This experiment was designed to determine what bovine milk media are most likely to produce antibiotic resistant bacteria and to characterize those which they produced. Bacteria were first inoculated into organic and inorganic milk media. Contrary to our original hypothesis that more bacteria would grow in organic milk than inorganic milk, no bacteria grew in the organic milk. As a result, we compared rotated and stationary inorganic milk media for the rest of the experiment. We hypothesized that anitibiotic resistant bacteria would be more common in the stationary inorganic milk media. Yet, we found that there was no significant difference in the number of antibiotic resistant patches between the two environments for the antibiotics ampicillin, kanamycin and tetracycline plates. The rest of the experiment focused on characterizing bacteria of particular interest within the surviving populations. MacConkey agar plates were used to show that bacteria stain number nine (Amp9) was able to ferment lactose, while strain two (Amp2) could not. A gram stains showed both of the strains to be coccus in shape, yet also showed Amp9 was gram-negative while Amp2 was gram-positive. A mini-prep protocol was used as well to isolate plasmids, which were used in a restriction digest to characterize the difference in plasmids. The mini-prep failed to produce environmental plasmids, and the restriction digest gave inconclusive results. This experiment contributes to the stores of knowledge about different antibiotic resistance in general; especially those which arise in milk when left to incubate. The inability of organic milk to harbor bacteria was most shocking of all and deserves further study with previously identified bacterial strains under more controlled conditions.
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The development of antibiotic resistance in bacteria is a serious issue facing the future of healthcare in both animals and humans. The beneficial uses of antibiotics were discovered in 1928 by Scottish physician Alexander Fleming with his discovery of penicillin (Neu, 1992). Since then, antibiotics have been used to treat various bacterial infections. In recent decades, they have been overused resulting in the evolution of widespread resistance. One source of misuse is the over-prescription of antibiotics, which selects for the evolution of antibiotic resistance (Neu, 1992). The abuse of antibiotics does not stop with over prescription at the hospitals, but also extends to livestock raised on ranches and factory farms (McEwen and Fedorka-Cray, 2002). Livestock such as cows are given antibiotics on a regular basis regardless of need. This is defended as a preventative measure against disease, widespread illness and helps them grow faster (McEwen and Fedorka-Cray, 2002). Bacterial resistance within the livestock themselves can be carried into the products from them, and when ingested by humans, can result in a widespread crisis as we further lose the ability to fight off bacterial infections (Tenhagen et al. 2006).
Antibiotics operate by chemically disrupting the life cycle of bacteria in a fatal fashion. Antibiotic resistance in a population can theoretically be traced back to a singular bacterium that possessed the original mutation that protected it from a particular antibiotic. When a population is exposed to an antibiotic, the antibiotic selects for those individuals which are resistant to the antibiotic and are able to survive. Most of these genes are found on plasmids within bacteria, but can also be found in chromosomal DNA (Cohen et al. 1972). As a result, the only bacteria remaining would be either the descendents of the original bacterium or those that were lucky enough to receive, via conjugation or transformation, a copy of the plasmid that codes for resistance. Plasmid conjugation is the process through which a bacterium transfers a plasmid to a receiving bacterium. Bacterial conjugation is so effective that it has been used to transfer DNA into eukaryotic cells as well (Jimenez and Davies, 1908). Bacterial conjugation adds to the already exponential growth of resistant in bacteric populations, which is due to binary fission. As long as the antibiotic remains in the environment the frequency of resistance will remain high in the population (Thiel, 1999).
One of the largest threats of causing wide spread antibiotic resistant infection is the food industry (McEwen and Fedorka-Cray, 2002). The widespread use of antibiotics on farms could lead to growing antibiotic resistant infections within our food supply, which can then easily be transferred to humans. In foods produced from livestock which are not heated prior to consumption, the risk of infection is particularly higher because heating foods is the last line of defense against most bacteria after antibiotics fail (Hamad et al., 2008; Cambell et al., 2008). The dairy industry is a perfect example of this problematic situation (Rodriguez-Alonso et al., 2009). Milk generally not heated directly prior to consumption, so any thermophobic bacteria present would be allowed to grow and thrive in transit from the cows to the consumers. Yogurt is an unusual exception, because it is heavily heat treated and then inoculated with bacteria (Bouzar et al., 1997). Yogurt differs from raw milk though, in the sense that the bacterial strains which are used for producing yogurt are heavily monitored and frequently tested, to confirm that they are not dangerous to consumers. With milk though, studies have even shown that some bacteria survive the brief heat of pasteurization, meant to kill bacteria, and make their way all the way to consumers (Grant et al., 2002).
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There is strong common belief that organic milk tastes better and is better for consumers (Hamad et al. 2008). Hamad et al. analyzed the sanitation of raw organic milk in Japan (2008). In their study, they examined cows that were determined to be healthy by the California Mastitis test and which had not been treated with antibiotics for at least three months prior to testing. After incubating milk collected from the cows for 24 hours at 35°C under agitation, they found a wide range of bacterial cultures within the milk. One strain within their samples of Klebsiella pneumoniae in particular, was resistant to oxyimino-cephalosporins and aztreonam. K. pneumonia antibiotic-resistant infections have become more wide spread throughout Japan in recent years without a conclusive explanation (Hamad et al., 2008). The study led by Hamad et al. (2008) suggests that raw milk could be the cause of the rapid spread of infection.
Japan is not alone in this threat of mass infection. Studies conducted in Ethiopia (Getahun et al., 2007), Italy (Rodriguez-Alonso et al., 2009), Germany (Tenhagen et al., 2006), California (Roesch et al., 2006) and Washington State (Raymond et al., 2006) have produced equally alarming results pertaining to the bacteria found in milk. The problem is escalating quickly. If changes are not made soon, antibiotic resistant bacteria could quickly spread throughout the world, resulting in a plague of diseases against which we would be unable to fight or defend ourselves.
The purpose of our experiment was to determine the effect of environment on the presence of antibiotic resistance in bacteria in organic U.S. non-organic milk. Cows that produce organic milk are treated with considerably less antibiotics than those used for the production of inorganic whole milk (Roesch et al. 2006). For this reason we hypothesized that more bacteria would grow in organic milk, but antibiotic-resistant bacteria would be more prone to thrive in inorganic.
In this study, we inoculated organic milk with bacteria by allowing samples to stand in the open air before incubation under stationary and oscillating conditions. However, both attempts to grow bacteria in organic milk failed (Figures A1, A2). As a result, we chose to study inorganic whole milk that was incubated standing in an LB only plate overnight and inorganic whole milk that was rotated in the milk media during incubation for the remainder of the study.
Oscillating conditions provides milk and any bacteria in it with a continuous supply of oxygen, distributed throughout the entire media, while oxygen in the stationary conditions is only available to bacteria living on the surface of the milk. As some antibiotics prevent and kill bacteria by removing the oxygen the bacteria need to survive, it was hypothesized that the bacteria would be more resistant to the antibiotics in the non-rotated inorganic whole milk because this environment provides less oxygenation of bacteria.
In order to study antibiotic resistance, bacteria swabbed from the rotated and non rotated organic milk were streaked onto three separate agar plates which each were treated with ampicillin, kanamycin, or tetracycline antibiotics. The only patches that grew on the tetracycline plates were bacteria taken from the rotated environment. However, there was no statistically significant difference in the number of antibiotic resistant patches that grew from the two different environments for any of the antibiotics. Patches nine and two (Amp9 and Amp2) were selected from the ampicillin patch plate that was inoculated from our second rotated non-organic whole milk environmental plate. The selected patches were streaked onto new agar plates containing the antibiotic they were resistant against to produce a greater population for further testing.
We performed a gram stain to identify the type of cell walls our bacteria possessed. Amp9 was gram-negative, Amp2 was gram-positive and both were coccus shaped. The KOH test produced similar results for determining the cell wall types of each of the strains. The MacConkey agar test also confirmed this and surprisingly revealed that Amp2 was incapable of fermenting lactose, while Amp9 could. This is shocking, considering that these bacteria initially were cultured from inorganic milk. All of our other attempts to perform a miniprep, restriction enzyme digest, transformation, and PCR failed. However, we were able to shed new light on the antibacterial qualities of organic milk.
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Initial Inoculation of Bacteria into Varying Environments
Two Petri-dishes of organic and inorganic milk were exposed to the open air over night in a dorm room. The following day we transferred each of the liquid milks to test tubes, which were kept in a rotating incubator at 37°C over night. The solidified milk at the bottom of the Petri-dishes was transferred to lysogeny broth (LB) plates and incubated at 37°C in a stationary incubator. Next, we streaked fresh LB plates using the rotated milk and refrigerated the non-rotated plates. After allowing the rotated milk to incubate for 24 hrs., we observed all of our plates, and found that bacteria had refused to grow on any of the plates containing organic milk. Due to this development, we decided to compare the bacteria grown in the rotated and non-rotated inorganic milk for the rest of the experiment.
Preparation of Antibiotic Patch Plates
The bacteria grown in rotated inorganic milk were streaked onto two petri-dishes containing LB, each with sixteen colonies, and bacteria grown in non-rotated milk were streaked onto two additional petri-dishes also containing sixteen sections of LB only. All four plates were incubated for approximately 24 hrs. at 37°C. After incubation, bacteria from each colony was then streaked onto the correlating grid on four petri-dishes, each containing one of the antibiotics ampicillin, kanamycin, tetracycline and one LB only media. The antibiotic petri-dishes were incubated for approximately 24 hrs. at 37°C. Bacteria that grew on an antibiotic petri-dish as well as on the LB only petri-dish were determined to be antibiotic resistant were then streaked onto another petri-dish containing the antibiotic to which they were determined resistant and further characterized.
Selection of Antibiotic Resistant Bacteria
The antibiotic resistant bacteria that were sampled and streaked were selected from separate colonies on the petri-dish containing ampicillin. These specific colonies were selected because of their successful growth and interesting characteristics such as varying textures and colony size. These new streak plates were then incubated for approximately twenty-four hours at 37 degrees Celsius.
Incubation of Selected Bacteria for further analysis
Our next step was to micropipette five micro liters of ampicillin into four loose top vials containing LB broth. Cultures chosen from the newly grown bacteria were then scraped from the streak plates with a sterilized loop. The bacteria were then swirled into each of the vials and placed into the rotator for approximately 24 hrs.
Gram Staining and KOH Test
The next step in this process was to complete a Gram Staining and KOH test (LBC Biology Staff, 2010). Five micro liters of 3% KOH were placed on a slide. Then, a sample of bacteria was scraped with a sterilized loop was stirred for a minute. The stickiness of the substance was used to indicate if the bacteria were Gram positive or Gram negative.
The gram stain was performed by inoculating and heat fixing our bacteria to two slides. We then flooded the slides with crystal violet, rinsed them with water, flooded them with Gram's iodine and rinsed them a second time with water, followed by decolorizer (95% ethanol). Once color no longer runs off of the slides, we flooded them with safranin and rinsed them with water. Finally, we observed the slides under a microscope to determine both the gram types and the structure of the bacteria.
Miniprep Plasmid Isolation
Once the bacteria were characterized, the next step was to isolate plasmid DNA from the liquid bacteria culture (Trevors, 1984). We used the Wizard ® Plus SV Minipreps DNA Purification System made by Promega. The cells were grown in a liquid culture and then centrifuged. The culture media was removed and then the cells collected at the bottom were resuspended and lysed. After being centrifuged again, the supernatant fluid was put in a spin column, which was placed into a collection tube. The collection tube was then washed and spun catching the plasmids in the silicon pad. Then the collection tube was rinsed a final time with nuclease-free water to pull the plasmids into a solution of water. After these plasmids were isolated, we separated the plasmid DNA by agarose gel electrophoresis. This allowed us to visualize and digest the gel under UV light.
Restriction Enzyme Digest
After the plasmids were separated we ran a couple of different tests. First we used restriction enzymes and gel agarose to help map the plasmid DNA. We used Eco RI and PST II restriction enzyme to strategically cut our plasmid DNA at strategic points for analysis. We then ran an agarose gel with the spliced plasmids along with a ladder, to determine the sizes of the plasmid segments.
We attempted to transfer our plasmids to viable cells by placing our plasmids into an environment with a previously non-resistant strain of viable E. coli to try to make the E. coli ampicillin resistant by transference of the plasmid gene.
Polymer Chain Reaction (PCR)
For our polymer chain reaction we prepared a master mix containing ddH2O, Taq Buffer, dNTP's, 11F, 1492R and Taq polymerase. We then added 30μL of master mix to three PCR tubes. Next, we added chromosomal DNA from our environmental bacteria, plasmid DNA from the blue control and water to the respective PCR tubes. After that, we left the tubes in the PCR machine over night and ran a gel of our DNA.
When organic and non-organic milk were initially incubated in the two different environments, the organic milk failed to produce any bacteria. As a result of this, the rest of the experimental bacteria came from the two different non-organic whole milk environments (Figure 1).
Bacteria from each cell on each environmental patch plate were streaked on a series of different antibiotic resistant patch plates to isolate resistant bacteria (Figure 2A-D). The chi test of independence produces a p-value of 13.07 based on the overall number of resistant bacteria in one environment against the other (Table 1).
Gram stain and KOH test
Amp9 responded negatively to the KOH test by failing to lyse while Amp2 responded positively. A gram-stain showed Amp9 to be gram-negative, while Amp2 was gram-positive (Table 2).
MacConkey agar and EMB plates
Further analysis of the sample strains was conducted by the use of MacConkey agar and EMB plates (Figure 3). In both sets of plates Amp9 bacteria grew while Amp2 did not.
The use of the miniprep protocol helped to isolate plasmids from the environment and red and blue control bacteria. These plasmids were analyzed by gel electrophoresis, which showed that the miniprep failed to produce plasmids for the environment, but successively isolated plasmids for both the red and blue control bacteria (Figure 5). Despite the absence of environmental plasmids, the control plasmids were further analyzed in a restriction digest. The digest showed band lengths of about 4,000 and 1,000 base pairs for lanes four and seven. Band lengths of about 6,000 base pairs were observed for lanes three and six. Lanes two and five were inconclusive (Figure 6).
Transformation of E. coli
The transformation plates show lawn growth on all of the LB only plates for blue control plasmid bacteria, environment plasmid bacteria, pLITMUS28i plasmid bacteria, and water bacteria (no plasmid). None of the ampicillin plates show growth. The blue control plasmid kanamycin plate shows growth (Figure 6A-D).
Polymer Chain Reaction (PCR)
The gel for our PCR showed no bands present for any of the test subjects, indicating that our DNA had not been duplicated at all by the PCR (Figure 7).
Bacteria were cultured in non-organic and organic whole milk to determine which environment was more hospitable for the growth of antibiotic resistant bacteria. Multiple tries failed to produce any viable bacteria in any of the treatments of organic milk, so the experiment was reduced to testing the differences of bacteria grown where air is readily available throughout the whole environment as opposed to just near the surface of the environment. It was hypothesized that bacteria would grow better in an oscillating environment that provided air throughout the entire medium.
The observation that organic milk failed to produce bacteria under the same test situations that allowed for plenty of bacteria in non-organic whole milk is one aspect of this experiment that needs further study. With respect to the idea that different quantities of bacteria could possibly stem from differences in production of organic and non-organic milk, this provides strong evidence towards the antibiotic properties of organic milk. However, stricter experimental conditions would be necessary to verify that the absence of bacteria was not due to other reasons. It would be beneficial to inoculate both environments with equal quantities of the same known bacteria to compare the true extent of the organic milk's properties on bacterial growth.
The colony counts produced a p-value of 13.07, which caused us to fail to reject the null hypothesis. Therefore, there was no statistically significant difference in the quantity of bacteria in the two different environments. This is not surprising since the bacteria that were transferred to the oscillating environment were inoculated from the stationary environment, and the stationary environment had already selected for bacteria that could survive with a smaller air supply. An interesting result none the less, was that none of the bacteria grown in the stationary environment were resistant to tetracycline, while several strains of bacteria grown in the oscillating environment were resistant to tetracycline.
To help understand our bacteria, samples were randomly selected for further study in the experiment. A gram-stain of the two different selected strains of bacteria showed Amp9 to be pink in color, and Amp2 to be purple. These slides were compared against a regrettably inconclusive control. This was more than likely was due to excessive or insufficient rinsing of the control slides after the addition of dyes. We were still able to compare the slides against pictures from previously stained bacteria from earlier experiments conducted in the lab. From previous results we were able to determine that the pink bacterium was gram-negative and the purple bacterium was gram-positive. This means that the gram-negative bacteria had the addition of an outer membrane to thir cell wall, while the outer layer of the gram-positive bacteria was made of peptidoglycan. During the course of the gram-stain, both bacteria were shown to be coccus in shape.
MacConkey agar plates are designed to allow bacterial growth if the bacteria have the ability to ferment lactose. The Amp9 displayed growth while the Amp2 did not. This is the first clear difference in metabolic processes of the strains of bacteria being studied. This also further confirmed that Amp9 was gram-negative and Amp2 was gram-positive. Amp2's failure to grow on the MacConkey agar plates revealed its inability to ferment lactose, which is surprising since all of our bacteria were originally cultured in milk.. Amp9 on the other hand, clearly was able to ferment lactose, since it thrived on the MacConkey agar plates.
Mini-prep results showed that plasmids were successfully isolated from the red and blue control bacteria, but not from the environment. Although the mini-prep was repeated many times there were never enough plasmids isolated for analysis. This suggests that plasmids were not present in all of the bacteria. It would be best to repeat the experiment with a greater quantity of bacteria, to isolate a greater concentration of plasmids.
The different DNA bands in the restriction digest gel were meant to indicate whether the bacteria contained pAmp or pKan. Unfortunately, the results of our digest were inconclusive. This is possibly due to our failure to isolate a large quantity of plasmid DNA. This experiment could have possibly yielded clearer results if the amount of DNA was amplified with a polymerase chain reaction prior to use in the restriction digest. A greater quantity of plasmid DNA would have caused the bands on the gel to appear more vibrantly and thus made it easier to determine the band lengths of our segments.
In order to sequence the chromosomes of Amp9, the cells were mixed in a medium and then underwent PCR to amplify the amount of DNA present in solution. PCR was not performed upon Amp2 due to the exhaustion of its population by previous tests. The PCR failed to produce results because the wrong mixture of thermopol buffer was used and there was not enough time to repeat the experiment with the correct chemicals.
This experiment serves to help better understand the overall trends of how antibiotic resistance develops in regard to the availability of oxygen. Above all else, this study shows the danger of consuming milk that has been left unrefrigerated and open to the air. It would be beneficial to further the effects of inoculating organic milk with bacteria. Despite our original hypothesis, organic milk proved to be a poor media for bacterial growth. It would also be interesting to repeat the tests for antibiotic resistance between the stationary and oscillating environments with a bacterium originally grown in a liquid culture, to see if inoculating the liquid environment in our experiment with bacteria from the stationary environment had any effect on our results. It would also be beneficial to duplicate our tests for antibiotic resistance between stationary and oscillating environments, to see if a significant difference would develop between the two environments for tetracycline resistance.