Chloe Roberts
Biology 110L
Section 001
12 March 2019
Introduction
Resistance genes are found on bacterial plasmids, and these genes are transferred to other
bacteria through conjugation. Some species have natural resistance, as bacteria are often
overuse of antibiotics. Antibiotics do not cause mutations themselves, but they create a selective
environment, killing the susceptible cells and leaving the resistant ones to reproduce (Burpee et
al, 2018).
In 2018, there were numerous outbreaks of E. coli-induced gastroenteritis. The FDA and
CDC conducted several studies to determine the possible causes of the outbreaks. They deduced
that they were a result of contamination of water used for irrigation and mingling of lettuce
during washing (FDA, 2018). A recent study also showed that other factors, such as nearby cattle
feedlots may affect the spread of E. coli (Berry et al, 2014). A separate study showed that
treatment of leafy greens during growth, harvest, processing, and packaging can affect
This particular experiment was designed to simulate these outbreaks and to determine
antibiotic resistance of E. coli on romaine lettuce grown on three test farms: Lucky’s Greens,
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Ella Farms, and Ollie’s Organics. The purpose is to determine which farms contain antibiotic-
resistant bacteria, determine the cause of contamination, and determine frequency and severity of
resistance on the farms. The goal is to determine how three distinct farms each had cases of
contamination, whether they were three separate cases or a single strain that infected the other
farms.
The bacteria being used are resistant to the antibiotic gentamicin. In a study of E. coli
found in the urinary tract, it was found that 59% of the E. coli studied were resistant to
gentamicin (Sabir et al, 2014). There are three known different genes that cause antibiotic
resistance to gentamicin. Bacteria containing any one of these genes will be resistant to the
antibiotic. These genes are different sizes, so gel electrophoresis, after serial dilutions and PCR,
will allow for the determination of which gene is present in the sample from each farm. It was
hypothesized that the three farms each had contamination from different strains of the bacteria,
which will be determined by the relative sizes of each sample’s resistance genes. It was
hypothesized that each farm’s sample would have different sized fragments, each bearing a
Cultures of bacterial samples from each farm were obtained, and the lab bench was
disinfected. Three nutrient agar plates containing antibiotic were obtained and labeled 10^-2,
10^-4, and 10^-6. Three hundred-fold dilutions were performed, and 100 microliters were plated
on each plate. Three nutrient agar plates without antibiotic were obtained and labeled 10^-2, 10^-
4, and 10^-6. Three hundred-fold dilutions were performed, and 100 microliters were plated on
each plate. Using countable plates without antibiotic, the amount of bacteria in the original
sample was determined. Using countable plates with antibiotic, the amount of resistant bacteria
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in the original sample was determined. The frequency of resistance was then calculated from
these data.
Using sterile toothpicks, three colonies from the agar plate with antibiotic were
transferred into tubes to run a PCR to amplify the DNA. The first colony was transferred to an
orange tube, the second transferred to a blue tube, and the third to a yellow tube. Three
remaining tubes (red, green, pink) contained control plasmids (A, B, and C respectively) that
A gel was prepared. 5 microliters of a DNA ladder was loaded into the first well. Loading
dye was added to each of the six tubes containing amplified DNA. 15 microliters from each tube
were extracted and loaded into the next six wells. The gel was run at 160 volts to determine
relative sizes of DNA fragments, and the gels were photographed. Sizes of fragments were
compared to known sizes of antibiotic resistant genes in order to determine the plasmids
Through these procedures, the frequency of resistance and the resistance genes present in
Results
Table 1 shows the colonies counted on the group’s plates (antibiotic and control). Table 1
shows the personal data of the group, which was later combined with data from other groups
with bacterial samples from Farm L. Table 2 shows this combined data, followed by a sample
# antibiotic resistant bacteria = [ (9197 colonies / 400 microliters plated /10^-2) + (300 colonies/
= 2.24 x 10^4
# total bacteria = [ (13,324 colonies / 400 microliters plated / 10^-4) + (366 colonies / 400
= 6.24 x 10^5
5
= 0.0359 = 3.59%
Table 3 shows the combined data of the groups with bacterial samples from Farm E,
Table 4 shows the combined data of the groups with bacterial samples from Farm O,
Volume Plated:
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Figure 1 shows the photographs of the gel electrophoresis runs that were done for each
sample. Both L and E (samples from Lucky’s Greens and Ella Farms, respectively) have the
same number of base pairs as resistance plasmid A. O (sample from Ollie’s Organics) has the
same number of base pairs as resistance plasmid C. This was reported in Table 2. Table 2 also
shows the frequency of antibiotic resistance, calculated above for each farm. Lucky’s Greens had
the highest frequency of resistance. Ollie’s Organics had the second highest, closely followed by
Ella Farms.
Discussion
The data show that the samples from Lucky’s Greens and Ella farms shared the same
resistance plasmid. This means that the outbreaks from the lettuce grown at these farms was due
to the same strain of E. coli. This could be due to sharing of contaminated irrigation sources or
mingling of lettuce during washing. This disproves the hypothesis that each farm’s outbreak was
caused by an entirely different strain of E. coli. Since the sample from Ollie’s Organics had the
genes for a different resistance plasmid, the outbreak on this farm was an entirely separate
occurrence.
Referring to Table 5 in the lab manual, it was determined that Ella Farms and Ollie’s
Organics did not reach the Low Level Contamination requirement, meaning that there are no
further recommendations for these farms (Burpee et al, 2018). However, Lucky’s Greens reached
Moderate Level Contamination. Recommendations for the farm include destroying available
romaine lettuce, replanting romaine lettuce, monitoring the farm for at least three months, and
Sources of error for the experiment may include non-calibrated instruments, such as a
PCR machine that is not running correctly. Another source of error could be contaminated agar
plates that were used for the serial dilutions. Another source of error could be a faulty DNA
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ladder that does not properly demonstrate the length of base pairs. Each of these has the potential
to alter data and give misinformation. The data could have been limited by any of these factors.
Also, the data could have been limited by a lack of more precise equipment. Even if everything
was calibrated correctly, limitations may have lain in the lack of more specialized instruments.
This experiment could be improved by running multiple gels for each farm sample to be
sure that there were no issues with the gel or DNA ladder. The experiment could also be
Works Cited
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Lopez-Velasco G., Millner PD. 2015. Effect of proximity to a cattle feedlot on Escherichia
coli O157:H7 contamination of leafy greens and evaluation of the potential for airborne
Burpee, D., Cyr, R., Hass, C., Ward, A. and D. Woodward. 2018. A Laboratory Manual for
Biology 110 Biology: Basic Concepts and Biodiversity. Department of Biology, The
Center for Food Safety and Applied Nutrition. (n.d.). Outbreaks - Environmental Assessment of
https://www.fda.gov/Food/RecallsOutbreaksEmergencies/Outbreaks/ucm624546.htm
Delaquis, P., Bach, S., and Dinu, L. 2007. Behavior of Escherichia coli O157:H7 in Leafy
Vegetables. Journal of Food Protection: August 2007, Vol. 70, No. 8, 1966-1974.
Sabir, S., Ahmad Anjum, A., Ijaz, T., Asad Ali, M., Ur Rehman Khan, M., & Nawaz, M. 2014.
Isolation and antibiotic susceptibility of E. coli from urinary tract infections in a tertiary care