Antimicrobial Susceptibility Testing Series: Traditional AST methods

There is no substitute for antimicrobial susceptibility testing (AST) data when you need it.  The sensitivity and resistance of pathogens to a wide range of antimicrobials is necessary so that the proper path to treatment can be determined. Several manual methods can be used, such as microbroth dilution, disk diffusion, and gradient diffusion. Microbroth dilution is considered the gold standard testing method. It involves filling microtiter plates with broth. Varying concentrations of antibiotics and bacteria to be tested are added to the plate. After incubation, plate is checked for bacterial growth. Miniaturization of the test incorporates ‘microdilution trays’, with standard trays containing 96 wells, allowing 12 antibiotics to be tested. Disk diffusion (syn: Kirby-Bauer test) uses antibiotic disks/discs made from filter paper that have been impregnated with antibiotics. The disks are are placed on an agar plate that has uniformly swabbed with a pure bacterial culture in buffer, standardized for turbidity. The compound concentration will be highest next to the disk, and will decrease as the distance from the disk increases. If the compound is effective against the bacteria at a certain concentration, no colonies will grow where the concentration in the agar is greater than or equal to the effective concentration ie zone of inhibition. This data, along with the rate of diffusion are used to estimate the bacteria’s susceptibility to that antibiotic. Larger zones correlate with smaller MIC of antibiotic for that bacteria. Gradient diffusion, such as the commercial test called Etest, incorporates thin plastic test strips that are impregnated on the underside with a dried antibiotic concentration gradient, and marked on the upper side with a concentration scale. The strips are placed in a radial fashion on the surface of an agar plate that has been inoculated with a standardized organism suspension. After overnight incubation, the tests are read by viewing from the top, and MIC is determined by the intersection of the lower part of the ellipse shaped growth inhibition area with the test strip.

Despite the differences in testing methods, all labs must ensure critical sampling and analysis so the results are accurate and reliable. Specimens include blood, urine, cerebrospinal fluid, sputum, wound, stool, and other body fluids and discharge.

Manual methods are labor intensive and complex, and are normally done in a reference laboratory. Several factors can affect the AST results including specimen quality, suspension preparation, media preparation, MIC panel quality, or issues related to protocols/training. Examples of commercial manual systems appear in Table 1.

Automated platforms (Table 2) are less labor intensive. They incorporate AST panels/cards, that incorporate dried antibiotics in custom configurations according to the manufacturer. AST panels are commercially available and offer speed and accuracy. Enhanced computer software incorporating MIC breakpoint information is used to interpret results including ‘expert systems’ for analyzing test results for atypical growth patterns and unusual resistance phenotypes.

In the next article of the series, we will look at how new and emerging ‘rapid’ AST methods, the various methods that are being used, and the commercial platforms that are revolutionizing sample-to-answer for AST.

Reference

Reller LB, Weinstein M, Jorgensen JH, Ferraro MJ (2009) Antimicrobial susceptibility testing: A review of general principles and contemporary practices. Clin. Infect. Dis. 49 (11):1749–1755 Link

Table 1. Examples of manual AST products

Table 2. Examples of automated platforms