The impact of agar depth on antimicrobial susceptibility testing by disc diffusion

Introduction. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) specifies the agar depth (4 +/- 0.5 mm) when performing antimicrobial susceptibility testing (AST). Since the infrastructure to produce standardized agar may be lacking in settings with limited resources, we wanted to examine to what extent variation in agar depth affects the inhibition zone diameters of quality control (QC) strains and AST of clinical isolates. Methods. The inhibition zone diameters on Mueller-Hinton II agar with different depths (2-6 mm) were tested for various QC strain-antimicrobial agent combinations using Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213. The relationship between zone diameters at different agar depths and MICs was investigated for 35 clinical isolates (E. E. coli, , Klebsiella pneumoniae, , S. aureus and P. aeruginosa) ) from Sierra Leone using MICs as the reference. Results. The inhibition zone diameters were within the acceptance ranges as defined by the EUCAST for the majority of QC strains and antimicrobials, independent of the agar depth. At extreme agar depths, inhibition zones were more frequently out of range. The accuracy of AST varied for clinical isolates at different agar depths for categorical agreement (85.8-94.6%), major error rate (0.4-2.1%) and very major error rate (VME: 3.3-12.5%). Conclusions. Even if the QC strains were in the acceptance range at different agar depths, this does not rule out unacceptably high VME rates (>3%) in clinical isolates.