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Lecture 05.2014

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MBC and MIC

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Lecture 05.2014

  1. 1. Lecture Five Topics: •Laboratory studies on antibiotic action Dr. G. Kattam Maiyoh 01/23/15 GKM/KISIIU/MBSM713 /BIOC.AMIC.AGENS.LEC05 1 MBSM 713: BIOCHEMISTRY OF ANTI MICROBIAL AGENTS
  2. 2. Laboratory studies on antibiotic action • In the laboratory, susceptibility is most often measured using a disk diffusion test. • Antibiotic solutions of particular concentrations are dried onto filter paper disks. • These are then applied to a lawn of the microbe under examination which has previously been inoculated onto an appropriate solid medium. 01/23/15 2 GKM/KISIIU/MBSM713 /BIOC.AMIC.AGENS.LEC05
  3. 3. Sensitivity tests • Susceptible or resistant to antibiotic • MIC = Minimum inhibitory concentration • MBC = Minimum bactericidal concentration • Minimum concentration required to inhibit growth – Disc diffusion – Agar dilution – Etest – Breakpoint MIC 01/23/15 3 GKM/KISIIU/MBSM713 /BIOC.AMIC.AGENS.LEC05
  4. 4. Diffusion of antibiotic from a paper disc After Incubation Zone of Sensitivity Concentration of antibiotic at periphery of zone equals the MIC Disc Area of Bacterial growth Disc Concentration Gradient 01/23/15 4 GKM/KISIIU/MBSM713 /BIOC.AMIC.AGENS.LEC05
  5. 5. 1 2 4 8 16 >16 Minimum Inhibitory Concentration (mg/L) 0 2 4 6 8 10 Number Breakpoint (mg/L) 0 4 1 8 2 16 Minimum Inhibitory Concentration (MIC) Determination 01/23/15 5 GKM/KISIIU/MBSM713 /BIOC.AMIC.AGENS.LEC05
  6. 6. MIC • Breakpoint: concentration above which the isolate is described at resistant and below which is susceptible e.g. S < 8mg/L R ≥ 8mg/L Breakpoint = 8mg/L • Range: Lowest to highest MIC for population • MIC50Median for series of MICs • MIC90 – MICs of population ordered from lowest to highest – MIC value of the strains that appears 90% up the series. – Antibiotic considered to be successful if > 90% of population inhibited. – Also show if resistance is emerging i.e. 10% of population resistant. 01/23/15 6 GKM/KISIIU/MBSM713 /BIOC.AMIC.AGENS.LEC05
  7. 7. Minimum Inhibitory Concentrations MIC50, MIC90 and Range MIC50 MIC90 01/23/15 7 GKM/KISIIU/MBSM713 /BIOC.AMIC.AGENS.LEC05
  8. 8. Etest Determination of MIC 16 8 4 32 24 12 6 3 2 1.5 1 CI 01/23/15 8 GKM/KISIIU/MBSM713 /BIOC.AMIC.AGENS.LEC05
  9. 9. Breakpoint Test to Determine Bacterial Sensitivity Trimethoprim (4mg/L) Inoculation of Plates Determination of growth at fixed concentrations shows resistanceAmoxycillin (4mg/L) Gentamicin (2mg/L)Ciprofloxacin (1mg/L) Cefotaxime (2mg/L) Ceftazidime (4mg/L) Control - No drug 01/23/15 9 GKM/KISIIU/MBSM713 /BIOC.AMIC.AGENS.LEC05
  10. 10. Evaluation of Laboratory Tests • MIC test on plates is the best – Time consuming and costly – Most detailed • Disc test/Etest is easiest – Requires more skill to interpret • Breakpoint – Least skill required – Technique must be exact – Can be read by computer – Large amounts of data 01/23/15 10 GKM/KISIIU/MBSM713 /BIOC.AMIC.AGENS.LEC05
  11. 11. Minimum Bactericidal Concentration MIC 64 32 16 8 4 2 1 mg/l Subculture onto drug-free agar 16 8 4 2 1 mg/l MBC 01/23/15 11 GKM/KISIIU/MBSM713 /BIOC.AMIC.AGENS.LEC05
  12. 12. Stokes controlled sensitivity test • A control organism is inoculated on part of a plate and the test organism is plated on the remainder. • Disks are placed at the interface and the zones of inhibition are compared. • The use of a sensitive control shows that the antibiotic is active, so that if the test organism grows up to the disk it may safely be assumed that the test organism is resistant to that drug. 01/23/15 12 GKM/KISIIU/MBSM713 /BIOC.AMIC.AGENS.LEC05
  13. 13. Stokes' sensitivity test •The bacterium in the diagram is susceptible to drug "x" but resistant to drug "y". • The disc containing drug "y" contains active antibiotic as shown by the zone of inhibition it causes in the control bacterium. 01/23/15 13 GKM/KISIIU/MBSM713 /BIOC.AMIC.AGENS.LEC05
  14. 14. MIC and MBC • An alternative measure of susceptibility is to determine the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of a drug. • A series of broths are mixed with serially diluted antibiotic solutions and a standard inoculum is applied. • After incubation, the MIC is the first broth in which growth of the organism has been inhibited. • The more resistant an organism is, then the higher will be the MIC. 01/23/15 14 GKM/KISIIU/MBSM713 /BIOC.AMIC.AGENS.LEC05
  15. 15. Minimum Bactericidal Concentration (MBC) • The MBC is measured by inoculating the broths used for MIC determinations onto drug-free medium. • The MBC is the first dilution at which no growth is observed. • Cidal drugs have MBC values that are close to the MIC value for particular organisms. • With static agents, the MIC is much lower than the MBC. 01/23/15 15 GKM/KISIIU/MBSM713 /BIOC.AMIC.AGENS.LEC05

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