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Comparison of Antibiotic Resistant Plasmid DNA to Ampicillin, Kanomycin, and Tetracycline in Gram-Positive Hand and Foot Bacteria using DNA transformation
This study investigates the resistance of bacteria collected from two different environments on human subjects, the hands and feet. Principally, the degree of resistance to antibiotics in the hands compared to the feet using Ampicillin (AMP), Kanomycin (KAN), and Tetracycline (TET). Multiple tests (including KOH test, MacConkey agar, and antibiotic resistance swabs) were run to determine the characteristics of the bacteria present from each colony. Further research such as a transformation of environmental plasmid was done with the bacteria collected to identify the means of plasmid transference between bacteria. For each process, DNA was collected through DNA mini-prep of environmental bacteria. A DNA polymerase chain reaction was run to copy the target DNA plasmid for further study. The end result of the study showed a significant difference in bacterial resistance to Kanomycin and Tetracycline, but each environment was only significantly resistant to one antibiotic. The hands of the subjects showed a significant resistance to the Ampicillin antibiotic while the feet showed a higher resistance to Tetracycline. Statistical tests concluded that these results were of greater significance in the Tetracycline antibiotic. There can be no conclusion on which environment is generally more resistant to antibiotics, as each contains bacteria resistant to different types of target antibiotics.
Bacteria are vital to all living organisms. Some of these are necessary and beneficial, while most will never directly affect us. There are, however, some that can be harmful and have become resistant to the drugs developed to control them. In recent years, antibiotic resistance has become an increasingly alarming threat to society.
Bacteria contain plasmids, which are extra circles of DNA that are copied and exchanged between cells that come in contact with each other. In some cases antibiotic resistance is spread to multiple bacterial cells through plasmid transfer. One way that many different types of bacteria can be transferred from place to place and have the opportunity to interact with a multitude of other bacteria is on human hands. It is expected that because of the varied student body of Michigan State University there will be a lot of different types of bacteria that have been exposed to many different types of antibiotics and environments.
Antibiotic use is as popular as ever and they are being given to humans every day. One of the most common everyday applications is antibacterial hand soap that mostly everyone uses when washing their hands. This is a daily habit of nearly every student at Michigan State. There are also many antibacterial sprays like Lysol and Windex that are used to clean in many students' rooms. The hands of students come in contact with these antibiotics many times a day. Students' feet, however, contain bacteria but are very rarely exposed to these daily antibiotics. Even showering may not expose feet to antibacterial soaps, as most body washes do not contain antibiotics. This was the base of the experiment conducted in order to compare and contrast antibiotic resistance in the hands and feet of students at MSU.
Samples were taken from the hands and feet of three different members of our lab team. They were instructed not to wash their hands or feet for a day before swabbing for bacteria. It is hypothesized that because the potentially antibiotic resistant bacteria were allowed to grow, the bacteria samples obtained from hands will contain more antibiotic resistant specimens than the samples obtained from feet. These types of bacteria will be restricted by their abilities to attach themselves to a foreign surface such as a hand. Based on a study presented in the Journal of Applied Microbiology, Gram-positive bacteria are transmitted to surfaces such as a hand more readily than Gram-negative (Rusin et al., 2002). From this study, there should likely be a high concentration of Gram-Positive bacteria found on the hands of the subjects. This study covered the transmission of bacteria from the environment to the hands of multiple human subjects and studied the bacteria present. The researchers also studied the transfer rates of bacteria from different surfaces to the hands. They found that a greater number of bacteria are transferred to hands off of hard non-porous surfaces compared to any other surface studied (Rusin et al., 2002). This indicates that there are significant amounts of bacteria present on the hands to study the species present.
Studying just the types of bacteria present is not the only important part in determining how harmful the bacteria actually are. The bacteria on the hands are exposed to much more antibiotic substances such as hand-soap and hand sanitizer. These products claim to kill 99.9% of bacteria on hands. The danger in this is the other 0.1% of bacteria on the hands (Levy, 1998). The bacteria are possibly resistant to the antibiotics being applied, due to a genetic mutation of the DNA or a plasmid present in the bacterium. The bacteria that are resistant to a certain type of antibiotic expand and multiply after being exposed to that antibiotic (Levy, 1998). Bacteria can pass on their resistant qualities to their offspring in a process called vertical transmission. This is dangerous because these resistant bacteria are allowed to grow freely in the newly empty, nutrient-rich environment and produce a high number of resistant bacteria than could potentially infect a person. Antibiotic resistant bacteria obtain their resistance by means of a plasmid within the cell. These plasmids that may code for resistance to a specific antibiotic can be transferred to the "offspring" that the bacterium may divide into, which is called vertical transmission. Bacteria can also obtain plasmids horizontally by conjugation or encountering bits of DNA left over from other dead bacteria cells. One other possible way of transmission is through a virus that would inject the DNA sequence into a bacterium and join a plasmid (Levy, 1998). These multiple ways for bacteria to receive antibiotic resistant genes make bacterial evolution very rapid and potentially harmful to people that use excessive amounts of antibiotics. This would cause a selection for resistant bacteria that may someday be beyond treatment.
In this experiment, we will be investigating the typical incidence of antibiotic resistant bacteria present on the human body, specifically on the hands and feet. We will isolate various strains and attempt to identify the mechanism by which the resistance is achieved and passed on. This study is relevant because the bacteria that pose the most risk to human safety are those that we come in direct contact with, such as the types that can be found on human hands. A similar study was conducted at a hospital where samples were taken from patients from all over their body to look for different kinds of bacteria. A heavy amount of MRSA (Methicillin-resistant Staphylococcus aureus) was found to be in areas often touched my hands (Gaspard et al., 2009). Places like pockets and waists were shown to be heavily populated. These are areas of the body that, especially in the line of work at a hospital, are often in contact with hands. These studies will provide evidence of whether hands or feet contain more antibiotic resistant bacteria. It also will give statistics on the amount of resistant bacteria to specific antibiotics (ampicillin, kanamycin, and tetracycline) from both hands and feet. This study on antibiotic resistant bacteria was furthered by plasmid transformations that can help to tell more about the types of bacteria present.
From the two different environments, each with three subjects, six swabs were taken. First, each sterile cotton swab was dipped into a sterile phosphate-buffered saline (PBS) to remove bacteria from subjects for sampling. The moistened PBS cotton swab was then streaked across the surface of either the hands or feet of the subject (between digits as well) and then was immediately swabbed back and forth across an LB agar plate. Samples were then incubated upside down at 37Â°C in the lab incubator for 24 hours.
Master Patch Plates:
From the environmental sample plate, several colonies were selected based on differing characteristics between the colonies. These different colonies were swabbed into sixteen different sections on each master plate. An inoculating loop used to streak the plate was sterilized in a flame between each bacterial streaking to avoid contamination. There was one master plate created for each environmental plate. A new patch plate had to be created every two weeks to ensure survival of the bacteria. Bacteria were then grown at 37Â°C for 24 hours in the incubator.
Antibiotic Patch Plates:
For each master plate, three antibiotic plates were made in a similar fashion. For each colony on the master plate, the bacteria were picked up and streaked in the corresponding colony locations on ampicillin, kanamycin, tetracycline, and an LB plate. The LB plate was streaked last to ensure there were sufficient bacteria on the loop as it was streaked on the other plates. The loop for streaking was not sterilized between each plate; the loop was streaked across all the plates before sterilizing. Bacteria grew at 37Â°C for 24 hours in the incubator in the lab.
Streak plates were prepared from four interesting bacterial colonies (any that appeared different) on the antibiotic plates compared to the other colonies. These colonies were also resistant as they were able to grow on the antibiotic plate. The colony was picked up and streaked across one third of the plate. The Inoculating loop was then used to streak the bacteria from the first third into the second, and then into the third. This process diluted the bacteria in the last third, causing segregated colonies to form. The streak plates were then incubated at 37Â°C for 24 hours.
Agarose Gel Electrophoresis:
For the agarose gel electrophoresis, a mixture of 70mL of 1X Tris Borate EDTA (TBE) and 0.7g agarose was used to create a 16-lane gel. The mixture was then heated in a microwave until completely mixed. Once the mixture cooled, 3ÂµL of Ethidium Bromide (EtBr) was added. This was fully mixed and the solution was poured into a gel mold with a comb and cooled for a half hour until it hardened. The gel was then placed into the electrophoresis rig and 1X TBE was used to submerge the gel. TBE was poured to a level approximately 1mm above the gel surface. The gel was run at 100 volts for 60 minutes. Pictures were taken under ultraviolet light.
For liquid cultures, only four different looking colonies (yellow compared to white and raised compared to peripheral colonies) from the streak plates were selected. For each colony, 5 ÂµL of the appropriate antibiotic concentrate was added to a provided liquid culture of LB medium. The liquid cultures of broth were then incubated in the same way the plates were, however they were homogenized during incubation using a shaker.
Plasmid Isolation (Mini Prep):
To begin, the Qiagen DNA Purification System was followed. The liquid culture was centrifuged into a pellet by slowly adding more of the liquid culture into a tube, centrifuging the culture for 5 minutes at a time, and decanting the supernatant. 250ÂµL of cell re-suspension solution was added and the pellet was again suspended in the solution. For all gram-positive bacteria, 63ÂµL lysozyme was added. Next, 250ÂµL of cell lysis solution was added to each sample, and the sample was inverted several times to mix. 10ÂµL of alkaline protease solution was added to the sample. The solution was then inverted several times to mix again. The solutions were let to incubate at room temperature for 5 minutes. After that, 350ÂµL of neutralization solution was added and the solution was once again inverted several times. This solution was centrifuged for ten minutes.
Each solution was inserted into a spin column and placed in a collection tube. These tubes were then centrifuged for 1 minute and decanted. The extra fluid was poured out and then 750ÂµL of wash solution was added. The new solution was then centrifuged for 1 minute. This step was repeated with 250ÂµL of wash solution. The dry spin column was transferred into a new microcentrifuge tube. Then, 50ÂµL of nuclease-free water was added through the spin column and was centrifuged for 1 minute. The DNA was stored in the microcentrifuge tube at -20Â°C.
The DNA solution was pipetted into a 1X TBE agarose gel was run using the four DNA plasmid solutions and a control of known plasmid content as a ladder. Ultraviolet pictures were taken to document the relation between the DNA in the bacterial colonies of interest and the known DNA. A control solution (from the Blue culture provided in the lab) of previously prepared miniprep DNA was also used in the electrophoresis run to ensure the miniprep was completed successfully.
Gram Identification-KOH test:
Colonies from the stored Antibiotic patch plates were tested for Gram identity. First, an inoculating loop was used to transfer a colony of bacteria from each plate onto a slide. Then, KOH solution was added to the bacteria smear and stirred with the sterilized loop. If the bacterial cell lyses and the solution became sticky, the bacteria were identified as Gram negative. If the solution remained highly viscous, the bacteria were identified as Gram positive.
Gram identity was again determined using Gram staining on the colonies from the environmental patch plates. First, an inoculating loop was used to sample a colony from each Antibiotic patch plate. Each colony was stirred into its own Eppendorf tube filled with 1 ml of dH2O. The solution was Vortexed to homogenize. Then, approximately 5 Î¼l of bacteria solution was pipetted onto a slide and allowed to dry. The slide was briefly held over a Bunsen burner to affix the bacteria. The slide was flooded with crystal violet and was let to stand for 60 seconds, then rinsed with dH2O for 5 seconds. Following, the slide was flooded with iodine solution and sat for 60 seconds, then was rinsed with dH2O for 5 seconds. Ethanol was added drop-wise to the slide as a de-colorizer until the majority of previously violet-stained bacteria turned clear, then the slide was rinsed with dH2O for 5 seconds. The slide was flooded with the counter-stain, safranin, for about 60 seconds and rinsed again with dH2O for 5 seconds. The slide was blot-dried with a Kimwipe and observed under a microscope. Immersion oil was used to view the specimens under higher magnifications (40x and 100x).
MacConkey Agar Plates
MacConkey agar plates were used as another tool for determining Gram Identity. The four target bacteria strains were streaked onto MacConkey Agar plates provided in lab. Growth on the plates would indicate Gram Negativity.
The restriction digest of the bacterial plasmids was used to map the plasmid DNA. Plasmid DNA was cut using restriction enzymes Pst I and Bam HI. The DNA was then analyzed virtually using NEB Cutter to develop a basic understanding of where the plasmids would be cut. For the Restriction digest, 10 ÂµL of DNA from the plasmid isolation miniprep, 1 ÂµL of each restriction enzyme, 2 ÂµL of NEB buffer 3, and 7 ÂµL of water were added to a 1mL tube. The digest tube was then incubated at 37Â°C for 18 hours.
Transformation of antibiotic resistant plasmids from environmental bacteria to competent E. coli was completed with a provided transformation procedure outside of the lab manual. 50 ÂµL of competent cells were thawed on ice. 22 ÂµL of these cells were then transferred into a new sterile microfuge tube. 5 ÂµL of plasmid DNA from the DNA miniprep were then added to the new sterile tube and mixed with the competent bacteria. A plasmid free control was made using 5 ÂµL of H2O and another 22ÂµL bacterial tube. For a positive control, 5 ÂµL of pLITMUS28i was added to the same volume of competent E. coli cells. The tubes were then set to rest on ice for 30 minutes before heat-shocking them for 30 seconds at 42Â°C in a warm water bath. The vials were then replaced on ice for 2 minutes before 250 ÂµL of warmed SOC medium (at 37Â°C) was added to each vial. The vials were then incubated in a shaker at 37Â°C for an hour. Once incubated, the bacteria were then streaked onto pre-warmed LB plates containing the Kanomycin and Ampicillin antibiotics and plain LB plates using a "hockey stick" bacterial spreader. Plates were then incubated at 37Â°C overnight.
Polymerase Chain Reaction
For the polymerase chain reaction, 80 ÂµL nuclease free water, 10 ÂµL Thermopol buffer, 3 ÂµL dNTPs, 2 ÂµL of 11F and 2 ÂµL of 1492R primers were added to a master 1mL microfuge tube. 1 ÂµL of Taq polymerase was then added to the master cocktail with help from the lab professionals. This mixture was then separated into each of the four 200 ÂµL microtiter PCR tubes. Separately, 10 ÂµL SOC medium was mixed with a selected bacterial colony. This mixture was mixed thoroughly and 1 ÂµL was added to the target microtiter tube. Bacteria were taken from Male 2's hand streak 3, Male 1's foot streak 7, E. coli from the lab, and water was added to the last tube. The tubes were then put into the PCR thermal cycler where the DNA went through denaturation at 94Â°C, annealing at 50Â°C, and extension at 72Â°C. This process was repeated 35 times mechanically to ensure precision.
The bacterial colonies collected from environmental sampling of Male 1, Male 2, and the Female subject's hands were found to have varying resistance to antibiotics (Figure 1). In Male 1's antibiotic test, 50% of bacteria were resistant to Kanamycin (KAN), no resistance was found for Tetracycline (TET), and 31% of bacteria were resistant to Ampicillin (AMP). In Male 2's hand antibiotic test showed a 69% resistance to Kanomycin, 75% resistance to Tetracycline, and a 44% resistance to Ampicillin. The female subject's hand showed a 19% resistance to Kanomycin, 31% resistance to Tetracycline, and 25% resistance to Ampicillin. The resistance found on the foot of Male 1 was 19%, 100%, and 38% to Kanomycin, Tetracycline, and Ampicillin, respectively. For Male 2's foot, resistance was found to be 19%, 100%, and 6% to Kanomycin, Tetracycline, and Ampicillin, respectively. The female subject's foot bacteria was found to have resistances of 25%, 19%, and 75% to Kanomycin, Tetracycline, and Ampicillin, respectively (Figure 1)(Table 1).
One factor that can affect the resistance to specific antibiotics is the Gram identity of bacteria. One way used to test this was the MacConkey agar growth test. From the MacConkey agar plates, all of the bacteria samples were found to be Gram-positive because all the colonies failed to grow on the plates with MacConkey. The bacteria were also found not to ferment lactose because of the lack of growth and instead use peptone (Figure 2)(Table 2).
The first colony used for determining Gram identity was from Male 2's hand, colony 3. The next colony used was from the female subject's hand, colony 8. Next, colony 7 was used from Male 1's foot. The last colony used was colony 12 from Male 1's foot. All of these colonies were found to be Gram-Positive by means of the Gram Staining along with the results from the KOH test (Figure 3)(Table 2).
The positive Gram identity found was used to appropriately isolate plasmid DNA from the cells in the mini-prep. From the mini-prep DNA isolation, plasmids were isolated and viewed with an agarose gel next to a ladder (Figure 4). The plasmid isolated was from the blue control bacterial liquid culture. After this, the restriction digest was run on the successfully extracted plasmid. The restriction digest of the Blue plasmid showed two different DNA lengths under ultraviolet imaging of gel electrophoresis. From the environmental digest, only one length of DNA showed up under ultraviolet light (Figure 5).
Transformation of DNA plasmids from the environmental bacteria was a success as the E. coli receiving the bacterial plasmids through horizontal transmission were able to grow on antibiotic plates. The plasmid from the blue control group was successful, as the competent E. coli strain was able to grow a single colony on the Kanomycin antibiotic plate (Figure 6).
After the successful transformation of DNA plasmids, a specific region of the chromosomal DNA was replicated using a polymerase chain reaction. In the polymerase chain reaction, the target strand of DNA (the 16s Ribosomal DNA) was successfully replicated from the Female subject's hand and run on an agarose gel (Figure 7). This electrophoresis gel was photographed using ultraviolet light, making it possible to view the replicated DNA.
The Chi-Squared goodness of fit test comparing the bacteria from the hand and foot environments showed that the bacterial growth from the Ampicillin plates had a p-value of 0.84. From the Kanomycin plates, the environments had a p-value of 0.101, and the p-value from Tetracycline plates was found to be 0.0221.
The major finding of this study is the degree of antibiotic resistance found in the bacteria from the hands of subjects compared to the resistance found on the feet. One major finding was that the feet of the majority of subjects seemed to have a higher resistance to the Tetracycline compared to the rest of the antibiotics used in the study. In the hands of the subjects, there was a higher resistance found to the Kanomycin antibiotic than Ampicillin and Tetracycline. The aim of this study was to find the degree of antibiotic resistance in the hands compared to the feet of human subjects. It has been found that the bacteria found in these two different environments posses different resistances, which could suggest two different plasmids present among different colonies.
All of the bacteria in this study were found to be Gram positive, which means the peptidoglycan layer is on the outside of the membrane of the cell. The results of the KOH test parallel the results of the MacConkey and the Gram staining tests for the Gram identity. These Gram-positive bacteria's plasmids could be isolated and then transformed into E. coli cells. The E. coli transformation results showed that the plasmid from the Blue control bacterial plasmid was transformed successfully into the E. coli, as the transformed bacteria were found to grow (although in a limited amount) on the Kanomycin antibiotic plate.
In the restriction digest, only one length of DNA was found from the digest of environmental DNA, which is most likely because of only a single cut in the plasmid, or possibly no cuts at all. The gel did show a single length of DNA, so a plasmid was present in the digest, but anything further than determining it is a plasmid is impossible.
The Chi-Squared goodness of fit test showed the only definite difference between environments was found to be on the Tetracycline antibiotic plates. This p-value of 0.0221 was appreciably low, suggesting that more Tetracycline resistant bacteria are present on the feet of subjects compared to the hands. The results of the test on the Ampicillin plates showed a p-value of 0.84, so no significant difference could be determined. The Kanomycin plates suggested that there was a significant difference in resistance between the two environments as more bacterial samples from the hands of the subjects were resistant, but with a p-value of 0.101 the difference was not significant enough. Since each environmental bacteria has it's own resistance to different antibiotics, it is not safe to assume that the strains of bacteria present in one environment are generally more resistant. Concerning the basic idea in question, that is antibiotic resistance on hands and feet, there is no significant evidence that one environment gives raise to any more antibiotic resistant strains of bacteria. Each environment has bacteria resistant to different antibiotics, so concluding which environment produces more hazardous bacteria is impossible. The initial hypothesis of this study was that hands would produce a higher degree of antibiotic resistant bacteria, but this was found not to be the case. This seems significant in the argument to reduce the amount of antibiotics used to clean hands everyday (Levy, 1998). If just as many antibiotic resistant bacteria exist in places unexposed to antibiotics, there could be less of a correlation present than previously expected. The initial concern in antibiotic resistance is the development of fully antibiotic resistant bacteria that "defy all antibiotics" (Levy 1998). The development of such bacteria may not have anything to do with the use of antibiotics, as found in this study. The main weaknesses in this line of research are sample size and availability of resources. Too few test subjects were used to determine confident results and only a limited number of tests on bacteria could be done. With more subjects (and more bacterial subjects) to work with, a better conclusion could be drawn about the general plasmid-borne resistance in hands and feet of human subjects. Further research could likely be done on these bacteria that were found to determine the resistance of many more species of bacteria present. In later experiments it would be important to use a larger sample size and allow more cultures to grow for each portion of the study, which was a definite weakness in the experiment conducted.