Comparing Antibiotic Resistant Bacteria Found on a Human and Objects of Contact by Lysis and Mapping of Plasmids

By:

Bradley Damm

Abstract

By: Bradley Damm

ÊÊÊÊÊÊÊ Bacteria are found all over the human body and places we come in contact with.Ê Whether useful or harmful, bacteria are becoming resistant to certain antibiotics.Ê To find whether similar resistant bacteria exist on the body as in places that the body part comes into contact with, a swab was taken of a human hand, mouth, and hair.Ê To coincide with these, a swab was also taken on a sink handle, toothbrush, and hairbrush.Ê The bacteria are picked up with a cotton swab and grown in LB Broth.Ê There are three antibiotics used: Ampicillin, Kanamycin, and Tetracycline.ÊÊ Once grown, the bacteria are spread onto 4 different Agarose gel plates each.Ê One plate for each antibiotic and another for control were used.Ê The resistant bacteria are harvested and lysed by alkali.Ê We prepared them by removing unwanted parts of the cell and with restriction enzymes to allow us to see the DNA and map the plasmid.Ê Will the body parts produce the same amount of bacteria and/or antibiotic bacteria as the places they frequently come into contact with?Ê Will the plasmids be similar?Ê I found Ampicillin resistant bacteria on the human hand and mouth.Ê There were Kanamycin resistant bacteria on the hairbrush and hair, the toothbrush, and the sink handle and hand.Ê No bacteria survived the tetracycline.Ê Due to the lack of time and success with alkali and restrictive enzymes, I was unable to map the plasmid to compare and identify them.Ê

Discussion

By: Bradley Damm

ÊÊÊÊÊÊÊ Bacteria exist most everywhere you can think of including the human body (Anonymous, 2005).Ê I predicted that body parts and places they frequently come into contact with will contain the same bacteria and antibiotic resistant bacteria.Ê I predict this because bacteria can develop a resistance to antibiotics such as soap and other things (Rock and Heath, 1999).Ê This led me to think that the hand or sink handle would generate more resistant bacteria.Ê I predicted the mouth and hair also contained bacteria because according to Crestâs website there is not any antibacterial agents in toothpaste (Anonymous, 2005) and the Pert Plus website also claimed to have no antibacterial agents in its shampoos (Anonymous, 2002).Ê Any place on the body should have antibiotic resistant bacteria because so many live on the body, and humans frequently take antibiotics to get rid of illnesses and infections (Bakalar, 2003).Ê From this information also, I predicted that the toothbrush and hair would not have antibiotic bacteria unless they came from the body.Ê Using my predictions and information collected, I hypothesized that the corresponding regions I sampled would contain the same plasmids.Ê In terms of the antibiotics used, my hypothesis was that the Tetracycline would have little or no resistant bacteria because it seems more severe and harder to get (Anonymous, 2004).Ê The Kanamycin is more widely used (Anonymous, 1994) and the Ampicillin is the easiest one of the three to get (Anonymous, 2005).Ê For that reason, I think the Kanamycin and Ampicillin will have more resistant bacteria against it, because the more the product is out, the better the chances a bacteria found a way to resist it (Rock and Heath, 2000).Ê

From the results, it is hard to show one location had more types of bacteria than the other.Ê The sink handle control group may, but it was hard to tell visually.Ê To me, it appeared the slight difference in color was due to the thickness of the bacteria.Ê More tests would have had to been done.Ê I could have found this out if I were able to get through the whole experiment.Ê Besides that, none of the locations sampled seemed to show different bacteria.Ê Every place sampled did have bacteria though.Ê The results also did not show a direct link between body parts and places of contact with other objects.Ê Not enough information was found to link body and object to each other. ÊHowever, Ampicillin resistant bacteria were found on the hand sample and Kanamycin resistant bacteria were found on the hand, sink handle, toothbrush, and hairbrush.Ê In order to show a link for example, the bacteria resistant to Ampicillin which was found on the hand should have also been found on the sink handle.Ê The same would hold true for the other antibiotics as well.Ê The plasmids were never mapped, though, because I ran out of time and had many negative results during the experiment forcing the procedure back.Ê

ÊÊÊÊÊÊÊ From the results, my predictions were not right.Ê Instead of having resistant bacteria on or in the body as well as on the toothbrush or hairbrush, the opposite occurred.Ê Kanamycin resistant bacteria were found on the toothbrush and hairbrush, but not in the mouth or on a piece of hair.Ê The sink handle and hand results came out close to my predictions.Ê They both contained Kanamycin resistant bacteria, and the hand also contained Ampicillin resistant bacteria as well.Ê

ÊÊÊÊÊÊÊ My hypothesis about which antibiotics would have more resistant bacteria was similar to the results.Ê It was not enough information however to say I my hypothesis was right.Ê More tests would need to be done.Ê Tetracycline did not have any resistance while Kanamycin had the most.Ê Ampicillin had just one.Ê This I would accredit to the accessibility to the latter two.Ê

ÊÊÊÊÊÊÊ I was unable to map the plasmid of my samples, which would have been useful in relating the data.Ê A plasmid is small, circular, and separate from the chromosome pieces of DNA.Ê They still replicate and carry genetic information like chromosomes.Ê A plasmid can kill an antibiotic by developing a plasmid that codes for a protein that targets the particular thing trying to kill the bacteria (Krha et al., 2005).

ÊÊÊÊÊÊÊ Upon collecting my samples and growing them, the lyses part I had the most trouble with.Ê I had to repeat the process three times to get any DNA and when I did, it was just in one sample.Ê The Ampicillin applied to the bacteria from the hand sample was the only one with DNA.Ê I decided due to the lack of time remaining to go with this sample and see if I couldnât map out the plasmid, so I could learn the other steps more efficiently and have something to show for my work.Ê However, after running the restrictive enzyme test, I did not have DNA anymore.Ê Upon collecting this information, I decided to end the experiment with a week left.Ê In order for me to harvest, lyses, and run restrictive enzymes again, it would have taken at least another week.Ê

ÊÊÊÊÊÊÊ After swabbing and growing my bacteria, I was surprised to find that the hand contained bacteria resistant to two antibiotics, but the sink handle bacteria were only resistant to one.Ê As the project progressed, I found other researchers that had found bacteria from the sink handle resistant to Ampicillin.Ê This could be accredited to a poor job swabbing on my part, time of day such as after itâd been cleaned, or a difference in locations.Ê Also, with doing humans and places they come into contact with, different people have different bacteria on them and have different habits that fluctuate what that bacteria may be or act like.Ê As for lyses, I had an inability to produce DNA.Ê The last time I was a lot more careful and I measured out everything before applying it, because I found my second time through lyses that the pipettes were not very accurate.Ê This helped to produce at least one DNA sample I could use.Ê I found that when I checked for DNA, using 1-1.5 μl of gel-loading buffer instead of 2 μl made it easier to tell if DNA was present under the light.Ê Too much made it dark and hard to see under the UV light.Ê After I found DNA with my Ampicillin sample with bacteria from the hand, I proceeded to the restrictive enzyme stage.Ê I chose HindIII and AvaII to use on my Ampicillin hand sample, because they cut on opposite ends of the map.Ê One cut at base pair 233 and the other at 2602.Ê When comparing this to the lamba latter, it would have been easier to see because the fragments would have been larger.Ê Here, I didnât have DNA left when I was done.Ê If I had time, I would go back and mix it a little differently.Ê I would use more DNA and less ddH20.Ê Maybe this would help.Ê Mistakes occur and many can occur with an experiment like this.Ê Simply breathing on the wrong thing or even heat changes can mess up your progress with the bacteria and DNA.Ê These things need to be considered in future experiments done.Ê ÊÊ Ê

DNA Found After Lyses in the Hand Sample Applied with Ampicillin

Figure 6: This shows that the sample on the end, which is the bacteria from the hand applied with Ampicillin still has DNA in it after Lyses.Ê Upon seeing this, I used the rest of this sample to proceed on with restriction enzymes.Ê