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Interactions of Salmonella Enterica and E. Coli O157:H7 with Protozoa from Fresh Produce

Berk, Sharon
Tennessee Technological University
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  1. The gene expression profile of S. enterica and E. coli O157:H7 while in Tetrahymena vacuoles, and identification of genetic determinants that have a role in this pathogen-protist interaction will be studied.
  2. The role of bacterial residence in Tetrahymena vacuoles and vesicles in the toxicity of E. coli O157:H7 will be determined.
  3. The effect of the sequestration of S. enterica and E. coli O157:H7 cells within Tetrahymena vesicles on survival of the bacteria during environmental stresses in vitro and in planta will be determined.
  4. We will investigate the behavior of human enteric pathogens in amoebae isolated from packaged fresh produce, and the role of amoebal encystment in their survival.
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NON-TECHNICAL SUMMARY: Fresh produce is one of the most important vehicles of food-borne illness, yet one of the most crucial questions that emerges from outbreaks is how the human pathogens survive the harsh environmental conditions on produce in the field and the sanitizer treatments. The purpose of this study is to understand how protozoa may influence the survival and virulence of enteric pathogens associated with produce.

APPROACH: Protozoa isolated from soil in CA and from a package of Dole baby spinach will be used to study several aspects of their interactions with foodborne pathogens. In general, vesicle production by the spinach protozoa will be studied, and additional studies will determine the degree to which cysts trap pathogens. Survival of the sequestered bacteria in vesicles from the protozoa compared with free bacteria will be examined under various environmental conditions, including desiccation, and exposure to UV light and chlorine. Gene expression profiles of S. enterica and E. coli O157:H7 will be established while the bacteria reside in the ciliate vesicles. Changes in virulence after passage through protozoa will be studied for the pathogens interacting with the ciliate.

PROGRESS: 2007/09 TO 2008/08
OUTPUTS: Experiments to determine enhanced survival of E. coli O157:H7 and Salmonella enterica Typhimurium by interactions with protozoa have been completed (part of Objective III). These experiments included survival of the pathogenic bacteria within expelled pellets of the ciliated protozoan in both the aqueous environment and under conditions of desiccation. In addition, experiments to determine the fate of bacteria within the expelled pellets have also been completed. Survival of both pathogen species in pellets exposed to the sanitizer, hydrogen peroxide plus peroxyacetic acid, has also been studied, and these experiments have been completed (also part of Objective III). Several experiments to determine bacterial gene expression in vacuoles of the ciliate have been initiated (Objective I). Development of the apparatus for UV light exposure of pathogens within expelled pellets has been completed (Objective III). Results from the first 8 months of the project were presented at the annual general meeting of the American Society for Microbiology, June, 2008 in Boston, MA.
PARTICIPANTS: The lead project director is Dr. Sharon Berk of Tennessee Technological University. A research assistant, Katherine Redding, was employed half-time on the project at Tennessee Technological University, and her role was to help maintain cultures and to carry out the testing of sanitizers against the pathogens within ciliate pellets. Dr. Barbara Goodson took over the technician role after Ms. Redding left for her advanced degree. Dr. Goodson is employed full time on the project and has developed the apparatus for the UV light studies and continues to carry out the work with UV light and ozone exposure. Charles Pannell is a graduate student who was supported by the Water Center of Tennessee Technological University, and who did the work on survival of pathogens under various enviromental conditions. A collaborator is Dr. Maria Brandl of the USDA/ARS, WRRC lab in Albany, California. She and her postdoctoral researcher have been developing and testing the microarray protocols for gene expression of bacteria in the protozoa.
TARGET AUDIENCES: Not relevant to this project.
PROJECT MODIFICATIONS: The only problem was the delay in the hiring of assistants for both institutions. The full time assistants (technician and postdoc) were hired several months after the activation of funds, primarily due to the length of the hiring process. Therefore, there is a slight delay in the start-up of certain objectives. However, we are close to our anticipated goals for this first year.

IMPACT: 2007/09 TO 2008/08
Studies of the long-term survival of the pathogens within the expelled protozoan pellets showed that those bacteria sequestered in the released pellets survived significantly longer than did free bacteria, not in pellets in the aqueous environment. When such pellets were dried, the E. coli O157:H7 within such pellets survived longer than did free E. coli O157:H7 not in pellets; however, the pellet form did not enhance the survival of the Salmonella in desiccation conditions. In addition, the ability of the pathogens to grow and escape the pellets was studied, and both foodborne species were able to grow and leave the pellets. These results show that interactions of protozoa with these particular pathogens can enhance the survival of the pathogens under certain environmental conditions. The impact of this work is that it has revealed a novel aspect of the environmental persistence of such foodborne pathogens in the environment, and that protozoa should be considered in the microbial ecology of such pathogens. The sanitizer, hydrogen peroxide plus peroxyacetic acid, was effective in killing all pathogens, both in pellets and free in the medium. Such information should be useful for reducing pathogen populations in food processing or food preparation areas.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Bacterial Pathogens
Natural Toxins