Quantitative phenotypic methods

Quantitative methods look beyond the binary R/S and estimate the minimum inhibitory concentration (MIC)

Quantitative methods can be tested on liquid medium (broth) or solid agar

Broth dilution method (macrodilution using culture tubes and microdilution using 96-well microtiter plates) is considered the gold standard reference method. In the microdilution method, serial 2-fold dilutions of an antibiotic are made in the broth media in the 96-well plate with each well containing a different concentration of antibiotic. This is followed by inoculation with standard inoculum of organism. After 18-24-h overnight incubation, depending on the organism, the lowest concentration with no visible growth is considered the MIC.

Agar dilution method is another quantitative reference method. Antibiotic is added to the agar medium in serial dilutions. The organism is inoculated onto the medium and this is incubated for 18-24 hours (depending on the organism). The lowest concentration with no visible growth is considered the MIC.

The agar gradient diffusion method, for example, using the Etest (bioMerieux) is a strip with predefined gradient of antibiotic concentrations. Just like the Kirby-Bauer disk diffusion method, when the strip is placed on the surface of an agar plate inoculated with a standard organism inoculum, the antibiotic in the strip diffuses into the agar forming a continuous gradient on the agar surface beneath and around the stip. After 16-24h incubation, depending on the organism, the inhibition along the test strip is observed and the MIC is the point where the margin of the organism intersects the edge of the calibrated strip.

The MIC is then matched to the breakpoints developed by CLSI or EUCAST. Importantly, both of these organisation consider broth dilution methods as the standard approach for generating MIC values. Just like in the qualitative methods, the antibiotic (type and concentration), the organisms, the culture media composition and the incubation conditions should be considered.

Pros and cons: The advantage of the quantitative AST methods is the ability to provide MIC values (the lowest concentration of an antibiotic which will inhibit the growth of the organism being tested). MIC values are useful in adjusting antibiotic dosages against non-susceptible organisms, especially for critically ill patients based on pharmacokinetic/pharmacodynamic (PK/PD) approaches. However, they are unable to show phenotypic resistance mechanisms of action.

Automated AST: Within healthcare settings, there also exist many (semi-) automated systems for determining antimicrobial susceptibility profiles. These use automatic spectrophotometry or turbidity or fluorescence to look for growth in the presence of the antimicrobial. We won’t outline these systems here but instead encourage you to look into these systems such as VITEK 2, BD Phoenix and Microscan.