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

Location: Produce Safety and Microbiology Research

2024 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 progress for project 2030-42000-055-000D, titled, “Elucidating the Factors that Determine the Ecology of Human Pathogens in Foods”, which started in February 2021. Under Sub-objective 1.A, progress continued in developing source attribution models for Campylobacter infections. The genomic sequences of Campylobacter coli from human and animal sources were used to test Machine Learning (ML) methods for probabilistic assignment of human cases of campylobacteriosis to possible source reservoirs. Genetic variation associated with adaptation to the most recent host was targeted using ML and probabilistic models to estimate the relative importance of different disease reservoirs. Probabilistic attribution identified poultry as the primary source of human clinical infections of C. coli in Peru over the past five years. To address Sub-objective 2.A, progress was obtained on the sampling of Salmonella enterica from major fresh produce growing regions for export to the United States. Fecal samples were collected from livestock in small rural farms, near rivers used for irrigation of agricultural fields for export produce, in Northwestern Mexico. The samples were subjected to enrichment and selective isolate recovery using different types of media to avoid culture bias. Long read sequencing identified Salmonella serovars Poona, Anatum, Minnesota and Typhimurium, which were previously implicated in multistate outbreaks in the United States linked to imported fresh produce. Preliminary analysis of functional gene categories in these serovars indicated a relatively large number of genes involved in DNA replication, recombination and repair and carbohydrate transport and metabolism within these serovars. Ongoing pangenome analyses are currently investigating the presence or absence variation of genes implicated in virulence and antimicrobial resistance in the recovered Salmonella isolates. Subsequent sampling of agricultural irrigation water revealed that established culturing and biochemical methods inaccurately speciated Salmonella and Citrobacter due to the shared environmental niches. Comparative genomic examination of the isolates from irrigation water identified Citrobacter werkmanii, an emerging and opportunistic pathogen, and documented for the first time a group of C. werkmanii, displaying highly pathogenic and multidrug-resistant genetic profiles. For Sub-objective 2.B, ARS researchers in Albany, California, continued metagenomic assemblies and analysis on 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 analysis should provide insight into the effects of the disinfectant calcium hypochlorite on the bacterial pathogens and the irrigation water microbial community. For Sub-objective 2.C, ARS researchers continued to investigate the role of bacteriophages in the microbial population of agricultural samples. The bacteria and viruses from different environmental samples, including animal feces, soil, and water, were collected and subjected to metagenomic sequencing. The composition and antibiotic-resistant genes (ARG) profile of bacterial and viral populations were determined by different bioinformatic pipelines, respectively. The analysis revealed that the viral population had much broader diversity than the bacterial population. Additionally, the antibiotic-resistant bacteria shared low similarities among different sample types. However, there were significant similarities in the profiles of viruses carrying ARGs across different sample types. Future studies will investigate the potential interactions and ARG transfer between bacterial and viral populations in agricultural environments. Under Objective 3, progress was made on Sub-objective 3A. Colicin immunity proteins (ImmE3, ImmE8, ImmD) whose genes are encoded in small plasmids were detected and identified in six of eight Shiga toxin-producing Escherichia coli (STEC) that had previously been genome sequenced. In addition to Shiga toxin, colicin immunity proteins were identified using antibiotic induction, MALDI-TOF-TOF mass spectrometry and top-down proteomic analysis. Five Salmonella Infantis strains (from FSIS and Clay Center having the pESI megaplasmid) were also tested for expression of plasmid-encoded proteins by exposure to different antibiotics. Unfortunately, no proteins were detected beyond highly conserved host proteins. Further analysis is ongoing. Predicted protein structures using Alphafold2 were obtained for 228 proteins of the pESI megaplasmid of SEE Infantis. The predicted structure of the heterotrimer (A:B:C) of cytolethal distending toxin (Cdt) was also obtained using Alphafold. Unfortunately, several attempts to identify Cdt by mass spectrometry from E. coli O157:H- strain 493/89 (H. Karch) were unsuccessful due to the lack of an inducible promoter or de-repressor. Progress was also made on Sub-objective 3B. Experiments continued on MALDI imaging of biofilms of pathogenic bacteria. A wild-type pathogenic strain was cultured for three to 13 days in broth culture at different incubation temperatures with different slides resulting in biofilm production at the air-liquid interface of a half-submerged ITO-coated glass slide. The strongest biofilm production occurred at 28°C for eight days. In addition, a protocol was successfully developed to analyze STEC strains for production of the B-subunit of Shiga toxin 2 (B-Stx2) using MALDI-imaging instrument which has significant design differences to that of the previous MALDI-TOF-TOF instrument for tandem mass spectrometry. Characteristic low and high energy fragment ions of B-Stx2 allow rapid top-down proteomic identification after culturing with antibiotic induction. In support of research conducted by the ARS Eastern Regional Research Center, ARS researchers in Albany, California, performed MALDI-TOF-TOF analysis of oligosaccharides from edible berries. In support of Objective 4, improvements on the Campylobacter detection threshold were obtained when testing chicken meat rinsate with the liquid crystal-based biosensor. By using prototype manual instrumentation, ARS researchers tested the use of various concentrations of surfactants for increasing the assay stringency by examining the aggregation of microspheres conjugated to an anti-Campylobacter C731 monoclonal antibody, generated by the PSM Research Unit. The research data was transferred by ARS to the industry stakeholders for developing an alternative conjugation strategy of the C731 monoclonal antibody to the magnetic microspheres for improving shelf-life of the reagent when employing the lyotropic liquid crystal detection platform for high-throughput onsite sampling in food processing facilities. Under Objective 5, research continued on the genomic comparison of lysogenic phages from different bacterial hosts. The lysogenic phage genomes were extracted from diverse STEC genomes using bioinformatic tools. The phylogenetic analysis of lysogenic phage genomes was conducted to determine the genomic difference and their correlation with bacterial hosts. The results showed that the lysogenic phages detected in the STEC O45, O111, and O121 strains were generally more conserved than those detected from other serotypes, such as O145 and O157. Moreover, several STEC strains contained multiple lysogenic phages carrying virulence genes with different genomic features. For lytic phage research, a phage-derived protein, depolymerase enzyme, was identified in a lytic phage UDF157lw with the characteristics of producing large plaques specifically targeting E. coli O157:H7 strains. Compared to the phage without encoding the depolymerase gene, UDF157lw had significant anti-biofilm activity, contributing to the dispersal of 48-h biofilm of E. coli O157:H7. The protocol of protein expression is being established to develop an anti-biofilm compound. Furthermore, the three-phage cocktail, EcoOut, was superior to a commercial phage cocktail product in mitigating E. coli O157:H7 in irrigation water, mung beans, and biofilm on a stainless surface; the information has been included in the provisional Patent application. In addition, ARS researchers in Albany, California, continued the encapsulation study to optimize the method for phage encapsulation with animal feed to facilitate the downstream application of E. coli and Salmonella phages. Additionally, a new member of the Tequintavirus phage was isolated and characterized for its antimicrobial potential against multidrug-resistant Salmonella Infantis.

Accomplishments
1. Accurate assessment of virulent Citrobacter species. The genomic similarity and shared environmental niches between Salmonella and Citrobacter pose a challenge in food safety since the established culturing and biochemical methods can misidentify both bacterial pathogens. By use of an ultrafiltration method with agricultural irrigation water in conjunction with high-resolution genome sequencing and bioinformatics, ARS researchers in Albany, California, identified the emerging and opportunistic pathogen Citrobacter werkmanii, which was initially mistaken for Salmonella by use of traditional methods. Subsequent comparative genomic analyses have documented for the first time a group in C. werkmanii strains displaying highly virulent and multidrug-resistant genetic profiles, and these findings provide essential data to regulatory agencies and the food processing industry on key molecular markers in C. werkmanii with a significant potential to cause illnesses in humans. Consequently, the insights gained from these analyses will enable the improved development of robust molecular assays for the rapid and accurate identification of closely related pathogens in agricultural samples.

2. New pipeline determined the microbial composition and antibiotic resistance genes (ARG) transfer within agricultural environments. The emergence and evolution of antibiotic-resistant bacteria have caused a serious food safety issue in the United States. ARS researchers at Albany, California, have established a pipeline of metagenomic sequencing and downstream bioinformatic analysis to determine the composition and antibiotic resistance genes (ARG) profile of microbial populations within agricultural samples. This pipeline provided a valuable method for researchers to investigate the microbial components and underlying ARG transfer among different microorganisms to prevent the spread of ARG in agricultural environments.

3. Plasmid-encoded colicin immunity proteins detected in Shiga toxin-producing E. coli (STEC) by top-down proteomic analysis. Colicins are bacterial proteins that function to destroy the DNA (or outer membranes) of neighboring bacteria that occupy the same environmental niche, providing a competitive survival advantage for the bacteria that produce the colicins. These bacteria also produce immunity proteins that neutralize the colicins they are producing. Immunity proteins for colicin E3, E8 and D were detected and identified in six of eight STEC strains by MALDI-TOF-TOF mass spectrometry and top-down proteomic analysis. SOS/LexA boxes were detected upstream of the colicin/immunity genes in small plasmid genomes consistent with gene repression by LexA until induction by DNA-damaging antibiotics causes self-cleavage of LexA and gene de-repression. Three of six strains having immunity proteins were missing the colicin/immunity genes. However, further plasmid DNA sequencing showed that the genes were encoded on small plasmids that were missed during initial genomic sequencing. Although bacterial colicins are not known to be harmful to mammalian cells, these proteins can increase the survival of pathogenic bacteria and thus the likelihood of their causing future outbreaks of foodborne illness.

4. Encapsulation provides additional protection for bacteriophage application in adverse environments. Although lytic phages are promising antimicrobial agents to improve the existing measurements in the food industry, maintaining the viability of these bacteriophages to increase bacterial encounters in adverse environmental conditions is critical. ARS researchers at Albany, California, have developed an alginate-based encapsulation method to improve the stability of a bacteriophage cocktail under simulated gut environments. This technology also provided an alternative delivery method for phages to reduce pathogen levels residing in the gastrointestinal tract of animals, such as chickens, and further prevent the contamination of final food products.

Review Publications
Schiaffino, F., Parker, C.T., Paredes Olortegui, M., Pascoe, B., Manzanares Villaneuva, K., Garcia Bardales, P.F., Mourkas, E., Huynh, S., Penataro Yori, P., Romaina Cachique, L., Gray, H.K., Salvatierra, G., Silva Delgado, H., Sheppard, S.K., Cooper, K.K., Kosek, M.N. 2024. Genomic resistant determinants of multidrug-resistant Campylobacter spp. isolates in Peru. Journal of Global Antimicrobial Resistance. 36:309-318. https://doi.org/10.1016/j.jgar.2024.01.009.
Liao, Y., Ho, K., Zhang, Y., Salvador, A., Wu, V.C. 2024. A new Rogue-like Escherichia phage UDF157lw to control Escherichia coli O157:H7. Frontiers in Microbiology. 14. Article 1302032. https://doi.org/10.3389/fmicb.2023.1302032.
Fagerquist, C.K., Shi, Y., Park, J. 2023. Unusual modifications of protein biomarkers expressed by plasmid, prophage, and bacterial host of pathogenic Escherichia coli identified by top-down proteomic analysis. Rapid Communications in Mass Spectrometry. 38(1). Article e9667. https://doi.org/10.1002/rcm.9667.
Goforth, M., Cooper, M.A., Oliver, A.S., Pinzon, J., Skots, M., Obergh, V., Suslow, T.V., Flores, G.E., Huynh, S., Parker, C.T., Mackelprang, R., Cooper, K.K. 2024. Bacterial community shifts of commercial apples, oranges, and peaches at different harvest points across multiple growing seasons. PLOS ONE. 19(4). Article e0297453. https://doi.org/10.1371/journal.pone.0297453.
Parker, C.T., Villafuerte, D.A., Miller, W.G., Huynh, S., Chapman, M.H., Hanafy, Z., Jackson III, J.H., Miller, M.A., Kathariou, S. 2024. Genomic analysis points to multiple genetic mechanisms for non-transformable Campylobacter jejuni ST-50. Microorganisms. 12(2). Article 327. https://doi.org/10.3390/microorganisms12020327.
Goforth, M., Obergh, V., Park, R., Porchas, M., Crosby, K.M., Jifon, J.L., Ravishankar, S., Brierley, P., Leskovar, D.L., Turini, T.A., Schultheis, J., Coolong, T., Miller, R., Koiwa, H., Patil, B.S., Cooper, M.A., Huynh, S., Parker, C.T., Guan, W., Cooper, K.K. 2024. Bacterial diversity and composition on the rinds of specific melon cultivars and hybrids from across different growing regions in the United States. PLOS ONE. 19(4). Article e0293861. https://doi.org/10.1371/journal.pone.0293861.
Schiaffino, F., Parker, C.T., Garcia-Bardales, P.F., Huynh, S., Manzanares Villaneuva, K., Mourkas, E., Pascoe, B., Penataro Yori, P., Paredes-Olortegui, M., Houpt, E.R., Liu, J., Cooper, K.K., Kosek, M.N. 2024. Novel rpsK / rpsD primer-probe assay improves detection of Campylobacter jejuni and Campylobacter coli in human stool. PLOS Neglected Tropical Diseases. 18(3). Article e0012018. https://doi.org/10.1371/journal.pntd.0012018.
Amezquita-Lopez, B.A., Soto-Beltran, M., Lee, B.G., Bon-Haro, E.F., Lugo-Melchor, O.Y., Quinones, B. 2024. Virulence and antimicrobial resistance profiles of Shiga toxin-producing Escherichia coli from river water and farm animal feces near an agricultural region in Northwestern Mexico. Microbiology Research. 15(1):385-403. https://doi.org/10.3390/microbiolres15010026.
Zhang, Y., Kitazumi, A., Liao, Y., de los Reyes, B.G., Wu, V.C. 2023. Metagenomic investigation reveals bacteriophage-mediated horizontal transfer of antibiotic resistance genes in microbial communities of an organic agricultural ecosystem. Microbiology Spectrum. 11(5). Article e00226-23. https://doi.org/10.1128/spectrum.00226-23.
Aguirre-Sanchez, J.R., Quinones, B., Ortiz-Muñoz, J.A., Prieto-Alvarado, R., Vega-Lopez, I.F., Martinez-Urtaza, J., Lee, B.G., Chaidez, C. 2023. Comparative genomic analyses of virulence and antimicrobial resistance in Citrobacter werkmanii, an emerging opportunistic pathogen. Microorganisms. 11(8). Article 2114. https://doi.org/10.3390/microorganisms11082114.
Carter, M.Q., Quinones, B., Laniohan, N.S., Carychao, D.K., Pham, A.C., He, X., Cooley, M. 2023. Pathogenicity assessment of Shiga toxin-producing Escherichia coli strains isolated from wild birds in a major agricultural region in California. Frontiers in Microbiology. 14. Article 1214081. https://doi.org/10.3389/fmicb.2023.1214081.
Park, J., Fagerquist, C.K. 2024. Exploring the fragmentation efficiency of proteins analyzed by MALDI-TOF-TOF tandem mass spectrometry using computational and statistical analyses. PLOS ONE. 19(5). Article e0299287. https://doi.org/10.1371/journal.pone.0299287.
Zhang, Y., Sharma, S., Tom, L., Liao, Y., Wu, V.C. 2023. Gut phageome—An insight into the role and impact of gut microbiome and their correlation with mammal health and diseases. Microorganisms. 11(10). Article 2454. https://doi.org/10.3390/microorganisms11102454.
Carter, M.Q., Quinones, B., He, X., Pham, A.C., Carychao, D.K., Cooley, M., Lo, C., Chain, P.S., Lindsey, R.L., Bono, J.L. 2023. Genomic and phenotypic characterization of Shiga toxin-producing Escherichia albertii strains isolated from wild birds in a major agricultural region in California. Microorganisms. 11(11). Article 2803. https://doi.org/10.3390/microorganisms11112803.
Zhang, Y., Chu, M., Liao, Y., Salvador, A., Wu, V.C. 2024. Characterization of two novel Salmonella phages having biocontrol potential against Salmonella spp. in gastrointestinal conditions. Scientific Reports. 14. Article 12294. https://doi.org/10.1038/s41598-024-59502-9.
Miller, W.G., Lopes, B.S., Ramjee, M., Jay-Russell, M., Chapman, M.H., Williams, T.G., Wood, D.F., Gruntar, I., Papic, B., Forbes, K.J. 2024. Campylobacter devanensis sp. nov., Campylobacter porcelli sp. nov., and Campylobacter vicugnae sp. nov., three novel Campylobacter lanienae-like species recovered from swine, small ruminants, and camelids. International Journal of Systematic and Evolutionary Microbiology. 74(6). Article 006405. https://doi.org/10.1099/ijsem.0.006405.