Enhanced survival of E. coli O157:H7 in Tetrahymena vesicles By: Tyler Pannell, Undergraduate Student (Botany Concentration), Dept. of Biology, Tennessee Technological University, Cookeville, TN 38505

Project Summary Problem: Do E. coli O157:H7 contained within Tetrahymena vesicles live longer than free floating E. coli? My objective is to show that E. coli O157:H7 survive longer in ciliate vesicles than do E. coli that are free- floating in the environment. I will accomplish this goal by feeding Tetrahymena ciliates E. coli O157:H7 and then observing the percentage survival approximately every other day. I expect to see enhanced survival of E. coli O157:H7 in vesicles as compared to the free-floating E. coli.

Introduction According to Rangel et. al cases of E. coli O157:H7 were reported to the CDC between 1982 and The research of Johnston et. al. indicates that E. coli contamination in produce increases during the packaging and processing of cantaloupe, but not in green leafy vegetables. Mubiru et. al. compared the survival rates of pathogenic versus nonpathogenic E. coli and discovered that there is no statistical difference in mortality between the two groups of E. coli grown under different physical and chemical soil environments. According to LeClerc et. al. antibacterial resistant pathogenic E. coli has a high percentage of mutator phenotypes as a result of defective methyl- directed mismatch repair gene sequences. This makes E. coli O157 hypermutable (i.e capable of surviving under a wider array of environmental conditions/disruptions). According to Gortz new methods for detecting intracellular bacteria have emerged and will likely lead to greater awareness of the diversity of intracellular bacterial symbionts – this awareness will help better predict how protozoa and symbiont bacteria will interact.

Literature Cited Gortz, Hanz-Dieter Intracellular bacteria in ciliates. International Microbiology 4: Johnston, L. M., L. Jaykus, D. Moll, M. C. Martinez, J. Anciso, B. Mora, C. L. Moe A field study of the microbiological quality of fresh produce. Journal of Food Protection 68: Leclerc, J. E., B. Li, W. L. Payne, T. A. Cebula High mutation frequencies among Escherichia coli and Salmonella pathogens. Science 274: Mubiru, D. N., M. S. Coyne, J. H. Grove Mortality of Escherichia coli O157:H7 in two soils with different physical and chemical properties. Journal of Environmental Quality 29: Rangel, J. M., P. H. Sparling, C. Crowe, P. M. Griffin, D. L. Swerdlow Epidemiology of Eschericia coli O157:H7 outbreaks, United States, Emerging Infectious Diseases 11:

Objective Because of the enhanced protection afforded by the vesicles, E. coli O157:H7 will survive longer inside them than free- floating within the environment. Null Hypothesis:There will be no difference in survival rates between free- floating E. coli O157:H7 and E. coli that are enclosed in vesicles.

Methods and Materials Methods –First, I will feed Tetrahymena with E. coli O157:H7. After waiting for vesicle formation (~24 hrs), I will then use BacLite® to determine the percenatage of live and dead bacteria inside vesicles and free floating in the environment. Live E. coli will fluoresce green and dead E. coli will fluoresce red. –I will count the bacteria inside the vesicles and (if free-floating) inside a grid on the microscope ~ every other day and use the Hypothesis Test to determine whether there is a significant difference between the bacteria growing inside vesicles compared to those free-floating in the environment. I will create graphs to more clearly illustrate my results. Data Form1Data Form1 Data Form 2

Expected Results and Benefits I expect that my research will provide evidence that E. coli survives longer in vesicles than free-floating in the environment. Hopefully this will show that when bacteria grow on produce (such as spinach) in concert with protozoa, they pose a greater health risk to humans than they would in the absence of protozoa.

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