Strong mucosal adhesion enhances the anti-infective effectiveness of T4-like phages.

Bacteriophage adhesion to intestinal mucus has been proposed as an important mechanism for preventing bacterial infection. However, whether differences in adhesion capacity among closely related phages translate into distinct protective outcomes remains unclear. In this study, we compared two T4-like bacteriophages, S143_2 and W143, to investigate how variation in mucosal adhesion influences antibacterial efficacy and host responses. Both phages exhibited comparable lytic activity against enteropathogenic Escherichia coli (EPEC143) but differed in adhesion capacity to intestinal epithelium. In vitro assays demonstrated that phages adhered to mucus-secreting IPEC-1 cells via a reversible mucus association, with S143_2 displaying significantly stronger adhesion than W143. This difference was further confirmed in vivo, where S143_2 showed higher mucosal retention and a significantly greater mucosa-to-digesta ratio of phage titers in the small intestine. Functionally, enhanced adhesion was associated with improved protective efficacy. In a prophylactic mouse model, pretreatment with S143_2 resulted in reduced body weight loss and lower intestinal pathogen load compared to W143. Furthermore, S143_2 induced a more robust systemic immune response evidenced by elevated serum cytokines and the enrichment of immune-related signaling pathways in the intestinal transcriptome. Together, these findings demonstrate that mucosal adhesion capacity is a critical determinant of phage performance in vivo. Our results highlight that, beyond host range and bactericidal capacity, adhesion properties should be considered a key criterion in the rational selection of bacteriophages for preventing and treating mucosa-associated infections.IMPORTANCEPhages are among the most promising antibiotic alternatives, yet their evaluation has largely focused on bacterial host range and lytic activity. Our findings highlight mucosal adhesion as a previously overlooked but decisive factor in phage efficacy when targeting mucosa-associated infections. Phages with stronger epithelial adhesion exhibit superior protection and immune modulation in the gut, underscoring that adhesion capacity should be integrated as a key criterion in the rational selection and engineering of therapeutic phages.