ENHANCED SURVIVAL OF E. COLI
O157:H7 IN TETRAHYMENA
PYRIFORMIS VESICLES
Charles T. Pannell
Tennessee Technological University
Cookeville, TN 38505
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.
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.
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 freefloating E. coli in TBSS incubated at 25°C.
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.
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.
Figure 1: DIC image of Tetrahymena pyriformis.
Figure 2: Tetrahymena vesicles with live E. coli O157:H7
Figure 3: Vesicle rotation movie.
Figure 4: Free-floating E. coli O157:H7 exhibiting live/dead pattern.
Incubation at 25 Degrees C
1.2
Percent Viable
1
0.8
Experimental
0.6
Control
0.4
0.2
0
1
2
3
4
5
6
7
8
9
10
11
Day
Figure 5: Comparison of percent viability between the control and experimental groups
Table 1: Mean and Standard Deviation of Data
Mean
Std Dev
Experimental
0.973
0.011
Control
0.628
0.086
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.
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.
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.
THE END