Avirulent Salmonella Strains and Their Use to Model Behavior of the Pathogen in Water, Composts, in and on Vegetables

Outbreaks of gastroenteritis linked to pre- and post-harvest contamination of produce continue to raise questions about the ecology of human enteric pathogens in the production environment. Although experiments using nonpathogenic indicator organisms have been instrumental in the development of the environmental safety metrics, important biological differences exist between Salmonella, Shiga toxin-producing E. coli (STEC), fecal coliforms and other organisms used as indicators. These differences are reflected in the pathogens’ persistence in the pre- and post-harvest production environments even when indicator organisms are not detected. Because all outbreak strains of STEC and Salmonella are pathogenic, field studies with these organisms in an unaltered state are not feasible. Two Shiga toxin-defective mutants of EHEC have been used in field studies in California and Georgia. No such avirulent Salmonella surrogate suitable for field studies was developed. Here, we will develop avirulent surrogate strains of Salmonella and test their suitability for field studies. Cifuentes et al. (2001) and our preliminary data demonstrate that Salmonella virulence features are not involved in persistence on or in plants. Therefore, it should be feasible to develop avirulent Salmonella suitable for field studies and incapable of harming humans and the environment. Several precautions will be taken to achieve this goal: Pathogenicity islands will be precisely excised, strains will not harbor virulence plasmids, they will have a genetic barrier to mating and acquisition of virulence plasmids, and they will be free of features increasing their resistance to antibiotics. <P>
Such surrogates will be engineered and their suitability for field experiments will be established with the following objectives: <P>

Objective 1. Construct avirulent surrogates in the sequenced model strain S. Typhimurium ATCC 14028, and strains of Salmonella isolated from tomato-producing fields (S. Newport), a cantaloupe outbreak (S. Poona), and the tomato isolate S. Braenderup. Verify safety in mouse and chick models.
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Objective 2. Determine whether surrogate strains can be detected with common isolation and identification protocols. Culture-based approaches for detecting surrogates and distinguishing them from the wild type salmonellae will be explored. Because the strains will lack virulence genes commonly used for the identification of Salmonella, we will validate a new set of PCR primers for the molecular detection of the surrogates and their differentiation from field isolates.
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Objective 3. Test the fitness of the avirulent strains during attachment to surfaces (stainless steel, plastics commonly used in vegetable production facilities, rubber gloves) and on red and green tomatoes, spinach and cantaloupes; and the persistence within tomatoes and cantaloupes. Test the strains’ sensitivity to common chemicals used in fruit and vegetable production (pesticides, herbicides) and post-harvest treatments (chlorine, produce washes).
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Objective 4. Test the field fitness of the surrogate strains in irrigation water, manure and compost.
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Upon completion of this project, we will have a series of avirulent strains of Salmonella suitable for on-site experiments aimed at defining fitness of the pathogen under the production conditions. Preliminary experiments conducted with mutants in S. Typhimurium ATCC14028 suggest feasibility of the proposed approach.