The Effects of Streptomycin and Ampicillin on Serratia Marcescens - Report Example

The Effects of Streptomycin and Ampicillin on Serratia marcescens March 2, While carrying out comparison tests on the effectiveness of antibiotics, specialists or professionals in the stated field have in the past usually made use of certain particular methods in performing the same. One such method is the Disc Diffusion method which measured the effects of any antimicrobial agent against bacteria growing in culture. Basically, it tested whether the said bacteria had any susceptibility to the specific antibiotics. In this study, while comparing the effectiveness of these two antibiotics under study- Streptomycin and Ampicilin- on the bacteria Serratia marcescens, the Disc Diffusion method was applied as an appropriate method in obtaining the strain resistance patterns of the bacteria. I grew a particular amount of bacteria on the agar plates provided supported by thin wafers that contained the relevant antibiotics as recognized in this study. From the results of the synthesis, it was apparent that some strains of the bacteria were resistant to certain plates thereby suggesting the presence of different antibiotic concentrations of resistant genes within a specific strain used in the experiment. The overall result of the experiment was that streptomycin is 24.4% more effective on Serratia marcescens than ampicillin; agar plates treated with streptomycin had an average inhibited bacterial growth diameter of 45.4mm, whereas those treated with ampicillin had an average of 36.5mm. Hence form the size of the observed zone inhibitions, the bacteria’s sensitivity to the antibiotics was estimated with a larger zone correlating with a small minimum inhibitory concentration (MIC) of the bacteria’s antibiotic. Introduction: Determination of the effects of the streptomycin and ampicillin antibiotics on the Serratia bacterium have been clearly shown by the agar plate tests performed. The Serratia marcescens, also known as the chromobecterium prodigiosin, is a gram-negative facultative anaerobe. Serratia marcescens emerged in the late centuries as a nosocomial pathogen. An opportunistic organism often functions in immunocompromised patients due to its invasive ability to tolerate the hospital instrumentations including endoscopes, catheters, and intravenous tubing (Davis, 1987). The Serratia marcescens is among the major nosocomial pathogens that are associated with respiratory and urinary track infections, meningitis, endocarditis, wound infections, osteomyelitis, septicemia, and eye infections (conjunctivitis). The Serratia marcescens is the only known pathogenic species in its genus. It inhabits varied ecological niches and it causes diseases in vertebrates, plants, and invertebrate hosts (Haddix et al., 2000). In determining the inhibition zone with regards to the measure of the compound’s effectiveness, this study noted that, the biological mechanisms that lead to the antibiotic resistance are often reversed. However, such phenomena can be classified into three main categories including the bacterial enzyme degradation of the drug. Under normal conditions, the antibiotic often functions as an irreversible trans peptidase enzyme inhibitor with a cross function linked to other bacterial cell walls. Secondly, the bacteria can initiate mutations thereby rendering the target module incapable of interacting with antibiotic. Streptomycin is an aminoglycoside antibiotic that is effective to for a wide range of bacteria (Pollack, Findlay, Mondschein, and Modesto, 2002). Effective laboratory conditions often enable the selection of the spontaneous streptomycin resistant E coli. Finally, it is worth noting that changes in the permeability of the bacterial cell to an antibiotic can confer antibiotic resistance. The Gram-negative bacteria emanated from the mutations of the membrane proteins a process that was facilitated by the resistance of Ampicillin. The outer membrane usually produces proteins thereby acting as a molecular sieve that in turn allows diffusion of molecules thereby allowing molecules into the cell depending on the charge and size of the molecules in question (Davis, 1987). Therefore, mutation of the membrane proteins or Ampicillin resistances reduced the ability of transportation of drugs across the cell membrane. Ampicillin’s mechanism of action is to attack the enzyme trans-peptidase, which is responsible for the synthesis of the bacterium’s cell wall, and since a gram-negative bacterium’s cell is coated with lipopolysaccharide, the effect of ampicillin on it is less. The disc diffusion is a sure classical test that can be employed quantitatively to measure the spontaneous mutation rate. The agar plate allows for the individual mutation into mutant colonies with the non-mutant cells from different cultures killed. This method can be related to the fluctuation test that is driven from the observation of what separates cultures. According to the observation, cultures often generate from minute inoculum that are defined by a small number of cells with each producing the different numbers of mutant colonies (Davis, 1987). The low number of fluctuation in the mutants indicates that there is a low probability of random occurrence. In this experiment, the effectiveness of the two antibiotics, Streptomycin and Ampicillin, is tested on the bacteria Serratia marcescens. Since this bacterium’s cell is coated with lipopolysaccharide, it can be expected that it will show more resistance towards Ampicillin than Streptomycin. Methods: The workstation was sterilized using 70 percent concentrated ethanol in order to reduce any form of contamination that may lead to error or inaccurate result of the experiment. The bacterial cultures were brought from Carolina Biological Supply and had a strain number of D1 #155455. The control was set up using two strains of ampicillin. The set up was checked for streptomycin resistance and this was followed by one positive control that was facilitated by checking the viability of the experiment. After determining that the experiment was viable, the discs were dipped into the deionized water after which they were placed on the control dishes that acted as a frame of reference or standard. This was followed by labeling 20 plates: 10-ampicillin, and 10-streptomycin. I transferred 100 microliter of the deionized water to facilitate transfer from the parent culture into the plates. After this, the plastic spreader was used to spread the bacteria sample across the plates evenly. Notably, the spreader removed any formed bubbles. The paper discs were dipped into the 100 mg/ml ampicillin or streptomycin using sterilized tweezers. The paper discs were placed in their corresponding discs at the center of the dish and was straightened to ensure the disc was completely in contact with the agar. Finally, the dishes were sealed in groups of five and incubated. It is worth noting that the incubation was 30 degrees Celsius in temperature and left to run without disturbing the setup for 24 hours after which the temperature was adjusted to 4 degrees Celsius until the data was collected. The antibiotic susceptibility test was conducted checking the antibiotic resistance of the ampicillin. On the other hand, the disc diffusion technique was employed to determine the susceptibility of the Serratia marcescens. Notably, the nutrient agar medium was used for bacterial isolation towards obtaining pure cultures. Basically, throughout this whole process, I measured the diameter of the clearance. Results: The results of this experiment can only be understood comprehensively if the information is computed into graphs as indicated in the graph below (Fig. 1). Fig. 1: Comparison of the average diameter of agar plates treated with the antibiotics, streptomycin and ampicillin. The error bars characterize one standard deviation above and below the mean of each diameter. It can be clearly seen from the graph that the average diameter of inhibition by the antibiotic streptomycin is higher than that by the antibiotic ampicillin. The agar plates treated with streptomycin had an average diameter of 45.4mm, whereas those treated with ampicillin have an average diameter of 36.5mm. These two numbers have a difference of 8.9mm. This indicates that streptomycin is 24.4% more effective on Serratia marcescens than ampicillin. Furthermore, it is notable from Fig. 3 (appendix) that the effectiveness of streptomycin is not only greater in average but also in each individual plate. In all the ten agar plates, streptomycin rated higher (Fig. 3. Discussion: In this experiment the effectiveness of each of the antibiotics, streptomycin and ampicillin, on the gram-negative bacteria, Serratia marcescens, was tested. The sensitivity of the bacteria to these antibiotics is directly proportional to the diameter of inhibited growth obtained from the disk diffusion method. In other words, if the diameter obtained was great, then the bacteria is sensitive to this antibiotic. As expected, the result of this experiment shows that the antibiotic streptomycin is more effective than ampicillin. This can be proven due to the fact that the average diameter of inhibited bacterial growth of plates treated with streptomycin was higher than those treated with ampicillin by 24.4%. Besides that streptomycin inhibits bacterial protein synthesis by binding to the 30S ribosomal subunits directly, the reason behind its higher effectiveness on gram-negative bacteria than ampicillin is that it has the ability to penetrate the outer membrane of gram-negative bacteria due to the presence of amino group in it structure (Chen, J. et al., 2003). Furthermore, according to a similar experiment carried by Werner et al. “Ampicillin Resistance in Serratia marcescens,” plates treated with ampicillin have a small white zone. Since our results were the same, this implies that the bacteria Serratia marcescens shows resistance towards ampicillin in general. These results can be best explained by the mechanism of action of ampicillin, where it attacks the enzyme trans-peptidase, which is responsible for the synthesis of the bacterium’s cell wall. It can be seen from the figure below (Fig. 2) that the outer membrane of a gram-negative bacterium is mainly composed of a lipopolysaccharide. Because of such structure, the effectiveness of ampicillin is less on gram-negative bacteria. Fig. 2: Composition of the cell wall of a gram-negative bacterium. (Source: http://classes.midlandstech.edu) Additionally, colonies of Serratia marcescens that escaped the effectiveness of ampicillin may show a mutation. Bacteria generally exchange their genetic information. Because each gene codes for a certain protein, if a bacterium picks up a new gene, this gene will code for a different protein. The same happened with ampicillin resistant bacteria; bacteria that show resistance to this antibiotic may have exchanged or picked up a gene from the environment that resulted in the formation of the enzyme beta lactamase. Beta lactamase is an enzyme that breaks down chemicals with beta-lactam ring, and since ampicillin has such ring in its structure, Serratia marcescens was able to degrade most of the ampicillin and reproduce (Bush, 1997). Appendix: Fig. 3: comparison of the diameter of each agar plate treated with the antibiotics, streptomycin and ampicillin. Table 1: Rank Sum Test on the effects of streptomycin and ampicillin on Serratia marcescens. Ho: There is no significant difference in the effectiveness of streptomycin and ampicillin on Serratia marcescens. Ha: There is a significant difference in the effectiveness of streptomycin and ampicillin on Serratia marcescens. Test Statistics = 55 Tabular statistics= (n1 = 10, n2 = 10; α = 0.01) = 71 Since the tabular statistics is larger than the test statistics, the null hypothesis is rejected; thus, there is a significant difference in the effectiveness of streptomycin and ampicillin on Serratia marcescens. *The mean was calculated using the formula: Xi = individual data items n = the number of data in the sample For Streptomycin, mean= 45.4 For Ampicillin, mean= 36.5 Percentage difference= [(45.4-36.5)/36.5]*100 = 24.4% *The standard deviation was calculated using the formula: Xi= individual data items Xi = mean n = the number of values For Streptomycin, S= 2.510478042 For Ampicillin, S= 1.841195264 References Bush, K. (1997). The evolution of beta-lactamases. In D. Chadwick (Ed.)., Ciba Foundation Symposium 207. Antibiotic resistance: origins, evolution, selection and spread (pp.152-166). New York: John Wiley and Sons. Chen, J. et al. (2003). Multidrug Resistance in Serratia marcescens and Cloning of Genes Responsible for the Resistance. Pharmaceutical Society of Japan. 26(3) 391—393. Davis, B., D. (1987). Mechanism of Bactericidal Action of Aminoglycosides. Massachusetts: Harvard Medical School. Haddix et al. (2000). Measurement of Mutation to Antibiotic Resistance: Ampicillin Resistance in Serratia marcescens. Laclede: Harris-Stowe State College, 26(1). Pollack, R. A., Findlay, L., Mondschein, W., & Modesto, R. R. (2002). Laboratory exercises in microbiology. New York, N.Y: John Wiley & Sons. Read More