Instituto de Cálculo, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.
Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.
BMC Infect Dis. 2021 Apr 29;21(1):394. doi: 10.1186/s12879-021-06069-9.
Whole-genome sequencing has shown that the Mycobacterium tuberculosis infection process can be more heterogeneous than previously thought. Compartmentalized infections, exogenous reinfections, and microevolution are manifestations of this clonal complexity. The analysis of the mechanisms causing the microevolution -the genetic variability of M. tuberculosis at short time scales- of a parental strain into clonal variants with a patient is a relevant issue that has not been yet completely addressed. To our knowledge, a whole genome sequence microevolution analysis in a single patient with inadequate adherence to treatment has not been previously reported.
In this work, we applied whole genome sequencing analysis for a more in-depth analysis of the microevolution of a parental Mycobacterium tuberculosis strain into clonal variants within a patient with poor treatment compliance in Argentina. We analyzed the whole-genome sequence of 8 consecutive Mycobacterium tuberculosis isolates obtained from a patient within 57-months of intermittent therapy. Nineteen mutations (9 short-term, 10 fixed variants) emerged, most of them associated with drug resistance. The first isolate was already resistant to isoniazid, rifampicin, and streptomycin, thereafter the strain developed resistance to fluoroquinolones and pyrazinamide. Surprisingly, isolates remained susceptible to the pro-drug ethionamide after acquiring a frameshift mutation in ethA, a gene required for its activation. We also found a novel variant, (T-54G), in the 5' untranslated region of whiB7 (T-54G), a region allegedly related to kanamycin resistance. Notably, discrepancies between canonical and phage-based susceptibility testing to kanamycin were previously found for the isolate harboring this mutation. In our patient, microevolution was mainly driven by drug selective pressure. Rare short-term mutations fixed together with resistance-conferring mutations during therapy.
This report highlights the relevance of whole-genome sequencing analysis in the clinic for characterization of pre-XDR and MDR resistance profile, particularly in patients with incomplete and/or intermittent treatment.
全基因组测序表明，结核分枝杆菌的感染过程比之前认为的更为复杂。分隔感染、外源再感染和微进化是这种克隆复杂性的表现。分析导致微进化的机制——即在短时间尺度上结核分枝杆菌的遗传变异——是一个重要的问题，但尚未得到完全解决。据我们所知，以前没有报道过对一个治疗依从性差的患者的原始菌株进行全基因组序列微进化分析。
在这项工作中，我们应用全基因组测序分析对一个来自阿根廷的治疗依从性差的患者体内的原始结核分枝杆菌菌株向克隆变体的微进化进行了更深入的分析。我们分析了 8 个连续的结核分枝杆菌分离株的全基因组序列，这些分离株是在间歇性治疗的 57 个月内从一个患者中获得的。出现了 19 个突变（9 个短期突变，10 个固定变异），其中大多数与耐药性有关。第一个分离株已经对异烟肼、利福平、链霉素耐药，此后该菌株对氟喹诺酮类药物和吡嗪酰胺耐药。令人惊讶的是，在 ethA 基因发生移码突变后，该菌株对其前体乙硫异烟胺仍保持敏感，ethA 基因是其激活所必需的。我们还发现了一个新的变异体，在 whiB7 的 5'非翻译区（T-54G），这是一个据称与卡那霉素耐药相关的区域。值得注意的是，携带这种突变的分离株先前在卡那霉素的经典和噬菌体药敏试验之间存在差异。在我们的患者中，微进化主要是由药物选择压力驱动的。在治疗过程中，罕见的短期突变与耐药性相关的突变一起固定。
本报告强调了全基因组测序分析在临床中的重要性，用于对预广泛耐药和耐多药的耐药谱进行特征描述，特别是在治疗不完整和/或间歇性的患者中。
