<ol> <li>Genome sequencing, annotation, and gene-indexing, of Campylobacter
species, Salmonella Enteritidis (SE) and pathogenic E. coli (Ec) to identify targets
for rapid detection and differentiation, and fitness and virulence factors.
<li>Develop DNA microarrays, and sequence-based typing methods to detect and
analyze multiple critical food-borne pathogens; validate assays with food samples.
<li>Develop new and/or improved multi-locus sequence typing (MLST) methods
for human pathogens. Combine MLST and microarray analysis to identify markers
associated with pathogen source and fitness, and relate to epidemiology and culture
<li>Develop specific capture and mass spectrometry (MS) methods to detect
and fingerprint foodborne pathogens and threat agents.
<LI>Evaluate methods for inactivating protein toxins.</ol>
<p>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).</p>
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). <BR><BR>