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<p>1. Conduct a functional and molecular characterization of Shiga-toxin producing Escherichia coli (STEC) with specific emphasis elucidating the responses to food-related stresses, and genomic and proteomic studies to assess changes in virulence and pathogenicity. 1A: Comparative phylogenomics and phenomics of non-O157 STEC. 1B: Examine and compare stress responses, including acid tolerance, in E. coli O157:H7 and non-O157 STEC. 1C: Role of SdiA in acid tolerance of STEC O157:H7 and non-O157 STEC. 1D: Molecular serotyping of E. coli. 1E: Methods for detection and identification of non-O157 STEC.</p>
<p>2: Conduct functional and molecular characterization of Campylobacter species with specific emphasis on responses to intrinsic and extrinsic stresses through genomic and proteomic studies, and examination of morphological and physiological changes. 2A: Determine the "mode of action" by which polyphosphates (extrinsic stress) enhance the survival of C. jejuni and C. coli strains. 2B: Use genomic and/or proteomic studies to molecularly characterize Campylobacter's physiological response to food additives under poultry processing conditions. 2C: Determine if members of the microbial ecology of chicken exudates provide survival advantages/disadvantages to Campylobacter. 2D: Determine if common food additives change the composition of the microbial ecology of chicken exudate and if these changes are responsible for enhancing the survival of Campylobacter under food processing and storage conditions.</p>
<p>3: Conduct functional and molecular characterization of Listeria monocytogenes serotypes with specific emphasis on elucidating responses to food-related stresses through proteomics and genomics; and determining virulence differences among L. monocytogenes serotypes through sequencing and comparative genomics. 3A: Determine genes that are essential for the survival and growth of L. monocytogenes under weak organic acid conditions. 3B: Determine genetic responses of a pressure-resistant L. monocytogenes mutant exposed to the food preservative nisin. 3C: Determine genes responsible for the differences in virulence and stress responses among L. monocytogenes serotypes through sequencing, gene expression, and comparative genomics.</p>
<p>Approach: The overall goal of this project is to apply comparative genomic/proteomic/phenomic technologies to understand how pathogens become resistant to food-related stresses and to uncover the genetic basis of their virulence. Three major food-borne pathogens will be investigated: Shiga toxin-producing Escherichia coli (STEC), Campylobacter species, and Listeria monocytogenes. A combination of "omics" techniques, including transcriptomics, comparative genomics, proteomics, and phenotypic arrays will be employed to analyze a large variety of strains of each of these pathogens to identify genes and proteins necessary for them to survive stresses encountered in food environments and to identify genes/mobile genetic elements necessary for them to cause human illness. Comparative genomic and gene expression techniques will be used to assess the virulence profiles of highly pathogenic non-O157 STEC strains and to determine genes responsible for the differences in virulence and stress responses among L. monocytogenes serotypes. STEC, Campylobacter spp., and L. monocytogenes will be exposed to food environments and food-processing related stresses, including acid, high pressure, exposure to antimicrobial compounds, and other stresses. In addition, we will investigate environmental stresses that affect the survival and persistence of Campylobacter spp. during poultry processing and the role that the microbial ecology of this environment plays in this process. The mechanism by which polyphosphates enhance the survival of C. jejuni and C. coli strains will be determined, and genomic and proteomic techniques will be used to molecularly characterize the physiological response of Campylobacter to food additives under poultry processing conditions. It will also be determined if members of the microbial ecology of chicken exudates provide survival advantages/disadvantages to Campylobacter. The microbiological and molecular data will aid in the development of practical preservation systems that minimize health risks and assist regulators in making science-based food safety decisions. The "omic" data will also reveal biomarkers useful for identification, molecular typing, and detection of the pathogens. Methods and platforms for molecular serotyping of E. coli and for detection and identification of non-O157 STEC will be developed. The research will expand our knowledge on the survival mechanisms of important food-borne pathogens, will provide insight into the evolution of pathogens, provide the tools to detect, identify, and type food-borne pathogens, and ultimately lead to better control strategies for STEC, Campylobacter, and L. monocytogenes in food.</p>