Ismail, Nazir AhmedOmar, Shaheed VallyIsmail, NabilaPeters, Remco P.H.2019-03-082018-10Ismail, N., Omar, S.V., Ismail, N.A. et al. 2018, 'In vitro approaches for generation of Mycobacterium tuberculosis mutants resistant to bedaquiline, clofazimine or linezolid and identification of associated genetic variants', Journal of Microbiological Methods, vol. 153, pp. 1-9.0167-7012 (print)1872-8359 (online)10.1016/j.mimet.2018.08.011http://hdl.handle.net/2263/68614Bedaquiline, clofazimine and linezolid are pertinent drugs for drug-resistant tuberculosis. Drug-resistant mutants provide insight into important resistance acquisition mechanisms. Methods for in vitro Mycobacterium tuberculosis mutant generation are poorly described. Induction (serial passaging) and spontaneous (adapted Luria-Delbrück assay) approaches using M. tuberculosis ATCC reference strains (one fully-susceptible, four unique mono-resistant) were performed. Mutant MIC values were confirmed (MGIT960) and resultant RAVs compared between approaches and to a catalog of previously published RAVs. Mutant MIC values showed a 3–4-fold (induced) and a 1–4-fold (spontaneous) increase compared to baseline. The pyrazinamide-resistant strain had higher baseline MIC values and acquired resistance (≥4-fold) in fewer passages than other strains (induction approach) for bedaquiline. Previously described and novel RAVs in atpE (8 vs. 1) and rv0678 (4 vs. 12) genes were identified in bedaquiline- and clofazimine-resistant mutants. No rv1979c and rv2535c RAVs were identified. Previously described RAVs were identified in rplC and rrl genes for linezolid-resistant mutants. Both approaches successfully led to in vitro mutants with novel RAVs being described in atpE and rv0678 genes. It was observed that pre-existing resistance may influence mutant phenotypic and genotypic characteristics and warrants further attention.en© 2018 Elsevier B.V. All rights reserved. Notice : this is the author’s version of a work that was accepted for publication in Journal of Microbiological Methods. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. A definitive version was subsequently published in Journal of Microbiological Methods, vol. 153, pp. 1-9, 2018. doi : 10.1016/j.mimet.2018.08.011.MutantsBedaquilineClofazimineIn vitroMycobacterium tuberculosis (MTB)LinezolidPostprint Article