Qualitative phenotypic methods

In the next three steps, we will focus on different phenotypic data approaches

The first type of phenotypic testing is qualitative approaches. Here we look for the binary resistant/sensitive readout.

Kirby-Bauer disk diffusion method is the most widely used standardized qualitative AST method, producing reliable and reproducible results to guide antimicrobial therapy.

Filter paper disks impregnated with a fixed amount of antimicrobial are placed on the surface agar plate. Next an inoculum of the organism of choice is prepared to a 0.5 McFarland turbidity standard and then inoculated and streaked on the agar plate. The antibiotic diffuses from the disk into the agar, inhibiting the organism when the concentrations are sufficient. A zone of inhibition is created around the disk. After 16–24-hour incubation, depending on the organism, the diameter of the zone of inhibition is measured.

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The zone diameters from antimicrobial susceptibility testing are categorized into breakpoints: susceptible, intermediate (susceptible increased exposure), and resistant, in accordance with international standards set by European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical Laboratory Standards Institute (CLSI). Several factors can influence the zone diameter, including the type and concentration of the antibiotic used, the susceptibility or resistance of the organism, the choice of agar media (e.g. media suitable for fastidious versus non-fastidious bacteria), and the incubation conditions.

Pros and cons: Disk diffusion method is cost-effective, flexible, and a preferred method for testing new antimicrobials. Additionally, by observing the zone diameters, one can determine various phenotypic mechanisms of resistance by deducing the presence of synergistic action between antibiotics and presence of organism populations with higher levels of resistance than the rest of the population in the same culture (heteroresistance). However, one of the main disadvantages of the qualitative method, in addition to the long turnaround time, is its inability to provide quantitative results in the form of minimum inhibitory concentrations (MIC) values unlike the quantitative methods.

Rapid detection of AMR using chromogenic agar

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Chromogenic agar has been introduced in the clinical microbiology laboratory to reduce the turnaround time in detecting resistance mechanisms. These media contain enzyme substrates that break down in the presence of an enzyme found in the organism. Colored dyes are released forming colored colonies on the media. There is no need to subculture the organisms for additional biochemical tests to test the presence of the enzymes. Methicillin resistant S. aureus, extended spectrum-beta lactamase- and carbapenemase-producing Gram-negative pathogens are among the organisms that can be detected rapidly using chromogenic agar.