Whole-genome sequencing of Staphylococcus aureus isolates after a decade-long decline in MRSA prevalence at a university referral hospital in Guadalajara, Mexico: clonal concentration in MRSA and greater genetic diversity in MSSA

Methicillin-resistant Staphylococcus aureus (MRSA) prevalence has declined in many settings, but genomic features of residual MRSA populations remain incompletely defined. We performed whole-genome sequencing (WGS) of 26 S. aureus clinical isolates (13 MRSA and 13 methicillin-susceptible S. aureus [MSSA]) collected by stratified random sampling during 2024–2025. Analyses included de novo assembly, species confirmation, MLST/ spa /SCC mec typing, resistance and virulence profiling, mobile-element analysis, genotype–phenotype concordance, and recombination-filtered/contextual phylogenomics. A secondary matched case-control analysis explored clinical correlates. MRSA was concentrated in ST5-t895-SCC mec II (CC5, New York/Japan lineage; 9/13, 69.2%), whereas MSSA was diverse (11 sequence types and 9 clonal complexes; Simpson’s 1 − D = 0.974 vs 0.500). SA023 (CC5-MSSA, ST5-t688) nested within the MRSA-CC5 clade and lacked SCC mec , a pattern consistent with putative SCC mec excision. WGS resolved lineage-specific resistance and mobile-element architectures: CC5-MRSA carried SCC mec II with ermA / aadD and quinolone resistance-determining region mutations, while SCC mec IV MRSA belonged to CC8/CC88 and lacked arginine catabolic mobile element/copper and mercury resistance. Panton–Valentine leukocidin genes occurred in six isolates (four community-associated MRSA and two community-associated MSSA). WGS-based resistance prediction showed 91.7% categorical agreement with phenotypes. In the exploratory clinical analysis, prior antibiotic exposure, intensive care unit admission, and healthcare exposure score were associated with MRSA. Contemporary MRSA in this hospital is clonally concentrated, while MSSA is diverse. WGS identifies lineage-specific resistance and mobile element architectures, as well as one observation consistent with SCC mec excision, generating hypotheses for longitudinal surveillance rather than establishing a mechanism for MRSA decline. Clinical associations are secondary and exploratory. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) infections have declined in many hospitals, but cross-sectional clinical data alone cannot explain the genomic structure of the MRSA populations that remain. This study uses whole-genome sequencing to show that contemporary MRSA isolates from a Mexican university referral hospital are concentrated in a CC5 New York/Japan lineage, whereas methicillin-susceptible S. aureus (MSSA) isolates are genetically diverse. The analysis identifies lineage-specific resistance determinants, mobile genetic elements, prophage content, defense systems, and one CC5-MSSA isolate showing a pattern consistent with SCC mec excision. These findings provide a descriptive genomic baseline for longitudinal MRSA surveillance. The clinical risk-factor analysis is included as exploratory context, not as the primary evidence for the genomic conclusions.