Diffusion Method Using Agar Disc

Question: Discuss the most common and widely used methods for the detection of antibiotics sensitivity against antimicrobial agent? Answer: Introduction One of the most common and widely used methods for the detection of antibiotics sensitivity against antimicrobial agent is the diffusion method using agar disc (Piddock, 1990, pp.307). The principle behind this method is to determine the extent of inhibition of microbial reproduction over the solid surface of medium plate in present of antimicrobial agent. These antimicrobial agents will be applied over a filter plate, which diffuses into the solid surface of medium (Acar Goldstein, 1996, pp.1). In a typical experiment of antibiotic disk sensitivity test, there will be a zone of inhibition around the target site impregnated with antimicrobial agent. This inhibition is referred to as inhibition of the reproduction of the microorganism, due to the antimicrobial action of the concerned agent. It is important to mention that the action and inhibitory effect of agent is specific and hence the resistance zone as well as the size cutoff for each antibiotic is different. The interpretation of the said test is thus referred to accurate calculation of this inhibitory diameter zone (Andrews, 2001, pp.43). The materials that are used for this test include more than 15 hours of culture for Staphylococcus epidermidis, Escherichia coli and Bacillus subtilis. A sterile cotton swab should be used for swabbing over the agar plates (Olsson-Liljequist, 1996, pp.13). The swabbing should be done in such a way the in case nothing is added to the plates; there should be a confluent growth over the plate. Antibiotics disc will be applied to the plate to experiment the inhibitory growth of the corresponding antibiotic agent (Reller, 2009, pp.1749). Once the said procedure will be over, the plates should be incubated for next 1-2 days at 370 C. The final measurement of the inhibition zone will be executed with the help of ruler (in millimeters). The pattern of antibiotics inhibitory effect should indicate the pathogenicity and corresponding natural habitat related characteristics (Wilkins, 1972, pp.451). Discussion With reference to above introductory information, the sensitivity of six individual antibiotics were obtained as referred to table. Table: Displaying the sensitivity of individual antibiotics. Antibiotics Inhibition zone (mm) Resistance/sensitivity Ampicillin 33 Sensitive Tigercycline 25 Resistance Ciprofloxacin 22 Sensitive Chloramphenic 27 Sensitive Gentamicin 22 Sensitive Penicillin 0 Resistance From the obtained results, it was revealed that culture disk were resistant against two antimicrobial agents, viz. Tigercycline and Penicillin. The remaining six culture disks were found to be sensitive with respect to antimicrobial agents, which are Ampicillin, Ciprofloxacin, Chloramphenic and Gentamicin. The concerned inhibition zone found in mm were indicative for the concerned results of resistance or sensitivity. Considering the ampicillin, the culture results are specific when considered against gram-negative rods and enterococci, while the same results with respect to inhibition zone is different when tested against staphylococci and penicillin sensitive organisms. In the former culture condition, the susceptible zone is 14 mm, which illustrates the sensitivity, whereas when it appears to be 20 mm, it is referred to as sensitive in later case (Thomson Sanders, 1992, pp.1877). The data referring in given table is 33mm, and hence can be depict to be tested against either of the two cases. Tigercycline is referred to as broad spectrum antibiotics, which is effective in case of certain tetracycline resistant gram positive as well as gram negative organisms, such as Streptococcus pneumonia, vancomycin-resistant Enterococcus and methicillin-resistant Staphylococcus aureus (Wang, 2012, pp.1240). In the present table, the inhibition zone appears to be 25 mm, and still the strain is referred to as resistance. It is possible that the used strain is metallo lactamase producing strains. Other possibility is that the strain used for disk inhibition test belongs to Campylobacter strains (Lehtopolku, 2010, pp.1232). Ciprofloxacin when tested at 5 g, was found to be resistant with inhibition diameter of 15 mm or less. Likewise, it is intermediate in terms of inhibitory activity, when the concerned value appears to be between 16 mm to 20 mm. The suspecibility of stains are resistant in case the inhibition zone diameter appears to be more than 21 mm (Kahlmeter, 2003, pp.145). The results are appeared in table depicts a value of 22, and hence is sensitive in the culture. These strains are aerobic gram positive and gram negative bacteria. Chloramphenic is also found to have activity criteria at disk potency concentration of 30 g. The concerned testing is referred to as resistant against the culture when the inhibition diameter appears to be 12 mm or less that this value. Likewise, it is referred to as intermediate potency against the culture disk, when the inhibition diameter value appears to be 13 mm to 17 mm. The disk inhibition value of 18 mm and above is referred to as value for susceptible against the culture. Owing to the characteristics of Chloramphenic, the culture stains must be gram negative in nature (Badal, 1995). The concerned values for gentamicin is valuable when the disk potency concentration adopted is 10 g in concentration. The value for inhibition zone of 12 mm or less is termed as resistant against the disk culture. Similarly, the value of disk inhibition zone diameter of 13 mm to 14 mm, is referred to as intermediate in action. The antimicrobial activity of gentamicin is referred to as susceptible against the culture, when the inhibition zone diameter appears to be more than a value of 18 mm. In the present table, the value appears to be 22. In general, it was known that gentamycin is susceptible against gram-negative bacteria (Wilkins Thiel, 1973, pp.350). Lastly, the said testing is observed against pencillin, which is found to be resistant against in genral upto a concentration of 10 units. It is hence the diskinhibition zone diameter was found to be zero. In general, it is known that lactamases (which are also referred to as penicillinases) are resistant against penicillin. The enzyme deactivates penicillin by destroying the lactam ring with the help of process called chemical hydrolysis (Brown Brown, 1991, pp.185). In conclusion, it can be said that the above table refers to the testing of disk inhibition diameter with respect to lactamases strain. References: ACAR, J. F., GOLDSTEIN, F. W. (1996). Disk susceptibility test. Antibiotics in laboratory medicine, 4, pp.1-51. ANDREWS, J. M., BSAC WORKING PARTY ON SUSCEPTIBILITY TESTING. (2001). BSAC standardized disc susceptibility testing method. Journal of Antimicrobial Chemotherapy, 48(suppl 1), pp.43-57. BADAL, R., KELLEY, R., SAND, T. T., BASCOMB, S. (1995). U.S. Patent No. 5,457,030. Washington, DC: U.S. Patent and Trademark Office. BROWN, D. F., BROWN, L. (1991). Evaluation of the E test, a novel method of quantifying antimicrobial activity. Journal of Antimicrobial Chemotherapy, 27(2), pp.185-190 KAHLMETER, G., BROWN, D. F., GOLDSTEIN, F. W., MACGOWAN, A. P., MOUTON, J. W., STERLUND, A., ... VATOPOULOS, A. (2003). European harmonization of MIC breakpoints for antimicrobial susceptibility testing of bacteria. Journal of antimicrobial chemotherapy, 52(2), pp.145-148. LEHTOPOLKU, M., NAKARI, U. M., KOTILAINEN, P., HUOVINEN, P., SIITONEN, A., HAKANEN, A. J. (2010). Antimicrobial susceptibilities of multidrug-resistant Campylobacter jejuni and C. coli strains: in vitro activities of 20 antimicrobial agents. Antimicrobial agents and chemotherapy, 54(3), pp.1232-1236. OLSSON-LILJEQUIST, B., LARSSON, P., WALDER, M., MIRNER, H. (1996). Antimicrobial susceptibility testing in Sweden. III. Methodology for susceptibility testing. Scandinavian journal of infectious diseases. Supplementum, 105, pp.13-23. PIDDOCK, L. J. (1990). Techniques used for the determination of antimicrobial resistance and sensitivity in bacteria. Journal of Applied Bacteriology, 68(4), pp.307-318. RELLER, L. B., WEINSTEIN, M., JORGENSEN, J. H., FERRARO, M. J. (2009). Antimicrobial susceptibility testing: a review of general principles and contemporary practices. Clinical infectious diseases, 49(11), pp.1749-1755. THOMSON, K. S., SANDERS, C. C. (1992). Detection of extended-spectrum beta-lactamases in members of the family Enterobacteriaceae: comparison of the double-disk and three-dimensional tests. Antimicrobial Agents and Chemotherapy, 36(9), pp.1877-1882. WANG, L., LIU, Y., YANG, Y., HUANG, G., WANG, C., DENG, L., ... SHEN, X. (2012). Multidrug-resistant clones of community-associated meticillin-resistant Staphylococcus aureus isolated from Chinese children and the resistance genes to clindamycin and mupirocin. Journal of medical microbiology, 61(Pt 9), pp.1240-1247. WILKINS, T. D., HOLDEMAN, L. V., ABRAMSON, I. J., MOORE, W. E. C. (1972). Standardized single-disc method for antibiotic susceptibility testing of anaerobic bacteria. Antimicrobial agents and chemotherapy, 1(6), pp.451-459. WILKINS, T. D., THIEL, T. (1973). Modified broth-disk method for testing the antibiotic susceptibility of anaerobic bacteria. Antimicrobial agents and chemotherapy, 3(3), pp.350-356.