Abstract

The overall goal of this study is to find antibiotic resistant bacteria and to identify the plasmids that are contained in the bacteria that confer this antibiotic resistance.  Four different sample sites were used in order to find bacteria.  The places that were swabbed were the IM East locker room of MSU, the Holmes Hall public men’s bathroom, the Totally Take-out café at McDonel Hall, and the janitor closet in Holmes Hall.  Each of the bacteria found in these places were grown in separate tubes containing LB broth.   Bacteria that grew were then inoculated on agar plates containing different antibiotics.  The three antibiotics that were used are ampicillin, tetracycline, and kanamycin.  A control plate was also used to make sure that there is nothing wrong the agar plate itself.  The bacteria that grew on the antibiotic containing plates was then transferred to new plates which still contain antibiotics and they were amplified by letting them grow into large colonies.  The plasmid DNA was then isolated and purified from the bacterial DNA.  The unknown plasmids will then be cut using restriction enzymes and then run on an agarose gel, using electrophoresis, in an attempt to see if there are any differences between plasmids obtained from different sites.  It is predicted that the plasmids isolated from the four different sites will be the same type of plasmid since all sites have been exposed to the same type of antibiotics.  An overall plasmid was not identified, but antibiotic resistant bacterial was found.  

Figure 4.  This is the first gel that was run.  It was used to check to see if DNA was present in the sample taken from the mini-prep.  There are no DNA bands present on the gel.  The two orange spots that are showing are traces of RNA that was left in the solution.  RNA was still present in the solution because no RNAse was present in the buffer that was used in the mini-prep.

Discussion

            Four sites around the Michigan State University’s Campus were sampled for bacteria with the intent of finding an antibacterial resistant plasmid inside of some of the bacteria that was found in at least one of the sites.  Even though no final plasmid was identified, a lot of good science was performed and meaningful, reproducible results were found. 

            It is shown that bacteria is abundant around the Michigan State University campus because all of the sites yielded at least some bacterial growth.  However, only two of the four sites contained bacteria that was resistant to either ampicillin, kanamycin, or tetracycline.  The other two sites could have had bacteria with resistances to other antibiotics that were not used.  Other antibiotics that bacteria might have a resistance to are Chloramphenicol, Nalidixic Acid, and Streptomycin (Krha et al., 2005).  Ampicillin, kanamycin, and tetracycline were used in this experiment because that is what was provided in the lab. 

The two sites that produced antibiotic resistant bacteria were the Janitor’s Closet in Holmes Hall and Totally Take-out café in McDonel Hall.  Bacteria will all be exposed to the same antibiotics because all of the collection sites are on campus and all of the sanitizers used at MSU are purchased from the same company, Ecolab® (Anonymous-1).  These sanitizers are located and stored in the Janitor’s Closet in Holmes Hall.  Since all of the antibiotics are purchased from the same place, bacteria in the area where the antibiotics are stored are going to have a higher resistance to more types of antibiotics.  This statement matches the data considering that the bacteria from the Janitor’s Closet was resistant to ampicillin.  The bacterial resistance from the bacteria at the Totally Take-out café has most likely gotten its resistance in the same sort of manner.  The Café is cleaned daily, and since all the cleaners at Michigan State University are the same, the bacteria present in the Café have a great chance to build up resistance to the antibiotics used. 

All of these resistant bacteria in common areas presents a problem for common citizens of the Michigan State University campus.  Many people live around and visit the MSU campus, with almost 45,000 students in about two square miles, the chance to spread bacteria is very large.  If the bacteria is antibiotic resistant, it will live longer, and wont be killed by certain antibiotics.  Students can pass it freely because the bacteria has resistances to the antibiotics that are used on Campus. 

In the first agarose gel, at the step in the procedure to check if plasmid DNA was present, no DNA appeared.  This is because in the previous step, the incorrect type of buffer was used.  The buffer that was used did not have any RNAse present, so the faint bands that were seen on the gel are actually bands of RNA, which is not useful in the identification of a plasmid.  Another situation that could account for the absence of DNA at this step would be if the bacterial resistance to a certain antibiotic was not contained in a plasmid, but was contained in the bacterial genome itself.  If this is the case, then no matter how many times that mini-prep is run, no DNA will show up, even though the bacteria have the antibiotic resistance. 

The final time that the bacteria was harvested did not produce any living bacterial colonies in the LB broth.  The procedure was altered in order to try to get a more concentrated sample of bacterial DNA.  This was done to try to ensure the presence of DNA on the gel, so the experiment could move on to the restriction digest step of the procedure.  It was also done because it would increase the number of replications of this part of the experiment.  This plan, however, did not work.  The problem was that less volume of LB broth was used for the same amount of bacteria.  The broth tubes containing the bacteria were placed in the shaker for less time than before, because the bacteria had less LB broth (i.e. food) to grow on.  Once the nutrients in the LB broth were used up, the bacteria died and mini-prep to isolate the DNA was no longer an option.  Given more time, this edited procedure would be attempted again with the further stipulation of drastically cutting down the time that the bacteria/broth vial spends in the shaker.