Background Drug-resistant tuberculosis is a growing public health threat, and early characterization of the resistance phenotype is essential for guiding treatment and mitigating the high mortality associated with the disease. However, the slow growth rate of Mycobacterium tuberculosis, the causative agent of tuberculosis, necessitates several weeks for conventional culture-dependent drug susceptibility testing (DST). In addition, there are no widely available molecular diagnostic assays for evaluating resistance to newer tuberculosis drugs or drugs with complex resistance mechanisms. Methods We have developed a luciferase-based reporter mycobacteriophage assay that can determine drug resistance within 48 hours. We engineered the TM4 mycobacteriophage to express green enhanced nanoluciferase (GeNL) cassette and optimized DST for bedaquiline, pretomanid, linezolid, clofazimine, and rifampicin using clinical M. tuberculosis isolates. Results To assess the feasibility of this assay, we conducted a proof-of-principle study using 53 clinical M. tuberculosis isolates. TM4::GeNL phage DST effectively distinguished between sensitive and resistant isolates for bedaquiline and rifampicin at a concentration of 0.125 μg/mL. Optimal differentiation between sensitive and resistant isolates for pretomanid, clofazimine, and linezolid was achieved at concentrations of 0.5 μg/mL, 0.25 μg/mL, and 1 μg/mL, respectively. Additionally, TM4::GeNL DST identified low-level rifampicin resistance in clinical isolates even though they were classified as sensitive by Mycobacteria Growth Indicator Tube DST. Conclusions TM4::GeNL reporter phage DST offers a rapid method to identify M. tuberculosis drug resistance, including resistance to newer tuberculosis drugs.

中文翻译：

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工程化噬菌体 TM4：：GeNL 快速测定结核分枝杆菌临床分离株中贝达喹啉、普雷托马尼、利奈唑胺、利福平和氯法齐明的敏感性


背景 耐药结核病是一个日益严重的公共卫生威胁，耐药表型的早期表征对于指导治疗和减轻与该疾病相关的高死亡率至关重要。然而，结核分枝杆菌（结核病的病原体）生长缓慢，需要数周进行常规培养依赖性药物敏感性试验 （DST）。此外，没有广泛可用的分子诊断检测方法用于评估对新型结核病药物或具有复杂耐药机制的药物的耐药性。方法 我们开发了一种基于荧光素酶的报告分枝杆菌噬菌体测定法，可以在 48 小时内确定耐药性。我们设计了 TM4 分枝杆菌噬菌体来表达绿色增强型纳米荧光素酶 （GeNL） 盒，并使用临床结核分枝杆菌分离株优化了贝达喹啉、普雷托马尼、利奈唑胺、氯法齐明和利福平的 DST。结果 为了评估该测定的可行性，我们使用 53 种临床结核分枝杆菌分离株进行了一项原理验证研究。TM4：：GeNL 噬菌体 DST 在 0.125 μg/mL 的浓度下有效区分贝达喹啉和利福平的敏感和耐药分离株。pretomanid、氯法齐明和利奈唑胺的敏感和耐药分离株在浓度分别为 0.5 μg/mL、0.25 μg/mL 和 1 μg/mL 时实现最佳区分。此外，TM4：：GeNL DST 在临床分离株中鉴定出低水平利福平耐药性，即使它们被分枝杆菌生长指示管 DST 归类为敏感。结论 TM4：：GeNL 报告基因噬菌体 DST 提供了一种快速鉴定结核分枝杆菌耐药性的方法，包括对新型结核药物的耐药性。