Methods of antimicrobial susceptibility test.
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CLSI antimicrobial susceptibility testing methods disk diffusion broth microdilution gradient diffusion automated systems
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Methods of antimicrobial susceptibility testing (AST)
AST determines whether a bacterial isolate is likely to respond to an antimicrobial agent in vitro. Results are interpreted using approved breakpoints as susceptible (S), intermediate or susceptible-increased-exposure (I), and resistant (R).
1. Disk diffusion method - Kirby-Bauer method
- A standardized bacterial suspension is spread over an agar plate, usually Mueller-Hinton agar.
- Paper disks containing fixed amounts of antibiotics are placed on the agar.
- After incubation, measure the diameter of the zone of inhibition around each disk.
- Compare zone sizes with CLSI or EUCAST breakpoint tables to report S, I, or R.
- It is simple, inexpensive, and widely used, but does not directly give an MIC.
2. Dilution methods
These determine the minimum inhibitory concentration (MIC), the lowest antimicrobial concentration that visibly inhibits growth.
a. Broth macrodilution
- Serial twofold concentrations of antibiotic are prepared in test tubes containing broth.
- Each tube receives a standardized bacterial inoculum.
- The lowest clear tube after incubation is the MIC.
- Accurate but labor-intensive and rarely used routinely.
b. Broth microdilution
- Same principle, performed in 96-well microtitre plates with small volumes.
- Each well contains a different antibiotic concentration.
- The first well without visible growth gives the MIC.
- This is a reference method and is commonly used for routine panels and automated systems.
c. Agar dilution
- Agar plates contain serial concentrations of an antimicrobial.
- Standardized bacterial inocula are spotted onto each plate.
- The lowest antibiotic concentration preventing growth is the MIC.
- Useful for testing many isolates against one drug and is a reference method for anaerobes, but is laborious.
CLSI recognizes broth macrodilution, broth microdilution, and agar dilution as standardized dilution approaches for aerobic bacteria (CLSI M07 standard). Tietz Textbook of Laboratory Medicine, 7th Edition, p. 3285.
3. Gradient diffusion method - E-test
- A plastic strip impregnated with a continuous gradient of antibiotic concentration is placed on an inoculated agar plate.
- After incubation, an elliptical inhibition zone appears.
- The MIC is read where the edge of bacterial growth intersects the scale on the strip.
- Convenient for individual isolates and for drugs requiring a precise MIC.
- More expensive than disk diffusion.
4. Automated susceptibility testing
Examples include automated broth-microdilution based systems such as VITEK, Phoenix, MicroScan, and Sensititre.
- They provide rapid organism-specific panels and usually report MICs plus S/I/R interpretation.
- Useful for high laboratory workload.
- Important resistance phenotypes or unexpected results may need confirmation by a reference method.
5. Molecular methods
- Detect known resistance genes or mutations directly, usually by PCR, sequencing, or other nucleic-acid tests.
- Examples: mecA/mecC for methicillin resistance in staphylococci, vanA/vanB for vancomycin resistance in enterococci, and carbapenemase genes such as KPC, NDM, or OXA-48-like.
- Very rapid, but they do not give an MIC and can miss resistance mechanisms not included in the assay.
- Therefore, molecular tests supplement rather than universally replace phenotypic AST.
6. Special phenotypic resistance tests
These are used when ordinary AST may not adequately detect a clinically relevant resistance mechanism:
- D-test for inducible clindamycin resistance in staphylococci and streptococci.
- Combination disk tests, inhibitor-based tests, or carbapenemase activity tests for beta-lactamase/carbapenemase detection.
- High-level aminoglycoside resistance testing in enterococci.
- Screening tests for ESBL, AmpC, methicillin resistance, or vancomycin resistance where indicated.
Key comparison
| Method | Main result | Main advantage | Main limitation |
|---|---|---|---|
| Disk diffusion | Zone size, S/I/R | Cheap and simple | No direct MIC |
| Broth dilution | MIC | Quantitative, reference method | Requires strict standardization |
| Agar dilution | MIC | Multiple isolates per antibiotic plate | Laborious |
| E-test/gradient strip | MIC | Easy individual MIC testing | Costly |
| Automated systems | MIC plus S/I/R | Rapid, high throughput | Some results need confirmation |
| Molecular tests | Resistance gene/mutation | Very rapid | Does not measure phenotypic MIC |
Important: Accurate AST requires a pure culture, standardized inoculum, correct medium, incubation conditions, quality-control strains, and current CLSI/EUCAST breakpoints. Disk diffusion is qualitative, whereas broth dilution, agar dilution, and gradient diffusion provide MIC values. Henry's Clinical Diagnosis and Management by Laboratory Methods, p. 116?
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