As an epidemiological tool
The emergence of resistant strains of major
pathogens (e. g. Shigellae, Salmonella typhi)
Continued surveillance of the susceptibility
pattern of the prevalent strains (e. g.
Staphylococci, Gram-negative bacilli)
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Introduction
Methods for antimicrobial susceptibility testing
Indirect method
cultured plate from pure culture
Direct method
Pathological specimen
e.g. urine, a positive blood culture, or a swab of
pus
Diffusion method
Put a filter disc, or a porous cup/a bottomless
cylinder containing measured quantity of drugs
on the a solid medium that has been seeded
with test bacteria
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Dilution Method
Broth dilution/ Agar dilution methods
Permit quantitative results:
Indicating amount of a given drug necessary
to inhibit (bacteriostatic activity) or kill
(bactericidal activity) the microorganisms
tested
Day 1
128 64 32 16 8 4 2 C1 C2
Add 1 ml of test bacteria
(1*106 CFU/ml) to tubes
containing 1 ml broth and
concentration of antibiotic
(mg/l)
64 32 16 8 4 2 1 C1 C2 Controls:
C1 = No antibiotic, check
Bacterial conc.= 5*105 CFU/ml viability on agar plates
immediately
Incubate 35 oC, o/n
C2 = No test bacteria
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Broth Dilution Method
Day 2
64 32 16 8 4 2 1 C1 C2
Record visual turbidity
Subculture non-turbid tubes
to agar plates (use 0.01 ml
standard loop)
0.01 ml (spread plate), Incubate 35 oC, o/n
MIC = 16 mg/l
Day 3
Determine CFU on plates:
At 16 mg/ = 700 CFU/ml >
0.1% of 5*105 CFU/ml
64 32 16
MBC = 32 mg/l
0.1%
= [(5*105)*0.1]/100 CFU/ml
= 500 CFU/ml
Solutions??
Agar dilution method
Disc diffusion method
Microbroth dilution method
Manually prepared
Commercially prepared
Frozen or Dried/ lyophilized
Consistent performance but high cost
May suffer from degradation of antibiotic during
shipping and storage
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Microbroth Dilution Method
Visualize turbidity
Light box/ mirror reader
Automated reader
MIC
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Diffusion Method
Disc diffusion method : The Kirby-Bauer test
Antibiotic-impregnated filter disc*
Susceptibility test against more than one
antibiotics by measuring size of “inhibition
zone ”
1949: Bondi and colleagues paper disks
1966: Kirby, Bauer, Sherris, and Tuck filter
paper disks
Demonstrated that the qualitative results of filter
disk diffusion assay correlated well with
quantitative results from MIC tests
0.5 standard.*
Streak the swab on the surface of the Mueller-Hinton a
gar (3 times in 3 quadrants)
Leave 5-10 min to dry the surface of agar
Susceptible
Intermediate susceptible
Low toxic antibiotics: Moderate susceptible
High toxic antibiotics: buffer zone btw resistant and
susceptible
Resistant
Aminoglycosides, erythromycin
Alkaline pH of mediu zones are larger
m
Subjective errors in determinin
Reading of zones g the clear edge
See Table 3.
MIC
“Synergistic”
Additive effect: increase in activity level
“Antagonistic”
Interfere effect: reduce activity level
“Antagonistic”
e. g. Penicillins and bacteriostatic drugs such
as tetracyclines are antagonistic, since
penicillins require actively growing cells