The EUCAST Disk Diffusion Method for Antimicrobial Susceptibility Testing of Oral Anaerobes

There is a need for standardized methods for antimicrobial susceptibility testing (AST) of anaerobic bacteria involved in oral and extra-oral infections. We tested the recently published EUCAST disk diffusion method for rapidly growing anaerobes on selected oral anaerobes. AST of 20 strains of Prevotella spp., 11 strains of Porphyromonas gingivalis, and six Fusobacterium nucleatum complex strains was performed with amoxicillin and metronidazole disks using EUCAST guidelines. Plates were incubated anaerobically, and inhibition zones were evaluated after 20 h (EUCAST recommendations) and again after 44 h. The recommended agar supported the growth of all 38 strains. Twenty-hour incubation was sufficient for the assessment of inhibition zone diameters of Fusobacterium strains. Although approved for Prevotella, an extended study of Prevotella species showed inconsistent growth within the EUCAST time limit of 20 h for some strains. All P. gingivalis strains required 44 h of incubation for the evaluation of inhibition zones. The EUCAST disk diffusion method for AST of rapidly growing anaerobes is applicable to members of the Fusobacterium nucleatum complex. P. gingivalis and several oral strains of Prevotella needed 44 h of incubation to enable reading of diffusion diameter. Further studies are necessary to validate the prolonged incubation of slow-growing anaerobes.