Triclosan Promotes Kanamycin Resistance:
Characterizing a Plasmid by Restriction Digestion and Electrophoresis
By Kevin Ogden
LBS 145 Spring 2005
Sunday Section
Rick Chalmers Jamie Mooney
29 April 2005
Website:
http://www.msu.edu/~ogdenkev/streamII.htm
Abstract
Antibiotics are an essential part of health care
today, however, overuse of antibiotics and antibacterial products has
drastically increased the amount of bacteria resistant to antibiotics (File,
1999). Given that antibacterial
resistance poses a major health concern, I am interested in examining the
effects of the antibacterial agent triclosan on the prevalence of antibacterial
resistance. For this investigation, I
examined the bathroom of suite 313 and 314 West Holmes Hall at Michigan State
University. I hypothesized that the use
of triclosan in antibacterial hand soap would lead to antibiotic resistant
bacteria. Because bacteria resistant to triclosan are also resistant to
ampicillin, tetracycline, and to a lesser extent, kanamycin, I predicted that
bacteria would be resistant to ampicillin or tetracycline. I swabbed the toilet handle, the faucet
handle, and the inside and outside handles of the door using one sterile cotton
swab. I placed the cotton swab in
Luria-Bertani (LB) broth in an incubator (37 ˚C) to grow the bacteria
overnight and then transferred the bacteria to Petri dishes containing media
with ampicillin, tetracycline, kanamycin, or no antibiotic to isolate
antibiotic resistant bacteria. After
harvesting the bacteria, I only found bacteria resistant to kanamycin. I isolated the plasmid DNA that confers
kanamycin resistance to the bacteria and digested the plasmid using the
restriction enzymes PstI and HindIII. I
separated the segments of DNA using agarose gel electrophoresis, and compared
the segments to DNA fragments from pKAN, another plasmid that confers
resistance to kanamycin, digested by Pst I and HindIII. I found that the plasmid did not have the
same DNA fragments as pKAN, and thus is probably a different plasmid.
Introduction
The discovery of antibiotics revolutionized medical
care, dramatically reducing illness from bacterial infections and significantly
increasing the average life expectancy (Yim, unknown). Antibiotic resistance poses a serious health
concern by making it more difficult to combat bacterial infections and
illnesses, which could lead to increased mortality rates and health care costs
(File, 1999). Adding to the threat of
antibiotic resistance, many bacteria are becoming resistant to multiple
antibiotics; in some cases, bacteria are resistant to as many as seven families
of antibiotics (Levy, 1998).
Bacteria become resistant to antibiotics when they can produce a protein that combats the antibiotic, usually by destroying the antibiotic or causing it to be excreted. If bacteria don’t already produce such a protein, they can acquire small, circular, extrachromosomal DNA, called a plasmid, which is replicated and transcribed like chromosomal DNA and codes for a protein. If the plasmid codes for more than one protein, or if the bacteria take in more than one plasmid, they can become resistant to multiple antibiotics.
Overuse or improper use of antibiotics promotes resistance because it creates an environment in which only antibiotic resistant bacteria can survive, thus eliminating any bacteria competing for energy or space. If the antibiotics do not kill all the bacteria in one locale, then it increases the likelihood that a plasmid that confers antibiotic resistance will be spread among other bacteria, increasing the number of antibiotic resistant bacteria. Thus, overuse of antibiotics in cleansing products can be a particularly serious concern. Many hand soaps, and even toothpastes and cosmetics, contain the antibacterial agent triclosan, which acts to block lipid synthesis in bacteria (Levy et al., 1999). Because it is so ubiquitous, many common bacteria are resistant to its activity, and bacteria that are less susceptible to triclosan may also be resistant to other antibiotics (Randall et al., 2003).
The above evidence suggests that, because triclosan was used in the hand soap in the bathroom of 313/314 West Holmes Hall, there might be some antibiotic resistant bacteria there. I hypothesized this was so, and investigated whether bacteria from that bathroom were resistant to ampicillin, kanamycin, or tetracycline. In addition, my aim was to characterize the plasmid that conferred resistance to the bacteria using restriction digestion enzymes and gel electrophoresis. To accomplish this, I swabbed places in the bathroom that frequently are exposed to users’ hands: faucet handles, toilet handle, and inside and outside of the doorknob. After culturing the bacteria, I identified antibiotic resistant bacteria and isolated the plasmid DNA via lysis. I then used the restriction digestion enzymes PstI and HindIII to characterize the plasmid that conferred resistance to the bacteria. Since bacteria that are resistant to triclosan are less resistant to kanamycin than to ampicillin or tetracycline (Randall et al., 2003), I predicted to find bacteria resistant to either ampicillin or tetracycline. Without further knowledge, however, it would merely be a guess as to which of those two would be more likely to have bacteria resistant to them.
Methods
Antibiotic
Resistant Bacteria Culture
Bacteria
for this investigation were collected from the dormitory bathroom of suite 313
and 314 West Holmes Hall at Michigan State University, East Lansing,
Michigan. The sink faucet handles,
toilet handle, and inside and outside door handles of the bathroom door were
swabbed using the same sterile cotton swab.
The cotton swab was placed in about 5 mL of sterile Luria-Bertani (LB)
broth and incubated overnight at 37 ˚C with shaking. Bacteria samples were then spread on LB/Agar
plates containing no antibiotics or ampicillin, kanamycin, or tetracycline and
allowed to grow for 48 hours at 37 ˚C.
Isolation of Plasmid DNA
Single colonies of
antibiotic-resistant bacteria, bacteria that grew on the LB/Agar plates with
ampicillin, kanamycin, or tetracycline, were harvested and lysed to isolate
plasmid DNA as described previously (Krha et al., 2005), except step 5
was omitted and supernatants were removed using a pipette rather than an
aspirator. The lysis product was tested
for DNA using gel electrophoresis, as described previously (Krha et al.,
2005). Agarose gels were made using 50
mL 1 x TBE electrophoresis buffer, 0.4 g agarose, and 4 μL ethidium
bromide (5 mg/mL). 6 μL of DNA and
2 μL loading dye were mixed and then added to the gel wells. Gels were run at 130 V for about one hour
and bands were visualized under an ultraviolet (UV) light.
Restriction Digestion of Plasmid DNA
Once it was confirmed that
lysis yielded DNA, DNA was digested using the restriction enzymes Pst I and
Hind III as described previously (Krha et al., 2005). Restriction digestion solution [14 μL
double-distilled water, 2 μL buffer (10 x KGB), 2 μL Pst I, 2 μL
Hind III, 2 μL DNA] was incubated at 37 ˚C for 90 minutes and kept
at 0 ˚C until gel
electrophoresis was run. DNA fragments
were separated using gel electrophoresis as previously described (Krha et
al., 2005). Gel loading dye (6
μL) was added to the restriction digestion product and 8 μL of this
solution was loaded into the wells of the gel. Gels were run at 80-100 V for
one hour and photographed with UV light.
Results
Identification
of Resistant Bacteria
To
determine if bacteria from the bathroom of 313/314 West Holmes Hall were
resistant to antibiotics, bacteria were grown on LB/Agar plates containing
ampicillin (Figure 1A), kanamycin (Figure 1B), or tetracycline (Figure 1C) or
no antibiotic (Figure 1D). Bacteria
growth was observed on the plates with kanamycin, but not on the plates with
ampicillin or tetracycline. Plates
without antibiotic were used as a control .
Isolation
of Plasmid DNA
Kanamycin
resistant bacteria were harvested and lysed to isolate and purify plasmid DNA
from the bacteria. Using agarose gel
electrophoresis, DNA was found in the lysed kanamycin resistant bacteria
(Figure 2).
Restriction
Digestion
After
isolating plasmid DNA from kanamycin resistant bacteria, the plasmids were
digested using the restriction enzymes PstI and HindIII. A distinct fragment of DNA was observed for
one of the lysis products, but not for the other (Figure 3). This fragment was calculated to be about
1950 base pairs, according to the 1 kb ladder.
To corroborate this finding, a second restriction digestion was
performed and a fragment of DNA at 1900 base pairs was observed (Figure
4). Digestion of the plasmid by PstI
did not reveal a fragment of DNA whereas digestion by HindIII revealed a
fragment of DNA at 1300 base pairs (Figure 4).
To characterize the plasmid isolated from bacteria from the bathroom of
313/314 West Holmes Hall, the plasmid pKAN was digested by PstI and HindIII and
a DNA fragment was observed at 3600 base pairs (Figure 4).
Discussion
Kanamycin Resistant Bacteria
Contrary
to my hypothesis, bacteria isolated from the bathroom of 313/314 West Holmes
Hall was found to be resistant to kanamycin, as bacteria grew on LB/Agar plates
with kanamycin, but did not grow on plates with ampicillin or tetracycline. It is reasonable to expect that some
bacteria from the bathroom would be resistant to antibiotics, since
antibacterial hand soap was used in the bathroom and would create an
environment that favors bacteria resistant to the antibacterial agents. However, it is difficult to explain why
bacteria from the bathroom would be selectively resistant to kanamycin over
ampicillin or tetracycline. Indeed,
bacteria with decreased susceptibility to the activity of triclosan were also
resistant to those three antibiotics (Randall et al., 2003), but out of
those three antibiotics, bacteria had the least resistance to kanamycin. However, several of my colleagues have also
found kanamycin resistant bacteria in restrooms in Holmes Hall where triclosan
is used in the hand soap, suggesting that perhaps another factor causes
kanamycin resistant bacteria to be prevalent in Holmes Hall.
Isolation of Plasmid
Because
my first attempt at lysis did not yield DNA, I did two lyses on my second
attempt. Both of these seemed to yield
DNA, as a band was observed on the agarose gel when the each of the lysis
products were run through electrophoresis.
However, for one of the products, no DNA was observed on the agarose
gels after restriction digestion. The
lack of DNA after restriction digestion could be because restriction digestion
was not optimized for the second product, though this seems unlikely, as both
were prepared in the same way and because no restriction digestion worked for
the second product. A more likely
explanation is that the DNA was not redissolved after lysis or that it broke
down, perhaps by DNase, after lysis and before restriction digestion.
Characterization of Plasmid
Restriction
digestion of plasmid that confers resistance to kanamycin with the enzymes PstI
and HindIII yielded at least one 1900 base pair fragment of DNA. PstI and HindIII were used to digest the
plasmid after using the program DNA Strider to simulate digestion of other
plasmids that confer resistance to kanamycin; PstI and HindIII digested other
plasmids to give at least two DNA fragments that differed by enough base pairs
to be distinguished by running gel electrophoresis. However, PstI and HindIII cut the unknown plasmid so that only
one band could be distinguished. It is
possible that the two enzymes cut the plasmid into multiple fragments of DNA
all around 1900 base pairs, thus making them indistinguishable. To determine if this was the case, the
unknown plasmid was digested with PstI alone and with HindIII alone. No DNA fragment was observed when the
plasmid was digested with PstI and this was probably because the deficient
lysis product was used for this digestion.
However, a 1300 base pair fragment of DNA was observed when the plasmid
was digested with HindIII. It is
expected that the fragment of DNA resulting from digestion with HindIII would
be different from the fragment resulting from digestion with both PstI and
HindIII. However, the fragment from
digestion with HindIII is smaller than the fragment from PstI and HindIII,
contrary to what is expected. One
explanation for this could be that the digestion with just HindIII ran for too
long, thus cutting the fragments into smaller pieces that would be expected,
but this does not seem likely, since the digestions all ran for approximately
the same time. Perhaps adding PstI acts
as an inhibitor to HindIII, preventing it from making its cuts in the DNA. It is reasonable that one restriction enzyme
would inhibit another since a bacteria may have more than one restriction
enzyme, in which case some control would be needed to prevent the enzymes from
digesting the bacteria’s own chromosomal DNA.
It is also possible that adding PstI with HindIII changed the
restriction digestion solution enough that HindIII activity was decreased,
preventing it from effectively digesting the plasmid.
Contrary
to the 1900 base pair DNA fragments from the unknown plasmid, DNA fragments
from pKAN digested by PstI and HindIII had about 3600 base pairs. This suggests that the unknown plasmid is
most likely not pKAN. pKAN was used as
a comparison to the unknown plasmid because both confer resistance to
kanamycin. However, it is possible that
the unknown plasmid is, or is similar, to a known plasmid that confers
resistance to kanamycin.
Perspectives
Though
I found the plasmid that confers kanamycin resistance to bacteria from the
bathroom of 313/314 West Holmes Hall not to be pKAN, further studies are needed
to compare the plasmid to other known plasmids that confer resistance to
kanamycin. Additionally, further
studies are needed to clarify what DNA fragments are produced when the plasmid
is digested with PstI and HindIII. An
important experiment to further strengthen the tie between kanamycin resistance
and triclosan would be to treat the kanamycin resistant bacteria with triclosan.
References
File, T. M., Jr. (1999). Overview of resistance in the 1990s. Chest,
115, 3S-8S.
Khra, M., Maleszewski, J., Wilterding, J., Sayed, M.,
Luckie, D. (2005). LBS-145: Cell
and Molecular Biology Lecture/Lab Spring 2005 Course Pack. East Lansing:
MSU Printing Services.
Levy, C, Roujeinikova, A, Sedelnikova, S,
Baker, P, Stuitje, A, Slabas, A, Rice, D,
Rafferty, J. (1999). Molecular basis of triclosan activity. Nature
398, 383-384.
Levy, S. B. (1998). Multidrug resistance: A sign of the times. New England
Journal
of Medicine. 338, 1376-1378.
Randall, L, Ridley, A,
Cooles, S, Sharma, M, Sayers, A,
Pumbwe, L, Newell, D,
Piddock, L, Woodward, M. (2003). Prevalence
of multiple antibiotic resistance in 443 Campylobacter spp. isolated
from humans and animals. Journal of Antimicrobial Chemotherapy , 52, 507-51.
Yim, G. Unknown. Attack of the Superbugs: Antibiotic Resistance.
http://www.bioteach.ubc.ca/Biodiversity/AttackOfTheSuperbugs/ Accessed 3/24/05.
Figures
A B D C
Figure 1. Bacteria from the bathroom of 313/314 West
Holmes Hall were resistant to kanamycin.
Bacteria were first grown in LB broth overnight and then transferred to
LB/Agar plates containing ampicillin (A), kanamycin (B), tetracycline (C), or
no antibiotic (D). Bacteria grew on
plates with kanamycin and with no antibiotic, but no growth was observed on
plates containing ampicillin or tetracycline.
Bacteria were grown on plates overnight to isolate single colonies of
antibiotic resistant bacteria for harvesting plasmid DNA. The plates are representative of two trials
of growing bacteria.
A B
Figure 2. Lysis of
kanamycin resistant bacteria yielded DNA.
Kanamycin resistant bacteria were harvested and lysed to isolate plasmid
DNA. To verify that lysis generated
DNA, the lysis product was run through agarose gel electrophoresis. DNA was observed in the product of two
different lysis of the bacteria. Gels
were run at about 130 V for 60 min and bands were visualized using ethidium
bromide under ultraviolet light.
B
Figure 3. Restriction
digestion using the enzymes PstI and HindIII yielded fragment of DNA (A, lane
1). Lane 2
also contained plasmid from kanamycin resistant bacteria digested with Pst I
and Hind III, but no DNA fragment was observed. Plasmids were digested for 90 min at 37 ˚C and then
run on agarose gel electrophoresis.
Bands were visualized using ethidium bromide and ultraviolet light. 1 kb ladder served
as a reference for molecular weight markers.
The fragment of DNA was calculated to be about 1950 base pairs,
according to the standard curve created from the 1 kb ladder (B).
A
B
Figure 4. Agarose gel electrophoresis of restriction-digested plasmid
DNA isolated from kanamycin resistant bacteria from the bathroom of 313/314
West Holmes Hall (A). Lanes 1 and 2
contained plasmid digested by PstI and HindIII and a 1900 base pair fragment of
DNA was observed. Lane 3 contained
plasmid digested by PstI; no band was observed. Lane 4 contained plasmid digested by HindIII and a fragment was
observed around 1300 base pairs. A 3600
base pair fragment of DNA was observed in lane 5, which contained the plasmid
pKAN digested by PstI and HindIII. 1 kb
ladder served as a reference for molecular weight markers; all DNA fragment
lengths were calculated according to the standard curve created from the 1 kp
ladder (B).
Appendix