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Antibiotic resistance (Abr) is a worldwide public health problem that was recognized soon after the discovery of antibiotics. Abr occurs when strains of bacteria in the human body become resistant to antibiotics due to improper use and abuse of antibiotics (American College of Physicians 2010). The effects that antibiotic resistant bacteria have on their environments can have detrimental results to living organisms' survival if there is no understanding of how the bacteria had acquired the antibiotic resistance (Levy et al. 2004). Once the acquired resistance is understood, this helps with future research in preventing or overcoming that resistance when it comes to harmful or unwanted bacteria.
Recently, it has been shown that the surfacing of antibiotic resistant (Abr) bacteria has weakened the effectiveness of many antibiotics that are used, in environments such as ours, to treat infectious diseases in humans and animals (Chander et al. 2007). Antibiotics are compounds that are produced by microorganisms such as bacteria or fungi and have inhibitory effects against other bacteria. The overuse and misuse of antibiotics in medicine are responsible for a rapid increase in the resistance of many pathogenic and non-pathogenic bacteria, which in turn makes in difficult and more expensive to treat infectious diseases, causing problems globally (Chander et al. 2007). To better understand how Abr bacteria came to existence, it is important to understand how antibiotics work in the bacterial cells to treat diseases and how the Abr bacteria have survived and adapted to the treatments (Andersson 2003).
The main purpose of this research was to characterize the antibiotic resistance of bacteria isolated from two different drains environments. a reverse osmosis water machine drain and a dormitory shower drain, on the Michigan State University campus, were tested for Abr bacteria. The experiment was done to investigate the pretense that the presence of Abr bacteria would be greater in one environment compared to the other environment. To investigate a step further, the Abr bacteria found in the 2 drain environments were isolated and characterized to give more insight on what different types of antibiotic resistant bacteria are present in these environments.
When dealing with Abr bacteria, it is important to undertstand how Abr bacteria emerged. Antibiotic resistance stems from a genetic mutation in a bacterium's genome which allows the bacterium to become unaffected by certain types of antibiotics (Neu 1992). Often, the bacterium's mutated resistance gene is located on the plasmid DNA, which is a circle of DNA located separately from the chromosomal DNA of the bacterium (Neu 1992). There are three different ways antibiotic resistant genes can be passed between bacterium including transformation, conjugation, and transduction (Chander et al. 2007). The passing of antibiotic resistance genes between bacteria is a concern not only because resistance can spread quickly through bacterial colonies, but it can also spread from non-pathogenic bacteria to pathogenic bacteria (Chander et al. 2007). As the non antibiotic resistant bacteria die off, the antibiotic resistant bacteria are able to prosper and multiply. With this understanding, research into the Abr bacteria present in drain environments is a good place to start because if antibiotic resistance is present and allowed to multiply, this could lead to emergence of new disease if gone unnoticed or cared for (Levy et al. 2004).
Plasmids have been show to play an important role in Abr because some Abr are considered to be plasmid-borne (Levin et al. 1997). Plasmid-borne simple means that the resistance gene is located on the plasmid. Plasmids in bacterial cells contain Abr genes that have been shown to follow the evolutions of group-beneficial traits, meaning the traits of that Abr gene will potentially benefit the whole population (Dugatkin et al. 2005). Group-beneficial traits act to preserve, or even increase, microbial genetic diversity by allowing a continuation of genotypes, which would be present in the plasmid, that would otherwise disappear under certain selective systems of treatment (Dugatkin et al. 2005). Yet the bacterial cell can still acquire resistance in the absence of plasmids through sequential mutations in their chromosome in the presence of an antibiotic (Chander et al. 2007). When searching for the cause of Abr it is important to take in to account all the possible ways the genes could have come about, but more importantly know where they can be found. Antibiotic treatment requires an extreme selection for bacteria carrying chromosomal- or plasmid-borne genes that code for resistance of Abr bacteria (Levin et al. 1997). It has been found that the Abr bacteria increase fitness to antibiotic treated environment can be attributed to an adaption by the bacterial chromosomal genes to the carriage of the plasmid (Levin et al. 1997).
Drain environments are all greatly effected by chemical product used for treating the drains (Levy 2000). Using household antibacterial products in cleaning surfaces and drains can increase the amount of resistance in the natural bacterial flora that are rinsed from the human body and resistance can then be passed to more harmful pathogenic bacteria in the water system (Chander et al. 2007). Knowingly this experiment also incorporates a water-containing environment, the drains. Water has constituted a way for Abr bacteria to spread widely among humans and animals (Baquero et al 2008). Research done previously found that water-containing environments contain environmental bacteria that act as an unlimited source of genes that when entered into a pathogenic organism like humans can become resistant genes (Baquero et al. 2008). The passing of antibiotic resistant plasmids from non-pathogenic bacteria to pathogenic bacteria through the use of a shower drain is a large contributor to the antibiotic resistance health scare. Testing for resistance in a shower and water fountain drain can lead to new ways to prevent resistance from spreading by targeting drain environment that can pose a heath threat (Chander et al. 2007).
Abr is studied in many different areas of human environment, including our area of study, drains, and many other experiments on antibiotic resistances in different types of foods. An experiment with similar hypothesis and procedures offers insight to our goals; in that experiment a study was done on antibiotic resistance in a specific type of sausage (Bacha et al. 2010). Combining the purpose of our research on drains, as well as other research done in all areas of our environment, helps improve our knowledge on antibiotic resistance and possible prevention techniques. Specifically to our experiment, in learning more about the antibiotic resistant bacteria present in various types of drains that humans come in contact with, we can hope to contribute to research based on the generation and spread of antibiotic resistant genes in common bacterial flora.
The two drains environments that were studied in this experiment are easily comparable because they are both a type of drain, but differ because of how the drains are used. For example, the bacteria present in a shower drain are exposed to human carried bacteria more readily than bacteria in the reverse osmosis drain. Furthermore, different treatment of the drains could cause differences in the levels of antibiotic resistant bacteria present in each. By studying the Abr bacteria present in the two environments, we can make conclusions about the types of environments Abr bacteria prefer and how they react to different treatment procedures.
In this experiment kanamycin, tetracycline and ampicillin are antibiotics that were used to investigate the presence of Abr bacteria in the bacterial samples taken from the two different environments. Whether or not they work is dependent on the type of antibiotic resistance, if any, in the bacterial colonies. The independent variables in the experiment are the bacteria obtained from the shower drain and the reverse osmosis water machine drain. In a similar experiment, ampicillin has been show to have little to no effect on the presence Abr bacteria especially on bacteria grown over shorter periods of time (Ito et al. 2009). In another experiment, using all three antibiotics that this experiment used, observed the frequency of Abr bacteria in a bacteria from an environment that had contact with humans, similar to the two environments that this experiment sampled from, and they found that resistance to ampicillin dominated, while resistance to tetracycline was next and resistance to kanamycin was poorest out of the three antibiotics used (Levin et al. 1997). This offers insight into understanding of the process of obtaining Abr, how it works, and which is more common.
For the experiment, it is hypothesized that the shower drain would have more antibiotic resistance bacteria than the reverse osmosis water machine because it has more contact with antibiotics and antiseptic cleaners used in the shower, creating an ideal environment for the antibiotic resistant bacteria to multiply and pass on their resistance genes (Chander et al. 2007). It was predicted that the shower drain samples would have a greater presence of bacteria on its antibiotic infused agar plates, based off the hypothesis that there is a larger presence of Abr bacteria, compared to the reverse osmosis water machine drain samples' antibiotic infused agar plates.
The focus of this experiment was aimed towards determining which of the two environments will have more Abr bacteria by treating the bacteria with three different antibiotics. Secondly, this experiment focused on characterizing different types of bacteria found in the shower drain and the reverse osmosis water machine drain by using a series of Gram test in order to find out as much as possible about the bacteria. Finally, this experiment aims focus on isolation of the bacteria found in the shower drain and the reversed osmosis water machine drain. This was done by performing a restriction digest and a polymerase chain reaction on the isolated plasmids from a mini-prep. And then running gel electrophoresis on all the products of those reactions for their results. This isolation of bacteria will give information on what types of bacterial strains are in those drain environment.
Â Â Â Â Â Â Â Â Â Â Throughout the duration of the lab sterile technique was used. Sterile technique includes washing hands before and after handling bacteria, wearing gloves when handling bacteria, sterilizing work areas with 70% ethanol solution before and after conducting experiments, and using a Bunsen burner to sterilize glass and metal instruments.
Â Â Â Â Â Â Â Â Â Â Two environments, a dorm shower drain and the drain of the reverse osmosis machine in East Holmes Hall, were sampled from to see if antibiotic bacteria were present. Using sterile swabs and sterile phosphate-buffered saline (PBS) three LB agar swab plates, LB agar plates were prepared by Lyman Briggs Lab (LBL) teaching staff, were created for each environment. After the plates were made they were incubated upside down at 37Â°C. After 24 hours the plates were covered in parafilm, labeled and placed in the refrigerator.
Creating Master Plates
Â Â Â Â Â Â Â Â Â Â LB agar plates were divided into 16 sections and each section was given a number (1-16) in order to keep track of separate colonies. Using a sterile loop, different colonies of bacteria were moved from our environmental plates to our master patch plates. Each colony was streaked into a numbered segment of the master plate. The plates were incubated at 37Â°C for 24 hours, removed from the incubator, covered with parafilm and put into the refrigerator.
Isolation of Antibiotic Resistant Bacteria
Â Â Â Â Â Â Â Â Â Â For each of the six master plates, four new plates LB plates were created, three of them containing antibiotics while one contained only LB. The three antibiotics used, each mixed in the LB on its own plate, and were ampicillin, tetracycline, and kanamycin.Â Â They were created in 600 mL amounts using 8.4 g agar, 12 g LB powder, 100Â Âµg/mL of ampicillin, 50Â Âµg/mL of tetracycline, and 50Â Âµg/mL of kanamycin for each respective set of antibiotic plates .The new plates were also sectioned (1-16) in order to known which colony that the new colony would have corresponded to on the original master plate. The antibiotic bacteria plates were made by taking the bacteria from the correct section on the master plate and using a sterile loop, the bacteria on the loop was touched to each corresponding section. The control LB plate, lacking any antibiotic, was always the last plate bacteria were added to. This insures the bacteria made it onto each of the plates, making sure there is no mistake in determining antibiotic resistance. The loop was then be sterilized again and that process was repeated for all section of the master grid that had colony growth. The plates were then incubated at 37Â°Â C for 24 hours.
Chi-squared test for independence
Â Â Â Â Â Â Â Â Â Â A statistical analysis of the independence of our variables, the number of resistant bacteria colonies on the antibiotic plate, and whether or not the antibiotic was present, was conducted using a chi-squared test. By comparing observed and expected values using the equation below, we found a chi-squared value at a significant p-value 0.05. If the p-value is less than 0.05, there is not a significant difference between the variables and they are dependent upon each other. Using a table to find the critical value, we found out if the chi-squared value is larger than the critical value. If the chi-squared value is larger than the critical value we know that the variables are independent. Equation for finding chi-squared (Ï‡2):
TheÂ VassarStats: Website for Statistical ComputationÂ was also utilized with the data to get Fisher Exact values were Chi-Squared Equation did not work (Lowry 2010).
Abr Streak Plates
Â Â Â Â Â Â Â Â Â Â From the ampicillin antibiotic plates, four Abr bacterial colonies were selected. Each colony was designated its own antibiotic plate. The ampicillin plates were divided into 3 sections, and a sterile loop was used to spread the Abr bacterial colony onto a third of the plate at a time, flaming the loop each time the bacteria were spread onto the plate. All four plates were incubated at 37Â°C for 24 hours and then each plate was parafilmed and refrigerated for later use of the bacteria grown on each plate.
Â Â Â Â Â Â Â Â Â Â Four 5 mL tubes with liquid medium were obtained; the lab directors made them. Then 5ÂµL of ampicillin was added to each of the liquid tubes and each tube was vortexed. We chose ampicillin because it had the most antibiotic resistant growth. With a sterilized loop, a single colony from an antibiotic steak plate was removed and then transferred to the liquid medium and mixed in. Then we capped the tubes loosely and placed them in the shaking incubator for 24 hours. We then applied this method using bacterial colonies from the four prepared ABr streak plates in separate tubes.
Differentiation of Gram-Negative and Gram Positive
Gram Stain:Â Â A bacterial solution was made using 90ÂµL of DI water, 5ÂµL of E. coli liquid culture, 5ÂµL unknown gram-positive solution, and a small colony ofÂ Staphylococcus aureusÂ bacteria.10ÂµL of the bacterial solution was applied to a glass slide. The bacterial solution was affixed to the slide using the flame of a Bunsen burner. The slides were flooded with three different dyes; each for 60 seconds and then each were rinsed with water for 5 seconds. The first dye used was crystal violet, the second was iodine solution and the third was safranin. Between the addition of the second and third dyes, ethanol was added sparingly for de-colorization.
KOH Test:Â 5Âµl of 3% KOH was added to a glass slide. Then a sterile loop full of bacterial cells from one colony was added to the KOH and stirred for about 1 minute. The consistency of the KOH and bacteria mixture was observed and recorded for results and their implications. This test was performed with a bacterial colony from all four Abr streak plate samples.
MacConkey's Agar:Â A MacConkey's Agar plate (LBL Staff) was streaked with a bacterial colony, from one of the Abr streak plates, using a sterile loop and the plate was incubated at 37Â°C for 24 hours. This test was performed with a bacterial colony from all four Abr streak plate samples on one MacConkey's Agar plate divided and labeled with the section that if was taken from off the Abr streak plate.
Eosin Methylene Blue (EMB) Agar:Â An EMB Agar plate was streaked with a bacterial colony from the Abr streak plates using a sterile loop and the plate was incubated at 37Â°C for 24 hours. This test was performed with a bacterial colony from all four Abr streak plate samples on two EMB Agar plates divided and labeled with the section that if was taken from off the Abr streak plate.
Performing a mini-prep
Â Â Â Â Â Â Â Â Â Â Liquid cultures from isolated bacterial cultures and two control samples were used to perform a mini-prep. The procedure provided by the WizardÂ® Plus SV Mini-preps DNA Purification System (Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711 USA) was followed. The bacterial cells from the liquid culture were lysated, Plasmid DNA was bond and washed and eluted in order to isolate them from the chromosomal DNA and stored at -20Â°C.
Â Â Â Â Â Â Â Â Â Â Gel Electrophoresis was used to determine if the DNA we extracted from our liquid cultures contained plasmids. 0.4 grams of agarose in 40 mL of TBE 1X was heated to dissolve. Once dissolved and cooled, 2Â ÂµL of Ethidium Bromide was added. This was then left to cool until the gel solidified and was placed into the gel electrophoresis apparatus. The first well of the gel was then loaded with 5mL of the 1kb ladder and the second was loaded with 10ÂµL of the gel-loading dye (New England Biolabs, 240 County Road, Ipswich, MA). The rest of the wells were then loaded with 2ÂµL of the gel-loading buffer and 10Â ÂµL of plasmid DNA. After conducting the electrophoresis at around 108 V for 45-60 min, the gel was viewed under UV light and photographed.
Restriction Digestion and Plasmid Maps
Â Â Â Â Â Â Â Â Â Â Using the NEB cutter V2.0 we identified the restriction site locations for the enzymes BamH1, HindIII, and PstI and produce a virtual restriction map.Â Â Began by taking 10mL of one plasmid DNA isolated from the mini-prep, and the plasmids of p-AMP, a control sample, and placed them in pipettes labeled by where the plasmids came from. Then to each of the pippettes 1mL of EcoR1 enzyme and 1mL of BamH1 were added (New England Biolabs ,240 County Road, Ipswich, MA). Then 2mL of buffer number 2 and 7Â mL of sterile water were added to both reactions in pipettes. The two pipettes were then placed in the incubator for 24 hours at 37Â°C. After that 24-hour period, 3.3mL of gel loading dye were added to both pipettes reactions. Agarose Gel Electrophoresis was performed, using the same procedure to make the gel, except the wells were then filled with the six reactions from the pipettes along with a ladder. pLITMUS28i, enzyme with known restriction digest reaction, was used as a control plasmid. The gel was then placed in the electrophoresis apparatus. After conducting the electrophoresis at around 108 V for 45-60 min, the gel was viewed under UV light and photographed.
Transformation of E. coli with plasmid DNA
Â Â Â Â Â Â Â Â Â Â Three sterile 1.5mL tubes were obtained and labeled as (+) plasmid and (-) plasmid and the last was labeled Script. 22mL of competent E. coli cells were added to each tube and put on ice. 3.3mL of p-AMP and PBlu-Script plasmid DNA were added to the (+) plasmid tube and Script tube, respectively, and was then pipeted repeatedly to ensure mixing. Then the tubes were returned to ice for 30 minutes. Created a (-) plasmid, a control, using 3.3mL of H2O in the (-) plasmid tube under the same procedure. While the tubes were incubating three LB plates and three LB/Amp plates were labeled with our group name and date. The cells were heat shocked by immersing the tubes directly into 42Â°C water bath for 90 seconds, and then returned to ice for 2 minutes. 250mL of pre-warmed (37Â°C) SOC medium was added to all three tubes. After the tubes were mixed and capped they were shaken horizontally at 37Â°C for 1 hour at 225rpm in a shaking incubator.Â Â 75mL of the cell suspension from each tube was pipetted on to the LB/Amp plates and another 75mL of the cell suspension from each tube was pipetted on to the LB only plates both using a "hockey stick" technique where the cell suspensions were spread over the surface of the plate using a sterile cell spreader and after allowing the plates to set for several minutes they were taped together and incubated at 37Â°C for 24 hours. While remaining transformation mixes were stored in a 4Â°C refrigerator.
Â Â Â Â Â Â Â Â Â Â The colonies on the plates were observed and photographed. The plates were then parafilmed and put into the refrigerator. The number of colonies on each plate were compared and contrasted in the following pairs: +LB and -LB, -LB/Amp and -LB, +LB/Amp and -LB/Amp, +LB/Amp and +LB.
Polymerase Chain Reaction
Â Â Â Â Â Â Â Â Â Â There were three polymerase chain reactions (PCR) that were conducted, one with pAMP, one with known E. coli control, and one with H2O control. A master cocktail of solution was made in a PCR tube using 80Â ÂµL nuclease-free H2O, 10Â ÂµL 10X PCR Buffer, 6Â ÂµL of 25 mM MgCl2, 16Â ÂµL of 1.25 mM dNTPs, 3Â ÂµL of 11F @ 10pmol/ÂµL, and 3Â ÂµL 529R @ 10pmol/ÂµL (Promega Corporation, 2800 Woods Hollow Road, Madison, WI USA and Integrated DNA Technologies, Inc., 6868 Nancy Ridge Drive, San Diego, California, USA). Then 30Â ÂµL of this cocktail was pipetted into three tubes, where the bacteria of the p-AMP, E. coli, andÂ Â ÂµL of H2O was added into the tubes separately with the ends of sterile tips. The PCR was then run for 3 hours, and the products were then used to run a gel electrophoresis on agarose gel. After the electrophoresis a picture of the gel was taken. A clean-up of the PCR product was done using a QIAquick PCR Purification Kit (QIAGEN Inc., 27220 Turnberry Lane Valencia, CA). 10ÂµL of eluted PCR product was used to run a agarose gel to assess the purified DNA. The created a sequence sample using 3ÂµL of 529R Primer, 7ÂµL of purified PCR Product and 2ÂµL of Nuclease-free water. Sequence was filled into a well of a sample plate in the lab and sent to MSU sequencing facility to be sequenced so that the sequence can be used for data.
The environmental swab plates had successful bacterial growth on all six plates made (Figure 1). Each plate had different levels of bacteria growth, with higher amounts of bacteria forming from bacteria taken from the shower drain environment (Figure 1). In all six of the master plates, the sectioned colonies had growth (Figure 2).
Upon isolation of the bacteria, the different strains of bacteria taken from the two different environments showed some antibiotic resistance. The water fountain drain environment bacteria had 39 out of 45 bacterial colony growths show antibiotic resistance to kanamycin, 45 out of 45 bacterial colony growths showed resistance to ampicillin, while no bacterial colonies showed any resistance to tetracycline (Tables 1). The bacteria from the shower drain environment had 40 out of 47 bacterial colony growths showed resistance to kanamycin, 44 out of 47 bacterial colony growths showed resistance to ampicillin, while, no bacterial colonies showed resistance to tetracycline (Tables 1).
A chi-squared statistical test was done, on the Abr bacteria colony count results for of each of the antibiotics used, to see if there was a significant association between where the bacteria came from, the shower drain or the reverse osmosis water machine drain, and whether or not the bacteria was Abr or not Abr bacteria. A Fisher's Exact Probability Test was done to calculate the p-values, using VassarStats, resulting in a two-tailed p-values of 1, 0.242117 and 1 for the kanamycin, ampicillin and tetracycline plates, respectively. For the ampicillin plates, the calculated observed Ï‡2 was 2.99488. For the tetracycline plates, the calculated observed Ï‡2 was 0 or undefined. And lastly for the kanamycin plates, the calculated observed Ï‡2 was 0.04614.
The antibiotic streak plates that were made using bacterial colonies from Abr bacterial colonies on the first and third shower drain ampicillin plates and the first and third reversed osmosis water fountain drain ampicillin plates. All streaked plates were successful indicated by the presence of single colonies on a portion of each of the plate (Figure 3).
The KOH Test was performed on the bacteria from the four created Abr streak plates. The sixth section from the first shower drain (SD1-6) bacteria was mixed into the KOH solution and the mixture remained fluid in consistency. The fourteenth section of the third shower drain (SD3-14) bacteria was also mixed into the KOH solution and the mixture was sticky in appearance when the solution was moved back and forth on the slide's surface with a loop. The test was performed on the twelfth section on the first reversed osmosis water fountain drain (RO1-12) bacteria and on the sixteenth section on the third reversed osmosis water fountain drain (RO3-16) bacteria, in both tests when the bacteria was mixed into the KOH solution and the mixtures were sticky in appearance when the solution was moved on the slide's surface with a loop (Table 2).
The results of the Gram Staining on bacteria from the four created Abr streak plates with the use of Crystal Violet chemical were viewed on microscope slides with a compound microscope at 1000x magnification (Table 2). The sixth Abr bacterial sample from the first shower drain sample Gram stain showed that the SD1-6 bacteria had bacilius bacterial shape with a streptococcus arrangement and the bacteria stained a strong violet purple color (Figure 4). The fourteenth Abr bacterial sample from the third shower drain sample Gram stain showed that the SD3-14 bacteria had coccus bacterial shape with a cluster arrangement and the bacteria stained a light purple-pink color (Figure 4). The twelfth Abr bacterial sample from the first reversed osmosis water fountain drain Gram stain showed that the RO1-12 bacteria had coccus bacterial shape with a cluster arrangement and the bacteria stained a reddish-pink color (Figure 4). And lastly, the sixteenth Abr bacterial sample from the third reversed osmosis water fountain drain Gram stain showed that the RO3-16 bacteria had coccus bacterial shape with a possible cluster arrangement and the bacteria stained a light pink-red color (Figure 4).
The SD1-6, SD3-14, RO1-12 and RO3-16 bacterial environments, all bacterial colonies grew on the MacConkey's agar plate and all of the colonies looked pinkish color (Figure 5). The MacConkey's agar also turned orange in color in comparison to color that the agar was before incubation.
The SD3-14, RO1-12, and RO3-16 bacteria grew on the Eosin Methylene Blue Agar plates and those colonies looked pink, milky appearance on the plates. In contrast, the SD1-6 bacterial colony's growth was considerably inhibited and was not pink in color (Figure 5).
The gel electrophoresis results came from the mini-prep performed on the isolated plasmids from the red Control 1, cultured by the lab's teaching staff, (C1), red Control 2, also cultured by the lab's teaching staff, (C2), SD1-6, SD3-14, RO1-12 and RO3-16 liquid cultures (Figure 6). The C1 and C2 had bands around 9,900 base pairs (bp) and around 7,900 bp. The SD1 and SD3 environments showed bands around 9,900 bp. And lastly the RO1 and RO3 environments showed bands around 9,800 bp(Figure 6).
The gel for the restriction digestion reactions performed on the red C1 and RO3-16 bacteria was unsuccessful. The agarose gel showed that that the EcoR1 and the Bam H1 enzymes failed to cut the plasmids from both the C1 and the RO3-16 samples (Figure 7).
The bacterial plasmids were present in the P-AMP bacteria and the PBLU-Script positive control showing that those plasmids were successfully transformed to "competent" E. coli cells signified by the growths of bacterial cell colonies on ampicillin treated plates and LB plates (Figure 8). The H2O control LB plate, the PBlu-Script LB plate and the p-AMP LB plate all had lawn colony growth. The H2O control ampicillin plate had no growth. The PBlu-Script and p-AMP ampicillin plates had single colony growth.
For the polymerase chain reaction, the gel contained reactions that were performed on plasmids from E. coli and RO3 bacteria and then H2O was used as a control (Figure 9). The all the polymerase reactions showed no bands on the gel.
Our experiment conducted many different tests to characterize the bacteria from the two environments. By identifying characteristics such as gram stain identity and morphology, one can make inferences on different types of bacterium and their antibiotic resistance. For the experiment, it was hypothesized that the shower drain would have more antibiotic resistance bacteria than the reverse osmosis water machine because it has more contact with antibiotics and antiseptic cleaners used in the shower, creating an ideal environment for the antibiotic resistant bacteria to multiply and pass on their resistance genes (Chander et al. 2007). The results, however, suggest that both environments contain Abr bacteria.
The water fountain drain environment bacteria contained antibiotic resistance to kanamycin, as well as resistance to ampicillin, while none showed any resistance to tetracycline. A similar thing occurred with the bacteria from the shower drain environment showed resistance to kanamycin, ampicillin, while, again, none showed any resistance to tetracycline. When looking at the main question in hand, whether or not one environment produces more or less Abr bacteria, we used a Chi-Squared test for independence. When analyzing the Chi-Squared test, the ampicillin plates gave an insignificant Ï‡2 value. In contrast to our hypothesis, this value is less than the critical value, deeming the results insignificant. This indicates that neither of the bacterial environments gave higher resistances to ampicillin. The kanamycin and tetracycline also gave insignificant Ï‡2 values through use of a Fisher's Exact Probability test, also showing the data to be insignificant, indicating that neither environment showed significantly higher levels of Abr bacteria. The tetracycline data were re-evaluated using a Fisher's Exact Probability test that resulted in an insignificant two-tailed p-value, further supporting the insignificance of the results. Overall, in the three different Chi-Squared tests for independence, our experiment failed to reject the null. Therefore our hypothesis that the shower drain would have more antibiotic resistance when compared to the reverse osmosis drain, failed to have significant results to support it.
Although the results failed to prove significant when discussing differences in the amount of Abr bacteria in the two different environments, the different tests conducted on the bacteria give some useful information on different characteristics of the bacteria. Four specific colonies, SD1-6, SD3-14, RO3-16, and RO1-12, two from each environment, were tested in four different ways to determine whether they were Gram-negative or Gram-positive. The purpose of the different tests is to offer information about the cell walls of the bacteria. If it is Gram-negative, it will have lipopolysaccharide layer in its cell wall, while Gram-positive will not. Using the KOH test it was determined that SD3-14, RO3-16, RO1-12, all showed the "sticky appearance", thus being Gram-negative. The SD1-6 colony however, did not display the sticky appearance when mixed with the KOH solution, therefore being Gram-positive. The sticky appearance is a result of the KOH destroying the lipid bilayer in the Gram-negative cells, thus resulting in the insides clumping in a stringy mess.
Another test, the Gram stain, was performed on the same colonies, giving the same results. The gram stain revealed the morphology of SD1-6 bacteria had bacilius bacterial shape with a streptococcus arrangement and the bacteria stained a strong violet purple color. The Gram-positive composition of the cell wall, lacking the lipopolysaccharide layer, results in its ability to retain the crystal violet dye, rather than the counter stain, safranin. The Gram stain showed that the SD3-14 bacteria had coccus bacterial shape with a cluster arrangement and the bacteria stained a light purple-pink color. The Gram stain also displayed that the RO1-12 bacteria had coccus bacterial shape with a cluster arrangement and the bacteria stained a reddish-pink color. Lastly, the Gram stain showed that the RO3-16 bacteria had coccus bacterial shape with a possible cluster arrangement and the bacteria stained a light pink-red color. All three colonies of these colonies did not retain the crystal violet dye because of their cell wall composition, resulting in a decision that they were Gram-negative.
The other two Gram-identity tests consisted of attempting to grow the bacteria on two different types of agar plates, Eosin Methylene Blue agar plates and MacConkyes Agar plates. The ability to grow on the plates indicates Gram-negative characteristic. If the bacteria do not contain the lipopolysaccharide layer, they will be unable to grow on the two different mediums. The Eosin Methylene Blue plates will also indicate whether or not the bacterial colonies ferment lactose. If the lactose is fermented, the dye will be absorbed and colored pink. The Eosin Methylene Blue agar plate test gave the same results as the other two; the same three colonies were Gram-negative, while one was Gram-positive. The three showed growth also displayed fermentation of lactose. In topic of the MacConkey agar plate tests, they gave all gram-negative results. And by the fact that the MacConky agar turned orange due to the presence of ammonium waste product that is the result of some of the bacteria being unable to ferment lactose. Overall, when looking at the results of the Gram-positive tests, you see the reasons for doing more than one test, also allowing for more knowledge about the bacteria. Information about the structure of the cell wall of these bacteria is known, and this allows for comparison to structure and antibiotic resistance, as to which bacterial shapes, arrangement, and Gram-identity will most likely produce Abr.
The gel electrophoresis results indicated that the performed mini-prep was successful in isolating plasmids from the red Control 1, red Control 2, SD1-6, SD3-14, RO1-12 and RO3-16 liquid cultures, due to the presence of bands. The bands represent, and are the types of plasmids that are present in the bacteria from each of the six liquids and the bands are compared to the ladder. Every location on the ladder represents a different sized plasmid or plasmid compound. The bands from the control are known so the plasmids of the bacteria became known because the DNA will show in the same location.
The bacterial plasmids were present in the P-AMP bacteria and the PBLU-Script positive control showing that those plasmids were successfully transformed to "competent" E. coli cells signified by the growths of bacterial cell colonies on ampicillin treated plates and LB plates. The transfer of the plasmid into the E. coli, allows the E. coli cells to grow on the ampicillin treated plates. Assuming a transformation is successful, if the antibacterial resistance genes were contained in the chromosomal DNA rather than the plasmid DNA, the transformed E. coli cells would not contain the Abr trait. Acknowledging this tells us that the bacterial resistance gene must be located in the plasmid DNA for the bacterial control sample that was used (Chander et al. 2007).
The experiment had a few problems with the transformations, the restriction digest, and the polymerase chain reactions. The transformation performed was originally intended to be done with the environmental plasmid DNA, however, in the end the only successful transformation done was with the controls with known ampicillin Abr. If the experiment on these environmental bacteria were re-performed, a transformation using the environmental DNA would allow us to conclude that the environmental Abr was actually plasmid borne.
The restriction digest performed on the environmental mini-prep plasmids was not successful because there were no bands present around 2137 bp, which would have indicated that the EcoR1 enzyme worked, and around 1017 bp, which would have indicated that the Bam H1 enzyme worked in the reaction. The restriction digest had technical problems, performed three times in which the plasmids were not being cut. The enzymes were too weak, or ill-prepared, resulting in no cutting of the plasmids. If the restriction digest had been successful, it would allow for insight on the size of the strand and the different places it would get cut. The restriction digest gives information on the composition of the plasmids, where the different enzymes are located, and where the different compounds are contained on the plasmid strand. If a more in depth study was performed on these environmental bacteria, it would be useful to perform numerous restriction digests. These would help in determining composition of the plasmids, along with possible location of the Abr resistance. The polymerase chain reactions also were also unsuccessful. The primers were suspected to be the reason for this malfunction, resulting in no magnification of the plasmid DNA. If the experiment were conducted again, the PCR, along with the restriction digest, would help with sequencing the environmental plasmids. This information would be especially useful in the determining the location of the Abr.
The results of the experiment, although somewhat insignificant when comparing each environment to each other, can be useful in creating awareness of the vast amounts of bacteria, even in places that have little antibiotic application, that are Abr bacteria. This information offers insight into the continuously growing knowledge of how bacteria have changed and are constantly changing. Whether the environment be the inside of a water drain, a shower drain, or a specific type of sausage, acknowledging that bacteria is growing more and more resistant, puts the world one step further towards predicting bacterial infection, manifestation, and possible pandemics (Bacha et al., 2010). The experiment overall was conducted on a small scale. It only gave information on two bacterial environments and three different antibiotics. This does not offer as much information as could be obtained if the same test were done with more starting variables. If research were to be done again or continued, there would hopefully be more variables in the experiment, along with the revisions stated earlier in the discussion. An example of a variable that would be useful to add would be the variety of antibiotics used. If a larger variety of antibiotics were used, there would be larger insight as to where different types of Abr occur. This would then lead to better identification of plasmid borne resistances, the location on the plasmid, so on and so forth. Overall the experiment gave useful information on the types of bacteria that develop in environments that people use everyday close to people. And the more research done on Abr resistance in bacteria within our society's environment can we start to find better way to prevent the emergence and presence of Abr resistant bacteria which will ultimately prevent the problem that are associated with their presence in our environment.