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Frequency and Type of Antibiotic Resistance of E. coli in the Spinach of Three Farms

Erin Kelly

November 29, 2017

BIOL 110H Lab Section 004

Partner: Kerry Abello

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1. Introduction

Bacteria uniquely contain two areas of genetic information within their cell body. The

first area is a larger central chromosome, and the second area is a plasmid which is a circular

DNA molecule (Burpee et al., 2017). The plasmids are incredibly important to the bacteria,

for as the bacteria replicate and transmit DNA to daughter cells during binary fission, the

plasmids can transfer their genes as well. The genes of the plasmid often code for special

actions like detoxification, virulence, ecological interactions, or antibiotic resistance (Smillie

et al., 2010). These genes within the plasmid often give the bacteria an advantage in the

environment they are in, allowing them to survive further and continue to pass on the

successful DNA.

When bacteria pick up a certain gene, via conjugation or transformation, that allows

them to fight and defeat antibiotics, they gain antibiotic resistance (Burpee et al., 2017).

Resistance is defined as “the likelihood of failure of therapy with a specific agent for a

specific organism at one or several alternative dosages" (Kahlmeter, 2016). Ultimately, when

bacteria develop resistance, antibiotics, that had previously succeeded, experience failure

because the bacteria have evolved to combat the antibiotic.

One bacteria, Escherichia coli, has been identified to carry antibiotic resistance to the

antibiotic gentamicin in certain strains (Burpee et al., 2017). This bacterium is often found on

farms, specifically spinach farms, and has been known to cause food poisoning if it is not

killed by the utilized antibiotic, gentamicin (Burpee et al., 2017). Gentamicin prohibits

bacterial growth by preventing protein synthesis in bacteria; however, if bacteria are

resistance to gentamicin they will continue to grow (Burpee et al., 2017). Generally,
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gentamicin resistance is caused by one of three different genes of differing sizes (Burpee et

al., 2017).

It has been noticed by research teams that three spinach farms show evidence of E.

coli contamination despite antibiotic use—Fields of Greens, Green Acres, and Popeye’s

Spinach. Consuming these bags of spinach have resulted in gastroenteritis, leading

researchers to believe that E. coli at these farms have developed antibiotic resistance. It is

important to discern which farm has the greatest frequency of contamination and what genes

are causing this resistance.

There are a few questions that aimed to be answered through experimental methods.

The first question is: what is the frequency of contamination at each farm? And is this

contamination low, moderate, or high? The next significant question is if the genes causing

the gentamicin resistance are all the same or different from farm to farm. Through a series of

experiments, antibiotic resistant bacteria will be cultured, counted, and tested. It is predicted

that each farm will contain bacteria of differing plasmids due to the varying environmental

factors at the differing locations of the farms.

To run this experiment, two smaller experiments must be conducted. First the

frequency of the bacteria must be calculated in order to determine the severity of the

contamination. To do so, a serial dilution will be manufactured for both antibiotic resistant

and non-antibiotic resistant bacteria of each farm. These frequencies will be later calculated

after the bacteria colonies are cultured and counted. Second, the type of plasmid of resistance

on each farm must be discovered. To find this, a sample colony of each farm will be taken

through PCR to expand the DNA amount, and then the DNA will be placed on a gel

electrophoresis along with the known genes of resistance and a DNA ladder to sort the DNA
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by size. The genes that run the same distance are the same size and the same genes, helping

to demonstrate which type of plasmid is causing gentamicin resistance in each farm colony

sample.

2. Methods

The severity of contamination must be calculated. This can be understood after finding

the frequency of the bacterial growth. A serial dilution is set up and both antibiotic resistant

bacteria and non-antibiotic resistant bacteria are diluted three times, tenfold, by a dilution

factor of 1:100. This will occur by pipetting 10l of mixed concentrated bacterial suspension

into 990l of sterile water then taking the result of the first dilution and pipetting 10l of the

first dilution on into 990l of sterile water. This will be done one more time. The final

outcome will be three microtubes of 10−2 , 10−4 , 10−6 dilution. These will be spread onto

agar plates via glass beads.

Figure 1: GROUP C Plating Configuration

Antibiotic Resistant Bacteria

10−2 10−4 10−6

Non-Antibiotic Resistant Bacteria

10−2
10−4 10−6
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This bacterial will be allowed to culture for a certain time span, and when desired growth

is reached after 48-72 hours the bacteria can be counted. Frequency can be determined by

counting up the bacteria and dividing the average number of colonies of antibiotic resistant

bacteria by the average number of initial antibiotic resistant bacteria as well as dividing the

average number of colonies of non-antibiotic resistant bacteria by the average number of

initial non-antibiotic resistant bacteria. The initial population can be determined by the

equation 𝐵 = 𝑁/𝐷 where B is the initial population, N is the number of colonies per added

amount of sample, and D is the dilution factor.

The type of gene causing the antibiotic resistance must also be determined. To do so, a

sample colony will be taken from one antibiotic resistant bacteria plate from each farm group

(A, B, C). Figure 2 demonstrates the mixture of primers and plasmids.

Table 1: PCR Mixtures

Tube PCR Primer Control Plasmids Resistant colony

color from farm’s “A”
plate
1 2 3 1 2 3 Popeye’s
Orange + - - - - - +
Blue - + - - - - +
Yellow - - + - - - +
Red + - - + - - -
Green - + - - + - -
Pink - - + - - + -

The samples in the microtubes will be put through a polymerase chain reaction, where the

DNA of the bacteria will become amplified through the process of denaturing the template

strand, annealing the primers, and elongating a copy strand. This will result in much more
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DNA that can then be placed in the well of a gel electrophoresis set up which is prepared by

mixing 0.3g of agarose, 30 ml of 1X TAE buffer, and 1l of ethidium bromide dye. The set-

up of the gel electrophoresis can be seen in Figure 3.

Table 2: Materials loaded into wells of agarose gel

Lane/ Well Sample Contents

1 DNA Ladder
2 PCR Sample 1 (Orange)
3 PCR Sample 2 (Blue)
4 PCR Sample 3 (Yellow)
5 PCR Sample 4 (Red)
6 PCR Sample 5 (Green)
7 PCR Sample 6 (Pink)
Molecular Weight Standard
Plasmid DNA from Colony 1
Plasmid DNA from Colony 2
Plasmid DNA from Colony 3
Standard of Plasmid A
Standard of Plasmid B
Standard of Plasmid C

The gel will run for about 30 minutes, and once viewed under a UV light, the genes of the

same size (same genes) will run the same distance, allowing for the identification of each

type of gene causing the antibiotic resistance at each farm.

(Burpee et al., 2017)

3. Results

Frequencies of Bacterial Growth

Table 3: Individual Data Farm C

Treatment 10^-2 10^-4 10^-6

Antibiotic 448 4 0

No Antibiotic Lawn Lawn 153

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Table 4: Farm C Data Average over Four Groups

Treatment 10^-2 10^-4 10^-6

Antibiotic 1664 27 0

448 4 0

1355 15 0

488 6 0

AVERAGE 988.75 13 0

No Antibiotic lawn lawn 153

lawn lawn 486

lawn 960 37

lawn lawn 191

AVERAGE lawn 960 214.25

Table 5: Class Data

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As seen in all the tables the bacterial growth was much lower for bacteria on the plates with

antibiotics, also known as the bacteria that developed antibiotic resistance. This occurred because

the antibiotic worked for a period of time to slow bacterial growth; however, the bacteria

developed antibiotic resistance by utilizing a certain gene in the plasmid and begin to grow

again. The bacteria on the blank plates did not encounter any aversion, making their growth

easier and therefore quicker. As seen Table 3, Green Acres (Farm B) had the greatest frequency

of antibiotic resistant bacteria at an average of 0.04379. Popeye’s (Farm C) had the second

greatest frequency with an average frequency of 0.002557. Finally, Fields of Greens (Farm A)

had the least contamination of antibiotic resistant bacteria with an average frequency of

0.001785. The frequency that the individual group C found was 0.001847 wish was relatively

close to the average frequency of 0.001785.

Type of Gene in Plasmid

Figure 2: Individual Gel Farm C

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Figure 3: Reference Gel Farm C (Philip)

Figure 4: Reference Gel Farm A (Philip)

Figure 5: Reference Gel Farm B (Philip)

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After the DNA of Farm C was placed in the well and the gel ran, the DNA did not

appear to leave the well as seen in Figure 2. Only the ladder ran. Possible reasons for

this error will be discussed later. As a substitute, Figure 3 is shown with all the DNA

of Farm C successfully run. In this image, it is clear to see that Farm C’s DNA ran to

about 300 Bp which is congruent with the distance run by known Plasmid C. In

Figure 4 it can be discerned that Farm A’s plasmid ran to about 500 Bp matching with

known plasmid A. Finally, in Figure 5 it can be seen that Farm B’s plasmid DNA ran

to about 350 Bp matching with known plasmid B.

Table 6: Summary of Antibiotic Resistance in Farms

Farm Antibiotic Resistance Gene Contamination Level

(Frequency x 100

1 2 3
1 Fields of yes no no 0.1785%
Greens
2 Green no yes no 4.379%
Acres
3 Popeye’s no no yes 0.2557%

4. Discussion

The contamination levels at each farm varies. Fields of Greens had a contamination

level of 0.1785%, which is considered less than low. Green Acres had a contamination

level of 4.379% which is considered moderate. Popeye’s had a contamination level of

0.2557% which is also considered less than low. These levels were determined by taking

the average frequencies calculated in the results section and multiplying the number by

100. This gave the percent contamination, and the danger level of the contamination was
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determined by referencing Table 5 in “A Laboratory Manuel for Biology 110 Biology:

Basic concepts and Biodiversity.”

Based on the gel electrophoresis, it can be understood that the E.coli in Fields of

Greens contained plasmid A, Green Acres contained plasmid B, and Popeye’s contained

plasmid C. This was determined because each of these farms plasmids ran the same

distance as the corresponding plasmid, proving their congruence because the same DNA

runs the same distance because it is the same size. Each farm was infected with a

different plasmid, or gene, of antibiotic resistance. This is interesting to note, for it means

that each farm’s bacterium took up a different gene of resistance from different sources.

The results supported the initial hypothesis that that each farm will contain bacteria of

differing plasmids due to the varying environmental factors at the differing locations of

the farms. The various sources that plasmids took up genes must have contributed to the

varying genes in each farm’s bacteria’s plasmids.

One issue that was encountered during this experiment was the lack of bands showing

up on Farm C’s gel agarose after gel electrophoresis—only the ladder ran. One possible

source of this error is that the DNA obtained from the colonies did not match the primers

during PCR. This would not allow for correct amplification and the DNA would not be

present enough to run on the gel during the electrophoresis. Another possible source of

error in this lab could be from the serial dilution being spread by the glass beads. These

beads were very hard to transfer in between plates, and often they fell out of the plates,

possibly skewing the sample numbers.

Ultimately the information obtained from this experiment is very important for the

consumer as well as the farmer. The consumer must recognize levels of contamination
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and prepare uniquely for the severity of it. At low levels of contamination, the consumer

must keep the spinach away from other produce and only use the spinach in cooked

preparations. In moderate levels of contamination, the famers must destroy the current

crop, monitor the new crop, and identify the source of contamination. In high levels of

contamination, the entire crop must be destroyed, the field must be left for a month

before replanting, and the fields must be monitored after replanting as well. The spinach

is not consumable at moderate or high levels. Identifying the gene that causes the

antibiotic resistance is critical to finding the source of contamination and fixing the issue.

Once the gene is identified, further tests can be performed to discover what

environmental factor the bacteria picked the gene up from. This factor can be removed

and perhaps the bacteria’s antibiotic resistance could be halted. Future experimentation

could allow for the preventative measure to be taken.

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5. References

Burpee, D., Cyr, R., Hass, C., Ward, A. and D. Woodward. A Laboratory Manuel for

Biology 110 Biology: Basic concepts and Biodiversity. 2017. Department of

Biology, The Pennsylvania State University, University Park, PA.

Kahlmeter, G. (2016). What is antibiotic resistance? Pharmaceutical Technology

Europe, 28(5), 8-9. Retrieved from

http://ezaccess.libraries.psu.edu/login?url=https://search-proquest-

com.ezaccess.libraries.psu.edu/docview/1792396475?accountid=13158

Philip, Chloe. Farm A Gel. 13 Nov. 2017.

Philip, Chloe. Farm B Gel. 13 Nov. 2017.

Philip, Chloe. Farm C Gel. 13 Nov. 2017.

Smillie, C., Garcillán-Barcia, M. P., Francia, M. V., Eduardo P. C. Rocha, & Fernando de

la Cruz. (2010). Mobility of plasmids. Microbiology and Molecular Biology

Reviews, 74(3), 434-452. doi:10.1128/MMBR.00020-10