Location: Produce Safety and Microbiology Research2011 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
This is the 1st year report for new project 5325-42000-047-00D, which was approved after termination of 5325-42000-045-00D. In 2011, we continued molecular and mass spectrometry characterization of important enteric food pathogens, with an emphasis on Shiga toxin-producing E. coli strains, which emerged as a critical public health issue. A) Campylobacter/Arcobacter genomics. Using next-generation sequencing, the genomes of 22 Campylobacter strains, including two novel species, were sequenced to draft level to provide genomic sequence data for all 30 accepted taxa within the genus Campylobacter. In addition, the genomes of three host-associated C. coli strains were sequenced to completion and the genomes of seven food-related Arcobacter species were sequenced to completion. B) Campylobacter typing methods. Using these next-generation Campylobacter sequence data, novel typing methods were developed for emerging Campylobacter species (e.g. C. hyointestinalis, C. sputorum and C. lanienae) and used to type > 1000 campylobacter strains isolated as part of an environmental survey. Two new species were identified. C) Development of multiplex assay for pathogen detection. Rapid methods are needed for characterizing the potential virulent types of E. coli. Multiplex polymease chain reaction (PCR) assays were implemented for the identification of O-group associated genes, flagellar antigen genes, and virulence factors that are characteristic of adherent-invasive, enterohemorrhagic, and enteroaggregative E. coli and for expanding the rapid microarray method developed in our previous plan for characterizing STECs and identifying virulent strains. D) Improved mass spectrometry (MS) for identification of protein biomarkers and toxins. A stand-alone software program was developed to construct in silico protein databases rapidly based on protein toxins, virulence and stress factors. We continued expanding our in silico database approach with new bacterial genome data for comparison to phenotypic and molecular results obtained by other methods. This provides detection of the products of genes and post-translational modifications for fuller understanding of pathogenesis. E) Analysis of extraintestinal E. coli strains isolated from Crohn's disease patients. In collaboration with researchers at Guy's and St. Thomas' Hospital (London, UK), commensal and Crohn's associated E. coli were analyzed by genotyping methods and MS to identify protein biomarkers unique to Crohn's associated E. coli and similarities to E. coli isolated from food and the agricultural production environments. Common strain types were identified and selected for genome sequencing in the UK.
1. Sequencing of Campylobacter fetus genomes. Campylobacter fetus contains two recognized subspecies important as veterinary pathogens in cattle and occasionally humans. In collaboration with Utrecht University, ARS scientists in Albany, CA sequenced the genomes of selected 23 strains of the two subspecies. The data revealed insertion sequences and prophage in one subspecies that may play a role in both the pathogenicity and host-restriction of the strains. These results will assist in development of new treatments.
2. Mass spectrometry (MS) identification of acid-stress chaperone proteins from E. coli K-12 and variants. E. coli pathogens must survive the acidity of the stomach to cause disease; acid-stress chaperone proteins, HdeA and HdeB, are important in E. coli acid-resistance. Top-down proteomic analysis was used to confirm the successful complementation of E. coli strain K-12 and variants with hdeA and hdeB genes that will be used for functional assays. Significant differences in acid-resistance of strains indicate strains may be more or less capable of causing illness. MS provides a rapid method to confirm protein expression. The results of the study provide fundamental molecular evidence that E. coli strains have evolved different mechanisms of response to stresses and that genes may function differently in different conditions/environments as part of a fitness and/or virulence profile.
3. Development of rapid, and inexpensive multiplex assays for pathogen detection and virulence characterization. Improved methods to rapidly detect and characterize virulent strains are needed. We expanded a low-density DNA microarray using a novel colorimetric method for enterohemorrhagic E. coli by adding new virulence factors and serotypes of interest and tested it with STEC isolates recovered from major produce production regions in California. The method will be expanded for identification of human noroviruses as part of a new USDA-NIFA funded project. Rapid and inexpensive methods for pathogenic bacteria and viruses will assist in environmental and outbreak investigations related to fresh produce production and in a deeper understanding of pathogen virulence.