Progress 10/01/23 to 09/30/24
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. 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. Artificial Intelligence (AI)/Machine Learning (ML) ARS scientists and collaborators used Machine Learning (ML) with a weighted random forest to examine source attribution of Campylobacter infections in Peru. Pattern distribution was scored using mutual information (MI) in each reservoir source population. Biologically, MI gives a measure of how effectively a DNA sequence pattern can distinguish the source in question from every other source. Models were trained to predict source populations using patterns. ML aided in the source attribution of Campylobacter infections in Peru from 2019-2023. The ML analysis was performed by collaborators in the United Kingdom. Structural analysis of foodborne pathogen proteins using Alphafold2 (Ceres/SciNet) was completed and compared to predicted structures previously obtained using mass spectrometry data. The analysis identified an unexpected influence of glycine residues on the backbone cleavage of proteins by the aspartic acid effect. This study was recently published in PLOS One. ARS researchers in Albany, California, also determined the predicted structures of 228 proteins of the pESI megaplasmid of SEE Infantis, the heterotrimer structure of cytolethal distending toxin (Cdt), the monomer and oligomer structures of CsgA (curli subunit) as well as CsgB (curli nucleator) using Alphafold and are performing molecular dynamics simulations on Atlas/SciNet that involve studying the dynamics and binding energies of protein complexes of foodborne pathogens including colicin/immunity proteins. ACCOMPLISHMENTS 01 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. 02 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. 03 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. 04 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.
Impacts
(N/A)
Publications
- 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.
- 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.
- 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.
- 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.
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Progress 10/01/22 to 09/30/23
Outputs
(N/A)
Impacts
(N/A)
Publications
- Lacombe, A.C., Quintela, I.A., Liao, Y., Wu, V.C. 2022. Shiga toxin- producing Escherichia coli outbreaks in California⿿s leafy greens production continuum. Frontiers In Food Science And Technology. 2. Article 1068690. https://doi.org/10.3389/frfst.2022.1068690.
- Fagerquist, C.K. 2023. Top-down identification of Shiga toxin (and other virulence factors and biomarkers) from pathogenic E. coli using MALDI-TOF/ TOF tandem mass spectrometry. In: Shah, H.N., Gharbia, S.E., Shah, A.J., Tranfield, E.Y., Thompson, K.C., editors. Microbiological Identification Using MALDI-TOF and Tandem Mass Spectrometry: Industrial and environmental applications. 1st edition. West Sussex, UK: John Wiley & Sons Ltd. p. 71- 96. https://doi.org/10.1002/9781119814085.ch3.
- Garcia Bardales, P., Schiaffino, F., Huynh, S., Paredes Olortegui, M., Penataro Yori, P., Pinedo Vasquez, T., Manzanares Villaneuva, K., Curico Huansi, G., Shapiama Lopez, W., Cooper, K.K., Parker, C.T., Kosek, M.N. 2022. ⿿Candidatus Campylobacter infans detection is not associated with diarrhea in children under the age of 2 in Peru. PLOS Neglected Tropical Diseases. 16(10). Article e0010869. https://doi.org/10.1371/journal.pntd. 0010869.
- Ndraha, N., Huang, L., Wu, V.C., Hsiao, H. 2022. Vibrio parahaemolyticus in seafood: Recent progress in understanding influential factors at harvest and food-safety intervention approaches. Current Opinion in Food Science. 48. Article 100927. https://doi.org/10.1016/j.cofs.2022.100927.
- Quintela, I.A., Vasse, T., Lin, C., Wu, V.C. 2022. Advances, applications, and limitations of portable and rapid detection technologies for routinely encountered foodborne pathogens. Frontiers in Microbiology. 13. Article 1054782. https://doi.org/10.3389/fmicb.2022.1054782.
- Gummalla, V., Zhang, Y., Liao, Y., Wu, V.C. 2023. The role of temperate phages in bacterial pathogenicity. Microorganisms. 11(3). Article 541. https://doi.org/10.3390/microorganisms11030541.
- Parker, C.T., Schiaffino, F., Huynh, S., Paredes Olortegui, M., Penataro Yori, P., Garcia Bardales, P.F., Pinedo Vasquez, T., Curico Huansi, G.E., Manzanares Villaneuva, K., Shapiama Lopez, W.V., Cooper, K.K., Kosek, M.N. 2022. Shotgun metagenomics of fecal samples from children in Peru reveals frequent complex co-infections with multiple Campylobacter species. PLOS Neglected Tropical Diseases. 16(10). Article e0010815. https://doi.org/10. 1371/journal.pntd.0010815.
- Aguirre-Sanchez, J.R., Valdez-Torres, J.B., Castro del Campo, N., Martinez- Urtaza, J., Castro del Campo, N., Lee, B.G., Quinones, B., Chaidez-Quiroz, C. 2022. Phylogenetic group and virulence profile classification in Escherichia coli from distinct isolation sources in Mexico. Infection, Genetics and Evolution. 106. Article 105380. https://doi.org/10.1016/j. meegid.2022.105380.
- Fagerquist, C.K., Wallis, C.M., Chen, J. 2023. Top-down proteomic identification of protein biomarkers of Xylella fastidiosa subsp. fastidiosa using MALDI-TOF-TOF-MS and MS/MS. International Journal of Mass Spectrometry. 489. Article 117051. https://doi.org/10.1016/j.ijms.2023. 117051.
- Fagerquist, C.K., Shi, Y., Dodd, C.E. 2023. Toxin and phage production from pathogenic E. coli by antibiotic induction analyzed by chemical reduction, MALDI-TOF-TOF mass spectrometry and top-down proteomic analysis. Rapid Communications in Mass Spectrometry. 37(10). Article e9505. https:// doi.org/10.1002/rcm.9505.
- Talukdar, P., Crockett, T.M., Gloss, L.M., Huynh, S., Roberts, S.A., Turner, K.L., Lewis, S.T., Herup-Wheeler, T.L., Parker, C.T., Konkel, M. 2022. The bile salt deoxycholate induces Campylobacter jejuni genetic point mutations that promote increased antibiotic resistance and fitness. Frontiers in Microbiology. 13. Article 1062464. https://doi.org/10.3389/ fmicb.2022.1062464.
- Liao, Y., Zhang, Y., Salvador, A., Ho, K., Cooley, M.B., Wu, V.C. 2022. Characterization of polyvalent Escherichia phage Sa157lw for the biocontrol potential of Salmonella Typhimurium and Escherichia coli O157:H7 on contaminated mung bean seeds. Frontiers in Microbiology. 13. Article 1053583. https://doi.org/10.3389/fmicb.2022.1053583.
- Sun, X., Liao, Y., Zhang, Y., Salvador, A., Ho, K., Wu, V.C. 2022. A new Kayfunavirus-like Escherichia phage vB_EcoP-Ro45lw with antimicrobial potential of Shiga toxin-producing Escherichia coli O45 strain. Microorganisms. 11(1). Article 77. https://doi.org/10.3390/ microorganisms11010077.
- Carter, M.Q., Laniohan, N.S., Pham, A., Quinones, B. 2022. Comparative genomic and phenotypic analyses of virulence potential in Shiga toxin- producing Escherichia coli O121:H7 and O121:H10. Frontiers in Cellular and Infection Microbiology. 12. Article 1043726. https://doi.org/10.3389/fcimb. 2022.1043726.
- Kirchner, M., Miller, W.G., Osborne, J., Badgley, B., Niedermeyer, J.A., Kathariou, S. 2023. Campylobacter colonization and diversity in young turkeys in the context of gastrointestinal distress and antimicrobial treatment. Microorganisms. 11(2). Article 252. https://doi.org/10.3390/ microorganisms11020252.
- Bolinger, H., Miller, W.G., Osborne, J., Niedermeyer, J., Kathariou, S. 2023. Campylobacter jejuni and Campylobacter coli from houseflies in commercial turkey farms are frequently resistant to multiple antimicrobials and exhibit pronounced genotypic diversity. Pathogens. 12(2) . Article 230. https://doi.org/10.3390/pathogens12020230.
- Meinersmann, R.J., Berrang, M.E., Shariat, N.W., Richards, A.K., Miller, W. G. 2023. Despite shared geography, Campylobacter isolated from surface water are genetically distinct from Campylobacter isolated from chickens. Microbiology Spectrum. 11(2). Article e04147-22. https://doi.org/10.1128/ spectrum.04147-22.
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Progress 10/01/21 to 09/30/22
Outputs
PROGRESS REPORT Objectives (from AD-416): 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-Barr� 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 (from AD-416): 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-Barr� 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 na�ve strains via transmissible plasmids. Matings between C. coli strains containing mobile elements and na�ve 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. 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, Per�. 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 01 Determining the prevalence and major source of multi-AMR isolates of Campylobacter in Amazonian Per�. Infections with Campylobacter (C.) jejuni and C. coli are endemic in infants in Amazonian Per�. 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. 02 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. 03 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. 04 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.
Impacts
(N/A)
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.
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Progress 10/01/20 to 09/30/21
Outputs
Progress Report Objectives (from AD-416): 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-Barr� 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 (from AD-416): 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-Barr� 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 na�ve strains via transmissible plasmids. Matings between C. coli strains containing mobile elements and na�ve 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. 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 Per�. 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 na�ve 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. Record of Any Impact of Maximized Teleworking Requirement: The maximized telework requirement prevented scientists in Albany, California, from performing basic microbiology procedures, from utilizing DNA sequencers, from utilizing proteomics equipment, from conducting phage characterization and antimicrobial activity tests, and from obtaining essential data for all project objectives. The maximized telework did not allow us to obtain new samples from collaborators and postponed field sampling trips until FY22. However, maximized telework allowed scientists in Albany, California, to adjust our focus on analyzing results, drafting unplanned manuscripts related to Objective 1, and developing protocols for all project objectives. ACCOMPLISHMENTS 01 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.
Impacts
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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.
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