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Given the significant role that fruits and vegetables produced along the border between the U.S. and Mexico have played on outbreaks of foodborne illness, and given the necessity to elucidate the factors that affect adherence of pathogenic contaminants on produce in the field, the proposed study will seek to test the following hypothesis: That persistence by pathogenic bacteria on the surface of produce, and the ability to remove these contaminants with antimicrobial application is a direct function of product surface properties such as surface tension, available nutrients, and the presence of specific microflora, and that these factors are themselves affected by seasonality and irrigation methods used in Texas and Mexico. <P>
To test this hypothesis, the following five objectives will be pursued: <OL> <LI> Conduct parallel field experiments in Texas and Mexico to determine the effect of season, and irrigation (method and frequency of application), on surface properties (topography, surface tension, and contact angle), nutrient concentration (sucrose), and composition of natural microflora on the surface of spinach, lettuce, leafy greens (cilantro or parsley), peppers, tomatoes, and cantaloupes. <LI> Determine the specific role of surface properties and available nutrients found on the surface of products in the field, as determined in Objective #1, on the migration to the stomatal region, and on biofilm formation, by E. coli O157:H7 inoculated on the surface of spinach, leafy greens, and lettuce, and by Salmonella Saintpaul inoculated on the surface of peppers, tomatoes, and cantaloupes in vitro. <LI> Evaluate the relative effect (agonistic vs. antagonistic) of the most populous genera and species of microflora found in products in the field, as determined in Objective #1, on the growth, biofilm formation, and migration to stomatal region by E. coli O157:H7 inoculated on the surface of spinach, leafy greens, and lettuce, and by Salmonella serovars inoculated on the surface of peppers, tomatoes, and cantaloupes in vitro. <LI> Determine the influences of season and irrigation on efficacy of water-based antimicrobial treatments applied at packing sheds in Texas and Mexico at removing field-inoculated non-pathogenic surrogate organisms from the surfaces of spinach, leafy greens, lettuce, peppers, tomatoes, and cantaloupes. <LI> Develop educational materials and training modules for industry and government inspectors, in both English and Spanish, based on results from this proposed study, to include recommendations on type of antimicrobial treatments that work best at removing E. coli O157:H7 and Salmonella from produce, according to the season and irrigation conditions, as well as application of natural microflora that are found to be antagonistic to pathogens as field inocula. <LI> Deliver the training in Texas, Arizona (as an additional border state), and Mexico. D<LI> evelop an evaluation instrument to determine effectiveness of the training, and develop a survey instrument to determine whether recommended practices are implemented by industry 6 months after training.
NON-TECHNICAL SUMMARY: In the past several years, there has been a significant increase in both the number and magnitude of disease outbreaks attributed to consumption of contaminated fruits and vegetables. The largest outbreak in U.S. history occurred in early 2008 from consumption of contaminated peppers grown in Mexico, which were then transported and sold in the U.S. through Border States such as Texas and Arizona. In spite of much research over the last decade, questions as to the factors that affect the persistence of harmful bacteria on the surface of certain fruits and vegetables remain unanswered. For example, how do the chemical and physical properties of the surface of produce, as well as the presence of nutrients, affect the ability of pathogens to invade and colonize it Do the types of non-harmful bacteria comprising the microbial community of fruits and vegetables affect the ability of pathogenic ones to thrive in these environments This project seeks to answer these and other questions by determining whether the persistence of Escherichia coli O157:H7 and Salmonella on the surface of produce and their ability to resist removal with antimicrobial treatments is affected by the surface properties of the product, the availability of nutrients, and by the presence of specific harmless bacteria on the product. Further, we will investigate whether these three factors are affected by seasonal changes and differences in irrigation methods used in growing fruits and vegetables in Texas vs. Mexico through parallel field studies in these locations during the Spring and Fall using spinach lettuce, leafy greens, peppers, tomatoes and cantaloupe. Knowledge gained from these studies will be used to develop education and training materials for industry and inspectors, delivering them as workshops and distance-education modules in Texas, Arizona, and Mexico.
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APPROACH: Two farms each in Texas and in the Sonora province of Mexico will be selected for sample collection during two seasons. Method and time of last irrigation will be documented for each farm and season, as well as any other information that may be deemed significant. To determine surface properties of each product sample, we will examine each sample's surface topography, as well as measure its surface tension, and contact angles. To determine nutrient availability on the surface, we will measure the concentration of sucrose. For each sample, we will enumerate aerobic mesophilic and psychrotrophic bacteria, coliforms, and lactic acid bacteria. Ten microbial isolates will be randomly chosen from the various plating media used, and screened for inhibitory action against E. coli O157:H7 and Salmonella Saintpaul. Isolates exhibiting antagonism towards one or both pathogens will then be used in Objective #3 to determine the effect of the presence of these organisms on growth, migration to stomatal region, and formation of biofilm by E. coli O157:H7 and Salmonella Saintpaul on product surfaces. Fresh spinach, lettuce, and cilantro will be inoculated with E. coli O157:H7. Migration of Bacteria to the Stomatal Region will be mesaured by Scanning Electron Microscopy and formation of biofilm by the crystal violet binding assay. To determine the effect of nutrient concentration on bacterial migration and on biofilm formation, product will be incubated in the presence of sucrose. The same procedures will be performed for Salmonella Saintpaul on the surface of fresh peppers, tomatoes, and cantaloupes. To determine the effect of surface properties, surfaces will be treated with various concentrations of sodium dodecyl sulfate, glycerol, or propylene glycol, inoculated with either E. coli O157:H7 or Salmonella Saintpaul, and tested for bacterial migration and biofilm formation. Fresh product (spinach, cilantro, lettuce, peppers, tomatoes, and cantaloupes) will be inoculated with each microfloral isolate followed by inoculation with either of the two pathogens. Biofilm formation will be measured by the crystal violet assay. We will assess efficacy of produce-applied antimicrobials (sodium hypochlorite, ethanolic suspension of carvacrol, acetic and citric acids) against multiple strains of non-pathogenic microbial organisms acting as surrogates for the enteric pathogens Escherichia coli O157:H7 and Salmonella. Antimicrobials will be prepared, either alone or in combination with sodium dodecyl sulfate, glycerol, and propylene glycol. We will develop education modules for training industry and Food & Drug Administration inspection officials. Delivery systems for disseminating the modules will include a variety of formats. Instructional materials, as well as the delivery methods, will be pilot-tested in Texas. Assessment of the effectiveness and adoption of appropriate practices will be accomplished through telephone surveys, online surveys, and personal observation, as well as through focus group research.