Unraveling Resistance and Thresholds in Phage Therapy Using In Vitro Staphylococcus aureus experiments and Pharmacokinetic/Pharmacodynamic Models.

Abstract

Phage therapy is a promising approach for infections caused by multidrug-resistant Staphylococcus aureus, but its clinical development is limited by knowledge gaps in pharmacokinetic/pharmacodynamic (PK/PD) and potential emergence of resistance. We performed in vitro time-kill assays to study the effect of three anti-staphylococcal phages (V1SA019, V1SA020, V1SA022) against two S. aureus strains (SH1000 and USA300). Resistance emergence was monitored through phenotyping and whole-genome sequencing of resistant clones.Bacteria and phage counts were analyzed by pharmacokinetic/pharmacodynamic (PK/PD) modeling with a non-linear mixed effects approach to quantify parameter variability. Phage-bacteria interactions followed a predator-prey pattern resulting in fast bacterial collapse. However, bacterial regrowth attributed to resistance was observed in some experiments and was associated with initial multiplicity of infection (MOI). All resistant clones harbored mutations in genes involved in teichoic acid biosynthesis, with associated growth defects. The PK/PD models adequately described bacterial and phage dynamics. Phage binding rate of resistant bacteria was 35 to 250-fold lower than that for susceptible bacteria, resulting in higher proliferation (45 to 310-fold) and higher inundation thresholds (34 to 190-fold). Parameters also varied between the two bacteria strains. Model-based simulations suggested that suppression of resistance require an initial phage dose exceeding the inundation threshold of both susceptible and resistant bacteria subpopulations. This study provides a quantitative in vitro framework for understanding the S. aureus and phage co-dynamics and resistance emergence. The identified threshold phenomena offer insights into the experimental dose-response relationship and may guide future investigations.