The Effects of the Artificial. Selection of Escherichia coli. for Resistance to Ampicillin

Transcription

1 1 William Fescemyer Lab Partners: Nolan Spahr, Levi Peterson, Emily Stankiewicz Bio 220W Sec.001 Dr. Julian Submitted on 3/28/2013 The Effects of the Artificial Selection of Escherichia coli for Resistance to Ampicillin

2 2 Abstract: In this experiment, we studied the relationship of the resistance of Escherichia coli to the antibiotic ampicillin. The goal of this experiment was to test if we could artificially select bacteria from E. coli colonies exposed to ampicillin to become more resistant to the ampicillin across a period of four weeks. Our hypothesis was that the bacteria would become more resistant over that period, and thus be able to grow closer to the ampicillin, causing a reduction in the zone of inhibition. To do this, we plated and grew multiple colonies, one group of bacteria from along the edges of the zone of inhibition, our selection group, and then plating another from bacteria furthest from the antibiotic, our control. This will show how the bacteria E. coli can evolve to ampicillin, an antibiotic often used to treat E. coli in patients, allowing pharmaceutical companies to better understand how it evolve, and find new ways of combating it. It also helps prove the theory that humans are capable of selecting and breeding traits over time, into the bacteria E. Coli. We did not find that the E. Coli bacteria was able to adapt effectively to the Ampicillin, and did not become more resistant over time. The final zone of inhibition measurements of our selection group ranged from 19 to 23 mm, showing no change from the beginning zone of inhibition measurements, ranging from 19 to 24 mm. The control and the selection s zone of inhibitions also remained about the same, the final control ranging from 19 to 24 mm showing that there was no development of resistance. Introduction: Escherichia coli are bacteria that cause a wide variety of afflictions in humans, mostly intestinal in nature. E. coli is a quick growing and hardy bacteria, which adapts

3 3 quickly to new environmental conditions, and so is ideal for a study such as this (E-coli 2011). We also chose E. coli, as in the bacteria resistance to Ampicillin is heritable. Ampicillin resistance is heritable through three genes. When the gene glutamine decarboxylase overexpressed it can provide resistance, but when expressed normally it does not affect the resistance of the bacteria (Adams 2008). In E. coli, the endogenous B- lactamase gene can provide resistance when it is activated by chance, and can provide very potent resistance to the ampicillin (Adams 2008). The gene DAM methylase, a gene that regulates the expression of genes, such as the multidrug efflux pimps, which can cause an increase in resistance (Adams 2008). As these genes can be passed on through reproduction, they can cause evolution as allele frequencies for these genes change. If the ampicillin creates a selective pressure on the E. coli bacteria, then selecting bacteria near the ampicillin to select the more resistant individuals of the E. coli to the antibiotic so that they may breed young with a similar resistance, and thus grow closer to the ampicillin the following generation, reducing the zone of inhibition over time. We expect this because a similar study was conducted, and found results that reflect this conclusion, which was conducted on the E coli bacterium, studying epigenetic inheritance of resistance (Adams 2008). The results can also be expected as we are selecting the bacteria with the higher expression of the genes that control for the resistance, as if they are living closer to the anti-biotic, they have a higher probability of expressing those genes. If this hypothesis is correct, then the zone of inhibition around the Ampicillin will decrease, as the bacterium is more resistant to the antibiotic, or at least is more likely to inherit genes. This experiment can add additional understanding to how bacteria inherit traits, and help increase the understanding of how bacteria reproduce (E-coli 2011).

4 4 Methods: In this experiment we grew colonies of the bacteria E. coli, through the use of the aseptic technique, using Muller-Hinton agar plates we grew four generations of the bacteria, with four plates of each of the selection and the control. Each plate had four ampicillin tablets emplaced in the agar, which each created a zone of inhibition. To achieve the goal of the experiment, we attempted to create a control by using the bacteria from the outer edges of the Petri dish, as they are the furthest from ampicillin, and so should be the least heavily affected by the antibiotic. The selection was selected from the edge of the zone of inhibition, from the bacteria closest to the ampicillin tablets. The two bacteria were then cultured at optimal temperature in test tubes over a twentyfour hour period, then plated onto the agar plate, which they were then allowed to grow on for another twenty-four hours. Then the plates were prepared for measurement. The zones of inhibition were measured the day after they were plated on the Petri dish. This was continued over four weeks, and over each generation the zone of inhibition was measured from the visually widest part. To create the final data for the lab, the four measurements of each of the four Petri dishes per week of the selection were averaged together to get an average for that group of the week. Then the standard deviation was taken to show if there were any outliers, and to show the precision of the results. The results were then graphed, and the standard deviation added as bars (Figure 1). The final data of the fourth week was also turned into a bar graph, with standard deviation bars to show the outliers. An ANOVA test was then conducted to test and see if the results were due to chance, or were because of the experiment.

5 5 Results: The difference between the control and selected was very small. The difference between the control and selection s zone of inhibition in the final generation was approximately 1 mm, with the selection being larger by that value. The graph shows a slight increase in the zone of inhibition, approximately 3 mm by the end of the experiment (Figure 1). The change was very small and so it will create a large P-value, greater than the target value of 0.05, due to the degrees of freedom for the experiment being at 23. The P-value for our experiment was.349, greater than the target value. According to the ANOVA test, the F-value is.915, indicating the difference between groups (Figure 4). The bar graph of the final generation of plating, showing the final difference between the control and the selection shows the final difference between the control and selected, the selected being larger by a margin (Figure 3). The bars showing the statistical deviation show that there is very little variation between the averages that we took of the data. Helping to confirm our results, another two groups, the blue and white, did an identical experiment, following the same method. Their data is very similar to our own, as shown by the table, with the data of the white and blue groups having no apparent outliers like our own data (Figure 5). The other group s data does show there is an outlier at the very end of our data. The other group s data shows similar results to our own, and helps confirm the accuracy of the data we gathered. Discussion:

6 6 With the ANOVA test showing the data having a p-value above 0.05, there is not enough of a difference to warrant a conclusion. If there is not enough of a difference to prove the hypothesis, then the null hypothesis must be accepted, meaning that E. coli cannot be artificially selected for resistance. The graph seems increase slightly, seeming to indicate that there is a slight increase, very different from the hypothesis which predicted a decrease in the zone of inhibition, possibly meaning that artificial selection is not possible for E. coli. This does match up with the other studies, as the other study on this topic found that the chemicals associated with resistance increased (Adam 2008). This study may show that ampicillin will be viable to drug companies for a much longer time, and will be able to continue to be used to treat E. coli infections for much longer to come. It will help pharmaceutical companies knowing that there is no risk of resistance being bred into E. coli (Ampicillin Oral 2010). Some possible explanations of these results could be related to the selective pressure of the antibiotic. It would be possible that the amount of anti-biotic used was too high, forcing bacteria to develop a very high level of resistance before being able to move into the zone of inhibition. Perhaps the amount of resistance the E. coli needed to develop creates an evolutionary tradeoff that is too much for the cell to survive. It could also be possible that the reversion rate is high enough that between plating s cause the bacteria that did not have resistance growing faster than the ones with resistance, and out competing them while we grew them in the test tubes (Adams 2008). The other study conducted on this bacterium said that they found reversion rates to be very high, so it is a conceivable that it was reverting during the generation gap (Adams 2008). As the other groups have similar data to ours, it precludes error, unless all three groups made an error. The hypothesis I have developed

7 7 that I believe caused this data is that there simply wasn t enough time for the bacteria to develop capable resistance to ampicillin. Four weeks is a very short time in terms of evolution, given the assumption that evolution occurs due to random chance (Adams 2008).

8 8 Figure 1: Graph of Zone of Inhibition at Each Week For Individual Group Figure # 3 Bar Graph of Week Four Averages

9 9 Week Control (mm) Selection (mm) Red White Blue Figure # 4 Average Zone of Inhibition for Class after 4 weeks

10 10 AveZOI Sum of Squares df Mean Square F Sig. Between Groups Within Groups Total Figure # 5 ANOVA test for our Groups Data Bibliography: Ampicillin oral. (2010, 09 01). Retrieved from E. coli (Escherichia coli). (2011, 08 26). Retrieved from Summers, A. (2006). Genetic linkage and horizontal gene transfer. Animal Biotechnology, 17(2), Retrieved from