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The goal of this project is to understand the physiology of stress-induced filamentation by Salmonella and to determine the role filaments play in acid tolerance and pathogenesis. This will be accomplished by the following specific objectives: <OL> <LI> Assess the ability of different stresses to induce filament formation <LI>Compare macromolecular synthesis between control cells and filaments <LI> Determine the mechanism of enhanced acid tolerance in filaments <LI>Determine the virulence of filaments in mice. </ol> Outputs: Results generated from this project will be presented at relevant national conferences such as annual meetings of the American Society for Microbiology, International Association for Food Protection, and Institute of Food Technology. In addition, as all three co-PIs are core members of the Food Research Institute (FRI), University of Wisconsin-Madison, the results will be presented at FRI annual meetings. Manuscripts describing the research will be submitted to relevant journals for publication.
NON-TECHNICAL SUMMARY: Salmonella causes an estimated 2-4 million cases of human gastroenteritis per year in the United States. Although most cases resolve in 5-7 days, the very young, elderly, and immunocompromised are susceptible to more severe infections that result in an estimated 500 deaths per year. It has been estimated that 95% of these cases result from consumption of contaminated food. Salmonella encounters myriad stresses in the pre-harvest and processing environments. Desiccation and temperature stress are a few of the commonly encountered stresses. In response, Salmonella triggers stress protection systems that render cells more tolerant to the inducing stress as well as cross protection to other stresses. As a consequence, there is greater survival and prolonged persistence of the organism in the environment or food. One of the long-term goals of our research is to define the role of stress-responses in the dissemination, fitness, and pathogenesis of salmonellae. One interesting response to stress that we and other groups have observed in salmonellae is the formation of filaments (elongated cells without septation) that can be greater than 200 microns long. The underlying processes and purpose of filament formation are unknown, but we have observed filament formation in response to several stresses including; desiccation, temperature, and ultraviolet light. This common response to different stresses points to a central system of importance to persistence and dissemination of this important human pathogen. Key questions center on whether filamentous salmonellae have enhanced survival properties and are infectious. Of equal significance is the formation of septa once encountering favorable conditions that results in a sudden burst in Salmonella numbers. Thus, filaments can affect tests to enumerate Salmonella in a food, which could influence retrospective assessments of the infectious dose and risk assessments. Likewise, the occurrence of filamentous Salmonella in the processing environment may influence the effectiveness of processing parameters. It is critical to understand under what conditions filaments are formed, whether filament formation affects the survival of Salmonella under other stress conditions, and if in turn these changes alter the virulence properties of the Salmonella cells. Our proposed research will provide insights into these important food safety questions.
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APPROACH: We will use two Salmonella enterica serovar Typhimurium strains, M-09-0001A-1 isolated from a peanut butter outbreak, and LT-2, a less robust isolate. We have shown that S. Typhimurium can form filaments over 100 microns long when grown under low water activity (aw) conditions, using NaCl as the humectant. Other stresses commonly encountered in the pre-harvest and food processing environment will be evaluated for their ability to induce filamentation. These include ultraviolet radiation, oxidative stress (e.g. bleach and oxidative sanitizers), and acid. DNA damage occurs after exposure to uv radiation, which induces the SOS response, ultimately leading to filament formation. The mechanism of filamentation under other stress conditions is unknown. We will determine whether DNA damage occurs and could be a cause of filamentation under these stresses. Ultraviolet treatment of bacteria results in inhibition of DNA synthesis but not RNA and protein, while DNA continues to be synthesized in cold-shock induced filamentous E. coli and is inhibited only slightly before inhibition of RNA and protein synthesis. We will monitor DNA, RNA, and protein synthesis in Salmonella grown under stress and non-stress conditions to determine if perturbation of any macromolecular synthesis can be related to filament formation. Cross protection is an important result of stress response. Our preliminary data showed that low aw induced Salmonella filaments are more tolerant to low pH than control cells. We will explore the mechanism of the enhanced acid tolerance in filaments by determining the expression levels of acid tolerance related genes such as gad, dps, and rpoS, enzyme activity levels of decarboxylase, urease, ATPase, and potential differences in membrane components such as cyclic fatty acids and outer membrane proteins OmpC and OmpF. We have shown that S. Enteritidis filaments are as virulent if not more in mice. Enhanced acid tolerance will also contribute to increased virulence. To test this, we will inoculate mice with and without pretreatment with bicarbonate with control and filamentous S. Typhimurium. Results generated will be presented at relevant conferences where the target audiences consist of members of academia, food industry, and food regulatory agencies. Knowledge gained on growth, survival, and virulence of Salmonella under stressful conditions will be used to make informed recommendations on interventions practices and risk assessments of Salmonella contamination of foods.