Antibiotic resistant bacteria from Free Range Organic Gallus Gallus and Tyson Gallus Gallus, characterized by gram identity tests and PCR show no difference in antibiotic resistance.
The purpose of this study was to determine differences in antibiotic resistance between Free Range Gallus Gallus and Tyson Gallus Gallus and then furthermore characterize and isolate bacteria from these Gallus Gallus's. Gallus Gallus (Chicken) as livestock have been fed antibiotics for a long time in order to produce the best and juiciest produce. There are however some Gallus Gallus that are not fed any antibiotics because of the dangers that antibiotic resistance in livestock presents to human consumers. The hypothesis of this experiment was that the chickens that have not been raised on antibiotics (Environment 2) are expected to have less antibiotic resistance than other chickens who have been raised on antibiotics (Environment 1) because previous exposure of the antibiotics leads to horizontal and vertical transmission of resistance. To determine if this was true, master patch plates of specific bacteria isolates were made and then transferred to master antibiotic patch plates of Ampicillin, Kanamycin, and Tetracycline. This resulted in p values of 0.8415, 0.0973, and 0.1648 corresponding to Ampicillin, Kanamycin and Tetracycline. Therefore this fails to prove that there is any difference of antibiotic resistance between Gallus Gallus that were fed antibiotics and ones that were not. Once this was done two isolates from the environments were further examined in order to characterize the bacteria that grew on the Gallus Gallus. Through running many tests like KOH, Gram-staining, MacConkey Agar streak plates, and Eosin Methylene Blue Agar streak plates it was determined that both 1.2 Tet patch 9 and 1.3 Kan patch 7 were gram-negative. Through restriction enzyme digest, transformations and PCR the bacteria isolate 1.3 Kanamycin was classified as Citrobacter Freundii.
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Recent anxiety has arisen regarding the increasing levels of antibiotic resistance in humans. Such antibiotic resistance is causing humans to become immunocompromised, with an inability to be treated for simple infections. Many link this problem to the overuse of antibiotics by humans, livestock, and agriculture (Levy, 1997). In past studies, drug use has generally led to the discovery of the increased use of antibiotics as being a main factor in drug resistance. The usage of antimicrobial agents as growth promoters or as treatment has influenced resistance levels in various bacteria (Costa et al., 2010). However, antibiotics not only treat the individuals themselves, but also treat the surrounding environment, affecting the society as a whole (Levy, 1997). This along with the abuse of antibiotics is what seems to be causing the new uproar in the health care system.
The over usage of antibiotics first became prevalent shortly after they were discovered. Their so-called miraculous effects made them popular and in high demand. Unfortunately, the same common antibiotics became used for humans, animals, and agriculture (Levy, 1997). This lack of variability amongst the antibiotics themselves may be a factor in the increasing rate of antibiotic resistance in the environment.
This new rise, however, has also given insight into the way mutant genes accounting for resistance travel amongst various species. It displays their ability for plasmids, extrachromosomal replicating genes, within the bacterial cells to transfer between different bacteria inhabiting many ecological niches. Gene transfer has no boundaries when it comes to species. This flexibility in genetics is another factor that strongly contributes to the acceleration of antibiotic resistance (Levy, 1997). Such problems arise, especially when antibiotics are misused. When patients do not take the proper amount of antibiotic prescribed, they allow for bacteria with antibiotic resistance to become stronger and replicate through plasmid transfer and conjugation (Levy, 1998). In order, however for such a spread to occur, both the resistant trait and an antibiotic must be present to allow for the selection of such drug resistant traits. Therefore, it is when an antibiotic is introduced into an environment that it eventually kills off all susceptible strains of bacteria, and leaves the host with only surviving resistant strains for multiplication (Levy, 1997). If no antibiotics are introduced, then resistant traits cannot prosper.
In order to reverse this rise in antibiotic resistance many methods have been proposed and considered. Some have thought of spraying environments with susceptible bacterial strains to compete with the resistant ones. Also, potentially, restoration of susceptible strains could be imposed through their implementation in the intestinal tract or skin. Even more, another proposal is to do more research on discovering new antibiotics to put into use, or inventing blockers for inactivating the resistant strains (Levy, 1997). Most, however, prefer simply to minimize the use of antibiotics to only emergencies. This would mean halting the use of antibiotics for growth factors in the food supply, as well as stopping the improper usage and abuse of antibiotics among humans. Specifically, one such attempt was made in December of 1998, when the European Council decided to ban the usage of four antimicrobial growth promoters due to fear of the weakening effects of antimicrobials in human medicine (Costa et al., 2010). This ban is hopefully, just the beginning of many to follow.
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As stated previously, the food supply is one of the main contributors to the rise in antibiotic resistance in humans. Animals, in particular, may be a reservoir of bacteria with increased resistance, raising even more concern about the transmission to humans through the food chain. Transmittance to humans through foods can be caused by consumption or mishandling. One such case supported this assumption, in which it was discovered that there was a high rate of colonization of chicken products by bacteria, representing the food chain as a public health problem. Not only that, but the study also revealed that there were increasing percentages of bacteria with multi-resistant phenotypes present in the chicken products (Avrian et al., 2003). In general, it seems more precautions need to be taken in the processing of the food supply.
There have been many other specific studies to test antibiotic resistance in animals, especially chicken. For example, the inclination of antibacterial growth on 69 E. Coli and 10 Salmonella isolates found from different retail chicken in Hiroshima prefecture, Japan markets were tested. Through the study it was found that the most common antibacterial resistant growth came from these antibiotics: ampicillin, streptomycin, spectinomycin, kanamycin, tetracycline, and trimethoprim/sulfamethoxazole (Ahmed, 2009). This is helpful knowledge since ampicillin, kanamycin and tetracycline are the three antibiotics being tested for resistance in the experiment at hand with Tyson chicken and Free Range chicken.
As for the experimental procedure of the Japanese chicken, the chickens were stored at 4ï‚°C and bacterial isolates were taken within 24 hours of retrieval. From there the isolates were then tested for susceptibility of antibiotic resistance. After a PCR was ran and conjugation experiments of plasmid transfer were completed, it was determined that 28 out of the 69 chickens had multidrug resistance to the above antibiotics. This proved to be very helpful because some of these antibiotics are among the most common used to prevent most infections, including E. coli (Ahmed, 2009). This study proves the relevance of studying the antibacterial resistance among animals and plants because it emphasizes how many multidrug resistances chickens have that could easily affect the human population if consumed incorrectly cooked. This is also why studies keep on being done to further understand how resistance to antibiotics is affecting the world we live in, for example through methods like plasmid transfer.
However, while the bacterial food safety of conventional animal products has been investigated, there is a large lack of research done on organic animal products. Since, there is a high rise in consumer interest with organic and all natural products, a better understanding of their food safety is now necessary. From the little research that has been completed on organic products, however, it has been discovered that there seems to be a significantly higher amount of antimicrobial resistance in bacterial isolates from conventional chicken products rather than organic. This could be due to the fact that prophylactic use of antibiotics and growth hormones is prohibited in organic animals. Particularly, in the United States, animals treated with any antimicrobials cannot be marketed as organic products (Young et al., 2009). Overall, results show that antibiotic resistance in food sources is partly dependent upon the type of husbandry applied to livestock.
Just as with the European ban on antimicrobial growth promoters, the effects of reducing the usage of antibiotics may not be obvious from their withdrawal, but the trends in resistance will become evident over time (Costa et al., 2010). One thing that is definitely clear is that more research needs to be done so the public can be educated on ways to improve the ecological imbalance that antibiotic resistance has created.
Therefore, it was decided that a comparative study would be done on antibiotic resistance in Tyson chickens (Gallus Gallus) and Free Range chickens. It was hypothesized in the study that environment 1 (Tyson Gallus Gallus), which was treated with antibiotics, would have more antibiotic resistant bacterial growth when treated with Ampicillin, Kanamycin, and Tetracycline, than environment 2 (Free Range Gallus Gallus). The reason for this is because chickens treated with antibiotics have been previously exposed to antibiotics, allowing only antibiotic resistant bacteria to survive and replicate (Levy, 1997). The null hypothesis was that there would be no difference between antibiotic resistant growth between environment 1 and environment 2.
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The goals set for this experiment were to determine the amount of antibiotic resistance in the two environments by creating patch plates. Then, the bacteria were to be characterized through Gram staining, KOH tests, MacConkey agar plates, and Eosin Methylene Blue agar plates. Then, from the bacteria collected, plasmids were to be isolated using miniprep isolation procedures and used in a restriction digestion. Transformation procedures tested whether or not a known set of plasmids in one bacterial cell could horizontally transfer into E.coli cells, which would transfer their antibiotic resistance from the plasmids. Finally a polymerase chain reaction (PCR) was conducted to further analyze antibiotic resistant bacteria identity and determine the bacterial species.
Collecting and Isolating Bacteria
First, two environments, Tyson Gallus Gallus and Free Range Gallus Gallus, were obtained from a local grocery store (Meijer) to test for antibiotic resistance. Each was selected as representative of chicken previously treated with antibiotics as well as representative of chicken that had never been exposed to antibiotics. Therefore, to gather sufficient amounts of bacteria from environments 1 and 2, the Tyson Gallus Gallus and the Free Range Gallus Gallus respectively, a 24 hour period was allotted for each type of chicken to be removed from refrigeration and allowed to sit out at a room temperature of approximately 27Â° C. After this incubation period, sterile swabs soaked in sterile phosphate-buffered saline (PBS) were passed over the top (.1), middle (.2), and bottom (.3) of each chicken and were swabbed onto consecutive 600 ml solution lysogeny broth (LB) agar plates. These plates were made with 8.8 grams of agar powder, 12 grams of LB broth, which were then placed on a hot plate with a stir bar until the mixture dissolved. They were then autoclaved for 30 minutes with the stir bar, were allowed to cool to approximately 55Â°C, and then were poured. The swab plates, when complete, were then incubated at 37Â° C for another 24 hour period and then were moved to a refrigerator at 5Â°C. Patch plates were then made to isolate different colonies of bacteria by marking petri dishes into 12 numbered sections. Small samples of different bacteria were gathered from the original swab plate and streaked within the numbered sections and then incubated again at 37Â° C for 24 hours. To isolate antibiotic resistant bacteria, 3 more types of LB plates were made with the antibiotics Ampicillin, Tetracycline, and Kanamycin. These new plates along with a control LB only plate were all numbered to match the previous patch plates and were streaked within the corresponding boxes and incubated for 24 hours. The colonies that survived and grew despite the presence of the antibiotics were then deemed antibiotic resistant and two bacteria colonies, Kanamycin 1.3, patch number 7, and Tetracycline 1.2, patch number 9, were chosen for further experimentation.
Gram Identity Tests
To characterize the bacteria, first a gram stain was performed to reveal the structure of the bacteria as Gram-positive or Gram-negative. For the procedure, a wax circle was drawn on a microscope slide, and within the circle a series of streaks was created with the bacteria using an inoculating loop. After the streaks dried, the slide was brushed over a flame of a Bunsen burner to affix the bacteria to the slide. Then, the chemical process began, in which the bacteria was first flooded with crystal violet for a primary stain. After sixty seconds, the slide was rinsed with distilled water for five seconds. Following that, the bacteria was flooded again, this time with the mordant, iodine, for sixty seconds, and then rinsed with distilled water. Next, the decolorizer, ethanol, was used and added drop-wise until the blue-violet color was no longer emitted from the slide. Again, it was rinsed with distilled water for five seconds. Finally, the counter stain, safranin, was flooded onto the bacteria for sixty seconds, and was cleansed with distilled water. After the slide had dried, a small amount of immersion oil was added to the slide, and it was placed under a microscope at 100x power for viewing (LBC Biology Staff 2010).
Another test performed to distinguish Gram-positive bacteria from Gram-negative was the KOH test. This was done with a microscope slide, in which 5 Î¼l of 3% KOH were placed on the slide. Then a loop full of the two different bacteria colonies selected were added to each drop and swirled for one minute. After one minute was complete, the solution was observed for a thick, "sticky" consistency. Even further, other tests completed on the bacteria to determine gram identity were a MacConkey agar and an Eosin Methylene Blue agar, in which the plates were streaked with the bacteria and incubated overnight (LBC Biology Staff 2010) to observe whether of not growth occurred.
After the structure of the bacteria was investigated, a Promega Wizard Miniprep was completed to isolate plasmids from the two streak plates' bacteria. To begin, liquid cultures were made for each sample and incubated overnight. Then, 5 ml of the liquid culture were centrifuged and the supernatant liquid was dispelled leaving a pellet. Next, the pellet was resuspended with 250 Î¼l of Cell Resuspension Solution, after which 250 Î¼l of Cell Lysis Solution was added to each sample and the solution was mixed through inversion. Then, 10 Î¼l of Alkaline Protease Solution was added, the solution was inverted, and it was incubated for five minutes at room temperature. Finally, 350 Î¼l of Neutralization Solution was added and the solution was inverted and centrifuged for ten minutes at 14,500 rpm. Now that the lysate formation was complete, a Spin Column was inserted into a Collection Tube and decanted the cleared lysate into the Spin Column. This was then centrifuged for one minute, and the flowthrough was discarded. After the Spin Column was reinserted into the Collection Tube, 750 Î¼l of Wash Solution was added and the Collection Tube was again centrifuged for a minute. The flowthrough again was discarded, and the entire step was repeated with 250 Î¼l of Wash Solution. After that was completed, the Collection Tube was again centrifuged, this time for two minutes. After the washing process was finished, the Spin Column was transferred to a new microcentrifuge tube and 50 Î¼l of Nuclease-Free Water was added to the Spin Column and centrifuged for a minute. Finally, 10 Î¼l of the final liquid product was placed in a new microcentrifuge tube with 2 Î¼l of 6x loading dye and it was thoroughly mixed.
Once the process of isolating the plasmid was complete, an agarose gel electrophoresis was performed to visualize the plasmid. First, 40 ml of clean TBE was placed into a 250 ml flask, and 0.4 g of agarose was added. The mixture was then heated for ninety seconds, cooled, and then 2 Î¼l of Ethidium Bromide was added. The mixture was swirled and then poured into a gel tray with stoppers and a comb was inserted to create wells. Then, the tray was refrigerated for twenty minutes to formulate a gel. Next, the stoppers and comb were removed, and the tray was placed inside of the gel apparatus. From there, used TBE was added to the apparatus until the surface of the gel was covered. Each well in the gel was then filled with the 6x loading dye solutions, and a 1 x Kb ladder was used as a control. Next, the apparatus was run for approximately thirty minutes at 120 volts, and finally, the plasmids were visualized under ultraviolet illumination using the Kodak imaging system (LBC Biology Staff).
Double Restriction Digestion
Furthermore, if plasmids were retrieved from the bacteria colonies, a double restriction digestion was performed using the plasmid DNA and restriction enzymes of choice based on the New England BioLabs (NEB) cutter website (Vincze, 2003). However, if no plasmids were retrieved, a control plasmid could be used instead. When using PstI and EcoRI, 50 Î¼l of NEB buffer three was added to 1 Î¼l of the blue control plasmid DNA. Then 1 Î¼l of each of PstI and EcoRI was added, followed by 0.5 Î¼l BSA if necessary. The BSA was only required to be added if the final volume of the reaction mixture did not reach 100 Î¼g/ml. The same process was repeated, this time with the restriction enzymes EcoRI and BamHI, using NEB buffer three. After the mixture was completed, it was then incubated for one hour at 37Â° C. At the end of this period, 3.3 Î¼l of 6x loading dye was added directly to each reaction mixture and 20 Î¼l of the final products were loaded into two wells of a gel. Additionally, a ladder was used as a means of comparison and the agarose gel was run for approximately 30 minutes (LBC Biology Staff 2010). The gel when completed displayed a specific fragment pattern that then could be used to create a plasmid map to identify the gene.
Transformation for Blue Control
Next to be accomplished in the experiment, was a transformation of E. coli with the plasmid DNA. To start, 150 Î¼l of cells was thawed and then pipette evenly into three sterile 1.5 ml tubes. Following this, 5 Î¼l of the blue control plasmid DNA was added and gently mixed into the first vial of cells. Next, 5 Î¼l of distilled water was added and gently stirred into the second vial, and finally 5 Î¼l of a "pLITMUS28i" positive plasmid control was added and mixed into the third vial of cells. After this the three tubes of cells were incubated on ice for 30 minutes. Once the incubation period was complete, the cells were heat-shocked for 45 seconds and then immediately placed on ice for 2 extra minutes. Then, 250 Î¼l of SOC medium was added to each tube of cells, and the mixture was shaken horizontally at 225 rpm for one hour at 37Â°C. Once this process was complete, using a sterilized "hockey stick", 75 Î¼l of the finished products containing the blue control plasmid DNA and the negative water control were each spread onto an LB only plate, as well as an LB + Ampicillin plate and an LB + Kanamycin plate. Then for the positive pLITMUS28i control, 75 Î¼l of the final product was spread on an LB only plate and an LB + Ampicillin plate. Finally the plates were placed upside down in an incubator of 37Â° C for 24 hours (LBC Biology Staff 2010). The following day, the plates were removed, and the colonies on each plate were counted.
Polymerase Chain Reaction
For the final process, a polymerase chain reaction (PCR) was done on the DNA. First, a reaction cocktail was made using a mixture of 80 Î¼l of Nuclease-free water, 10 Î¼l of Thermopol buffer, 3 Î¼l of dNTPs, 2 Î¼l of primer 11F, and 2 Î¼l of primer 529R. Then, 1 Î¼l of Taq polymerase was added to the cocktail and this mixture was vortexed for one to two seconds three times. Next, the resulting mixture was divided evenly into three PCR tubes. From there, a small bit of the environmental bacteria was added to one tube using a micropipetter tip, a small bit of E. coli was added to another using another tip, and finally 1 Î¼l of water was added to the final tube to serve as a control. After this, each tube was loaded onto the thermal cycler to be treated with a variety of temperatures for three hours. Following this process, 10 Î¼l of the PCR products were run on an agarose gel with 2 Î¼l of 6x loading dye, and viewed. A ladder was used again as a control. Since the 16s rRNA gene was present in the gel at approximately 1500 base pairs, a PCR clean up was completed, in which 125 Î¼l of PBI buffer was added to 25 Î¼l of the successful PCR sample and mixed. From there, a Spin Column was placed in a Collection Tube and the sample was applied and centrifuged for one minute. Furthermore, the flowthrough was discarded and 750 Î¼l of PE buffer was added to the Spin Column and centrifuged for one minute. Again, the flowthrough was discarded and the Spin Column and Collection Tube were centrifuged for one minute. Once this was done, the Spin Column was placed into a new 1.5 ml microcentrifuge tube, and 50 Î¼l of EB buffer was added and centrifuged for one minute. Finally, 10 Î¼l of eluted sample and 2 Î¼l of 6x loading dye were ran in a second PCR gel, along with a ladder, which in the case that it was successful, allowed the eluted sample to be submitted to the MSU sequencing facility (LBC Biology Staff 2010).
In order to interpret the data received, the colonies of the master antibiotic patch plates were counted and observed. The master antibiotic patch plates were the observed values which were entered into the Vassar stats website and given expected values, which were calculated using the Fisher Exact Probability Test (Lowry, 2010). From here the observed values were subtracted from the expected values, squared, then dived by the expected, and summed together for each antibiotic, Ampicillin, Kanamycin, and Tetracycline. For each antibiotic there was three plates per ï£2 value. These values then resulted in p values by using an excel worksheet that was made.
For the experiment, there was a large amount of growth on both environmental swab plates (Figure 1). Furthermore, growth was found in the master patch plates (Figure 2). However, this was not the case for the antibiotic patch plates for both environments (Figure 3). Also, the environment 2.1 Ampicillin plate had continuous growth throughout the majority of the plate, in which the colonies overgrew into one big body of colonies (Figure 4). Based on the colony counts of the antibiotic patch plates, it was determined that there were ï£Â² values of 0.04 for Ampicillin, 2.75 for Kanamycin, and 1.93 for Tetracycline, with corresponding p-values of 0.8415, 0.0973, and 0.1648 (Table 1).
As for bacterial structure, it was found that Kanamycin 1.3, patch number 7, and Tetracycline 1.2, patch number 9, showed gram-negative results for all of the completed tests (Table 2). The KOH test showed a "sticky" consistency for both bacterial colonies. The MacConkey agar test produced growth on both plates. For the Kanamycin 1.3 bacterial isolate a bright pink color was observed and for the Tetracycline 1.2 bacteria the plate concluded in a pinkish-orange color (Figure 5). The Eosin Methylene Blue (EMB) agar test resulted in growth for both plates, with a green metallic sheen appearance for the Kanamycin 1.3 colony, and a pink appearance for the Tetracycline 1.2 colony (Figure 6). Furthermore, Gram staining on the two bacterial isolates displayed only pink bacteria cells using a light microscope at 100x objective (Figure 7 and 8). Finally, after three trials of running the mini prep product with gel electrophoresis, no plasmids were found in the gel (Figure 9).
Regarding further bacterial research and plasmid analysis, it was discovered from the transformation that there was no growth on any of the negative control plates, no growth on the LB + Ampicillin plate for the control plasmid, lawns on the LB only plates, and bacterial growth on the control plasmid LB + Kanamycin plate, as well as the Ampicillin pLITMUS28i plate (Figure 10).
For the restriction digestion, the blue control was treated with two double digestions. For lane 3, which was treated with the restriction enzymes PstI and EcoRI, the results showed three fragments with sizes of approximately 2845, 923, and 420 base pairs. Then, for lane 4, which was treated with the restriction enzymes EcoRI and BamHI, it displayed one fragment of approximately 4173 base pairs (Figure 11).
Lastly, from the polymerase chain reaction (PCR) results for the environmental bacteria Kanamycin 1.3, patch 7, the gel confirmed the 16s rRNA gene in the DNA (Figure 12). After which, the second gel, resulting after the 16s rRNA gene was isolated for submission to the MSU sequencing facility, repeatedly confirmed the gene (Figure 13). From the MSU sequencing facility results and previous research collected it was suggested that the bacteria specimen was Citrobacter freundii.
The purpose of this research was to discover the difference in antibiotic resistant growth between previously antibiotic treated Gallus Gallus and Free Range Gallus Gallus, as well as to further characterize the bacteria that grew on the environmental swabbed plates. From the results, it was determined that there is no significant difference when it comes to antibiotic resistance in Tyson Gallus Gallus and Free Range Gallus Gallus. These results contradicted the hypothesis that previous exposure to antibiotics would influence the amount of antibiotic resistant bacterial growth, therefore the null hypothesis was failed to be rejected. This is noteworthy because it was thought that conventional chicken would have a greater resistance to antibiotics than organic chicken, which in this study appears to be false.
The reason the hypothesis was negated was based off of the resulting p-values for the antibiotics Ampicillin, Kanamycin, and Tetracycline from a ï£Â² statistical test. Since the corresponding p-values were 0.8415, 0.0973, and 0.1648, there is an 84%, 9%, and 16% probability that the results obtained deviated from the expected results by chance alone, compared to the 5% standard, which means that there is a 5% chance that the null hypothesis is failed to be rejected. Based on these results it was discovered that there was no significant difference in antibiotic resistant growth among Tyson Gallus Gallus and Free Range Gallus Gallus.
Even though the hypothesis was refuted, there is some research that does support the null hypothesis. This research found that the bacterial isolates of S. aureus and L. monocytogenes showed no significant difference between conventional Gallus Gallus and organic Gallus Gallus. However, this was not the case for a bacterial strain of E. coli. This strain displayed more antibiotic resistant growth with conventional Gallus Gallus (Miranda 2008). So, although E. coli showed results that would disagree with the results at hand, all other bacteria S. aureus and L. monocytogenes inferred that conventional Gallus Gallus and organic Gallus Gallus had no substantial differences of antibiotic resistance.
From the results of the two bacterial isolates (Kan 1.3 and Tet 1.2) from the Tyson Gallus Gallus, bacteria characterization was analyzed. The "sticky" consistency of the KOH test results signified that both bacteria isolates were Gram-negative. This is due to the fact that Gram-negative cells have a thinner peptidoglycan layer than Gram-positive, which allows the KOH to lyse the cells and release their DNA and proteins. The MacConkey agar results also indicated that the bacterial isolates were Gram-negative, because only gram-negative colonies can grow in the presence of biosalts found in the MacConkey agar. The Kan 1.3 plate was hot pink, which conveys that it ferments lactose, while the Tet 1.2 plate's pinkish-orange color could convey that the bacteria has a low fermentation level. Even more, the Eosin Methylene Blue agar results verified that the isolates are Gram-negative, while the Kan 1.3 plate's green metallic sheen suggests that it ferments lactose and the Tet 1.2 plate's pink colored colonies confirms that it ferments lactose at a much lesser degree. Once and for all, the Gram-staining results also corroborated that the bacterial isolates were in fact Gram-negative. Both isolates had pink-stained bacterial cells, because they have a thinner peptidoglycan layer and an additional outer membrane containing lipids that are separated from the cell wall. These lipids react with the decolorizer in the procedure, and remove the initial crystal-violet color. Therefore, when the safranin was added, the Gram-negative cells picked up the pink color and appeared pink under the microscope lens.
Furthermore the two bacterial isolates were examined for plasmids. Therefore a mini prep was run to isolate plasmids in the bacteria samples and blue control. After three trials of running the mini prep products on gel electrophoresis', it can be inferred that the environmental bacteria had no plasmids. On the other hand, the blue control displayed plasmids under UV light.
From these results it can be speculated that the antibiotic resistant gene is located in the chromosomal DNA of the bacterial samples.
Since plasmids were found only in the blue control, a double restriction digestion was completed to determine if the control was pKAN or pAMP. The results obtained verified that the blue control is pKAN. For the double digestion in lane 4, EcoRI and BamHI, two fragments were supposed to appear. However, only one fragment of around 4000 base pairs showed on the gel. The other fragment would have been 21 base pairs, but the likelihood of it running off of the gel before both double digestions were complete is high. Therefore, it can still be assumed that the blue control is pKAN. The blue control was also proven to be pKAN from a successful transformation showing growth on the LB only agar and the LB + Kanamycin plates.
Through running a PCR, the environmental bacteria Kanamycin 1.3, patch 7, was characterized to be Citrobacter freundii. This bacterium is a gram-negative bacterium in the Enterobacteriaceae family. The morphology of this bacterium tends to be in the Bacilli (rod-shaped) form (Rollins 2000). Additionally, Citrobacter freundii has the ability to ferment lactose, which agrees with the results obtained from the MacConkey and EMB agar streak plates (Lipsky 1980).
This bacterium is typically found in the soil, water, and decaying matter, therefore it usually only compromises individuals with open wounds (Rollins 2000). These locations may increase antibiotic resistance in the bacteria because most plants and animals are treated with antibiotics allowing drainage into the soil and water. Also, from a previous study it was discovered that this bacterium had a 67.5% sensitivity level to Kanamycin, therefore based on these results it has around a 30% chance of being antibiotic resistant to Kanamycin (Lipsky 1980). Common diseases where this bacterium is found are in urinary tract infections, respiratory tract infections, and meningitis cases (Rollins 2000).
Due to mistakes in the procedures, some of the results of the experiment could be faulty. For example, the bacteria that grew together could have affected the results of the colony counts and further the p-values determined from the ï£Â² statistical test. For instance, if the observed values were greater than they actually were, then the ï£Â² value would be greater. Additionally, in the plasmid mini prep and gel electrophoresis procedures, errors could have been made in the process, such as the DNA may not have bound properly to the Spin Column, affecting the results of the gel. This could result in a gel with no plasmids present. Further verification could be made regarding if there is any difference in the antibiotic resistance levels in antibiotic-treated chicken versus Free Range chicken by repeating the experiment. Through repetition, if the same results persisted, the validity of the conclusions will become stronger. Also, more research could be done on the background of the Gallus Gallus, to determine if the Free Range Gallus Gallus' diets and surrounding environments may have prior exposed them to some sort of bacteria allowing them to acquire certain antibiotic resistant genes. This could be accomplished by testing the soil where the food sources for livestock are grown for the presence of antibiotics. To test this experiment, the soil could be diluted into a solution and then grown on antibiotic patch plates to test for resistance. Overall, more research needs to be completed on the increasing antibiotic resistance levels found in humans, livestock, and agriculture, so that the ecological imbalance that antibiotic resistance has created can be corrected. The research completed throughout this lab has proven to be significant to society for determining antibiotic resistance between conventional and organic Gallus Gallus, which could threaten human consumption.