Prophage and metabolic determinants of Staphylococcus aureus survival to vancomycin identified via TraDIS screening

Reduced vancomycin susceptibility phenotypes in Staphylococcus aureus contribute to treatment failure, yet the genetic determinants of survival under inhibitory vancomycin exposure remain incompletely defined. We performed transposon-directed insertion-site sequencing (TraDIS) on a methicillin-resistant S. aureus (MRSA) ST398 mutant library following exposure to vancomycin at its minimum inhibitory concentration, identifying 52 genes whose disruption was associated with loss of population survival at inhibitory drug concentrations. Prophage-associated loci were the largest functional group, spanning predicted structural and regulatory genes, as well as multiple conserved hypothetical proteins. Targeted testing of defined transposon mutants in a USA300 background confirmed that disruption of selected loci impaired growth under vancomycin exposure. Our results highlight the contribution of diverse physiological processes, including metabolism and stress responses, as well as a prominent role for prophage-associated functions, rather than discrete resistance pathways. Together, these findings indicate that vancomycin tolerance is shaped by the general physiological state of the bacterial cell, including metabolic capacity and stress adaptation.IMPORTANCETreatment failure in Staphylococcus aureus infections often occurs in the absence of known antibiotic resistance determinants, suggesting that additional survival mechanisms influence therapeutic outcomes. In this study, we identify genetic determinants required for survival during inhibitory vancomycin exposure, revealing a broad role for metabolic functions, stress adaptation, and prophage-associated loci. The prominence of these diverse processes highlights that survival reflects global physiological adaptation rather than discrete resistance pathways. This insight underscores the need to consider cellular physiology and stress responses when developing strategies to prevent antibiotic tolerance and improve treatment efficacy.