Department of Medicine, Division of Infectious Disease, Ruy V. Lourenco Center for the Study of Emerging and Re-emerging Pathogens, Rutgers New Jersey Medical School, Newark, New Jersey, USA.
Department of Hydrology, Indian Institute of Technology, Roorkee, Uttarakhand, India.
mBio. 2022 Dec 20;13(6):e0279522. doi: 10.1128/mbio.02795-22. Epub 2022 Nov 8.
Initial responses to tuberculosis treatment are poor predictors of final therapeutic outcomes in drug-susceptible disease, suggesting that treatment success depends on features that are hidden within a small minority of the overall infecting Mycobacterium tuberculosis population. We developed a multitranswell robotic system to perform numerous parallel cultures of genetically barcoded M. tuberculosis exposed to steady-state concentrations of rifampicin to uncover these difficult-to-eliminate minority populations. We found that tolerance emerged repeatedly from at least two subpopulations of barcoded cells, namely, one that could not grow on solid agar media and a second that could form colonies, but whose kill curves diverged from the general bacterial population within 4 and 16 days of drug exposure, respectively. These tolerant subpopulations reproducibly passed through a phase characterized by multiple unfixed resistance mutations followed by emergent drug resistance in some cultures. Barcodes associated with drug resistance identified an especially privileged subpopulation that was rarely eliminated despite 20 days of drug treatment even in cultures that did not contain any drug-resistant mutants. The association of this evolutionary scenario with a defined subset of barcodes across multiple independent cultures suggested a transiently heritable phenotype, and indeed, phase variation mutants were associated with up to 16% of the resistant cultures. Drug tolerance and resistance were eliminated in a Δ mutant, consistent with the importance of bacterial stress responses. This work provides a window into the origin and dynamics of bacterial drug-tolerant subpopulations whose elimination may be critical for developing rapid and resistance-free cures. Tuberculosis is unusual among bacterial diseases in that treatments which can rapidly resolve symptoms do not predictably lead to a durable cure unless treatment is continued for months after all clinical and microbiological signs of disease have been eradicated. Using a novel steady-state antibiotic exposure system combined with chromosomal barcoding, we identified small hidden Mycobacterium tuberculosis subpopulations that repeatedly enter a state of drug tolerance with a predisposition to develop fixed drug resistance after first developing a cloud of unfixed resistance mutations. The existence of these difficult-to-eradicate subpopulations may explain the need for extended treatment regimen for tuberculosis. Their identification provides opportunities to test genetic and therapeutic approaches that may result in shorter and more effective TB treatments.
初始的结核病治疗反应是耐多药疾病最终治疗结果的不良预测指标，这表明治疗成功取决于在整个结核分枝杆菌感染人群中的一小部分隐藏特征。我们开发了一种多transwell 机器人系统，对暴露于利福平稳定浓度下的遗传条形码结核分枝杆菌进行许多平行培养，以揭示这些难以消除的少数人群。我们发现，耐受性至少从两个条形码细胞亚群中反复出现，一个是不能在固体琼脂培养基上生长的亚群，另一个是可以形成菌落的亚群，但它们的杀灭曲线分别在药物暴露后 4 天和 16 天与一般细菌群体不同。这些具有耐受性的亚群可重复性地经历一个特征为多次未固定耐药突变的阶段，随后在一些培养物中出现耐药性。与耐药性相关的条形码鉴定出一个特别有利的亚群，即使在没有任何耐药突变体的培养物中，即使经过 20 天的药物治疗，该亚群也很少被消除。这种进化情景与多个独立培养物中定义的亚群相关联表明存在一个短暂的可遗传表型，实际上，与多达 16%的耐药培养物相关联的是相位变异突变体。在Δ突变体中消除了药物耐受性和耐药性，这与细菌应激反应的重要性一致。这项工作为了解细菌药物耐受性亚群的起源和动态提供了一个窗口，这些亚群的消除可能对开发快速和无耐药性的治疗方法至关重要。 结核病在细菌疾病中是不同寻常的，因为能够迅速缓解症状的治疗方法并不能预测性地导致持久的治愈，除非在所有临床和微生物学疾病迹象都被消除后，再继续治疗数月。我们使用一种新的稳定抗生素暴露系统结合染色体条形码，鉴定出小的隐藏的结核分枝杆菌亚群，这些亚群在首先产生一团未固定的耐药突变后，会反复进入药物耐受状态，并容易发展为固定耐药性。这些难以消除的亚群的存在可能解释了需要延长结核病治疗方案的原因。它们的鉴定为测试可能导致更短和更有效的结核病治疗的遗传和治疗方法提供了机会。
