paediatric group. The common clinical presentation included fever 100%, abdominal pain 38%, diarrhoea 19%, and vomiting 38%. The clinical

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that are expressed only intracellularly at high levels. This double-selection, promoter-trap system has proven to be a powerful tool for identifying intracellularly-induced genes, which should lead to the identification of novel virulence attributes.

155(B)

USE OF ASD-BALANCED LETHAL HOST-VECTOR SYSTEM TO EXPRESS SHIGELLA SONNEI FORM 1 POLYSACCHARIDE IN A SALMONELLA VACCINE STRAIN

D. Xu, D. J. Kopecko; FDA-CBER, Bethesda, MD. The use of attenuated mutants of Salmonella as vaccine carrier strains implies the introduction of foreign genes, usually on recombinant plasmids, into the vector bacterium. These plasmids generally contain an antibiotic resistance gene in order to keep selective pressure in favor of plasmid retention. For this study, we used the previously reported asd-deletion mutant of Salmonella strain SL7201 and the asd+ plasmid pYA292 (Galan, J.E.et al.,Gene 1990,94:29-35) to express the S. sonnei form 1 polysaccharide antigen. Plasmid pYA292 contains a functional asd gene encoding aspartate b­semialdehyde dehydrogenase which is a component of a balanced lethal host-vector system. In this system, the asd gene from S. typhimurium, a non-drug resistance selectable marker, is present on the plasmid vector and complements an asd deletion present in the host bacterial chromosome. Since absence of the recombinant plasmid is lethal for the cells grown without diaminopimelic acid (DAP), the cloned genes that code for the heterologous antigen are stably maintained both in vitro and in vivo. The use of this system eliminates the need for antibiotics to stabilize the expression-cloning vector. In this study, the gene cluster involved in synthesis of the S. sonnei form 1 antigen was cloned into vector pYA292. Genetic analysis demonstrated that the resulting construct pXK51 contains the necessary sequences to assemble S. sonnei form 1 polysaccharide antigen in the Salmonella typhimurium asddeleted strain. Western immunoblot of polysaccharide obtained from cells containing pXK51 showed a similar profile compared with the recombinant plasmid pXK65(Xu DQ, et al., Infect Immun. 2002,70:4414-23)carrying the same O antigen sequence but tested in S. typhi Ty21a. Our data demonstrate the stable expression of a polysaccharide antigen from an asd+ plasmid vector in its cognate host strain, which together may serve as a model Salmonellavectored vaccine.

154(A)

CONSTRUCTION OF A LIVE, ATTENUATED SALMONELLA TYPHI TY21A VECTOR EXPRESSING ANTHRAX PROTECTIVE ANTIGEN

S. P. Bhattacharyya 1, M. Osorio1, M. Bray1, S. Leppla2, R. Walker 1, D. Kopecko 1; 1CBER/FDA, Bethesda, MD, 2NIAID/ NIH, Bethesda, MD. Bacillus anthracis is a gram-positive spore-forming bacterium that causes the disease anthrax. The disease is typically initiated by introduction of Bacillus anthracis endospores into the body and protection is afforded by an antibody response against the protective antigen (PA) component of anthrax toxin. Recent terrorist activities in Washington DC involving the mailing of anthrax spores have now shown that anthrax can serve as an effective bio-terrorist weapon. The only FDA-approved anthrax vaccine is toxoid based, requires multiple doses to stimulate immunity, causes moderate adverse effects Clearly, there is a need for new anthrax vaccine approaches. We have chosen to test the feasibility of using an attenuated Salmonella typhi strain expressing PA to induce an immune response to PA and thus protect against anthrax exposure. The advantages of live attenuated vaccines are: (i) limited multiplication in vivo (aborted infection), thereby inducing an immune response resembling that seen after natural infection; (ii) the need for only one or a few doses to confer long-lasting immunity against certain diseases; (iii) the ability of orally-administered live, attenuated carrier strains such as Salmonella typhi to stimulate both humoral and cellular arms of the immune response; and (iv) self-administration of vaccine, eliminating the need/expense of administration by skilled health care personnel. In these studies, the anthrax PA was cloned behind an inducible promoter in a genetically stable low copy plasmid vector. The live attenuated Salmonella vector containing the PA plasmid construct expresses PA intracellularly and under condition of oxygen limitation. The anthrax PA is expressed at high levels in this system as demonstrated by western blot analysis using a polyclonal anti-PA antibody. Preliminary mouse studies have shown PA-specific immune stimulation. Currently dosing schedules and administration methods are being optimized. Given the safety record of attenuated salmonella vaccine vectors in humans, a vaccine construct that induces a strong response to PA could form the basis of a safe and effective vaccine against anthrax.

156(C)

STRENGTHENING LABORATORYBASED SURVEILLANCE

A. Aidara-Kane 1, B.C. Imhoff2, P. Braam1, H.C. Wegener 3, M. Jouan 4, Jaap Wagenaar5, A. Ellis1, Danilo Lo Fo Wong3, F. J. Angulo2, and WHO Global Salm-Surv; 1 World Health Organization, Geneva, Switzerland, 2 Centers for Disease Control and Prevention, Atlanta, GA, 3 Danish Veterinary Institute, Copenhagen, Denmark; 4 Institut Pasteur, Paris, France; 5 Animal Sciences Group, Netherlands Background: World Health Organization (WHO) Global Salm-Surv is a global network of national and regional public health, veterinary, and food reference laboratories and individuals involved in Salmonella surveillance. WHO Global

Pathogenesis, Epidemiology, and Vaccine Development

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Salm-Surv strives to enhance the capacity and quality of Salmonella isolation, identification, serotyping, and antimicrobial susceptibility testing throughout the world and to support local interventions that reduce the human health burden of Salmonella and other foodborne diseases. Initiated in January 2000, WHO Global Salm-Surv is coordinated by a Steering Committee including WHO Headquarters (Geneva, Switzerland), the Centers for Disease Control and Prevention (Atlanta, Georgia, U.S.), Institut Pasteur (Paris, France), Health Canada (Ottawa, Canada), the Animal Sciences Group (The Netherlands) and the Danish Veterinary Institute (Copenhagen, Denmark). Methods: WHO Global Salm-Surv program elements include: regional training courses, a moderated electronic discussion group, an external quality assurance system (EQAS), a country databank containing annual Salmonella surveillance summaries, a web site, and reference testing services. Results: WHO Global Salm-Surv currently has 654 general members and 127 participating member institutions from 138 countries. Among the member institutions, 38% test non-human specimens, 45% test human specimens, and 17% test both human and non-human specimens. WHO Global Salm-Surv members live in the following regions: Africa (6%), the Eastern Mediterranean (10%), Europe (33%), the Americas (27%), South-East Asia (9%), and the Western Pacific (15%). WHO Global Salm-Surv Regional Centers have been established in Asia (Thailand) and South America (Argentina). Between January 2000 and July 2003 we conducted 18 regional training courses, resulting in training more than 160 microbiologists on Salmonella isolation, identification, serotyping and antimicrobial susceptibility testing methods. WHO Global Salm-Surv sent 110 electronic discussion group messages between 2000 and 2002 and thus far, has conducted 3 cycles of EQAS (2000, 2001, 2002). A web-based country databank for Salmonella surveillance summaries has been created. These data and other information, including the WHO Global Salm-Surv Strategic Plan and laboratory training protocols, is available on the WHO Global Salm-Surv web site at www.who.int/salmsurv . Conclusion: Future WHO Global Salm-Surv activities include regional training courses in Western Europe and Eastern Africa, and continuing training activities in Argentina, Cameroon, China, Mexico, Poland, Russia, Thailand, Trinidad and Tobago, and the Eastern Mediterranean. Through these and other activities, WHO Global Salm-Surv continues to strengthen the capacity of local, national and regional laboratories, with the ultimate goal of reducing the global burden of Salmonella and other foodborne diseases and contributing to the global effort of the containment of antimicrobial resistance in foodborne pathogens.

157(A)

AUTOMATED RIBOPRINTING: A TOOL FOR MOLECULAR SEROTYPING OF NON-TYPABLE SALMONELLA STRAINS

J. Sonne-Hansen, V. Fussing; The Danish Salmonella Centre, Statens Serum Institut, Copenhagen, DENMARK. Background: Traditional serotyping by slide agglutination is the golden standard for characterization and monitoring of Salmonella. The method is easy to perform; cheap and reliable when specific antisera are available. Some strains are nontypable, when the O-antigens are lost or H- antigens are not expressed. Non-typable strains are rarely obtained from human stool-, urine or blood-specimens, while they have more frequently been isolated from production environments such as slaughterhouses. Objectives:The purpose of this study was to evaluate the automated ribotyping by a RiboPrinter, as a tool for molecular serotyping of "non-typable strains". Methods: 50 non-typable strains were ribotyped by use of the restriction enzyme pvuII. The strains were further analysed by serogroup PCR, amplification of fljB, fljB H1complex multiplex PCR and sequence analysis of fliC. Results 38 strains, representing five different serotypes could be identified using the RiboPrinter: 24 strains were identified as S. Typhimurium, six as S. Derby, four as S. Infantis and three strains as S. Enteritidis. RiboPrint identification of serovars of seven strains could not be confirmed by PCR. Five strains were not recognized by the RiboPrinter, but by visual interpretation of the banding patterns two of these strains could be identified. Conclusion: The results indicate that ribotyping with pvuII is a promising tool for identification and characterisation of "non-typable" Salmonella strains, but an enlargement of the database with more strains and serotypes are needed.

158(B)

SALMONELLA ENTERICA SEROVAR TYPHIMURIUM PRODUCES A TYPE IV PILUS THAT INFLUENCES INTESTINAL COLONISATION

E. Morgan1, J. D. Campbell1, D. J. Maskell 2, T. S. Wallis1; 1 Institute for Animal Health, Berkshire, UNITED KINGDOM, 2 University of Cambridge, Cambridge UNITED KINGDOM. Type IV pili are filamentous appendages produced by several gram-negative pathogenic bacteria. Recently a type IV pilus was shown to be encoded by Salmonella Pathogenicity Island7 of serovar Typhi and influences invasion of human epithelial cells. The sequence of serovar Typhimurium LT2 suggests that this serovar does not carry SPI-7. During a screen of signature-tagged mutants of serovar Typhimurium 4/74 to identify genes influencing colonisation of bovine intestinal mucosa, we identified a mutant with a Tn5 insertion in a gene almost identical to the pilO gene of serovar Typhimurium plasmid R64. Analysis showed that a pil operon is carried on an R64-related plasmid in serovar Typhimurium 4/74. The pil operon of R64 encodes a type IV pilus which was previously thought to be required only for attachment of donor to recipient bacteria during liquid ASM Conference on Salmonella

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conjugation. The pilO mutant was attenuated following oral infection of ityS mice, but virulent following intra-peritoneal infection of ityR mice. A calf ileal loop model, however, showed that the pilO mutant elicited an intestinal secretory and inflammatory response identical to that of the wild-type strain. These results suggest that the type IV pilus of serovar Typhimurium is required during colonisation of the gastrointestinal tract but does not contribute to Salmonella-induced enteropathogenesis or systemic pathogenesis.

160(A)

SALMONELLA TYPHIMURIUM INVADES MULTIPLE EPITHELIAL CELL TYPES IN PIG JEJUNUM

K. Schauser1, J. Olsen2, L.-I. Larsson1; 1 Department of Anatomy and Physiology, 2 Department of Veterinary Microbiology, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark. Infection of swine with Salmonella Typhimurium represents a serious problem. However, most studies on Salmonella infection have been carried out on other species. We have studied Salmonella infection in pigs aged 8-10 weeks, i.e. the time when Salmonella enteritica serotype Typhimurium infection commonly occurs in this species. The infection process was followed using immunocytochemistry for Salmonella and for cytokeratin-18 (a proposed M cell marker) as well as by HE staining. Although cytokeratin-18 has been proposed to be a specific M cell marker, several other cell types, including brush and goblet cells as well as enteroendocrine cells stained for this marker. Importantly, Salmonella invasion was observed both in cytokeratin-18 positive cells and in cytokeratin-18 negative cylindrical absorptive cells. Invasion of cytokeratin-18 positive, tentative M cells as well as of cytokeratin-18 negative cylindrical absorptive cells situated in the follicle-associated epithelium of the dome were observed already after 5-10 min in the oral and somewhat later in the caudal jejunum. Additionally, invasion of the subepithelial compartment was evident. Invasion of epithelial cells situated on ordinary villi occurred slightly later (5 min) in a characteristic patchy pattern and appeared first in the oral jejunal loops. In general, invasion was always most marked and occurred earliest in the oral jejunal loops. These results document that in the present pig model Salmonella infection occurs more rapidly and is more severe in the oral than in the caudal jejunum and that both cytokeratin-18 negative and positive cells are invaded.

159(C)

IDENTIFICATION OF SALMONELLASPECIFIC SEQUENCES REQUIRED FOR COLONISATION OF CATTLE AND CHICKENS BY SIGNATURE-TAGGED MUTAGENESIS

E. Morgan 1, J. D. Campbell1, K. L. Page 1, P. A. Barrow 1, D. J. Maskell2, T. S. Wallis1; 1Institute for Animal Health, Berkshire, UNITED KINGDOM, 2University of Cambridge, Cambridge UNITED KINGDOM. Salmonella enterica serovar Typhimurium infects a broad range of animals. The nature and severity of such infections varies depending on the host species. Numerous virulence genes have been identified in serovar Typhimurium, largely from experimental infection studies in mice. However the roles of such genes in the infection of other animal species remains unclear. We have performed a study using 1,045 signaturetagged transposon mutants of serovar Typhimurium strain 4/74 and identified 75 mutants which were deficient in their ability to colonise calf intestinal mucosa, 61 different mutants which could not colonise chick caecae and 52 mutants which could not colonise the intestines of either animal species. The transposon insertion sites were determined for all but four of the 188 mutants, and 144 chromosomal and 5 plasmid genes were found to have been mutated. Of the chromosomally-encoded genes, 42 had orthologues in Escherichia coli K-12 and most were deemed to be required for "housekeeping", 40 were in the previously identified Salmonella pathogenicity islands SPI-1 to SPI-5, 9 genes were in the rfa or rfb LPS operons and 5 were in integrated phages. The remaining 48 genes were mapped to 34 islands or prophages, ranging in size from insertion of a single gene, to acquisition of regions encoding up to 37 genes. 24 of these islands were not present in E. coli K-12, and 1 island appears to have been lost from serovar Typhi since the divergence of S. enterica from E. coli. Three islands were not present in K-12 or in serovar Typhi; one of these islands encodes 27 genes, including a putative integrase, and is adjacent to the Fels-2 prophage. The other two regions appear to be insertions of 2 and 4 genes into the serovar Typhimurium chromosome. The remaining 6 islands are mosaic in structure due to various independent insertion/ deletion events. Although some of these islands carry metabolic genes which, when mutated, lead to a general virulence defect which affects colonisation of both calves and chicks, some islands appear to encode specific virulence factors which are required for colonisation of a single animal species. Further characterisation of these islands and functions of the encoded genes is underway. Pathogenesis, Epidemiology, and Vaccine Development

161(B)

PATHOLOGY OF SALMONELLOSIS AMONG OSTRICH CHICKS IN EGYPT

S. M. Aly; Suez Canal University, Ismailia, EGYPT. Ostrich (Struthio camelus) have been raised intensively to produce meat, hides, feathers and eggs. Ostrich farms started in Egypt since 1997. By 2000, Ostrich chicks and layers were reared in 55 farms with a total population of 4000. Mortality due to infectious diseases is greatest in the first six weeks of chick's life. Little is known not only about infectious diseases that occur in farmed Ostrich and also about the microorganisms in healthy birds. The bacterial infections are major health concern of Ostrich production and enteric infections by salmonella are most commonly seen in Ostrich chicks up to 4 month of age. The present article aimed to determine the prevalence of salmonellosis among ostrich chicks and their associated pathological lesions. The study was carried out on 861 Ostrich chicks collected from several farms in different localities of Egypt. The age of the chick in this study ranged from one day to 3 months old. Samples 89

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were subjected for bacteriological examination for the isolation and identification of salmonella species. Tissue specimens were collected from lungs, hearts, spleens, intestines, kidneys, livers and air sacs from dead Ostrich chicks and processed for histopathological examinations. Out of 861 ostrich chicks; 600 were died, 221 showed clinical signs of infection and 40 were apparently healthy. Bacteriological examinations were positive for 73 died chicks (12.2%), 122 diseased chicks (55.0%) and 15 of healthy one (37.5%). Out of these bacterial isolates; 36 were identified as salmonella sp. Including 22 isolates of S. dorgana (61%) and 14 isolates of S. typhimurium (38%).Salmonella were isolated from 22 ostrich (1day ­1week of age), 5 ostrich (1-2 weeks of age) and 1 ostrich (2 weeks ­ 3 months of age). Clinically infected chicks showed weakness and white mucoid diarrhea. The postmortem examination revealed hydroperitoneum with fibrinous pericarditis, air-sacculitis and ulcerative enteritis. Pneumonia was noticed and necrotic foci were seen in the liver surface. Histopathologically, the heart showed edema, congestion and mononuclear leukocytes in the pericardium. The myocardium was edematous and showed focal areas of hyaline degeneration. The intestine was congested with mucinous degeneration in their epithelium. In other cases, the intestine showed in the epithelial lining and the lumen contain epithelial debris and mononuclear leukocyte. The liver section showed congestion and focal areas of necrosis. The necrotic hepatocytes were either ruptured or lacked there nuclei and other hepatic cells showed vacuolar degeneration. The kidney revealed tubular nephrosis with marked edema, congestion, and hemorrhage. In some cases, the glomeruli were either atrophied or showed vacuolated endothelium with swollen Bowmen's capsule. We concluded that, salmonella was responsible for enteritis and mortality especially in the first week of life and S. dorgana was isolated for the first time from ostrich chicks in Egypt. system encoded by SPI4. SirA also represses flagellar gene expression. SirA orthologs in other genera are also required for virulence (gacA, varA, letA, uvrY, expA). We have purified SirA and the cytoplasmic portion of its sensor kinase BarA (BarA198). BarA198 autophosphorylates in the presence of ATP and transfers this phosphate to SirA. SirA-P alters the gel mobility of hilA, hilC, and csrB promoter DNA fragments, but not those of the hilD, invF, csrA, or flhD promoters.

163(A)

IDENTIFICATION OF A NEW LOCUS INVOLVED IN THE VIRULENCE OF SALMONELLA ENTERITIDIS

I. Virlogeux-Payant, M. Amy, D. Senocq, E. Bottreau, F. Mompart, P. Velge; INRA de Tours-Nouzilly, Nouzilly, FRANCE. Salmonella enteritidis continues to be a major cause of foodborne infections, with poultry being a major source of this infection in industrialized countries. An important virulence factor of this bacteria is its ability to gain access to host cells in humans as in poultry. Contact of Salmonella with cultured epithelial cells results in the transient assembly on the bacterial surface of structures termed invasomes. The assembly of these structures is dependent on an intact type III secretion system (TTSS) encoded by the Salmonella pathogenicity island 1 (SPI-1) that translocates effector proteins like the Salmonella invasion proteins (Sip). We have identified a new locus involved in S. enteritidis invasion. The nucleotide sequence of this chromosomal locus revealed four open reading frames (ORFs), yfgM, yfgL, engA and yfgJ all transcribed in the same orientation. A polar mutation in yfgL exhibited a reduced entry into human Caco2 enterocytes (p < 0.01). Examination of proteins secreted by this mutant, by microsequencing of these proteins and by western-blot using an anti-SipA or an anti-H: g,m antiserum, revealed that this strain secreted a reduced amount of the SipA and SipC effector proteins as well as decreased amounts of FliC, FliD and FlgL flagellar proteins. In agreement with this, the yfgL mutant was less motile than the wild type strain. All these defects could be overcome by inserting the yfgM, yfgL, engA and yfgJ ORFs into the mutant on a recombinant plasmid. In addition, the yfgL mutant colonized significantly less the ceca (p < 0.05) and the spleens (p < 0.01) of one-day old chicks at 2 and 5 days after oral inoculation. Taken together, these results suggest that the yfg-eng locus is involved in the virulence of S. enteritidis by regulating the secretion of proteins by the SPI-1 and the flagellar-encoded TTSS. Molecular mechanisms involved in this regulation are currently being investigated in our laboratory.

162(C)

DETECTION OF THE HOST ENVIRONMENT: SDIA AND SIRA

M. Teplitski, B. M. M. Ahmer; The sdiA and sirA genes are encoded adjacent to one another at 42 cs of the S. typhimurium chromosome. SdiA is a LuxR homolog that detects the N-acylhomoserine lactone (AHL) production of other bacterial species. In response, SdiA activates the rck operon on pSLT and the srgE gene (STM1554) at 33.6 cs. Rck is a small outer membrane protein that confers resistance to complement killing and adhesion to epithelial cells and extracellular matrix. Within the rck operon are two genes that affect Pef fimbriae expression, pefI and srgA. PefI is a regulator of the pef operon while srgA is a dsbA paralog that catalyzes disulfide bond formation within the PefA fimbrial subunit. SdiA is the first exclusively interspecies signaling system. Although it seems likely that interspecies microbial signaling will turn out to be common, there are significant technical challenges involved in their identification. SirA is a response regulator of the two component family that activates the Type III secretion system encoded by SPI1 and the Type I secretion

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ASM Conference on Salmonella

INDEX INDEX

Aabo, S. 68 Aarts, H. 119, 13, 21 Abbas, Z. 125 Abee, T. 13, 21 Abuhelfaia, A. 48 Adams, L. 23, 37 Adams, L. G. 134 Adeishvili, E. G. 98 Ahmer, B.M.M. 162 Aidara-Kane, A. 156 Aggarwal, P. 152, 24 Alam, K. 132 Almeida, I. C. 104 Aly, S.M. 161 Alyapkina, Y. S. 121 Ammendola, S. 130 Ammon, A. 58 Amy, M. 163 Andersen, J. S. 120 Andrews-Polymenis, H. 134 Andrews-Polymenis, H. L. 136 Andriopoulos, P. 110 Angulo, F.J. 156 Anriany, Y. A. 151 Are, B. 71 Arnold, C. 65 Attridge, S. R. 85 Azara, A. 71 B. Jensen, B. 88 Babic-Erceg, A. 42 Bacciu, D. 102, 89 Bader, M. W. 56 Baggesen, D. L. 120 Bakanidze, L. G. 98 Baker, S. 44 Ban, S. 91 Banda, H. T. 31 Banser, P. 81 Barchiesi, J. 118 Barrow, P. 141, 54 Barrow, P. A. 34, 43, 63, 159 Battistoni, A. 130 Bäumler, A. J. 134 Baumler, A. J. 136 Bäumler, A. J. 146 Baumler, A. J. 19, 23 Bäumler, A. J. 37 Beal, R. K. 34 Beeston, A. L. 66 Ben Aissa, R. 48 Ben Ali, M. 48 Bennett, A. M. 26 Berggren, I. 108 Berk, P. 13 Berk, P. A. 18 Bernardes, E. S. 104 Berndt, A. 59 Bertini, A. 122 Bessa, M. C. 72 Beuling, A. 21 Bhattacharyya, S. P. 154 Bhutta, Z. A. 125 Bishop, A. L. 44 Blanc-Potard, A. 64 Boisivon, A. 76 Bongaerts, R. J. 86 Bottreau, E. 163 Bossi, L. 128, 130 Boyen, F. 73 Bramm, P. 156 Brandão, L. G. 67 Brandelli, A. 79 Braoudaki, M. 61, 8 Bray, M. 154 Brenner Michael, G. 69 Breslin, M. 14, 15 Brocchi, M. 103, 104, 67 Brown, N. F. 149, 4 Brumell, J. H. 149, 4 Bueno, S. M. 17 Bumann, D. 100 Caccuri, R. 106 Cado Bessa, M. 69 Campbell, J. 131 Campbell, J.D. 158, 159 Canu, N. 72 Canu, N. A. 147 Carattoli, A. 122 Cardona-Castro, N. 27 Cardona-Castro, N. M. 94 Cardoso, M. 69, 72 Caron, E. R. 79 Cartolano, G. 75, 76 Casadesús, J. 49, 50 Castagna, S. 72 Cerquetti, M. C. 106 Chadfield, M. S. 68 Chakraborty, T. 97 Chang, C. 115 Chauhan, S. K. 36 Chen, T. R. 5 Chen, Z. 117 Chessa, D. 89 Chien, M. 117 Chitsatzo, O. 147 Chiu, T. H. 111, 113, 5 Cho, S. 91 Christensen, J. P. 68 Clifton-Hadley, F. A. 129 Collard, J. 73 Cook, N. 40 Cooles, S. W. 129 Coombes, B. K. 149 Cordone, A. 95 Correa-Ochoa, M. 27 D'Aoust, J. Y. 46 Damborsky, J. 70, 99 Danino, V. 116 Davies, R. H. 14, 15 De Buck, J. M. 20 De Felice, M. 95 de Jonge, R. 18 De Keersmaecker, S. 109, 142 De Keersmaecker, S. C. 144 De Moor, B. 109 Detweiler, C. S. 144 Devi, S. 9 Dias, A. V. 67 Digiannatale, E. 122 Dionisopoulou, M. 110 Dorman, C. J. 116 Dorsey, C. W. 146 Dougan, G. 44, 92 Droleskey, R. 146, 23 Ducatelle, R. 20, 60, 73, 74 Dufani, M. 48 Dulal, K. 135 Dutta, D. K. 132 Echeita, M. A. 133 Edgeworth, J. D. 33 Ehrbar, K. 96 Ellis, A. 156 Escalante-Semerena, J. C. 55 Espariz, M. 118 Falchi, G. 102, 147, 89 Faldynova, M. 99 Falkow, S. 101, 143, 144 Feng, X. 107 Fernandes, S. A. 103 Ferraz Castagna, S. M. 69 Fierer, J. 55 Figueiredo, J. 134, 23 Figueroa-Bossi, N. 128, 130 Finlay, B. 149 Finlay, B. B. 105, 4 Fokas, S. 110, 110 Folb, J. E. 33 Fookes, M. 44, 7 Fuchs, T. M. 2, 3 Fuentes, J. A. 17 Fussing, V. 157 Gaasbeek, E. J. 123 Gaind, R. 152, 24 Gallagher, M. P. 53 Gandhi, M. 16 Gao, S. 62 García Véscovi, E. 118 Garcia-Del Portillo, F. 83 Garmory, H. S. 26 Garza, L. R. 136 Geddes, J. K. 150 Geimba, M. P. 79 Gericke, B. 58 Ghenghesh, K. S. 48, 84 Giacomodonato, M. N. 106 Gibson, D. 81 Gibson, D. L. 139 Gilks, C. F. 31 Gintsburg, A. L. 121 Giri, C. P. 153 Godard, C. 60 Goh, Y. 10 Goldberg, M. 101, 116 Goldberg, M. D. 45 Gomes, D. J. 132 Gordon, M. A. 31, 52 Graham, S. M. 39, 41 Grant, G. 47 Gregorova, D. 124, 43 Griffin, G. E. 33 Griffin, K. F. 26 Guerra, B. 6 Gwanzura, L. 147 Hackett, J. 32 Haesebrouck, F. 20, 60, 73, 74 Halphen, C. 75 Halvorson, D. A. 148 Hamilton, M. S. 86 Hanna, E. S. 104 Hapfelmeier, S. 96 Hardt, W. 93 Hardt, W. D. 96 Hart, C. A. 31 Hasan, R. 125 Hassan, F. 132 Hau Yang, T. 12 Havlickova, H. 70 Heckler, K. S. 79 Helmuth, R. 40, 6 Hermans, A. 13, 21 Hermans, K. 74 Herrera-León, S. 127, 133 Heyndrickx, M. 60, 73, 74 Hilbert, F. 35 Hilton, A. C. 61, 8 Hinton, J. 25, 7 Hinton, J. C. 101, 116, 45, 86 Hobolt Høegh-Andersen, K. 68 Holden, D. 92 Holden, D. W. 101 Hoorfar, J. 40 Hossain, M. A. 132 Houghton, S. B. 129 House, D. L. 92 Hsien-Yee, H. 12 Huang, J. 57 Huang, S. 115 Hulme, S. D. 63 Humphreys, S. 7 Humphries, A. D. 134, 146, 19, 23 Hung, C. L. 5 Hur, S. 91 Hwang, W. Z. 111, 113 Imhoff, B.C. 156 Imnadze, P. G. 98 Inchley, C. J. 53 Irshad, A. 125 Isaacson, R. E. 82 Islam, M. A. 132 Ismail, A. 11 Isogai, E. 38 Ivanis, V. A. 28 Ivens, A. 44, 7 Jenks, S. 44 Jer-shan, L. 12 Jiao, X. 57, 62 Joseph, S. W. 151 Jouan, M. 156 Jovanovic, M. 145 Kaiser, P. 141, 54, 63 Kalkani, M. 110 Kam, K. 11 Karpiskova, R. 124, 99 Kay, W. W. 81 Keestra, A. M. 123 Kekelidze, M. G. 98 Kelly, A. 138 Kelly, A. E. 116 Kelly, A. P. 77 Kenney, L. J. 107 Khare, S. 134, 23 Kidgel, C. 44 Kim, C. 143 Kim, D. 91 Kim, W. 80, 81 Kim, Y. 55 Kingsley, R. A. 146, 19, 23 Knodler, L. A. 105, 4

Pathogenesis, Epidemiology, and Vaccine Development

91

INDEX INDEX

Kolackova, I. 99 Kopecko, D. 154 Kopecko, D. J. 153, 155 Korhonen, T. K. 112, 114 Kormanec, J. 7 Kovalchuk, N. I. 29, 30 Kramer, T. T. 22 Kraska, A. M. 148 Kubota, Y. 38 Kuhar, I. 123 Kujat-Choy, S. 4 Kukkonen, M. 114 Kyllönen, P. 112, 114 L. Mikkelsen, L. 88 Lafay, B. 64 Lähteenmäki, K. 112, 114 Lai, K. 11 Lång, H. 114 Lappin-Scott, H. M. 86 Larsson, L.I. 160 Lashkarashvili, M. G. 98 Lawhon, S. 134 Lazzerine, A. N. 141, 54 Leclerc, C. 75 Lee, H. 91 Lee, S. 91 Lee, W. 117 Lee, Y. 91 Lemire, S. 128 Lemmens, K. 109 Leori, G. 102 Leppla, S. 154 Liao, C. 117 Liao, M. H. 111, 113 Liebana, E. 14 Lin, C. 117 Lin, J. 115 Lin, Y. 117 Liu, C. 115 Liu, W. 62 Liu, X. 57, 62 Logan, J. 65 Lopes, V. C. 148 Lucchini, S. 101, 25, 45, 86 Luzzi, I. 122 Madajczak, G. 78 Maida, I. 71 Malorny, B. 40 Marchal, K. 109, 144 Marsh, L. 97 Martel, A. 60, 73 Martinez, R. 103 Martynova, A. V. 1 Maskell, D. J. 87, 158, 159 Matiasovicova, J. 124, 99 Matsui, H. 51 Matthews, K. 16 Mauriello, E. 95 Mayrhofer, S. 35 McDaniel, J. 153 McDermott, P. 101 Mehta, R. 152, 24 Mendoza, M. C. 6 Methner, U. 43, 59 Michael, G. B. 72 Miller, S. I. 56 Mitchell, E. K. 138 Mogensen, M. M. Mols, M. Molyneux, M. E. Mompart, F. Monsieurs, P. Mora, G. C. Morgen, E. Morona, R. Morris, C. Mortimer, C. K. Morton, M. Moulies, M. Muresu, E. Murillo, A. A. Murray, G. L. Mwenechanya, J. Nagaraja, K. V. Nagy, I. Nair, G. B. Nashnoush, H. A. Naughton, P. J. Nunes, J. O'Dowd, A. M. O'Gaora, P. Oliveira, F. A. Olsen, J. E. Osorio, M. Page, K.L. Pan, Z. Pang, J. C. Pang, T. Panunto-Caslelo, A. Park, J. Partanen, L. Parween, Z. Pasmans, F. R. Pathmanathan, S. Patterson, S. K. Paul, P. Paula, C. D. Paulin, S. M. Paulsen, P. Pena-López, M. J. Perkins, S. D. Peters, T. Pezzella, C. Piana, A. Pickard, D. Pietro, C. Porter, J. D. Powers, C. M. Pozo, J. Prager, R. Pravcova, M. Prieto, A. I. Pucciarelli, M. Puthucheary, S. Quessy, S. Rabsch, W. Raffatellu, M. Rakov, A. V. Ramos-Morales, F. Raupach, B. Raybourne, R. B. Read, R. C. Reinicke, A. T. Reis, B. P. 53 13 31, 41 163 109 17 158, 159 85 32 65 26 76 71 17 85 41 148 142 132 84 47, 88 23 26 44 79 68, 160 154 159 57, 62 111, 113 9 104 91, 91 112 125 60, 73, 74 27 82 152, 24 79, 79 131 35 133 26 65 122 71 44 147 86 34 127 58 99 50 83 10, 11, 27 126 58 134, 146, 23 28 49, 50 101 153 52 77 37 Rheault, N. 126 Ricca, E. 95 Ricci, A. 122 Roberts, M. 7 Rolfe, M. 25 Rollenhagen, C. 100 Romanova, Y. M. 121 Roque-Barreira, M. C. 104 Rossetti, C. 134 Rothwell, L. 63 Rowley, G. 7 Rubino, S. 147, 72, 89 Rubio, P. 127 Rychlik, I. 124, 43, 70, 99 S. Hedemann, M. 88 Sack, D. A. 132 Safa, A. 132 Sahlström, L. 108 Sales, A. L. 103 Sanchez-Jimenez, M. M. 94 Santos, R. L. 37 Santos, V. R. 103 Sarnacki, S. H. 106 Schauser, K. 160 Scholtens, I. 119 Schoofs, G. 142 Schouwenberg, J. 136 Schwarz, P. 69 Schwarz, S. 69 Sebkova, A. 124 Séguier, J. 76 Segura, I. 49 Senocq, D. 163 Seok, S. 91 Sevcik, M. 70 Sharma, V. D. 36 Shubin, F. N. 28, 29, 30 Siddiqui, A. A. 125 Silveira, W. D. 103, 67 Sisak, F. 70 Sisti, F. 106 Skov, M. N. 120 Smith, A. L. 34 Smulders, F. J. 35 Soerensen, M. 100 Solnik, H. 90 Soncini, F. C. 118 Sonck, K. A. 142 Sonne-Hansen, J. 157 Sotgiu, G. 71 Soto, S. M. 6 Spazziani, A. 102 Stecher, B. E. 93, 96 Steele-Mortimer, O. 105 Stein, M. A. 140 Stevens, M. P. 131 Stevenson, A. 7 Strugnell, R. A. 87 Sultana, J. 132 Suomalainen, M. 114 Surette, M. G. 66, 80, 81 Suvarnapunya, A. E. 140 Syrzova, N. A. 1 Takaya, A. 38, 51 Talukder, K. A. 132 Tam, C. 32 Tan, W. 9 Tang, S. 9 Tarkhashvili, N. V. 98 Taylor, T. E. 41 Tedin, K. 25 Tembo, M. 41 Teplitski, M. 162 Tevzadze, L. P. 98 Thomas, R. 153 Thompson, A. 101, 25, 7 Thomson, N. 44 Thong, K. 10, 11, 27, 9 Titball, R. W. 26 Tobgi, R. S. 84 Tomova, A. S. 121 Tomoyasu, T. 38, 51 Tondo, E. C. 79 Trombert, A. N. 17 Trowsdale, J. 138, 77 Tsanava, S. A. 98 Tschäpe, H. 58 Tsen, H. Y. 111, 113, 5 Tsironi, M. 110 Tsolis, R. M. 19, 23, 37 Turcutyuicov, V. B. 1 Turner, A. K. 26 Unsworth, K. 92 Usera, M. 127 Usera, M. A. 133 Uzzau, S. 102, 147, 89 Valdezate, S. 127, 133 Vallance, B. A. 4 Valle-Sousa, M. 104 van Boxel, N. 109 van den Bosch, J. F. 129 van Hoek, A. 119 Van Immerseel, F. 60, 73, 74 Van Immerseel, F. M. 20 van Putten, J. P. 123 Van Rooijen, N. 87 Vanderleyden, J. 109, 142, 144 Velayudhan, B. T. 148 Velge, P. 163 Verhoeven, T. L. 144 Vidal, A. 127 Villa, L. 122 Villarreal-Ramos, B. 131 Virkola, R. 114 Virlogeux-Payant, I. 163 Volf, J. 70 Wagenaar, J. 156 Walia, M. 152, 24 Walker, R. 154 Wallis, T. S. 131, 158, 159 Walsh, A. L. 31, 41 Walthers, D. 107 Wang, L. 9 Weening, E. 23 Weening, E. H. 146, 19 Weinberger, M. 90 Wegener, H.C. 156 Welker, Y. 75 Werber, D. 58 Wessells, K. R. 151 White, A. P. 81 Wigley, P. 34, 63 Wijburg, O. L. 87 Wildemauwe, C. 60

92

ASM Conference on Salmonella

INDEX INDEX

Winter, S. Won, H. Wong, D.L.F. Woodward, M. J. Wybo, I. Xu, D. Yamamoto, T. Yaron, S. Yasin, R. Youderian, P. yousefi-mashouf, R. Yuo, C. Zaidi, A. K. Zhang, S. Zhang, X. Zhgenti, E. G. Zwietering, M. 23 91 156 129 73 155 38, 51 90 10, 11 17 137, 137 115 125 23, 37 57 98 13, 21

Pathogenesis, Epidemiology, and Vaccine Development

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