Location: Produce Safety and Microbiology Research
Project Number: 2030-42000-055-000-D
Project Type: In-House Appropriated
Start Date: Feb 25, 2021
End Date: Feb 24, 2026
Objective:
Objective 1: Identify and characterize factors associated with virulence and/or environmental adaptation of human bacterial pathogens using genomic and transcriptomic analyses.
Sub-objective 1.A: Develop source attribution models for Campylobacter infections using frequency matching and population genetics-based approaches.
Sub-objective 1.B: Identify ganglioside-like structures associated with Guillain-Barré syndrome in non-jejuni Campylobacter taxa.
Sub-objective 1.C: Identify specific Campylobacter factors that contribute to the development of post infectious-Irritable Bowel syndrome (PI-IBS) and links to host response.
Sub-objective 1.D: Identify the transcriptional network patterns of bacterial pathogens under stress and during adaptation to different environments.
Sub-objective 1.E: Characterize mobile elements linked to the transfer of antimicrobial resistance (AMR) genes in Campylobacter.
Objective 2: Evaluate microbiomes of produce production sites and their role in antimicrobial resistance gene reservoirs and bacterial pathogen fitness.
Sub-objective 2.A: Investigate the utilization of fecal microbiomes to determine the role of indigenous fauna in the spread of Salmonella and AMR.
Sub-objective 2.B: Evaluate the effects of irrigation water treatment on the microbial community and foodborne pathogens.
Sub-objective 2.C: Evaluate the microbiomes of produce production environments to identify the role bacteriophages play in the development of AMR in bacteria.
Objective 3: Assess virulence and antimicrobial resistance of foodborne pathogens using mass spectrometry-based proteomics.
Sub-objective 3.A: Perform top-down proteomic identification of toxins, antibacterial and antimicrobial resistance proteins expressed by plasmids and bacteriophage carried by foodborne pathogens.
Sub-objective 3.B: Investigate biofilms of pathogens using MALDI MSI, MALDI-TOF-TOF-MS/MS and top-down proteomic analysis.
Objective 4: Characterize biomarkers for the development of automated detection platforms for onsite monitoring of foodborne pathogens.
Sub-objective 4.A: Develop and evaluate immuno-biosensors for the detection of C. jejuni and C. coli using a liquid crystal-based biosensor.
Sub-objective 4.B: Characterize outer membrane antigens in C. jejuni as a novel single ligand for detecting Shiga toxins.
Objective 5: Elucidate the interplay between bacteriophages and their bacterial hosts in the environment to enhance the safety of food products and the prevention of emerging foodborne pathogens.
Sub-objective 5.A: Determine the induction parameters and the mechanisms of transduction through lysogenic bacteriophages that contribute to the potential emergence of new pathogens.
Sub-objective 5.B: Investigate the role of lytic bacteriophages against their host strains and other serogroups.
Approach:
Objective 1: Campylobacter from poultry may be the source of infection in infants in low- and middle-income countries. Whole genome sequencing (WGS) of Campylobacter from various animals will be used in source attribution of infected infants. Non-jejuni Campylobacter may produce human ganglioside-like structures associated with Guillain-Barré syndrome. Using antisera, dot blot assays will use antibody binding to establish the presence of such structures. Campylobacter associated with post infectious-irritable bowel syndrome (PI-IBS) may have observable genomic signatures. WGS and gene-by-gene analysis will be compared between Campylobacter isolated from infections resulting in PI-IBS or no PI-IBS. Transcriptional patterns of C. lari may be altered under salt and oxidative stress. RNA sequencing will be used to determine the patterns that correlate with adaptation. C. coli mobile elements are potentially transferred into naïve strains via transmissible plasmids. Matings between C. coli strains containing mobile elements and naïve recipients will test lateral transfer of mobile elements.
Objective 2: Microbiome WGS from animal feces might detect the presence of Salmonella and antimicrobial resistance (AMR) genes. Short- and long-read WGS of microbiomes from feces near produce will be used to determine presence and transmission of Salmonella and AMR genes. Irrigation treatments may affect the diversity of microbial communities and pathogens. WGS of irrigation samples will be used to learn the effects of disinfection on microbial communities and pathogens. Some bacteriophages may be associated with the transfer of AMR genes. WGS of environmental samples and metagenomic analysis will be used to understand transmission of AMR by bacteriophage.
Objective 3: Induced toxins and AMR proteins may be identified by mass spectrometry (MS) and analysis. MS will be employed to determine conditions that cause the expression of toxins and AMR proteins. Also, mass spectrometry imaging and proteomic analysis will be used to spatially map Shiga toxin-producing Escherichia coli (STEC) biofilm-associated molecules.
Objective 4: Campylobacters may potentially be detected in poultry products through use of liquid crystal system methodology. Monoclonal antibodies (mAb) that bind both C. jejuni and C. coli will be evaluated for sufficient selectivity and sensitivity. Using these mAb, a liquid crystal detection platform will be developed where the mAb-Campylobacter complex causes an observable deformation of lyotropic liquid crystals. The expression of certain LOS by C. jejuni may act as biosensors to detect Shiga toxins. In vitro binding assays will be used to identify C. jejuni strains that express LOS that mimic P-blood group antigens and quantify Shiga toxin (Stx)-binding ability.
Objective 5: Stx-converting bacteriophage released by STEC may infect other bacteria to form new pathogens. Phages containing Stx genes will be used to lysogenize other E. coli. Bacteriophage cocktails may be developed into biocontrol alternatives to antibiotics. Lytic phages will be developed into multi-bacteriophage cocktail formulae for the reduction of target pathogens.
