Unraveling Resistance and Thresholds in Phage Therapy Using <i>Staphylococcus aureus</i> In Vitro Models

Abstract Background Phage therapy is a promising approach against multidrug-resistant Staphylococcus aureus , but its clinical development is limited by gaps in pharmacokinetic/pharmacodynamic (PK/PD) understanding, especially concerning phage self-replication and resistance dynamics. Methods We combined in vitro time-kill assays with PK/PD modeling to study three anti-staphylococcal phages (V1SA019, V1SA020, V1SA022) against two S. aureus strains (SH1000 and USA300). A system of differential equations captured the co-dynamics of susceptible, resistant and infected bacteria and free phages. Nonlinear mixed-effect modeling quantified parameter variability. Resistance emergence was monitored through phenotyping and whole-genome sequencing of resistant clones. Results Phage–bacteria interactions followed a predator–prey pattern, with early bacterial growth, rapid phage amplification, and subsequent bacterial collapse. However, resistant subpopulations emerged, regrew over time, in a multiplicity of infection (MOI) dependent way. The model accurately described bacterial and phage dynamics and estimated kinetic parameters including adsorption and burst size. Proliferation and inundation thresholds varied by strain and phage. All resistant clones harbored mutations in genes involved in teichoic acid biosynthesis, with associated growth defects. Simulations demonstrated that only phage doses exceeding both susceptible and resistant bacterial inundation thresholds fully suppressed regrowth. Conclusion This study provides a quantitative framework for understanding phage– S. aureus co-dynamics and resistance emergence. It emphasizes the importance of considering both proliferation and inundation thresholds when designing phage dosing regimens. These findings inform the rational development of phage therapy and support translation toward in vivo and clinical applications.