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United States Department of Agriculture

Agricultural Research Service

Research Project: Molecular Identification and Characterization of Bacterial and Viral Pathogens Associated with Foods

Location: Produce Safety and Microbiology Research

Project Number: 2030-42000-051-00-D
Project Type: In-House Appropriated

Start Date: Feb 25, 2016
End Date: Feb 24, 2021

Objective:
The overall objective of this project is to develop novel typing methods to identify foodborne pathogens and characterize: bacterial foodborne pathogens through genomics, transcriptomics and proteomics; virulence factors and bacterial toxins; and antibiotic resistance in food production. Specifically, during the next five years we will focus on the following objectives: Objective 1: Develop improved identification technologies for human bacterial and viral pathogens to replace current testing methodologies. Sub-objective 1A: Develop a fast, simple and high throughput array-based method fortyping pathogens. Sub-objective 1B: Validate the array genotyping tool for the identification of viral and bacterial pathogens in samples from agricultural environments. Sub-objective 1C: Develop novel Campylobacteraceae species identification methods. Objective 2: Identify and characterize genetic factors associated with virulence and/or environmental adaptation of human bacterial pathogens using genomic, transcriptional and proteomic analyses. Sub-objective 2A: Identify the transcriptional network patterns of bacterial pathogens during environmental adaptation and modulation of their stress response. Sub-objective 2B: Identify genes involved in host/environmental adaptation and investigate variation in virulence potential through in-depth genome sequencing of selected taxa. Sub-objective 2C: Identify the genetic and epigenetic alterations or factors involved in the environmental adaptation of foodborne pathogens through genomic and methylome analyses. Sub-objective 2D: Quantitative proteomic and transcriptomic analysis of virulence factors of foodborne pathogens can be used to elucidate transcriptional vs. posttranscriptional control of virulence in foodborne pathogens. Sub-objective 2E: Top-down proteomic characterization of bacterial virulence proteins or toxins. Objective 3: Characterize molecular mechanisms contributing to the potency of bacterial toxins. Sub-objective 3A: Identification and characterization of Shiga toxin 2 (Stx2) subtypes in environmental STEC strains. Sub-objective 3B: Characterization of Stx2 expression levels and functional activities in environmental STEC strains. Sub-objective 3C: Characterization of pathogenic mechanisms associated with Stx2 subtypes produced by E. coli strains. Sub-objective 3D: Investigate toxin-inactivation mechanisms by natural plant compounds. Objective 4: Identify antimicrobial resistance gene reservoirs in the food production ecosystem and characterize the fitness and virulence of resistant pathogens. Sub-objective 4A: Complete genomic sequencing and functional metagenomic analyses of antibiotic-resistant Campylobacter. Sub-objective 4B: Characterization of the fitness and virulence of antimicrobial-resistant Campylobacter jejuni and Campylobacter coli.

Approach:
Objective 1: A fast, simple and high throughput array-based method for typing pathogens will be developed. Capture probes will be designed to target norovirus and hepatitis A virus, clinically-important Salmonella serovars and Campylobacter spp. To evaluate probe specificity, viral RNA or bacterial DNA will be extracted from clinical samples or cultured strains. A Cooperative Research and Development Agreement (CRADA) has been established with Arrayit Corporation to develop a fast, simple, and cost-effective test, in conjunction with inexpensive instrumentation. The array genotyping method will be validated using samples from agricultural environments. Also, MALDI-TOF-MS will be assessed as a faster, more accurate and reliable identification of Campylobacteraceae taxa, when compared to current phenotype-based approaches. Objective 2: The transcriptomic patterns that correlate with environmental adaptation and stress modulation for Campylobacter, Salmonella and E. coli will be determined, using RNA-Seq under distinct and relevant environmental conditions. Gene content or alleles that tentatively correlate with niche preference, environmental adaptation or pathogenicity will be identified by sequencing Campylobacter and Arcobacter isolates from a more diverse strain set. Alleles or methylation patterns within a population that correlate with environmental adaptation or pathogenicity will be identified through next-generation genomic analysis. Also, proteomic and transcriptomic analysis will be used to investigate transcriptional/post-transcriptional control of virulence factors and to characterize bacterial toxins and virulence determinants. Objective 3: A genotypic and proteomic screen for identifying and classifying Shiga toxin subtypes, harbored by strains recovered from different sources and locations in a major agricultural region, will be conducted. Using enzyme-linked immunosorbent assay and cell-based assays, the amounts and functional activities of Shiga toxin 2a and 2c subtypes will be determined. Using surface plasmon resonance, the mechanisms contributing to the cytotoxicities associated with the Shiga toxin 2a and 2c subtypes will be characterized, by investigating their role in the inhibition of protein synthesis in mammalian cells, thus providing a better understanding of the toxin’s mode of action. Natural plant compounds, specifically polyphenolics, will be investigated as potential inactivators of bacterial toxins. Objective 4: Genome sequencing of antimicrobial-resistant Campylobacter, isolated from poultry farms, will be performed to identify (potentially novel) antibiotic resistance genes. Metagenomic analysis of bacteria isolated from samples (such as litter, insects and fecal droppings) from these same poultry farms will be performed to identify the pool of ‘available’ antibiotic resistance genes that could potentially be transferred into Campylobacter. The fitness and virulence of resistant Campylobacter will be measured, to determine if increased fitness explains the persistence of resistant strains. Fitness metrics will include survival in insects and on poultry, and fecal colonization.

Last Modified: 06/28/2017
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