Stop Waiting for Tomorrow: Early Disk Diffusion—An Accurate Method for Antimicrobial Susceptibility Testing With Reduced Turnaround Time

Background Disk diffusion is a slow but reliable reference method for measuring antimicrobial susceptibility. Our objective was to improve the turnaround time for this method by reducing the culture incubation period prior to disk diffusion testing (referred to as early disk diffusion [eDD] testing). Methods Clinical isolates (n = 13) and quality control (QC) strains (n = 8) of bacteria, including 6 Staphylococcus aureus, 3 Enterococcus faecium, 3 Enterococcus faecalis, 3 Escherichia coli, 3 Pseudomonas aeruginosa, 2 Klebsiella pneumoniae, and 1 Enterobacter cloacae, were inoculated on blood agar by quadrant streaking and were incubated at 35○C for 6 hours (eDD6), 10 hours (eDD10), or 24 hours (standard [sDD]) before disk diffusion testing was set up in accordance with CLSI guidelines using Mueller Hinton Agar (Hardy Diagnostics) and clinically appropriate antimicrobial agents (total: 24). Experiments were performed sequentially in triplicate with zones measured by two independent readers. Results Examination of 6-hour blood agar plates showed limited growth in the first 1 to 2 quadrants while 10-hour growth plates had 4-quadrant growth and individual colonies in most cases. Despite limited growth on 6-hour plates, there were adequate bacteria to produce a 0.5 McFarland standard for disk diffusion testing for all 126 eDD replicates. Results were evaluable for 1,206 of 1,206 (100%) of eDD and 603 of 603 (100%) of standard disk diffusion (sDD). A comparison of early vs standard disk diffusion showed that eDD6 had 3 of 154 (1.9%) very major errors and 6 of 449 (1.3%) major errors, whereas eDD10 had no very major errors and 3 of 449 (0.7%) minor errors. Very major errors in eDD6 included a 1-mm difference between eDD6 and sDD for cefoxitin inhibition of S aureus as well as 4- and 6-mm differences between eDD6 replicates and sDD for nitrofurantoin inhibition of E cloacae. There was similar categorical agreement of eDD6 (96.7%) and eDD10 (96.7%) with sDD. Overall, there was a very good correlation of eDD6 (r2 = 0.98) and eDD10 (r2 = 0.99) with sDD. When grouping results by bacterial species, there was no more than 1-mm difference in median antimicrobial inhibition between eDD6, eDD10, and sDD. Of note, 5 of 7 bacterial species had the same median zone size when comparing eDD6 or eDD10 with sDD. There was also very little difference between early and standard methods when examining bacteria-drug combinations, with a similar zone size (difference in mode <2 mm) in 67 of 69 (97%) eDD6 and 66 of 69 (96%) eDD10 compared to sDD. Test results for QC isolates agreed with the CLSI recommended ranges in 97 of 99 (98%) eDD6, 97 of 99 (98%) eDD10, and 96 of 99 (97%) sDD. Conclusion Early disk diffusion testing is a simple and accurate method of antimicrobial susceptibility testing that can reduce time to results by as much as 18 hours with no additional cost.