Division of Pulmonary Medicine, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical Universitygrid.412896.0, Taipei, Taiwan.
School of Respiratory Therapy, College of Medicine, Taipei Medical Universitygrid.412896.0, Taipei, Taiwan.
Microbiol Spectr. 2022 Aug 31;10(4):e0075422. doi: 10.1128/spectrum.00754-22. Epub 2022 Aug 4.
Drug resistance in Mycobacterium tuberculosis (MTB) has long been a serious health issue worldwide. Most drug-resistant MTB isolates were identified due to treatment failure or in clinical examinations 3~6 months postinfection. In this study, we propose a whole-genome sequencing (WGS) pipeline via the Nanopore MinION platform to facilitate the efficacy of phenotypic identification of clinical isolates. We used the Nanopore MinION platform to perform WGS of clinical MTB isolates, including susceptible (=30) and rifampin- (RIF) or rifabutin (RFB)-resistant isolates (=20) according to results of a susceptibility test. Nonsynonymous variants within the gene associated with RIF resistance were identified using the WGS analytical pipeline. In total, 131 variants within the gene in RIF-resistant isolates were identified. The presence of the emergent Asp531Gly or His445Gln was first identified to be associated with the rifampin and rifabutin resistance signatures of clinical isolates. The results of the minimum inhibitory concentration (MIC) test further indicated that the Ser450Leu or the mutant within the rifampin resistance-determining region (RRDR)-associated rifabutin-resistant signature was diminished in the presence of novel mutants, including Phe669Val, Leu206Ile, or Met148Leu, identified in this study. Current approaches to diagnose drug-resistant MTB are time-consuming, consequently leading to inefficient intervention or further disease transmission. In this study, we curated lists of coding variants associated with differential rifampin and rifabutin resistant signatures using a single molecule real-time (SMRT) sequencing platform with a shorter hands-on time. Accordingly, the emerging WGS pipeline constitutes a potential platform for efficacious and accurate diagnosis of drug-resistant MTB isolates.
结核分枝杆菌（MTB）的耐药性一直是全球范围内的严重健康问题。大多数耐药 MTB 分离株是由于治疗失败或感染后 3~6 个月的临床检查而确定的。在这项研究中，我们提出了一种基于纳米孔 MinION 平台的全基因组测序（WGS）工作流程，以促进临床分离株表型鉴定的效果。我们使用纳米孔 MinION 平台对临床 MTB 分离株进行 WGS，包括药敏试验结果为敏感（=30）和利福平（RIF）或利福布汀（RFB）耐药的分离株（=20）。使用 WGS 分析工作流程鉴定与 RIF 耐药相关的 基因中的非同义变体。在 RIF 耐药分离株中，共鉴定出 基因内的 131 个变体。首次鉴定出利福平耐药和利福布汀耐药的临床分离株中出现的 Asp531Gly 或 His445Gln 与 rifampin 和 rifabutin 耐药特征有关。最小抑菌浓度（MIC）试验的结果进一步表明，在本研究中鉴定出的新型突变体（包括 Phe669Val、Leu206Ile 或 Met148Leu）的存在下，与 rifampin 耐药决定区（RRDR）相关的 rifabutin 耐药特征中的 Ser450Leu 或突变体的耐药性降低。目前诊断耐药 MTB 的方法耗时较长，因此导致干预效率低下或进一步疾病传播。在这项研究中，我们使用单分子实时（SMRT）测序平台在更短的操作时间内，为差异 rifampin 和 rifabutin 耐药特征相关的编码变体编制了列表。因此，新兴的 WGS 工作流程构成了一种有效的、准确的耐药 MTB 分离株诊断的潜在平台。
