in Attempt to Locate

By: Devin Murphy

LBS 145L Section W2

April 27, 2005

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Abstract

ÊÊÊÊÊÊÊÊÊÊÊ Most all surfaces are cleaned on a regular basis with antimicrobial cleaning agents. The cleansers defend against the growth and spread of bacteria. The cleansing process has progressed into a cycle of evolutionary competition. Bacteria containing certain plasmids are able to formulate a resistance to the cleaning agents. This resistance causes for the need to strengthen the chemicals used in the cleaning products. East Holmes Hall on the Michigan State campus is cleaned on a regular basis with such antimicrobial cleaning agents. This thus gave rise to the question as to whether or not bacteria found in Holmes Hall contained plasmids allowing for them to build up a resistance. This experiment looks to see if the bacteria have built up a resistance to three antibiotics: ampicillin, kanamycin, and tetracycline, via plasmids. If so, what are these plasmids? To test for this, the bacteria was collected and incubated overnight to multiply in LB broth. The bacterium was grown against antibiotic containing agar plates. Colonies were removed and purified through the process of lysis to leave behind DNA. The DNA undergoes restriction digest and is then run through gel electrophoresis to determine the plasmid present. A resistance to the three antibiotics tested was found to be present amongst the bacteria from the different locations tested. This concluded due to the colony growth on the antibiotic agar plates. The plasmid which caused such resistance was unable to be identified due to the lack of plasmid DNA found via gel electrophoresis.

Discussion

This experiment tested for antibiotic resistant bacteria in a few commonly encountered areas of Holmes Hall on the Michigan State University campus located in E. Lansing, Michigan. The null hypothesis, Ho, is that there will be no difference in the resistance of the bacteria to the different antibiotics. The alternative hypothesis is that there will be a difference in the resistance between the bacteria. The locations swabbed were a classroom, an elevator, a study lounge, and a stairwell. These areas are cleaned on a regular basis using an array of antibacterial detergent products. Over a period of time it would be expected that these microbes would develop a resistance to the cleaning products (Oliveira, S et al, 2005). This resistance comes in a form known as a plasmid. A plasmid is the DNA that will be a code for the production of specific proteins. These proteins then degrade the antibiotics and cause them to become useless in the fight against bacterial growth (Krha et. al, 2005).

First tested was the bacterial resistance to the three different types of antibiotics: ampicillin, tetracycline, and kanamycin. To test this resistance the bacteria was first amplified in the LB broth to obtain a large concentration of bacteria in the tube, which could be seen by the overwhelming cloudiness of the broth after removal from the overnight incubation. The tubes were incubated at approximately 98¡F and shaken continuously. The temperature was as such since bacteria grow ideally around that temperature, the shaking was used to speed up the replication process. The bacteria was streaked onto separate agar plates which were made to contain one of the three antibiotics to be tested against. If the bacteria were resistant to the antibiotic it would be able to grow on the antibiotic containing gel (Krha et. al, 2005). Just as the tubes were kept in a warm climate for ideal growth conditions so were the plates. Over the three trials of plate growth the first and second trials results were fairly similar to one another, where as, the third trials plates had the significant difference in the growth of bacteria on the tetracycline plates more so then the ampicillin plates. This discrepancy could be due to the fact that the swabbings took place at different times on different days. The locations may have been cleaned recently to the swabbing being done and thus killing the non ampicillin resistant bacteria. There may have also been a switch in the cleaning agents used, and due to the fast replication time of DNA it would be possible for a tetracycline resistant plasmid to quickly become spread amongst the bacteria (Todar, 2002). These colonies that grew were thus resistant to the antibiotic and therefore most likely contained a plasmid making it resistant (Krha, 2005). The colonies that grew were then harvested to undergo the lysis process to leave behind purified bacterial DNA. Only one colony from each plate was to be harvested to reduce the probability of contamination by other bacteria that may have grown on the plate forming its own colonies. The process of lysing involves a multitude of steps which could lead to difficulty in getting results if done improperly. This thus helps explain the possible reasoning for the lack of DNA found during the test gel electrophoresis. In lysis a latter step calls for 95% ethanol and another step slightly thereafter calls for 70% ethanol (Krha, 2005). During the first lysing process the ethanol percentages were switched and thus human error may have lead to the lack of DNA. The solutions used in lysis also have specific storage temperatures, some must be kept frozen in order to work; this includes the TB solution that contains RNAase. If the solution is not kept in the freezer the solution can go bad and thus not denature the RNA. The use of TB solution that was at room temperature is believed to have been the mistake which lead to the manifestation of RNA in the third test gel. The exact reason for the large occurrence of no DNA found in the test electrophoresisâ could be due again to human error in the removal of the supernatant to leave behind the pellet; due to its small and somewhat invisible size it is easy to accidentally remove the pellet. During the lysis by alkali process the linear DNA is disrupted and denatures. This does not happen to the plasmid DNA though because it is entangled with one another, this allows for the purification of the chromosomal DNA to take place without harming the plasmid DNA (Krha, 2005). The removal of the washes and supernatant liquid is vital and if it is not all removed can lead to disruption of the DNA purification and to further problems in restriction digest (Krha, 2005). The DNA found on the gel shows up as a bright pink florescence because of the use of the ethidium bromide used in the gel. The ethidium binds to the DNA and when the UV light is shown upon the gel the DNA absorbs it but is then radiated via the ethidium bromide. This is why the DNA does not just show up as huge streaks when being viewed in the UV light (Krha, 2005).Ê Once DNA was found from the test gels; restriction digest could be run on the lysed bacteria. To begin restriction digest the restriction enzymes needed to be chosen.

To do so the antibiotic that the bacteria was resistant to was taken into play. In the LBS 145 lab manual by Krha et al there are several plasmid maps, which show the types of enzymes that will cut it and at how many base pairs. The bacteria resistant to the kanamycin used the plasmid map pKAN to determine to use the Bam HI enzyme which cuts the plasmid at 2095 base pairs. For the bacteria resistant to tetracycline the map pACYC177 was used to determine to use the enzyme Hind III to cut the plasmid at 2472 base pairs. Knowing where the plasmid is cut helps in the comparison of the distance traveled by plasmid DNA found through gel electrophoresis against a known Hind III ladder to determine whether or not the plasmid in the lab book represents the plasmid found in the bacteria. In this experiment no plasmid DNA was recovered after the restriction digest. This may have occurred for many reasons. One being that if not all of the liquid was properly removed in lysis which will cause for the DNA to be resistant to cleavage. Another potential problem was thought to be that the restriction digest was run to long, having been run for 18hrs. This could have caused for the DNA to have been overly cleaved to a length of base pairs to small to have been stopped by the gel. The time thus was reduced to 7hrs; however, plasmid DNA was still not obtained. Due to the lack of time the experiment was unable to proceed further to run more tests to obtain plasmid DNA.

In conclusion, the results of this experiment were enough to reject the null hypothesis. There was a difference in the antibiotic resistance of the bacteria as seen in the trials of plates run. It can be discerned, through background information, that there were plasmids present in the bacteria causing for the resistance to the antibiotics. The plasmids however were unable to be located identified during the time allotted for the experiment. If this experiment were to be produced again some alternatives to be made would be to get more background information on the cleaning agents used, to vary the time allowed for the restriction digest to run, also to try lysis on a large scale to thus reduce the risk of removing the DNA pellet.ÊÊ

Figure 5: In this DNA test gel electrophoresis DNA was found in three of the tetracycline resistant bacteria wells. In the well labeled B, which contained bacteria from swab B of the lounge, showed the most prevalent pink line in front of the well, indicating the presence of DNA. The well labeled A in the photo contained bacteria off swab A of the lounge and had a faint line of DNA in front of the well. The well labeled C contained bacteria off swab B from the elevator and also has and extremely faint line of DNA.