1a.Objectives (from AD-416):
1. Concurrently evaluate the persistence of three foodborne pathogens (E. coli O157:H7, Salmonella, and Norovirus) on foliar surfaces of plants (lettuce and spinach), under the same conditions to facilitate a direct comparison of the survival characteristics of these organisms in the phyllosphere.
2. Investigate plant defense pathways during attachment and internalization of E. coli O157:H7, Salmonella and noroviruses. Determine if plants respond to these human pathogens using one or more plant-specific defense strategies.
3. Evaluate the metabolic pathways used by pathogenic bacteria to attach to plants, specifically using a novel approach to identify the major proteins of pathogenic bacteria using proteomics.
1b.Approach (from AD-416):
ARS will acquire the basic knowledge of how E. coli O157:H7, Salmonella, and norovirus survive under the exact same growing conditions, and to use culture and molecular methods to evaluate the persistence of these pathogens on foliar surfaces and plant responses to these pathogens. This information will be used by both ARS and the Cooperator to jointly develop a comprehensive understanding the interaction of bacterial and viral pathogens with leafy green crops and possibly develop antimicrobials or Good Agricultural Practices that can limit the contamination of produce. The Cooperator will specifically evaluate the infectivity of norovirus inoculated on leafy greens, and will determine methods using quantitative real time PCR (qPCR) targets to measure the expression of plant defense responses to pathogens.
Wild-type and rpoS-deficient strains of Salmonella and E. coli O157:H7 have been modified with chromosomal or plasmid-based antibiotic resistances so that their persistence on spinach and lettuce can be assessed by culture media. Strains were then differentiated on E. coli O157:H7-agar or Salmonella-selective agar to determine that all four strains could be identified. Preliminary inoculations procedures of leafy green commodities have been identified for a combined inoculum of wild-type Salmonella and E. coli O157:H7 and rpoS-deficient E. coli O157:H7 and Salmonella. In consultations with collaborators, the several different varieties of spinach and lettuce have been identified and selected so that they can be grown at three institutions participating in this study (University of Delaware, University of Wisconsin and USDA-ARS).