Institute of Ecology and Genetic of Microorganisms, Perm Federal Research Center, Ural Branch RAS, Perm, Russia.
Int J Mycobacteriol. 2020 Apr-Jun;9(2):156-166. doi: 10.4103/ijmy.ijmy_61_20.
Mycobacterium smegmatis and other nontuberculous mycobacteria (NTM) are widely distributed in the environment, but a significant increase of NTM infections has taken place in the last few decades. The objective of this study was to determine the role of toxin-antitoxin (TA) vapBC and mazEF systems that act as effectors of persistence in the stress response of NTM.
The growth ability and the biofilm formation of NTM were evaluated by conventional methods. Bacterial cell viability was determined using MTT staining, agar plating, or the method of limiting dilutions. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antibiotics were estimated using broth and agar dilution methods.
Despite a comparable growth dynamics and biofilm formation on solid/liquid interface with the wild type, a M. smegmatis vapBC, mazEF, and vapBC × mazEF deletion mutant produced more abundant pellicle and were more susceptible to heat shock. Significant differences were also found in the resistance wild type of NTM to isoniazid and ciprofloxacin reflected by higher MBC/MIC ratios. The proposed method of cultivation of agar blocks without visible growth after MIC determination into a liquid medium allows us to detect transition of all wild type of NTM strains to a dormant state in the presence of subMICs of isoniazid and ciprofloxacin while all deletion mutants failed to form dormant cells.
Our data suggest that both vapBC and mazEF TA systems putatively involved in the heat and antibiotic stress response of NTM via their key role in transition to the dormant state.
耻垢分枝杆菌和其他非结核分枝杆菌（NTM）广泛分布于环境中，但在过去几十年中，NTM 感染的发生率显著增加。本研究的目的是确定毒素-抗毒素（TA） vapBC 和 mazEF 系统在 NTM 应激反应中作为持久效应子的作用。
采用常规方法评估 NTM 的生长能力和生物膜形成能力。使用 MTT 染色、琼脂平板或有限稀释法测定细菌细胞活力。采用肉汤和琼脂稀释法估计抗生素的最小抑菌浓度（MIC）和最小杀菌浓度（MBC）。
尽管 vapBC、mazEF 和 vapBC×mazEF 缺失突变体与野生型相比在固体/液体界面上具有相似的生长动力学和生物膜形成能力，但产生了更丰富的菌膜，并且对热休克更敏感。在异烟肼和环丙沙星对 NTM 野生型的耐药性方面也发现了显著差异，表现为更高的 MBC/MIC 比值。在 MIC 测定后，将琼脂块培养物培养在没有可见生长的液体培养基中，这种方法可以检测到所有 NTM 野生型菌株在亚 MIC 异烟肼和环丙沙星存在下向休眠状态的转变，而所有缺失突变体均无法形成休眠细胞。
我们的数据表明，vapBC 和 mazEF TA 系统可能通过在向休眠状态的转变中发挥关键作用，参与 NTM 的热应激和抗生素应激反应。
