Project : USDA ARS

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

Location: Produce Safety and Microbiology Research

2025 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 report documents FY 2025 progress for project 2030-42000-055-000D, “Elucidating the Factors that Determine the Ecology of Human Pathogens in Foods”, which began in February 2021. Under Sub-objective 1.A, progress continued with the sequencing, assembly, annotation and submission of over 500 Campylobacter genomes isolated from human and animal sources into publicly available databases. Genetic variation of Campylobacter associated with adaptation to the most recent host was targeted using machine learning and probabilistic models to estimate the relative importance of different disease reservoirs for human infection. Probabilistic attribution identified poultry as the primary source of human clinical infections of Campylobacter in Peru over the past five years. To address Sub-objective 2.A, progress continued on the sampling of Salmonella enterica from Mexican fresh produce to be exported to the United States. Samples were collected from livestock in small rural farms, near rivers used for irrigation of agricultural fields and were subjected to enrichment and selective isolate recovery using different types of media to avoid culture bias. Long read sequencing identified multiple Salmonella serovars that were all previously implicated in multistate outbreaks in the United States. The results analysis of functional gene categories revealed serovar specific determinants in the examined Salmonella isolates, recovered from livestock in farms proximal to the fresh produce fields. In particular, the S. Weltvreden and S. Typhimurium strains were found to harbor the largest gene repertoire related to carbohydrate transport and metabolism, which would consequently confer fitness traits enhancing the pathogen’s survival in the domestic animal and environmental niches. Moreover, Salmonella strains belonging to serovars Minnesota, Poona, Typhimurium, Havana, and Weltvreden were found to harbor membrane systems involved in the transport of metal ions, conferring a dual functional role in resistance to heavy metals as well as resistance to antimicrobials. Heavy metal resistance in Salmonella can lead to multidrug-resistant strains with an impact on public health by contributing to the spread of these pathogens in the environment as well as posing a significant threat to public health. For Sub-objective 2.B, ARS researchers continued metagenomic assemblies of whole metagenomic sequences from samples of irrigation water that were treated or not treated with the disinfectant calcium hypochlorite. The irrigation samples had been spiked with different levels of foodborne pathogens, Shiga toxin-producing E. coli (STEC) and Salmonella. The metagenomic analysis was performed using the University of Arizona high performance computer cluster (HPC). The analysis will provide insight into the effects of the disinfectant calcium hypochlorite on the bacterial pathogens and the irrigation water microbial community. In support of Sub-objective 2.C, ARS researchers continued to investigate the potential interactions and antibiotic-resistant genes (ARG) transfer between bacteria and bacteriophages in agricultural environments. The bacterial and viral populations from animal fecal and agricultural environmental samples on an organic farm were subjected to metagenomic sequencing and prediction of phage-bacterial interactions via bioinformatic tools. The predicted phage-bacterial interactions on bipartite ARG transfer indicated that phages belonging to the order Caudovirales, particularly in the Siphoviridae family, contained diverse ARG types in different samples. Their interactions with various bacterial hosts further implied the important role of bacteriophages in ARG transmission across bacterial populations. Future studies will investigate the potential mechanisms of phage-mediated ARG transmission and their correlation with the resistome evolution of certain antimicrobial resistance (AMR) pathogens in natural agricultural environments. Under Sub-objective 3.A, ARS researchers analyzed for the presence of colicin plasmids in Shiga toxin-producing E. coli (STEC) and Salmonella enterica by top-down proteomic analysis using MALDI-TOF-TOF as well as high resolution Orbitrap mass spectrometry. Genomically and non-genomically sequenced strains were analyzed. If a colicin was detected by top-down proteomic analysis, but its gene (and the plasmid) was not sequenced or was overlooked during genomic sequencing, small plasmid sequencing was performed to confirm top-down identification of the colicin/immunity gene(s) as well as the SOS gene regulation upstream of these genes. ARS researchers found that some pathogenic strains have multiple plasmids, each with their own colicin nuclease (E3, E8 or D). A pore-forming colicin plasmid was also identified from the identification of a 7.8 kDa protein biomarker it expressed. Stx1 and Stx2 were also detected as well as bacteriophage proteins, some of which were only detected after chemical reduction, suggesting that such proteins were bound to a larger bacteriophage complex via disulfide bonds. Progress was made on Sub-objective 3.B. csgA gene knockouts of a biofilm-producing pathogenic STEC strain were constructed. CsgA is an amyloid protein, and the primary component of curli which are thin fiber-like structures that extend from the surface of the bacterial cell anchoring it to biotic and abiotic surfaces. Curli and biofilm formation are often linked by gene regulation. The csgA knockout strain was cultured in broth for several days at a temperature (28 degrees C) that maximizes biofilm and curli formation. Biofilm formation occurs on a half-submerged ITO-coated glass slide primarily at the air-liquid interface. The biofilm-coated slide was analyzed using MALDI imaging mass spectrometry. Unlike the wild-type strain, the knockout strain showed the complete absence of the CsgA protein as expected. Analysis is on-going to distinguish other differences in biofilm features between knockout versus wild-type pathogenic strains. To address Objective 4, several tests were conducted to improve the Campylobacter detection threshold when testing chicken meat rinsate with the liquid crystal-based biosensor on the development of a commercially viable Campylobacter assay. By using prototype manual instrumentation, ARS researchers optimized the use of various concentrations of surfactants for increasing the assay stringency when using the anti-Campylobacter C731 monoclonal antibody. The research data, transferred by ARS researchers to industry stakeholders, provided supporting evidence for the replacement of the conjugation method of the C731 monoclonal antibody to the magnetic microspheres for improving the shelf-life of the reagent, a critical issue that would need to be addressed when commercializing the detection assay. Additionally, ARS researchers provided industry stakeholders some guidance on the limitations of the current version of the assay that resulted in reduced detection sensitivities in the presence of background microflora when testing enrichments derived from chicken meat rinsates. ARS researchers also submitted recommendations to industry stakeholders on future studies aimed at evaluating alternative antibodies and enrichments modifications when designing the optimized method with the stakeholder’s new automated detection platform for use by food processing facilities. In support of Objective 5, research continued to understand the role of lysogenic phages in the emergence and evolution of foodborne pathogens. Salmonella Infantis strains isolated from the poultry production environment were subject to antimicrobial susceptibility tests and whole-genome sequencing. The bioinformatic analysis revealed that each multidrug-resistant (MDR) S. Infantis strain contained one mega-plasmid. Most ARGs were located in the mega-plasmids and bordered by diverse mobile genetic elements and prophages, in particular. One intact prophage within S. Infantis mega-plasmid that contained six intact ARGs was detected. This prophage was distinct from other prophage genomes and classified into a single clade based on the phylogenetic tree of prophages within mega-plasmid, indicating the potential role of prophages carrying ARGs within mega-plasmids in contributing to the emergence of new MDR pathogens. For lytic phage research, a phage-derived protein, endolysin, was identified in two E. coli-infecting phages (Ro145clw and G157lw) and a Salmonella-infecting phage (SIA3lw). Protein expression was conducted to develop an alternative antimicrobial agent. The protocol of protein expression was further optimized through the endolysin expression process. One newly isolated phage specific to O103 was characterized with depolymerase enzyme activity, demonstrating antibiofilm activity in degrading extracellular polymeric substances (EPS) of STEC O103 biofilm. Another newly isolated lytic phage with host specificity for O157 also demonstrated anti-biofilm potential against STEC O157. All these phages can be used to further enhance the antimicrobial and antibiofilm efficacy of our patented three-phage cocktail, EcoOut. Moreover, a new Epseptimavirus phage was isolated to control antibiotic-resistant S. Infantis. Some bacteriophage-insensitive mutants (BIMs) obtained after single phage treatment became sensitive to a specific antibiotic, to which the wild-type Salmonella strain was resistant. Most importantly, all selected BIMs were susceptible to infection by a different phage. In addition, ARS researchers continued to test various polymers (gellan gum, emulsion, etc.) to improve the efficiency and stability of the encapsulated phages and enhance phage delivery and effectiveness in different conditions.

Accomplishments
1. Adaptation and survival traits in Salmonella from surface waters near leafy greens. Non-typhoidal Salmonella enterica is the costliest causative agent of bacterial foodborne disease in the United States, and the economic burden of non-typhoidal Salmonella is estimated at $17.1 billion. Salmonella enterica serovar Infantis (S. Infantis) is a very common serotype associated with human illness linked to the eating contaminated leafy greens in the United States. ARS researchers in Albany, California, conducted a comparative genomic analysis of S. Infantis recovered from water sources adjacent to leafy greens farms in Central California strains. The computer-based analysis automatically identified adaptation and survival determinants in S. Infantis strains recovered from water sources adjacent to leafy greens farms in Central California. The scientific data showed indications of persistence of Salmonella traits associated with growth, colonization, and resistance to stress and to harmful compounds such as antibiotics and metals. These findings have set a precedent for the development of new predictive applications, targeting the contributing factors that promote enhanced fitness of Salmonella in these agricultural environments.

2. A new deep-learning-based tool predicts bacteriophage lifecycles via DNA sequences. Bacteriophages (phages) are viruses that infect bacteria. Phages are classified into lytic and lysogenic life cycles. Lytic phages infect and promptly kill bacteria, while lysogenic phages integrate into the bacteria and often provide the bacteria with toxins that increase their virulence in humans. Identifying the lifecycle utilized by a phage is essential in determining their practical use for stakeholder applications, in which, lytic phage are useful to kill human pathogens and lysogenic phage are problematic. ARS researchers at Albany, California, developed a deep learning-based bioinformatic tool—DeepPL— to predict phage lifecycles via phage DNA sequences. The test results showed that DeepPL is very accurate (accuracy of 94.65%), sensitive (sensitivity of 92.24%), and specific (specificity of 95.91%). DeepPL also performs better on phage lifecycle classification than currently available tools. This tool provided a valuable method for researchers to create phage applications to kill bacterial pathogens that stakeholders can utilize in agricultural, clinical, and processing settings.

U.S. DEPARTMENT OF AGRICULTURE

Produce Safety and Microbiology Research: Albany, CA