Population-scale analysis of prophages and defense systems reveals distinct evolutionary routes for virulence and antimicrobial resistance in Staphylococcus aureus

Staphylococcus aureus possesses a highly dynamic genome shaped by both mobile genetic elements and bacterial defense systems. However, the population-scale relationships among prophages, defense systems, and functional genes remain poorly understood. We analyzed 1678 high-quality complete S. aureus genomes to identify prophages, defense systems, anti-defense systems, virulence factor (VF) genes, and antimicrobial resistance (AMR) genes. Their distribution, co-occurrence patterns, and associations were systematically evaluated using comparative genomic and statistical approaches. A total of 5249 prophages were predicted, including 3439 classified as intact, and 95.2% (1598/1678) of strains carried at least one predicted intact prophage. These intact prophages comprised 76 distinct types and exhibited marked sequence type (ST)-associated distributions. In parallel, 45 defense systems comprising 14,137 loci were identified, with restriction–modification systems being the most abundant. Multiple defense systems frequently co-occurred within individual genomes, and anti-defense systems, predominantly associated with anti-CRISPR, were also detected. Intact-prophage abundance showed a weak but statistically significant positive correlation with the number of defense systems (Spearman’s ρ = 0.25), which persisted after excluding prophage-encoded defense systems. Functional annotation revealed that 56.3% of intact prophages carried VF genes, whereas only 4.3% harbored AMR genes. VF genes showed frequent associations with specific prophage types, whereas AMR genes were less frequently detected in prophages and exhibited more restricted association patterns. Network analysis further revealed that VF genes were frequently linked to prophage nodes, whereas most AMR genes were associated with defense-system nodes without an intervening prophage node. However, some observed associations may be influenced by bacterial lineage structure. Intact prophages were substantially more frequently associated with VF gene carriage than with AMR gene carriage, suggesting distinct patterns of prophage involvement in the genomic distribution of these functional genes. Defense systems exhibited heterogeneous, system-specific associations with prophages and functional genes. Together, these findings provide population-scale insights into the relationships among prophages, defense systems, virulence, and antimicrobial resistance in S. aureus , while highlighting the need for further experimental validation of their potential functional and evolutionary implications.