Evaluation of a phage-inclusive multimodal strategy for recurrent urinary tract infections.

Urinary tract infections (UTIs) are among the most common bacterial infections worldwide and are associated with high recurrence rates and substantial morbidity. New therapeutic approaches are urgently needed, especially for recurrent UTIs (rUTIs), among which phage therapy represents a promising option. Here, we systematically evaluated the performance of six E. coli phages under urinary tract-relevant conditions and established a quantitative area-under-the-curve (AUC)-based framework for the rational selection of therapeutic phages. Phage lysis behavior was tested under oxic and anoxic conditions, revealing modulation by oxygen availability. Phages were also screened for biofilm-degrading activity, and in silico depolymerase predictions were compared with in vitro results, showing strong correlation. Phage-antibiotic interactions and refined treatment strategies were further investigated. Phages G9062, G10400, and MM02 emerged as the most promising candidates, remaining stable and active in urine under physiological pH fluctuations and exhibiting synergistic effects with gentamicin and fosfomycin, but not with the bacteriostatic nitrofurantoin. Importantly, we demonstrate that treatment sequence is a critical determinant of efficacy, with phage administration prior to antibiotic exposure consistently producing the strongest synergistic effects. These findings highlight the potential of phages as an integral component of a holistic therapeutic strategy for rUTIs.Importance Recurrent urinary tract infections (rUTIs) are a major clinical challenge characterized by repeated episodes, increasing antimicrobial resistance, and a substantial socioeconomic burden. Although bacteriophages represent a promising alternative or adjunct to antibiotics, their clinical translation has been hindered by the lack of standardized criteria for phage selection, dosing, and combination with antibiotics. This study addresses these gaps by defining key determinants of phage efficacy under conditions relevant to the urinary tract. Our findings highlight that phage performance is strongly shaped by the treatment environment and by interactions with antibiotics, and that inappropriate combinations or treatment sequences may compromise efficacy. By providing quantitative and transferable frameworks for evaluating phage effectivity, this work advances the rational development of phage-based therapies and helps bridge the gap between experimental phage research and clinically actionable treatment strategies for rUTI.