<b>Characterization of the </b><b>v</b><b>irulence </b><b>s</b><b>haping and </b><b>ad</b><b>aptability in the </b><b>methicillin-resistant </b><b>Staphylococcus aureus </b><b>ST9 </b><b>l</b><b>ineage</b><b>.</b>

Staphylococcus aureus is a major pathogen responsible for hospital- and community-acquired infections, with the accessory gene regulator (agr) system playing a central role in virulence control. The ST9 lineage, commonly associated with livestock, exhibits multidrug resistance but remains understudied in terms of virulence. In this work, we examined nine clinical ST9 MRSA isolates and identified two strains carrying spontaneous frameshift mutations in the agr locus, resulting in the loss of RNAIII and α-toxin expression. These agr-deficient strains exhibited reduced hemolytic activity, enhanced biofilm formation, and attenuated virulence in a Galleria mellonella infection model, underscoring critical roleof agr system in ST9 virulence.Comparative genomic analyses revealed distinct pathogenicity islands in ST9, differing from those in other common clinical lineages. Notably, we identified three novel prophage islands (φST9-A, φST9-B, and φST9-C), with φST9-B carrying the recently characterized tarP virulence gene, suggesting that recombination events within livestock-associated MRSA could contribute to the genetic diversification of ST9. These findings demonstrate the significant virulence potential of the ST9 lineage and the importance of agr-mediated regulation. Moreover, the presence of new prophages highlights the evolutionary adaptability of this lineage. Collectively, our results underscore the clinical threat posed by ST9 MRSA and the necessity for vigilant monitoring to control its spread.