Decoding the genomic repertoire of fosfomycin resistance genes in staphylococci

Fosfomycin is regaining clinical relevance for multidrug-resistant staphylococcal infections, yet the diversity, evolutionary origin and nomenclature of staphylococcal fosfomycin resistance genes remain inconsistently defined. We surveyed 17,024 publicly available genomes representing 14 clinically common Staphylococcus species to curate a catalogue of fosfomycin-modifying fos homologues. By integrating amino-acid identity, phylogenetic topology, genomic context and population-level distribution, we established a reproducible classification framework that resolved species- or lineage-associated intrinsic genes, including fosSA, fosSE, fosSC, fosSCp and fosSL, from acquired determinants, including fosB, fosD, fosY and ten prophage-associated families designated fosSΦA-J. Intrinsic genes showed conserved local contexts, whereas prophage-borne determinants occupied diverse insertion sites, supporting distinct evolutionary trajectories. Functional assays showed variable effects on fosfomycin susceptibility. Deleting fosSA in MRSA reduced the fosfomycin MIC 16-fold, and complementation restored resistance. This study provides a unified nomenclature and evolutionary framework for staphylococcal fos genes and highlights the need to monitor both lineage-associated and mobile fos determinants.