Optimizing phage-antibiotic combinations: impact of administration order against daptomycin non-susceptible (DNS) MRSA clinical isolates.

The rise of bacterial resistance has driven the exploration of novel therapies, such as bacteriophage-antibiotic cocktails (PACs), which have shown in vitro promise against resistant pathogens, including daptomycin non-susceptible-methicillin-resistant Staphylococcus aureus (DNS-MRSA) strains. While daptomycin has been a cornerstone for treating MRSA bacteremia and vancomycin-refractory infective endocarditis, the emergence of DNS-MRSA presents a significant challenge due to high morbidity, mortality, and rapid intrinsic resistance development. Phages, Intesti13 and Sb-1, were selected for their unique host range and activity against sixteen DNS-MRSA strains. Synergy with antibiotics was assessed via growth suppression curves and 24-hour time-kill assays (TKAs) across varying administration sequences and minimum inhibitory concentration (MIC) increments. Selected regimens were further assessed in an ex vivo simulated endocardial vegetation (SEV) model, with pharmacokinetic analyses confirming target antibiotic concentrations. In the ex vivo SEV model, simultaneous PAC administration using daptomycin ± phage showed superior bactericidal activity over sequential treatments in isolate C6 (P < 0.01). Similarly, in the same model, C2 reached detection limits within 48 h and remained suppressed for 120 h (P < 0.0037). Sequential outcomes varied by phage-antibiotic order and antibiotic choice. Simultaneous and phage-first regimens outperformed antibiotic-first, especially in 24 h TKAs, but showed variability at lower MICs and between in vitro and ex vivo settings. This study highlights PAC's potential for DNS-MRSA treatment, emphasizing the importance of administration timing. The observed differences across clinical strains emphasize the need for strain-specific evaluations and a deeper understanding of phage-antibiotic interactions to optimize therapy. Future research must focus on expanding phage diversity, refining protocols, and clinically validating sequential strategies to enhance PAC efficacy.