Shortening the incubation time for antimicrobial susceptibility testing by disk diffusion for Enterobacteriaceae: how short can it be and are the results accurate?

- Clear growth is visible after 6-7 h of incubation.
- Categorical error rates between early disk zone reading and 20 h reading are low.
- Established disk diffusion breakpoints can be used with early reading.
- Turnaround time is reduced by early disk zone reading.

The standard incubation time for antimicrobial susceptibility testing (AST) by disk diffusion is primarily based on laboratory working hours rather than growth and kill characteristics of bacteria. Faster AST results could result in better patient outcomes and reduced costs by initiating earlier appropriate therapy. The earliest possible reading moment for disk diffusion using established disk zone diameter breakpoints for Enterobacteriaceae was determined with a special focus on the accuracy of the results. A total of 88 Enterobacteriaceae challenge isolates, including isolates with specific resistance mechanisms such as extended-spectrum β-lactamase (ESBL), were subjected to disk diffusion with 15 antibiotics. Hourly images were automatically produced by an incubator/camera combination from 1 h to 20 h. Disk zones were plotted over time for all combinations. Essential and categorical agreement rates using the final reading after 20 h of incubation as a reference were calculated for every hour. In total, 1320 antibiotic-micro-organism combinations were tested. Clear growth with readable inhibition zones was visible after 6 h of incubation for the majority (95.8%) of plates and after 7 h for all incubated plates. However, zone sizes changed significantly after those time points for a number of strains. After 10 h of incubation, minor, major and very major error rates were 1.6% (n = 21), 0.2% (n = 1) and 0.7% (n = 4), respectively. The results of this study clearly indicate that early reading of inhibition zones to 10 h after incubation is feasible and accurate and thus may save significantly on turnaround time.