Mycobacteriology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.
Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium.
Sci Rep. 2019 Aug 13;9(1):11760. doi: 10.1038/s41598-019-48289-9.
Heteroresistance - the simultaneous presence of drug-susceptible and -resistant organisms - is common in Mycobacterium tuberculosis. In this study, we aimed to determine the limit of detection (LOD) of genotypic assays to detect gatifloxacin-resistant mutants in experimentally mixed populations. A fluoroquinolone-susceptible M. tuberculosis mother strain (S) and its in vitro selected resistant daughter strain harbouring the D94G mutation in gyrA (R) were mixed at different ratio's. Minimum inhibitory concentrations (MICs) against gatifloxacin were determined, while PCR-based techniques included: line probe assays (Genotype MTBDRsl and GenoScholar-FQ + KM TB II), Sanger sequencing and targeted deep sequencing. Droplet digital PCR was used as molecular reference method. A breakpoint concentration of 0.25 mg/L allows the phenotypic detection of ≥1% resistant bacilli, whereas at 0.5 mg/L ≥ 5% resistant bacilli are detected. Line probe assays detected ≥5% mutants. Sanger sequencing required the presence of around 15% mutant bacilli to be detected as (hetero) resistant, while targeted deep sequencing detected ≤1% mutants. Deep sequencing and phenotypic testing are the most sensitive methods for detection of fluoroquinolone-resistant minority populations, followed by line probe assays (provided that the mutation is confirmed by a mutation band), while Sanger sequencing proved to be the least sensitive method.
异耐药性——同时存在对药物敏感和耐药的生物体——在结核分枝杆菌中很常见。在这项研究中，我们旨在确定基因分型检测方法检测实验混合种群中加替沙星耐药突变体的检测限（LOD）。将氟喹诺酮敏感结核分枝杆菌母株（S）及其体外选择的耐药子株（携带 gyrA 中的 D94G 突变的 R）以不同比例混合。测定对加替沙星的最小抑菌浓度（MIC），同时采用基于 PCR 的技术包括：线探针分析（基因型 MTBDRsl 和 GenoScholar-FQ+KM TB II）、Sanger 测序和靶向深度测序。液滴数字 PCR 用作分子参考方法。断点浓度为 0.25mg/L 可用于表型检测≥1%的耐药杆菌，而 0.5mg/L 时可检测到≥5%的耐药杆菌。线探针分析可检测到≥5%的突变体。Sanger 测序需要存在约 15%的突变体杆菌才能被检测为（异源）耐药，而靶向深度测序检测到≤1%的突变体。深度测序和表型检测是检测氟喹诺酮耐药少数群体的最敏感方法，其次是线探针分析（前提是突变通过突变带得到确认），而 Sanger 测序被证明是最不敏感的方法。
