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

Location: Produce Safety and Microbiology Research

2022 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
For Sub-objective 1.A, progress was made in determining the potential source of Campylobacter infections among infants in low- and middle-income countries. Campylobacter was cultured and sequenced from stool samples from infants enrolled in the study. Also, total DNA was extracted from the stool samples and sequenced to determine the intestinal microbial communities of the infants. Campylobacter was isolated and sequenced from chickens sold by poultry vendors and household animals from the community associated with this project in Iquitos, Perú. Core genome multilocus sequence typing (cgMLST) and antimicrobial resistance (AMR) markers were determined for all samples. 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. For Sub-objective 2.A, sampling was expanded to include irrigation water from major rivers close to agricultural fields in Northwestern Mexico, which has become an important region for the production of fresh produce commodities imported into the United States. The combinatorial use of a size-exclusion ultrafiltration method was employed with an enrichment step and selective media to enable the improved detection of Salmonella when present at low concentrations in river water samples. Subsequent analysis of water parameters indicated a negative effect of pH and salinity and a positive effect of river water temperature on Salmonella levels. Using genome sequencing and bioinformatics tools, molecular subtyping revealed Oranienburg, Anatum and Saintpaul were the most predominant S. enterica serovars in river water. The classification of the recovered S. enterica isolates based on genetic differences showed variability in genes required for S. enterica adaptation and survival in the environment. Future studies will further evaluate fitness traits in S. enterica that confer increased survival in surface water habitats. Additional studies were also conducted to evaluate the prevalence of S. enterica in animal fecal samples collected from small rural farms close to fresh produce fields. Preliminary analysis of genome sequencing identified S. Weltevreden, S. Typhimurium, S. Sandiego, and S. Havana, as the predominant serovars detected in pig fecal samples. Subsequent characterization of antimicrobial resistance genes in these pig S. enterica isolates revealed multidrug resistance to macrolides, tetracycline, and cephalosporins, as well as aminoglycosides, fluoroquinolone and phenicols. For Sub-objective 2.B, researchers at Albany, California, performed whole metagenomic sequencing on irrigation samples. Metagenomic assemblies were performed on these sequences to determine the microbial communities for each sample. In support of Sub-objective 2.C, research continued to investigate the role of bacteriophages associated with the development of bacterial antimicrobial resistance. Antimicrobial resistance gene profiles and the potential transfer between bacterial and viral populations in various environmental samples, including animal feces, agricultural water, and soil, were determined using metagenomic sequencing technology. The results showed that certain antimicrobial-resistant genes, like macrolides, lincosamides, streptogramins, and aminoglycosides, were encoded in both bacterial chromosome and phage genomes. The findings indicate the potential transfer of these genes between bacterial and phage populations in the environmental samples. Further genomic analysis and manuscript are in progress. Under Objective 3, progress was made on Sub-objective 3.A involving top-down proteomic identification of proteins produced from plasmid-carrying Shiga toxin-producing E. coli (STEC) strains using antibiotic induction and MALDI-TOF-TOF mass spectrometry. Although plasmid proteins were not detected, several protein biomarkers (including Stx2a) were identified such as stress proteins (HdeA, HdeB, HPr) as well as the acyl carrier protein (ACP) with its attached prosthetic linker for fatty acid transport. Under disulfide bond reducing conditions, we also identified two tail fiber phage proteins suggesting that disulfide bonds play a critical role in the structural assembly of bacteriophage. Our in-house USDA software (Protein Biomarker Seeker), which iteratively cleaves in silico residues from the N- and C-termini, was able to identify protein biomarkers having sequence truncations as well as attachments that add mass to the biomarker. For example, the ACP protein was identified that has both removal of its N-terminal methionine as well as attachment of a prosthetic linker. For Sub-objective 3.B, an E. coli biofilm was analyzed by MALDI imaging; however, the imaging protocol needs further refinement to maximize ion intensity of biofilm biomolecules. Under Objective 4, significant progress was obtained on the development of a liquid crystal-based biosensor for the detection of Campylobacter spp. in food samples. For the specific identification of campylobacters, microspheres of different sizes were conjugated to anti-Campylobacter monoclonal antibodies, generated by the ARS researchers, for use with the biosensor. By using prototype manual instrumentation provided by industry stakeholders, results identified C731 monoclonal antibody to be the most efficient in detection capability by promoting microsphere aggregate formation when testing cultures of C. jejuni, C. coli and C. lari. The results obtained with the manual instrumentation by ARS will be adapted by industry stakeholders to develop a method using the automated liquid crystal-based instrumentation for high-throughput onsite sampling in food processing facilities. Ongoing research is currently aimed at optimizing procedures for the cost-effective and stable conjugation of antibodies to microspheres that are used with the liquid crystal-based platform. In addition, inclusivity and exclusivity tests will be expanded with pure cultures and spiked food samples by using the selected combination of microspheres with the lyotropic liquid crystal and associated instrumentation for data analysis. Under Objective 5, research continued to study the association between lysogenic bacteriophages (a type of phage that can infect a bacterium and incorporate the phage DNA into the bacterial DNA without killing the bacterial host) and the potential emergence of new pathogens. Stx-converting bacteriophages (lysogenic bacteriophages) were induced from different Shiga toxin-producing Escherichia coli (STEC) strains for the whole-genome sequencing and comparative genomic analysis. Approximately 40% of genes of the Stx-converting phages were associated with the fitness of the phages and their bacterial hosts to environmental stress. Phylogenetic analysis revealed that Stx-converting phages had high genomic diversity, especially those induced from E. coli O157:H7. Additionally, a lysogenic phage found in a STEC strain isolated by ARS researchers was similar to a lysogenic phage isolated from a reference STEC isolate of clinical origin, suggesting the potential dissemination of Stx-converting bacteriophages among the E. coli population. For lytic phages, the antimicrobial activities of various phage combinations were determined with strong lytic effects. Those phages were isolated from different environmental sources. Additionally, one polyvalent phage, capable of infecting STEC O157 and Salmonella Typhimurium, resulted in approximately 2.5 log reduction of E. coli O157:H7 and 1.5 log reduction of Salmonella Typhimurium on contaminated mung bean seeds after 1-h of the phage treatment. Two STEC O157-infecting phages, one isolated from bovine feces and one from agricultural water, were found to have the most synergistic effect against STEC O157 in liquid media, with more than 5 log reduction at 30C for 3 h. Several Salmonella phages were also isolated from sewage water samples and characterized using whole-genome sequencing. Those phages have a wide host range and strong antimicrobial activities for the potential development of phage cocktails against S. Typhimurium.

Accomplishments
1. Determining the prevalence and major source of multi-AMR isolates of Campylobacter in Amazonian Perú. Infections with Campylobacter (C.) jejuni and C. coli are endemic in infants in Amazonian Perú. Diarrhea caused by multi-antimicrobial resistance (AMR) Campylobacter is problematic in this region since there are few accessible antibiotics for treatment including ciprofloxacin and azithromycin. ARS researchers in Albany, California, in collaboration with scientists at the University of Virginia and the University of Bath in the United Kingdom, have identified AMR genes and AMR allele markers that have been incorporated into source attribution models. AMR alleles and genes identified among the C. jejuni and C. coli strains included gyrA alleles (ciprofloxacin resistance), 23S rRNA alleles (azithromycin resistance), aph genes (kanamycin and gentamycin resistance), and tet(O) gene (tetracycline resistance). Resistance to both ciprofloxacin and azithromycin was much more common in C. coli with 5% of C. jejuni isolates and 35% of C. coli isolates resistant to both. However, multi-AMR Campylobacter were not confined to specific genotypes within these species. The major source of multi-AMR Campylobacter was market poultry, and this finding provides a foundation for examining poultry practices that reduce AMR and provide safer poultry products.

2. Prevalence and diversity of Salmonella enterica in irrigation river water. The bacterial foodborne pathogen Salmonella enterica is a leading cause of human gastrointestinal infections worldwide from foodborne and waterborne sources. Due to the high demand for year-round availability of fresh produce in the United States, Mexico has become a leading agricultural supplier of various fresh produce commodities to be exported into the United States. ARS researchers at Albany, California, in collaboration with scientists at the National Research Laboratory in Food Safety (LANIIA) with the Center for Research in Food and Development (CIAD), located in Mexico, evaluated the prevalence and genotypic diversity of S. enterica isolates recovered from major rivers used for irrigation in the Culiacan Valley, an important agricultural region in Northwestern Mexico. An efficient filtration method was employed in combination with selective culturing to enable the identification of Salmonella when present at low concentrations in river water samples. By employing whole genome sequencing and bioinformatics tools, molecular subtyping revealed Oranienburg, Anatum and Saintpaul were the most predominant S. enterica serovars detected in irrigation river water. Subsequent genomic characterization of the recovered S. enterica isolates showed variability in genes required for S. enterica adaptation and survival in the environment. These findings have set the foundation for further characterization of traits conferring in S. enterica an increased fitness in surface water habitats by many microbiologists and will help identify produce growing conditions that may reduce S. enterica in fresh produce.

3. Shiga toxin-producing E. coli (STEC) strains biomarkers were identified by antibiotic induction and MALDI-TOF-TOF mass spectrometry. The ability of foodborne pathogens to respond to environmental challenges (including antibiotics) reflect their robustness, longevity, and potential to cause foodborne illness in the future. Identifying stress-induced protein biomarkers demonstrates the likelihood that a pathogen may survive and persist in the environment. Researchers in Albany, California, have identified several stress-response proteins in two genomically sequenced STEC strains using antibiotic induction and top-down proteomic analysis. Acid stress proteins, as well as a general stress response protein were identified. These biomarkers were rapidly characterized by in-house software (Protein Biomarker Seeker) that identified post-translational modifications (PTM) demonstrating the usefulness of this program for pathogen proteomics and STEC detection.

4. Lysogenic bacteriophages enhance the ability of bacteria to cause disease. Shiga toxin-producing Escherichia coli (STEC) infection contributes to more than 63,000 foodborne illnesses and 2,000 hospitalizations yearly in the United States. Although various antimicrobial interventions have been used in the food industry, the incidence of foodborne outbreaks does not decrease. ARS researchers in Albany, California, investigated the roles of STEC lysogenic phages (a type of virus that only infect a bacterium and can insert the phage DNA into the bacterial DNA without killing the bacterial host). The results showed that a lysogenic phage containing a toxin gene was released from a pathogenic E. coli after it was exposed to environmental stresses. This phage then infected a non-pathogenic bacterial strain, which then became a new pathogen and produced the toxin. Additionally, viral populations, particularly bacteriophages, were found to contain common antimicrobial-resistant genes, as seen in the bacterial population in the pre-harvest environment. The finding suggests that environmental stresses should be carefully evaluated by the food industry to prevent the release of lysogenic phages from pathogenic bacteria to reduce the emergence of new pathogens.

Review Publications
Nothaft, H., Bian, X., Shajahan, A., Miller, W.G., Bolick, D.T., Guerrant, R.L., Azadi, P., Ng, K.K., Szymanski, C.M. 2021. Detecting glucose fluctuations in the Campylobacter jejuni N-glycan structure. ACS Chemical Biology. 16(11):2690-2701. https://doi.org/10.1021/acschembio.1c00498.
Lu, L., Quintela, I.A., Lin, C., Lin, T., Lin, C., Wu, V.C., Lin, C. 2021. A review of epidemic investigation on cold-chain food-mediated SARS-CoV-2 transmission and food safety consideration during COVID-19 pandemic. Journal of Food Safety. 41(6). Article e12932. https://doi.org/10.1111/jfs.12932.
Shu, X., Singh, M., Karampudi, N., Bridges, D.F., Kitazumi, A., Wu, V.C., De los Reyes, B.G. 2021. Responses of Escherichia coli and Listeria monocytogenes to ozone treatment on non-host tomato: Efficacy of intervention and evidence of induced acclimation. PLoS ONE. 16(10). Article e0256324. https://doi.org/10.1371/journal.pone.0256324.
Soto-Beltran, M., Lee, B.G., Amezquita-Lopez, B.A., Quinones, B. 2022. Overview of methodologies for the culturing, recovery and detection of Campylobacter. International Journal of Environmental Health Research. https://doi.org/10.1080/09603123.2022.2029366.
Luna, E., Parkar, S., Kirmiz, N., Hartel, S., Hearn, E., Hossine, M., Kurdian, A., Mendoza, C., Orr, K., Padilla, L., Ramirez, K., Salcedo, P., Serrano, P., Choudhury, B., Paulchakrabarti, M., Parker, C.T., Huynh, S., Cooper, K.K., Flores, G.E. 2021. Utilization efficiency of human milk oligosaccharides by human-associated Akkermansia is strain-dependent. Applied and Environmental Microbiology. 88(1). Article e01487-21. https://doi.org/10.1128/AEM.01487-21.
Rane, B., Lacombe, A.C., Guan, J., Bridges, D.F., Sablani, S., Tang, J., Wu, V.C. 2021. Gaseous chlorine dioxide inactivation of microbial contamination on whole black peppercorns. Journal of Food Safety. Article e12948. https://doi.org/10.1111/jfs.12948.
Fagerquist, C.K., Dodd, C.E. 2021. Sequestration of the ionizing proton in singly charged metastable protein ions generated by MALDI. International Journal of Mass Spectrometry. 471. Article 116736. https://doi.org/10.1016/j.ijms.2021.116736.
Fagerquist, C.K., Dodd, C.E. 2021. Top-down proteomic identification of plasmid and host proteins produced by pathogenic Escherichia coli using MALDI-TOF-TOF tandem mass spectrometry. PLoS ONE. 16(11). Article e0260650. https://doi.org/10.1371/journal.pone.0260650.
Guan, J., Lacombe, A.C., Rane, B., Tang, J., Sablani, S., Wu, V.C. 2021. A review: Gaseous interventions for Listeria monocytogenes control in fresh apple cold storage. Frontiers in Microbiology. 12. Article 782934. https://doi.org/10.3389/fmicb.2021.782934.
Quintela, I.A., Hwang, A., Vasse, T., Salvador, A., Zhang, Y., Liao, Y., Wu, V.C. 2022. Whole-genome analysis of Escherichia phage vB_EcoM-S1P5QW, isolated from manures collected from cattle farms in Maine. Microbiology Resource Announcements. 11(4). Article e00041-22. https://doi.org/10.1128/mra.00041-22.
Liao, Y., Zhang, Y., Salvador, A., Harden, L.A., Wu, V.C. 2022. Characterization of a T4-like bacteriophage vB_EcoM-Sa45lw as a potential biocontrol agent for Shiga toxin-producing Escherichia coli O45 contaminated on Mung Bean seeds. Microbiology Spectrum. 10(1). Article e02220-21. https://doi.org/10.1128/spectrum.02220-21.
Bridges, D.F., Lacombe, A.C., Wu, V.C. 2022. Fundamental differences in inactivation mechanisms of Escherichia coli O157:H7 between chlorine dioxide and sodium hypochlorite. Frontiers in Microbiology. 13. Article 923964. https://doi.org/10.3389/fmicb.2022.923964.
Heikema, A.P., Strepis, N., Horst-Kreft, D., Huynh, S., Zomer, A., Kelly, D.J., Cooper, K.K., Parker, C.T. 2021. Biomolecule sulphation and novel methylations related to Guillain-Barre syndrome-associated Campylobacter jejuni serotype HS:19. Microbial Genomics. 7(11). Article 000660. https://doi.org/10.1099/mgen.0.000660.
Achtman, M., Van den Broeck, F., Cooper, K.K., Lemey, P., Parker, C.T., Zhou, Z. 2021. Genomic population structure associated with repeated escape of Salmonella enterica ATCC14028s from the laboratory into nature. PLoS Genetics. 17(9). Article e1009820. https://doi.org/10.1371/journal.pgen.1009820.
Crippen, C.S., Zhou, B., Andersen, S., Patry, R.T., Muszynski, A., Parker, C.T., Cooper, K.K., Szymanski, C.M. 2021. RNA and sugars, unique properties of bacteriophages infecting multidrug resistant Acinetobacter radioresistens strain LH6. Viruses. 13(8). Article 1652. https://doi.org/10.3390/v13081652.
Peters, S., Pascoe, B., Wu, Z., Bayliss, S.C., Zeng, X., Edwinson, A., Veerabadhran-Gurunathan, S., Jawahir, S., Calland, J.K., Mourkas, E., Patel, R., Wiens, T., Decuir, M., Boxrud, D., Smith, K., Parker, C.T., Farrugia, G., Zhang, Q., Sheppard, S.K., Grover, M. 2021. Campylobacter jejuni genotypes are associated with post-infection irritable bowel syndrome in humans. Communications Biology. 4. Article 1015. https://doi.org/10.1038/s42003-021-02554-8.
Gonzalez-Lopez, I., Medrano-Felix, J.A., Castro-del Campo, N., Lopez-Cuevas, O., Gonzalez-Gomez, J.P., Valdez-Torres, J.B., Aguirre-Sanchez, J.R., Martinez-Urtaza, J., Gomez-Gil, B., Lee, B.G., Quinones, B., Chaidez, C. 2022. Prevalence and genomic diversity of Salmonella enterica recovered from river water in a major agricultural region in northwestern Mexico. Microorganisms. 10(6). Article 1214. https://doi.org/10.3390/microorganisms10061214.
On, S.L., Miller, W.G., Biggs, P.J., Cornelius, A.J., Vandamme, P. 2021. Aliarcobacter, Halarcobacter, Malaciobacter, Pseudarcobacter and Poseidonibacter are later synonyms of Arcobacter: transfer of Poseidonibacter parvus, Poseidonibacter antarcticus, ‘Halarcobacter arenosus’, and ‘Aliarcobacter vitoriensis’ to Arcobacter as Arcobacter parvus comb. nov., Arcobacter antarcticus comb. nov., Arcobacter arenosus comb. nov. and Arcobacter vitoriensis comb. nov. International Journal of Systematic and Evolutionary Microbiology. 71(11). https://doi.org/10.1099/ijsem.0.005133.
Hanafy, Z., Osborne, J.A., Miller, W.G., Parker, C.T., Olson, J.W., Jackson III, J.H., Kathariou, S. 2022. Differences in the propensity of different antimicrobial resistance determinants to be disseminated via transformation in Campylobacter jejuni and Campylobacter coli. Microorganisms. 10(6). Article 1194. https://doi.org/10.3390/microorganisms10061194.