Impact on Daptomycin-Resistance Using Combination of Daptomycin Plus Ceftaroline in Daptomycin-Susceptible and -Resistant <i>Staphylococcus aureus</i>

Abstract Persistent Staphylococcus aureus infections treated with prolonged daptomycin (DAP) can select for DAP resistance (DAP-R), reinforcing the need for combination strategies that both improve killing and constrain resistance evolution. Prior work suggests the DAP+ceftaroline (CPT) combination can deliver synergistic killing, prevent emergence of DAP-R, and resensitize DAP-R subpopulations toward a DAP-susceptible (DAP-S) phenotype. Here, using a clinically derived, MSSA isogenic DAP-S (616)/DAP-R (703) strain pair, we evaluated DAP+CPT activity across in vitro assays, ex vivo models, and an in vivo experimental infective endocarditis (IE) model, and integrated ultra-deep targeted sequencing to link phenotypic responses to genomic adaptation. DAP+CPT produced enhanced killing of both strains in vitro and ex vivo, improved target-tissue clearance of the DAP-R strain in vivo, prevented emergence of DAP-R in the DAP-S parental strain in vitro and ex vivo, and resensitized the DAP-R strain toward a DAP-S phenotype ex vivo. Genomically, ultra-deep sequencing of resistance loci (thousands-fold coverage) identified fixed background divergence versus the N315 reference and revealed regimen-dependent selection in membrane-stress pathways, including a high-frequency mixed mprF subpopulation consistent with DAP-driven heterogeneity under monotherapy, contrasted by distinct locus-level changes under combination exposure. In addition, coverage profiling detected a large, combination-associated mobile-element/prophage gene-content event in the 703 background affecting an immune-evasion/β-hemolysin–converting region, highlighting that antibiotic pressure can couple resistance dynamics with pathogenesis-relevant genome remodeling. Together, these data show that DAP+CPT provides potent activity beyond synergistic killing—improving clearance while constraining or reshaping resistance evolution—and they define genomic signatures that help explain divergent evolutionary trajectories under DAP alone versus DAP+CPT.