Evolution under vancomycin selection drives divergent collateral sensitivity patterns in <i>Staphylococcus aureus</i>

<i>Staphylococcus aureus</i> bacteremia is typically treated empirically with vancomycin, with therapy later tailored based on susceptibility results. However, these tests occur before vancomycin exposure and do not account for adaptation during empiric treatment that can alter <i>S. aureus</i>' susceptibility to first-line drugs. To investigate these collateral drug responses, we experimentally evolved 18 methicillin-susceptible <i>S. aureus</i> (MSSA) populations under increasing vancomycin concentrations until they achieved intermediate resistance. Genomic sequencing revealed two distinct adaptive pathways characterized by mutations in the WalKR regulon, affecting cell wall metabolism, or <i>rpsU</i>, impacting translational stress responses. These pathways correlated with divergent collateral sensitivity profiles to first-line antibiotics. By developing a Collateral Response Score (CRS), we quantified the probability and magnitude of these responses, demonstrating that evolutionary dynamics critically influence resistance outcomes. Our findings suggest a probabilistic approach to antimicrobial therapy, advocating for rapid genomic diagnostics alongside susceptibility testing to better anticipate and respond to evolutionary changes.