Tracing Sources of Bacterial
Contamination Using Genes for
Antibiotic Resistance
Biology 110, Section 40
TA Lauren Chaby
December 6th, 2012
By: Margaret Culver
Introduction:
When bacteria become resistant to antibiotics, doctors have very few other ways
to fight bacteria and the infections they cause—making antibiotic resistant bacteria a
serious problem. Bacteria can become antibiotic resistant when they are continually
exposed to antibiotics over a long period of time.2 When a mutation occurs with
resistance to antibiotics, then it is advantageous for the bacteria to possess this gene and
natural selection causes that strain to survive. The genes for antibiotic resistance are
usually contained within a plasmid, a circular bit of DNA, separate from the majority of
chromosomal DNA. This gene can be passed on from parent to daughter cells through
reproduction or conjugation. In reproduction, bacteria separate and grow rapidly through
binary fission. This leads to a short generation time that allows evolutionary changes to
become evident very quickly. Conjugation can also spread the plasmid around quickly
by two bacteria cells exchanging genetic material across an appendage called a pilus.
This results in both bacteria having the new plasmid. These two mechanisms allow the
antibiotic resistance gene to spread to an entire population in a short amount of time.1
This cycle of continual exposure to antibiotics, that creates the selective pressure
for antibiotic resistance, is prevalent in chickens, who are bred for their meat or eggs.
Farmers and feed suppliers will add antibiotics to the chicken feed to get rid of bacteria in
the food and keep the chickens healthier. One common type of antibiotic used in chicken
feed is kanamycin. Kanamycin works by interfering with translation and inhibiting
protein synthesis in the ribosomes. There are three genes known to be responsible for
resistance to this antibiotic. These three different genes are different sizes and also code
for different enzymes, which affect the kanamycin differently, but detrimentally. The
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enzymes reside between the inner and outer membrane of a bacteria, and modify the
kanamycin as it passes through the outer membrane. The change to the kanamycin
renders it no longer able to perform it’s duty of interfering with translation.2
Our lab was made aware of some recent outbreaks of gastroenteritis, caused by
eating raw or undercooked eggs. When further examined, the cases led back to
Salmonella enteriditis bacteria, in which kanamycin resistance genes were present. This
is dangerous, because if the bacteria are antibiotic resistant, there are few ways to get it
out of the food. The infected eggs were traced back to three different farms in different
geographical locations. It is important to know if the same gene caused this
contamination, so that the industry can take the first steps towards solving the problem.2
Our hypothesis was that there was some shared source of contamination. Even
though the farms are sufficiently far apart, many farmers follow the same practices and it
could be that one of those practices or procedures led to this contamination. The purpose
of this lab will be to determine which gene caused each contamination and to calculate
the frequency of kanamycin resistance bacteria so the severity of the problem can be
understood. After these two steps, a recommendation can be made to try and solve the
problem.
The first part of this experiment will be carried out using a substance called a
polymerase chain reaction (PCR), which amplifies the resistance gene. Then agarose gel
electrophoresis will be used, so that the lengths of the DNA can be compared to known
lengths for each gene and the correct gene from each farm identified. The second part of
the experiment involves serially diluting plates to find the right amount of countable
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colonies on plates with and without the kanamycin resistance gene. From that data, the
frequency of the kanamycin resistance gene can be determined.2
Methods:
Before the kanamycin could be tested in our lab to determine the frequency and
what gene the contamination was from, steps were taken to isolate the gene. The
kanamycin resistance gene from each farm was isolated and placed in non-pathogenic E.
coli. This was necessary to test specifically the kanamycin resistance gene through PCR
and not other genes. It was also safer to use a nonpathogenic form.2
The first step done by our lab was to create serial dilutions of bacteria with the
kanamycin resistant genes and without the kanamycin resistant genes. We prepared
dilutions of 10-2, 10-4 and10-6 of the bacteria. These dilutions were added to 3 agar petri
plates with the kanamycin resistance gene and 3 without, creating a total of 6 plates.
Sterile glass beads were rolled around the inside of the petri dishes to distribute the
bacteria. Serial dilutions were completed to find the right density of colonies. We didn’t
know how much E. coli would produce the right number of colonies so they could be
counted. It also helped determine the concentration of bacteria that have the resistance
gene and the concentration of bacteria that do not have the resistance gene. This is the
initial step in isolating bacterial colonies with antibiotic resistance. The plates were
incubated for 24 hours and then stored in a cold room for 6 remaining days.2
The next step is to amplify the specific genes for kanamycin resistance through
PCR. We used pipettes to extract DNA from three different colonies on the kanamycin
plates and added those to three colored tubes with PCR reaction mix. These were run
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through the PCR machine. Three additional tubes of different colors, containing primers
with control plasmids were also run through the PCR machine. The controls were used to
have a comparison value when evaluating the unknown kanamycin genes.2
While the PCR machine was running, the colonies on the plates were counted. If
there was an unreadable smear of bacteria, it was referred to as a lawn and not counted.
Distinct colonies were counted and where large numbers existed, a small portion of the
plate was counted and multiplied by the number of portions to get the total. Equation 1
(see below) was used to calculate the original population size. Then using the answer to
that for both kanamycin and no kanamycin plates, the frequency of kanamycin resistance
gene was calculated using equation 2 (see below). This is important to determining how
serious the problem is. A gel solution was then prepared by heating 1X TAE buffer and
agarose in a flask, pouring the solution into the gel electrophoresis tray and letting that
harden for ten minutes.2
Once the PCR machine was finished running and the agarose gel was set, the gel
was loaded and prepared to run. The six samples of DNA from the PCR machine were
mixed thoroughly with a loading dye, composed of bromophenol blue and xylene cyanol
ff. The loading dye weighs down the DNA until the current passes through and also
allows you to see how far the gel has elctrophoresed. A seventh sample called a DNA
ladder was also loaded into the gel electrophoresis tray. The DNA ladder is present so
that the lengths of unknown DNA can be measured against the ladder and lengths
determined. The control DNA plasmids from known gene type A, B, and C were also
included to compare the unknown DNA to known DNA lengths. The current was
supplied at 160 volts and when the gel had migrated half of the way, the current was
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switched off and the gel taken under a UV light to be photographed. The photograph
revealed how far each DNA sample had migrated and the values could be compared.2
Data Analysis:
Analysis Equations:
Equation 1: Calculate original bacteria population size:
N
P0 =
DV
- where P0 = initial population, N = number of colonies, D = dilution factor, V =
volume plated in mL
Sample Calculation from Farm A:
P0 =
P0 =
N
DV
1456
(10-6 )(.1)
P0 = 1.456 x 1010
Equation 2: Calculate frequency of kanamycin resistance gene:
Fk =
-
Pk
Pnk
where Fk = frequency of kanamycin resistance gene, Pk = population of bacteria
with kanamycin resistance gene, Pnk = population of bacteria without kanamycin
resistance gene
Sample calculation for frequency of kanamycin resistance gene:
Fk =
Pk
Pnk
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Fk =
1.90 × 107
1.456 × 1010
x 100%
Fk = .130 %
Results:
The results from the gel electrophoresis reveal that Plasmid A, caused the
contamination on farm A. My experiment was performed on farm A and combined with
data from groups who performed the experiment on the other two farms. As seen in
Figure 1, the middle two samples of unknown DNA did not electrophorese. However,
the first sample did. It’s length is exactly equal to that of Plasmid A, confirming its
identity. Farm C was also contaminated by Plasmid A. However, farm B did not follow
the pattern and was contaminated by Plasmid B. The frequency of kanamycin resistance
gene from each farm was: Farm A = .130%, Farm B = .250 %, and Farm C = .026 %.
Data Table 1: Numbers of E. coli Bacterial Colonies on +/- Kanamycin Dilution Plates
Dilution
10-2
10-4
10-6
Volume Plated
.1 mL
.1 mL
.1 mL
Farm A
Kanamycin
480
190
3
(# of Colonies)
No Kanamycin
Lawn
Lawn
1456
Farm B
Kanamycin
100
2
0
(# of Colonies)
No Kanamycin
6400
400
11
Farm C
Kanamycin
712
15
1
(# of Colonies)
No Kanamycin
Lawn
4832
151
Farm C
Kanamycin
392
12
0
(# of Colonies)
No Kanamycin
Lawn
3056
90
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* The numbers highlighted are the numbers that were used to calculate kanamycin
frequency
Data Table 2: Kanamycin Resistance Gene and Frequency
Farm
Kanamycin resistance
gene
Frequency of kanamycin
resistance gene
A
Type A
.130%
B
Type B
0.250%
C
Type A
.026%
Figure 1: Gel Electrophoresis Results for Farm A
C
B
A
U1 U2
U3 Ladder
Where C, B, and A are the control plasmids and U1, U2, and U3 are the unknown genes
from Farm A.
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Discussion:
The frequencies of kanamycin resistance gene were relatively insignificant, with
all the percentages below .3 %. The sources of contamination were not all from the same
source. However farm A and farm C had the same contamination source of plasmid A,
while farm B had a source of plasmid B. This means that the contamination was not all
caused by the same event, because each of the resistance genes likely arose independently
of one another. This is contrary to the initial hypothesis that the contamination could
have been caused by the same type of farming practice or procedure.
Because the sources of contamination for each farm are different, they will need
to be addressed individually. Each farm will need it’s own separate investigation of the
farm and the food supply to determine how each was contaminated. There is a chance
that farm A and C were contaminated in the same way, however since B was different all
the farms need to be separately examined. Since the level of bacterial contamination was
below 5 %, there are a couple steps that need to be taken to ensure egg and farm
decontamination. The eggs must be sent to a pasteurization center and remain there until
the contamination levels are below 1% for 8 weeks. The farm should also be watched
carefully over the next couple of months to be sure this outbreak would not occur again.
As stated above, each farm will need a separate investigation of its contamination and the
level and/or type of antibiotic will need to change as well. If they lowered the levels of
antibiotics or frequency with which they were given to the chickens, the selective
pressure for bacteria to be antibiotic resistant will decrease as well.
This was a complicated procedure and there are different parts where there could
have been error introduced. Our levels of kanamycin resistance gene frequency were
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extremely low indicating there could have been a problem somewhere. The
transformation procedure, performed before the samples reached our lab could have been
ineffective. When the plasmids samples from the bacteria were inserted into the E. coli
plasmid, that plasmid could eject the new piece of plasmid, accept it, or the sample
bacteria might not make it into the E. coli plasmid at all. If it is ejected, or doesn’t enter
it to begin with, then our results will be skewed because there will be less kanamycin
resistant E. coli, lowering the frequency.
Another source of error was that we had multiple groups working on different
farms and even one group working on two samples from the same farm. This introduces
a lot of variability with individual pipetting techniques and transfer techniques. With so
many different hands working on separate parts of the lab, people could have performed
steps a little bit differently, which would have affected the results. Lastly, when the farm
bacteria were added to the agarose plates, there could have been other bacteria from the
environment that was added as well, contaminating the sample.
In the future, the farming industry has to more carefully regulate its antibiotic
regime to prevent bacterial outbreaks like this one.
References:
1. Cyr, R., 2002. Prokaryotes I – Cellular and Genetic Organization. In, Biology 110:
Basic concepts and biodiverity course website. Department of Biology, The Pennsylvania
State University. http://www.bio.psu.edu/
Cyr, R., Hass C.,
2. Woodward D., and Ward A., 2010. Using Genes for Antibiotic Resistance to Trace
Source(s) of Bacterial Contamination. In, Biology 110: Basic concepts and biodiverity
course website. Department of Biology, The Pennsylvania State University.
http://www.bio.psu.edu/
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