Determining Sources and Recommending Containment Strategies of Kanamycin Resistant Bacteria
1
Introduction:
The Center for Disease Control has recently begun an investigation into reports of
sickness due to severe gastroenteritis resulting from consuming raw or undercooked eggs. The
team investigating has isolated the strain of bacteria responsible for the outbreak. The strain,
found within eggs from three farms geographically separate from one another, has been found
to be resistant to the antibiotic Kanamycin. Kanamycin is a commonly used antibiotic used in
bird feed to raise healthier chickens and kill invading bacteria. The team is investigating
whether or not the strains of bacteria from each farm (Acme Eggs, Big Al's Poultry Farm,
Clucky's Chickens) have arisen separately or are somehow related, whether from a common
bird feed, cage, etc. (Hass,2010)
Kanamycin works by preventing the ribosomes within the bacteria's inner membrane
from functioniong properly; impeding protein synthesis and translation of mRNA. Each strain of
bacteria has become resistant by means of altering the kanamycin molecule so as not to be
taken up into the inner membrane of the bacteria. There are known to be three different genes
that code for this method of resistance. Each gene produces a different enzyme used to alter
the kanamycin molecule. Thus, determining which genes are used by each strain of bacteria will
determine which farms have common strains, and by what methods the CDC should control the
outbreak. (Hass,2010)
Three methods will be used in these experiments to; determine frequency of
kanamycin bacteria in a population, and determine which gene is used by each population for
kanamycin resistance. The first method, serial dilutions, takes a highly concentrated sample of
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Determining Sources and Recommending Containment Strategies of Kanamycin Resistant Bacteria
2
bacteria and through a stepwise process, dilutes the bacteria sample to a usable point. The
second method, Polymerase Chain Reaction is used to rapidly replicate large amounts of DNA
fragments. After the DNA is replicated a process known as gel electrophoresis will be used to
separate the DNA fragments and determine which plasmids are present in each of the three
populations.
Materials and Methods:
Materials

Agar Plate

PCR Machine

Kanamycin resistant and nonresistant
bacteria

Gel Electrophoresis Unit

taq Polymerase

DNA ladder

Control Plasmids

Plasmid Standards

Agarose Powder


Pipette
DNA Template
Methods:
The experiment was first started with serial dilutions (dilutions of 10-6, 10-4 and 10-2) of
bacteria from a population plated out onto agar plates. Three were treated with and three
were treated without without kanamycin. The cultures without kanamycin were used as a
control. After waiting a week to allow for the cultures to grow, the percent frequency of
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Determining Sources and Recommending Containment Strategies of Kanamycin Resistant Bacteria
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resistant bacteria found within the given population was found by dividing the average number
of resistant colonies by the average number of non k-resistant colonies.
The gene determining portion of the lab was started by filling six vials with solutions
that would participate in the PCR process. In three of the DNA primers and dilutions of
kanamycin resistant bacteria from each of the three dilutions (10-6, 10-4 and 10-2) were
prepared. In the remaining 3 vials were added solutions of DNA primers and plasmids A B and C
that would act as standards for each of the three gene mutations. The six vials were then added
to the PCR machine to start the PCR process. After completing the PCR process each sample
was added to agarose gel wells in the gel electrophoresis unit as well as a DNA ladder, used as a
standard for distance traveled by DNA fragments. Electricity was passed through the gel which
migrated the DNA fragments in each of the wells toward the positive electrode. This enables
finding which gene for kanamycin resistance is expressed by comparing plasmid distance of the
sample to the plasmid standards. This process is repeated for each of the three farms.
(Hass,2010)
Results:
Group
Big Al's Poultry Farms
K Colonies
Number of Colonies
Number of Bacteria
10 ^-6
11
5.5 X 10^7
10^-4
9
4.5 X10 ^5
10^-2
136
6.8 X 10^4
Non-K Colonies
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Determining Sources and Recommending Containment Strategies of Kanamycin Resistant Bacteria
10 ^-6
1794
8.97 X10 ^9
10^-4
5280
5.28 x 10^6
10^-2
N/A(lawn)
N/A (lawn)
4
#𝑜𝑓 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠 𝐾
= 𝑓𝑟𝑒𝑞𝑢𝑒𝑛𝑐𝑦 𝑟𝑒𝑠𝑖𝑠𝑡𝑎𝑛𝑡 𝑏𝑎𝑐𝑡𝑒𝑟𝑖𝑎
#𝑜𝑓 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠 𝑛𝑜𝑛 − 𝐾
10(−6) 𝐾 𝑐𝑜𝑙𝑜𝑛𝑦 =
11𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠𝐾
𝑥100 = .61315%
1794𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠𝑛𝑜𝑛𝐾
10(−4) 𝐾 𝑐𝑜𝑙𝑜𝑛𝑦 =
9𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠𝐾
𝑥100 = .1515%
5280𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠𝑛𝑜𝑛𝐾
Section
Name
Acme Eggs
Big Al's Poultry Farm
Clucky's Chickencs
Frequency %
43.3
.32
.51
Name
Acme Eggs
Big Al's Poultry Farm
Clucky's Chickens
Plasmid
A
B
C
Results indicate that both Big Al's Poultry Farm and Clucky's Chickens have low levels of Kanamycin
resistant bacteria. Acme Eggs has a very large frequency of kanamycin resistant bacteria. Results also
indicate that all three farms have different Plasmids encoding for kanamycin resistance.
Discussion:
Acme Eggs
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Determining Sources and Recommending Containment Strategies of Kanamycin Resistant Bacteria
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Control Plasmid A has a weight of 700
BP which matches with the
Kanamycin resistant bacteria plasmid.
The bacteria from Acme uses the A
Plasmid
Big Al's Poultry Farm
Control Plasmid B has a weight of 1KB
which matches with the Kanamycin
resistant bacteria plasmid. The
bacteria from Big Al's uses the B
plasmid.
Clucky's Chickens
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Determining Sources and Recommending Containment Strategies of Kanamycin Resistant Bacteria
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Control Plasmid C has a weight of
1.5KB which matches with the
Kanamycin resistant bacteria plasmid.
The bacteria from Clucky's uses the C
plasmid.
The Polymerase Chain Reaction shows that each of the three strains of Kanamycin resistant
bacteria are of different Weights, indicating that each strain contains a different plasmid. This explains
that the mutations within each strain arose independently from one another and do not share a
common source, but rather are unique to each farm. Cross contamination of the agarose gel well during
electrophoresis however could have affected results. It is recommended that due to the sensitive nature
of this case, that these experiments be repeated so as to confirm these findings. Also it is pertinent that
an investigation be done into where these farms aquired these strains. Because each farm is
independent to one another, it is possible to do three separate investigations.
Recommendations as to the results of the serial dilutions and subsequent calculations of the frequency
would be that due to the low percentage of resistant bacteria at both Clucky's Chickens and Big Al's
Poultry Farm the following measures should be taken: Divert eggs to pastuerization facility until
contamination levels have been <1% for 8 weeks, monitor farms weekly, idetify sources of
contamination, and change antibiotic regimen. At Acme farms the following measures should be take:
Cull entire population, disinfect entire facility, identify sources of contamination, and submit
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Determining Sources and Recommending Containment Strategies of Kanamycin Resistant Bacteria
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contamination prevention plan. Cross contamination of serial dilutions could have resulted in incorrect
readings ho ver, and due to sensitive as well as economic implications of these measures, these tests
should be done again to confirm findings.
References:
Cyr, R., Hass C., Woodward D., and Ward A. (2010). Using genes for antibiotic resistance to
trace source(s) of bacterial contamination.
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