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 and developed methods to deconvolute operon-specific epitranscriptomic changes. Together, this platform uncovered a previously unrecognized, operon-specific pathway of resistance 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 uniquely contains nine tRNA genes. RNA direct nanopore Sequencing generated a comprehensive and updated rRNA modification map for S. aureus and revealed extensive remodeling of rRNA modifications in the resistant strain. Differentially incorporated modifications included pseudouridine, dihydrouridine, and 5-hydroxycytidine at functionally relevant positions within the ribosome. Upon mapping these epitranscriptomic changes, we noted they were operon specific. This likely gives rise to ribosome heterogeneity with potential for selective translation of stress-response genes that favor resistance. Collectively, these findings establish nanopore Sequencing as a powerful platform for resolving coupled genomic and epitranscriptomic adaptations, providing molecular insight into how bacteria evolve resistance to Antibiotics.