ÊÊÊÊÊÊÊÊÊÊÊ Searching at ãHolmesä for an Antibiotic-Resistant Plasmid by Isolation, Amplification, and Purification

By: Nidhi Atri W1 LBS 145 Spring 2005

ÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊ AÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊ B

Figure: Elevator Button Kanamycin Plate and Telephone Handle Kanamycin Plate. Figure A shows the bacterial colonies that grew on the LB agar plates that were made with kanamycin. You can see the distinct areas where the plate was obviously swabbed and the pattern in which the bacteria grew. The same is true for the bacteria on the telephone handle on the kanamycin plate of the telephone handle in figure B. There is heavy growth here of individual colonies. These colonies were then harvested, purified through lysis, and gel electrophoresis was run in order to see if there was indeed DNA present after the purification process.

Abstract

ÊÊÊÊÊÊÊÊÊÊÊ The purpose of this study was to amplify, isolate, and identify a piece of environmental plasmid DNA. This was done by obtaining bacterial swabs from areas of suspected high traffic areas of not only bacteria, but areas that may come into contact with antibacterial substances. The swabs were taken in four locations: the telephone handle on the public phone in front of East Holmes Hall, a keyboard in the LBS 145 laboratory, the basement elevator button of West Holmes Hall, a fish tank, and an eyeglasses plate.

ÊÊÊÊÊÊÊÊÊÊÊ The first process used in order to identify the plasmid of DNA was the isolation. Q-tips were used to swab the locations and then placed into test tubes containing 2mL of Luria-Bertani broth. After substantial growth was noted, they were transferred onto plates containing Ampicillin, Kanamycin, Tetracycline, and a control plate, which was used to determine which antibiotics the bacteria samples were resistant to. After this, it was noted that the bacteria samples obtained from the elevator button and telephone handle had a resistance to kanamycin. Single colonies from each plate were swabbed with a wire loop and placed into a test tube with 12μL of kanamycin and 3mL of LB broth. After a growth was noted and the single colonies were harvested and allowed to sit overnight, the cell lysis was carried out.

ÊÊÊÊÊÊÊÊÊÊÊ Lysing was done in order to extract genetic information and separate the genomic DNA from the plasmid DNA.Ê Once the lysing process was completed, a quick gel was run in order to see if there was indeed DNA present. After the harvesting and lysing processes were conducted five times and gels were run with no DNA, it was determined that somewhere in the lysing process, the plasmid DNA was lost each time. The successive trials were done in order to hopefully attain a perfection in which a plasmid could be achieved. Unfortunately, it was unsuccessful and no plasmid was ever obtained so experimentation was stopped there.

Discussion

ÊÊÊÊÊÊÊÊÊÊÊ Disease causing bacteria are rapidly increasing in not only number, but in species. Studies are now being done and it has been found that there are viruses in hospitals that are now resistant to all antibiotic drugs that they are now being treated with potentially harmful experimental drugs (Anonymous-2, Unknown). This resistance to antibiotics is sometimes referred to as antimicrobial resistance. The problem began just after penicillin, the first antibiotic, was introduced. Penicillin-resistant infections developed which were caused by Staphylococcus aureus, or ãstaph.ä The resistant bacteria can cause infections such as urinary tract infections and pneumonia. Methicillin, developed to combat these resistant strains has now even been combated itself. Now, vancomycin, the most lethal drug against the resistant strains is even unable to treat some strains now. This is a very serious problem because soon, these bacteria will be stronger than any antibiotic that we have to treat them (Bren, 2002). The purpose of this study was to determine just where these antibiotic-resistant bacteria could be.ÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊ

Obtaining and Culturing Samples of Bacteria/ Culturing Antibiotic Resistant Bacteria Colonies

ÊÊÊÊÊÊÊÊÊÊÊ From the sampled sites, bacteria were present in the telephone handle, elevator button, laboratory keyboard, and fish tank. As predicted, prevalent growth was seen in all four of the bacterial cultures so all of these sampled locations were plated onto Petri dishes with LB agar and each with a different antibiotic. There was lawn growth on all of the control plates, as there should have been. There was heavy growth on the kanamycin plates of the fish tank and keyboard. It was very thick and lawn like, indicating that it was not spread as separately as it could have been. This made it impossible to harvest individual colonies from these plates and therefore they were not used in the harvesting process. There was no growth on the ampicillin or tetracycline plates on any of the four sample locations. The elevator button and telephone handle yielded growth with individual colonies on the kanamycin plates and therefore these were the plates and sample locations used for harvesting.

ÊÊÊÊÊÊÊÊÊÊÊ The bacterial growth seen on these sample sites can be attributed to the fact that these sites are areas that see high traffic. The fish tank can be explained by the chemical used in the cleaning process. However, the telephone handle, elevator button, and computer keyboard in the lab are all touched several times a day by many different people. These people all come into contact with various objects and use different soaps and antibacterial products in their daily lives. With the continued exposure, the bacterial strains grow stronger and stronger and eventually can develop a resistance to different things (Doolittle, 2000).

Harvesting and Lysis of Bacterial and Plasmid DNA (Miniprep)

ÊÊÊÊÊÊÊÊÊÊÊ After seeing that there were bacterial strands from the elevator button and telephone handle that were resistant to kanamycin, single colonies of these were harvested and put in the shaker overnight and allowed to grow. It had originally been predicated that there would be bacterial strains at each location that would be resistant to at least one antibiotic, if not more.

This was suspected due to the fact that areas such as those sampled are places that are cleaned on some sort of schedule because they are public locations with cleansers that are usually antibacterial. The harvesting process is done in order to hopefully attain a purified sample of plasmid DNA. It is neither the bacteria nor the plasmid itself, however, that are resistant to antibiotics. The resistance is due to specific proteins that can fight off the antibiotics on a cellular level. The proteins are produced from DNA sequences which become part of the bacterial DNA sequence when a Ring of Plasmid DNA is inherited. The plasmid DNA is able to replicate itself with the genomic DNA and thereby incorporate itself into the DNA and be passed on to offspring. Therefore, the proteins are what cause the resistance that is seen, not that bacteria or plasmid alone (Krha et al., 2005).ÊÊÊ

ÊÊÊÊÊÊÊÊÊÊÊ After the harvested bacteria were allowed to grow, the lysis process allowed for the purification of the plasmid DNA. The step following lysis was to conduct gel electrophoresis to see if there was indeed plasmid DNA present in the resulting sample. Five quick gels were run and all proved to be unsuccessful. After the initial centrifugation of the sample in the lysing stage, a distinct white pellet was seen; indicating that at that stage in the experiment there was DNA. However, this does not necessarily indicate DNA. Also, even when the white pellets were seen in later steps of lysis that does not indicate that there is DNA present in the pellet.

ÊÊÊÊÊÊÊÊÊÊÊ The set up of the electrophoresis process was correctly run because the dye traveled down the gel as it was supposed to. However, when the gel was then put under UV analysis there were no glowing bands, which indicated that there was no plasmid DNA present in the sample. This could be for a number of reasons. One of which is that the sample was lost somewhere in the lysing process. There were several steps in lysing where the procedure was very precise, and if it was done with the slightest imperfection, all DNA could be lost.

ÊÊÊÊÊÊÊÊÊÊÊ However, bacteria can be resistant to antibiotics from other things. They can become resistant to antibiotics from genetic mutations. Mutations can cause resistant strains of various microorganisms. Also, conjugation, which is the process where genetic material is exchanged between two different bacterial cells, can lead to resistance. The process of conjugation plasmids from one organism can be transferred to other organisms. However, plasmids are not the only way to explain why a specific bacteria is resistant to antibiotics. Bacteria have the ability to have their genetic material available by two means of DNA transposition. In transformation, environmental DNA, such as DNA from other microorganisms, can be absorbed into the cell of the bacteria. In the process of transduction, pieces of DNA can be carried into the cell from a virus. With this new genetic material, an organism can be resistant to an antibiotic (Thompson, 1994).