Ismail Nabila, Sirgel Frik, Omar Shaheed Vally, Omar Shatha, de Kock Marianna, Spies Claudia, Folkerts Megan, Theron Grant, Engelthaler Dave, Metcalfe John, Warren Robin M
South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa.
Centre for Tuberculosis, National Institute for Communicable Diseases, Johannesburg, South Africa.
bioRxiv. 2025 Mar 10:2025.03.09.642295. doi: 10.1101/2025.03.09.642295.
Phenotypic drug susceptibility testing (pDST) remains the gold standard for complex drug resistance determination. Next generation sequencing technologies can identify heteroresistant populations at low frequencies, but little is known about the impact of heteroresistance on bedaquiline (BDQ) pDST results. We simulated heteroresistance using generated mutants mixed with the progenitor strain at various percentages (1-20%) and did pDST using MGIT960 culture (1 and 2 μg/mL BDQ concentrations). Targeted Next Generation Sequencing (tNGS) was used to quantify the mutant sub-population in growth control tubes, which were expected to maintain the mutant: wild type proportion throughout the assay. Growth units of these growth control tubes were also comparable with minor differences in time-to-positivity between ratio mixtures. Only when intermediate results were considered could BDQ heteroresistance be detected at frequencies of approximately 1% by pDST at a critical concentration of 1 μg/mL using BACTEC MGIT960 coupled with EpiCenter TBeXiST software. The ability of pDST, a widely available DST technique, to reveal the presence of BDQ-resistant subpopulations at the phenotypic testing stage could improve resistance determination and potentially reduce time to effective treatment.
表型药敏试验（pDST）仍然是确定复杂耐药性的金标准。新一代测序技术可以识别低频存在的异质性耐药群体，但关于异质性耐药对贝达喹啉（BDQ）pDST结果的影响知之甚少。我们使用生成的突变体与亲代菌株按不同比例（1%-20%）混合来模拟异质性耐药，并使用MGIT960培养法（BDQ浓度为1和2μg/mL）进行pDST。使用靶向新一代测序（tNGS）对生长对照管中的突变亚群进行定量，预期在整个检测过程中突变体与野生型的比例保持不变。这些生长对照管的生长单位也具有可比性，不同比例混合物之间的阳性时间略有差异。只有将中间结果纳入考虑时，使用BACTEC MGIT960和EpiCenter TBeXiST软件，在1μg/mL的临界浓度下通过pDST才能检测到频率约为1%的BDQ异质性耐药。pDST作为一种广泛应用的药敏试验技术，在表型检测阶段揭示BDQ耐药亚群存在的能力，可改善耐药性判定，并可能缩短有效治疗所需时间。
