Bacteria Do Not Need R-plasmids In Order To Be
Resistant To Antibiotics
Paul Tomaszycki
LBS 145 Cell and Molecular Biology ö Spring 2005
Lyman Briggs School of Science, College of Natural Science
Michigan State University, East Lansing, MI
Instructors:
Dr. Douglas Luckie
Rebecca Degraaf
Angela Wright
Abstract:
The purpose of this experiment was to find antibiotic-resistant bacteria that contain plasmids which give antibiotic-resistance (called R-plasmids) and to identify the specific plasmids that cause this to happen.Ê To compare different sites of bacteria, four locations at Michigan State University were swabbed five times for samples of bacteria: the IM East men's locker room (IM), the first-floor men's bathroom of East Holmes Hall (HB), the Totally Take-Out food line in Mcdonel Hall's cafeteria (TT), and the janitor's closet in West Holmes Hall (JC).Ê These swabs were cultured in LB broth and transferred onto LB agar plates each containing ampicillin, kanamycin, tetracycline, or no antibiotics at all (control plates).Ê Bacteria from all four sites thrived in all of the control plates.Ê The TT bacteria thrived in the plate containing kanamycin, and the JC bacteria thrived in the plate containing ampicillin.Ê From this point on, the main responsibility was to study the bacteria from Totally-Take Out food line.Ê Individual bacterial colonies were transferred into LB broth containing one of the antibiotics to check for double resistance.Ê The DNA from the bacteria was lysised (extracted) and tested for the presence of DNA by using gel electrophoresis.Ê If DNA was present, restriction enzyme would have been used in order to break up the plasmid DNA, and that would have gone through another gel electrophoresis to identify the plasmids present in the bacteria.Ê Results showed that there was no plasmid DNA present.
Discussion:
It was predicted in the beginning that the four locations tested for antibiotic-resistant bacteria, especially the Totally Take-Out food line for this specific study, would have unique plasmid DNA because different bacteria can be found everywhere and not just in individual places.Ê Also, different bacteria thrive better in certain environments over others.Ê For example, there are bacteria that can live in temperatures above boiling point, while there are bacteria that can live temperatures that can freeze human blood (American Society for Microbiology, 2005).Ê Although this example is a little extreme in this experiment, it does point out that there is not one single bacterium that will dominate four different places.Ê It turns out that there were bacteria resistant to ampicillin in the janitor's closet in West Holmes Hall, while there were bacteria resistant to kanamycin in the Totally Take-out food line in Mcdonel Hall.Ê This could have happened because two different kinds of cleaners could have been used to clean each of those locations.Ê If this was true, then the two cleaners would probably contain different antibiotics, which would prove wrong that the same antibiotics would be in all of the cleaners provided by Ecolab¨.Ê There were no antibiotic-resistant bacteria in East Holmes Hall's bathroom as well as the locker room of the IM East building possibly because the cleaners used in there contain no antibiotics.Ê Again, this would prove wrong that the same antibiotics would be in all of the cleaners provided by Ecolab¨.Ê None of the bacteria were resistant to tetracycline, which probably shows that none of the used cleaners contain tetracycline as an antibiotic.
During the first harvest, a janitor's closet bacterial colony that was resistant to ampicillin in the agar plate was also resistant to kanamycin in the LB broth, and a Totally Take-Out food line's bacterial colony that was resistant to kanamycin in the agar plate was also resistant to ampicillin in the LB broth.Ê There were so many bacterial colonies on each of the plates that there was the possibility of a few having more than one form of antibiotic resistance.Ê Actually picking the colonies that were to be harvested was probably caused by luck.Ê During the second harvest (when only the Totally Take-Out food line's bacteria were being tested), the bacteria still survived in the kanamycin-containing broth, but did not survive in the ampicillin-containing broth.Ê Again, this was probably because of luck of finding an individual colony with those properties.
When the focus of the experiment shifted from all four locations to just the Totally Take-Out food line, finding actual plasmid DNA was the challenge.Ê It was predicted that there would be a unique plasmid in the tested bacteria since there was only one colony being tested.Ê If there were multiple colonies being tested at one time, then there could have been more than one plasmid because different plasmids can give the same resistance effects in different kinds of bacteria (Pasquali, et al., 2005) (Yang, et al., 2003).Ê However, after testing for the presence of plasmid DNA four times, there was no plasmid DNA to be identified based on the results of finding no orange bands in the gels.
This absence of plasmid DNA and the presence of antibiotic resistance could have happened for several reasons.Ê In some bacteria, the genomic DNA can code for antibiotic resistance.Ê For example, in Staphylococcus aureus, part of its genomic DNA codes for β-lactamase, an enzyme that breaks down and detoxifies certain antibiotics (Etz, et al., 2002).Ê Another example of this would be in Streptococcus gallolyticus, where an aphA-3-like gene in the genomic DNA [and not plasmid DNA] codes for high resistance to kanamycin (Leclercq, et al., 2005).Ê It could be hypothesized that the genomic DNA could also code for antibiotic resistance similar to this situation.
There could be different organelles in the bacteria that could cause the organisms to have antibiotic resistance.Ê For example, Mycobacterium tuberculosis encodes truncated hemoglobins, which protects the bacteria from nitric oxide, which is toxic to certain bacteria (Ouellet, et al., 2002).Ê Although nitric oxide is structurally not an antibiotic, more research and investigation could be accomplished to see if bacterial organelles can protect bacteria from other toxins like antibiotics.
There was also room for human error.Ê After running the first gel electrophoresis test, it was realized later that the incorrect lysis buffer was used to break down the RNA still in solution.Ê Also, small drips of chemicals needed for the experiments dripped out of the micropipette tubes while inserting them into the solutions needed for lysis to work.Ê This was not taken into account during the experiment mainly because of the lack of experience needed for this lab to effectively work.Ê Also, during the final lysis process, the centrifuge for the microfuge tubes might have not centrifuged the particles to the bottom of the tube enough in order to work with clean supernatant containing the DNA, creating impurities in the tested chemicals.
These results show that further research is needed in order to find different causes of antibiotic resistance.Ê Future research could include the investigation of resistance coded from genomic DNA versus resistance coded from plasmid DNA.Ê Also included in the investigations could be the testing of organelles and their effects on detoxifying antibiotics.Ê Another investigation could include the use of cleaners containing antibiotics as well as the necessary restriction enzymes to break down the plasmids that code for antibiotic resistance in bacteria.
Figure:
ÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊ AÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊ B
ÊÊÊÊ 
Figure.Ê The two plates, each containing one antibiotic, in which bacteria survived and multiplied on.Ê Because these bacteria survived on agar plates containing an antibiotic, they are considered as resistant to the specific antibiotic.Ê (A) Bacteria from the janitor's closet in West Holmes Hall that are resistant to ampicillin.Ê Since the lights above the plate created a glare on the plate, the bacteria can be identified by the dark region inside the plate.Ê (B) Bacteria from the Totally Take-Out food line in Mcdonel Hall's cafeteria that are resistant to kanamycin.Ê Unlike the other plate, these bacteria grew in individual colonies more so than the bacteria resistant to ampicillin.Ê (Pictures taken on March 24, 2005)
References:
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Ouellet, H., Ouellet, Y., Richard, C., Labarre, M., Wittenberg, B., Wittenberg, J., and Guertin, M.Ê 2002.Ê Truncated Hemoglobin HbN Protects Mycobacterium bovis from Nitric Oxide.Ê Proceedings of the National Academy of Sciences of the United States of America 99 (9): 5902-5907.
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Wang, N.S.Ê Unknown.Ê Experiment No. 8B, Aseptic Culture Techniques, Petri Dish Preparation.Ê http://www.glue.umd.edu/~nsw/ench485/lab8b.htm/Ê Accessed 3/16/05.
Yang, S., Clayton, S. R., and Zechiedrich, E. L.Ê 2003.Ê Relative contributions of the AcrAB, MdfA and NorE efflux pumps to quinolone resistance in Escherichia coli.Ê Journal of Antimicrobial Chemotherapy 51 (3): 545-556.
2005Ê Paul Tomaszycki
contact: tomaszyc@msu.edu