Multicentre validation of EUCAST disk diffusion for frequently encountered anaerobic bacteria (excluding Cutibacterium acnes) in clinical microbiology laboratories in the Netherlands

Objectives:

The European Committee on Antimicrobial Susceptibility Testing (EUCAST) disk diffusion method on Fastidious Anaerobe Agar with horse blood (FAA-HB) was developed for antimicrobial susceptibility testing of common anaerobic bacteria. This study evaluated its performance in routine clinical microbiology laboratories using FAA-agars and disks from various manufacturers. 

Methods:

Thirty isolates of Bacteroides spp., Prevotella spp., Fusobacterium necrophorum , and Clostridium perfringens were tested using FAA-HB agar manufactured by Liofilchem, EO Labs, and Xebios Diagnostics according to the EUCAST manual. Cutibacterium acnes , another common anaerobic bacterium for which EUCAST provides breakpoints, was not included due to insufficient growth on the agar in preliminary testing. Six laboratories participated, each selecting at least one manufacturer. Agar dilution MICs, interpreted with EUCAST breakpoints, served as the reference. Categorical agreement (CA), very major (VMD) and major discrepancy between disk diffusion and reference method were calculated. 

Results:

For F. necrophorum and C. perfringens , the CA for all antibiotics across FAA-HB agar was above 90% (range between agar manufacturers 91.2–100%). For Bacteroides spp., CA for clindamycin, metronidazole, and meropenem exceeded 85% across all agars, with VMD for clindamycin ranging between 3.2% and 7.9%. CA for piperacillin/tazobactam and amoxicillin/clavulanic acid for Bacteroides spp. was between 55.5% and 61.8%, and 84.9% to 93.1%, respectively, with VMD for amoxicillin/clavulanic acid for Bacteroides spp. varied between 5.6% and 8.1%. For Prevotella spp., 30% to 40% of disk diffusion inhibition zones were uninterpretable (i.e. no inhibition zone could be read) across tested antimicrobial–organism combinations. After excluding these unreadable results, FAA-HB performance was >90% (range: 91.7–100%). 

Conclusion: 

In general, FAA-HB demonstrated acceptable performance for disk diffusion antimicrobial susceptibility testing of frequently isolated anaerobic species. However, its performance was not acceptable for β-lactam and β-lactamase inhibitors, especially for piperacillin/tazobactam in Bacteroides spp., and its use is limited in Prevotella spp. due to frequent insufficient growth.