Determining The Minimum Inhibitory Concentration Biology Essay

Determining The Minimum Inhibitory Concentration Biology Essay The basic structure of penicillin.The first two steps in cell wall synthesis specifically the formation of acetylglucosamine to from peptidoglycan chains, cannot be inhibited by penicillin. However, in the final step where the crosslinking between peptidoglycan by side peptide chains, the penicillin inhibits this process. This is because penicillin has similar structure to the terminal D-alanine-D-alanine if the pentapeptide, which binds covalently to the active site of the transpeptidase enzyme (Gorbach et al., 2003). Chloramphenicol is another antibiotic and it has the ability to penetrate though cellular membranes and be easily in engagement with the bacteria colonising in human cells to allow its antimicrobial characteristics to take place. Chloramphenicol has a simple structure and the mechanism depends on the propanoeidol moiety and dichloracetamide chain. C:Usersctlee11Desktop12.jpg Figure 2.0: The basic structure of chloramphenicol.What chloramphenicol does is that it inhibits protein synthesis in bacteria, however nucleic acid synthesis in unaffected. Chloramphenicol binds reversibly to the larger 50S ribosome subunit of the 70S ribosome. This inhibits the protein synthesis by preventing aminioacyl-transfer RNA from attaching to the 50S binding site (Gorbach et al., 2003). However, it has been recorded that chloramphenicol affects the mitochondrial protein synthesis. This is because the mammalian mitochondrial protein has strong similarity to bacterial ribosome where both are of 70S, with the mitochondria of the bone marrow especially susceptible (Riviere Papich, 2009). Protein synthesis plays a major role in bacterial growth as there are various cofactors and enzymes needed for multiple metabolic pathways. Minimum inhibitory concentration (MIC) is the minimum concentration of a drug which inhibits bacterial growth, but does not kill the microorganism. Minimum bacteriostatic concentration (MBC) is the minimum concentration of a drug which kills the bacterial and thus, no longer grows. AIMS This investigation was done to determine Minimum inhibitory Concentration (MIC) and the Minimum Bacteriocidal Concentration (MBC) of Penicillin and Chloramphenicol of Escherichia coli as well as; to determine the antibiotics sensitivity against the Staphylococcus aureus and Pseudomonas aeruginosa. METHODS AND MATERIALS The methods and materials are as per laboratory manual of Medical Microbiology BTH3722 of Monash University Semester Two, Year 2012 of pages 17 20. RESULTS SESSION/DAY ONE There are two sections to this investigation, where there is (i), the MIC and MBC determination of antibiotic Penicillin G and Chloramphenicol against Escherichia coli, and then there is (ii) antibiotics testing towards two microorganisms which are Staphylococcus aureus and Pseudomonas aeruginosa. In the determination of MIC, serial dilutions of antibiotics are done with known concentrations. Then, each tube containing different antibiotic concentration is the inoculated with bacterial culture and incubated overnight. This goes the same for the determination of MBC. Then, for each serial dilution of antibiotic, there would be two control tubes, one positive where the tube is inoculated with bacterial culture and the incubated without antibiotics and then, negative control where the tube is not inoculated but theres presence of antibiotics. For the antibiotic sensitivity testing, there are two Mast rings used and both of them are ready-made device that can simultaneously test for antibiotic susceptibility testing using 8 types of antibiotics. The Mast Rings used in this investigation is MAST M43 to test against MG8 culture: Staphylococcus aureus and MAST M14 to be used to test against MG53 culture: Pseudomonas aeruginosa. All these are done on the MHA plate using spread plate technique. SESSION/DAY TWO The MIC and MBC for each antibiotic type (Penicillin G and Chloramphenicol) is determined by observing the turbidity in the test tube. A turbid medium signifies for bacteria growth. Then all the tubes that are sterile are then poured into respective Trypticase Soy Broth (TSB) bottles including the tube that has the minimum positive bacteria growth to determine the MBC. These bottles are then incubated overnight. Results are on Table 1.0. For the antibiotic susceptibility testing, the annular radii of the clear zones are measured for each antibiotic type and these measurements are recorded. The measurements and interpretation are on Table 2.0 and Table 3.0. Tube Concentration of Antibiotics (c),  µg/ml Growth of Escherichia coli Penicillin G Chloramphenicol 1 125.00 + 2 62.50 + 3 31.25 + + 4 15.63 + + 5 7.81 + + 6 3.91 + + 7 1.95 + + 8 0.98 + + 9 0.49 + + 10 0.24 + + 11 0.12 + + 12 (+ CTRL) 0.00 + + 13 (- CTRL) 0.00 Table 1.0: The table below shows the results of the from the MIC determination of Penicillin G and Chloramphenicol on E. coli. In Table 1.0, it is shown that there are 13 tubes where the 12th and 13th are controls, 12th as the positive (+) and the 13th as negative (-). Positive control is the control which is inoculated without antibiotics and Negative control as the control which not inoculated. On the growth column, + signifies theres growth and thus turbidity in the tubes, while (-) signifies negative growth. It is also that has been determined that the MBC value is 125  µg/ml for Chloramphenicol. Table 2.0: The table below shows the annular radius that was measured form the MHA plate cultured with Pseudomonas aeruginosa using MAST Ring M43. MASTRING ANTIBIOTICS M43 Antibiotics Abbreviation Annular Radius Sensitivity PG Penicillin G 0 R CD Clindamycin 1 R GM Gentamycin 10 S FC Fusidic Acid 20 S E Erythromycin 0 R TM Trimetroprim 0 R SMX Sulphamethoxazole 0 R T Tetracyclin 0 R The general rule is that if the annular radii are more than 6mm in length, then the bacteria are susceptible to that particular antibiotic. However, if the annular radii are 6mm or less than, it is considered resistant to the particular antibiotics. Table 3.0: The table below shows the annular radius that was measured form the MHA plate cultured with Staphylococcus aureus tested using MAST Ring M14. MASTRING ANTIBIOTICS M14 Antibiotics Abbreviation Annular Radius Sensitivity AP Ampicillin 0 R KF Cephalothin 1 R CO Colistin Sulphate 7 S GM Gentamycin 8 S S Streptomycin 8 S ST Sulphatriad 1 R T Tetracyclin 0 R TS Cotrimoxazole 0 R The general rule is that if the annular radii are more than 6mm in length, then the bacteria are susceptible to that particular antibiotic. However, if the annular radii are 6mm or less than, it is considered resistant to the particular antibiotics. DISCUSSION The MIC and MBC value of penicillin G cannot be determined as at all concentrations of the serial dilutions, the bacteria is resistant to the Penicillin G (PG) even from the concentration of 125  µg/ml. This is because there was growth observed from the turbidity of all the serial dilution tubes. However for the antibiotic Chloramphenicol, the MIC is at 31.25  µg/ml while the MBC is at 62.6  µg/ml. At these concentrations, in treating infections that are E. coli related, a rather heavy dosage of PG is needed to be administered. Thus, PG is not really effective in killing E. coli. Besides, the MBC value is always expected to be higher than the MIC value is higher dosage is needed to kill a microorganism (Prescott et al., 2005). As it has been previously mentioned, PG targets the peptidoglycan structure of the cell wall. If the bacteria were able to resist PG, it would be because that the bacteria have a mechanism to counter such actions. True enough, E. coli could be resistant t o PG because it could produce beta-lactamase that inactivates penicillin (Sorbo Marshall, 2006). Beta-lactamase which is also known as penicillinase, acts by hydrolysing the CO-N bond in the beta lactam ring of the penicillin molecule and then the amidase group cleaves the CO-NH bond found within the side chain and the six amino acid groups of penicillic acid group of the penicillin molecule (English et al., 1960). However, not only E. coli is not the only one bacteria that possesses the beta-lactamase gene which can caused by the fact that certain bacteria passed on their DNA to another bacteria during conjugation, or from a virus to a bacteria during transduction or also that the bacteria can take in foreign DNA from their surroundings (Karp, 2009). In the antibiotic testing on the bacteria lawn, it is found that the Staphylococcus aureus (S. aureus) is less resistant than Pseudomonas aeruginosa (P. aeruginosa) as S. aureus is susceptible to three kinds of antibiotics (susceptible: CO, GM and S; resistant: AP, KF, ST, T and TS) compared to P. aeruginosa which is susceptible to two types (susceptible: GM and FC; resistant: PG, CD, SMX, T and EM). The reason P. aeruginosa is so particularly so resistant is to most of the modern antibiotics is because of its intrinsic resistance that arises from the permeability and secondary resistance mechanisms such as energy-dependent multidrug efflux and chromosomally encoded periplasmic ÃŽÂ ²-lactamase. At such level of natural resistance, mutational resistance to most classes of antibiotics can readily arise (Hancock Speert, 2000). For instance, the resistance of P. aeruginosa towards PG is because of de-repression of chromosomal ÃŽÂ ²-lactamase and also the overexpression of the MexA B-OprM multidrug efflux pump due to a NalB mutation. Specific plasmid-mediated ÃŽÂ ²-lactamases also infer that the resistance to PG could be a form of introduction of foreign DNA from surroundings or via conjugation with other bacteria (Hancock Speert, 2000). As for S. aureus, the bacteria is resistant towards ampicillin and that would probably due to the act that most strains of S. aureus are able to produce ÃŽÂ ²-lactamase to breakdown the beta-lactam ring of the penicillin-derivate antibiotics. CONCLUSION The MIC and MBC values are important in determining the correct dosage of antibiotics that is supposed to be administered to the patient. However, E. coli was particularly resistant to Penicillin G and thus, was unable to produce any MBC or MIC values. In such cases, an AST can be ran first before testing E. coli against its susceptible drug for MIC and MBC values. IX. REFERENCES English, A.R., McBride, T. J. and Huang, H. T. (1960). Microbial Resistance to penicillin as related to penicillinase or penicillin acylase activity, Proceedings of the Society or  Experimental  Biology and  Medicine, Vol. 104, pp. 547 549. Gorbach, S.L., Barlett, J.G. and Blacklow, N.R. (2003).Treatment of Infectious Disease, Infectious Disease, Lippincott Williams Wilkins, pp. 184 186. Hancock, R. E. W. and Speert, D. P. (2009). Antibiotic Resistance in Pseudomonas aeruginosa: mechanisms and impact on treatment, Drug Resistance Updates, 3, pp. 247 255. Karp, G. (2009). The Growing Problem of Antibiotic Resistance, Cell and Molecular Biology: Concepts and Experiments, John Wiley Sons, pp. 105 106. Riviere, J.E. and Papich, M. G. (2009). Chemotherapy of Microbial Diseases, Veterinary Pharmacology and Therapeutics, John Wiley Sons, pp. 944 946. Sorbo, L. D. and Marshall, J.C. (2006) Antibiotic Resistance in the Intensive Care Unit, Intensive Care Medicine: Annual Update 2006, Springer, pp. 582 583.