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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Research Project #438551

Research Project: Human Pathogens within the Produce Production Continuum; their Detection, Mechanisms for Persistence, and Ecology

Location: Produce Safety and Microbiology Research

2023 Annual Report

Objective 1: Elucidate microbial and plant factors and molecular mechanisms that affect fitness characteristics related to survival and growth of enteric pathogens in the produce production continuum. 1.A: Screen lettuce cultivars for genotypes exhibiting a wound response inhibitory to EcO157. 1.B: Assess the role of enzymatic browning in the survival of EcO157 on fresh-cut lettuce. 1.C: Determine whether chemoattractants in lettuce leaves induce the ingression of EcO157 into cut tissue. 1.D: Identify and characterize genetic determinants and physiological traits contributing to colonization of crop plants by STEC and Salmonella enterica. 1.E: Determine the fitness of various genomic subtypes of L. monocytogenes in the colonization of produce. 1.F: Compare fitness and physiology of different STEC serotypes, Salmonella, and L. monocytogenes on produce growing in conventional vs organic soil. 1.G: Isolation and characterization of HuNoV and TV ligands from mammalian cells and bacteria for the development and validation of viral ligand-based assays for determination of viral infectivity. Objective 2: Identify environmental and bacterial factors that affect the persistence and transmission of enteric pathogens in the produce production environment for risk assessment. 2.A: Perform an epidemiological investigation of STEC from the Salinas, CA region with whole genome sequencing. 2.B: Monitor the persistence and distribution of Salmonella and L. monocytogenes genomic types in the Central California Coast agricultural region. 2.C: Determine the survival of EcO157 in water-sediment microcosms from public access watershed sites near a leafy greens-growing region in Central Coastal California. 2.D: Identify environmental reservoirs of enteric pathogen strains exhibiting enhanced pathogenicity potential and environmental persistence by comparative genomics and functional genomics. 2.E: Monitor the transport of pathogenic bacteria in various Salinas watersheds using ddPCR and wgMLST. Objective 3: Develop methods to detect, subtype, and distinguish bacterial pathogen strains from produce production environments to aid surveillance and epidemiological investigations. 3.A: Develop improved media for equivalent enrichment of Salmonella serotypes, and CRISPR-SeroSeq for rapid serotype detection. 3.B: Develop and validate viral ligand-based assays for infective HuNoV and surrogate TV and to determine viral infectivity.

The project will address mechanisms for the detection, persistence, and ecology of human pathogens within the produce production continuum. Objective 1 will address microbial and plant factors that affect the fitness of human pathogens to survive and grow on plants. Objective 2 will focus on the ecology of human pathogens in the plant pre-harvest environment by studying factors that allow the pathogens to survive and be transported through the Central California Coast region. Finally, Objective 3 will concentrate on methods for effective detection of human pathogens for use in surveillance laboratories. Under Objective 1, lettuce cultivars will be screened for genotypes exhibiting a wound response to Escherichia coli O157:H7 (EcO157) to identify cultivars that would reduce potential EcO157 contamination of lettuce in modified atmosphere packaging. The role of browning on fresh cut lettuce regarding EcO157 survival will be measured. Chemoattractants in lettuce leaves will be assessed to determine if they induce the ingression of EcO157 into cut lettuce tissue. EcO157 and two serovars of Salmonella enterica will be studied to determine the genetic determinants that contribute to those human pathogens colonizing spinach, romaine lettuce, cucumbers, tomatoes, cantaloupe, and watermelon. The fitness of Listeria monocytogenes subtype ST382, which has been responsible for several produce-related outbreaks, will be compared with other subtypes on lettuce, cantaloupe, and sprouts. The fitness of various Shiga toxin producing E. coli (STEC) strains, Salmonella, and L. monocytogenes on plants growing in organic and conventional soils will be measured. For Human Norovirus (HuNoV) studies, ligands that bind the virus will be isolated from mammalian cells and bacteria to develop better assays for HuNoV detection. Under Objective 2, an epidemiological investigation of STEC from the Central California Coastal region will be done to track movement and determine contamination routes. The persistence of Salmonella in the same region will be assessed using strains isolated from waters in the area. The ability of EcO157 to survive in soil/water microcosms will be studied using sediment/water samples from the California Central Coast. EcO157 survival and water chemistry will be monitored in these microcosms for up to 3 months. Environmental reservoirs of STEC and Salmonella will be studied by comparing genomic information and epidemiological history of outbreak and environmentally isolated strains. Phenotypic traits that contribute to environmental persistence will be examined, and environmental reservoirs of hypervirulent STEC strains will be identified. Transport of STEC, Salmonella, and L. monocytogenes in Central California watersheds will be monitored using genomic subtyping and detection of virulence genes. Under Objective 3, improved enrichment culture media will be developed to equally enrich various Salmonella serovars for improved Salmonella surveillance. Whole genome methods for detection of relevant serovars will be developed. Assays to detect infectious HuNoV will be developed and validated using ligands for HuNoV isolated under Objective 1.

Progress Report
As part of work under Objective 1, additional enzymatic assays were performed to identify a broader range of lettuce cultivars that inhibit E. coli O157:H7 (EcO157) survival on cold-stored shredded lettuce more than others. This inhibition was linked to lettuce tissue biochemical traits and even more broadly associated with lettuce resistance mediated via jasmonic acid signaling during the response to wounding. This indicates that lettuce phenotypes and genotypes currently used for breeding against specific pathogens and pest injuries may be the focus also of lettuce breeding against the high survival of foodborne pathogens on cold-stored fresh-cut lettuce. A research article is currently being prepared for submission to a journal in fiscal year (FY) 2023. This work is in support of Sub-objective 1A. In continuing work on lettuce cultivars under Sub-objective 1B, more cultivars that are prone to browning were identified during a screen of shredded lettuce to complement the six cultivars that were proposed originally to be tested. Activity of the polyphenol oxidase enzyme (PPO) and peroxidase (POD) responsible for lettuce browning in cut leaf tissue was measured in 32 lettuce cultivars, and results were used to assess the association of their activity with EcO157 survival on cut lettuce. Two E. coli donor strains, one carrying the barcoded Tn5 transposons and the other one carrying the barcoded mariner transposons, were used to generate transposon mutant libraries in representative strains of Shiga toxin-producing E. coli (STEC), Salmonella Newport, and S. Javiana. The conjugation condition for each recipient strain was optimized and the quality of the 20 mutant libraries is currently evaluated by Tn-Seq at Lawrence Berkeley National Laboratory. This work supports Sub-objective 1D. For Sub-objective 1E, eight strains of Listeria monocytogenes of serotypes 1/2a and 4b were screened for fitness on cut green lettuce tissue from lettuce purchased at retail. Strains were inoculated in 3-strain combinations at 1:1:1 levels and the amount recovered after 24 hours and one week was calculated by a real-time polymerase chain reaction (PCR) assay. In Human Norovirus (HuNoV) work, a ligand mining platform for norovirus using a bacterial cell surface display system (BSDS) was constructed in collaboration with Shanghai JiaoTong University. The system uses Escherichia coli (E. coli) as a cellular vector, the N-terminal end of icosahedral nucleoprotein as an anchoring protein to express the P protein of HuNoV on the surface of the transformed E. coli cell to fish the candidate ligand for HuNoV binding. The complexes of the P domain and ligand candidates were isolated, released by thrombin and identified by a nanoliter liquid chromatography tandem with a quadrupole orbital trap mass spectrometer. A proteinaceous oyster heat shock protein 70 (oHSP 70), was isolated and showed a strong ability in HuNoV binding. Recombinant oHSP70 was constructed and used to concentrate and capture HuNoV. The recombinant oHSP70 showed a higher binding ability to HuNoV than commonly used pig stomach mucin (PGM) which contains known HBGA receptors for HuNoV by ELISA assay and the ISC-RT-qPCR assay. This research supports Sub-objective 1G. Under work for Objective 2, 40 strains of Salmonella serovar Infantis strains isolated from water surveys of the California Central Coast were screened for large plasmids. Eighteen strains contained plasmids, and genome sequencing of those strains is underway to determine the nature of the plasmids in these Infantis strains. This research supports Sub-objective 2B. Two clinical STEC strains, one linked to the 2018 romaine lettuce-associated outbreak and the other one linked to a 2019 romaine lettuce-associated outbreak in the United States were obtained from a collaborator at the California Department of Public Health. In collaboration with another ARS researcher in Clay Center, Nebraska, the complete genome sequences of both strains were determined and deposited in GenBank. Spontaneous rifampicin-resistant mutants of both strains were generated. These modified strains and another rifampicin-resistant mutant of a clinical STEC strain linked to the 2006 spinach-associated outbreak were used to test the survival of STEC in field water to determine the impact of the field water microbiome on STEC survival and persistence. The field water samples were collected from the Salinas River, California, in November 2022 and April 2023. The survival of STEC was monitored for two months by plate count only, and resuscitation of Viable But Not Culturable (VBNC) cells was followed by plate count. Total genomic DNA from the water samples collected in April 2023, before the inoculation, and after the two months of incubation in a temperature-controlled incubator were prepared and shipped to a collaborator at Oklahoma University for metagenomic sequencing. This work was in support of Sub-objective 2C. More than 300 virulence genes and 15 pathogenicity islands common to E. coli were screened systematically in STEC environmental strains. Hybrid pathotypes appeared to be common in STEC strains isolated from wild birds, including STEC-ETEC (Enterotoxigenic E. coli), STEC-EAEC (Enteroaggregative E. coli), and STEC-EPEC (Enteropathogenic E. coli). Novel combinations of virulence factors were also detected in several avian STEC strains, suggesting the emergence of “high-risk” (increased likelihood to cause severe human disease) STEC strains in preharvest environments. A manuscript was submitted to Journal Frontiers in Microbiology. This research supports Sub-objective 2D. In work under Objective 3, 10 strains of Salmonella serovars Give, Kentucky, Typhimurium, and Enteritidis were screened in the common pre-enrichment media Buffered Peptone Water (BPW) to study culture bias. Mixtures of strains were co-cultured in various formulations of the medium to test the effect of different buffers on culture bias. Phosphate buffer, HEPES, and MOPS buffers had little effect on alleviating culture bias in mixed cultures. Predicted amino acid sequences of genes in the Pho regulon were compared, and there were differences noted in the sequence of the PhoR protein that may be related to serovar. Additional work on culture bias was done with collaborators at ARS in Clay Center, Nebraska, and the University of Georgia. In this work, strains of Salmonella serovars Infantis, Kentucky, and Enteritidis were inoculated in combinations of 1:1, 1:10, and 10:1 into Tetrathionate Broth (TT) and Rappaport Vasiliadis Soya Peptone Broth (RVS) to determine if serovar bias was affected in the different media when in direct comparison. This works supports Sub-objective 3A. In HuNoV work on Sub-objective 3B, the standard curves for digital droplet reverse transcriptase quantitative polymerase chain reaction (ddRT-qPCR) and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) were established. The TCID50 infectious unit in the viral binding assay was established. Validation of cellular captured and blood group antigen captured viral genomic copies and TCID50 infectious units are ongoing. The project was significantly impacted by the renovation of the office and lab.

1. New mathematical model to predict foodborne pathogen colonization of pre-harvest leafy greens. The lettuce industry in California is valued at over $2B annually and outbreaks of Escherichia coli (E. coli) O157:H7 (EcO157) associated with lettuce grown on the California Central Coast and in the Imperial Valley show seasonality. ARS researchers in Albany, California, in collaboration with Cleveland State University, developed a mechanistic model based on publicly available weather data obtained from California weather stations (CIMIS) in order to predict EcO157 trends on lettuce leaves after contamination in the field. The model accurately predicted EcO157 survival on young romaine lettuce plants measured in previous field experiments in Salinas, California. The model was published and may be used to develop a weather-based risk assessment tool for the lettuce industry.

2. Identification of novel molecular targets for detection of hypervirulent STEC strains. Shiga Toxin producing Escherichia coli (STEC) is estimated to cause 2.8 million cases of acute illnesses globally each year. Genetic markers that enable differentiating the high-risk STEC strains are critically needed by regulatory agencies and the food industry for rapid detection and risk assessment. ARS researchers at Albany, California, performed comparative pathogenomic analyses and phenotypic assays to systematically evaluate the virulence potential of STEC strains isolated from various environmental samples. The study provides additional molecular targets for the development of a systematic approach for STEC detection and identification, which is of use for modelers, regulators, and developers of detection tests.

3. Corn starch-coated activated carbon (CSCAC) enhances the detection limit for human norovirus. Human Norovirus (HuNoV) is the most common cause of human foodborne illness. Common polymerase chain reaction (PCR) methods for detecting HuNoV from seafood, produce, and environmental samples are hindered by the presence of PCR inhibitors in the samples, leading to decreased sensitivity of detection and false negatives. ARS researchers in Albany, California, in collaboration with scientists at JiangXi Agriculture University, developed a highly sensitive pretreatment method to remove inhibitors prior to reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) for virus detection in shellfish and produce. Corn starch-coated activated carbon (CSCAC) was used in the elution buffer during a pretreatment step. The modified assay is 10-100 times more sensitive than the standard method (ISO 15216-2:2019). The lower limit of detection of these viruses by the new method was 100 Genome Copies per gram of material. Therefore, this method could be effectively used to detect low levels of foodborne viruses in shellfish and other samples, which is of interest to regulators and public health laboratories.

4. Identification of oyster heat shock protein 70 (oHSP70) as a good ligand for binding of HuNoV and Tulane virus. Human Norovirus (HuNoV) is the most common cause of human foodborne illness. There are limitations in the current methods for detecting HuNoV from food samples with false positive results due to degraded viral RNA and false negative results due to low virus titers on the foods and/or polymerase chain reaction inhibitors in the food sample. ARS researchers in Albany, California, in collaboration with scientists at Shanghai JiaoTong University, developed a system to isolate ligands for norovirus binding to be used to concentrate HuNoV in food samples. The oyster heat shock protein 70 (oHSP 70) was identified as a high-affinity binding ligand for HuNoV compared with the commonly used pig stomach mucin (PGM) in previously developed assays. The new capture method is promising for detecting a wide array of genotypes of HuNoV and simplifies the steps of virus concentration and virus extraction with higher sensitivity and accuracy than the conventional to reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) assay. This is of interest to food surveillance laboratories and regulators.

Review Publications
Liao, N., Tang, M., Chen, L., Tian, P., Wang, D., Cheng, D., Wu, G. 2023. Soluble extracellular polymeric substance (SEPS) of histo-blood group antigen (HBGA) expressing bacterium Sphingobacterium sp. SC05 influences the survival and persistence of norovirus on lettuce. Food Microbiology. 109. Article 104126.
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.
Brown, P., Kucerova, Z., Gorski, L.A., Chen, Y., Ivanova, M., Leekitcharoenphon, P., Parsons, C., Niedermeyer, J., Jackson, J., Kathariou, S. 2023. Horizontal gene transfer and loss of serotype-specific genes in Listeria monocytogenes can lead to incorrect serotype designations using a commonly-employed molecular serotyping scheme. Microbiology Spectrum. 11(1). Article e02745-22.
Brandl, M., Mammel, M., Simko, I., Richter, T., Gebru, S., Leonard, S. 2023. Weather factors, soil microbiome, and bacteria-fungi interactions as drivers of the epiphytic phyllosphere communities of romaine lettuce. Food Microbiology. 113. Article 104260.
Brandl, M., Ivanek, R., Allende, A., Munther, D. 2023. Predictive population dynamics of Escherichia coli O157:H7 and Salmonella enterica on plants: A mechanistic mathematical model based on weather parameters and bacterial state. Applied and Environmental Microbiology. 89(7). Article e00700-23.
Devarajan, N., Weller, D., Jones, M., Adell, A., Adhikari, A., Allende, A., Arnold, N., Baur, P., Beno, S., Clements, D., Olimpi, E., Critzer, F., Green, H., Gorski, L.A., Gruber, A., Kovac, J., McGarvey, J.A., Murphy, C., Murphy, S., Navarro-Gonzalez, N., Owen, J., Pires, A., Richard, N., Samaddar, S., Schmidt, R., Scow, K., Shariat, N., Smith, O., Spence, A., Stoeckel, D., Tran, T., Wall, G., Karp, D. 2023. Evidence for the efficacy of pre-harvest agricultural practices in mitigating food-safety risks to fresh produce in North America. Frontiers in Sustainable Food Systems. 7. Article 1101435.