1a. Objectives (from AD-416)
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. 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. 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.
1b. Approach (from AD-416)
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.
3. Progress Report
The goal of this project is to use molecular technologies to understand how STEC, Campylobacter, and Listeria monocytogenes, become resistant to food-related stresses and to uncover the genetic basis of their disease causing potential (virulence). Researchers at the ARS in Wyndmoor, PA are investigating a variety of strains of each pathogen isolated from cases of human illness, and from food, animals, and the environment by comparative analyses to understand which genes and proteins are necessary to survive stresses encountered in food environments and to cause human illness. Also, genes that are biomarkers of virulence and genes specific to E. coli serogroups are being utilized to develop methods for STEC detection and for developing methods for molecular serotyping of E. coli. The USDA FSIS has begun monitoring for non-O157 STEC serogroups of major public health significance in ground beef. ARS researchers in Wyndmoor, PA developed methods for detection and isolation of the non-O157 STEC, which are being used in national surveys by the FSIS to determine the presence of these important emerging pathogens in beef. Researchers in Wyndmoor have also made substantial progress in understanding the genes that are important for STEC virulence by comparative analyses using DNA microarrays and by using bioinformatics techniques. Studies to examine acid tolerance of a variety of STEC strains have also been initiated, and platforms for molecular serotyping of E. coli are being developed since the DNA sequences of sets of genes important for identification of the ca. 180 different serogroups of E. coli were determined. The effect of food-grade polyphosphates, added to processed chickens to enhance moisture retention, on survival of Campylobacter was assessed. Polyphosphates enhanced the survival of Campylobacter during typical poultry processing and storage conditions; therefore, their use increases the opportunity for disease via cross contamination or by consumption of improperly cooked chicken. Polyphosphates caused subtle pH shifts of poultry exudates resulting in a more optimal pH for Campylobacter growth; however, use of some polyphosphates was less of a food safety risk. Studies to understand the effects that polyphosphates and pH changes have on Campylobacter survival in chicken at the molecular level are ongoing. This work provides information to the poultry industry to help in understanding how to control growth and survival of Campylobacter. Although there has been considerable microbiological research aimed at understanding stress responses for L. monocytogenes in foods, there is only a rudimentary understanding of these responses at the molecular level, which is needed to design effective control strategies. ARS researchers in Wyndmoor have made substantial progress in molecular studies to understand the growth of L. monocytogenes in apple juice and other foods and to determine the dose responses of nisin (a compound known as a bacteriocin that inactivates L. monocytogenes under certain conditions), and they have begun to obtain a collection of strains from various sources to study virulence in L. monocytogenes.