OBJECTIVES 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

中文翻译：

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临床分离的结核分枝杆菌中降低了对苯达喹啉的敏感性和耐药性。

目的贝达喹啉是一种有效的药物，用于治疗MDR和XDR结核病，在复杂的治疗方法中具有较高的治愈率。mmpR（rv0678）和atpE基因的突变与对苯达喹啉的敏感性降低有关，并且已在体外选择的菌株和临床分离株中得到鉴定。但是，由于从接受含苯达喹啉治疗方案的患者中收集了少量临床分离株，因此尚未建立用于检测苯达喹啉耐药性的表型标准。方法分析74例接受苯达喹啉治疗的患者的182个临床分离株，以及107例未接触苯达喹啉的患者的163个分离株。在7H11琼脂平板和Bactec MGIT 960系统上使用系列稀释液测试了苯达喹啉MIC。通过Sanger测序对mmpR和atpE基因进行测序。结果7H11琼脂法可快速区分突变型和野生型分离株以及暴露和未暴露的分离株。相较于参考菌株（89％的MIC≤0.03 mg / L），暴露于苯达喹啉的分离株中有73％以及突变的菌株中有91％的苯达喹啉MIC（在7H11培养基上≥0.12 mg / L）升高。 L）。在临床分离物中观察到先前报道的体外选择的突变体（E61D和A63P）和新型AtpE替代物（G25S和D28G）。AtpE密码子63的取代可能与较高的bedaquiline MIC有关。确定了五例新存在的对苯达喹啉的敏感性降低的病例，在大多数分离物中伴有mmpR突变，没有苯达喹啉的治疗史。结论贝达喹啉治疗可导致苯达喹啉MIC升高，并导致结核菌菌株获得mmpR和atpE基因突变。基于我们研究与以往研究之间的差异以及固体和液体介质MIC测定的差异，迫切需要贝达喹啉表型药敏试验的标准化。