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The overall goal of the New York Food Safety Research Consortium (FSRC) is to conduct and coordinate food safety research, which provides critical new knowledge on foodborne pathogens and which leads to the development of new tools and innovative food safety intervention strategies for application by the food industry. We propose to use 2008 CSREES Special Research Grant funds supporting the FSRC to (i) further expand and maintain a comprehensive foodborne pathogen subtype database (www.pathogentracker.net) with a particular focus on L. monocytogenes and Salmonella, including support of specific research projects on molecular and phenotypic characterization of these pathogens and (ii) to implement rapid and affordable full genome sequencing methods to allow for improved subtyping of L. monocytogenes that will allow for validation of traditional subtyping data in traceback and outbreak investigations. <p>
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<li> Characterize L. monocytogenes phenotypic diversity relevant to foodborne transmission and risk assessments. <li> Perform full genome sequencing of selected Listeria and L. monocytogenes isolates using new rapid sequencing methods and characterize the epidemiological relevance of full genome sequencing data. <li> Continue a comprehensive collection as well as molecular and phenotypic characterization of human, food, and animal isolates of Salmonella <li> Continue a comprehensive collection as well as molecular and phenotypic characterization of human, food, and animal isolates of L. monocytogenes <li> Enhance and maintain the PathogenTracker database, including development of improved data analysis tools and user interfaces, and distribution of strains and isolates. </ol>
NON-TECHNICAL SUMMARY: Estimates published by the US Centers for Disease Control and Prevention in 1999, estimated 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. L. monocytogenes was estimated to cause about 2,500 human foodborne listeriosis cases and 500 deaths annually, while Salmonella was estimated to cause 1.4 million cases of foodborne disease annually in the US, including approximately 550 deaths. Thus, these two foodborne pathogens have a significant negative impact on human health and well being in the US. Recent estimates published in 2008 suggest that there has been limited progress in recuing foodborne illnesses in the US over the last 2 - 3 years. In particular, the number of foodborne infections caused by Salmonella seems to have shown limited reductions over the last 10 years. This project will develop improved molecular characterization and subtyping methods to further enhance our understanding of the transmission and ecology of Salmonella and Listeria monocytogenes and to more accurately detect and identify foodborne disease outbreaks and outbreaks sources. Strain collections, subtyping and characterization methods, and protocols will be made broadly available to facilitate application of the methodologies developed. Application and use of the molecular subtyping methods developed here will provide critically needed knowledge and tools to reduce foodborne illnesses in the US.
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APPROACH: Methods to be used are detailed to correspond to the five main objectives of our project. Objective 1. L. monocytogenes isolates from human listeriosis outbreaks as well as virulence attenuated L. monocytogenes not associated with human cases will be characterized by (i) growth assays uisng broth culture formulated to simulate food-processing stresses (e.g., refrigerated storage, salt, low pH and growth in-hibitors) using a low inoculum cold growth assay and RTE deli meats (i.e., turkey, ham and beef) formulated with and without growth inhibitors; (ii) intracellular growth assay, and (iii) intragastric guinea pig infections. <P> Objective 2. We will initially perform genome sequencing of selected L. monocytogenes strains using, most likely, the 454 GS20FLX (Roche) and the Illumina Solexa. Full genome analyses to be performed will include (i) full genome alignments to identify gene presence/absence patterns and single nucleotide polymorphisms (SNPs), (ii) analyses for recombination, and (iii) analyses for positive selection. <P> Objective 3. Human and animal Salmonella isolates will be obtained through collaborations with the NYS Department of Health (NYSDOH) and the NYS Animal Health Diagnostic Laboratory (AHDL) at Cornell. All isolates will be characterized by 3-gene multilocus sequence typing (MLST) approach and by PFGE using the standard CDC PulseNet protocol for PFGE (CDC, 2003). Selected isolates will further be screened for the presence/absence of the most common Salmonella antibiotic resistance genes using PCR and microarray assays. <P>Objective 4. Human clinical L. monocytogenes isolates will be obtained through continuation of our collaborations with the NYS Department of Health. Food L. monocytogenes isolates will be obtained through collaborations with the NYS Department of Agriculture and Markets. Isolates will be characterized by automated EcoRI ribotyping, MLST, and PFGE typing using the CDC standard Pulsenet protocol. Isolate information and subtype data will be entered into the PathogenTracker database. <P>Objective 5. We will work with a network of collaborators to get feedback on improvements and additions to be made to improve the PathogenTracker database, including redesign WWW-based interfaces for search functions, to add new .data fields, and re-design data fields. We will also continue enhanced efforts to identify data sets and publications that use isolates already in Pathogen-Tracker and add appropriate links and data information to make this information more broadly accessible. As part of the FSRC we will further continue to distribute strains and isolates in our collection to other researchers.