Contamination of Fruits, Nuts, and Vegetables by Filamentous Salmonella; Persistence and Virulence

NON-TECHNICAL SUMMARY: Recent outbreaks associated with nuts, peanut butter, toasted oats, and vegetables in the U. S. have increased concerns of Salmonella contamination, survival, and infectivity in or on products with reduced water activity. These products may become contaminated with Salmonella at one or more stages of pre-harvest, harvest, processing, or storage. The organism does not normally grow in low-moisture environments but some serovars can survive for prolonged periods. Salmonella encounters a myriad of stressful conditions in the food production chain that can trigger specific and general stress-protection systems that render cells more tolerant to stress and promote persistence in the environment or food. One interesting response to stress is the formation of filaments that can reach >100 I?m in length. Much of the basic biology of filamentation is unknown including triggers, production on foods, survival characteristics, and virulence. The formation of Salmonella filaments is significant to food safety because under favorable conditions the filaments will form septa and divide the filament into multiple, typical cells that can impact estimations of pathogen numbers, the effectiveness of intervention practices (i.e., critical control points), and risk assessments. This proposal will study Stress-Induced Filamentous Salmonella (SIFS) on fruits, nuts, and vegetables and specifically determine there: 1.) pathogen load assessments and persistence on foods, 2) formation and characterization, 3) mitigation measures, 4) infectious dose and virulence. This project will provide fundamental information on stress response in Salmonella that impacts persistence on fruits, nuts, and vegetables and define effective harvest and/or production practices that prevent formation or inactivate these stress-induced cells.

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APPROACH: Salmonella present on the surface of fruits, nuts, and vegetables will likely encounter one or more of the stress conditions identified in our preliminary studies trigger formation of SIFS. Five serovars of Salmonella enterica will be studied for filament formation. Filaments will also be used to inoculate tomato, peanuts, lettuce and stainless steel surfaces and cell morphology and viable numbers monitored over time. The growth and survival properties of filaments will be compared with normal cells in five different Salmonella serovars. Microarray comparisons of filaments and normal cells will be conducted with three strains; Typhimurium, Typhi, and Agona. The mean hybridization signal intensities for each gene will be calculated from the multiple probe targets per gene. Genes will be considered differentially expressed when the signal-to-noise ratios exceed 3, the paired Student's t test of the average RNA levels are significantly different from controls (P<0.05), and the mean RNA level changes are at least twofold. The results from this objective will determine whether filamentous cells are more or less robust than normal cells and provide insights into their formation, survival, and virulence. The preceding objectives will characterize the formation and persistence of SIFS. Results from these experiments will be used to set the specific parameters of storage and the basis for formulating control strategies by modifying storage or process interventions. This objective will evaluate conditions that failed to support or repressed filament formation (i.e., storage temperature, storage humidity, time) as an intervention step during the storage of in-shell peanuts, tomatoes, and lettuce to determine if these conditions will render Salmonella more sensitive to subsequent roasting (peanuts) and sanitizing (tomatoes and lettuce). The roasting process will be modified (i.e., time and temperature of roast, blanch-roast) as needed to determine the necessary time and temperature to achieve a significant reduction of Salmonella numbers. The free chlorine concentration, temperature, and exposure time will be tested to determine the most effective combination for Salmonella reduction on tomatoes and lettuce. To assess virulence, the ability of SIFS and normal cells of Salmonella to attach to, invade and multiply within intestinal epithelial cells, using the Caco-2 human intestinal epithelial cell line, will be compared. The virulence of SIFS will be tested in a mouse model of gastrointestinal bacterial infection using the genetically susceptible C57Bl/6 strain of mice. Salmonella cells will be inoculated directly into the gastrointestinal tract to mimic the natural route of infection. Results from this project will disseminated in peer-reviewed publications, presentations at scientific meetings, and through extension and outreach activities.