Project Number: 5325-42000-045-00
Project Type: Appropriated

Start Date: May 16, 2006
End Date: May 15, 2011

Objective:
Objective 1: Genome sequencing, annotation, and gene-indexing, of Campylobacter species, Salmonella Enteritidis (SE) and pathogenic E. coli to identify targets for rapid detection and differentiation, and fitness and virulence factors. Objective 2: Develop DNA microarrays, and sequence-based typing methods to detect and analyze multiple critical food-borne pathogens; validate assays with food samples. Objective 3: Develop new and/or improved multi-locus sequence typing (MLST) and multi-locus variable tandem repreat analysis (MLVA) methods for human pathogens with emphasis on enterohemorrhagic E. coli. Combine MLST, MLVA and microarray analysis to identify markers associated with pathogen source and fitness, and relate to epidemiology and culture method bias. Objective 4: Develop specific capture and mass spectrometry (MS) methods to detect and fingerprint foodborne pathogens and threat agents. Objective 5: Evaluate methods for inactivating protein toxins. Problem to be Addressed: Through the use of genomics and proteomics develop multiplex assays to detect, identify and differentiate foodborne pathogens on fresh produce (leafy vegetables) to derive fundamental data to increase the safety and security of this commodity. FY07 Objectives of Research: Genome sequencing, annotation, and gene-indexing, of pathogenic E. coli to identify targets for rapid detection and differentiation, and fitness and virulence factors, with special emphasis on E. coli in the environment of produce production. Use fundamental genomic and proteomic information produced to develop microarray or other multiplex immunoreagent methods to identify and analyze genera, species and strains of critical food-borne pathogens. Identify single nucleotide polymorphism hot-spots in "clonal" pathogens for high resolution fingerprinting. Characterize E. coli O157:H7 strains associated with outbreaks and to identify potential virulence factors and other factors that may enhance fitness in produce production environments (plants, animal hosts, environment).

Approach:
Our objectives address fundamental research to develop high-resolution genotyping methods for characterizing and tracking multiple pathogens related to food. Multiple approaches to methods development are described as contingencies to ensure success. The recent sequence data we have collaborated in producing for Campylobacter and Arcobacter species and collaborations on S. enterica, Ec O157:H7 and Lm genotyping will be invaluable for this work. Recent PSMRU involvement in two outbreak investigations of pre-harvest produce and tree-nuts contaminated with Ec O157:H7 (letter, J. Farrar) and S. Enteritidis (letter, J. Adams), respectively, has confirmed the need for improved methods for tracking pathogens in complex environments, determining their relatedness, and the relevance of these studies also to addressing potential intentional contamination events. The objectives we describe have been organized with the following strategy in mind: (i) Emphasize Campylobacter species, especially emerging species, because they remain underappreciated as food pathogens and causes of serious illness. Recent progress in sequencing and MS analysis facilitate comparative genomics and proteomics, and the expertise gained will be beneficial for development of similar approaches for other pathogens; (ii) Develop microarrays specific for genotyping, to learn as much as possible about virulence factors and fitness characteristics that might be beneficial to interventions during production or processing; (iii) Expand, as appropriate, the microarray approach to more comprehensive DNA microarrays for detection of many pathogens simultaneously; (iv) Develop methods useful for addressing objectives of PSMRU CRIS-040 (¿Biology and Control of Human Pathogens on Fresh produce¿) to leverage methods and discoveries and increase productivity; (v) Collaborate with other groups who have access to productions systems and/or strains for assessing the robustness of genotyping or protein typing; (vi) Use the novel methods developed to address whether culture bias is affecting the ability to obtain meaningful data about reservoirs, food sources and epidemiology. Replacing 5325-42000-041-00D (4/06).