OBJECTIVES: Brucellosis in humans is mainly caused by Brucella melitensis and is associated with a risk of chronic infections and relapses. For appropriate patient treatment decisions and outcomes, clinical breakpoints as well as standard antimicrobial susceptibility testing (AST) procedures, are needed. In this study, a Europe-wide network of Brucella reference laboratories aimed, in close collaboration with the European Committee on Antimicrobial Susceptibility Testing (EUCAST), at establishing standardized AST methods, wild-type (WT) minimum inhibitory concentrations (MICs), and zone diameter distributions, and to set epidemiological cutoff (ECOFF) values for nine therapeutically relevant antimicrobial agents.

METHODS: A total of 499 B. melitensis strains were tested at six study centres by broth microdilution (BMD) and disc diffusion (DD). MICs and inhibition zone diameters were curated according to EUCAST standard operating procedure (SOP) 10.2, and the results were submitted to EUCAST for ECOFFs and clinical breakpoint determination.

RESULTS: BMD and DD data distributions revealed putative wild-type distributions for the tested antimicrobial agents. MIC ECOFFs were determined for all agents based on five to six distributions, encompassing 249 to 499 observations. Six isolates showed MIC values slightly above the ECOFFs, indicating the presence of a potential resistance mechanism to rifampicin, streptomycin, and trimethoprim-sulfamethoxazole. Zone diameter ECOFFs were established for rifampicin and ceftriaxone, whereas tentative (t)ECOFFs were determined for ciprofloxacin, levofloxacin, gentamicin, and streptomycin.

DISCUSSION: Standardized BMD and DD methodologies for B. melitensis were validated, and AST results were used by EUCAST to set ECOFFs. Based on these, clinical breakpoints were released in v14.0 of the EUCAST clinical breakpoints table, enabling sensitive detection of resistance mechanisms and monitoring of resistance development. Genetic changes in isolates with slightly elevated MICs remain to be investigated.