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The problem being addressed in this study is the resistant of antibiotic bacteria are becoming more prevalent in our society because of misuse and will have devastating consequences if nothing is done to stop the overuse of antibiotics. This was studied in depth by looking at two specific environments; the mouth of a canine and the mouth of a feline. The canine was hypothesized to have more antibiotic resistant bacteria because the dog was on antibiotics for an ear infection during the swabbing. Four samples were selected from our antibiotic patch plates. In order to characterize these bacteria multiple tests were performed; potassium hydroxide (KOH), MacConkey agar, Eosin Methylene Blue agar (EMB), Gram stain, restriction digest, transformation, and PCR. KOH test showed that the dog ampicillin (DAMP) and dog tetracycline (DTET) resistant strains were Gram negative, and cat tetracycline (CTET) and dog kanamycin (DKAN) were Gram positive. For the MacConkey agar, only DAMP grew indicating it as Gram negative; the other three showed no growth indicating Gram positive. The Gram stain test showed that DAMP and DTET were Gram negative, and CTET and DKAN were Gram positive. Restriction digestion showed that the cat was the only one that cut using the enzymes Pst1 and EcoR1. The dog had more antibiotic resistant bacteria which supported our hypothesis. None of our environmental antibiotic resistant bacteria grew when their plasmids were transferred to E. coli. The PCR showed that the canine had Staphylococcus and the feline had Citrobacter freundii. These results helped to show that the widespread use of antibiotics could lead to more antibiotic resistant bacteria.
Bacteria are microscopic prokaryotic organisms present in virtually all environments. Antibiotic resistant bacteria are becoming more prevalent in our society because misuse of antibiotics, which will have devastating consequences if nothing, is done to stop this overuse. Antibiotics are chemical agents that are used to kill or prevent bacterial growth; some of these include tetracycline, kanamycin, and ampicillin (Levy, 1998). Antibiotic resistance evolves through natural selection, this is acquired by applying evolutionary stress on a population, which will be discussed in more depth later (Wish, 2008). Bacteria have evolved ways to combat antibiotics, including the production of enzymes which are coded by genes that degrade antibiotics, or chemically alter and deactivate the antibiotics (Levy, 1998). Resistant genes are in the bacterial chromosome and more often plasmids of bacteria (Levy, 1998). Plasmids are small circular pieces of DNA that can be shared with other bacteria through conjugation (Levy, 1998).
In this experiment plasmids were isolated from the bacteria taken from the oral cavity of a canine and a feline. Scientists studied the addition of sewage sludge to farmland impacted the number of bacteria that were resistant to ampicillin (Kunberger, 2004). They picked three locations, one site that was treated with sludge, one that was upstream from the sludge treatment location, and one that was downstream from the treatment site (Kunberger, 2004). It was concluded that the runoff from the farmland was highly correlated with a higher resistant to ampicillin (Kunberger, 2004). This is significant because it showed that sludge and ampicillin resistance were correlated, so the antibiotic ampicillin is starting to become ineffective against bacteria. Ampicillin is a drug that is used to fight bacterial infections. This experiment was looking at only ampicillin while our experiment was looking at three different antibiotics including ampicillin, kanamycin, and tetracycline. The sludge allowed the bacteria to gain resistance to antibiotics, which is what we expect to see in the case of the canine which is on antibiotics.
In another experiment, Acinetobacter baumannii, which is a multi drug resistant bacterium, has been known to cause several infections, including pneumonia, urinary tract infections, wound infections, meningitis and bactermia (Nucleo et al., 2009). This was studied for 60 years in Italy by collecting samples from hospitals. Samples were collected and 69 of the 73 showed identical antibiotic resistance to fluoroquinolones, aminoglycocides and most beta-lactams, it remained susceptible to carbapenems, tetracycline, and ampicillin (Nucleo et al., 2009). Carbapenems must be monitored carefully or else bacteria will start to develop resistance to this family of antibiotics (Nucleo et al., 2009). This is evidence that a select number of bacteria are already resistant to most antibiotics that are currently in use and some that are already resistant to all known antibiotics.
We hypothesized that our cat will have a fewer number of resistant antibiotic bacteria but will have a greater number of bacteria. This was because the dog that we got our samples from was on antibiotics when we took the swabs while the cat was not. We believe that the cat will have more bacteria because dogs have a more hygienic mouths compared to cats.
Various tests identifying Gram identity were performed allowing us to gain basic knowledge about the bacteria allowing other tests to be performed, to determine if the antibiotic resistance was located on the plasmids of the bacteria being tested. These tests indicated that our bacterial samples from the canine and feline were Staphylococcus and Citrobacter freundii.
Bacteria were obtained from two environments, the oral cavities of both a domestic canine and feline. A sterile swab was dipped into phosphate-buffered saline (PBS) and then inserted into the oral cavity of the feline, and a sample was retrieved. The swab was then used to streak a Lysogeny broth (LB) agar plate, to grow bacteria from the cat's mouth. This procedure was repeated three times for the canine and feline, each time utilizing a new LB agar plate and sterile swab. The LB plates were then incubated for 24 hours at a temperature of 37 degrees Celsius. After the 24 hour time period they were removed from the incubator, sealed with parafilm, and placed into a refrigerator. Master patch plates were then created from our original four environmental swab plates. We had two master patch plates for each animal, for a total of four. We created a grid of 15 sections on our master patch plates. We then selected one bacterium from each of our four environmental patch plates to swab from our original patch plates onto each place of the grid of our master patch plate.
Once we had bacterial growth on our master patch plates we created our antibiotic plates. We had four plates, ampicillin, tetracycline, kanamycin, and LB which was our control. We then gridded out plates to match the numbers that we had on our master patch plates for each antibiotic and control plate. We then swabbed individual bacteria that grew on our master patch plates into all antibiotic plates as well as the control. The plates were then placed into an incubator for 24 hours at 37 degrees Celsius. One or two colonies were selected for each member of the group, yielding a total of eight new plates. Then we inoculated liquid LB media and antibiotic, and cultured these overnight.Â We then compared the number of bacteria colonies in both the AB patch plates and the LB patch plates to see how many bacteria are resistant to the three antibiotics we tested in both the feline and the canine patch plates.Â If the bacteria are resistant to the antibiotics they will appear on both the antibiotic plate and LB plate, which acted as the control.
The bacteria were also tested to determine if they are Gram positive or Gram negative.Â This was prepared by making a streak for each sample on a slide.Â The slide was then flooded with crystal violet for 30 seconds, and rinsed with cold water.Â The slide was flooded a second time with iodine for 1 minute and rinsed once again with cold water. Then ethanol was be added, acting as a decolorizer, and the slide was flushed with water.Â Finally the slide was flooded with safranin for one minute and then rinsed. MacConkey and EMB were done the exact same way using two different agar plates, one with MacConkey agar and the other EMB agar. Each sample was streaked onto the both plates and then incubated at 37 degrees Celsius. The KOH test was also used to determine the Gram identity based on the cell wall. One drop of 3% KOH was put on a glass slide along with a sample of our bacteria. The mixture would then be thickened Gram- or non thickened Gram+.
For detailed instructions of how to do mini-prep protocol used Promega Wizard SV mini-prep kit (Promega). An agarose gel electrophoresis was then run to separate nucleic DNA from plasmids based off their size and their electrical charge; both affect the rate of movement through the gel. The preparation of the gel is 1g of agarose and 100ml of TBE which is heated in a microwave until the solution becomes clear.Â This solution is then poured into the gel plate and allowed to cool.Â Then 17ml of dye and 83ml of water are added to the gel and loaded into wells.Â Each sample bacterial DNA, control DNA, and ladder were given its own individual well and dye was added which acted as a tracer for DNA. Ethidium bromide is a fluorescent dye used to stain the DNA, allowing the bands of DNA fragments to fluoresce beneath a UV light.
We digested our plasmids with three specific enzymes, BamHI, AvaII and EcoR1. These digestive enzymes cut our plasmids in two different ways depending on whether we were given p-ampicillin (pAMP) or p-kanamycin (pKAN). For pAMP the enzymes created six bands that can be seen with gel electrophoresis, while pKAN created four bands on our gel. Dye was added again in order to see DNA fragments under UV light.
Obtain two 50 Î¼L aliquots of cells from our environmental samples. Pipette 22 Î¼L of cells from each tube into a new sterile micro centrifuge tube. Added 1 Î¼L of plasmid DNA into CTET4, 1Î¼L to DKAN9 and 5Î¼L DAMP and DTET8 and then stirred (different amounts due to results plasmid mini-prep). A control was made by substituting water for plasmid DNA. Also make a positive control pLITMUS28i for environmental samples. Incubated tubes of cells for 30 minutes on ice and heat shocked cells for 45 minutes at 42 degrees Celsius without shaking. Put on ice for 2 minutes and added 250Î¼L of SOC medium added to each tube. Tubes shook for an hour at 225 rpm and 75Î¼L of each transformation was spread onto LB plates and corresponding antibiotic plates. Plates incubated at 37 degrees Celsius.
PCR and PCR clean-up
Master cocktail consisted of 80Î¼L of nuclease-free water, 10Î¼L thermopol buffer, 3Î¼l dNTPs, 2Î¼ of primer 11F, and 2Î¼L of 1492R. 1Î¼L of Taq polymerase and centrifuged for 2 seconds. This was split into three reaction tubes made of 30Î¼L of mixture. Our environmental sample added to a tube, E. coli added to one, and water added to one. Put into the thermo cycler, in thermo cycler underwent denaturation for minutes at 94 degrees Celsius. 35 cycles were completed of denaturation, annealing and extension (30 seconds 94 degrees Celsius, 30 seconds at 50 degrees Celsius, and 45 seconds at 72 degrees Celsius). After this, reaction undergoes final extension for 7 minutes at 72 degrees Celsius. Results were viewed by gel electrophoresis. The clean-up of the PCR was performed using a QIA quick PCR Purification Kit (QIAGEN). This was sent to the MSU sequencing facility to be processed.
In this experiment, 28 bacterial colonies from each environment were selected and placed on antibiotic plates (Fig 1). Environment 1, the canine, resulted in having 28, 24, and 12 bacterial colonies growing in the ampicillin, kanamycin, and tetracycline respectively. Environment 2, the feline, had 14, 9, and 6 bacterial colonies growing in the ampicillin, kanamycin, and tetracycline respectively. Our control grew on LB in all 30 plates for both environments. From these we picked bacteria that grew on our antibiotic patch plates and LB plates. DKAN9, DAMP1, DTET8, CTET4, DKAN4, and CTET11 were selected. CTET11 did not grow and was removed from the plasmid selection process and DKAN4 was rejected because of poor streaking, leaving DKAN9, DAMP1, DTET8 and CTET4 to be investigated further (Fig 2). A chi-squared test was performed on the abundance of antibiotic resistant bacteria between the two environment patch plates and was determined that there is a statistically significant difference between the two environments (Table 1).
MacConkey agar showed that we had growth on DAMP1 showing it is Gram-, while CTET4, DTET8 and DKAN9 showed no growth indicating Gram+. EMB showed that we did not have growth on DKAN9 indicating it as Gram+, CTET4, DTET8, and DAMP1 had growth indicating Gram- (Fig. 3). Our gram stains showed that DKAN9 and CTET4 were Gram positive, giving a purple color. DAMP1 and DTET8 were Gram negative, giving a pink color (Fig 4 & Table 2). Our KOH test also showed the same results, the Gram+, DKAN9 and CTET4 were not thickened while Gram-, DAMP1 and DTET8 were thickened.
From there, DKAN9, DAMP1, DTET8, CTET4 was prepared using the Wizard Plus SV Miniprep and put in an electrophoresis gel to visualize plasmids. All four samples had plasmids which were used for transformation into Escherichia coli (Fig 5).
A restriction digest using three specific enzymes BamHI, AvaII and EcoRI were used to cut the plasmids to make a plasmid restriction map. PAMP the plasmid for AMP was predicted to be cut in 6 pieces, while pKAN would be cut into 4 pieces. One control, "red" was cut into 6 pieces, suggesting it is pAMP, and the other control group, "blue," was inconclusive (Fig 6). The transformation resulted in no growth in environmental samples or the "blue" samples. However, there was growth in Red Amp1, Red Amp2, Red LB1, Red LB2, +Amp, and +LB (Fig 7).
Our results of our PCR clean up showed that we had Staphylococcus for DKAN9 and C. freundii in CTET4 (Fig 8).
Environment 1, the canine, had more antibiotic resistance than environment 2, the feline. We predicted that that the canine would have more antibiotic resistant bacteria because it was using antibiotics for an ear infection. The Chi squared test performed allowed us to reject our null hypothesis in that there was no significant difference between the amount of antibiotic resistant bacterial growth in environments 1 and 2. Since environment 1 was using antibiotics, the question posed is whether or not the canine had more antibiotic resistance than the feline, which, as the results show, is true, thus verifying our hypothesis. The environments chosen for this experiment were used because environment 1 is more likely to have antibiotic resistance while environment 2 was unknown. The results obtained in this experiment are similar to other findings since when antibiotics are present more antibiotic resistant bacteria are present. For example the A. baumannii that was studied in the Italian hospitals (Nucleo et al., 2009). Another study tested the runoff of sewage sludge to the addition of farmland which increased the number of bacteria resistant to ampicillin (Kunberger, 2004).
The environmental plates, master patch plates, antibiotic patch plates and antibiotic streak plates all showed bacterial growth. This means that both had antibiotic resistant bacteria, but the canine had a greater number of antibiotic resistant bacteria. Gram stain and KOH tests had consistent results, while both MacConkey agar and EMB showed different results. This could be a result of improper streaking methods. Mini-prep showed that plasmids were found in all 4 of the randomly selected antibiotic resistant colonies. These plasmids were tested to see if the antibiotic resistance is passed through conjugation or located on the chromosomal DNA. The restriction digest for our environments was an incomplete digest meaning that the results are inconclusive. The transformation was unsuccessful for our environmental bacteria, meaning that the antibiotic resistance is located on the chromosomal DNA or that the transformation protocol was performed incorrectly. The PCR results were Staphylococcus for DKAN9 and C. freundii CTET4. Staphylococci in most cases are harmless and reside in mucous membrane and on skin and common cause of food poisoning because it can grow when improperly stored. Its capable of growing both aerobically and anaerobically and can grow in the presence of bile salts, which allows it to grow in MacConkey agar even if it is Gram+ (Hurst). C. freundii are found almost everywhere including the human intestine, they are rarely the source of illnesses, except for urinary tract infections and sepsis. Strains have ampC genes encoding resistance to AMP; in addition it may be resistant to multiple antibiotics as a result of plasmid encoding genes (Oelschlaeger). The PCR cleanup showed that we had
To get more accurate results more samples should have been taken and the various tests could have been performed on all antibiotic resistant bacteria acquired. This would make the results more specific in determining exactly which type of bacteria will grow in this type of environment and whether or not the bacteria transfer their antibiotic resistance through conjugation. A test between two canines with one being on antibiotics and one not using antibiotics would help explain more about the type of antibiotic being used. Also more types of antibiotics could be tested. Overall, from this simple experiment one can see the effects of overuse of antibiotics could lead to mass antibiotic resistance and antibiotic use should be monitored more carefully to prevent this.
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Figure 1. Environmental swab plates from the oral cavities of a feline and a canine. The pictures above show the amount of growth of bacteria swabbed form the oral cavities of a canine and feline. For every environment three swabs were performed and streaked onto the plates above. Swab plates A-C are for dog environment, while D-F are cat environment. These were then incubated for 24 hours at 37 degrees Celsius. These two environments provided the antibiotic resistant bacteria used throughout the experiment.
Figure 2: Antibiotic streak plates. We streaked from our antibiotic plates onto these antibiotic streak plates. Bacteria shown above are resistant to the type of antibiotic used on its patch plate. We randomly selected these four antibiotic resistant bacteria to use for the rest of our experiment. The four plates shown here were randomly selected from our antibiotic growth plates. We then randomly selected individual bacterial colonies from each of these patch plates that were used later during the experiment.
Figure 3: MacConkey agar and EMB plate. In figure A, the bottom left portion of the MacConkey agar plate shown is DAMP1. This grew in the MacConkey agar because it is Gram- since Gram+ cells do not grow in the agar because of the presence of salts and crystal violet. The DAMP1 is pink in color due to the acid byproduct that is produced as they ferment lactose. In figure B, all of the 4 sections had growth except the bottom left which is DKAN9 this indicates that DKAN9 is Gram+ while the other 3 are Gram-.
Figure 4: Gram stains of environmental bacteria. A and D are Gram negative, meaning that these bacteria have a thin wall layer and the outer membrane stains pinkish red. C and B are Gram positive have a thicker wall layer but have no outer membrane and stain violet.
Figure 5: Mini-prep of our environmental plasmids. Lanes: 1: ladder, 2: dog tet 8, 3: blue 2, 4: dog amp 1, 5: red 2, 6: gel control p litmus28i, 7: ladder, 8: cat tet 4, 9: red 1, 10: dog kan 9, 11: blue 1. This showed that we did have plasmids in each of our environments that were in lanes 2, 4, 9, and 13. The cat tet 4 showed in this figure had the most plasmids and the dog tet 8 showed the least. These plasmids were then used throughout the rest of the experiment.
Figure 6: Restriction digest agarose gel. Lane 1 has red control, lane 2 has blue control and lane 3 is the ladder. Our restriction digest showed a complete digestion for our red control. This cut 6 times and is therefore p-amp. While blue is p-kan and was predicted to have been cut 4 times but it is difficult to see in the figure. The restriction enzymes used were BamHI, AvaII and EcoRI for restriction digest. Then put under UV light to see the dye that was added to each lane to help visualize plasmids.
Figure 7: Transformation plates. A-D had no growth, these were our environmental samples. In the Blue control our E and G grew which were our positive and negative controls. In the red group K, N, M and P grew. The growth on these plates indicates that the transformation was successful.
Figure 8: PCR clean up. Lanes 2 and 5 showed that we had a successful PCR clean up because you can see bands from our two environments, of DKAN and CTET. Lane 1, 1 kb ladder; Lane 2, Cat Tet 4; Lane 3, positive control (E. coli); Lane 4, negative control (Hâ‚‚O); Lane 5, Dog Kan 9; Lane 6, positive control (E. coli).
Table 1: Antibiotic resistant colony count and chi squared results.
Table 2: Gram stain results for dog kanamycin 9, dog tetracycline 8, dog ampicillin 1 and cat tetracycline 4.