Location: Produce Safety and Microbiology Research2013 Annual Report
1a. Objectives (from AD-416):
Objective 1: Comparative genomic analyses of Campylobacter, Arcobacter, E. coli and S. enterica to identify novel genetic elements or polymorphisms that are associated with virulence, niche specialization or other adaptive traits. Objective 2: Develop sequence-based typing methods to detect and analyze multiple critical food-borne pathogens from multiple sources. Objective 3: Generate gene expression profiles for E. coli, S. enterica, and Campylobacter from varying sources and in response to various environmental stresses to identify factors contributing to virulence and survival in diverse habitats. Objective 4: Develop rapid, simple and inexpensive multiplex assays for pathogen detection and virulence characterization using novel technology for use in surveillance and outbreak epidemiology. Objective 5: Establish proteomic approaches for detecting and typing foodborne pathogens and toxins, and measure pathogen response to environmental stresses by mass spectrometry methods. Objective 6: Investigate mechanisms of bacterial toxicity and evaluate novel methods for inactivating Shiga toxins by developing cell-based assays for assessing toxicity and comparing the relative toxicity of Shiga toxin 1 and 2 variants.
1b. Approach (from AD-416):
Our objectives address fundamental research for comparative genomic analyses of Campylobacter and Arcobacter species, and pathogenic Escherichia coli and Salmonella enterica serovars to identify novel genetic elements or polymorphisms associated within virulence, niche specialization or other adaptive traits. We will analyze the genomic data to develop sequence-based typing methods to detect and analyze multiple critical food-borne pathogens from multiple sources for purposes of improved source tracking to identify reservoirs in food production environments. The genomic data will allow us to develop methods for gene expression profiling of pathogens from different sources and their responses to various environmental stresses to identify factors contributing to virulence and survival in diverse habitats; and develop rapid, simple and inexpensive multiplex assays for pathogen detection and virulence characterization using novel technology for use in surveillance and outbreak epidemiology. The genomic data facilitate developing “top-down” and “bottom-up” mass spectrometry approaches for proteomic analysis for detecting and typing foodborne pathogens and toxins, and measuring pathogen response to environmental stresses. Non-O157 Shiga toxin-producing E. coli (STEC) strains have emerged as a source of more human illness than appreciated previously. Data for isolating and detecting them are a critical need and will be provided through this research. We also will be researching mechanisms of bacterial toxicity to develop and evaluate novel methods for inactivating Shiga toxins by developing cell-based assays for assessing toxicity and comparing the relative toxicity of Shiga toxin 1 and 2 variants. In summary, we will use the latest analytical tools to obtain data for identifying foodborne pathogens, differences in strains related to fitness and virulence, novel interventions related to toxins and, ultimately, approaches for minimizing illness associated with food.
3. Progress Report:
We made significant advances towards major objectives of our 5-year research plan: the establishment of a framework for comparative genomic analyses; identification of novel genetic elements; and the development of sequence-based methods that type and detect foodborne pathogens. Closure, analysis, and annotation were performed on the genomes of multiple Campylobacter species, several strains of Shiga toxin-producing E. coli (STEC), and Salmonella enterica strains associated with multiple outbreaks. We achieved major progress in Campylobacter genomics, including the identification of 15 new taxa as part of multiple national and international collaborations. Comparative analysis revealed Campylobacter taxa clustering based on gene content that correlated with the environmental source and phenotypes of these taxa. We developed a multiplex PCR method from the genome sequences of the pathogen C. jejuni to quickly and accurately type strains. The genomes of several serotype O111 STEC strains from a survey of the Salinas Valley and from clinical sources were sequenced and gene content comparisons are proceeding as part of identifying the most virulent STECs from environmental and clinical sources. We completed the assembly and annotation of the first closed genome of S. enterica serovar Thompson, a contaminant of many foods. In collaboration with the USDA Natioan Institute of Food and Agriculture (NIFA) Food Virology Project, a focused microarray was designed and validated in conjunction with rapid and inexpensive colorimetric technology for the specific identification of a subset of norovirus strains that are associated with foodborne illness in humans. Finally, research employing whole genome sequencing is advancing to identify key regulatory genes responsible for increased Shiga toxin (Stx) levels, detected in STEC strains from an agricultural region in California. By using a quantitative cell-based assay, these STEC strains, expressing either the Stx2a or Stx2c variant, were identified to have an enhanced ability to inhibit protein synthesis in mammalian cells. We have made further significant progress in proteomics and the characterization of antibacterial compounds from plants. Top-down proteomic identification was combined with RT-PCR to analyze a clinical E. coli strain that produces two variants Stx2a and Stx2c, which are distinguishable and identifiable by MALDI-TOF-TOF mass spectrometry. Protein biomarkers of three S. enterica serovars (Newport, Kentucky, Thompson) were also identified using MALDI-TOF-TOF mass spectrometry. Enzymatic surface shaving experiments were conducted on E. coli O157:H7 and a strain of Listeria monocytogenes. As part of a collaborative project, a protein from L. monocytogenes, that appears to be the antigenic target for a monoclonal antibody, was identified by top-down proteomic identification. In other collaborative projects, we have demonstrated the ability of specific plant compounds alone and in edible films to inactivate S. enterica. These findings provide an excellent scientific basis for the application of plant compound interventions in food packaging.
Sheppard, S.K., Didelot, X., Jolley, K.A., Darling, A.E., Kelly, D., Colles, F.M., Strachen, N.J., Ogden, I.D., Forbes, K.J., French, N., Carter, P., Miller, W.G. 2012. Progressive genome-wide introgression in agricultural Campylobacter coli. Proceedings of the National Academy of Sciences. 22:1051-1064.