Examining Antibiotic Resistance to Determine Sources of Bacterial Contamination
Maya Evanitsky*, Deirdre Carlson, Katie Zinn, Allison Ng, Samantha Fortier
Group 1
BIOL 110, Section 905
TA: Rachel Cortese
6 December 2012
Introduction
Certain bacteria populations possess plasmids, which are small circular DNA molecules
that are separate from the main bacterial chromosome1. Plasmids can be replicated independently
from the main chromosome and transmitted to daughter cells during cell division or to other
bacteria through conjugation. Conjugation occurs when a bacterium with an appendage known as
a pilus physically joins with another bacterium lacking a pilus. Genetic material can then be
transferred from the bacterium with the pilus to the other bacterium. This will result in the
second bacterium developing a pilus. While not necessary for survival, plasmids may contain
genes useful to bacteria under certain conditions, such as antibiotic resistance or toxin immunity,
and can give bacteria a selective advantage1.
In today’s world, bacteria that are antibiotic resistant have become increasingly more
common due to the increased human use of antibiotics2. This is part of natural selection – since
antibiotics are naturally derived from fungi and other organisms, it is logical that bacteria will
evolve to resist them. Chicken farms and chicken feed suppliers add antibiotics to chicken feed
to prevent bacteria contaminating meat and eggs. This can then lead to antibiotic resistant
bacteria populations specific to the antibiotic used on the chickens. The antibiotic kanamycin
interferes with inhibits protein synthesis in the bacteria by interfering with translation in the
ribosome2. Translation is the second part of the process used to synthesize proteins and takes
place in the ribosome3.
Reports of severe gastroenteritis linked to eating raw or undercooked eggs have led the
FDA to investigate. Salmonella enteriditis bacteria have been located from the batches of eggs
that caused the food poisoning. These bacteria were found to have plasmids containing
kanamycin resistant genes. The eggs are from three different chicken farms in three different
states2.
Thus, the purpose of this lab is to determine whether the farms have a shared source of
bacterial contamination or if each case of kanamycin-resistant bacteria is distinctive to each
farm. There are three different genes responsible for kanamycin resistance and each gene codes
for a different enzyme that alters the chemical make-up of the kanamycin molecule in different
ways. These enzymes occur in the area between the inner and outer bacterial membranes
(periplasmic space). The kanamycin is disabled once it passes through the outer membrane and
cannot be taken up by the inner membrane in order to reach the ribosomes and inhibit protein
synthesis. Therefore, bacteria with one of these genes can grow in presence of kanamycin and
administering kanamycin to chickens will not prevent bacterial infection. The different genes
that cause antibiotic resistance are each a different size and are usually found in different
bacterial populations. The genes can then be identified by size and their identity used to trace the
source of the bacterial strain responsible for the infection2.
The experiment was done in two parts. One was done by other researchers prior and
involved creating serial dilutions and plating them on plates with and without kanamycin. The
colonies were then counted and the data used to determine the frequency of kanamycin resistant
bacteria in population. The kanamycin resistance gene was then transferred from the plasmid of
Salmonella enteriditis into a specially modified E. coli plasmid. This was done so that research
could be completed using non-pathogenic bacteria and the E. coli plasmid provides sites
recognized by particular PCR primers needed for the PCR reactions2.
PCR stands for polymerase chain reaction and is the process by which the DNA sample is
amplified in order to be analyzed4. In this process, the sample DNA is first heated so it separates
into two separates strands of DNA. After this, the enzyme Tag polymerase functions as DNA
polymerase would to synthesize two new strands of DNA by adding complementary nucleotides
to the original strands. This is repeated numerous times and results in over one billion copies of
the particular DNA segment4.
The second part dealt with the E. coli cultures containing the kanamycin resistance gene2.
Each group was assigned to the bacteria from one of the three farms. Serial dilutions were then
performed and plated with and without kanamycin present. After the bacteria grew, the colonies
were counted to determine the frequency of the resistance gene. A DNA sample was then taken
from the colonies and PCR performed. The final step was analyzing the DNA via electrophoresis
in order to identify the specific kanamycin resistance gene at each farm2.
The expected results were that the bacterial contamination was most likely due to
different genes. This is based on the fact that the different genes appear to have arisen
independently and usually occur in different bacteria populations2. Also, a frequency of
kanamycin resistance greater than five percent was predicted since bacteria are capable of
rapidly transmitting genetic material through reproduction and conjugation1.
The actual results of the experiment were neither the original bacteria population size for
the 10-2 dilution nor the kanamycin resistance gene frequency couldn’t be determined since a
lawn was formed; the 10-4 dilution had a population size of 2.13×108 cells/mL with a frequency
of .28% for the kanamycin resistance gene; the 10-6 dilution had a population size of 6.2×108
cells/mL and 0% frequency for the kanamycin resistance gene.
Materials and Methods
Materials
The materials used for the research prior to the experiment involved with examining the
E. coli cultures were plates with kanamycin and without, microtubes, Salmonella enteriditis
bacteria found at the farms, and engineered E. coli bacteria. For the next part of the experiment,
plates with kanamycin and without, microtubes, sterile glass beads, the genetically engineered E.
coli bacteria, and an incubator were used. The PCR aspect required the use of DNA primers,
three distinctive control plasmids, and the machine that performed the PCR. In the final phase,
DNA ladder, agarose gel, buffer, ethidium bromide, loading dye, a gel electrophoresis rig, and a
UV light with a hood were utilized. Throughout the experiment pipettes and disposable tips were
made use of.
Methods
Throughout the experiment, sterile technique was consistently used to prevent
contamination of the sample2. Gloves were worn at all times and a new pipette tip was used for
each solution and substance. The amount of time that containers were open was minimized and
the plate lids were only opened slightly rather than taken all the way off. Additionally,
everything was properly sterilized and sealed before and during the experiment2.
Serial Dilutions
The first step was done by researchers prior the experiments done involving the E. coli
cultures. First serial dilutions of 10-2, 10-3 10-4 10-5 of the original bacteria samples from the
chicken farms were prepared. 100 microliters were then plated onto plates with and without
kanamycin present. Once the bacteria had grown, the number of bacteria cultures was counted.
After this, the kanamycin resistance gene found at the farms was transferred from the original
pathogenic bacteria plasmid to an engineered E. coli plasmid2.
The next main step involved plating the E. coli bacteria cultures for further analyzing. For
this part, three serial dilutions (10-2, 10-4, 10-6) were done on the bacteria sample. These were
done by adding ten microLiters of concentrated bacterial suspension to the 10-2 microtube along
with 990 microLiters of water. Ten microLiters of the 10-2 dilution were then pipetted into the
10-4 microtube and diluted. The 10-6 dilution was done using ten microliters from the10-4
dilution. Each dilution was then plated onto two plates: one with kanamycin present and one
without. This involved inoculating each plate with the correct dilution and using sterile glass
beads to evenly distribute the bacteria across the plate. Two sets of beads were used; one for the
kanamycin plates and one for the plates lacking kanamycin. The beads were first placed on the
plate with the smallest concentration and transferred to the next smallest and to the largest. Once
this was done, the bacteria were incubated for twenty-four hours and stored in a cold room until
they could be counted. The final step was to count the bacteria colonies2. For this part only half
of the 10-4 concentration of bacteria on the plate without kanamycin was counted and doubled.
PCR
The next section of the lab dealt contributed to identifying the gene of kanamycin
resistance present in the bacteria2. The initial step was adding a bacteria colony from each plate
containing kanamycin to three separate microtubes. These tubes held primers to make the sample
replicable so the polymerase chain reaction (PCR) could occur. Each tube held a different primer
that corresponded to a different gene so only one would work to amplify the DNA. These were
then placed in the PCR machine along with three different control plasmids which served to
compare to the bacteria samples. PCR was then run and the DNA sample amplified.
Gel Electrophoresis
The final step was to set up and perform gel electrophoresis on the amplified DNA2. The
process of gel electrophoresis works to separate DNA fragments5. This is done by passing an
electric current through the solution. Since DNA backbone is made up of negatively charged
phosphate groups, the DNA moves towards the positively charged pole of the electrophoresis
tray. The DNA separates because smaller DNA fragments travel through the gel pores more
quickly than the larger DNA fragments5.
Gel electrophoresis was done by dissolving agarose into a buffer and heating until the
solution was clear2. Once it had cooled, ethidium bromide was added. The mixture was then
poured into the gel tray (see Figure 5) and hardened. Once the gel had solidified, buffer was
poured so that the gel was completely covered. The PCR DNA ladder was then added to the first
well to function as a size standard. Loading dye was added to the three samples and three
controls that had run through PCR. The dye functioned to allow the samples to sink to the bottom
of the wells and allow observation of how far the DNA travelled. The gel electrophoresis unit
was connected to the power supply and the process run until the bromophenol tracking dye has
migrated at least half way. The gel was then taken out and placed under a UV light for
analyzing2.
Figure 1: Diagram of gel
electrophoresis6
Results
Table 1: Numbers of Salmonella colonies from infected chicken farms growing on +/kanamycin plates
Chicken Farm
Kanamycin
A
B
C
No Kanamycin
A
B
C
Dilutions
10-4
10-2
10-3
10-5
Lawn
252
Lawn
122
26
428
9
3
41
1
0
5
Lawn
Lawn
Lawn
Lawn
Lawn
Lawn
107
96
121
11
8
13
Table 2: Numbers of E. coli bacterial colonies on dilution plates with and without kanamycin
(Farm B)
Treatment
Dilutions
-2
Volume Plated
(mL)
Kanamycin
No Kanamycin
-4
10
.1
10
.1
10-6
.1
859
lawn
6
2130
0
62
Table 3: Frequency of Kanamycin Resistant Gene and Original Bacteria Populations of
Salmonella enteriditis
Chicken
Farm B
Frequency of
Kanamycin
Resistant
Gene
Original
Population
Size
(cells/mL)
10-2
1.82%
K1.38×107
10-3
2.26%
Dilutions
10-4
3.13%
K+
KK+
K2.52×105 1.15×107 2.60×105 9.60×106
10-5
0%
K+
KK+
3.00×104 8.00×106 0
Table 4: Frequency of Kanamycin Resistant Gene and Original Bacteria Populations of E. coli
10
-2
Dilutions
10-4
10-6
Frequency of
Kanamycin
Resistant Gene
Original
Population Size
(cells/mL)
1.17%
0.28%
K7.32×107
0%
K+
KK+
8.59×105 2.13×108 6.00×105
K6.2×108
K+
0
Table 5: Class Plasmid Data
B
Kanamycin resistance
gene
A
B
C
x
A
x
Chicken
Group
Farm
1
Data from “orange” well matched “red”
well  control #1. Also had to use stock
gel image.
Had to use group 1’s image
2
3
4
B
C
Comments
X
x
Figure 7:
Photograph of
electrophoresis
results for Farm B
Table one shows the number of Salmonella enteriditis colonies that grew for each
dilution plate. The bacteria that grew in the presence of kanamycin were the antibiotic resistant
bacteria and had significantly lower numbers than the bacteria that did not grow in the presence
of kanamycin. The next table displays the colony counts for the engineered E. coli bacteria. The
E. coli had larger colony counts when comparing the corresponding dilution plates. Table three
shows the frequency of the kanamycin resistance gene for the bacteria taken from the chicken
farms along with the calculated original bacteria population size for each plate. These numbers
were calculated using the data in Table one. Each dilution had a kanamycin resistance gene
frequency of less than five percent, equating to a relatively low frequency overall. Table four
contains the kanamycin resistance gene frequency and original population size for each plate for
the E. coli cultures. These quantities were calculated using the information from Table two.
Comparing Tables three and four shows that the E. coli bacteria had larger original populations
than the Salmonella enteriditis for each plate, but the S. enteriditis had higher kanamycin
resistance gene frequencies.
The last table exhibits a summary of the electrophoresis results for the class and indicates
that the three farms each had a different kanamycin resistance gene. Figure seven is a photograph
taken of the electrophoresis gel while under the UV light. This was analyzed to compare the
DNA sample from Farm B to the three control plasmids and identify which gene was responsible
for the contamination. Figure seven indicates the DNA from Farm B matched the B kanamycin
resistance gene.
Sample Calculations
1) Original population size
B = Number colonies = 2130/10-4 = 2.13×108 cells/mL
Dilution factor
2) Predicted Original Population Size (Lawn)
% difference = 2130 colonies x 100% = 3435% larger
62 colonies
B = (% difference) × (closest colony count) = (3435%)(2130 colonies) = 7.32×107 cells/mL
Dilution factor
10-2
3) Frequency of resistance gene
number of colonies on Kanamycin plate
number of colonies on no kanamycin plate
× 100% =
6 colonies
× 100% = .28%
2130 colonies
Discussion
The results exhibited that Salmonella enteriditis had a significantly higher frequency of
the kanamycin resistance gene than the E. coli bacteria, even though the latter had a higher
colony count. This discrepancy can be accounted for by the fact that the E. coli bacteria used for
this experiment were genetically engineered to be incapable of conjugation2, which limited their
means of dispersing the resistance gene throughout the population. Overall, though, both bacteria
had a frequency lower than five percent for the kanamycin resistant gene found at Farm B. This
rejects the stated hypothesis that there would be a kanamycin resistance gene frequency higher
than five percent for the bacteria population at Farm B. The second part of the hypothesis, which
predicted that each farm would have a different kanamycin resistance gene, was consistent with
the results of the gel electrophoresis. This suggests that each contamination arose independently
and there was no shared source of contamination, such as a chicken feed supplier or source of
laying hens, between the three farms.
Since the bacterial contamination level at Farm B was found to be less than five percent,
but greater than one percent, there are guidelines the CDC should follow when handling the
bacterial contamination. The recommended plan based on this level of contamination is to diver
the eggs to a pasteurization facility until the contamination levels have been less than one percent
for eight consecutive weeks2. This will also involved monitoring the farm weekly and
determining the original source of the contamination. The antibiotic regimen for the chickens
will have to be changed as well2.
During the course of this experiment, there were several possible sources of error that
could have affected the results. One such source is the fact that the original bacteria from the
chicken farms were not used. Instead, the kanamycin resistance gene was transferred to specially
modified E. coli bacteria which were then analyzed. While this prevented the use of infectious
bacteria in the lab, it also introduced error. In this process, the resistance gene was transmitted to
the E. coli bacteria via transformation2. Transformation is the process by which bacteria acquire
DNA from their surrounding environment1. The first step of this process was to extract the DNA
from the plasmid containing the kanamycin resistant gene in the original bacteria2. The DNA
fragments were then inserted into an E. coli plasmid vector.
After this the E. coli plasmid was introduced into the non-infectious E. coli which were
then grown on non-kanamycin broth2. There are several steps that could have failed to
successfully transfer the antibiotic resistant gene from the S. enteriditis into the E. coli. First, the
enzyme used to cut out the DNA from the plasmid might not have successfully extracted the
DNA fragments. In this case transformation wouldn’t have occurred. Second, the E. coli plasmid
might not have inserted properly into the non-infectious E. coli leading to this bacteria
population not obtaining the kanamycin resistant gene. Additionally, assuming the non-infectious
E. coli received the resistance gene, the bacteria may have lost this gene since they were grown
in an environment without kanamycin present. This is because bacteria must use energy to keep
the plasmids containing this gene and if the gene is not actively used since the antibiotic is not
present, the bacteria can expel the gene to save energy2. Furthermore, both the Salmonella
enteriditis and E. coli cultures may not have grown on the plates due to other factors.
As a result of the inherent error possible in the process of transferring the kanamycin
resistance gene to the E. coli bacteria, there are several limitations to this experiment. One is the
accuracy: this is limited by the aforementioned error that arises from the transformation process.
Another is the inability to obtain an exact colony count for several of the plates – due to the
formation of a “lawn” on the plates, an accurate count was unobtainable. Thus a reasonable
estimation based on corresponding data was made. This also was a cause of error in the
experiment – neither an accurate original population size nor frequency could be calculated.
Despite this, there are significant advantages to conducting the experiment in this
manner. One is not having to use the original bacteria found at the farms. This lessens the risk of
contamination and further spreading the pathogenic bacteria beyond the lab. The use of the serial
dilution method is also a reliable and easy method to obtain a reasonable approximation of the
number of viable bacteria in the sample. Serially diluting the sample means that most of the
plates will yield countable results, if not all.
While this experiment did establish that each of the three farms had a unique gene
responsible for the kanamycin resistance, further experimentation needs to be done to determine
the sources of the contamination at each farm. This will require examining each of the facilities
and their suppliers. Additionally, further experiments focusing solely on the transformation of
genetic material between bacteria in a lab should be done. This could establish a rate of success
for this process in order to calculate a precise relative error. A third experiment could also focus
on the time it would take a bacteria population to dispose of their advantageous plasmids that are
not actively in use. This could be used to aid in an accurate error approximation for experiments
utilizing this technique.
References
1. Cyr, R., 2002. Tutorial 4: prokaryotes 1 – cellular and genetic organization. In, Biology 110:
Basic concepts and biodiversity course website. Department of Biology, The
Pennsylvania State University. http://www.bio.psu.edu/
2. Cyr, R., Hass C., Woodward D., and Ward A., 2010. Using Genes for Antibiotic Resistance to
Trace Source(s) of Bacterial Contamination. In, Biology 110: Basic concepts and
biodiversity course website. Department of Biology, The Pennsylvania State University.
http://www.bio.psu.edu/
3. Cyr, R., 2002. Tutorial 35: from gene to protein. In, Biology 110: Basic concepts and
biodiversity course website. Department of Biology, The Pennsylvania State University.
http://www.bio.psu.edu/
4. National Human Genome Research Institute (2012). Polymerase chain reaction (PCR).
Retrieved from: http://www.genome.gov/10000207.
5. Cold Spring Harbor Laboratory. Biology animation library: gel electrophoresis. Retrieved
from: http://www.dnalc.org/resources/animations/gelelectrophoresis.html
6. DNA sequencing: gel electrophoresis. [Art]. In Encyclopedia Britannica. Retrieved from
http://www.britannica.com/EBchecked/media/40224/In-gel-electrophoresis-an-electricfield-is-applied-to-a