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Angel Li
BSCI105 (0101)
7/6/06
Kanamycin Found to Inhibit the Growth of the Washington 1 Strain of
Escherichia coli
ABSTRACT:
The emergence of a new strain of Escherichia coli (E. coli) containing the
Washington 1 plasmid has been causing severe diarrhea in patients and was found to be
resistant to many of the usual antibiotic treatments. The hypothesis was that the
Washington 1 plasmid contained genes coding for the resistance of certain antibiotics. In
order to find an appropriate treatment for the new strain, transformation was performed to
incorporate the Washington 1 plasmid into laboratory strains of E. coli, and these cells,
along with non-plasmid cells, were then cultured and treated with ampicillin and
kanamycin. The results of the positive and negative controls were as expected, and
kanamycin, but not ampicillin, was shown to be effective in inhibiting E. coli growth.
However, further testing on the actual Washington 1 strain must be conducted to validate
the use of kanamycin as an effective treatment for patients.
INTRODUCTION:
Escherichia coli, or E. coli, is a bacterium that is prevalent in the intestinal tract of
humans as well as other warm-blooded animals (Feng, 2006). E. coli accounts for about
0.1% of the total bacteria within an average adult’s intestines, but its presence is essential
for a properly functioning digestive system (Brown, 2006). The bacteria help to digest
food by converting glucose into necessary macromolecules and by supplying certain
vitamins that the human body can absorb (Todar, 2006). However, each organism hosts a
different strain of E. coli and these bacteria have localized functions, so it is the
emergence of an unfamiliar strain or colonization in an incorrect region of the body that
leads to illness (Brown, 2006). These different strains can be transmitted through various
means, but fecal contamination is by far the most common method (Todar, 2006).
Different strains of bacteria come about by the acquisition and incorporation of
extra genetic information, often in the form of plasmids through a process called
transformation (Brown, 2006). A plasmid is a piece of DNA, similar to a parasite, which
enters the cell and uses its resources to replicate (Campbell, 2002). Oftentimes, the cell
can benefit from the incorporation of plasmids because the foreign DNA may contain
genes coding from certain antibiotic resistance (Wassenaar, 2006). These genes can
either be translated into antibiotic-destroying proteins or enzymes that act to cut and
inactivate antibiotics, which are drugs used specifically to kill bacteria, through the
modification of the target site or the creation of an alternate metabolic pathway (Tenover,
2006). E. coli can easily acquire drug resistance because plasmids are readily passed
between bacteria (Gross, 2006). It is extremely important to be prudent in the use of
antibiotics because over-prescription can select for resistance and lead to the proliferation
of harmful and difficult to treat bacteria (Murchan, 2006).
It was recently observed that a new strain of E. coli has been plaguing hospitals
by causing severe diarrhea in patients. Labeled Washington 1, this E. coli was found to
be resistant to the usual antibiotics, penicillin, chloramphenicol, and neomycin, and has
therefore been difficult to treat. Furthermore, this new strain cannot be grown under
normal laboratory conditions, but its plasmid can be extracted from the cells. Based on
the known information, it was hypothesized that the Washington 1 plasmid confers
resistance to certain antibiotics. Therefore, in order to discover an antibiotic that will be
effective in treating the E. coli, it was necessary to culture plasmid-incorporated
laboratory E. coli and treat those cells with different antibiotics – in this case, ampicillin
and kanamycin – to see whether either drug will inhibit bacterial growth (Gross, 2006).
METHODS:
Knowing that the Washington 1 strain of E. coli is resistant to penicillin,
chloramphenicol, and neomycin, it was necessary to treat the bacteria with different
antibiotics in order to determine which antibiotics would be effective in killing this
particular strain. However, it was found that the Washington 1 strain cannot be cultured
in a laboratory setting. Therefore, the experiment first required the incorporation of the
Washington 1 plasmid into a laboratory strain of E. coli. To perform this transformation,
competent E. coli cells treated with CaCl2 were separated into 2 tubes of 100 µl each.
The cells were allowed to just thaw and were then put on ice immediately to stabilize the
phospholipids membrane. Ten µl of plasmid was added to one of the tubes and the other
tube was used as the non-plasmid control. The plasmid-containing tube was swirled and
placed on ice for 30 more minutes. After 30 minutes, both tubes were placed in a 42º
water bath for 90 seconds in order to heat shock the cells and allow the plasmids to be
properly incorporated. They were then placed on ice for one to two minutes to restabilize
the cells and their membranes. For the purpose of diluting the CaCl2 and providing food
for the cells, 800 µl of growth media was added to each tube. Following that, the tubes
were incubated in a 37º (normal body temperature) water bath for 20 minutes to allow the
genes on the plasmid to be transcribed and translated into the necessary drug resistance
proteins.
Once incubation was completed, the cells were plated onto Petri dishes along with
the antibiotics they were treated with. Three plates were each added with 200 µl of
plasmid-containing cells and three different plates with 200 µl each of non-plasmid cells.
Each set of 3 plates included one containing no antibiotics, one containing ampicillin, and
one containing kanamycin. Under a hood, the bacteria were spread across the surface of
each of the plates using sterile cotton swabs. The plates were then turned upside down,
sealed with Para film, and placed in a 37º incubator for 24 hours. After 24 hours, the
plates were examined for growth and/or contamination in order to determine whether the
antibiotics were effective against the Washington 1 strain (Gross, 2006).
RESULTS:
After 24 hours of incubation, the 6 plates were observed for growth. Colonization
was apparent in both of the positive controls containing no antibiotics, representing cell
viability. There was no growth in either of the negative controls containing non-plasmid
cells and antibiotics, eliminating the possibility of contamination having occurred. As for
the experimental plates, there was growth in the plasmid-containing cells treated with
ampicillin and no growth in the cells treated with kanamycin, as shown in Table 1.
Table 1: Growth of E. coli bacteria with and without Washington 1 plasmid under
different antibiotics
No Plasmid
Plasmid
No antibiotics
Growth
Growth
Kanamycin
No Growth
No Growth
Ampicillin
No Growth
Growth
Half of the E. coli cells were treated with the Washington 1 plasmid while the other half
were not. The two plates with no antibiotics acted as positive controls to check for cell
viability and the two plates containing antibiotics and non-plasmid cells acted as negative
controls to check for contamination. Antibiotic effectiveness was tested with the two
experimental plates containing Washington 1 E. coli and antibiotics.
DISCUSSION:
Based on the growth patterns observed in this experiment, it can be concluded that
the antibiotic kanamycin can be a possible treatment for the Washington 1 strain of E.
coli. As expected, growth was observed for the positive controls, signifying that the cells
used were viable. Also expected was the lack of colonization in the negative controls.
This ruled out any possible occurrence of cross contamination, environmental
contamination, or spontaneous mutation and helped to validate the results obtained from
the experimental plates. The plasmid-containing cells treated with ampicillin still grew
and colonized, signifying that the Washington 1 plasmid most likely contained genes
coding for the resistance of ampicillin, which was not unexpected since it was
hypothesized that the plasmid conferred resistance to certain antibiotics not limited to the
ones already known. However, there was no growth in the cells treated with kanamycin,
meaning that the isolated plasmid may not contain kanamycin-resistant genes and the
antibiotic could be a possible treatment for the patients suffering from E. coli infection.
The testing of specific antibiotics against bacterial strains is necessary in order to
prevent the further spread of multi-drug resistance. Exposing patients to a plethora of
antibiotics can actually lead to the elimination of useful bacteria while promoting the
growth of harmful, resistant bacteria. Same is true of using an incorrect antibiotic.
Although bacterial cells are quite different from human cells and the antibiotic will have
little or no effect on the rest of the body, it will kill all susceptible bacteria that it comes
in contact with. This selects specifically for the antibiotic-resistant bacteria, which can
then reproduce rapidly without having to compete with other bacteria for space and
resources. These bacteria can then confer their resistance upon other bacteria through the
transmittance of DNA, causing those bacteria to be immune to antibiotics that they might
not even have previously come in contact with. The result is a limited availability of
future bacterial treatments (Irwin, 2006).
The next step would be to further test the effectiveness of kanamycin against the
actual Washington 1 strain of E. coli. A possible hypothesis could be that kanamycin is
an effective antibiotic treatment against E. coli infections caused by the Washington 1
strain. Since bacteria acts differently in different environments, as do antibiotics, the
usefulness of kanamycin must be tested against the actual strain versus the laboratory
engineered cells in this study. This might even call for the use of kanamycin in treating
select patients to determine whether individuals respond similarly to the antibiotic.
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Campbell, N.A. and Reece, J.B. 2002. The Structure, Function, and Reproduction of
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Gross, P. and Lanford, P.J. 2006. Demonstrating Drug Resistance in Bacteria. In:
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Murchan, S and Cunney, R. 2006. Rise in antimicrobial resistance in invasive isolates of
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