Antibiotic Resistance Lab Report. Bacterial plasmids are small, circular molecules of DNA that can be found in

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1 Adil Sabir Bio 110H 24 November 2014 Antibiotic Resistance Lab Report I. Introduction Bacterial plasmids are small, circular molecules of DNA that can be found in bacteria (Hass, Richter, Ward, 2014). Plasmids are physically separate and can replicate independently from the bigger bacterial chromosome; they are replicated by enzymes and transmitted to daughter cells during the process of cell division. Plasmids can be transferred by the process of transformation and conjugation (R. Giraldo, 2014). In the presence of some antibiotics, which are used to treat diseases, plasmids that have a selective advantage where some can contain genes that are resistant to the antibiotic (Hass, Richter, Ward, 2014). Through humans frequent use of antibiotics, we have significantly increased the frequency of antibiotic resistant genes. Cattle farmers and growers feed their livestock sub therapeutic amounts of antibiotics to function as a growth enhancer (Hass, Richter, Ward, 2014). One of the most frequently used antibiotics is tetracycline. Tetracycline is a drug that is commonly used to treat bacterial infections such as urinary tract infections, chlamydia, and acne (Drugs.com tetracycline antibiotics, 2014). Tetracycline affects bacterial growth because it interacts and interferes with a group of ribosomes that are responsible for translation therefore, inhibiting protein synthesis

2 (Hass, Richter, Ward, 2014). Since plasmids can spread by transformation and conjugation, bacteria that are resistant to antibiotics can reproduce rapidly in the presence of antibiotics. Reports of severe gastroenteritis caused by consumption of raw or undercooked meat led the FDA to investigate this outbreak (Hass, Richter, Ward, 2014). Scientists discovered that the Escherichia coli bacteria, which contains tetracycline resistant genes, was responsible for the outbreak. Three cattle farms, Speedy s Beef Farm (Farm A), Calli s Cows and Bulls (Farm B), and Green Acres Cattle (Farm C) were all contaminated by the Escherichia coli bacteria. A pressing question we must ask is whether each occurrence of tetracycline resistant bacteria is unique to each farm, or whether there is a shared source of contamination for these three farms. Scientists already know that three different genes are commonly known to be responsible for tetracycline resistance. These genes have risen independently of each other and can be differentiated by their individual sizes (Hass, Richter, Ward, 2014). It is important for us to know which specific gene is present at which farm because they require different treatments. Bacteria reproduce rapidly and can grow to very high concentrations which can sometimes be uncountable and are referred to as a lawn. For this reason, we used the technique of serial dilution to easily count the number of colonies. Serial dilution is multiplicative; with each dilution, we added 10 ul of bacteria to 90 ul of solution. Using the equation B=N/D where B is the initial population size, N is the number of colonies, and D is the dilution factor, the original concentration of bacteria can be calculated and can be found in the results section (Hass, Richter, Ward, 2014). To examine the plasmids containing Tetracycline resistant genes, we used a polymerase chain reaction (PCR) to replicate large amounts of specific DNA fragments. PCR utilizes the

3 DNA polymerase in bacteria and allows their enzymes to function at extreme temperatures which they would normally be denatured in. The DNA polymerase we used in this experiment was called Taq polymerase. PCR occurs in three steps; in the first, called denaturation, DNA is heated and separated into two strands. In the next step, annealing, the strands are cooled down and DNA primers bind to each end. Finally, in elongation, Taq polymerase binds free nucleotides to their base pairs and elongates the strand (Hass, Richter, Ward, 2014). To determine the tetracycline resistant genes from the samples of the cattle farms, we used gel electrophoresis to create distinct DNA fragments and compare them to the known tetracycline resistant genes. To do this, we ran a voltage across a gel with DNA placed into different wells. The DNA will travel from the negative end to the positive end; larger molecules will travel shorter distances while shorter molecules will travel further. We can then compare the sizes of these samples to the known sizes of Tetracycline resistant genes. The two main purposes in performing this lab and that guide our procedure is to determine whether the tetracycline resistant bacteria present was unique to each farm or came from a shared source of contamination as well as to determine the frequency of tetracycline resistant bacteria at each farm (Hass, Richter, Ward, 2014). By comparing factors such as tetracycline resistance frequency and gene size, we can determine if the tetracycline resistant bacteria came from a common source. By determining the frequency of tetracycline resistant bacteria, we can then determine the severity of contamination and take the necessary further steps.

4 After understanding the function that plasmids serve in bacteria, we developed a hypothesis that the tetracycline resistant bacteria is due to differing genes located within a plasmid that neutralize enzymes which regulate the tetracycline antibiotic. II. Materials and Methods The methods for this lab are explained in the Biology 110 Lab Manual. We begin by taking three tetracycline plates and labeling each adequately with our name, date, section, and dilution factor (10^ 2, 10^ 4, and 10^ 6). We then took three empty, sterile microtubes, added 990 ul of water to each, and performed our appropriate dilutions with each different dilution factor. We then lined up our three petri dishes and using a pipettor, inoculated each dish with 100 ul of sample bacteria based on appropriate dilution factor and using a new pipette tip for each. Then, using glass beads, we shook the dish back and forth starting with the 10^ 6 dish and working down to effectively spread the bacteria throughout the agar. After disposing of the beads in the biohazard bag, we stacked our petri dishes, taped them together, and labeled them. During the next lab, we put the bacterial colonies from the plates through PCR to replicate fragments of DNA. We began by using the orange, blue, and yellow primers that were provided and mixed these primers with the colonies; orange to colony one, blue to colony two, and yellow to colony three. We then proceeded to load these tubes into the PCR. Next, we counted the number of colonies growing on the tetracycline plate as well as the non tetracycline plate. By comparing these two totals, we can determine the percentage of cells resistant to tetracycline.

5 After PCR, the products underwent gel electrophoresis. We made the gel by weighing out 300 mg of agarose and mixing it with 30 ml of 1x TAE buffer, measured in a graduated cylinder, into a 125 ml Erlenmeyer flask. After swirling the mixture, we heated it for 35 seconds and let the solution cool for two minutes. After cooling, we put on disposable gloves and added 1 ul of ethidium bromide to the solution. We then poured our molten agarose into the gel tray after making sure the dams were snugly wedged into place. After allowing the gel to harden, we poured the electrode buffer into the unit and removed the comb. We loaded 5 ul of PCR DNA ladder into the first well, and then 15 ul from our six colored tubes into the preceding six wells and ran a current through it. Once the DNA fragments had traveled halfway across the gel, a photograph of the gel was taken. From the photo, we could tell the sizes of the tetracycline resistant genes from the three farms and compare this to the sizes of the known resistant genes. We then determined the frequency of tetracycline resistance and made a recommendation to the FDA on how to treat the problem (Hass, Richter, Ward, 2014).

6 III. Results Each lab group took pictures of individual plasmids after undergoing gel electrophoresis. This picture shows our plasmid sample from Farm B, or Calli s Cows and Bulls. Table 1: Number of Bacterial Colonies on +/ Tetracycline Dilution Plates (Farm B) Treatment Dilution Dilution Dilution 10^ 2 10^ 4 10^ 6 Volume Plated 100 ul 100 ul 100 ul Tetracycline No Tetracycline Lawn (Hass, Richter, Ward, 2014)

7 Table 2: Number of Countable Bacteria (Farm B) Number of Bacteria in Original Sample (B=N/D) Number of Bacteria in Original Sample (B=N/D) Number of Bacteria in Original Sample (B=N/D) Dilution Factor 10^ 2 10^ 4 10^ 6 Tetracycline 0 9.2x10^5 1.5x10^7 No Tetracycline Lawn 5.0x10^6 6.6x10^7 B=N/D Frequency= (B tetracycline/b non tetracycline)x100 =((N tetracycline/d tetracycline)/(n non tetracycline/d non tetracycline))x100 Sample Calculation for Farm B Group 1: ((92/10^ 4)/(66/10^ 6))x100 =1.39% Farm B Group 2=.016% Farm B Group 3=.028% Avg for Farm B=.48%

8 Table 3: Summary of Tetracycline Resistance in Beef Farms Group Beef Farm Tetracyclin e Resistant Gene Tetracyclin e Resistant Gene 1 (A) 2(B) 3(C) Tetracyclin e Resistant Gene Frequency of Resistance Comments 1 A X.11% Image did not show up well but a better image was given 2 B X.48% 3 C X.13% 4 B X.09% Band lined up with plasmid but also with another band that had no reference. (Hass, Richter, Ward, 2014)

9 Table 4: Recommendation Guidelines for Bacterial Contamination at Meat Producing Facilities Level of Contamination Standard Recommendations If the Bacterial contamination is < 1% Send all meat to a pasteurization facility elsewhere until contamination levels are leveled out to < 1% for 8 weeks or longer Farm must be monitored weekly The source of contamination must be identified Antibiotic regime must be changed If the Bacterial contamination is between 2% and 30% All meat must be disposed of safely until contamination levels are reduced to <1% for 8 or more weeks All those infected must be identified and properly treated Farm must be monitored weekly The source of contamination must be identified Antibiotic regime must be changed If the Bacterial contamination is > 31% Entire infected population must be identified and treated All meat must be disposed of in an orderly, safe fashion Entire facility must be completely disinfected The source of contamination must be identified A contamination prevention plan that entails how appropriate steps will be taken to hinder contamination in the future must be submitted (Hass, Richter, Ward, 2014)

10 IV. Discussion After analyzing the data present in our results, we can then answer and explain the research questions: Is the bacterial contamination at each farm due to the same gene or genes independent of each other? And what is the frequency of tetracycline resistant bacteria at each farm? We can answer the first question by interpreting the images of the gel electrophoresis of the plasmids from the three different farms. Since the photo for Farm B was presented in the results section, we can use this image as our example. By comparing the distance that the plasmid from Farm B traveled to the distance that the tetracycline resistant gene B traveled, we can see that these distances are identical. We know that the distance traveled is based off size; smaller plasmids will travel further distances and vice versa. We can infer that the these plasmids are the same size and therefore, the same gene. In farms A and C, we observed different band patterns than Farm B, however the distance traveled in Farm A matched that of Gene A and the distances in Farm C matched that of Gene C. With this information, we can conclude that all of three farms matched with different tetracycline resistant genes and did not come from a common source of contamination. This conclusion supports our hypothesis that the tetracycline resistant bacteria from the three farms all come from different genes.

11 To address the second question, we can reference Table 4 which provides the frequency of resistance for each individual farm. We were able to calculate the frequency by using the formula: Frequency= (B tetracycline/b non tetracycline)x100 and then expanding it to Frequency=((N tetracycline/d tetracycline)/(n non tetracycline/d non tetracycline))x100. While our lab group only calculated the frequency for Farm B, the other groups in the class calculated the frequencies for Farm A and Farm C. While one group of Farm B had the lowest frequency at.09%, the other group of Farm B had the highest frequency at.48%. Using Table 4, we can determine the severity of the level of contamination and the necessary steps we will have to take once we know these levels. All three farms had a level of contamination that was less than 1%, therefore all farms are recommended to follow the same steps. They should all move the meat that is currently in at the farm to an alternative facility until the contamination levels at at a constant 1% or less for 8 or more weeks. The farm should also be monitored weekly and change its antibiotic regime. Most importantly, the source of contamination should be identified and eliminated to ensure that it does not cause contamination again (Hass, Richter, Ward, 2014). While all three farms had different sources of contamination and different tetracycline resistant genes, they all must take similar steps to prevent further or future outbreaks. Sources of error in this lab could have existed in numerous places. Most obviously, we could have forgotten to wipe down our lab station before performing the experiment. This could have led to contamination and could have skewed our results. A common source of human error could have simply been in counting the colonies of

12 bacteria. This would have skewed the numbers for the frequency of resistant genes and the level of contamination. Another error that could have occurred is in the precise measurement we had to take during serial dilutions, PCR, and gel electrophoresis. If we measured too much or too little of something, it could have affected our DNA replication in PCR or how far the plasmids traveled in electrophoresis. This could have a great influence on our results. For future experiments, we can further test and examine the frequency of antibiotic resistant plasmids and genes. This is a very important topic because if the frequency for an antibiotic resistant gene increases to a very high number, then we have no method of treating the infected organism or product. One possible experiment could be to examine a gene with antibiotic resistance and try to determine what exactly makes it resistant and if there are any selective pressures to make the gene act a certain way, and also if there are ways that we can manipulate the gene to make it non resistant ( AUPA, Tufts). If we can figure out why genes are resistant and nonresistant and manipulate them to behave a certain way, it would be a huge breakthrough for the medical community and we could treat infected patients more effectively.

13 Works Cited Biology 110 Laboratory Manual 2014 Del Solar, Gloria, Rafael Giraldo, Maria J. Ruiz Echevarria, Manuel Espinosa, and Ramon Diaz Orejas. "Replication and Control of Circular Bacterial Plasmids." N.p., n.d. Web. 22 Nov Nature.com. Nature Publishing Group, n.d. Web. 29 Nov "Tetracycline (Antibiotics) Uses, Dosage, Side Effects Drugs.com." Tetracycline (Antibiotics) Uses, Dosage, Side Effects Drugs.com. N.p., n.d. Web. 29 Nov