A temperate phage acts as a symbiotic fitness enhancer for UPEC but surrenders to combination therapy through lysogeny suppression

ABSTRACT Temperate phages are increasingly recognized as key drivers of bacterial evolution. Here, we isolated a myoviral prophage, vB_EcoM_P899, from a clinical uropathogenic Escherichia coli (UPEC) isolate and characterized how lysogenization shapes host biology. Lysogenization significantly enhanced biofilm formation, swarming motility, oxidative stress resistance, and adhesion to macrophages (up to 100-fold) without altering antibiotic susceptibility or overall 72-h lethality in the Galleria mellonella larval infection model. Mechanistically, this is associated with upregulation of the fimbrial gene FimA and enhanced inducibility of the oxidative stress response genes KatE and OxyR under oxidative stress. Crucially, combining P899 with ciprofloxacin or kanamycin produced synergistic killing and suppressed new lysogen formation in an antibiotic-class-dependent manner. In contrast, ceftazidime, while synergistic, failed to prevent lysogeny. Our findings demonstrate that while prophage vB_EcoM_P899 promotes bacterial niche adaptation, its lysogenization frequency is significantly reduced under specific antibiotic pressure, facilitating clearance of the challenged UPEC population. This work provides a strong rationale for optimizing phage–antibiotic combination therapies to treat drug-resistant infections by simultaneously enhancing bacterial killing and limiting lysogenic survivors. IMPORTANCE This study reveals that lysogenization by a temperate phage can enhance the niche adaptation of uropathogenic Escherichia coli without increasing its virulence in the Galleria mellonella larval model or altering antibiotic resistance. Crucially, we demonstrate that combining this phage with select antibiotics (e.g., kanamycin and ciprofloxacin) not only kills the bacteria effectively but also actively suppresses the formation of new lysogens in an antibiotic-class-dependent manner. These findings uncover a novel role for temperate phages as symbiotic regulators within the host and provide direct experimental evidence for a phage–antibiotic combination strategy. This approach holds promise for combating chronic, recurrent urinary tract infections and offers a clinically viable pathway to address antibiotic tolerance in chronic, recurrent urinary tract infections.