Spontaneous wbbL and waaG mutations selected by phage vB_EcoP_P61425 confer resistance and attenuate virulence in...

Bacteriophage therapy is a promising strategy against multidrug-resistant (MDR) extraintestinal pathogenic Escherichia coli (ExPEC), yet bacterial resistance evolution remains a major clinical challenge. In this study, we isolated and comprehensively characterized a novel lytic phage, vB_EcoP_P61425, belonging to the genus Suseptimavirus. The phage has a latent period of 15 min and a burst size of approximately 68 PFU/cell. Phage vB_EcoP_P61425 exhibits stability across temperatures of 4 °C to 50 °C and a pH range of 3 to 10, displaying potent lytic activity against common ExPEC serotypes including O25 and O75. Genomic analysis revealed a 77933 bp double-stranded DNA genome with a GC content of 42.60% and 118 predicted coding sequences (CDSs), devoid of known lysogeny-associated, toxin, or antimicrobial resistance genes. In vitro assays confirmed its strong bactericidal efficacy, and it significantly reduced bacterial colonization in a medical catheter model. To elucidate the genetic mechanisms underlying bacterial resistance, spontaneous phage-resistant mutants were isolated and analyzed using whole-genome resequencing, targeted gene deletion, and genetic complementation assays. We demonstrated that mutational inactivation or directed deletion of the lipopolysaccharide (LPS) biosynthesis genes wbbL and waaG significantly impaired phage adsorption, thereby conferring resistance to vB_EcoP_P61425 in EC1649. Crucially, these resistant mutants exhibited phenotypic and virulence-associated trade-offs, manifested by altered cell morphology, markedly impaired adherence to host epithelial cells, and significantly attenuated virulence in a Galleria mellonella larval infection model. Collectively, this study demonstrates that mutations in the LPS biosynthesis genes wbbL and waaG in E. coli EC1649 confer phage resistance while imposing an evolutionary trade-off that attenuates bacterial pathogenicity, providing new insights for optimizing phage therapy regimens against ExPEC.