Molecular Mycobacteriology, Research Center Borstel, Borstel, Germany.
PLoS One. 2013 Dec 6;8(12):e82551. doi: 10.1371/journal.pone.0082551. eCollection 2013.
Multidrug-resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains represent a major threat for tuberculosis (TB) control. Treatment of MDR-TB patients is long and less effective, resulting in a significant number of treatment failures. The development of further resistances leads to extensively drug-resistant (XDR) variants. However, data on the individual reasons for treatment failure, e.g. an induced mutational burst, and on the evolution of bacteria in the patient are only sparsely available. To address this question, we investigated the intra-patient evolution of serial MTBC isolates obtained from three MDR-TB patients undergoing longitudinal treatment, finally leading to XDR-TB. Sequential isolates displayed identical IS6110 fingerprint patterns, suggesting the absence of exogenous re-infection. We utilized whole genome sequencing (WGS) to screen for variations in three isolates from Patient A and four isolates from Patient B and C, respectively. Acquired polymorphisms were subsequently validated in up to 15 serial isolates by Sanger sequencing. We determined eight (Patient A) and nine (Patient B) polymorphisms, which occurred in a stepwise manner during the course of the therapy and were linked to resistance or a potential compensatory mechanism. For both patients, our analysis revealed the long-term co-existence of clonal subpopulations that displayed different drug resistance allele combinations. Out of these, the most resistant clone was fixed in the population. In contrast, baseline and follow-up isolates of Patient C were distinguished each by eleven unique polymorphisms, indicating an exogenous re-infection with an XDR strain not detected by IS6110 RFLP typing. Our study demonstrates that intra-patient microevolution of MDR-MTBC strains under longitudinal treatment is more complex than previously anticipated. However, a mutator phenotype was not detected. The presence of different subpopulations might confound phenotypic and molecular drug resistance tests. Furthermore, high resolution WGS analysis is necessary to accurately detect exogenous re-infection as classical genotyping lacks discriminatory power in high incidence settings.
耐多药（MDR）结核分枝杆菌复合体（MTBC）菌株对结核病（TB）控制构成重大威胁。MDR-TB 患者的治疗时间长且效果较差，导致大量治疗失败。进一步的耐药性发展导致广泛耐药（XDR）变异体。然而，关于治疗失败的个体原因（例如诱导的突变爆发）以及患者体内细菌的进化的数据仅很少可用。为了解决这个问题，我们研究了来自三位接受纵向治疗的 MDR-TB 患者的连续 MTBC 分离物的患者内进化，最终导致 XDR-TB。连续分离物显示出相同的 IS6110 指纹图谱模式，表明不存在外源性再感染。我们利用全基因组测序（WGS）分别筛选来自患者 A 的三个分离物和来自患者 B 和 C 的四个分离物中的变异。随后通过 Sanger 测序在多达 15 个连续分离物中验证获得的多态性。我们确定了八位（患者 A）和九位（患者 B）的多态性，这些多态性在治疗过程中以逐步的方式发生，并与耐药性或潜在的补偿机制相关。对于这两个患者，我们的分析表明在长期共存的克隆亚群中存在不同的耐药等位基因组合。在这些亚群中，最耐药的克隆在种群中固定。相比之下，患者 C 的基线和随访分离物分别由十一个独特的多态性区分，表明存在未通过 IS6110 RFLP 分型检测到的 XDR 菌株的外源性再感染。我们的研究表明，纵向治疗下 MDR-MTBC 菌株的患者内微进化比以前预期的更为复杂。然而，没有检测到突变体表型。不同亚群的存在可能会干扰表型和分子药物耐药性测试。此外，需要进行高分辨率 WGS 分析以准确检测外源性再感染，因为经典基因分型在高发地区缺乏区分力。
