Prophage and SaPI diversity in local clinical Staphylococcus aureus reveal hidden drivers of virulence.

Prophages are key drivers of bacterial evolution, genomic variability, virulence, and ecological adaptation. Here, we examined the prophage diversity of 109 Staphylococcus aureus clinical isolates from four tertiary care hospitals in Mexico City, integrating comparative analyses with 993 international genomes. Prophages were detected in 97% of the local strains, mirroring the near-ubiquitous presence (99%) observed globally. We identified 216 genomic regions encoding putative prophage functional elements. Using Mitomycin C induction, we recovered 17 temperate phages, 12 of which were functionally active and capable of lysogeny-lysis switching and reinfection. Induced phages lacked virulence or antibiotic resistance genes, in contrast to 55% of the predicted prophages in local S. aureus isolates that harbored known virulence factors. Moreover, 19% of the predicted prophages were phage-inducible chromosomal islands (PICIs) related to SaPI1, SaPI2, and SaPIpt1028. These PICIs encode anti-phage defense systems (63%) and virulence genes (27%), such as tsst-1 and hlb. Our findings reveal a complex prophage and PICI landscape in S. aureus circulating in Mexican hospitals, with implications for phage- host dynamics, horizontal gene transfer, and the evolution of pathogenic potential across geographically diverse populations. ImportanceStaphylococcus aureus is a major hospital pathogen whose evolution is driven by mobile genetic elements including prophages and phage-inducible chromosomal islands (PICIs). Although computational analyses predict prophages in nearly all S. aureus genomes, we demonstrate that only a small fraction is functionally active. Through experimental induction of clinical isolates from Mexican hospitals, we recovered active prophages that surprisingly lacked virulence genes, in contrast to the predicted prophages that frequently harbor virulence factors. Importantly, we discovered that many predicted "prophages" are PICIs carrying anti-phage defenses and toxins. These findings challenge the current prophage prediction methods and reveal the complex prophage-PICI landscape that shapes horizontal gene transfer and pathogenic evolution in clinical S. aureus populations.