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Characterization of antibiotic resistant bacteria in bathrooms vs. heavily trafficked areas using antibiotic agar plates, plasmid isolation procedures, and E. coli transformation
With bacteria becoming increasingly resistant to antibiotics, it is crucial to understand where higher concentrations of antibiotic resistant bacteria reside. This experiment attempted to determine whether bathrooms of Meijer, Burger King, and Déjà vu (ENVA) or the shelves at Meijer, the play-structure at Burger King, or the bar at Déjà vu (ENVB), are home to the highest concentration of antibiotic resistant bacteria. Due to their frequent cleanings and contact with humans, we predicted ENVA would contain more antibiotic resistant bacteria than ENVB. The experiment began by culturing environmental bacteria on LB media with ampicillin, kanamycin, and tetracycline incorporated separately with a control LB only group. A chi squared test failed to find a statistical difference in the number of antibiotic resistant bacteria between the two environments. A selection of antibiotic resistant bacteria from ENVA and ENVB were further characterized via standard procedures, including a Gram straining procedure, a KOH test, and EMB and MacConkey Agar selective media. An attempt was made to isolate plasmids from the environmental bacteria for transformation into E. coli. No environmental plasmids were extracted so a pKan control plasmid was used. The transformation of the pKan plasmid confirmed resistance to kanamycin. An attempt to identity of the environmental bacteria was made via the polymerase chain reaction (PCR) of a portion of the 16S rRNA gene; however, the PCR product was unable to be sequenced due to a failed clean-up procedure.
Antibiotic resistant bacteria have become increasingly more hazardous to the well being of the world's human population. This is due to the inability of modern medicine to keep up the ability of bacteria to evolve resistance to antibiotics through natural selection. Therefore, it is important to understand where antibiotic resistant bacteria tend to congregate. The purpose of this experiment was to determine whether areas of heavy human traffic at popular locales, such as a grocery store, a child's play area, and an adult entertainment facility (ENVB), or the bathrooms at these locales (ENVA) are reservoirs of antibiotic resistant bacteria. We predicted that the exposure of these places to antibiotics and frequent cleanings with antiseptics, we would find a greater number of antibiotic resistant bacteria in ENVA than in ENVB.
In modern society, antibiotics are commonly prescribed without medical evidence they are needed. Historically, antibiotics have been considered as a "cure all", and prescribed to anyone whom they could help, as well as those who could just not accept being sick without a cure (Campbell et al. 2008). Moreover, when given these antibiotics for true medical reasons, people often do not take the full dose of the prescription and stop treatment at a stage where the bacteria are not completely killed. The bacteria that are still alive are resistant to the antibiotics that they had been exposed to, and free of competition from non resistant strains, could then propagate (Campbell et al. 2008). Thus failure to complete the full round of antibiotic treatment selects for antibiotic resistance.
Resistance in bacteria to antibiotics is spread through bacterial populations via two main methods; vertical and horizontal transmission. Vertical transmission is the transmission of an antibiotic resistant gene from the parent cell to the daughter cells following binary fission. Because the DNA from the mother cell is replicated, all of the daughter cells that come from the original bacteria will contain the same antibiotic resistance as the parent cell if the resistance is chromosomally or plasmid-bourne (Campbell et al. 2008). The second method of antibiotic resistance dispersion is a process called conjugation. In one form of vertical transmission, conjugation, a donor bacterium transfers to a non-resistant bacteria a resistance gene bourne on a plasmid. If the plasmid is incorporated into the chromosomal DNA, the bacteria can become more resilient to antibiotic threats (Campbell et al. 2008).
High traffic locations, such as bathrooms and other places where people tend to travel, are constantly treated with antibiotics and disinfectants. The constant exposure to antibiotics has led to selection of resistant forms of the bacteria in these types of location (Campbell et al. 2008). A study was done showing that bacteria at a veterinary hospital, a medical hospital, and an animal nursery found that 93.9% of bacteria were resistant to tetracycline and 74.9% were resistant to ampicillin (Lu, 2009). These places are repeatedly cleaned and exposed to antibiotics, much like common bathrooms. An experiment performed by the Indian Institute of Science in 2009 demonstrates that increased antibiotic resistance in bacteria can occur in places that have high densities of the bacteria such as bathrooms and heavily trafficked areas. The results of this study suggest that heavy use of antibiotics in heavily trafficked areas selects for mobile, swarming bacteria that are able to evade exposure (Butler et al. 2009). This defense system is commonly found in bacteria that are found in areas that have higher concentrations of antibiotics (Butler et al. 2009).
Places like bathrooms, such as ENVA, are breeding grounds for high-density colonies of bacteria due to the high concentrations of antibiotics from waste and cleaning products (Haapapuro et al. 1997). Therefore, it is predicted that these types of bacteria are more likely to be resistant to the various forms of antibiotics as compared heavily trafficked areas (ENV B). A study also showed the harmful effects of feeding animal excrement to other livestock due to the adverse effects of antibiotics laced in stool due to overmedicated animal procedures. A study by Haapapuro et al. (1997) provided evidence that livestock excrement laced with antibiotics causes an increase in antibiotic resistant bacteria. The amount of antibiotics in human excrement poses a similar problem in places like bathrooms. In many cases, humans, like the animals, have taken in a large amount of antibiotics. These antibiotics are released with the excrement. Bathrooms could therefore house a sufficient amount of bacteria that has a resistance to antibiotics.
In addition, areas designed for children's entertainment or activity have been shown to be breeding grounds for bacteria, especially if not properly cleaned. Play area ball pits are a shining example of an area where there is a high amount of human activity and problematic cleaning conditions. A study was conducted where three random ball pits were chosen, two indoors and one outdoors. Samples were taken at the center of each pit and from four points equidistant of each other. The outcome of this experiment yielded disturbing results. Eleven different species of bacteria were discovered in at least one of the ball pits (Davis et al. 1999). Among these species was Acinetobacter Iwoffi, a bacteria associated with pneumonia, endocarditis, meningitis, skin and wound infections, peritonitis and urinary tract infections. The Acinetobacter Iwoffi was resistant to penicillin and ampicillin, as well as cephalotin and chloramphenicoi (Virella, 1997). The researchers found an antipsuedemonal resistant strain of Stentrophomonas (Walker, 1998), as well as the bacteria Yersinia entercolitica, which causes outbreaks of diarrhea, lymphadenopathy, pneumonia, and spontaneous abortions in animals. These bacteria came about due to the high number of children that play in it and the difficulty in cleaning these frequently used areas. A comparison can be drawn between this and other heavily trafficked areas such as a bar at an adult entertainment club, a shelf at the local grocery store, and especially the play-structure at the local fast food restaurant, which tends to host a similar cliental.
The experiment began by collecting bacteria from environments A and B of Déjà vu, Meijer, and Burger King respectively. The bacteria were then cultivated on an LB plate and then moved to antibiotic infused patch plates. Plasmids were then extracted from four of the bacteria cultivated and then transformed into E.coli. While extracting the plasmids, the Gram identity was found using a combination of the KOH test, the Gram staining procedure, and EMB and MacConkey Agar plates. Our results revealed that there is no significant difference in the antibiotic resistance of the bacteria of ENVA compared to ENVB.
Sample collection and plate preparation
Samples of the bathrooms of Meijer, Déjà vu, and Burger King were collected, as well as samples of the shelf, the bar, and the play structure respectively. This was carried out by taking a sterile swab saturated in sterile phosphate buffered saline (PBS). The swab was passed over the experimental surface several times, and then swabbed onto the entire surface of a Lysogeny broth (LB) plate. Plates were incubated for 24 hrs. At 37Â°C and then sealed with parafilm and refrigerated until further characterization.
Antibiotic patch plates and Chi squared test
Each of the six environmental samples (3 from ENVA, 3 from ENVB) into patch plates. Master plates were divided into sixteen cells, and sixteen separate colonies from each environmental swab plate were transferred, to a single cell on the patch plate. To insure the maintenance of the colonies, these plates were redone every one to two weeks. After incubating for 24 hrs. at 37Â°C, each cell of the six master patch plates were , one cell at a time, a kanamycin patch plate, a tetracycline patch plate and an ampicillin patch plate, and finally an LB patch plate to act as a control to make sure bacteria was transferred to all plates. These were incubated for 24 hrs. At 37Â°C. and then the frequency of antibiotic resistance of the bacteria from each environment was analyzed by counting the number of cells on each antibiotic patch plate that contained bacteria, then the totals for each of the antibiotics was tallied, and using a Chi-squared test was employed to compare the relative frequency of antibiotic resistance in the two environment types. In total, 36 plates were used. The Chi-squared calculations provided a P-value for each antibiotic, showing whether the difference in antibiotic resistance between the two environments was significant.
Gram identity testing and Gel electrophoresis
Week three began with the Gram stain, KOH test, and growth on selective media was employed to determine, and then confirm, the Gram identity of the antibiotic resistant bacteria from our environments. The protocol for gram staining is as follows: A colony of bacteria was diluted in water then smeared on a microscope slide. After heat fixing it, the slide is flooded with crystal violet for 60 seconds to stain the positive bacteria. After rinsing with water it will then be flooded with iodine to fix the stain for 60 seconds, then rinsed again with water and diluted with ethanol until no excess color shows. After rinsing again the slide is flooded with safranin to counter stain for gram-negative bacteria for 60 seconds and again rinsed with water. The slide will then be air-dried and if gram negative, then the bacteria would take on a more pink color while the gram positive will take on a more purple color. The slide is then immersed in oil and put under the microscope at 100x magnification to be viewed.
The KOH test was conducted to find out if the bacteria is gram positive or negative by using a different technique other than the Gram staining. Once the bacteria were put on the slide, it was immersed in KOH. If the bacteria become sticky and stringy then it we interpreted this as Gram negative and if it remains more watery, the bacteria were Gram positive.
During the fourth week of the experiment, plasmids were isolated from the enviromental samples. The samples after being streaked onto LB plates were converted to liquid cultures and incubated in the shaker for 24 hrs. at 37Â°C. After incubation, we used the procedure Promega WizardÂ® Plus SV Minipreps DNA Purification System to isolate plasmids from our bacteria. In brief, the protocol was as follows: 10 ml of the sample was placed in a centrifuge five minutes at 14000 rpm to remove the bacteria from the solution, then re-suspended in a resuspension solution. The bacteria were then lysed with cell lysis and alkaline protease solutions to remove the genetic material and proteins and to release the plasmid and chromosomal DNA from the cell. The sample was then centrifuged one minute at 14000 rpm to remove the unwanted cellular material (cellular membranes, chromosomes, etc.). The supernatant containing the plasmids was placed on a spin column, washed with wash solution and then eluted in 50 Î¼l of nuclease free water. The isolated plasmids are then run through a gel electrophoresis, and using the NEB cutter V2.0 program from New England Biolabs, a plasmid map can be produced.
Agarose gel electrophoresis was used to separate DNA strands into bands based on their length. The gel is made out of highly purified agarose, which is cooled and forms a matrix. To make the gel, 40ml of the TBE is mixed with 0.4g of agarose and then cooled. Following that, 2 ïL of ethidium bromide is added. If done properly, the DNA will run from the negative end of the cell to the positive end and form columns of bands that were compared to a NEB 1Kb ladder to determine how many base pairs long the DNA fragment shown by each band is.
Isolated plasmids from the resistant strains of bacteria from the environmental samples were transformed into chemically competent E. coli cells. Three samples, two containing plasmids and one with PBS as a control were heat shocked for ninety seconds, mixed with warm LB broth, and incubated at 37 degrees Celsius for one hour in the shaker, at 225 rpm. The samples were then spread onto an LB+antibiotic plate to determine if the antibiotic resistance in the environmental bacteria was plasmid-bourne. An LB only plate was used as a control (growth on the LB only plate ensures that growth was possible if no growth occurred on the antibiotic plate).
Restriction digest of the plasmids
A restriction digest was performed to characterize the plasmids isolated from our bacteria. The restriction enzymes PSTI and PVUII from New England Biolabs in Massachusetts were used for the digest. For a 20ïL reaction, a microfuge tube was filled with 7 ïL of distilled water, one ïL of each restriction enzyme to be used, and 2 ïL of the buffer that maximizes the restriction enzyme's activity (for this experiment, buffer number 3 was used). Ten micro liters of the isolated plasmid DNA to be sampled were then placed in the solution, which was then incubated for 24 hrs at 37Â°C. After incubation, 10 ïL of the restriction digest reaction was visualized via agarose gel electrophoresis through a gel. The size of the restriction fragments was determined via comparison to a 1kb DNA standard.
Polymerase chain reaction and clean up
A polymerase chain reaction, or PCR was performed in order to amplify copies of a 1500 bp portion of the 16S rRNA gene. To begin, 55ïL of nuclease free water is placed in a microfuge tube. 10ïL of 10X PCR buffer and 6 ïL 50 mM MgCl2 were added to a 0.2 ml tube, followed by 16 ïL of 1.25 mM dNTPs. Finally, 3ïL of 11F and 1492R primers, and 1 ïL of Taq DNA polymerase were added. This cocktail was kept on ice throughout the experiment. Once the cocktail was complete, 22 ïL of the master mixture was placed into four PCR tubes. Two different environmental samples were diluted in SOC media and transferred by pipette, each to one of the tubes. The final two tubes contained each a negative (5ïL of water), and a positive (E. coli) PCR control. The PCR reaction proceeded for 35 cycles of denaturation (94Â°C, 5 min initially, then 30 seconds for each cycle), annealing (50Â°C, 30 seconds) and extension (72Â°C, 30 seconds) along with a final extension cycle at 72Â°C for seven minutes. After the cycles were complete, the PCR products were visualized on an agarose gel.
PCR reactions were cleaned using a Qiagen QIAquickÂ® PCR purification kit following the kit protocol. The cleaned PCR product was eluted in 50Î¼L of water. A 5Î¼L sample of the cleaned PCR product was examined via agarose electrophoresis.
Environmental Swab plates and Antibiotic patch plates
The original swab plates showed a great deal of diversity in colony type, size, color and texture (Figure 1). The bathroom samples as well as the Meijer shelf showed a large lawn colonization pattern, with an off-white bacteria with a rough texture, while the Burger King play structure and the Déjà vu bar had isolated circular colonies with as smooth texture. The Burger King play structure was orange colored, while the Déjà vu bar had white colonies. The antibiotic streak plates worked as expected (figure 2), with all four strains of bacteria tested growing. The four strains tested were ampicillin resistant strains from the Déjà vu locations, and the Meijer bath, as well as a tetracycline resistant strain of the bacteria from the Burger King play structure.
Gram identity testing
The plates from the Déjà vu Bathroom and Burger King Play structure grew on MacConkey agar, indicating that the bacteria were Gram negative, while the Meijer Bathroom and Déjà Vu bar did not grow indicating that they were Gram positive. Based on the color of the plate and the colonies, it became clear that the bacteria sampled from the Burger King Play structure cannot digest lactose but does digest peptone. The EMB agar however, did not support significant growth of these bacteria (Figure 3b), suggesting that the samples were actually gram positive. The Gram stain procedure was also used to test the Gram identity (Figure 4). The results showed that only the Déjà vu Bathroom was Gram Negative and the rest were Gram positive. All bacteria samples were cocci shaped except for the Déjà vu bar, which was streptobacilli shaped.
Gel electrophoresis of the DNA isolated from the environmental samples showed that no plasmids were extracted from the four bacterial samples previously listed (Figure 5). The positive control group however did contain plasmids. This gives concrete evidence that the procedure itself works and that there were no plasmids from the environmental samples, thus the antibiotic resistance of the environmental bacteria was likely not plasmid-bourne.
Restriction digest of the plasmids
Due to the lack of environmentally obtained plasmids, pKan plasmid was used in its place for the restriction digest and transformation procedures. The restriction digest was successful, showing four bands at 2000, 1100, 900, and 400 base pairs (Figure 6). The transformation showed that the positive control, the pLITMUS28i plasmid grew on the LB plate and the ampicillin plate, showing that the bacteria were capable of receiving plasmids (Figure 7a). The negative controls grew on the LB plates only (figure 7b), indicating a complete lack of antibiotic resistance prior to the introduction of plasmids. The transformation of the blue control plasmids into E. coli showed that the blue control was resistant to Kanamycin (figure 7c); however it proved to lack a resistance to Ampicillin.
Polymerase chain reaction and clean up
The polymerase chain reaction was only partially successful, with only one of the environmental samples working, the Burger King play structure. The Déjà vu bath and the E. coli control failed to yield results. The water control did not yield results, however this was expected, if anything had been visible in the negative control, it indicates contamination of the PCR. (Figure 8).
The Chi-squared results were obtained for each of the antibiotic treatments from the antibiotic patch plates for ampicillin, kanamycin, and tetracycline (Table 1). The Chi-squared values for these treatments were 0.31, 3.77, and 2.12 respectively. The P-values for these treatments were 0.48, 0.05, and 0.15, respectively. Based on these results, the null hypothesis was accepted and the original hypothesis was refuted.
After analyzing the data produced by the antibiotic patch plates of the kanamycin, tetracycline, and ampicillin, it was found that the p-values were greater than 0.05 for ampicillin, kanamycin, and tetracycline. Therefore, the null hypothesis, that there is no difference in antibiotic resistance between heavily trafficked areas (environment B) and bathrooms (environment A) of Meijer, Burger King, and Déjà vu, was accepted meaning that our original hypothesis was incorrect. The question being addressed in this experiment was whether bacteria samples found in bathrooms had more antibiotic resistance than samples found in areas where there is a high amount of human contact. The hypothesis of the overall experiment was that the samples found in bathrooms would contain more antibiotic resistance than the samples from the heavily trafficked areas due to the constant exposure to antibiotic infused excrement and the overuse of inadequately concentrated anti-septic and antibiotic soaps and cleaning agents. The null hypothesis being that there is no significant difference in the antibiotic resistances for the heavily trafficked areas and the bathroom samples.
The environmental samples, showing large amounts of growth throughout the plates, proved to have a large amount of genetic diversity, based on the diverse amount of coloration and colonization behaviors. They were marked by a multitude of types of visible consistencies and tints on the patch plates cultured later in the experiment. Based on the results of the KOH test, the Gram staining test and the MacConkey agar tests, it was found that the Déjà vu bath was Gram negative and metabolized lactose, The Burger King play structure was gram negative and digested peptone, and the Meijer bath and déjà vu bar were Gram positive. The Gram staining procedure, however, did contradict the results of the Burger King play structure, possibly due to misinterpretation or procedural error. The EMB plate further disagreed with the other tests and showed no significant growth, suggesting that the bacteria were all gram positive. This was most likely due to improperly prepared EMB plates Based on the color of the MacConkey's agar on the Burger King Play-structure plate, the bacteria digested peptone instead of the lactose. The significance of the metabolism tells us more about the bacteria's type and in what environments it can survive. During the mini prep of the environmental bacteria samples, we were unable to find any plasmids, suggesting that the resistance was not plasmid based. A pKan plasmid was used as a control to insure that the mini prep procedure worked. While the environmental samples did not yield plasmids, the pKan mini prep was successful. Because plasmids were obtained from the pKan, the experiment was continued with the pKan as our sample for the transformation and restriction digest procedure.
The restriction digest procedure was successful, with the gel showing faint but noticeable bands at the approximate lengths of the four predicted largest fragments (2000, 1100, 900, and 400 base pairs). Due to the small size of the fragments, the size of the gel, and the detail of the ladder, the smaller fragments did not show up on the image of the gel.
During the transformation of competent E. coli cells with the plasmid, the cultures spread on the kanamycin antibiotic plate and the LB plate showed growth, while the culture spread on the ampicillin plate showed no growth at all. Based on these results, it was accepted that the plasmid contained the gene for kanamycin resistance. In addition to the actual plasmid, the water (negative) control did not grow on either kanamycin or ampicillin, as expected, while still growing on the LB plate. Our positive controls with pLITMUS28i grew on both the ampicillin and LB plates.
The bacteria found in the bathrooms, a similar environment due to the frequent cleanings by antiseptics and constant exposure to antibiotics, found analogous results. The bacteria found in the bathroom was also resistant to ampicillin and tetracycline, as well as resistant to kanamycin. The results obtained in this experiment agree with the research previously mentioned. As previously stated, a study was done showing that bacteria found at a veterinary hospital, a medical hospital, and an animal nursery. It found that 93.9% of bacteria were found to be resistant to tetracycline and 74.9% were found resistant to ampicillin (Lu, 2009).
Moreover, it was found that the children's play area found at Burger King and the ball pit from the report previously mentioned shared similar results. As previously stated, a wide variety of bacteria were found inside the ball pit due to difficult cleaning conditions (Davis et al. 1999). The play-structure found at Burger King was also riddled with bacteria due to un-ideal cleaning conditions. The majority of these bacteria were antibiotic resistant and therefore maybe even more harmful than the samples found in the ball pit.
Although the experiment was an overall success, there were several errors along the way. In the very beginning, some of the antibiotic plates were not viable due to uneven amounts of antibiotics. This forced us to redo the original patch plates and recollect the data. The second major roadblock in the experiment came when there were no plasmids to be found in any of the environmental samples. This forced us to use the pKan bacteria instead of our own samples for the restriction digest and transformation procedures. In addition, the EMB plate procedure did not produce the expected results, possibly because of subjectivity on behalf of the experimenter, in the process skewing the Gram identity results. With differences in the mini prep procedures for Gram-positive and Gram-negative bacteria, the lack of adequate identity data may have resulted in the improper procedure being used, rendering the plasmids unobtainable. Due to this, we were unable to determine the vector of antibiotic resistance of the bacteria gathered in this fashion. Another issue of this experiment was when the PCR only yielded one successful sample out of the original four environmental samples. This meant that we would only be able to determine the species of one of the four environmental bacteria samples, taking away from the results of the experiment. The final error that occurred was when preparing the cleanup procedure for the only sample of bacteria that worked on the PCR, an Elution Buffer (EB) buffer was used instead of the nuclease free water as the final step in the procedure. To counteract this, the cleanup procedure was run again on the tainted PCR product, this time using the nuclease free water instead of the EB buffer. Unfortunately this did not correct the error, and the cleanup had failed, shown by the lack of banding in the gel electrophoresis of a sample from the cleanup procedure.
The significance of our research was to determine the hidden dangers of antibiotic resistance in locations where we often take safety for granted. Often times we are careless with where we allow our kids to play, or where we walk barefoot. As more and more antibiotics are used, bacteria that are already resistant to them will survive, causing the antibiotics to always be one step behind the natural selection of more resistant strains. Eventually many of the antibiotics that are currently prescribed without medical evidence will become ineffective and obsolete due to the overuse and over-exposure to overprescribed antibiotics.
If this research were to continue, it would be wise to determine how much the antibiotics found in waste effects the bacterial population in the common bathroom. If the results proved that the effect was minimal, it would give an explanation as to why there was no significant difference between the antibiotic bacterial population of the bathrooms and heavily trafficked areas. Moreover, it would be beneficial to determine how the antibiotic resistance was found in environment B if they were only cleaned with antiseptics as opposed to only exposed to antibiotics. The results could show a link between frequent cleanings with antiseptics to antibiotics. Moreover, it would give further evidence as to why there was no significant difference in antibiotic resistance between the two areas.