Plasmid in antibiotic-resistant
bacteria
isolated by lysis, cut by restriction enzymes, gauged by gel
electrophoresis
Samantha
Salvia
LBS
145
Thursday
1
Dr.
Luckie
ABSTRACT:
Recently, there has been an increase in antibiotic-resistant bacteria (Lewis, 1995). When bacteria become resistant to antibiotics, they’re more harmful to humans because it becomes harder to stop any infection they may cause. One year, various resistant bacteria lead to the death of 13,300 hospital patients (Lewis, 1995). It is important to find where resistant bacteria are so we can better understand their optimal growing conditions. This research was conducted to identify the plasmids in antibiotic-resistant bacteria from different locations to determine which plasmids cause bacteria to be resistant to certain antibiotics.
Research was started by swabbing
several public locations, including a weight room, elevator, and trash
compactor in Holmes Hall. These places
were chosen because bacteria are so prevalent that it can be assumed
that they
can be found on virtually every surface (Anonymous-1, 1999). The bacteria was cultured on agar plates
containing three different antibiotics: kanamycin, tetracycline, and
ampicilin. Next a colony was amplified by
being cultured
in LB broth, and then the cells were lysed to obtain the individual
plasmids. Last the plasmids were cut
with restriction enzymes and then a gel electrophoresis was performed
to find
the number of base pairs in the plasmid fragments.
However, even after each step was performed
several times, no plasmid could be identified.
DISCUSSION
Since bacteria are omnipresent, it was predicted that bacteria would be located at all the locations that were swabbed. This was true for eleven of the twelve sites, which was seen by the cloudiness in the tubes containing the LB broth and q-tip swabs. It is not clear why no bacteria grew from the swab of the mouth, considering there are plenty of bacteria found in the mouth (Vanhoeck, 2004). The person tested was chewing Big Red gum prior to the swabbing, and it has been found that this can act as an antibiotic (Anonymous-5, 2005). Although this may have killed some of the bacteria in his mouth, it is not likely that it would kill all the bacteria, so this, in all probability, does not account for the entire reason that bacteria did not grow in the LB broth containing swabs from the mouth.
In each location it was expected to find bacteria that were resistant to the various antibiotics we used. This is because several locations are often cleaned, and this increases the chances that the bacteria have become resistant to any antibiotics that they are regularly exposed to (Anonymous-6, 2005). All the bacteria that grew in the LB broth were also resistant to the ampicilin; there were bacteria from eight locations that grew in the kanamycin, and only four sites had bacteria that grew in tetracycline. If bacteria continues to grow when put in antibiotic solutions, this means it is resistant to the antibiotic; likewise, it dies if it did not contain resistant qualities. Each of the resistant bacteria contains one or more plasmids that give them this resistant characteristic (Krha, 2005). According to these results, ampicilin-resistant bacteria were found at the most swab sites, followed by kanamycin-resistant bacteria, and only a few sites had bacteria resistant to tetracycline.
Next, bacteria was transferred from the tubes to the LB agar plates (Figure 1). One plate was made for each site that had antibiotic-resistant bacteria. Because the bacteria was added to a plate consisting of the same antibiotic that was in the LB broth in the tube, it was predict that this will not hinder any growth since it had already been verified that the bacteria grew in that specific antibiotic and was therefore resistant to it. However, no plates with tetracycline had growth, and four plates with ampicilin did not have growth, including plates with bacteria from the weight room, shoes, bike room and cafeteria door handles. There might not have been growth on these plates because there might not have been growth in the original tubes containing the antibiotics. The cloudiness may have been something other than bacterial growth and could have been mistake for growth. And so if there was no original growth, there would be no growth on the plates. Another place for error would be in the streaking methods used. If the bacteria was not streaked correctly on the plate, none may have reached the plate, and therefore none would grow.
After harvesting the cells to obtain an isolated colony, lysis was performed to obtain the plasmid found in each bacterium. The way lysis works is it denatures the chromosomal and plasmid DNA strands, and then attempts to put the pieces back together. However, since the chromosomal DNA is composed of millions of base pairs, it will not renature in the correct order. The plasmid DNA, however, is much smaller and therefore the renaturing process is more successful (Krha, 2005). When gel electrophoresis was run to test for DNA after lysis, DNA was obtained only the second and fourth times lysis was performed. The pink band on the gel indicated the presence of plasmid DNA. Any chromosomal DNA was too large to move through the gel, and therefore remained in the gel. During the first and third times lysis was performed, there were steps that could have gone wrong. When the supernatant was transferred to a new tube, part of the pellet at the bottom may have been accidentally kept, and this is chromosomal DNA. Also, after the ethanol was added and the tubes were centrifuged, many times it was hard to see the new pellet at the bottom. Often the pellet looked more like a smudge on the tube. So when the liquid was being removed, it is possible that the pellet was removed with it. Also, if the plasmid pellet was not secured to the side of the tube, when the tube was inverted, the pellet may have been lost. All these steps could have contributed to missing plasmid DNA.
Next restriction enzymes were added to cut the plasmid into fragments. The results of these various cuts could not be seen until the gel electrophoresis was run. The gel electrophoresis was used to determine approximately how many base pairs were in each DNA piece. The buffer solution containing the DNA was put into the wells near the cathode, which has a negative charge. Since DNA is also negatively charged, when the current went through the gel, the DNA traveled away from the cathode towards the anode at the base of the gel plate. This is because the positively charged anode attracted negative charges. In gel electrophoresis, smaller pieces travel farther due to the fact that they are not hindered as much as the larger fragments are when traveling through the agarose gel. When the gel electrophoresis was run, the HindIII lambda ladder and 1 kb DNA ladder obtained from New England Bio Labs Inc. were used to compare the base-pair length of the unknown plasmid pieces to the base-pair length of known DNA pieces.
In the first trial of restriction enzymes, which used HindIII and PstI to cut the plasmid, the pink band of DNA was still in the well. This was DNA from the kanamycin-resistant sewer bacteria. This DNA may have remained in the well because it was chromosomal DNA, so the restriction enzymes wouldn’t have much effect since the chromosomal DNA is so large, and therefore this DNA would be too big to go through the gel. In the second trial of restriction enzymes again HindIII and PstI were used. Kanamycin-resistant sewer plate bacteria and bike room bacteria and ampicilin-resistant compact room bacteria were tested. The only pink bands that were visible were in the wells of the gel. Once again, these bands may have been chromosomal DNA. Another possibility for these first two trials is that the plasmids were overly digested. Two restriction enzymes were used in both trials, and both were incubated overnight. Even though the enzymes only cut at certain sequences in the DNA, they only make a certain amount of cuts in a given amount of time. So after 15 hours of incubation, the DNA fragments might have been more digested than if it was only incubated for 3 hours. This may have caused the DNA to be small, and then it might not have been visible on the gel.
In the third trial of restriction enzymes, two tests were run. Bacteria from the sewer plates that was resistant to kanamycin and bacteria from the compact room that was resistant to ampicilin were digested with both HindIII and PstI, and then with just HindIII. Since there were pink bands present when just the HindIII was used, but no bands when both were used, this supports the over-digestion hypothesis. When only one restriction enzyme was used, it may not have cut the plasmid as much, and therefore the fragment sizes may have been large enough to see. Also, during this trial, the DNA only incubated for about 4 hours, so this may have also been a factor in the amount of cutting the enzymes did. One thing that does not fit with this hypothesis is the size of the fragment. The band was at the 10,000bp band on the 1 kb ladder. This indicated that the fragment was about 10,000 base pairs long. However, the plasmids diagramed in the LBS 145 Lab Book were under 6,000 base pairs. So 10,000bp seems very large. And the longer the DNA strand becomes, the harder it is for it to renature during lysis.
In the fourth trial or restriction enzymes, when PstI was used on kanamycin-resistant sewer plate bacteria and ampicilin-resistant compact room bacteria, there were again pink bands at the 10,000bp mark. There was also a band at this distance for the DNA from ampicilin-resistant bacteria from the compact room that was cut with HindIII. During this trial, the DNA was only incubated for 2 hours. This may not have been enough time for the restriction enzymes to cut, which may have been the reason that the plasmids were so large still.
So even though no plasmids were identified
after these procedures were performed, I believe that further research
may be
successful in discovering which plasmids cause these bacteria to be
resistant
to ampicilin and kanamycin.
Figure 1. Plate containing growth
with kanamycin antibiotic.
This is a picture of a plate of
bacteria obtained from the bike room. It contains colony growth
that is resistant to
kanamycin.