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

Thomas Schön, Jim Werngren, Diana Machado, Emanuele Borroni, Maria Wijkander, Gerard Lina, Johan Mouton, Erika Matuschek, Gunnar Kahlmeter, Christian Giske, Miguel Santin, Daniela Maria Cirillo, Miguel Viveiros, Emmanuelle Cambau

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51 Citations (Scopus)


Scope: Several methods are used worldwide for antibiotic susceptibility testing (AST) for the Mycobacterium tuberculosis complex (MTBC). The variability in the results obtained with these methods hampers to set epidemiological cut-off (ECOFF) values and clinical breakpoints according to EUCAST guidelines. Methods for susceptibility testing and determination of the minimal inhibitory concentrations (MIC) need to be standardized for MTBC isolates for old and new agents. Our objective was to establish a standardized reference method for MIC determination of the MTBC.

Methods: The EUCAST antimycobacterial susceptibility testing subcommittee (AMST) compared protocols of MIC determination with regard to medium, inoculum preparation, antituberculous agent preparation, incubation, reading of the results and interpretation.

Recommendations: The EUCAST reference method of MIC determination for MTBC is the broth microdilution method in Middlebrook 7H9-10%OADC medium. The final inoculum is a 105 CFU/ml suspension, from a 10-2 dilution of a 0.5 McFarland suspension prepared after vortexing bacterial colonies with glass beads before suspending them in water. The culture is maintained in a U-shaped 96-well polystyrene microtiter sterile plate with a lid incubated at 36°+1°C. Reading is done using an inverted mirror as soon as the 1:100 diluted control (i.e. 103 CFU/ml) shows visual growth. The MIC, expressed in mg/L, is the lowest concentration which inhibits visual growth. M. tuberculosis H37Rv ATCC 27294 is used as the reference strain and its targeted MIC values are within the range 0.03-0.12 for isoniazid, 0.12-0.5 for levofloxacin and 0.25-1 mg/L for amikacin.

Conclusions: The EUCAST reference method for MTBC was endorsed by EUCAST after public consultation and will from now on be used to define EUCAST ECOFFs and clinical breakpoints. This reference method is not primarily intended to be used under routine conditions and the AST methods need to be calibrated against this reference method to be used with EUCAST breakpoints.
Original languageEnglish
Pages (from-to)1488-1492
Number of pages5
JournalClin. Microbiol. Infect
Issue number11
Early online date1 Aug 2020
Publication statusPublished - Nov 2020


  • Broth microdilution
  • MIC
  • Tuberculosis
  • Reference method


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