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The purpose of this project is to investigate foodborne pathogens and their ability to survive in food and food production/handling systems using integrated applied and basic research approaches. The objectives and approaches described hereafter have been developed to investigate human foodborne pathogens (e.g. Salmonella, E. coli, Listeria monocytogenes) in an effort to improve food safety and enhance public health.<P> The specific objectives of this project include: (i) Objective I. Determine foodborne pathogen prevalence and contamination levels in food and food production and handling environments and(ii) Objective II. Characterize the stress response mechanisms, molecular subtypes and pathogenic potential of foodborne pathogens isolated from food and food production and handling systems. <P> Outcomes: Objective I. Upon completion of this objective, we will have determined the prevalence and levels of foodborne pathogens (e.g., L. monocytogenes, E. coli, Salmonella) in food and food production and handling environments. These data will be used to update U.S. FDA/CFSAN, USDA/FSIS, and CDC risk assessments. By engaging an advisory board, this study will better meet the needs of the major stakeholder groups, notably industry, and will integrate our research and extension-based approach. Producer input will provide invaluable information on production practices and insight into feasibility of potential control strategies. As we will be collecting food and environmental samples from food production and handling systems, the food industry has a vested interest in understanding foodborne pathogen prevalence in products and their establishments. Engaging experts in policy and risk assessment will facilitate discussions between industry and regulators to ensure that data are interpreted accurately. Outreach/extension specialists will help us achieve our long-term goal of communicating the results to industry.<P> Objective II. Upon completion of this objective, we will have identified environmental niches in food processing and handling environments that are persistently contaminated by foodborne pathogens and identified potential routes of transmission that contribute to cross-contamination of foods.
Non-Technical Summary: <BR>Characterizing foodborne pathogens and their ability to survive in food and food production/handling systems using integrated applied and basic research approaches is important to improving public health. This project will engage stakeholder advisory groups to consult studies aimed to investigate the human health risk attributable to foodborne pathogens in food and food production and handling systems, determine foodborne pathogen prevalence and contamination levels in food and food production and handling environments, and characterize the stress response mechanisms, molecular subtypes and pathogenic potential of foodborne pathogens isolated from food and food production and handling systems. Results will be communicated to stakeholders to encourage development of intervention strategies to prevent disease. <P> Approach: <BR> Objective I. Stakeholder advisory groups would be composed of representatives from food production, food processing, retail (grocery), government/policy, and academia/outreach sectors. The goals of advisory group meetings will be to discuss previous and ongoing research in foodborne pathogen prevalence and persistence along the food chain, discuss industry needs for pathogen control, and to identify additional long-term goals including intervention and control strategies Objective I. Detection and isolation of human foodborne pathogens. The FDA Bacterial Analytical Manual methods will be used to detect and isolate foodborne pathogens in food and environmental sponge samples (www.cfsan.fda.gov/~ebam/bam-10.html Objective III. RNA-Seq. RNA-Seq technology will be used to identify differences in genes expression among strains exposed to stress compared to a control. Specifically, RNA will be extracted and processed for sequencing as previously described by Oliver et al. 2009. Sequencing will be carried out on the Illumina Genome Analyzer or equivalent sequencer. To identify genes that showed significantly different transcript levels in the control strain and the test strain, statistical analyses will be performed using the normalized RNA-Seq coverage of each coding gene. Normalized RNA-Seq coverage (i.e. the number of reads that match an annotated CDS after normalization across runs) will be used for statistical analyses. Pulsed field gel electrophoresis (PFGE). PFGE typing will be performed using the standardized CDC PulseNet protocol (http://www.cdc.gov/pulsenet/protocols/pulsenet listeria protocol%20. pdf). DNA from isolates will be digested separately with the restriction enzymes which act as "molecular scissors" to yield DNA fingerprint patterns. PFGE patterns will be analyzed and compared using Bionumerics software (Applied Maths, Austin, TX). Single nucleotide polymorphism (SNP) genotyping. A multiplex SNP genotyping assay (SNaPshot Multiplex SNP Genotyping Kit; Applied Biosystems, Foster City, CA) will be used to determine the proportion of L. monocytogenes isolated from produce and other plant-based products and the retail environment that pose reduced human health risk. Briefly, the SNaPshot assay identifies changes (mutations) in DNA sequence that lead to nonfunctional proteins (typically virulence factors). Objective VI. Presentations of outcomes at professional and industry meetings. We will present our findings from Objectives I and II at professional meetings (e.g. International Association for Food Protection) and at relevant industry meetings (e.g. Food Marketing Institute) to reach a wider range of constituents. Webinars. We will conduct a series of webinars to disseminate knowledge on foodborne pathogen prevalence persistence, and virulence in foods and food processing and handling environments to producers and retailers. Delivery of webinars will allow us to reach a broad target audience across the U.S. (and beyond), including small and very small producers that would not typically attend symposia or workshops.