Antimicrobial susceptibility testing of mycobacterium tuberculosis complex isolates - the EUCAST broth microdilution reference method for MIC determination

Scope:Several methods are used worldwide for antibiotic susceptibility testing (AST) for theMycobac-terium tuberculosiscomplex (MTBC). The variability in the results obtained with these methods hamperssetting epidemiological cut-off (ECOFF) values and clinical breakpoints according to EUCAST guidelines.Methods for susceptibility testing and determination of the minimal inhibitory concentrations (MICs)need to be standardized for MTBC isolates for old and new agents. Our objective was to establish astandardized reference method for MIC determination for MTBC.Methods:The EUCAST antimycobacterial susceptibility testing subcommittee (AMST) compared pro-tocols of MIC determination with regard to medium, inoculum preparation, antituberculous agentpreparation, incubation, reading of the results and interpretation.Recommendations:The EUCAST reference method of MIC determination for MTBC is the broth micro-dilution method in Middlebrook 7H9-10% OADC medium. Thefinal inoculum is a 105CFU/mL suspension,obtained from a 10 2dilution of a 0.5 McFarland suspension prepared after vortexing bacterial colonieswith glass beads before suspending them in sterile water. The culture is maintained in a U-shaped 96-well polystyrene microtitre sterile plate with a lid incubated at 36 ±1 C. Reading is done using aninverted mirror as soon as the 1:100 diluted control (i.e. 103CFU/mL suspension) shows visual growth.The MIC, expressed in mg/L, is the lowest concentration that inhibits visual growth.MycobacteriumtuberculosisH37Rv ATCC 27294 is used as the reference strain and its targeted MIC values are within therange 0.03e0.12 for isoniazid, 0.12e0.5 for levofloxacin and 0.25e1 mg/L for amikacin.Conclusions:The EUCAST reference method for MTBC was endorsed by EUCAST after public consultationand will from now on be used to define EUCAST ECOFFs and clinical breakpoints. This reference methodis not primarily intended to be used under routine conditions and the AST methods will need to be