Antibiotic Effectiveness Comparison - Research Paper Example

Antibiotic Effectiveness Comparison: To Compare the Effects of Streptomycin and Ampicillin on Serratia Marcescens Using Disc Diffusion Abstract Metal tolerance and antibiotic patterns of Serratia marcescens can be obtained from different sources for analysis. Using the standard minimum inhibitory concentrations (MIC) for these samples, the Kerby Bauer disc diffusion methods are applied as an appropriate method to obtain the antibiotic Serratia strain resistance patterns and the MIC of the metals. The metals that may be detected in these samples include chromium, cobalt, copper, cadmium, and lead. Plasmid curing was also conducted for the specific metal and antibody resistance to confirm the plasmid born transfer within the resistance genes. From the results, it is apparent that the multi drug resistant (MDR) strains of Serratia are resistant to certain plates or metal thereby suggested the presence of certain specific metal antibiotic resistant genes within different strains. Key words: Disc diffusion, Multi drug resistance, minimum inhibitory concentration, and plasmid curing Introduction Microorganisms often display some ubiquitous nature despite being involved in barely all biological processes of human life among other lives. Rapid natural and urbanization processes, the heavy metal are increasing in proportion in the natural habitat of the microbe. Metals are known to play a significant role either indirectly or directly in the essential metabolic processes including growth and development of microorganisms (Pollack, Findlay, Mondschein, and Modesto, 2002). Nonetheless, the elevated concentration of metals beyond certain tolerance levels often forces these organisms to adapt to different biological mechanisms so that they can cope with the changed environmental conditions (South African Association for the Advancement of Science, 1903). Therefore, under new environmental conditions created by increased metal concentrations, the microbes often develop new mechanisms such as metal influx systems, reduction in metal ions, complexation, or the utilization of metal as terminal electron receptors in the anaerobic respiration in cases of accumulation of heavy metals. The Bacteria that may create resistance to the heavy metals often play a vital role in their biological cycling thereby make them to have great potentials in the bioremediation of cells (Versalovic and American Society for Microbiology, 2011). The tolerance of heavy metals have been considered or observed in Serratia marcescens a member of the Enterobacteriaceae, therefore, Serratia marcescens has been thought of to be contributing to the resistance of plasmid borne genes (Pollack, Findlay, Mondschein, and Modesto, 2002). Different isolated microorganism strains have since been used to test the tolerance of different heavy metals against numerous metal salts with the aims of identifying specific strains that can be used to isolate certain metals from environments including water where they are often present as pollutants (Willey, Sherwood, Woolverton, Prescott, and Willey, 2011). The antibiotic susceptibility has also proved to a major health concern in that it has ever-increased health hazard because of the indiscriminate use of the antibiotics. This state of events has led to severe complication in many patients especially with diagnosed with gram-negative bacterial infections since numerous drugs there are limited drugs to combat this condition (Pollack, Findlay, Mondschein, and Modesto, 2002). Additionally, the multidrug resistance (MDR) may be may also be attributed to by an alternative mechanism of accumulation of the multiple antibiotic resistance genes. In most cases, these are often code for a single antibiotic within specific resistance (R) plasmids. Notably, the Multi Drug Resistance organisms have proved to a major challenge since they have been posing a huge threat in most of the treatment procedures since the presence of plasmid borne mobile resistance genes spread readily throughout the entire efflux system and population towards encountering the third and the fourth generation cephalosporin (BioSciences Information Service…, 1971). The Serratia marcescens, also known as the chromobecterium prodigiosin (BioSciences Information Service…, 1971) 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. 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 (Willey, Sherwood, Woolverton, Prescott, and Willey, 2011). 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. For instance, the enzyme beta lactamase can interact the resistance of the beta lactam antibiotic ampicillin (Ap). Under normal conditions, the Ap often functions as an irreversible trans peptidase enzyme inhibitor with a cross function linked to other bacterial cell walls (Excerpta Medica (Firm), 1971). 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. Therefore, any resistance to the Streptomycin Protein Synthesis Inhibitor leads to mapping of rpsl gene that encodes the ribosomal protein (Willey, Sherwood, Woolverton, Prescott, and Willey, 2011). 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 can emanate from the mutations of the membrane proteins a process that can be facilitated by the resistance of Ampicillin (Apr) (BioSciences Information Service…, 1971). 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. Therefore, mutation of or on Apr reduces the ability of transportation of drugs across the cell membrane. The disc diffusion is a sure classical test that can be employed quantitatively to measure the spontaneous mutation rate. The separation of plate into different media allows the individual mutation of 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 (American Chemical Society, 1907). 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. The low number of fluctuation in the mutants indicates that there is a low probability of random occurrence. From this principle in the experiment, it is clear that factors that will affect the surface of the membrane shall affect migrations either into or out of the cell (Willey, Sherwood, Woolverton, Prescott, and Willey, 2011). Materials and Methods The materials that were used in the experiment include 23 plates and three-sterilized tweezers (they are sterilized to avoid trace contamination since the experiment is qualitative. There was also plastic spreaders, ethanol (70 percent concentrated), microleters pippeters ranging from 20 to 200mg, deionized water, paper discs, and Serratia marscens parent culture. Methods The workstation was sterilized using the 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 vortexed. The control was set up using two-negative 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 lighting the methanol lab. This was followed by labeling 20 plates, 10-ampicillin, and 10-streptomycin. The micropipeter was set at 100 microliter to facilitate transfer from the parent culture into the plates. After this, the plastic spreader was used to spread the bacteria sample cross that 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 at the center of the dish that 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 set of standard conditions of 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. Incubation process was involved in the experiment to facilitate bacterial growth. Result and Discussion Goroar 1 Goroar 2 Goroar 3 Jazi 1 Jazi 3 Chaker 1 Chaker 2 Chaker 3 Column1 36 35 36 36 34 36 35 36 35.4444444 37 40 37 39 40 38 39 39 38.4444444 32 33 32 31 32 32 35 32 32.3333333 38 38 39 38 38 39 39 39 38.5555556 38 37 38 38 38 37 39 38 37.8888889 36 38 37 36 37 35 37 37 36.5555556 36 37 35 39 35 34 36 35 35.8888889 38 37 35 36 34 36 36 37 36 37 36 35 36 37 37 37 35 36.1111111 40 37 37 38 36 38 36 39 37.6666667 45 45 43 44 45 43 47 45 44.5555556 45 42 42 43 43 44 44 42 43.1111111 42 42 43 41 42 42 42 42 42.2222222 45 43 42 45 44 43 43 46 43.8888889 45 45 45 44 46 45 46 46 45.4444444 46 44 45 44 46 46 47 46 45.4444444 45 44 45 44 45 43 45 44 44.7777778 49 50 50 50 50 49 49 50 49.7777778 44 45 45 44 46 44 46 45 45 50 50 49 50 49 49 50 51 49.7777778 Fig. 1 These results in this table can only be understood comprehensively if the information is computed into graphs as indicated in the graph below: Fig. 2 Fig. 3 The graphs in fig. 2 and fig. 3, it is apparent that Apr colonies per culture show a general decrease as the number of plates decrease. Despite the perception and interpretation of the data above, it is clear that these twenty-three plates generated or populated bacterial growth that led to the generation of million cells (BioSciences Information Service…, 1926). The used of culture in the experiment led to the limited logical constrains. The result from the experiment can be analyzed from different perspectives (American Chemical Society, 1907). For instance, if the analysis aims at examining the fluctuation in the concentration of mutant colonies, then it is better to plate higher number of cells. However, if there is need to calculate the rate of mutation, then lower cell numbers must be plated to ensure that certain plates will not indicate any form of growth. Notably, the best scoring plates for mutant colonies are only realized after 24 hours incubation at the temperatures of 30 degrees Celsius (BioSciences Information Service…, 1971). Cells that were mutant at the time the plate was in the incubator indicated a rise to the colonies up to 1 mm in diameter after the 24 hours of incubation. However, the pigmentation of the mutants was never developed after the full time of incubation; however, after sometimes after the mark of 24 hours the Apr colony pigmentation showed variation, but most of the large colonies were either white or red while the small ones were just white all through. Additionally, the plates that did not indicate any form of growth after the 24-hour mark were small colonies and it is likely that such colonies were driven from Apr at the plating time; hence, such plates cannot be used for qualitative analysis (American Chemical Society, 1907). Therefore, it is worth noting that system selection well applies in the development of micro colonies especially at the initial points of selecting plates; thus, the growth also takes place on selected plates in the incubator. Nonetheless, the current mechanism allows cell amplification especially of the non-mutant genes that will automatically produce low but sufficient mutant protein levels. This mechanism can only be possible through translation error; otherwise, the incubation period allows creation of cells that are capable to undergo rapid growth under certain conditions that are also viable for colony development. Conclusion From the experiment, it is apparent that Serratia marcescens fluctuates in measuring ampicillin’s mutation frequency. The process is quantified; however, the process is quite spontaneous in mutation and random in occurrence. Moreover, it clears from the experiment that mutation rates can be measured from different cultures. This experiment is immensely adaptable. References American Chemical Society. (1907). Chemical abstracts. Columbus, Ohio, etc.: American Chemical Society. BioSciences Information Service of Biological Abstracts., & Union of American Biological Societies. (1926). Biological abstracts. Philadelphia: BioSciences Information Service of Biological Abstracts [etc.. Excerpta Medica (Firm). (1971). Excerpta medica: Section 4. Amsterdam, the Netherlands: Excerpta Medica. Pollack, R. A., Findlay, L., Mondschein, W., & Modesto, R. R. (2002). Laboratory exercises in microbiology. New York, N.Y: John Wiley & Sons. South African Association for the Advancement of Science. (1903). South African journal of science: Suid-Afrikaanse tydskrif vir wetenskap. Marshalltown [etc.: South African Association for the Advancement of Science. Versalovic, J., & American Society for Microbiology. (2011). Manual of clinical microbiology: Vol.2. Washington, D.C: ASM Press. Willey, J. M., Sherwood, L., Woolverton, C. J., Prescott, L. M., & Willey, J. M. (2011). Prescott's microbiology. New York: McGraw-Hill. Read More