Comparing the Effect of Different Antibiotics on Gram-Negative Bacteria Culture - Research Paper Example

 Comparing the Effect of Different Antibiotics on Gram-Negative Bacteria Culture I. Abstract This paper details the results of an experiment comparing the effect of two antibiotics, ampicillin and streptomycin, on bacterial cultures of Serratia marcescens. Two sets of ten agar plates each with the bacterial culture are tested versus filter paper discs containing either ampicillin or streptomycin, and the resulting sizes of the discs created by the reaction of the bacteria to the antibiotics are measured. The data is subjected to statistical tests to determine mean sizes, differences between the means of the two sets of data, and the statistical significance of the differences. The experiment takes the hypothesis in line with the predictions in the literature, that streptomycin has the greater effect on the bacteria culture, owing to the developed resistance of serratia cultures to ampicillin. The tested prediction of the hypothesis is that owing to the nature of the bacteria culture, which has less cell wall, it is streptomycin which will have the greater impact and greater efficacy on the bacteria culture compared to ampicillin. This flows from the hypothesis that the greater efficacy belongs to streptomycin, and this also matches the expected results of the study, which is that the culture disc diameter for streptomycin will be of bigger in comparison with the corresponding disc diameter for ampicillin cultures. The results of the experiment validate the hypothesis as well as the findings in the literature (GraphPad; Schaefler; Haddix and Werner; Olexy et al.; Davis). II. Introduction The objective of the exercise is to be able to compare the effect of two different types of antibiotics on gram-negative bacteria culture. These two antibiotics are streptomycin and ampicillin. (Davis; Haddix and Werner) Serratia marcescens, the bacteria used for this experiment, is said to be ideal for the study of bacterial resistance to antibiotics for a number of reasons. One is that the literature has documented varying ampicillin resistance and streptomycin resistance abilities of the bacteria. This means that an experiment can validate contrasting resistance for the two antibiotics. Two is that it is relatively common, and its modes of actions and effects on humans have been widely studied and discussed in the literature. For instance, it is known that it has differing activities for a number of antibiotics outside of ampicillin and streptomycin too. It is known to infect people almost exclusively in hospital settings. It targets mainly the urinary tract, and wounds and the respiratory system to a lesser extent. The bacteria is also known to be localized in their location in the human body too. For instance it is not found in the human stool almost always, and is limited in their location to the infection sites listed above (Schaefler et al. 339). Elsewhere in the literature, serratia marcescens is also singled out as an excellent bacteria for use in studies on antibiotic resistance, because its mutations are easily identified, isolated, and characterized (Haddix and Werner 21). It is also known to be very adaptable and malleable for a number of classroom and laboratory, educational purposes (Haddix and Werner 31). Streptomycin is considered to be the first among a class of antibiotics known as aminoglycosides. It was discovered in 1944, and has been thoroughly investigated in terms of its antibiotic actions (Davis 341). The mechanism of action of streptomycin is varied and is now established to occur in many stages. The literature describes four such ways or sets of effects/stages. These are the blockade of the ribosomes effect; the effect of translation misreading; the damage to membranes; and uptake of streptomycin by the bacteria that cannot be reversed. Translation misreading, as a highlight stage, has to do with bacteria misreading protein and then including that protein into its own membrane. This action eventually compromises the internal consistency and integrity of the bacterial organism. On the other hand, while damage to bacterial membranes had been identified, together with the other three effects listed above, the exact mechanisms of action of streptomycin were poorly misunderstood in early research. This was the state of things until insights into such mechanisms yielded a new hypothesis. This hypothesis is that the way streptomycin effects antibiotic action happens in a number of stages, with the four effects listed above all playing a role in the multi-stage process (Davis 341). On the other hand, ampicillin is considered a beta-lactam antibiotic, with a beta-lactam ring that is the active antibiotic factor. It targets cellular transpeptidase enzymes in bacteria. These enzymes are essential to the integrity of bacterial cell walls. The mode of action of ampicillin is said to be related to its ability to inhibit the synthesis of such cell walls, via its actions on the transpeptidase enzymes (Haddix and Werner 27). This effect is curious for the way it targets specific cells. The literature notes that this effect is pronounced in bacterial cells that have no mutation, while not as pronounced in bacterial cells that have mutation. In this way its action is selective. That selective action is shown to enhance the proliferation of mutated bacterial cells. This can be a mechanism that has resulted in bacterial mutations of serratia cultures to resist the antibiotic action of ampicillin (Haddix and Werner 29). We expect ampicillin to not show as large an effect on the bacteria culture. This expectation springs from the fact that the literature has thoroughly documented resistance of serratia marcescens cultures to ampicillin (Haddix and Werner; Olexy et al.: Schaefler et al.). In one study for instance, it has been found that for several hospitals surveyed and examined in one geography, there are similarities in the strains of bacteria that yielded resistance to a host of antibiotics. Among these antibiotics is ampicillin. This study underlines the susceptibility of patients to infection from bacterial agents that are able to resist the antibiotic actions ampicillin and other antibiotics. This also stresses the need to use other antibiotics to which bacterias of note are unable to develop resistance. This particular study focused on the relationship between the use of antibiotics like ampicillin and the development of resistance to such antibiotics by serratia marcescens (Schaefler et al.). This finding of the resistance of ampicillin to the bacteria culture that we used is not isolated to that one study above, but is confirmed in other studies as well. Ampicillin is not the only antibiotic to which serratia has grown a tolerance and resistance for. It is one of several prominent antibiotics (Olexy et al.; Haddix and Werner). The literature includes studies where samples of serratia marcescens from thirteen different samples were found to have developed resistance to a host of antibiotics as well. Those include ampicillin and tobramycin. These findings confirm that some mutations of serratia are resistant to ampicillin (Olexy et al). III. Methods For this experiment, ten agar plates were allocated for streptomycin, ten for ampicillin, and one control agar plate was reserved as control. These add up to a total of 21 agar plates. The lone control agar plate did not have an antibiotic, and it served the purpose of ensuring that the serratia bacterial culture is alive and able to thrive. The agar plates were spread with the serratia culture making use of a micropipettor, with the calibration at 200 microliters. The liquid bacterial culture was then retrieved from the bacterial source and then placed on each of the 21 plates of agar. To make sure that the bacterial culture is spread all over the agar surfaces, a spreading rod was used. Each of the agar plates were rotated two times and the spreading rod was used each time to spread the culture across the surfaces, for rigor. Grade 5 Watman Filter paper was cut into 20 discs each measuring 15 mm in diameter. Ten discs were dipped into 2mL of 100mg/mL of ampicillin making use of a pair forceps, and then drained, before being placed individually in the ten plates allocated for ampicillin, at the center of each of the ten plates. Using another set of forceps to avoid the mixing of the two antibiotics, the same procedure is repeated for streptomycin. For the positive control agar plate, a disc of the same size is dipped in water that is distilled, and then placed in the same manner at the top of the control plate, as in the twenty plates with antibiotics-dipped filter paper discs. IV. Results The prediction from the hypothesis and from a review of the literature conducted earlier is that those bacterial culture plates containing the discs with streptomycin would have larger measured disc diameters in comparison to those plates to which ampicillin-soaked filter papers were placed. This is because we expect the size of the resulting discs on the plates to correlate with bacteria control. Given that we hypothesized the greater efficacy of streptomycin and the reduced efficacy of ampicillin due to documented resistance of the serratia culture to it, the predicted observation is that the sizes of the discs for the streptomycin plates will be larger compared to the ampicillin discs (Davis; Schaefler; Haddix and Werner; Olexy et al.) The data shows us that there are significant differences in the sizes of the resulting discs for the streptomycin plates and for the ampicillin plates, with the sizes of the discs being larger for the former plates, as predicted (Davis; Schaefler; Haddix and Werner; Olexy et al.) We take the mean of the averages for each of the two sets of plates above and come up with an average disc size of 2.899 for the ampicillin plates and an average of 4.592 for the streptomycin plates. From these two averages we get a difference in resulting disc size of 1.698. We perform an unpaired t-test statistical analysis on the two data sets to find out if the differences in the means of the averages for the two data sets are significant. We take the first nine averages for streptomycin and compare that with the nine averages available for ampicillin and input those as data for an unpaired t-test. The following are the resulting intermediate values: t = 21.765; df = 16; standard error for the difference = 0.078 (GraphPad) For these values, the resulting value of P is revealed to be lower than 0.0001. This implies that the statistical test reveals that the difference in the means of the averages is very significant from a statistical point of view (GraphPad). Looking at the resulting confidence interval, meanwhile, we get that, as stated above, the differences in the means of the two sets of averages as reflected in the tables is 1.698. The 95 percent confidence interval for this figure is found to range from 1.53314 to 1.86398 (GraphPad). The following is an overview of the standard statistics for the data table above, with the data being imputed into the t-test that was conducted, and whose results have been discussed, above (GraphPad): Group Group One Group Two Mean 4.59811 2.89956 SD 0.15121 0.17871 SEM 0.05040 0.05957 N 9 9 Source of Computation Results: GraphPad V. Discussion The results of the study validate the predictions from the literature and the predictions of the hypothesis with regard to the greater efficacy of streptomycin as compared to ampicillin for controlling the growth of the chosen bacterial culture for this experiment, namely serratia marcescens. The data and the resulting statistical analysis shows that the differences in the means of the averages for the disc sizes for the two sets of plates show that the disc sizes for the streptomycin plates are larger in comparison to those for the ampicillin plates Moreover, the t-test shows that the differences between the two means are very highly significant, meaning that indeed, there is a significant difference in the size of the discs for the streptomycin plates compared to the ampicillin plates, Moreover, the t-test confirms that we can make the conclusion that streptomycin better controls serratia marcescens. Put another way, there is less resistance to streptomycin by serratia marcescens in comparison to ampicillin (GraphPad; Davis; Schaefler; Haddix and Werner; Olexy et al.). VI. Literature Cited GraphPad. “QuickCalcs Unpaired t-test results”. GraphPad Software. 2013. Web. 26 February 2013. Davis, Bernard. “Mechanism of Bactericidal Action of Aminoglycosides”. Microbiological Reviews. September 1987. Haddix, Pryce and Terry Werner. “Measurement of Mutation to Antibiotic Resistance: Ampicillin Resistance in Serratia marcescens”. Bioscene 26 (1). February 2000. Web. 26 February 2013. Olexy, VM et al.. “Hospital isolates of Serratia marcescens transferring ampicillin, carbenicillin, and gentamicin resistance to other gram-negative bacteria including Pseudomonas aeruginosa.”. PubMed/Antimicrob Agents Chemotherapy. 1979. Web. 26 February 2013. Schaefler, S. et al. “Specific Distribution of R Factors in Serratia marcescens Strains Isolated from Hospital Infections”. Applied Microbiology 22 (3). 1971. Web. 26 February 2013. VII. Appendix See Excel sheet for data Read More