Jan. 1, 2013 - Dec. 31, 2014Award Number
Max Teplitski, Ph.D.
University of Florida
M. McClelland, M. DanylukResources
Coliforms and other indicator organisms are poor predictors of behavior of human pathogens (like Salmonella, pathogenic E. coli and Listeria) in the crop production environment. The purpose of this project is to generate suitable surrogates that could be used to safely model persistence of Salmonella in pre- and post-harvest environment. Such surrogates can then be used for field experiments on the effectiveness of different production practices and post-harvest treatments in reducing Salmonella. Therefore, we will develop safe strains of Salmonella (derived from isolates recovered from produce-linked outbreaks) suitable for environmental release. These strains will lack virulence genes and plasmids, they will not be able to acquire virulence plasmids, nor carry antibiotic resistance genes. The safety of these strains will be validated in mouse and chick models. To establish whether these safe surrogate strains behave similarly to their virulent parents in the environment, their fitness will be tested under conditions that mimic pre- and post-harvest treatment of produce. Once biological safety and environmental fitness of these strains is established, detection protocols will be designed to ensure that the surrogate strains do not trigger false-positive responses, yet are easy to identify.
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.
Such surrogates will be engineered and their suitability for field experiments will be established with the following objectives:
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.
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.
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).
Objective 4. Test the field fitness of the surrogate strains in irrigation water, manure and compost.
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.