1. ENHANCED SURVIVAL OF E. COLI O157:H7 IN TETRAHYMENA PYRIFORMIS VESICLES Charles T. Pannell Tennessee Technological University Cookeville, TN 38505

2. Introduction The fresh produce industry has expanded remarkably in the last ten years in response to health-conscious consumers’ desires to eat a more balanced diet including leafy greens (US FDA 2001). Although, fresh produce can become contaminated with bacteria, viruses, and parasites, bacterial contamination poses the greatest threat to consumers in terms of both serious infection and number of infections (Beuchat 1995). Between 1982 and 2002 approximately 8598 cases of E. coli O157:H7, most of which resulted from the consumption of contaminated beef and produce, were reported to the CDC (Rangel et al 2005), and as recently as the Fall of 2006 an outbreak of E. coli O157:H7, linked to cattle feces-contaminated soils, sickened 199 people in the United States (US FDA 2006). Studies by Brandl et. al. (2005) show that enhanced protection is afforded to Salmonella enterica when Tetrahymena species ingest and expel them in vesicles.

3. Introduction (cont’d) Another study shows that pathogens that are internalized in their protozoan predators exhibit enhanced survival against the chemical agent colistin (Shona et al 2003). Studies by Johnston et al (2005) show that produce does not become more contaminated during packaging and processing; thus E. coli contamination originates in the fields and orchards where produce is grown. The purpose of this study is to determine whether Tetrahymena pyriformis, a bacterivorous protozoa species isolated from spinach, can increase the viability of E. coli O157:H7 by ingesting and then expelling this dangerous pathogen in membrane-bound vesicles. The null hypothesis is there will be no difference in percent survival between E. coli O157:H7 in vesicles and the control.

4. Methods and Materials Organisms and Growth Conditions. Tetrhaymena pyriformis, which was originally isolated from spinach, was procured from the American Type Culture Collection (ATCC) and grown axenically according to the procedure of Berk et al (1998) at 25° C. Green fluorescent E. coli O157:H7 were also obtained from the ATCC and grown on nutrient agar at 35° C, then aseptically transferred to a 75 mL nutrient broth solution along with 225μL KAN and incubated at 35° C. Production of Vesicles. Both bacteria and protozoa were washed according to the procedure of Berk et al (1998). Both E. coli and Tetrahymena were washed three times to ensure adequate removal of growth media. Using differential interference contrast (DIC) microscopy, the concentrations of both bacteria and protozoa in the original suspensions were determined. Tetrahymena were combined with E. coli in a ratio of 1:100,000 along with TBSS and incubated in a microwell at 25°C. The control consisted of free- floating E. coli in TBSS incubated at 25°C.

5. Methods and Materials (cont’d) Observations. On the day the cocultures were prepared, an initial count of E. coli was conducted to determine the ratio of live to dead bacteria. This was performed by combining BacLite® with the bacteria in a 1:1 fashion and incubating for 25 minutes at 35°C. Afterward, the bacteria were counted using an epi-fluorescent microscope; bacteria that fluoresced red were dead and those that fluoresced green were alive. –After 24 h the cocultures were observed under the light microscope for vesicle production. When vesicles were observed, bacterial counts were conducted using BacLite® and epi-fluorescence microscopy. Counts were also performed for the control. These counts continued for a period of ten days. Statistical Analysis. To determine whether a significant difference exists between the control and experimental group, the t-test will be used to compare average values of live versus dead bacteria. Significant differences were reported at the P<0.05 level.

6. Results After 24 h Tetrahymena (Fig 1) produced vesicles containing live E. coli O157:H7 (Figs 2 and 3). Die-off in the free floating bacteria was observed immediately, and with the exception of Day 4, continued throughout the experimental period (Fig 4). Figure 5 shows the difference between the control and experimental groups. I then calculated the mean and standard deviation for each group (Table 1). Using the t-test, I concluded that there is a significant difference between the control and experimental groups (p=0.018); thus I reject the null hypothesis - that there is no difference between the two groups.

7. Figure 1: DIC image of Tetrahymena pyriformis.

8. Figure 2: Tetrahymena vesicles with live E. coli O157:H7

9. Figure 3: Vesicle rotation movie.

10. Figure 4: Free-floating E. coli O157:H7 exhibiting live/dead pattern.

11. Figure 5: Comparison of percent viability between the control and experimental groups

12. Table 1: Mean and Standard Deviation of Data MeanStd Dev Experimental0.9730.011 Control0.6280.086

13. Discussion As is consistent with the work of Shona et al (2005) and Brandl et al (2005), it appears that the existence of membrane-bound vesicles provides enhanced protection for the bacteria contained within them. Similar observations on bacterial viability within respirable vesicles have also been made using Acanthamoeba and Legionella spp (Berk et al, 1998) Past studies on the microbiological quality of fresh produce have focused primarily on assessing the bacterial quality of different types of produce and have overlooked the effects of produce being grown under different agricultural conditions (Johnston et al 2005). Gong et al (2005) conducted research to test the viability of E. coli during composting and found that most but not all bacteria are killed during the heating process of composting.

14. Conclusions E. coli that are excreted in vesicles remain viable Such bacteria exhibit enhanced survival compared to bacteria free-floating in the environment Future research should be conducted to determine whether E. coli, once inside vesicles, can emerge from them and reproduce. Other research studies should be used to discover whether organic versus traditional methods of agriculture affect the microbiological quality of produce.

15. Acknowledgements I would like to thank the TTU Water Center for allowing me to use its facilities. I would also like to thank Poornima Gourabathini, Lalitha Janaki, and Dr. Berk for guiding and assisting me in my research.

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