Ismail N, Omar S V, Ismail N A, Peters R P H
Department of Medical Microbiology, Faculty of Health Sciences, University of Pretoria, 0002 Prinshof, Gauteng, South Africa.
Centre for Tuberculosis, National Institute for Communicable Diseases, National Health Laboratory Service, Sandringham, Gauteng, South Africa.
J Microbiol Methods. 2018 Oct;153:1-9. doi: 10.1016/j.mimet.2018.08.011. Epub 2018 Aug 28.
Bedaquiline, 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.
贝达喹啉、氯法齐明和利奈唑胺是治疗耐药结核病的相关药物。耐药突变体有助于深入了解重要的耐药性获得机制。关于体外结核分枝杆菌突变体产生的方法描述甚少。使用结核分枝杆菌ATCC参考菌株（一株完全敏感、四株独特的单耐药菌株）进行了诱导（连续传代）和自发（改良的卢里亚-德尔布吕克试验）方法。确认了突变体的最低抑菌浓度（MGIT960），并比较了两种方法产生的耐药性增加值（RAV）以及与先前发表的RAV目录的差异。与基线相比，突变体的最低抑菌浓度显示出3至4倍（诱导）和1至4倍（自发）的增加。对于贝达喹啉，耐吡嗪酰胺菌株的基线最低抑菌浓度较高，并且在比其他菌株更少的传代次数中获得了耐药性（≥4倍）（诱导方法）。在贝达喹啉和氯法齐明耐药突变体中鉴定出了先前描述的以及atpE（8个与1个）和rv0678（4个与12个）基因中的新RAV。未鉴定出rv1979c和rv2535c RAV。在利奈唑胺耐药突变体的rplC和rrl基因中鉴定出了先前描述的RAV。两种方法均成功产生了体外突变体，并且在atpE和rv0678基因中描述了新的RAV。观察到预先存在的耐药性可能会影响突变体的表型和基因型特征，并值得进一步关注。
