{"doi":"10.5281/zenodo.18096987","title":"Nanopore sequencing reveals operon-specific ribosome remodeling accompanying naphthyridone resistance in Staphylococcus aureus","abstract":"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.","journal":"Zenodo (CERN European Organization for Nuclear Research)","year":2025,"id":587715,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9466,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":765972,"name":"Aaron M. Fleming","orcid":"0000-0002-2000-0310","position":0,"is_corresponding":true}],"reference_count":0,"raw_metadata":null,"created_at":"2026-07-19T02:59:39.958043Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}