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<p>The goal of this proof-of-principle study is to use anti-pathogen endophytes to reduce food safety risks of fresh and fresh-cut vegetables in situ via colonization of the plants with endophytic strains of Bacillus subtilis that produce a broad spectrum of antimicrobial substances against the foodborne pathogens. The specific aims of this pilot study are the followings: </p>
<p>1) to verify endophytic ability of the newly isolated strains of anti-human-pathogen B. subtilis and </p>
<p>2) to evaluate the efficacy of the endophytic strains of B. subtilis on preventing or reducing the internalization and surface contamination of selected foodborne pathogens in/on lettuce from pre- to post-harvest of the vegetable.</p>
<p>NON-TECHNICAL SUMMARY:<br/> Endophytic bacteria can live inside plants from pre-harvest to post-harvest without causing any overt negative effect. The goal of this proof-of-principle study is to test our hypothesis that anti-human-pathogen endophytic strains of Bacillus subtilis can be used as bio-control agents in-situ to reduce food safety risks of fresh and fresh-cut vegetables. Our specific objectives are to verify the endophytic ability of anti-human-pathogen B. subtilis isolates and to evaluate the efficacy of the endophytic isolates on preventing or reducing the internalization and/or surface contamination of selected foodborne pathogens in/on the vegetable from pre-harvest to post-harvest. Lettuce is selected for this study as it has been identified to be the pathogens carrier responsible for several recent outbreaks of foodborne illness in the U.S.. The B.
subtilis isolates will be bioengineered to become constitutive expression of FP so that their internalization in lettuce can be evidenced via visualization. The inhibitory efficiency of endophytic B. subtilis against the internalization and growth of pathogens in/on vegetables will be determined before, at, and after harvest by enumeration of the pathogen on the edible portions of vegetables and the cut surface. Avirulent and fluorescent-protein (FP) expressing strains of Escherichia coli O157: H7, and Salmonella enteric will be used as the surrogates of foodborne pathogens throughout this project, while pathogen Listeria monocytogenes will be used for the post-harvest test. This study addresses the critical need of devising effective methods for reducing pathogen load, while maintaining food quality of produce, consistent with the objectives of the Program Area A4181.
<p>APPROACH:<br/> Lettuce and spinach are selected for this study as they have been identified to be the pathogens carriers responsible for several recent outbreaks of foodborne illness in the U.S.. Avirulent and green fluorescent-protein (GFP) expressing strains of E. coli O157: H7 and Salmonella typhimurium will be used as the surrogates of foodborne pathogens for their safety and detection advantages. Comprehensive experiments are designed to fulfill the objectives and will be conducted in the growth chambers or greenhouse. The inhibitory efficiency of Bacillus spp. bacteria and the major factors affecting this efficiency against the internalization, transmission, growth, and survival of the surrogate pathogens in/on vegetables will be determined before, at, and after harvest by enumeration of the surrogate pathogens on the edible portion of vegetables. The tested
vegetables will be grown in sterile or conventional soils mixed with composted or aged manure (spike with the surrogate pathogens). The optimization experiments will be designed to identify the major affecting factors. The affecting factors to be evaluated will include: 1) the timing of (at different growth phases of vegetables) and the cell form (vegetative cell vs. endospore) for introducing endophytic Bacillus spp. bacteria to the plants; 2) the use of indigenous vs. alien strains of the microbes; 3) the use of constitutive- vs. inducible-antimicrobial-producing strains of the bacteria; 4) the use of single vs. mixed multiple strains of the microorganisms. The result of the optimization test will be used to develop a vegetable farming practice and tested in the greenhouse.