Nair Rashmi Ravindran, Sharan Deepti, Ajitkumar Parthasarathi
Department of Microbiology and Cell Biology, Indian Institute of Science, Bengaluru, India.
Front Microbiol. 2019 Aug 13;10:1842. doi: 10.3389/fmicb.2019.01842. eCollection 2019.
Antibiotic-exposed bacteria produce elevated levels of reactive oxygen species (ROS), to which either they succumb or get mutated genome-wide to generate antibiotic resisters. We recently showed that mycobacterial cultures contained two subpopulations, short-sized cells (SCs; ∼10%) and normal/long-sized cells (NCs; ∼90%). The SCs were significantly more antibiotic-susceptible than the NCs. It implied that the SCs might naturally be predisposed to generate significantly higher levels of ROS than the NCs. This in turn could make the SCs more susceptible to antibiotics or generate more resisters as compared to the NCs. Investigation into this possibility showed that the SCs in the actively growing mid-log phase culture naturally generated significantly high levels of superoxide, as compared to the equivalent NCs, due to the naturally high expression of a specific NADH oxidase in the SCs. This caused labile Fe leaching from 4Fe-4S proteins and elevated HO formation through superoxide dismutation. Thus, the SCs of both and inherently contained significantly higher levels of HO and labile Fe than the NCs. This in turn produced significantly higher levels of hydroxyl radical through Fenton reaction, promoting enhanced antibiotic resister generation from the SCs than from the NCs. The SCs, when mixed back with the NCs, at their natural proportion in the actively growing mid-log phase culture, enhanced antibiotic resister generation from the NCs, to a level equivalent to that from the unfractionated whole culture. The enhanced antibiotic resister generation from the NCs in the reconstituted SCs-NCs natural mixture was found to be due to the high levels of HO secreted by the SCs. Thus, the present work unveils and documents the metabolic designs of two mycobacterial subpopulations where one subpopulation produces high ROS levels, despite higher susceptibility, to generate significantly higher number of antibiotic resisters from itself and to enhance resister generation from its kin subpopulation. These findings show the existence of an inherent natural mechanism in both the non-pathogenic and pathogenic mycobacteria to generate antibiotic resisters. The presence of the SCs and the NCs in the pulmonary tuberculosis patients' sputum, reported by us earlier, alludes to the clinical significance of the study.
接触抗生素的细菌会产生更高水平的活性氧（ROS），它们要么会因此死亡，要么会在全基因组范围内发生突变以产生抗生素抗性菌。我们最近发现，分枝杆菌培养物包含两个亚群，短细胞（SCs；约10%）和正常/长细胞（NCs；约90%）。SCs比NCs对抗生素更敏感。这意味着SCs可能天生就比NCs更容易产生更高水平的ROS。反过来，这可能使SCs比NCs更容易受到抗生素的影响，或者产生更多的抗性菌。对这种可能性的研究表明，与同等数量的NCs相比，处于对数中期活跃生长阶段的培养物中的SCs由于SCs中特定NADH氧化酶的自然高表达，自然会产生显著高水平的超氧化物。这导致不稳定的铁从4Fe-4S蛋白中浸出，并通过超氧化物歧化作用增加羟基自由基（HO）的形成。因此，和的SCs固有地比NCs含有显著更高水平的HO和不稳定铁。这反过来又通过芬顿反应产生显著更高水平的羟基自由基，促进SCs比NCs产生更多的抗生素抗性菌。当SCs以其在对数中期活跃生长阶段培养物中的自然比例与NCs重新混合时，会增强NCs产生抗生素抗性菌的能力，使其达到与未分离的全培养物相当的水平。在重新构建的SCs-NCs自然混合物中，发现NCs产生抗生素抗性菌的能力增强是由于SCs分泌的高水平HO。因此，目前的研究揭示并记录了两个分枝杆菌亚群的代谢设计，其中一个亚群尽管敏感性较高，但会产生高水平的ROS，从而从自身产生显著更多数量的抗生素抗性菌，并增强其同类亚群产生抗性菌的能力。这些发现表明，在非致病性和致病性分枝杆菌中都存在一种固有的自然机制来产生抗生素抗性菌。我们之前报道的肺结核患者痰液中存在SCs和NCs，暗示了该研究的临床意义。
