Stop Waiting for Tomorrow: Disk Diffusion Performed on Early Growth Is an Accurate Method for Antimicrobial Susceptibility Testing with Reduced Turnaround Time

Disk diffusion is a slow but reliable standard method for measuring the antimicrobial susceptibility of microorganisms. Our objective was to improve the turnaround time for this method by reducing the time that cultures are incubated before setting up disk diffusion testing. For initial method development, clinical isolates (n = 13) and quality control strains (n = 8) of bacteria were inoculated on blood agar and were incubated at 35°C for either 6, 10, or 24 h before performing disk diffusion testing, in triplicate, using a panel of clinically appropriate antimicrobial agents. Disk diffusion zone sizes were interpreted using Clinical and Laboratory Standards Institute (CLSI) guidelines. Compared to standard 24 h of incubation, early 6-h growth had 1.3% major errors (MEs) and 1.9% very major errors (VMEs), whereas 10-h growth yielded 0.7% MEs and no VMEs. Categorical agreement with standard incubation was similar for both 6 h (96.7%) and 10 h (96.7%) growth. Inhibitory zone size from 6 h (r² = 0.98) and 10 h (r² = 0.99) growth correlated well with results from standard conditions. Based on these results, we performed disk diffusion under optimized conditions (6 h growth), using 100 additional clinical isolates, demonstrating a high level of categorical agreement (917 of 950 measurements [96.5%]; 95% confidence interval [CI], 95.2 to 97.5%), as well as no VMEs or MEs. Using early growth for disk diffusion testing is a simple and accurate method for susceptibility testing that can reduce time to results by as much as 18 h, compared to standard incubation, with no additional supply costs or equipment/instrumentation.