Bleick, C.; Van Helden, S. R.; Berti, A. D.; Richa, R.; Lehman, S. M.; Bayer, A.; Rybak, M. J.

Background: 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 (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. Methods: Phages Intesti13 and Sb-1, were selected for their unique host ranges and activity against sixteen DNS-MRSA strains. Synergy with antibiotics was assessed via growth suppression curves and 24-hour time-kill assays (TKAs) across administration sequences and MIC increments. Selected regimens were further assessed in an ex-vivo simulated endocardial vegetation (SEV) models, with pharmacokinetic analyses confirming target antibiotic concentrations. Results: 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 48h and remained suppressed for 120h (p<0.0037). Sequential outcomes varied by phage-antibiotic order and antibiotic choice. Simultaneous and phage-first regimens outperformed antibiotic-first, especially in 24h TKAs, but showed variability at lower MICs and between in-vitro and ex-vivo settings. Conclusion: 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.