Abstract：
Mycobacterium abscessus is a non-tuberculous mycobacterial pathogen known to cause pulmonary and skin infections worldwide. Renowned for its multidrug resistance, M. abscessus infections often result in unfavorable clinical outcomes. Clarithromycin plays a pivotal role in treating M. abscessus infections, with resistance commonly leads to treatment failure. While canonical mutations in 23S rRNA residue 2270/2271 are recognized as a major mechanism for acquired clarithromycin resistance, resistant isolates devoid of such mutations have been widely reported. In this study, we conducted a comprehensive investigation into acquired clarithromycin resistance using spontaneous mutants derived from two parental strains characterized by erm(41) T28 and C28 sequevars respectively. A total of 135 resistant mutants were selected from the parental strains. Sequencing of the 78 mutants lacking canonical 2270/2271 mutations identified mutations within the peptidyl-transferase center and in hairpin loops 35, 49, and 74 of the 23S rRNA. Moreover, these noncanonical mutations were identified in 57 out of 1875 genomes of clinical isolates. Thirteen representative mutations were introduced into the bacterial genome via site-directed mutagenesis, and their contribution to macrolide resistance was verified. Mapping these mutations onto the three-dimensional structure of 23S rRNA revealed their localization at the entrance of the nascent peptide exit tunnel, potentially contributing to resistance by disrupting the macrolide binding pocket. The identification of these noncanonical 23S rRNA mutations advances our understanding of macrolide resistance in M. abscessus and underscores their importance as potential markers for detecting clarithromycin resistance.
摘要：
脓肿分枝杆菌是一种非结核分枝杆菌病原体，已知在全球范围内引起肺部和皮肤感染。以其多重耐药性而闻名，脓肿分枝杆菌感染通常导致不利的临床结果。克拉霉素在治疗脓肿分枝杆菌感染中起关键作用，抵抗通常导致治疗失败。虽然23SrRNA残基2270/2271中的典型突变被认为是获得性克拉霉素抗性的主要机制，没有这种突变的抗性分离株已被广泛报道。在这项研究中,我们使用来自两个亲本菌株的自发突变体分别以erm（41）T28和C28序列为特征，对获得的克拉霉素抗性进行了全面调查。从亲本菌株中选择总共135个抗性突变体。缺乏经典2270/2271突变的78个突变体的测序鉴定了23SrRNA的肽基转移酶中心和发夹环35、49和74中的突变。此外，在临床分离株的1875个基因组中,有57个发现了这些非经典突变.通过定点诱变将13个代表性突变引入细菌基因组，并验证了它们对大环内酯抗性的贡献。将这些突变映射到23SrRNA的三维结构上，揭示了它们在新生肽出口隧道入口处的定位，可能通过破坏大环内酯结合袋而导致抗性。这些非规范23SrRNA突变的鉴定促进了我们对脓肿分枝杆菌中大环内酯抗性的理解，并强调了它们作为检测克拉霉素抗性的潜在标记的重要性。
