The purpose of the experiment is to determine whether there are a higher number of antibiotic resistant bacteria in one environment compared to another. The selected environments were shoes worn in a dormitory dish room and shoes regularly worn not in the dormitory dish room. Since more chemicals and antibiotics are used in the dormitory dish room, which in turn selects for antibiotic resistance through horizontal and vertical gene transfer. We hypothesized that there would be more antibiotic resistant bacteria in the dormitory dish room environment than in the control. The antibiotic resistance of the bacteria in the selected environments was tested through bacterial growth on patch and streak plates, gram stain identification, bacterial transformation, and polymerase chain reactions. The results of the growth on the antibiotic plates showed a greater amount of resistant strains in the dormitory dish room environment compared to the control environment. Further tests indicated a statistically significant difference between the two environments (p=0.01). The chemicals and antibiotics used in the dormitory dish room environment may have been the cause of the greater prevalence of resistant strains due to general and broad use of the chemicals and antibiotics. Antibiotic resistance in food service facilities is especially dangerous because of the potential threat to infect a large number of people in a short time. Environments that employ similar bacteria management techniques may also be at risk of increased prevalence of antibiotic resistant bacteria, such as hospitals, health care facilities, or large scale food service facilities or restaurants.
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Antibiotic resistance occurs when an antibiotic is used on a population of bacteria and is mostly effective. Antibiotic resistance is formed from random mutation, and is then passed on via horizontal and vertical gene transfer. The gene that codes for the antibiotic resistance has can found to be on plasmids, chromosomal DNA, and on transposable elements. Plasmid-borne resistance means that the gene resides on the plasmid, which allows the insertion of the resistance gene into other bacteria (Campbell et al., 2008). The infection can spread rapidly in these environments due to little competition from other bacteria, allowing for rapid transference of the resistance.
The broad general use of antibacterial materials, using antibiotics when they are not needed, not using a large enough dose of antibiotics and not using the antibiotics completely even when no more symptoms are noticed, are all factors that contribute to the sharp rise of antibiotic resistant bacteria. The resistant bacterium increases the risk that already harmful bacteria, such as tuberculosis, would get the resistant DNA from harmless bacteria and become a dangerous threat. By studying the mechanisms for how antibiotic resistant bacteria emerge, scientists and researchers can determine ways to minimize the threat of a "super-bacteria" that is resistant to many different types of antibiotics and also minimize the risk of that resistance transferring to other bacteria (Levy, 1998).
An option presented by one source is to use older antibiotics that have not been used frequently because they were thought to be less effective or toxic to humans (Falagas 2008). These would be effective against strains of antibiotic resistant bacteria because the bacteria have not yet developed any resistance to the older antibiotics. These could be very valuable treatment options for strains that have developed many different types of resistances (Falagas and Grammatikos 2008).
Thousands of strains of MRSA obtained from various clinical samples in Kuwait over the course of ten years were tested to determine where the gene for antibiotic resistance was located, the bacteria's Gram identification, cultural characteristics, positive tube coagulase and DNase (Udo et al., 2006). The strains were streaked onto various antibiotic plates and incubated to specify which antibiotics they were resistant to. The plasmids were isolated and separated by gel electrophoresis and then again tested for antibiotic resistance, in order to see if any resistance loss occurred. All of the strains were found to contain the mecA gene for methicillin resistance, which was amplified using polymerase chain reactions. Many of the strains were susceptible to vancomyacin and teicloplanin, with reduced numbers of susceptibility during the later years of testing (Udo et al., 2006).
The experiment presented in the study of MRSA in Kuwait relates directly to Stream II in our lab because of the data collected and the methodology explained. Similar methods include the streaking of bacteria onto antibacterial plates, plasmid isolation and identification by gel electrophoresis. This article outlines the implications of finding out whether the resistance gene resides on a plasmid or in chromosomal DNA. Also, the article about older antibiotic use also explains how current antibiotic use in the world is the reason for so many strains of antibiotic resistant bacterium, which is general and broad use, and not taking prescription medications for the entire prescribed period of time.
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Antibiotics are classified into three different types in relation to how they attack and neutralize bacterium. There are protein synthesis inhibitors, cell wall synthesis inhibitors, and protein uptake inhibitors. Kanamycin and tetracycline are protein synthesis inhibitors, meaning they react with different subparts of the rRNA in the bacterium to stop the synthesis of the proteins that the bacterium needs to survive. Ampicillin, on the other hand is a cell wall inhibitor. It works by interacting with the transpeptidase in the cell wall. The ampicillin mimics the peptide bridge that exists between the two membranes that make up the cell wall of bacterium. This weakens the cell wall and makes it very easy to break the cell wall down and kill the bacteria. Even within these three main types there still lies a variety of different ways each species reacts to prevent antibiotics from working. For example, some bacteria simply lack the transport protein that allows the antibiotic to enter the cell (Bower and Daeschel, 1999). There is also a link between resistance to antibacterial or disinfectants and resistance to antibiotics. Antibacterial or disinfectants work by attacking multiple different sites in a bacterium with no definite process that destroys the cell (Hoff and Akin, 1986).
The aim of this experiment is to determine the differences between the bacterial samples found in two different environments. We hypothesized that the bacteria grown from the samples taken from the dish room shoes would have more antibiotic resistant bacteria on them due to the high and continuous use of a variety of strong chemicals. In the dish rooms, there are many chemicals used in a small space, making an ideal environment for antibiotic resistant bacterial growth. Different chemicals are used to soak silverware and coffee mugs, and those are both different chemicals than what is used in the dish machine itself. The only bacteria that can possibly survive are those that are resistant to all the chemicals and those bacteria would thrive because they also have such little competition. This environment is ideal for these bacteria to grow because it is warm and moist and the only competition would be with the other resistant bacteria.
Samples were taken from the shoes worn in the Holmes Hall dish room by three cafeteria workers. Each worker produced two samples, one from shoes worn in the dish room and one from shoes not worn in the dish room. These samples were collected using swabs that were then used to swab each shoe with one swab per sample. The swabs were then used to streak agar plates, each sample having its own agar plate. These agar plates were made with ampicillin, tetracycline, kanamycin and lysogeny-broth (LB) mixed into the agar. The LB plate was used as a control to ensure that there was actually some bacterium on the first three plates.
To study the antibiotic resistance of bacteria in the dormitory dish room versus resistant bacteria in other environments, bacterial samples were collected from the front toes of six separate shoes that had been exposed to the two different environments and grown on LB agar plates. A sterile swab was dipped into sterile phosphate-buffered saline (PBS), and wiped along the inside toe of the shoe. The swab was then used to inoculate a 100x15mm LB agar plate. The agar plates were incubated at 37o C for 24 hours.
The 6 original swab plates were marked with 16 spots. Then 6 LB agar plates were divided into 16 squares to create a patch plate for each of the swab plates. A metal loop was sterilized and a single spot on the swab plate was collected. Then a one-centimeter streak was placed in a section on a patch plate, and repeated for each of the 16 spots that were preselected on the swab plate. This procedure was done for each of the six swab plates to create 3 patch plates for each of the two environments. The plates were incubated at 37o C for 24 hours and any and all growth was photographed and recorded. The LB agar plates were then wrapped in Parafilm and stored at 4°C to prevent additional growth.
Antibiotic Resistance Tests
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Agar plates with Tetracycline (TET), Kanamyacin (KAN), and Ampicillin (AMP) were created using 2.4 ml, 1.2 ml, and 2.4 ml, respectively, in 600 ml of LB. The concentrations used for the antibiotics were 50 Î¼g/mL, 50 Î¼g/mL, and 100 Î¼g/mL for the TET, KAN, and AMP, respectively. For each of the six patch plates, a TET, KAN, AMP, and LB plate was prepared with a grid identical to the patch plates. A metal loop was sterilized to transfer a bacterial colony from one section of the patch plate and streak it on each of the antibiotic agar plates and finally on the LB plate, or the control (without re-sterilization), in the corresponding section. The plates were incubated at 37o C for 24 hours. Five strands were isolated for further characterization.
With a sterile loop, a colony of bacteria was added to 40 microliters of water. A drop of water was placed onto a slide, and the water slowly evaporated so that only the bacteria remained on the slide. The slide was flooded with crystal violet, iodine and safranin, rinsing with water in between each addition. The slide was blotted dry with a Kim wipe and viewed under the microscope at 100x objective lens under immersion oil. If the bacterium was dyed pink, then it was Gram-negative. If it was dyed violet, then it was Gram-positive (LBC Biology Staff 2010).
KOH Test/MacConkey Agar/EMB Agar
A drop of 3% potassium hydroxide was added to a bacteria sample. If the sample turned sticky, then the bacterium was considered to be Gram-negative. On the other hand, if the consistency did not change, it was considered to be Gram-positive. The bacterial colonies were also streaked on MacConkey Agar for further Gram stain identification. The plate was incubated for 24 hours and results were recorded. Similarly, bacterial colonies were streaked onto Eosin Methylene Blue (EMB) agar plates. Plates were incubated for 24 hours and results were recorded.
Liquid cultures containing single colonies from the streak plates were prepared and incubated for 24 hours. Using Wizard Plus SV Minipreps DNA Purification System, the centrifugation protocol was followed to collect plasmids. The liquid cultures prepared 24 hours previously were pelleted. Then the supernatant fluid was removed and the pellet resuspended. The cells were then lysed and neutralized. The product was centrifuged and the clear lysate containing plasmids was poured through a spin column. The plasmid columns were washed with wash solution and then diluted. An agarose gel for electrophoresis was created just as previously done in the lab. The agarose gel was then filled with DNA ladder and the plasmids, then run for 30-60 minutes at 100 Volts. After the time had elapsed, a picture of it was taken using the Kodak imaging system. In this picture the plasmids were compared to the DNA ladder to see the DNA the plasmid was composed of.
Restriction Enzyme DigestÂ Â Â Â Â Â Â
A restriction digest is used to cut the DNA at predetermined sites along the strand in order to isolate a plasmid. The digest solution was created using 1Î¼L of restriction enzyme, 10uL of DNA from the plasmid, 3uL of 10X NEBuffer depending on the enzyme, and 100ug/mL of 1X BSA. At this point, nuclease-free water was added so that the total solution was 30Î¼L. The solution was placed into an incubator at 37°C for an hour.Â Â Â Â Â Â Â Â Â Â
Transformation of E. coli
In order to transform the E. coli with a plasmid, 10 Î¼L of the plasmid was added to a tube containing 200 Î¼L of competent cells, 200 Î¼L of 50mM CaCl. The tubes were set on ice to lower their temperature for 10 minutes. To heat shock the cells, the tubes were placed in an incubator and then immediately cooled again with an addition of LB. The resulting transformed cells were plated on the chosen antibiotic and a control LB plate in the incubator for 12-24 hours to check for growth and a successful transformation.
The environmental swab plates grew bacteria that were swabbed directly from the environments. The plate CW had many different types of bacterial colonies compared to the other plates. AW and AO seemed to only grow single, small, and smooth colonies unlike the fuzzy colonies found on all of the other plates (Figure 1). The environmental patch plates yielded successful bacterial growth from the individual colonies on the environmental swab plates. Every selected colony that was streaked onto the patch plate grew, which allowed for easy colony identification (Figure 2). From these environmental patch plates, colonies were streaked onto antibiotic plates. The different antibiotic plates grew different colored and different textured bacteria. On plate A, there were opaque, beige colonies of bacteria, which looked similar to the opaque and yellow colonies on plate C. Plate E had large colonies on varying color while all of the colonies on plate B were thin and light in color (Figure 3).
The MacConkey agar and EMB plates showed full growth on all of the sections except for one section, which was BO KAN. The sections of no growth can be seen in the figure as sections 5 and 6 (Figure 4). The gel electrophoresis containing the plasmid isolates did not show any bands that would indicate the presence of plasmids. The only strong band that appeared was in lane 2, which was the positive control (Figure 6). The restriction digest contained strong, bright bands in lanes 3 and 5, which was the blue control and buffers PstI and EcoRV, respectively. Lane six contained more bands than 3 or 5, but they were much lighter (Figure 7). The transformation plate containing E. coli bacteria with resistance from pLitmus 28i showed growth of many individual colonies (Figure 8).
To determine whether there was a significant difference between the two environments, a Chi-Squared test was performed. A p-value of less than 0.05 implies the rejection the null hypothesis, which was that there was no significant difference between the numbers of antibiotic resistant strains between the environments. The data entered was from the number of colonies that grew well, somewhat well, and not at all on the different antibiotic plates (Table 1). Growth on the plates was classified by full resistance, partial resistance, or no resistance. Full growth was when there was an opaque, uniform, and visible growth on the plate, as seen in B2 (Figure 2). Partial resistance was awarded to colonies that exhibited minimal growth, which was when colonies were not opaque or had very thin colonies unlike the full growth colonies, such as in A2 (Figure 2). No resistance colonies were those that did not show any growth, such as in A1 (Figure 2). There was a significant difference between the two environments, including all of the antibiotics, with a p-value of 0.01. Both the ampicillin and kanamycin turned out to have a significant difference whereas the Tetracycline did not show a significant difference (Table 1).
Gram staining was also performed on six isolated strains of possibly resistant bacteria (Table 2). They all tested Gram negative except for CO, because they all held a pink stain instead of the violet stain that a Gram positive would have shown (Figure 5). The KOH test contradicted some of those results of the Gram stain. The KOH test supported the results of AO and LW being negative, but it contrasted with the other three samples (Figure 3). This test showed that CO was negative and AW in addition to LO was positive. A MacConkey agar was also used to test the bacterial strains due to the contradiction found by the first two tests. The MacConkey agar test completely supported the results of the Gram stain test (Figure 4). The six bacterial strains went through plasmid isolation testing and the only plasmids that were found were those of the control and the ladder (Figure 6).
Everyday, strains of antibiotic resistance bacteria are emerging all over the world. The tactics used in order to prevent and treat sickness provide the optimal environments for antibiotic resistance bacteria to emerge. By not completing prescriptions or not using strong enough medications that would completely wipe out the bacteria, only the resistant strains survive (Levy 1998). Essentially, humans are selecting for antibiotic resistant bacteria. In environments with a higher usage of antibiotics, there will also be a higher chance of more antibiotic resistant strains. This experiment is necessary in trying to find alternative ways to handle bacteria and disease today, and can help diminish the alarming rate of antibiotic resistant bacteria emergence.
Some antibiotic resistance was found on all of the antibiotic patch plates used. This means that the bacteria carrying the resistance gene can pass it along to other bacteria and endanger the humans are in the vicinity of the environments tested. Through horizontal and vertical gene transfer, entire environments can quickly acquire the resistance gene (Levy 1998). Bacterial strains that do not have any competition from other bacterial strains can grow very quickly since they have easy and constant access to all of the resources in an environment. Although it may seem contradictory, sometimes using an antibiotic or chemical may allow resistant strains to flourish. In the two environments, we do not think that all the bacteria except for the resistant strains survive, because there were strains of bacteria from the environmental patch plates that did not survive on the antibiotic patch plates (Figure 2). Since many of the strains of bacteria did survive on the antibiotic plates, the use of the antibiotics and chemicals in the dormitory dish room does seem to influence the prevalence of resistant strains of bacteria. Although the antibiotics and chemicals are necessary to maintain a clean dormitory dish room, different methods should be adopted to prevent antibiotic resistant bacteria. Stronger concentrations of the chemicals and antibiotics should be used in order to kill all strains of the bacteria and disinfect the dishes and silverware. Also, a variety of antibiotics should be used once in a while in order to kill any colonies of bacteria that have mutated into resistant strains.
The results support the hypothesis that there would be more resistant strains of bacteria in the shoes worn in the dormitory dish room than in shoes not worn there. The data indicates statistical significance (p=.01) in the difference between the two environments. Statistical significance can be assumed when p<0.05. A significant difference in antibiotic resistance was reported with the use of the antibiotics AMP and KAN in the two different environments. There was no significant difference in the TET resistance in the two environments. Although antibiotic resistance is randomly generated, environments with non-resistant strains that are killed allow for easy resistant strain growth. The chemicals used in the dormitory dish room, along with other environmental factors such as the high volume of people and food in a confined area, provided an environment with a high chance of antibiotic resistant bacteria to emerge for AMP and KAN (Bower and Daeschel 1999).
The Gram stain identification resulted in mostly Gram negative bacteria. Although the mechanisms of all of the antibiotics and chemicals used in the dormitory dish room were not studied, it can be assumed that they are able to kill Gram positive bacteria more easily.
The dormitory dish room is a highly sanitized area because the risk of an outbreak of disease is high due to all of the fresh foods, and the risk factor is very high because of the large number of people that work and eat in the cafeteria. Just as many strains of antibiotic resistance bacteria were found in the hospitals in Kuwait, more strains were found in the dormitory dish room compared to the control (Udo et al., 2006). Many of the chemicals used in the dormitory dish room and cafeteria are designed to kill the bacteria in many different ways, not through just one specific method. The purpose of this is to kill the bacteria even if it has acquired a mechanism of resistance. The problem with this approach is that bacteria that survive are going to have several mechanisms of defense (Falagas and Grammatikos 2008). If a strain of antibiotic resistant bacteria emerged in the dormitory dish room that was resistant to many of the generally used chemicals, it would be extremely dangerous. This was seen with two particular strains found in the experiment, as they were resistant to all three of the antibiotics used.
One of the problems that we found in the experiment was that although some bacteria grew on the antibiotic plates, plasmids were not able to be isolated. Furthermore, we observing antibiotic resistance on the plates, we believe that the some of the colonies of the bacteria may have been highly dense, which could have possibly mislead us into believing that the bacteria were antibiotic resistant, when in fact they were not. This is because some of the bacteria in the dense colony may have not been treated with antibiotic from the plate. Another problem in the first plating of the patch plates was that the concentrations of the antibiotics were too weak. This was the case with all of the antibiotics because almost all of the strains of bacteria grew on the plates.
If this research were to continue, more samples would need to be taken from different areas around the cafeteria and all of the mechanisms of the chemicals and antibiotics used in the cafeteria would be studied (Bower and Daeschel 1999). Knowing the mechanism that the chemical uses to kill certain bacteria, and the mechanism of resistance, is necessary to create an alternative mechanism. Also, different practices can be adopted so that the environment is not as suitable for antibiotic resistant bacteria. Another relevant test that could be performed is to test whether there is a difference between chemical resistance and antibiotic resistance in the strains of bacteria found in the environments.
Bower, C. K, M. A. Daeschel. 1999. Resistance responses of microorganisms in food environments. International Journal of Food Microbiology 50: 33-44.
Brumfit W. and Hamilton-Miller. 1989. Methicillin-resistant Staphylococcus aureus.
N Eng J Med 320:1188-1196
Campbell, N., Reece, J.B. 2008. Biology - 8th ed. Pearson Education, Upper Saddle River, NJ.
Falagas, ME, & Grammatikos, AP. (2008). Potential of old-generation antibiotics to address current need for new antibiotics. Expert Review of Anti-infective therapy 6(5), 592-600.
Hoff, J. C., E. W. Akin. 1986. Microbial Resistance to Disinfectants: Mechanisms and Significance. Environmental Health Perspectives 69: 7-13.
LBC Biology Staff. 2010. LB145 Spring 2010 Lab Manual, Allegra Print and Imaging, Saline, MI.
Levy, Stuart. "The Challenge of Antibiotic Resistance." Scientific American. (1998): 46-53.
Udo, E. and Al-Sweih, N. 2006. Antibacterial resistance and their genetic location in kjk MRSA isolated in Kuwait hospitals, 1994-2004. BMC Infectious Diseases, 6(168)
Figure 1. Environmental Swab Plates. A, B, and C are the master swab plates of the bacteria cultured from three individuals. F, D, E, and are the master swab plates of the bacteria cultured from the shoes not worn to work, but rather worn every day, which is environment B in this experiment whereas environment A is shoes worn to work in the dish room. The plates were used to both ensure that bacteria had actually been cultured and to use for the making of patch plates. The plates were incubated for 24 hours at 37Â° C.
Figure 2. Environmental Patch Plates. The main patch plates were made by taking small samples from the master plates at sixteen different points and making a small streak in a designated section on the patch plate. This was done for each of the six master plates. These patch plates were then used to make antibiotic patch plates. The plates were incubated for 24 hours at 37Â° C.
Figure 3. Antibiotic patch plates. A is the Ampicillin patch plate for B.W. B is the Kanamycin patch plate for B.O. C is the Ampicillin patch plate for C.O. D is the Kanamycin patch plate for A.W., and E is the Ampicillin patch plate for A.W. These plates show what was considered to be fully resistant, partially resistant and not resistant. Plate C is the best example of each of the three different classifications. C 10 is considered to be partially resistant, whereas C 14 would be completely resistant, and C 9 would be not resistant. The plates were incubated for 24 hours at 37Â° C. Each antibiotic plate was made by transferring the bacteria from the main patch plate to the new one; section one bacteria is moved to section one on the Ampicillin, Kanamycin, Tetracycline and LB only plates without going back to the original patch plate for more bacteria.
Figure 4. MacConkey Agar and EMB plates. The MacConkey agar is shown on the left and the EMB plate is shown on the right. Both the MacConkey agar and the EMB plate show whether bacteria are gram negative or positive. Both agars inhibit the growth of gram positive cells, therefore if any colonies grow, they are likely to be gram negative. Section 1 is AW Kan, 2 is BW Amp, 3 is CO Amp, 4 is AW Amp and 5 is BO Kan. All of these are on the MacConkey agar. Section 6 is BO Kan, 7 is AW Kan, 8 is AW Amp, 9 is BW Amp and 10 is CO Amp. These sections are on the EMB plates. All of the MacConkey sections had growth except for 5. The EMB showed growth on all sections except for 6. This means that they are all gram negative except for the two sections without growth.
Figure 5. Gram stain of environmental bacterial colony compared to control colonies. A is a picture of the gram stain done on A.W. bacteria that grew on the Kanamycin antibiotic plate. B is a picture of the control gram stain. C is of a gram stain performed on B.O. bacteria that grew on the Kanamycin antibiotic plate. D is a of the gram stain done on B.W. bacteria that grew on the Ampicillin antibiotic plate. E is of the gram stain performed on C.O. bacteria that grew on the Ampicillin antibiotic plate. F is the gram stain of A.W. bacteria grown on the Ampicillin antibiotic plate. The pink hues found in A, C, D and F indicates gram negative bacteria, whereas the purple hues found in B and E indicates gram positive bacteria. Gram negative bacteria is pink because the peptidoglycan layer is between two cell walls which stops the crystal violet dye from attaching and staining the bacteria purple like it does in gram positive bacteria, which has the peptidoglycan layer above the cell wall.
1 2 3 4 5 6 7
Figure 6. Plasmid Isolation by Gel Electrophoresis. Wells one and five contain the ladder which is used as a reference point for reading the results from the other samples run in the gel. The second well contains the positive control. Well three contains BO Kan bacteria. Well four contains AW Amp bacteria. Well six contains CO Amp bacteria. Well seven contains BW Amp bacteria and well eight contains AW Kan bacteria.
1 2 3 4 5 6 7 8
Figure 7. Restriction Digest Gel Electrophoresis. A restriction digest was run to determine the best combination of buffers to use for PCR. The second and fourth wells contain the environmental controls. Well three contains the blue control. Well five contains the buffers PSII and ECORV run with blue control bacteria. Well six contains blue control bacteria and the buffers BAMHI and PVUII. Well seven contains the buffers PSTI and ECORV with the environmental bacteria BW Amp. Well eight contains pLitmus 28i bacteria and the buffers PSTI and ECORV.
Figure 8. Transformation Plate. This transformation plate, done on pLitmus28, showed antibiotic resistance to Ampicillin.
Table 1. Chi Squared Test Results. A Chi squared test was performed on the results of the antibiotic patch plates.
Partially Ampicillin Resistant
Not Ampicillin Resistant
X2 = 8.75
p = 0.012
Reject Null Hypothesis
Partially Kanamycin Resistant
Not Kanamycin Resistant
X2 = 7.05
p = 0.0295
Reject Null Hypothesis
Partially Tetracycline Resistant
Not Tetracycline Resistant
X2 = 3.67
p = 0.1596
Fail to Reject Null Hypothesis
Partially Antibiotic Resistant
Not Antibiotic Resistant
X2 = 8.39
p = 0.0151
Reject Null Hypothesis
Table 2. Gram Stain Identification Test Results.
C Other Ampicillin 14
A Work Kanamycin 00
B Other Kanamycin 6
A Work Ampicillin 00
B Work Ampicillin 7