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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
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
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
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
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
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
Minimum Inhibitory Concentrations
MIC50, MIC90 and Range
MIC50
MIC90
01/23/15 7
GKM/KISIIU/MBSM713
/BIOC.AMIC.AGENS.LEC05
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
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
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
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
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
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
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
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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Lecture 05.2014

  • 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. 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. 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. 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. 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. 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. Minimum Inhibitory Concentrations MIC50, MIC90 and Range MIC50 MIC90 01/23/15 7 GKM/KISIIU/MBSM713 /BIOC.AMIC.AGENS.LEC05
  • 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. 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. 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. 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. 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. 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. 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. 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

Notas do Editor

  1. Hamburg 2 The conventional method of sensitivity testing is by the disc sensitivity test or agar dilution method. In the correct hands this is a powerful technique that is capable of showing the relative sensitivities of a range of antibiotics to a specific pathogen. The size of the zone of sensitivity is dependent on the MIC and the concentration of the interface between growth and no growth is the MIC.