Barriers to Antimicrobial Resistance Gene Exchange in Methicillin Resistant Staphylococcus aureus Cluster into Transfer Islands

Abstract

Horizontal gene transfer (HGT) via generalised transduction is a major driver of antimicrobial resistance (AMR) in Staphylococcus aureus, yet the genetic barriers regulating phage-mediated transfer remain poorly defined. Using a co-culture gene transfer rate assay (COGTRA) that quantifies phage-dependent resistance gene exchange under competitive, antibiotic-free conditions, we screened 1,920 mutants from the Nebraska Transposon Mutant Library for elevated transfer. We identified 32 validated high-transfer mutants that act as HGT barriers, 66% of which clustered within two chromosomal regions that we term S. aureus Transfer Islands (SauTI1 and SauTI2). SauTI loci restricted generalised transduction bidirectionally, and SauTI1 genes were widespread across S. aureus genomes yet showed lineage-specific variation. SauTI1 encodes an SMC2-Cbf1 condensin-nuclease module whose disruption markedly increases generalised transduction and plasmid transfer, resembling Wadjet-like restriction systems. Together, these findings identify clustered defence loci that restrict HGT in S. aureus and identify SauTIs as key regulators of AMR evolution.