Location: Produce Safety and Microbiology Research
Project Number: 2030-42000-052-000-D
Project Type: In-House Appropriated
Start Date: Dec 28, 2020
End Date: Dec 27, 2025
Objective:
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.
