The Moscow Research and Clinical Center for Tuberculosis Control of the Moscow Government Health Department, Moscow, Russia.
Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia.
J Infect. 2020 May;80(5):527-535. doi: 10.1016/j.jinf.2020.01.007. Epub 2020 Jan 23.
Bedaquiline is an effective drug used to treat MDR and XDR tuberculosis, providing high cure rates in complex therapy. Mutations in the mmpR (rv0678) and atpE genes are associated with reduced susceptibility to bedaquiline and have been identified in both in vitro selected strains and clinical isolates. However, the phenotypic criteria used to detect bedaquiline resistance have yet to be established due to the collection of few clinical isolates from patients receiving bedaquiline-containing treatment regimens.
One hundred eighty-two clinical isolates from 74 patients receiving bedaquiline and 163 isolates from 107 patients not exposed to bedaquiline were analysed. The bedaquiline MICs were tested using serial dilutions on 7H11 agar plates and the Bactec MGIT 960 system. The mmpR and atpE genes were sequenced by Sanger sequencing.
The 7H11 agar method allowed for rapid discrimination between mutated and wild-type isolates and between exposed and non-exposed isolates. Seventy-three percent of bedaquiline-exposed isolates, as well as 91% of isolates with mutations, had an elevated bedaquiline MIC (≥ 0.12 mg/L on 7H11 media) compared to the reference isolates (89% had an MIC ≤ 0.03 mg/L). Previously reported in vitro-selected mutants (E61D and A63P) and novel AtpE substitutions (G25S and D28G) were observed in the clinical isolates. Substitutions in codon 63 of AtpE were likely associated with a higher bedaquiline MIC. Five new cases of pre-existing reduced susceptibility to bedaquiline, accompanied by mmpR mutations in most isolates, without a history of bedaquiline treatment were identified.
Bedaquiline treatment leads to an elevated bedaquiline MIC and the acquisition of mmpR and atpE gene mutations in tuberculosis strains. The standardisation of bedaquiline phenotypic susceptibility testing is urgently needed based on observed discrepancies between our study and previous studies and differences in solid and liquid media MIC determinations.
贝达喹啉是一种有效的药物，用于治疗耐多药和广泛耐药结核病，在复杂治疗中提供高治愈率。mmpR（rv0678）和 atpE 基因突变与贝达喹啉的敏感性降低有关，在体外选择的菌株和临床分离株中均有发现。然而，由于从接受贝达喹啉含药治疗方案的患者中收集的临床分离株很少，因此尚未建立用于检测贝达喹啉耐药性的表型标准。
分析了 74 名接受贝达喹啉治疗的患者的 182 株临床分离株和 107 名未接触贝达喹啉的患者的 163 株分离株。使用 7H11 琼脂平板的连续稀释法和 Bactec MGIT 960 系统检测贝达喹啉 MIC。通过 Sanger 测序对 mmpR 和 atpE 基因进行测序。
7H11 琼脂法可快速区分突变型和野生型分离株以及暴露型和非暴露型分离株。与参考分离株（89%的 MIC 为≤0.03mg/L）相比，73%的贝达喹啉暴露分离株和 91%的突变分离株的贝达喹啉 MIC 升高（7H11 培养基上的 MIC≥0.12mg/L）。在临床分离株中观察到了先前报道的体外选择突变（E61D 和 A63P）和新型 AtpE 取代（G25S 和 D28G）。AtpE 密码子 63 中的取代可能与更高的贝达喹啉 MIC 相关。确定了 5 例新的贝达喹啉固有低敏感性病例，这些病例在大多数分离株中均存在 mmpR 突变，且无贝达喹啉治疗史。
贝达喹啉治疗导致结核菌株的贝达喹啉 MIC 升高和 mmpR 和 atpE 基因突变的获得。根据我们的研究与之前研究之间的差异以及固体和液体培养基 MIC 测定之间的差异，迫切需要标准化贝达喹啉表型药敏试验。
