Xu Jin-Tian, Li Kun, Lin Yi, Cheng Tao, Gu Jing, Chen Yao-Kai, Yu Ji-Fang, Deng Jiao-Yu
Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; University of Chinese Academy of Sciences, Beijing 100049, China.
Department of Infectious Diseases, Chongqing Public Health Medical Center, Chongqing 400036, China.
EBioMedicine. 2025 May 30;117:105776. doi: 10.1016/j.ebiom.2025.105776.
Since the discovery of streptomycin in the 1940s, more than a dozen drugs have been continuously introduced into tuberculosis (TB) therapy. However, limited attention has been paid to the collateral effects of drug resistance evolution in Mycobacterium tuberculosis (Mtb). Recently, we observed a clear discordance between the capreomycin (CAP) susceptibility of rifampicin-resistant (RR) Mtb clinical isolates and the adverse outcomes associated with CAP treatment, indicating potential collateral effects between rpoB mutations and CAP. To explore this relationship, we integrated clinical isolate data, experimental evolution data, phenotypic data, sequencing data, and genome-wide association studies (GWAS).
We analysed the correlations between CAP resistance and rpoB mutations at various loci based on phenotypic drug susceptibility testing (pDST) profiles and rpoB sequencing data from 565 RR Mtb isolates collected in southwestern China. To validate the clinical observations, we screened RR mutants of Mtb H37Rv and conducted rpoB sequencing to characterise the mutation sites. Additionally, we constructed various rpoB mutants in Mycobacterium smegmatis (Ms). We then examined the impact of these mutations on the efficacy of CAP through minimum inhibitory concentration (MIC) tests and time-kill assays in both Mtb and Ms rpoB mutants. Furthermore, we investigated the influence of three major rpoB mutations on the frequency of occurrence of rrs A1401G-associated with CAP resistance-using a GWAS of 607 Mtb genomes from a global dataset.
By analysing 565 clinical isolates from southwestern China, we found that the CAP resistance in isolates with a single mutation at rpoB site 445 was significantly lower than in those with a single mutation at other sites (P < 0.05, Pearson chi-square test and Fisher exact test; odds ratio = 0.272). In contrast, the opposite trend was observed in isolates with a single mutation at rpoB site 435 (P < 0.001, Pearson chi-square test and Fisher exact test; odds ratio = 3.067). Subsequently, using laboratory-evolved RR mutants, we demonstrated that mutations at rpoB site 445 or site 441 enhanced the bactericidal effect of CAP. However, the opposite result was observed in mutants with mutations at rpoB site 435. Furthermore, we found that the occurrence frequency of the rrs A1401G mutation was significantly lower in clinical isolates with rpoB mutations at site 445, but significantly higher in those with mutations at site 435.
Although rpoB mutations in Mtb did not affect the MIC of CAP, they influenced its bactericidal effect, highlighting the need for time-kill assays when investigating collateral effects. Different rpoB mutations may exert diverse impacts on the bactericidal effect of CAP-or CAP tolerance-underscoring the complexity of collateral effects and supporting the use of targeted sequencing in the molecular diagnosis of RR Mtb. As RNA polymerase plays a central role in bacterial RNA transcription, it regulates most metabolic processes in Mtb. Thus, different rpoB mutations may elicit distinct gene expression profiles upon CAP treatment, a hypothesis warranting further investigation. Additional clinical studies are needed to verify whether the adverse outcomes of CAP treatment are associated with infections caused by strains harbouring rpoB mutations at site 435. If so, such outcomes could be mitigated through rational drug regimens guided by precise molecular diagnosis. This study provides insights into the collateral effects of drug resistance mutations and advances the case for precision medicine in treating infections caused by drug-resistant bacteria.
Funding for this study was provided by the National Key Research and Development Program of China (Grant no. 2021YFA1300901).
自20世纪40年代发现链霉素以来，已有十多种药物不断被引入结核病（TB）治疗。然而，对结核分枝杆菌（Mtb）耐药性演变的附带效应关注有限。最近，我们观察到耐利福平（RR）Mtb临床分离株对卷曲霉素（CAP）的敏感性与CAP治疗相关的不良结局之间存在明显不一致，表明rpoB突变与CAP之间存在潜在的附带效应。为了探究这种关系，我们整合了临床分离株数据、实验进化数据、表型数据、测序数据和全基因组关联研究（GWAS）。
我们根据中国西南部收集的565株RR Mtb分离株的表型药物敏感性测试（pDST）结果和rpoB测序数据，分析了不同位点的CAP耐药性与rpoB突变之间的相关性。为了验证临床观察结果，我们筛选了Mtb H37Rv的RR突变体并进行rpoB测序以表征突变位点。此外，我们在耻垢分枝杆菌（Ms）中构建了各种rpoB突变体。然后，我们通过最低抑菌浓度（MIC）测试和时间杀菌试验，研究了这些突变对Mtb和Ms rpoB突变体中CAP疗效的影响。此外，我们利用来自全球数据集的607个Mtb基因组的GWAS，研究了三个主要rpoB突变对与CAP耐药性相关的rrs A1401G发生频率的影响。
通过分析来自中国西南部的565株临床分离株，我们发现rpoB位点445处有单个突变的分离株对CAP的耐药性明显低于其他位点有单个突变的分离株（P < 0.05，Pearson卡方检验和Fisher精确检验；优势比 = 0.272）。相反，在rpoB位点435处有单个突变的分离株中观察到相反的趋势（P < 0.001，Pearson卡方检验和Fisher精确检验；优势比 = 3.067）。随后，使用实验室进化的RR突变体，我们证明rpoB位点445或位点441处的突变增强了CAP的杀菌效果。然而，在rpoB位点435处有突变的突变体中观察到相反的结果。此外，我们发现rrs A1401G突变在rpoB位点445处有突变的临床分离株中的发生频率明显较低，但在rpoB位点435处有突变的分离株中明显较高。
虽然Mtb中的rpoB突变不影响CAP的MIC，但它们影响其杀菌效果，突出了在研究附带效应时进行时间杀菌试验的必要性。不同的rpoB突变可能对CAP的杀菌效果或CAP耐受性产生不同的影响，强调了附带效应的复杂性，并支持在RR Mtb的分子诊断中使用靶向测序。由于RNA聚合酶在细菌RNA转录中起核心作用，它调节Mtb中的大多数代谢过程。因此，不同的rpoB突变在CAP治疗后可能引发不同的基因表达谱，这一假设值得进一步研究。需要更多的临床研究来验证CAP治疗的不良结局是否与rpoB位点435处有突变的菌株引起的感染有关。如果是这样，可以通过精确分子诊断指导的合理药物方案来减轻这些结局。本研究为耐药性突变的附带效应提供了见解，并推动了在治疗耐药细菌引起的感染中精准医学的发展。
本研究由中国国家重点研发计划（项目编号：2021YFA1300901）提供资金支持。
