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Chloe Roberts

Biology 110L

Section 001

12 March 2019

Antibiotic Resistance of Escherichia coli in Romaine Lettuce

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

surrounded by antibiotic-producing fungi and microbes. Resistance can also be a result of

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

contamination levels (Delaquis et al, 2007).

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

different plasmid with resistance genes.

Materials and Methods

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

were also run in the PCR machine to amplify the DNA.

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

contained in each sample (Burpee et al, 2018).

Through these procedures, the frequency of resistance and the resistance genes present in

each sample were determined.

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

calculation for antibiotic resistance frequency on the farm.

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Table 1: Number of bacterial colonies on Farm L (Personal Data)

Treatment Dilution: 10^-2 10^-4 10^-6

Volume Plated: 100 microliters 100 microliters 100 microliters

per plate per plate per plate

Antibiotic 543 (2 plates) 1 (2 plates) 0 (2 plates)

No Antibiotic Lawn 7506 (2 plates) 265 (2 plates)

Table 2: Bacterial Colonies on Farm L (Combined Data)

Treatment Dilution: 10^-2 10^-4 10^-6

Volume Plated: 100 microliters 100 microliters 100 microliters

per plate per plate per plate

Antibiotic 9197 (4 plates) 300 (3 plates) 11 (2 plates)

No Antibiotic Lawn 13,324 (4 plates) 366 (4 plates)

Antibiotic Frequency = (# antibiotic resistant bacteria) ÷ (# total bacteria)

# antibiotic resistant bacteria = [ (9197 colonies / 400 microliters plated /10^-2) + (300 colonies/

300 microliters / 10^-4) + (11 colonies / 200 microliters / 10^-6) ] ÷ 3

= 2.24 x 10^4

# total bacteria = [ (13,324 colonies / 400 microliters plated / 10^-4) + (366 colonies / 400

microliters plated /10^-6)] ÷ 2

= 6.24 x 10^5
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Frequency of antibiotic resistance

= (2.24 x 10^4) / (6.24 x 10^5)

= 0.0359 = 3.59%

Table 3 shows the combined data of the groups with bacterial samples from Farm E,

followed by the calculated antibiotic frequency:

Table 3: Bacterial Colonies on Farm E (Combined Data)

Treatment Dilution: 10^-2 10^-4 10^-6

Volume Plated: 100 microliters 100 microliters 100 microliters

per plate per plate per plate

Antibiotic 1829 (4 plates) 857 (3 plates) 0

No Antibiotic lawn 17,822 (4 plates) 910 (3 plates)

Antibiotic Frequency: 0.834%

Table 4 shows the combined data of the groups with bacterial samples from Farm O,

followed by the calculated antibiotic frequency.

Table 4: Bacterial Colonies in Farm O (Combined Data)

Treatment Dilution: 10^-2 10^-4 10^-6

Volume Plated:
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100 microliters 100 microliters 100 microliters

per plate per plate per plate

Antibiotic 2152 (4 plates) 24 (4 plates) 1 (1 plate)

No Antibiotic Lawn 12,204 (4 plates) 198 (4 plates)

Antibiotic Frequency: 0.928%

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.

Figure 1: Gel Electrophoresis Photos

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Table 2: Summary of Antibiotic Resistance in Farms

Farm Antibiotic Frequency of
Resistance Gene Resistance
1 Lucky’s Greens Plasmid A 3.59%
2 Ella Farms Plasmid A 0.834%
3 Ollie’s Plasmid C 0.928%
Organic’s

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

identifying the source of contamination.

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

improved by applying the techniques to multiple kinds of bacteria, to demonstrate how

conjugation of resistance plasmids can occur between multiple species.

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Works Cited

Berry ED., Wells JE., Bono JL., Woodbury BL., Kalchayanand N., Norman KN., Suslow TV.,

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

transmission. Appl Environ Microbiol 81:1101–1110.doi:10.1128/AEM.02998-14.

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

Pennsylvania State University, University Park, PA.

Center for Food Safety and Applied Nutrition. (n.d.). Outbreaks - Environmental Assessment of

Factors Potentially Contributing to the Contamination of Romaine Lettuce Implicated in a Multi-

State Outbreak of E. coli O157:H7. Retrieved from

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

hospital. Pakistan Journal of Medical Sciences, Vol. 30, 389-92.

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