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

Location: Produce Safety and Microbiology Research

2021 Annual Report

Objectives
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.

Approach
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
This is the first progress report for the bridge project 2030-42000-052-00D, which replaces the terminated project 2030-42000-050-00D. Out of a collection of 500 different lettuce cultivars, 37 were selected for their potential to display a strong defense response to wounding. Cultivars were grown in the laboratory, and assays were conducted to characterize and compare the production of major plant defense compounds made in response to wounding. A subset of cultivars that have distinct profiles of phenolics, anthocyanins, and reactive oxygen species in cut leaf tissue was identified. This work is in support of Sub-objective 1A. In support of Sub-objective 1G, a system to isolate ligands that bind to Human Norovirus (HuNoV) was developed in collaboration with scientists at Shanghai Jiao Tong University and the Shanghai Institute of Technology. In this system, E. coli bacteria express on their surface the protein necessary for binding of HuNoV to host cells. The system will be tested on the HuNoV surrogate, Tulane Virus (TV). Culture of TV in MK2 cells was established. In relation to Sub-objective 2D, the complete genome sequences of environmental Shiga toxin-producing Escherichia coli (STEC) strains of serotype O121:H10 and O121:H7 were determined. Comparative genomics were performed to identify important Single Nucleotide Polymorphisms (SNPs) and genetic loci in defining the virulence lineage of O121:H19. Comparative genomics between clinical and environmental STEC O145 strains were carried out to identify genes that may confer niche-specific traits, including genetic loci contributing to colonization of humans, plants, or other environmental reservoirs by STEC. This work is in support of Sub-objective 1D. Related to Sub-objective 1A, a collaboration with the Food and Drug Administration Center for Food Safety and Nutrition was established to begin subtyping over 4800 strains of STEC. These strains were isolated during surveys of the Central California Coastal region over the last 15-20 years. Previous work to subtype these strains based on Multi Locus Variable Repeat Analysis revealed hundreds of subtypes but was not conclusive. The strains will be subjected to Whole Genome Sequencing (WGS) and then Multi Locus Sequence Analysis (wgMLST) to prepare for an analysis of STEC transport. More than 500 STEC strains from this collection have been sequenced and typed to date. In support of Sub-objective 2B, serotype analysis of over 2000 Salmonella strains was completed by a combination of traditional serotyping methods and WGS. WGS was done on approximately 100 Salmonella strains isolated from the Central California Coast environment. In a collaboration with scientists at North Carolina State University, over 80 Listeria monocytogenes strains were screened for the presence of phage in their genome. These isolates were originally isolated from the environment in the Central California Coast.

Accomplishments

Review Publications
Silva, C.J., Onisko, B.C., Dynin, I.A., Erickson-Beltran, M.L., Requena, J.R. 2021. Time of detection of prions in the brain by nanoscale liquid chromatography coupled to tandem mass spectrometry is comparable to animal bioassay. Journal of Agricultural and Food Chemistry. 69(7):2279-2286. https://doi.org/10.1021/acs.jafc.0c06241.
Xu, M., Lu, F., Wu, Q., Zhang, J., Tian, P., Xu, T., Wang, D. 2021. Broad-range and effective detection of human noroviruses by colloidal gold immunochromatographic assay based on the shell domain of the major capsid protein. BMC Microbiology. 21. Article 22. https://doi.org/10.1186/s12866-020-02084-z.
Guan, J., Lacombe, A.C., Tang, J., Bridges, D.F., Sablani, S., Rane, B., Wu, V.C. 2021. Use of mathematic models to describe the microbial inactivation on baby carrots by gaseous chlorine dioxide. Food Control. 123. Article 107832. https://doi.org/10.1016/j.foodcont.2020.107832.
Zhang, Y., Liao, Y., Salvador, A., Wu, V.C. 2021. Genomic characterization of two Shiga toxin-converting bacteriophages induced from environmental Shiga toxin-producing Escherichia coli. Frontiers in Microbiology. 12. Article 587696. https://doi.org/10.3389/fmicb.2021.587696.
Gorski, L.A., Walker, S., Romanolo, K.F., Kathariou, S. 2021. Growth and survival of attached Listeria on lettuce and stainless steel varies by strain and surface type. Journal of Food Protection. 84(5):903-911. https://doi.org/10.4315/JFP-20-434.
Hnasko, R.M., Jackson, E.S., Lin, A.V., Haff, R.P., McGarvey, J.A. 2021. A rapid and sensitive lateral flow immunoassay (LFIA) for the detection of gluten in foods. Journal of Food Chemistry. 355. Article 129514. https://doi.org/10.1016/j.foodchem.2021.129514.