Tracking and Understanding Foodborne Transmission of L. Monocytogenes, E. Coli, and Salmonella using Genomics and Molecular Approaches

NON-TECHNICAL SUMMARY: Recent estimates project that 76 million cases of gastrointestinal foodborne illnesses occur in the US on an annual basis, resulting in at least 325,000 hospitalizations and 5,000 deaths (Mead et al., 1999). Thus, foodborne illnesses and their medical sequelae have a significant negative impact on human health and well being. This project will develop and apply molecular characterization and epidemiological methods to provide an improved understanding of the transmission and ecology of selected bacterial foodborne pathogens, including Salmonella, Listeria monocytogenes, and E. coli. Strain collections, subtyping and characterization methods, and protocols will be made broadly available to facilitate application of the methodologies developed.
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APPROACH: The FSRC program will be administered by faculty and staff in the Department of Food Science at Cornell. They will administer the funds and conduct research as specified and involve faculty at other universities and organizations as formal or informal collaborators. The progam will interface with and draw on the existing 'Cornell Food and Water Safety Program' (see http:/www.foodscience.cornell.edu/fws/fws.htm), which includes more than 30 faculty members at different colleges with research, teaching and extension efforts and interests in food and water safety. We will use the following specific approaches to accomplish the objectives described above: (1) Obtain human and animal associated Salmonella isolates and characterize isolates by multilocus sequence typing, and antimicrobial resistance testing. Analyze data using evolutionary and statistical approaches to describe the diversity of food associated Salmonella and their distribution among human and food isolates. (2) Obtain human, animal, and food associated L.monocytogenes isolates, including testing of prospectively collected samples from retail establishments. Characterize isolates by multilocus sequence typing and other molecular subtyping methods and analyze data to probe ecology and transmission of different L.monocytogenes subtypes along the food system. (3) Analyze 12-16 L.monocytogenes genome sequences using evolutionary approaches and construct and evaluate appropriate L.monocytogenes mutants using tissue culture and other assays to probe the importance of strain variation on virulence. (4) Obtain food associated E.coli isolates and characterize isolates by mulitlocus sequence typing. Analyze data using evolutionary and statistical approaches to describe the diversity of food associated E.coli isolates. (5) Develop a user network to evaluate user friendliness of the existing PathogenTracker network and revise PathogenTracker interface to become more user-friendly and intuitive.

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PROGRESS: 2005/07 TO 2008/06 <BR>
OUTPUTS: As part of this project activities were conducted to (i) expand and maintain a comprehensive foodborne pathogen subtype database (www.pathogentracker.net) with a particular focus on L. monocytogenes and Salmonella and to (ii) develop and apply molecular and genomics-based subtyping methods for these pathogens to develop a better understanding of their transmission. As of 06/30/2008, PathogenTracker included>6,500 L. monocytogenes and >1,500 Salmonella isolates. Information on these isolates, including subtype data, has been deposited into our publicly available foodborne pathogen subtype database, PathogenTracker. In particular, we have added to PathogenTracker a number of L. monocytogenes isolates, which have been isolated from retail operations through a collaborative project with the New York state Department of Agriculture. Subtyping of these isolates has provided critical new information on L. monocytogenes persistence and transmission in retail operations. In addition, PathogenTracker now contains >400 E. coli isolates, including >100 isolates added by an outside laboratory. Specific research efforts on Salmonella have focused on (i) characterization of isolates representing the emerging Salmonella serotype 4,5,12:i:- and (ii) analysis of full genome Salmonella sequences to identify highly variable genes that are under positive selection. Through our research efforts we have expanded our collection of Salmonella 4,5,12:i:- isolates to include isolates from various US states and Spain. Initial full genome analyses of various Salmonella genomes has been completed and we have also completed follow-up studies that characterize highly variable genes identified in these studies in a larger set of isolates from various sources that are found in our strain collection. These studies have identified highly variable genes that are under particularly rapid evolution (i.e., positive selection) and may thus contribute to emergence of new Salmonella subtypes. These genes could also potentially be used to develop more sensitive Salmonella subtyping approaches. We have also completed two separate subprojects that analyze available L. monocytogenes genome sequences. Full genome analyses showed that recombination and positive selection contributed to the evolution of L. monocytogenes and these evolutionary forces are more prominent among lineage II strains. These data will help provide suitable gene targets for highly sensitive subtyping of L. monocytogenes. Additional genome analyses of L. monocytogenes isolates associated with a single plant, but isolated about 12 years apart, showed that genome diversification over short time frames occurs predominantly in pro-phage sequences; these results will help to better understand instances where genetically closely related isolates that are also possibly epidemiologically related show distinct, but similar DNA subtypes. In addition, this work has laid the foundation for the use of full genome sequencing methods for subtyping of foodborne pathogens, particularly if highly sensitive subtyping methods are needed for investigations following-up on initial PFGE studies. <BR>
PARTICIPANTS: Martin Wiedmann, Principal Investigator: He provided project oversight. K. Abou-Zeid, Post Doctoral fellow: performed L. monocytogenes transmission modelling S. Chaturongakul, Post Doctoral fellow: performed L. monocytogenes characterization A. Moreno-Switt, Post Doctoral fellow: performed molecular subtyping and Salmonella characterization A. Pradhan, Post Doctoral fellow: performed L. monocytogenes transmission analyses A. Roberts, Academic Professional: performed L. monocytogenes characterization B. Bowen, Research Support Specialist: performed L. monocytogenes characterization Y. Chan, Graduate Research Assistant: performed L. monocytogenes characterization H. Franks, Graduate Research Assistant: performed L. monocytogenes characterization R. Ivanek, Graduate Research Assistant: provided bioinformatics support and performed modeling of L. monocytogenes and Listeria transmission C. Lucas, Graduate Research Assistant: performed Listeria characterization and isolation S. Milillo, Graduate Research Assistant: performed Listeria characterization and genome comparisons J.Ollinger, Graduate Research Assistant: performed Listeria characterization R.Orsi, Graduate Research Assistant and Postdoctoral fellow: performed Listeria genome comparisons M.Palmer, Graduate Research Assistant: performed Listeria characterization S.Raengpradub, Graduate Research Assistant: performed Listeria characterization M. Ranieri, Graduate Research Assistant K. Vongkamjan, Graduate Research Assistant: performed Listeria isolation and characterization S. Roof, Technician: provided strain collection and database support and performed molecular subtyping L. Rodriguez, Temporary Technician: performed molecular subtyping and Salmonella characterization J. Badamo, Temporary Technician: performed molecular subtyping and L. monocytogenes characterization J. Corron, Temporary Technician: performed Listeria isolation and characterization <BR>
TARGET AUDIENCES: The target audience for the project includes food microbiologists and food safety professionals in industry, academia, government agencies, and non-government organizations (e.g., research institutes, commodity groups etc.). <BR>
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IMPACT: 2005/07 TO 2008/06<BR>

This project has developed and applied molecular characterization and epidemiological methods to provide an improved understanding of the transmission, evolution, epidemiology, and ecology of selected bacterial foodborne pathogens, including Salmonella and Listeria monocytogenes. In particular, development of improved subtyping methods and better basic understanding of the biology behind some subtyping methods provides for improved subtype data for bacterial isolates and improved data interpretation, including during outbreak investigations. A unique aspect of this project is that all subtype data are made publicly available through a database (www.pathogentracker.net) developed as part of this project and that isolates are freely distributed to the research community. This project thus will assure the accessibility of molecular technologies and improved knowledge on key foodborne pathogens to the food industry and regulatory agencies throughout the US, which will enhance integrated farm-to-table food safety efforts. Through our WWW-based strain database, subtyping and characterization data are made broadly available to facilitate application of the methodologies developed. In particular, our work has laid foundation for use of full genome sequencing methods for molecular subtyping of foodborne pathogens; these highly sensitive subtyping methods will allow for improved subtype discrimination in outbreak investigations. The impact of our project is supported by the fact that the PathogenTracker database has 673 registered users and also 304 log-ins from different IP addresses, indicating broad use of the information generated through this project. This project also contributed considerably to the training of graduate students and staff in molecular techniques, including a number of students that now work in industry and academic institutions, thus providing critically needed personnel resources in the area of food safety.