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.