Nanopore sequencing reveals operon-specific ribosome remodeling accompanying naphthyridone resistance in Staphylococcus aureus

Antimicrobial resistance (AMR) threatens global health; however, the molecular adaptations underlying resistance to emerging antibiotic classes remain poorly defined. Here, we applied long-read DNA and direct RNA nanopore sequencing to deconvolute operon-specific rRNA sequence and modification changes. Together, this platform uncovered a previously unrecognized, operon-specific pathway of resistance not resolvable with traditional sequencing approaches in Staphylococcus aureus to the naphthyridone antibiotic A-692345. Genomic nanopore sequencing identified a single 23S rRNA mutation (T1732C) confined to one of the six rRNA operons (operon 2), which is uniquely associated with nine tRNA genes. Direct RNA nanopore sequencing generated a comprehensive and updated rRNA modification map for S. aureus, revealing extensive remodeling of rRNA modifications in the resistant strain upon exposure to A-692345. Modification patterns for pseudouridine, dihydrouridine, 5-hydroxycytidine, and N4-methyl-2′-O-methylcytidine at functionally relevant positions within the ribosome changed as a function of A-692345 dose. Mapping these epitranscriptomic changes revealed that they were operon-specific. This operon-restricted remodeling likely gives rise to ribosome heterogeneity, with the potential to enable selective translation of stress-response genes that favor resistance. These findings establish nanopore sequencing as a powerful platform for resolving coupled genomic and epitranscriptomic adaptations and providing molecular insight into how bacteria can evolve resistance to antibiotics through operon-specific ribosome remodeling.抗菌药物耐药性（Antimicrobial resistance, AMR）对全球公共健康构成重大威胁，但目前针对新型抗生素类别产生耐药性的分子适应机制仍未得到清晰阐明。本研究采用长读长DNA与直接RNA纳米孔测序（nanopore sequencing）技术，解析各操纵子（operon）特异性的核糖体RNA（ribosomal RNA, rRNA）序列与修饰变化。该技术平台成功发现了一种此前未被报道的操纵子特异性耐药通路——这一通路无法通过传统测序方法在针对萘啶类抗生素A-692345的金黄色葡萄球菌（Staphylococcus aureus）中被解析。基因组纳米孔测序鉴定出一处仅存在于6个rRNA操纵子中的第2号操纵子内的23S rRNA突变（T1732C），该突变仅与9个转运RNA（transfer RNA, tRNA）基因相关联。直接RNA纳米孔测序为金黄色葡萄球菌构建了一套全面且更新的rRNA修饰图谱，揭示了耐药菌株在暴露于A-692345时，其rRNA修饰发生了广泛重塑。假尿苷（pseudouridine）、二氢尿苷（dihydrouridine）、5-羟基胞苷（5-hydroxycytidine）及N4-甲基-2′-O-甲基胞苷（N4-methyl-2′-O-methylcytidine）在核糖体功能相关位点的修饰模式，会随A-692345的给药浓度发生动态变化。对这些表观转录组（epitranscriptomic）变化进行定位分析后发现，其均具有操纵子特异性。这种受操纵子限制的修饰重塑可能会引发核糖体异质性（ribosome heterogeneity），进而有望实现对利于耐药性形成的应激反应基因（stress-response genes）的选择性翻译。本研究结果证实，纳米孔测序是一种极具潜力的研究平台，可用于解析基因组与表观转录组的协同适应机制，并为细菌如何通过操纵子特异性的核糖体重塑进化出抗生素耐药性提供分子层面的认知。