Antibiotic Resistance

in a Beef Farm Lab

Joy Li
TA: Christina Peters
Section Number: 043
Lab Partner: Alonna Brumbaugh
12/5/14

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Introduction:
In this study, the cultures of antibiotic resistant bacteria from three different beef farms
were diluted, amplified, and analyzed to determine the frequency of the resistant bacteria and
whether or not the contamination was due to the same plasmid (Hass, Richter, and Ward, 2014).
Bacteria contain plasmids, or smaller rings of extra-chromosomal DNA (CSUN, 2002). These
plasmids are replicated by the bacteria and transmitted to daughter cells. Many of them contain
genes that have the potential to be useful in certain situations (Cyr, 2002). The most familiar
being antibiotic resistance genes. Antibiotic resistance occurs when an antibiotic has lost its
ability to effectively kill bacterial growth, resulting in bacterium that continue to multiply even in
the presence of an antibiotic (Tufts University, 2014). This gives the bacterium a selective
advantage, allowing for them to survive and reproduce (CSUN, 2014). A common problem as a
result of the antibiotic resistant bacteria can be found in the use of antibiotics, typically
tetracycline, in cattle feed by cattle growers and feed suppliers for use as an enhancer. By
interfering with the translation stage, the tetracycline inhibits protein synthesis, and as a result,
interferes with bacterial growth (Cyr, 2002). This study dealt with the contamination of batches
of meats, that caused gastroenteritis, and were found to carry plasmids with tetracycline resistant
genes. The meats were traced back to three different cattle farms from three different states,
prompting the first research question: Is the bacterial contamination at these three farms due to
the same plasmid or different plasmids? (Hass, Richter, and Ward, 2014). Another concern
focused on recommendations for dealing with the contamination, which required knowledge of
the extent of contamination, subsequently prompting the second research question: What is the
frequency of tetracycline resistant bacteria in the beef farm cultures? The task was, therefore, to
determine the types of genes responsible for the tetracycline resistance and to make

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recommendations to prevent future outbreaks (Hass, Richter, and Ward, 2014). Based upon the
fact that the meats originated at three different farms that were not near one another, it was
hypothesized that the contamination was not due to the same tetracycline resistant gene.
Three different genes are known to be commonly responsible for tetracycline resistance,
with each one coding for different proteins with the capability of preventing the antibiotics from
binding to the ribosome. The three different genes have been discovered to display different sizes
that can be used to determine their identity. The experiment first involved diluting the cultures to
create concentrations that avoided lawns and were countable so that the frequency of tetracycline
resistant bacteria at the three cattle farms could be determined from the data of the original
bacterial population. The diluted cultures were counted for resistant and non resistant colonies,
and were used to calculate the concentration and frequency of tetracycline resistance to
determine the levels of contamination per farm. This was done using the formulas N=[Colonies]/
[Volume] (where N=number of colonies, and volume is in mL); B=N/D (where B=initial
population size, N=number of colonies, and D=dilution factor); and Frequency=[Population of
Resistant]/[Population of Non-Resistant] to calculate the levels of contamination. These levels of
contamination were then used to make appropriate recommendations for the prevention of
further outbreaks.
In exploration of the origins of the contamination, samples of the bacterial colonies from
the dilution plates were taken and placed into PCR reaction mixture. PCR, or Polymerase Chain
Reaction, is a method based on the ability of DNA polymerase to amplify regions of DNA. DNA
polymerase synthesizes complementary strands of new DNA to the offered template strand and
allows for the detection of specific DNA sequences from minute amounts of starting material
(NCBI, 2014). The experiment utilized a type of polymerase evolved in thermophilic bacteria,

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called Thermus aquaticus, or Taq polymerase. As an enzyme derived from organisms adapted to
live in lower temperatures, Taq has a thermoresistant nature that allows it to "function under
repetitive heating cooling cycles" (Hass, Richter, and Ward, 2014). It adds onto the 3' end of a
growing strand of DNA with monomeric nucleotides. The components of PCR include the DNA
template, DNA polymerase, oligonucleotide primers, and free nucleotides. The template is the
sample DNA that contains the target sequence. The primers are short pieces of single-stranded
DNA composed typically of about 20-30 nucleotides that are required since the polymerase
cannot synthesize the new strand of DNA without a free 3' end (DNALC, 2014). They bind to the
ends of the segment being amplified on the template, and through cycles of heating and cooling,
nucleotides are added to produce identical copies of the particular DNA template segment. The
DNA sequences can then be analyzed for their length through Agarose Gel Electrophoresis.
Agarose Gel Electrophoresis is a method to separate DNA fragments to compare and
determine their sizes. The length of migration of DNA sequences can be used to determine the
size of the sample. The farther away from the negative pole the DNA sample is, the quicker the
migration, and the smaller the DNA fragment is. The closer the sample electrophoreses, the
slower the migration, and the larger the fragment is. This can be used to identify which resistance
gene it has, as the genes can be identified by their different sizes. The negative charges of the
phosphate groups of DNA cause the sample to migrate through the gel with the aid of volts of
electricity toward the positive electrode of the gel. Contained within the gel is ethidium bromide,
a DNA-binding dye which gives the fragments visibility when bound to DNA once
electrophoresed. By comparison to the ladder, the respective sizes of the tetracycline resistance
genes in the sample can be determined with size markers, which can be used to identify the
particular resistance gene at each of the farms (Hass, Richter, and Ward, 2014).

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Method:
[Procedure found in Biology 110 Laboratory Manual 2014, pages 63-99]
First, the serial dilutions of the bacterial cultures were prepared by taking three
tetracycline plates and three non-tetracycline plates and labeling one of each with 10 -2, 10 -4, and
10 -6. Three empty sterile microtubes were similarly, and 990 l was added to each of the
microtubes with sterile technique. The bacterial suspension microtube was mixed with a gentle
flicking motion before 10 l was removed to be used as the starting sample of the three 100-fold
serial dilutions. Pipettors were used to inoculate the Petri dishes with 100 l of their respective
samples. The bacteria was distributed evenly with sterile glass beads, starting from 10 -6 and
moving to 10 -2, before sliding the Petri dish side-to-side to distribute the bacteria evenly. The
beads were disposed of, and the plates were left to incubate. The resulting cultures were used for
counting purposes in frequency identification, as well as PCR amplification for size comparison.
The plates with individual bacteria colonies were counted and recorded for frequency
calculation purposes. The PCR samples were then prepared to amplify the specific genes by
identifying three antibiotic-resistant colonies on the tetracycline plates and adding one to each of
the three tubes of primer (orange, blue, and yellow) with a pipette. The red, green, and pink tubes
contained primers with control plasmid. All six tubes were loaded into the PCR machine and a
gel was prepared while it was running, by combining 300 mg of Agarose with 30 mLs of 1X
TAE buffer. The mixture was swirled and microwaved for about 35 seconds before 1 l of
ethidium bromide was added after the 2 minute cooling process. The gel was cast and loaded first
with 5 l of PCR DNA ladder, and then with 15 l of a mix of the samples from the six tubes
combined with 2 l of 6x loading dye. After electrophoresis, The gels were photographed under

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UV light. The results of these two processes were then used to calculate the frequency of the
tetracycline resistant gene and identify the specific resistant genes from each farm.
Results:
Table 1: Numbers of bacterial colonies on +/- tetracycline dilution plates of our data

-2

Dilutions
10 -4

10 -6

Treatment

10

Volume Plated

100 l

100 l

100 l

Tetracycline

378

No Tetracycline Lawn
1920
50
The table features the counted colonies of each of the dilutions of Farm "C" of our group's plates
The data from the 10 -4 was the only one usable for frequency calculation
Table 2: Numbers of bacterial colonies on +/- tetracycline dilution plates of lab table data
Treatment

10 -2

Dilutions
10 -4

10 -6

Volume Plated

100 l

100 l

100 l

Tetracycline

182+378+180= 740

3+1+8= 12

0+0+0= 0

No Tetracycline

lawn+lawn+1216= lawn

3188+1920+20= 5128

8+50+1= 59

The table features the combined counted colonies of each of the dilutions of Farm "C" of the
entire lab table's plates
Only the 10 -4 data would be suitable for frequency calculation

Table 3: Numbers of bacterial colonies on +/- tetracycline dilution plates of all type C
Treatment

10 -2

Dilutions
10 -4

10 -6

Volume Plated

100 l

100 l

100 l

Tetracycline

1478

19

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No Tetracycline

Lawn

9184

280

The table features the combined counted colonies of the plates of both lab tables of Farm "C"
The 10-4 dilution was the one that featured colonies that could be used in frequency calculation
Table 4: Summary of Tetracycline Resistance in Beef Farms
Group
1
2

Farm
A
B

Tetracycline Resistance
1
2
0.28
0.93

Gene
3
0.47

0.21

Frequency
of Resistance
0.28%
0.71%

Comments
Gene 1; poor sample; 600 bp
Gene 2; control 1 did not

0.21%

show up; 500 bp

Gene 3; unknown band;
contamination from more

than one gene; 400 bp

The table shows the frequency of resistance from each of the three farms, what gene each farm
contamination originated from, and the size of the respective genes
Resistant Farm C:
N=[Colonies]/[Volume]= (19)/(100mL)= 0.19
B=N/D= (0.19)/(10-4)= 1.9 x 103
Frequency=[Population of Resistant]/[Population of Non-Resistant]=
(1.9 x 103)/(9.184 x 105)= 0.002069= 0.2069%= 0.21%
The frequency of resistance for all three farms was less than 1%, indicating the lowest
level of bacterial contamination.
Gel Electrophoresis for Farm C

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Plasmid DNA

Standard fof

PCR Ladder
Standard fof

The first well contained a ladder; the second well contained plasmid DNA from Colony 1; third
Plasmid DNA

Plasmid DNA

Standard fof

contained plasmid from Colony 2; fourth contained plasmid from Colony 3; fifth contained
standard of Plasmid A; sixth contained standard of Plasmid B; and seventh contained standard of
Plasmid C.
The gel demonstrated that Farm C was based on Gene 3, at 400 bp, with the fourth well
of Plasmid DNA from Colony 3 matching the fragment of well seven of the Standard of Plasmid
C. *the third well was broken during the casting, but did not end up affecting the results of the
experiment
Discussion:
The serial dilutions allowed for the counting of the colonies, and those numbers were
then used to calculate the frequency of the tetracycline resistance for each of the Beef Farms in
response to the research question: What is the frequency of tetracycline resistant bacteria in the
beef farm cultures? The frequencies of all the farms were extremely low (less than 1% with Beef
Farm A at 0.28%, Beef Farm B at 0.71%, and Beef Farm C at 0.21%), demonstrating a very low
contamination level. The low contamination level corresponded with the standard
recommendations to divert meat to pasteurization facility until contamination levels have been

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<1% for 8 weeks, monitor farm weekly, identify source(s) of contamination, and change
antibiotic regime. These standards indicated that the levels of contamination were not at an
incontrollable state, and were fairly manageable for the three farms, so long as they followed the
instructed recommendations.
The PCR process and gel electrophoresis answered research question: "What is the
frequency of tetracycline resistant bacteria in the beef farm cultures?" by demonstrating that
Farm A's contamination originated from Gene 1 (600 bp), Farm B from Gene 2 (500 bp), and
Farm C from Gene 3 (400 bp). By analyzing the gels, the DNA fragments were compared to the
ladder and the controls to determine the corresponding gene and base pair fragment size. Based
on these results of the experiment, the hypothesis that the contamination was not due to the same
tetracycline resistant gene was proven true, since each of the Farms showed different fragment
sizes, indicating the origins by three different tetracycline resistant genes.
A source of error included contamination of the sample, resulting in an unknown band
Farm C's DNA fragment that did not correspond with any of the three resistant genes. While, in
this particular case, it did not impact the results very much as the expected gene was still able to
be identified, it could have greatly resulted in stray data and results for the identification of the
resistance genes. Another source of error could have resulted from the concentrated bacterial
suspension not being evenly distributed throughout the sample. The bacteria may have settled
within the tube between the time of flicking and the removal of the sample. This would result in
uneven amounts of bacteria for each starting sample, leading to an unrepresentative sample of
bacteria.
In conclusion, the contamination for the three farms did not occur from the same gene,
and the frequency levels were rather low. Future experiments could explore the relationship

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between the frequencies and the resistant gene types in exploration of whether or not there
existed correlation between the different types of resistant genes and the different levels of
resistance frequencies. The mutations that caused for the existence of the resistant genes could
also be explored in efforts to eliminate this problem.

References:
Antibiotic Resistance Lab. Written by Hass, C., Richter, K., and Ward, A. 2014. Department of
Biology, The Pennsylvania State University, University Park, PA.
"Bacterial Plasmids." Bacterial Plasmids. N.p., n.d. Web. 04 Dec. 2014.
<https://www.csun.edu/~hcbio027/biotechnology/lec2/PL/pl.htm>.

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Cyr, R. 2002. Prokaryotes I: 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/
"General Background: About Antibiotic Resistance." Tufts University. Alliance for the Prudent
Use of Antibiotics, 2014. Web. 04 Dec. 2014.
<http://www.tufts.edu/med/apua/about_issue/about_antibioticres.shtml>.
"Reagents for Functional Genomics." National Center for Biotechnology Information. U.S.
National Library of Medicine, n.d. Web. 02 Dec. 2014.
<http://www.ncbi.nlm.nih.gov/probe/docs/techpcr/>.
"Polymerase Chain Reaction." DNALC Blogs. Cold Spring Harbor Laboratory, n.d. Web. 03
Dec. 2014. <http://www.dnalc.org/resources/animations/pcr.html>.