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USING GENES FOR ANTINBIOTIC RESISTANCE TO TRACE SOURCE (S) OF
BACTERIAL CONATMINATION IN A BEEF PRODUCTION FARM
Mallori Kolar
Biology 110, Sec 003
Anthony Sangregorio
December 1, 2014
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Introduction
Antibiotics are used to treat illnesses or ailments cause by a bacterial
infection that your body cannot treat on its own. Along with this, antibiotics can help
decrease your severity of symptoms and decrease the time in which you feel sick.
The ability of antibiotic use can be very helpful in most situations of bacteria or
contamination problems. However, because antibiotics interfere with the genetic
makeup of bacteria, if the antibiotic is used too often, the strain of bacteria could
ultimately build a resistance to the specific antibiotic and start to have no effect.
There are many reasons as to the cause of this resistance. The main reason as
to why resistance occurs is a spontaneous or induced genetic mutation. A genetic
mutation occurs during crossing over in meiosis. (Cyr) Bacteria seem to be the most
common organism that becomes resistant to antibiotics because they have a “high
rate of replication […] and free plasmids that exist in a bacterial cell separate from
the chromosome” (“Antibiotic resistance”).
The antibiotic resistance genes are most commonly found on the bacterial
plasmid. Genes that are carried on the bacterial plasmids often present resistance.
These plasmids also can be passed through binary fission, conjugation,
transformation and transduction in wild populations. Most bacteria use binary
fission for propagation, but the bacteria we use can use all four types. (Angert) We
can identify the different tetracycline resistance genes by their sixe and the identity
of a particular gene for tetracycline can be used to trace the source of a particular
resistance. (Hass and Ward)
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In this lab, there is an outbreak of gastroenteritis linked to the consumption
of raw or partially cooked meats. The source can be traced back to three local farms,
Speedy’s Beef Farm, Calli’s Cows and Bulls, and Green Acres Cattle. Our goal is to
determine whether there is a shared source for the bacterial contamination among
the three farms, that could have resulted from either shared feed producer or share
supplier of building materials, or whether each occurrence of tetracycline-resistant
bacteria is unique to each farm. It can be hypothesized that each of the three farms,
Speedy’s Beef Farm, Calli’s Cows and Bulls, and Green Acres Cattle do not share the
same source for the antibiotic resistant bacterial contamination.
To test our hypothesis we used several different methods to ultimately
follow the contamination. We used the techniques of serial dilution, Polymerase
Chain Reaction (PCR), electrophoresis and DNA replication in order to get a solid
foundation of which the source of contamination could be identified. (Hass and
Ward)
Materials and Methods
Several techniques were used in this experiment to help determine if each
farm had the same source of contamination. The first method was to determine the
frequency of antibiotic resistant bacteria using the serial dilution technique. First we
labeled the six petri dishes with our information and a dilution number of 10-2, 10-4,
or 10-6. After labeling three micro tubes with 10-2, 10-4, or 10-6, we added 990ul of
water to each of the three micro tubes.
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Taking 10ul of the starting sample, we made three, 100-fold serial dilutions
starting with 10ul of the concentrated bacterial suspension. Next we pipetted 100ul
of the each of the 10-2, 10-4, and 10-6 solution into matching petri dishes. Finally, we
coated the petri dishes using the glass beads and taped the dishes shut. (Hass and
Ward)
The second method was used to amplify specific genes for tetracycline
resistance using PRC. First, we pipetted three different colonies into the orange, blue
and yellow tubes. The red, green and pink tubes contained primers with a control
plasmid. These six micro tubes were then placed in the PCR machine, where they
would repeatedly be heated and cooled in order to multiply the DNA template of
each sample. For the duration of the time in which the DNA was processing, the
colonies in each petri dish sample was counted. For easier counting, one quadrant
was counted and then multiplied by four to get an estimate of the number of
colonies. Those with too much bacteria growth were labeled as “lawn.” In order to
get the frequency, we divided the number of colonies in the dish with tetracycline by
the number of colonies in the non-tetracycline and multiplied by 100.
While the DNA processed, we prepared out gel solution and then placed the
solution in the electrophoresis unit. Once the gel and the PCR samples were ready,
we placed the DNA ladder into the first well. We then continued to mix 2ul of 6x
loading dye into each of the six tubes containing our samples. After they were
mixed, we load 15ul from each colored tube in the six wells after the DNA ladder.
After turning the power on and the tracking dye reached halfway, we removed the
gel and placed it up the UV light box where we took a picture. It was explained that
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the picture would show how far our plasmid moved across the gel, being pulled to
the positive pole.
Results
Table 1: Numbers of bacterial colonies on +/- tetracycline dilution plates
Dilutions
Treatment
-2
10
10-4
10-6
100ul
100ul
100ul
Volume Plated
Tetracycline
~250
2
1
No Tetracycline
Lawn
~500
~75
This table shows the number of colonies counted in each of the six petri dishes.
These numbers are used to determine the frequency of the antibiotic resistant
bacteria in each of the 3 serial dilutions. In order to get the frequency, we divided
the number of colonies in the dish with tetracycline by the number of colonies in the
non-tetracycline and multiplied by 100. (Hass and Ward)
The frequency determined for our resistance strain was averaged between two
groups in our lab to ultimately be 1.17%.
PCR Pictures
Figure 1: Speedy’s Beef Farm (Strain
A)
It can be observed that the molecular
weight of this DNA strand is ~600BP
Figure 2: Calli’s Cows and Bulls (Strain
B)
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It can be observed that the molecular
weight of this DNA strand is ~300KB
It can be observed that the molecular
weight of this DNA strand is ~500BP
Figure 3: Green Acres Cattle (Strain C)
These pictures represent each of the three strains of bacteria’s for each farm. The
pictures show the results of the PCR testing. Our lab strain was C, from Green Acres
Cattle. When looking at the individual farms, it can be observed that each farm had
different strains of bacteria.
Discussion
In conclusion, our gel image showed that the strain we had was comparable
with tetracycline resistance gene C. (Figure 3) In Figure 1, it can be observed that
the resistance strain was from tetracycline resistance gene A. Figure 2 represented
beef farm B and they contained tetracycline resistance gene B. This experiment
helped to prove my hypothesis, being that every beef farm held a different
tetracycline resistance gene. Since none of the three samples represented different
genes, it proves that each of the samples must have come from farms that were not
infected by the same source of contamination.
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Our farm was considered diseased by the tetracycline resistance gene C with
a resistance frequency of 1.17%. (Table 1) In order to help fix this bacterium
contamination, there are a multitude of recommendations. Green Acres Cattle Farm
should identify source of contamination and treat infected individuals by changing
their antibiotic regimen. Next, they should destroy all meat until the levels have
been tested to be less than 1% for 8 weeks, monitoring weekly. Following this plan
of standard recommendations should ultimately eliminate the contamination and
prevent future outbreaks.
Although each group was able to identify their tetracycline resistance gene
clearly that resulted in each farm containing a different source of contamination, our
group did have a few sources of error. Our tetracycline data in the petri dish labeled
10-4 and 10-6 had only 1 and 2 colonies, respectably. This being said, if the dilution
series was more accurate there should have been more visible and usable colonies.
For future experiments, I would recommend a more sterile environment and lab
conditions that are not put under a time limit, allowing for my accurate results.
Our lab method results were clear enough to come to the conclusion that the
contamination in each of the local farms was not from the same bacteria strain. Also,
with a resistance frequency of 1.17% at Green Acres Cattle, the problem is easily
fixable and should be a quick return to normal.
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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 biodiverity course website. Department of Biology, The Pennsylvania
State University. http://www.bio.psu.edu/
Cyr, R., 2002. Heredity and Life Cycles. In, Biology 110: Basic concepts and
biodiverity course website. Department of Biology, The Pennsylvania State
University. http://www.bio.psu.edu/
"Antibiotic resistance." Encyclopedia Britannica. Encyclopedia Britannica Online.
Encyclopedia Britannica Inc., 2011. Web. 02 Dec. 2011.
<http://www.britannica.com/EBchecked/topic/1027479/antibioticresistance>.
Angert, Esther R. "Alternatives to Binary Fission in Bacteria." Nature Reviews
Microbiology 3.3 (2005): 214-24. Web. 30 Nov. 2014.