Research Project: Elucidating the Factors that Determine the Ecology of Human Pathogens in Foods

Location: Produce Safety and Microbiology Research

2021 Annual Report

Objectives
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.

Progress Report
This is the initial report for project 2030-42000-055-00D, “Elucidating the Factors that Determine the Ecology of Human Pathogens in Foods” which started in March 2021 and expands research from project 2030-42000-051-00D “Molecular Identification and Characterization of Bacterial and Viral Pathogens Associated with Foods." For Sub-objective 1.A, progress was made in determining the potential source of Campylobacter infections among infants in low- and middle-income countries. Sampling protocols and DNA extraction methods were determined. Samples were collected, and Campylobacter was cultured from chickens sold by poultry vendors and household animals from the community associated with this project in Iquitos Perú. In support of Sub-objective 1.C, significant progress was made in determining observable genomic differences between Campylobacter isolates associated with post-infection irritable bowel syndrome (PI-IBS) and strains not linked to PI-IBS. A collection of 120 DNA samples from clinical Colorado Campylobacter, representing most Campylobacter infections from that state in 2020, were whole genome sequenced and assembled. The whole genome draft sequences were deposited into the PubMLST database. Sequence data analysis provided discriminatory features, including serotypes, lipooligosaccharide classes and multilocus sequence types for each isolate. Additionally, a collaboration with scientists at University of Bath in the United Kingdom was expanded to examine a separate C. jejuni strain set associated with PI-IBS. For Sub-objective 1.E, progress was made by generating antibiotic resistance-marked Campylobacter coli and Campylobacter jejuni recipient strains. The recipient strains will be used to test whether mobile elements can be transferred from a donor C. coli strain into a naïve recipient strain via transmissible plasmids. To address Sub-objective 2.A, a sampling study was initiated to assess the composition of fecal microbiomes and to determine the role of indigenous fauna in the prevalence of Salmonella enterica in agricultural regions that export produce. Fecal samples were collected from various locations proximal to agricultural fields in the Culiacan Valley, Mexico, which has become the single most important agricultural region for various fresh produce commodities exported into the United States. Initial analysis of the metagenomics data, generated using a high-throughput sequencing platform, examined the relative abundance of operational taxonomic units. The findings revealed a high abundance of Eubacteriaceae and Bacteroidales in the cow and pig samples and Enterobacterales in the chicken samples. Ongoing research is currently assessing the sequence reads in low abundance and additional analyses are being conducted to evaluate phylogenetic relationships, with the goal of identifying the prevalence of Salmonella in these fecal samples. By testing various selective enrichment media, S. enterica isolates were recovered from the fecal samples, and whole genome sequencing is currently being performed to identify virulence factors and antimicrobial resistance genes in these isolates. For Sub-objective 2.B, significant progress was made to determine the effects on the natural bacterial communities of the leafy greens, rhizosphere (soil associated with the plant roots) and topsoil (soil not associated with the plants) following the treatment of irrigation water with peracetic acid (PAA) 21 days prior to leafy green harvesting. Over 1,200 samples were collected, processed, and DNA sequenced for the study to compare impacts on the microbiome community composition, using PAA treated irrigation water versus untreated irrigation water. Preliminary results suggest that there are major changes to the bacterial communities of the leafy greens and rhizosphere, but very little change to bacterial communities of the topsoil. In support of Sub-objective 2.C, research began on the study of microbiomes in produce production environments to identify the role of bacteriophages associated with antimicrobial resistance in bacteria. Preliminary metagenomic results showed that bacteriophages belonging to the Caudovirales order could infect Shiga toxin (Stx)-producing E. coli (STEC) and Salmonella. This group of bacteriophages was found in all sample types. Additionally, antimicrobial-resistant genes were present in these bacteriophage genomes, indicating that these bacteriophages may play a critical role in transferring antimicrobial-resistant genes among bacteria. Further metagenomic analyses are in progress. Under Objective 3, progress was made on Sub-objective 3.A involving top-down identification of Stx produced from STEC strains (from major outbreaks in Arizona and Belgium) using matrix assisted laser desorption/ionisation - time of flight/ time of flight mass spectrometry (MALDI-TOF-TOF MS). Expression of the stx gene was induced by antibiotic exposure and the Stx B-subunit was identified from the supernatant of an unfractionated sample and analyzed by tandem mass spectrometry. Progress was made on Sub-objective 3.B. A MALDI sprayer was installed and tested, and development protocols were initiated for using this MALDI sprayer in MALDI MS imaging of biofilms. In support of Sub-objective 4.A, progress was made on the characterization of biomarkers in C. jejuni and C. coli for the development of a liquid crystal-based biosensor as an automated detection platform for onsite monitoring of pathogens. For the specific detection of C. jejuni and C. coli, monoclonal antibodies C731, C740, and C791 were evaluated by conducting inclusivity and exclusivity tests to assess the specificity for detecting the targeted Campylobacter species. For the inclusivity tests, a larger and comprehensive subset of strains from C. jejuni and C. coli were evaluated from food or food-related sources, including poultry, cattle, swine, and food processing facilities. The results from these tests showed that specific signals detecting targeted species were 10-fold higher when compared to non-targeted strains belonging to the Campylobacteraceae and Enterobacteriaceae families. Ongoing studies are currently assessing the binding of the anti-Campylobacter monoclonal antibodies C731, C740, and C791 by using both titrated antigen and purified antibody independently. In addition, the monoclonal antibodies are being evaluated by the two-bead method for formation of the sandwich complex with the chromogenic liquid crystal system in the presence of the targeted C. jejuni, C. coli and C. lari strains from various geographical locations and sources. In support of Objective 5, research began to investigate the role of lytic bacteriophages against their host strains and other serogroups. Bacteriophage cocktails using various fully characterized lytic phages are being developed. A bacteriophage cocktail for STEC O157, containing different bacteriophages, was formulated based on different genetic features, host range, and antimicrobial activities. The preliminary results showed that the bacteriophage cocktail's antimicrobial effect was significantly increased compared to that of a single bacteriophage, and the chance for bacteria to develop bacteriophage resistance was significantly reduced. To improve the efficiency of bacteriophage application under adverse environmental conditions, encapsulation technology is being developed.

Accomplishments
1. Campylobacter jejuni genotypes associated with post-infection irritable bowel syndrome. Irritable bowel syndrome (IBS) is a chronic, disabling gastrointestinal disorder that affects up to 15% of people worldwide. Acute gastroenteritis due to Campylobacter jejuni infections is a risk factor for the development of chronic IBS post-infection. ARS researchers at Albany, California, in collaboration with scientists at the University of Bath in the United Kingdom, the Mayo Clinic in Minnesota, and Iowa State University, have identified genetic markers within strains of C. jejuni associated with post-infection IBS (PI-IBS) using genome-wide association studies. Phenotypic analyses of PI-IBS associated strains demonstrated that they had greater virulence properties than strains not associated with PI-IBS. These findings pave the way for the identification of high-risk C. jejuni infections that can be amenable for preventative strategies and will lead to additional studies to significantly advance the understanding of PI-IBS pathophysiology.

Review Publications
Zhang, Y., Liao, Y., Salvador, A., Lavenburg, M.V., Wu, V.C. 2021. Characterization of two new Shiga toxin-producing Escherichia coli O103-infecting phages isolated from an organic farm. Microorganisms. 9(7):1527. https://doi.org/10.3390/microorganisms9071527.
Parisi, A., Chiara, M., Caffara, M., Mion, D., Miller, W.G., Caruso, M., Manzari, C., Florio, D., Capozzi, L., D'Erchia, A.M., Manzulli, V., Zanoni, R.G. 2021. Campylobacter vulpis sp. nov. isolated from wild red foxes. Systematic and Applied Microbiology. 44:3. https://doi.org/10.1016/j.syapm.2021.126204.
Cornelius, A.J., Huq, M., On, S.L., French, N.P., Vandenberg, O., Miller, W.G., Lastovica, A., Istivan, T., Biggs, P.J. 2021. Genetic characterization of Campylobacter concisus: strategies for improved genomospecies discrimination. Systematic and Applied Microbiology. 44. Article 126187. https://doi.org/10.1016/j.syapm.2021.126187.
Parker, C., Cooper, K.K., Schiaffino, F., Miller, W.G., Huynh, S., Olortegui, M., Bardales, P.G., Trigoso, D.R., Kosek, M.N. 2021. Genomic characterization of Campylobacter jejuni adapted to the guinea pig (Cavia porcellus) host. Frontiers in Cellular and Infection Microbiology. 11. Article 607747. https://doi.org/10.3389/fcimb.2021.607747.
Parker, C., Huynh, S., Alexander, A., Oliver, A.S., Cooper, K.K. 2021. Genomic characterization of Salmonella typhimurium DT104 strains associated with cattle and beef products. Pathogens. 10(5):529. https://doi.org/10.3390/pathogens10050529.
Quinones, B., Yambao, J.C., Deguzman, V., Lee, B.G., Medin, D. 2021. Genomic analysis of high copy-number sequences for the targeted detection of Listeria species using a flow-through surveillance system. Archives Of Microbiology. 203:3667-3682. https://doi.org/10.1007/s00203-021-02388-2.
Fagerquist, C.K., Rojas, E.N. 2021. Identification of antibacterial immunity proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and top-down proteomic analysis. Journal of Visualized Experiments. 171. Article e62577. https://doi.org/10.3791/62577.
On, S.L., Miller, W.G., Yee, E., Sturgis, J., Patsekin, V., Lindsay, J.A., Robinson, J.P. 2021. Discrimination of shellfish-associated Arcobacter species by Elastic Light Scatter analysis. Current Research in Microbial Science. 2. Article 100033. https://doi.org/10.1016/j.crmicr.2021.100033.