Max Louis Isabelle

BIO110H
Jesse Weber
11/26/15
Concentration of Tetracycline resistant bacteria in Beef
producing facilities and the origins of their resistance.
Introduction:
In this lab, the author and a group of researchers will determine
the source of an outbreak of gastroenteritis. This experiment will focus
on three different meat farms- Bovines R Us, Rebels of Renovo and
Jumping Steer Farm. The contamination is linked to the concentration
of tetracycline resistant bacteria found in the meat at each farm.
Tetracycline is an antibiotic that is administered as a growth enhancer
to cows (South Dakota University). Along with trying to determine the
source of the outbreak, the researchers will also be finding the
concentration of tetracycline resistant bacteria at each farm, then
make recommendations to each farm on what their course of action
should be to combat the bacteria.
For this investigation, the author and his team will be using
different techniques to approach and examine this problem. Each
technique has a different purpose. A serial dilution is the first
technique used to conduct this experiment. This is a simple way to
reduce the concentration of bacteria in a sample so that specific

colonies can be isolated. In this experiment, serial dilutions will be

used to determine the frequency of tetracycline resistant bacteria and
isolate tetracycline resistant colonies to be used in PCR.
PCR(Polymerase Chain Reaction) is a process that allows small
amounts of DNA to be replicated very fast. PCR uses a heat resistant
DNA polymerase, commonly Taq polymerase for this process due to the
fact Taq polymerase will not denature under high heat. Taq DNA
polymerase is heat-stable and will synthesize DNA at elevated
temperatures from single-stranded templates (Thermo Fisher
Scientific). This process is used in this experiment to amplify the
tetracycline resistant bacteria DNA, so that it can be run in a gel
electrophoresis. Through gel electrophoresis it will be determined
which tetracycline resistant plasmid came from which farm, by
comparing the tetracycline resistant bacteria DNA to known
tetracycline resistant plasmids.
Bacteria plasmids carry DNA distinct from the main bacterial
chromosome. The fact that these plasmids are distinct from the main
chromosome makes it easy to transfer them between bacteria through
all means of reproduction; binary fission, conjugation transformation,
and transduction. Plasmids that contain antibiotic resistant genes will
be spread quickly in a population that is exposed to antibiotics. There
are three known antibiotic resistant plasmids the author will be testing
for in this lab.

It is important to avoid contamination issues through the

experiment. Bacteria are everywhere so good sterilization techniques
will need to be used to limit contamination. Techniques such as
keeping a sterile work environment, while also reducing the time of air
exposure to the bacteria, by being prepared and working efficiently will
limit contamination.
The author hypothesizes that when the three bacteria strains
from the different farms are exposed to tetracycline; different antibiotic
resistant plasmids will be present from each farm.

Material and Methods:

The first step in the beginning of the investigation is to create
serial dilutions of the bacteria samples from each farm. Making serial
dilutions is a process that allows the researcher to count specific
colonies when a sample is diluted enough, to prevent the growth of a
Lawn on the growth medium. A Lawn is when there is so much
bacteria growth on a plate, that no individual colonies can be counted.
For this experiment, the author created three dilution levels 10-2, 10-4
and 10-6. Serial dilutions are created by reducing the number of
bacterial per ml in a stepwise fashion. For example, the author
accomplished his dilution by adding 10ul of sample to 990ul of dilution.
This first dilution creates a dilution level of 10-2. The process is
repeated two more times to get dilution levels of 10-4 and 10-6.

After the serial dilutions are completed, they are plated on six
different growth mediums. Three of the growth medium plates will
have tetracycline (an antibiotic) and the other three will be normal
control plates. Each of the three dilution levels will be plated on
tetracycline plates and non-tetracycline plates. 100ul of dilution will be
added to each plate and then spread by glass beads. The author
started with the 10-6 dilution and moved to a lower dilution level to
prevent substantial bacterial exchange between the plates caused by
the glass beads. Once plated, the bacteria was left to grow for a week
under standard growth conditions.
After a week of growth the six bacteria plates were examined,
the author recorded the frequency of colonies on each plate at each
dilution level. The plates with tetracycline were also grown to set up
PCR (Polymerase Chain Reaction). Three individual tetracycline
resistant bacteria colonies were taken from the plates and put into
tubes containing PCR reaction mix. PCR is used to amplify the samples
of bacteria plasmid DNA. This is done in preparation for running gel
electrophoresis.
After the PCR tubes are run in the PCR machine there is enough
DNA to run a gel electrophoresis. Before running the gel with
tetracycline resistant bacterial DNA, the gel apparatus has to be set
up. To create the gel, weigh 300mg of agarose and place it in a 125ml
Erlenmeyer flask. Then pour 30mLs of 1X TAE buffer into the flask.

Mix and then place in microwave for 35seconds, let cool for two
minutes and add 1ul of ethidium bromide dye. Once the gel liquid is
created, it needs to be poured into the apparatus. Before pouring,
wedge the dams into their slots and then pour the molten gel. Finally,
place the comb in the gel to creating the loading slots and let cool for
10 minutes.
Once cooled, the gel is ready to be loaded. The gel will be
loaded with seven different samples. One sample is the DNA ladder
and the other six are the three tetracycline resistant bacteria and the
three standard plasmids that went through PCR. Once the samples are
loaded, the gel is turned on to 160 volts and let run until the dye has
gone around half the gels length. The gel is now complete and ready
to be viewed under UV light (Burpee).

Results:
All of the results were gathered by compiling information from the
authors own work and the other groups working on this problem. The
authors results are specific to Farm C, while there are also results from
Farm A and B. The results for Farm A and B came from sharing
of data with the other groups working on this experiment. For all of the
gels, stock images were used.
Stock gel for Farm A Picture 1

This is Farm A gel electrophoresis, which shows that the A

antibacterial plasmid is present.
Stock gel for Farm B Picture 2

This is Farm B gel electrophoresis, which shows that the B

antibacterial plasmid is present.

Stock gel for Farm C Picture 3

This is Farm C gel electrophoresis, which shows that the C

antibacterial plasmid is present.

Farm C #1: Frequency of Bacteria Colonies(Table 1)

Treatment

Dilution Levels
10-2
10-4
10-6
Volume Plated
100ul
100ul
100ul
Tetracycline
132
6
4
No Tetracycline
Lawn
1300
76
As the dilution level increase the frequency of bacteria decreases,
which would be expected.
Farm C #2: Frequency of Bacteria Colonies (Table 2)
Treatment

Dilution Levels
10
10-4
10-6
Volume Plated
100ul
100ul
100ul
Tetracycline
144
9
0
No Tetracycline
Lawn
Lawn
636
There were very few bacterial resistant colonies for this group. Their
-2

ratio between tetracycline resistant and non tetracycline resistant

bacteria is indeterminable, because it is 0/636.

Antibiotic resistant bacteria percentages (Table 3)

Group#1
Farm
Farm
Farm
Farm

Group#2

Average

A
1.19%
0.468%
0.83%
B
N/A
6.91%
6.91%
C
1.91%
1.78%
1.845%
A had a low level of contamination, Farm B had the most and

Farm C had the middle amount of contamination.

% of tetracycine reistent Bacteria(Graph 1)

% of tetracycine reistent
Bacteria

From the Data, it is evident that all three farms possess

tetracycline resistant bacteria that could be the cause of the outbreak.
Since all the farms bacteria have at least a 0.83% resistance (Table 3).
The fact that all three farms posses different level of resistance shows
that different antibacterial resistant plasmids are more effective than
others, or just more widespread across the bacterial population at each
farm.
Through the process of running gel electrophoresis it was found
that each Farm possessed a different strain of tetracycline bacteria.
The difference between each farm was which plasmid was present in
the tetracycline resistant populations. Farm A had plasmid A(Picture
1), Farm B had plasmid B(Picture 2), and Farm C had plasmid
C(Picture 3). These finding from the gel electrophoresis help explain

why each farms bacteria was more or less resistant to tetracycline

(Table 3). It is because each farm possesses a different strain.

Discussion:
The author hypothesized that when the three bacteria strains
from the different farms were exposed to tetracycline, different
antibiotic resistant plasmids would be present from each farm. This
hypothesis is supported by the findings in the gel electrophoresis as
each farms bacteria population carried a different antibacterial
resistant plasmid and at a different concentration level.
The significance of the results is that based on the level of
contamination of antibiotic resistant bacteria, different
recommendations are made. For Farm A their contamination level
was <1%. At that level, Farm A is recommended to change
antibiotics, monitor the farm weekly, identify contamination, and not
ship meat until contamination has been <1% for eight weeks. For
Farm B and C they fall into the 2-30% contamination range. The
only difference in this range from Farm A is that they have to destroy
all meat in the facility until contamination levels hold constant at <1%
for eight weeks. This process is used by the CDC (Center for Disease
Control) to recommend how to handle outbreaks of tetracycline
resistant bacteria, in an attempt to prevent future outbreaks (Burpee).

Each farm possessed a different antibacterial resistant plasmid.

Running the gel electrophoresis and comparing the results to the
known tetracycline resistant bacteria strains found this fact. Farm A
had plasmid A, Farm B had plasmid B and Farm C had plasmid
C. Each farm possessing a different plasmid could be why
contamination levels were different at each farm. Certain plasmids
may be more effective than others at resisting tetracycline and
spreading through a population.
Sources of error in this experiment arise during many steps of
this experiment. During the step of making the dilutions and plating
them is where the most potential for error arose. Through the process
of dilution, the bacteria was exposed to the air for extended periods of
time, Air can play a central role as a reservoir for microorganisms
which always runs the risk of contaminating samples(Napoli). Once
the dilutions were made and plated, the process for spreading the
bacterial dilution on the growth plate was using glass beads. The glass
beads were used for all three-dilution levels. The glass beads may
have been ineffective at spreading the bacteria over the growth plates.
The authors group had beads bounce out onto the table. This could
also have contaminated the plates.
These sources of error could have affected the growth of the
bacteria on their plates, either increasing or decreasing frequency at
each dilution level. The frequency of bacterial colonies at each level of

dilution is what was used to determine the concentration of antibiotic

resistant bacteria. These sources of error could cause the wrong
recommendation to be made.
To reduce this source of error, the author could have used new
beads for each plate, reducing the contamination between each. A
less conservative use of beads would allow the author to not be forced
to use beads that had been dropped on the table or floor.
Future experiments that could be done include changing what
antibiotic is used. Bacteria from the meat farms population may be
resistant to other antibiotics that are not being tested for by the farms
staff. Also the strain of bacteria being tested could be changed. For
this experiment, the author used E.coli bacteria, but there are many
different strains of bacteria present in a meat farm that could be a
potential source of contamination.
In conclusion, the bacterial contamination at these three farms is
due to different strains of bacteria. By running the gel electrophoresis,
it is found that each farms antibiotic resistant bacteria population
posses a different plasmid that gives it this characteristic. While the
bacteria are all E.coli, they are different strains. The concentration of
tetracycline resistant bacteria in cattle on Farm A is 0.83%, Farm B
is 6.91% and Farm C is 1.845%.
Sources:
Burpee, D., Cyr, R., Hass, C., Ikis, D., Richter, K., Ward, A. and D.
Woodward, eds. A
Laboratory Manual for Biology 110 Biology:

Basic Concepts and Biodiversity.

2015. Department of Biology, The
Pennsylvania State University, University Park, PA
Napoli, Christian, Vincenzo Marcotrigiano, and Maria Montagna. "Air
Sampling Procedures to Evaluate Microbial Contamination: A
Comparison between Active and Passive Methods in Operating
Theatres." BMC Public Health 12.1 (2012): 594. Web. 1 Dec. 2015.
"South Dakota State University." South Dakota State University. N.p.,
n.d. Web. 03
Dec. 2015.
"Taq DNA Polymerase." Taq DNA Polymerase. ThermoFisherScientific,
n.d. Web. 03
Dec. 2015.