Validation of EUCAST 2026 breakpoints for Brucella species using routine laboratory methods: A comprehensive French National Reference Centre study

Brucellosis, caused by various species of the bacterial genus Brucella, represents a significant global health burden in many parts of the world. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) recently established standardized clinical breakpoints (version 16.1, 2026) for Brucella to support global surveillance and evidence-based treatment. This study validates EUCAST 2026 breakpoints using routine laboratory methods in a large national reference collection. We retrospectively analyzed 234 Brucella isolates from the French National Reference Centre between January 2017 and June 2026, using MALDI-TOF mass spectrometry and multiplex PCR for species identification, and E-test strips and disk diffusion for antimicrobial susceptibility testing (AST) according to EUCAST guidelines. Brucella melitensis predominated (n = 210, 89.7%), with smaller numbers of B. suis, B. abortus, and B. amazoniensis. Universal susceptibility was observed across all 234 isolates for eight tested antibiotics. Notably, 49.6% of isolates displayed rifampicin minimum inhibitory concentrations (MICs) within two doubling dilutions of the EUCAST epidemiological cut-off (ECOFF) value, highlighting the need for continued surveillance of rifampicin susceptibility in this genus. A critical methodological finding emerged regarding streptomycin assessment. E-test strips indicated elevated streptomycin MICs in a substantial proportion of isolates, with results consistent across different lots. However, disk diffusion performed on identical isolates demonstrated universal susceptibility for all 234 isolates, with inhibition zone diameters ranging from 21-35 mm. This study validates that EUCAST 2026 breakpoints are clinically appropriate and epidemiologically sound when applied using appropriate routine laboratory methods. Disk diffusion should be considered the reference method for streptomycin susceptibility determination in Brucella species. These findings provide essential practical guidance for clinical microbiology laboratories, demonstrate that France remains free from antimicrobial resistance, and support standardized AST testing for optimized management of human brucellosis in both endemic and non-endemic regions worldwide.