In this experiment, bacterial cultures were drawn from two different environments and compared to see if there was any difference in the amount of antibiotic resistant bacteria in each environment. The two environments chosen were the door handles of three bathrooms of West Holmes Hall and Intramural Sports East (IM East). We hypothesized that there would be more antibiotic resistant bacteria coming from IM East than that of Holmes because there is a greater number of people from more diverse locations that use the facilities at IM East, and thus there should be a greater diversity of bacteria contained in this area and a greater amount that would be resistant. The results of the chi-squared analysis were a p-value of 0.671 for tetracycline, 0.791 for ampicillin, and 0.823 for kanamycin. This suggests that there was no significant difference in the amount of antibiotic resistant bacteria between West Holmes Hall and IM East. This contradicts our hypothesis that there would be more bacteria in IM East. We chose to study one bacteria further because of its kanamycin resistance. This bacteria was from Holmes bathroom 2, #11 on the original streak plate. The strain appeared to be Gram-negative as a result of a Gram-stain, but Gram-positive with the KOH, EMB agar, and MacConkey agar tests. We attempted to isolate a plasmid from the bacteria to see if the resistance was as a result of genetic information on a plasmid but were not successful. This could mean that the resistance is coded in the bacterial chromosome, but it could also mean that we just were unable to isolate a plasmid from the bacteria. The results of the study show that there are more factors influencing the prevalence of antibiotic resistance in a community of bacteria than just the amount or diversity of people that use the facilities in that environment.
As a result of the over-administration and misuse of antibiotics and antiseptics, bacterial resistance to antibiotics is a growing problem. One of the leading causes of the spread of antibiotic resistance is due to the distribution of bacteria that contain resistance genes. Bacteria that contain resistance genes can give them to other bacteria in the population in a variety of ways, making potentially the whole population resistant to antibiotics. With their unresponsiveness to many types of drugs, antibiotic-resistant strains of bacteria present a serious hazard to human health and the public should be kept informed. Due to the considerable health risks presented by antibiotic-resistant bacteria, it is necessary to explore where the occurrence of resistant strains is greatest.
Antibiotic resistance is a growing problem for several reasons that are mostly related to the medical community overprescribing antibiotic medication and patients not using them properly. An example this is when antibiotics are prescribed to people that do not require them for the illness that they have, such as a viral infection. Not only will this not affect the sickness that they have, but it will also potentially allow for bacteria that are already living in their bodies that have the resistance to the drug and thrive. Also, when prescribed with an antibiotic properly some people do not heed the directions and only take some of the proper dose. Essentially what this does is kill some of the bacteria that are in their body but not enough for the immune system to finish off the remainder of the bacteria. This allows for the numbers of bacteria to rise once again, with an increase in the amount of bacteria that were resistant (Levy 1998).
Another problem contributing to the trend of increasing antibiotic resistant bacteria is the popular antiseptics hand sanitizers and soaps that have antibiotics right in the formula. Hand sanitizers and antiseptics kill the majority of bacteria that are on surfaces. However, the bacteria that do survive are generally those that are predisposed to surviving in such conditions and their competition for resources has been eliminated (Levy 1998). This allows them to reproduce and grow rapidly, eventually creating large amounts of bacteria that are also predisposed to resisting such actions against them. The excessive use of such products is breeding a whole new set of bacteria that are resistant to treatments that usually would kill them. A goal of this research is to find out where such bacteria are living in the world around us.
Another goal of the research is to explore the ways that bacteria actually resist the antibiotics. There are several ways that bacteria can inherit genes that code for the proteins making them resistant. First would be chromosomal inheritance, where generations of bacteria are naturally predisposed to having resistant characteristics and they pass these characters to their progeny. Another way that bacteria can give others this resistant gene is through additional genetic fragments called plasmids. Plasmids contain DNA that does not occur in the chromosome of the bacteria, and the plasmid DNA can directly give this information to others in the environment around it through a process called conjugation (Levy 1998).
Due to abundant moisture and the prevalence of mold and mildew, bathrooms are commonly thought of as some of the most bacteria-rich environments. A study done in Japanese households found that the amount of bacteria found in bathrooms was second in number only to those found in kitchens (Ojima et al. 2002). It is because of these findings that the inside door handles of two different bathrooms were swabbed for microbes. Then, the resistance and the variety of bacteria from each location were analyzed and compared. Testing for antibiotic resistance in different environments may provide information on where antibiotic resistant strains of bacteria are most likely to occur. Hopefully, this will allow a greater understanding of how antibiotic resistant strains of bacteria get into the environment in which they live.
Public bathrooms are places where a variety of different people enter each day, each with differing hygienic habits. The people that visit these restrooms bring a many bacteria with them. The most common places to find antibiotic resistant bacteria on a college campus, according to one study, were in cafeterias, bathrooms, and computers (Shanks, 2009). Bathrooms were an easy and accessible place to swab for this experiment, and this seemed like a reasonable place to search for antibiotic resistant strains of bacteria.
Although microbes thrive in dark moist places, they are not necessarily the prime location for bacteria; instead most bacteria are found on surfaces with high levels of tactile traffic (Ojima 2002). As one of the most touched surfaces in any building, outgoing bathroom door handles were our testing environments. The two environments tested in this study were public bathrooms in West Holmes Hall and public bathrooms in Intramural Sports East. Although a previous study found no significant difference between the number of antibiotic-resistant bacteria on surfaces in medical and domestic locations (Blouin, 1998), our hypothesis is that there will be more resistant bacteria from the handles at the Intramural fitness center than at West Holmes. Our hypothesis is based on the idea that the greater diversity of locations from which the visitors to the fitness center are coming means that there will be a greater diversity of bacteria brought in to the facilities by those people.
Methods
Written by: Curtis Abell and Kris Kutskill
Bacteria collection and pouring plates
Bacteria were collected by swabbing the door-handles of three different bathrooms in West Holmes Hall using a cotton swab dipped in phosphate-buffered saline. This solution allows for the bacteria to be transferred from one surface to another without killing them. Two of these were men's bathrooms and one was a women's bathroom, and combined these are group 1 of the research. Samples of bacteria were also collected from Intramural (IM) Sports East in the same fashion; two men's bathrooms and one women's bathroom for experimental group 2. The environmental samples were placed onto a LB agar plate and incubated at 37°C overnight.
Plates of agar medium were made by creating a solution containing 8g of bacto-agar, 12g LB powder, and 600mL of water. These solutions were autoclaved to ensure that they were not contaminated by any other microbes from the environment. Six of the plates were allowed to remain with just the LB agar and were used for patch plates, while three other groups of six plates had antibiotics added to them. The three antibiotics used were kanamycin, ampicillin, and tetracycline at a concentration of. Patch plates were made by streaking a single colony of bacteria onto an LB plate that had labeled with sixteen different coordinates on it. These colonies were chosen randomly from the original swab plate and patched on to these plates in one coordinate. These plates were then transferred into the incubator for 20 hours at 37°C.
Making Patch Plates
A total of 24 patch plates were made to determine antibiotic resistance in bacteria to one or more of the antibiotics ampicillin, kanamycin, and tetracycline. An LB agar only plate was also used as the control when streaking. The LB only plate was streaked last to ensure that bacteria had still been on the loop through the antibiotic plates prior to streaking the LB plate. The plates were marked as before with 16 quadrants matching the master plates. A sterile loop was then used to scrape up bacteria from the master plate and then streak each antibiotic plate and the LB only plate last without going back to the master plate or re-sterilizing the loop. The plates were patched corresponding to their appropriate numbers. For example quadrant one on the master plate was streaked on quadrant one for the antibiotic and LB only plates. Once all 24 plates were streaked, the plates were grouped according to environment, turned upside down, and incubated at 37oC for 24 hours.
Agarose gel electrophoresis
Rubber stoppers were placed on a gel caste that serves as the mold for the gel. A comb was put in the slots with the dots facing in to make the lanes in the gel. Then 0.4g ultrapure agarose and 40mL TBE were mixed in a 125mL Erlenmeyer flask. The mixture was microwaved for one minute. Once it cooled, 2µL of ethidium bromide (EtBr) was added and poured into the gel caste. The gel box was then filled half of the way with used TBE solution. The finished gel was then placed in the gel box with the TBE and the lanes are ready to be loaded. The gels were loaded with 5 µL of 1kb ladder for the control lane. The other lanes were loaded with 5 µL of 6x loading dye and 1 µL of the solution being tested. The gels were run under about 100 volts of electricity for about 30-45 minutes; until the ladder passed through two thirds of the gel.
Gram stain
For the Gram stain, a bacteria colony was spread into 50µL of water and then affixed with a flame onto a slide. The colony was treated sequentially with crystal violet, iodine, ethanol and safranin. The crystal violet dye was allowed to stand on the slide for about a minute before being rinsed. Then the iodine was added for a minute and rinsed with water again. Ethanol was applied until there was no more blue dye dripping off of the slide. Finally the safranin was added to the slide and was allowed to set on the slide for one minute and rinsed with water. Then the slide was observed through a compound microscope to designate whether the bacteria were Gram-positive or Gram-negative. If the colonies were pink, they would be considered Gram-negative. If the colonies appeared violet, they were recognized as Gram-positive.
KOH test
Five µL of 3% KOH solution were placed on a slide in a drop. Then a loop full of bacteria from environmental colonies was introduced to the solution and mixed for about a minute. Gram-negative cells will lyse and the solution thickens as it is pulled away from the slide. Gram-positive cells do not lyse and the solution containing the cells and KOH will not become sticky.
MacConkey Agar test and Eosin Methylene Blue (EMB) test
The MacConkey Agar test, environmental samples of bacteria were placed on MacConkey's Agar medium and placed in an incubator for 24 hours at 37°C. Only Gram-negative bacteria will grow on this medium. Environmental samples of bacteria were also placed on EMB agar and incubated at 37°C for 24 hours. Gram-negative bacteria generally grow on this agar exclusively.
Plasmid isolation and agarose gel electrophoresis analyzation
Positive Red control plasmid from E. coli bacteria was purified using Promega's Wizard Plus Miniprep kit and protocol. The environmental bacteria were harvested from the antibiotic streak plate and plasmid DNA was purified from the bacteria also using Promega's Wizard Plus Miniprep kit and protocol (Promega, 2009). The absorbance of the purified plasmid DNA was measured using a spectrophotometer. The number of base pairs in the plasmid DNA was determined by agarose gel electrophoresis when compared to a 1kb ladder solution. The values were compared to the ladder solution because the number of base pairs is known on the ladder as it goes through the agarose gel.
Restriction enzyme digestion
We intended to find out more information about our Red control plasmid by digesting it with restriction enzymes. We used a solution of 1µL of the restriction enzymes PST1 and Eco RV along with 2µL of buffer 3 and 7µL of nuclease-free water for our first test. The gel electrophoresis for this procedure is determined in figure 7. For the second restriction we used a solution similar to the first only with Eco RI and PST1 restriction enzymes instead of PST1 and Eco RV. The solutions were incubated for 20 hours at 37°C. After incubation the solutions were used in the lanes of an agarose gel electrophoresis.
Results
Written by: Kris Kutskill and Curtis Abell
Figure 1 shows the bacteria from both environments that were collected for further study. These plates were the original swab plates that were used to collect colonies to be tested for antibiotic resistance. The bacteria were counted from the plates in figure 2 to see how many of the colonies from each environment had antibiotic resistance and statistical analysis was done on it. Table 1 shows that there were less antibiotic resistant bacteria in the Intramural Sports East building bathrooms than those in West Holmes. The total number of bacteria in IM East was 44 and the total number of antibiotic resistant strains in West Holmes was 33 (table 1). However, a chi-squared analysis revealed that the difference between the two environments was insignificant. The p-value for kanamycin was 0.823, ampicillin was 0.791, and tetracycline was 0.671(table 1).
The results of the electrophoresis on the agarose gel are that no plasmids occurred in our environmental specimens (figure 3). However, figure 3 shows that the red control plasmid had a positive result. Figure 4 shows the result of a Gram-stain test on bacteria from Holmes bathroom 2 #11, and when compared with the control stain it appeared to be Gram-negative. However, the results of the KOH test, EMB agar test, and MacConkey agar test (figure 6) were negative, as in the bacteria in the test appeared to be Gram-positive (table 2).
The red plasmid contains the information for the ampicillin resistance gene, as shown by the results of a transformation test on E. coli in figure 5. The results of this test show growth in E. coli bacteria that previously were not antibiotic resistant in plates that contain the antibiotic ampicillin. The pLitmus28i was a positive control in this test, and water was used as a negative control. The negative control was to make sure that the solution was not contaminated and to see that the antibiotic in the agar was working. Bacteria added to the water control plates were not transformed, and thus they did not grow on the ampicillin plate.
The results of the restriction digestion of the Red control plasmid are inconclusive because the ladder in the sample ran faster than normal (figure 7). When compared to the control group in the agarose gel, it is clear that the digestion of the plasmid occurred in the second lane. This lane had the restriction enzymes EcoRV and PST I, and the bands indicated are similar to our prediction of there being three fragments with base pair lengths of 2860bp (base pairs), 1150bp, and 529bp. Lane 1 had a digest with enzymes Eco RI and PST 1 and had two fragments and had predicted base pair lengths of 1591 and 2984bp.
Discussion
Written by: Kris Kutskill and Curtis Abell
We found quite a few bacterial colonies on each of the door handles of the bathrooms which is consistent with the study done on Japanese households (Ojima et al. 2002). When we compared the amounts of resistant strains from each environment, found that there were more antibiotic resistant strains of bacteria in the bathrooms West Holmes Hall (moderate tactile traffic) when compared to the bathrooms in Intramural Sports East (high traffic). However, the results of the chi-squared analysis on each of the antibiotic types revealed that there was not a significant difference between the amounts of antibiotic resistant strains in each environment. This was because the p-values of each of the tests were above 0.5. These results are contrary to our original hypothesis that there would be more antibiotic resistant strains in IM East than in Holmes Hall because of the diversity of locations that people that use the facilities come from. This result could have several possible explanations; first it is possible that the facilities are cleaned more often at IM East than in Holmes Hall as a result of the knowledge that many people use the bathrooms each day. Additionally, the cleaning techniques could be more thorough at IM East when compared to those used at Holmes Hall. Finally, we may have underestimated the amount of people that use the bathrooms in Holmes. There actually could be more people using the bathrooms there than at IM East. As a result of the assumption that more people use the bathrooms at IM East from a greater diversity of locations, we could have overlooked the fact that many people could also use the bathrooms in Holmes outside of the homogeneous group contained within the residential college. In other words, many more people other than the people that live in the building of West Holmes could be using the bathrooms during the day.
Another potential problem with the study was sampling and plating. The original samples that were patched onto antibiotic plates were discarded after an unusual amount of bacteria was growing on them. All of the plates testing for antibiotic resistance had to be re-patched from our environmental patch plates; this could have altered the results. It is not clear whether the patch plates grew initially as a result of faulty plates or if the bacteria were resistant.
We chose to study one strain of antibiotic resistant bacteria that was obtained from the environment further. These bacteria were a strain found on Holmes bathroom 2, #11 on the original patch plate. These bacteria were studied further as a result of its resistance to kanamycin. The bacteria appeared to be Gram-negative as a result of the Gram-stain test, but seemed to show characteristics of Gram-positive bacteria under the KOH, EMB, and MacConkey agar tests. It is unclear whether this strain is Gram-negative or positive as a result of this contradiction. Colonies of Holmes 2 #11 were white in color on the original patch plates, and the colonies formed small circles with clear edges. Under magnification (during the Gram-stain test) the individual bacterium appeared to have cocci shape.
During the two different tries at isolating a plasmid using the mini-prep protocol and agarose gel electrophoresis, we were unable to isolate a plasmid from our environmental bacteria. This result could indicate that the gene for resistance to kanamycin is on the bacterial chromosome rather than on a plasmid. On the other hand, the plasmid that codes for the antibiotic resistance could have existed, but we were just unable to isolate it. If time would have permitted we could have continued to try to isolate a plasmid from the bacteria, but the amount of time allotted only allowed for two tests.
As a result of not being able to isolate an environmental plasmid, we chose to use the control Red plasmid for further use in restriction digestion and transformation. The results from the transformation test that was done on E. coli bacteria allowed us to infer which antibiotic the control Red plasmid coded. This information was deduced by looking at the control pLitmus28i plates in this test that were guaranteed to grow in the presence of ampicillin if the transformation was successful. The E. coli that were transformed with the Red plasmid were not previously resistant to ampicillin, as shown by the negative control with just water added to the cells, but they became resistant to the antibiotic after the transformation. Due to the fact that the bacteria became resistant after the transformation process, it was concluded that the Red plasmid contained the genetic information to give the E. coli bacteria resistance to the antibiotic ampicillin.
A key thing that was learned from this experiment is that just because a location is frequented by a more diverse group of people, it does not necessarily mean that the amount of antibiotic resistant strains of bacteria will be higher. The number of antibiotic resistant bacteria is influenced by many factors, and a place such as Holmes Hall that has the same people using the bathroom facilities could contain even more antibiotic resistant bacteria than a place that has many different people from a large variety of places. Antibiotic resistance could be created by a closely knit group of people as well as people from diverse locations.
Due to the conclusions drawn from this experiment, it would be interesting to see the results of a similar experiment done in a different but similar situation. It would be interesting to see if there is a greater variety of bacteria coming from the different environments. To understand the problem even better, it would be necessary to find out specifically which bacteria were coming from these environments and see where the most bacteria in these environments occurs. Bacteria live in other parts of the bathroom and not just on the door handles. The most common places to find bacteria in bathrooms are in the sinks or on toilet seats (Shanks, 2009). More bacteria may live in such areas of the bathroom and may give a more accurate representation of the bathroom environment.
References
Blouin, S. A. 1998. "A comparison of antibiotic resistance in bacteria found in medical and domestic environments." Bios. 69: 109-118.
Levy, S.B. 1998. "The Challenge of Antibiotic Resistance." Scientific American. March 1998. 46-53.
Ojima, M., Y. Toshima, E. Koya, K. Ara, H. Tokuda, S. Kawai, F. Kasuga, N. Ueda 2002. "Hygiene measures considering actual distributions of microorganisms in Japanese households." Journal of Applied Microbiology. 93(5): 800-809.
Promega Corporation. 2009. Wizard Plus SV Minipreps DNA Purification System. Retrieved (4/25/10), from Promega Web Site: http://www.promega.com/tbs/tb22 5/tb225
Shanks, C. R. and M. A. Peteroy-Kelly. 2009. "Analysis of Antimicrobial Resistance in
Bacteria Found at Various Sites on Surfaces in an Urban University." Bios. 80(3): 105-113.
Tables and Figures
A) B) C)
D) E) F)
Figure 1. Bacteria collected from three locations within two different environments. The bacteria were obtained from the door handles leaving each bathroom. A-C are a samples from three different West Holmes bathrooms (environmental group 1). The bacteria from environmental group 1 are representative of moderate tactile traffic. D-F are samples from three different bathrooms in IM Sports East (environmental group 2). Environment group 2 represents high tactile traffic.
a) IM East #2
b) Holmes #3
Figure 2. Antibiotic patch plates from environmental samples. The left column are control LB only plates, the second column are plates containing LB agar and kanamycin antibiotic, the third column contains plates with LB agar and tetracycline, and the fourth column contains plates with LB agar and ampicillin. Rows a, d, and e contain bacteria from environment 2 (IM East), and rows b, c, and f contain bacteria from environment 1 (Holmes Hall).
f) Holmes #2
e) IM East #3
d) IM East #1
c) Holmes #1
1 2 3 1 2 3 4 5 6 7 8
a) b)
Figure 3. Agarose gel with Red plasmid control and Environmental mini-prep results. a) An agarose gel with 1kb ladder in the left lane and Red plasmid control in lanes 2 and 3. b) Agarose gel made by electrophoresis with 1kb ladder in the left lane and red control plasmid in lane 2. Lanes 3 and 4 contained sample from environment Holmes bathroom 1 #1. Lanes 5 and 6 contained Holmes bathroom 2 #11, and lanes 7 and 8 had sample from IM East bathroom 1 #12.
a) b)
Figure 4. Determination of the Gram identity of environmental isolate Holmes 2 #11. a) Control Gram stain containing Gram positive and Gram negative bacteria. The Gram positive Staphylococcus aureus bacteria (stained purple) appear in the upper center of the photo, while the Gram negative E. coli (stained pink) appear around the periphery. b) Gram stain of bacteria strain #11 isolated from Holmes bathroom 2. Based on a comparison to the Gram positive and Gram negative controls, we concluded that Holmes 2 #11 was Gram negative. Cells from both a) and b) were observed with a compound microscope with the 100x objective (1000x magnification) under oil immersion.
3
2
1
4
7
6
5
Figure 5. Test of transformation utilizing control Red plasmid on E. coli bacteria.
These plates show a successful transformation of E. coli bacteria to have antibiotic resistance to ampicillin after transforming them with the control Red plasmid. Plate 4 is a plate that had the transformation, but is growing on an LB agar plate without any antibiotic. Testing growth of the E. coli without transformation (adding just water) was used to show that the antibiotic ampicillin was working (3 and 7). The pLITMUS28i plates serve as a positive control for transformation of the bacteria (5 and 6). We concluded from the positive pLITMUS28i test that the Red plasmid contains the genetic information for ampicillin resistance due to the growth of the E. coli on the plate 2 containing this antibiotic. The bacteria did not grow on kanamycin after the transformation (1), so we concluded that the plasmid did not code for kanamycin resistance.
Figure 5. These transformation plates show a successful transformation of E. coli bacteria to have antibiotic resistance to ampicillin after transforming them with the control Red plasmid.
a) b)
Figure 6. Eosin Methylene Blue(EMB) agar and MacConkey agar tests on Holmes 2 #11. a) The sample of bacteria from Holmes 2 #11 did not grow on the EMB agar plate after being incubated at 37°C for 24 hours. Bacteria that are Gram-negative grow on such agar, so this test would lead us to conclude that the bacteria are Gram-positive. b) Holmes 2 #11 was streaked onto a plate containing MacConkey agar and incubated at 37°C for 24 hours. The bacteria did not grow in this agar which would suggest a result of Gram-positive bacteria.
1 2 3 4
Figure 7. Restriction digestion of Red control plasmid using Eco RV, Eco RI, and PST 1 restriction enzymes. Lane 4 contains the 1kb ladder, and it ran extremely fast in this instance. It is unknown why it ran faster than usual. Lane 3 contains an undigested Red control plasmid. Lane 2 contains a Red control plasmid digested with EcoRV and PST I restriction enzymes. Lane 1 contains Red control plasmid digested with EcoRI and PST I restriction enzymes.
Table 1. Colony count of antibiotic resistant strains of bacteria
Holmes 1
Holmes 2
Holmes 3
IM East 1
IM East 2
IM East 3
Tetracycline
5
3
4
1
5
1
Ampicillin
2
6
3
6
3
0
Kanamycin
3
10
8
5
11
1
Total
10
19
15
12
19
10
X2 results:
Tetracycline: p-value = 0.671
Ampicillin: p-value = 0.791
Kanamycin: p-value = 0.823
Table 2) Results from four different Gram-identity tests on Holmes 2 #11
Gram Identity Test
Gram Stain
KOH Test
MacConkey's Agar
Eosin Methylene Blue (EMB) Agar
Identity Indicated
Negative
Positive
Positive
Positive
