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United States Department of Agriculture

Agricultural Research Service

Research Project: Ecology and Detection of Human Pathogens in the Produce Production Continuum

Location: Produce Safety and Microbiology Research

Project Number: 2030-42000-050-00-D
Project Type: In-House Appropriated

Start Date: Dec 28, 2015
End Date: Dec 27, 2020

Objective:
1:Elucidate biological factors and molecular mechanisms that enhance or reduce fitness characteristics related to survival and growth of enteric pathogens in the produce production continuum.(A)Phenotypic characterization of shiga toxin-producing E. coli O111 strains of environmental & clinical origins & (B)traits contributing to fitness of Salmonella strains on pre-harvest lettuce.(C)Evaluate formation of STEC persisters & factors contributing to population size in produce production continuum.(D)Compare fitness & physiology of different STEC serotypes & associated indigenous microbiota on produce growing in conventional vs organic soil.(E)EcO157 biofilm formation under stress conditions in MAP lettuce.(F)Identify genes involved in attachment of L. monocytogenes to produce & role of biofilm formation in survival on produce.(G)Evaluate interaction of EcO157 with various lettuce accessions.(H) Investigate evolution of EcO157 in MAP lettuce & (I)interactions between norovirus & native plant-associated bacteria. 2:Identify environmental factors that affect the persistence and transmission of enteric pathogens in the produce production environment for risk assessment.(A)Develop & deploy quantitative assessment of enteric pathogens in surface water in several watersheds (WS)in Salinas region to enhance current incidence data supplied to FDA for risk assessment model.(B)Determine survival & fitness characteristics of enteric pathogens in water & sediment samples from central California coastal (cCc) produce production regions & (C)the impact of indigenous microbial community on environmental persistence of STECs in sediment from WS locations often positive for STEC strains in Salinas region using metagenomics approach.(D)Comparative genomics & transcriptomics applied to characterize environmental Shiga toxin-producing E. coli. (E)Investigate survival & persistence of Salmonella & L. monocytogenes in Central California WS to measure prevalence, identify subtypes & regional traits.(F)Characterize F+ RNA Coliphages in water samples from cCc produce production regions & determine feasibility as indicators for source-tracking enteric pathogens & (G)enteric pathogens transported through aerosols & determine seasonal fluctuations in cCc produce production regions.(H)Determine if wild pigs are hosts for human norovirus (HuNoV). 3:Develop methods for the detection and subtyping of enteric bacterial and viral pathogens from produce production environments; to aid epidemiological investigations and to distinguish pathogenic from non-pathogenic strains. (A)Develop FT-IR identification libraries for serotype analysis.(B)Improve STEC subtyping using sequence of CRISPR & other hypervariable genes to augment existing MLVA method.(C)Examine bacterial & produce-associated factors impacting genome & STEC virulence evolution.(D)Develop better assay to determine inactivation status of Tulane virus (TV) & HuNoV caused by viral genomic damage & (E)accurate, fast, & low-cost multi-amplicon in situ capture qRT-PCR assay determining HuNoV infectivity. 4:Study the ecology of Shiga toxin-producing E. coli (STEC) bacteriophages and its association with bacterial hosts.

Approach:
Plant-microbe model systems in combination with population studies, ecology, molecular methods, genomics, and microbiology will be used to investigate the interaction of human bacterial and viral pathogens with plants and plant-associated bacteria, as well as to develop improved methods for detection and subtyping of human on produce. Pathogenic E. coli is a foodborne pathogen that has been linked to numerous outbreaks of foodborne illnesses, and the illnesses are primarily attributed to the ingestion of Shiga toxin-producing E. coli (STEC). Previous research has indicated the virulence markers such as stx genes, of STEC strains are conferred to stx-encoding bacteriophages and can be transduced into the susceptible bacterial hosts. In order to understand the interplay between STEC-specific phages and their bacterial hosts in the environment to enhance the safety of food products and the prevention of new emerging foodborne pathogens, the initial focuses of the phage research are to isolate, collect and characterize STEC phages and to understand the relationship between phages and their hosts in the environment. Efficient methods for isolation of STEC bacteriophages will be utilized. Characterization of STEC bacteriophages will be established using genomic sequencing and proteomic analyses. The association of fecal contamination with the population of STEC bacteriophages in the environment will be determined. Environmental factors that influence the geographical distribution of STEC bacteriophages will be identified. This will establish a foundation to study biological interactions between phages and their hosts and the association of phages with bacterial evolution as well as to utilize collected phages to develop biosensors and pre-harvest biological controls for STEC to improve the microbiological food safety of the food supplies.

Last Modified: 06/28/2017
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