Isolation and characterization of phage vB_KpnS_SXK7 against multidrug-resistant Klebsiella pneumoniae and the mechanism of O-antigen-associated phage resistance.

The increasing prevalence of multidrug-resistant (MDR) Klebsiella pneumoniae necessitates alternative therapeutic strategies. Here, we isolated and characterized a lytic phage, vB_KpnS_SXK7, from hospital sewage and investigated the host's resistance mechanism. Morphological analysis identified it as a siphovirus-type phage. It exhibited a 30-min latent period, a burst size of 270 PFU/infected cell, and stability across pH 5-12 and temperatures up to 60 °C. Host range testing against 25 K. pneumoniae strains showed that vB_KpnS_SXK7 lysed 9 strains (36%). Whole-genome sequencing revealed a 49.1-kb circular dsDNA genome lacking tRNA, virulence, or antibiotic-resistance genes, confirming its safety profile. In vitro, vB_KpnS_SXK7 reduced bacterial viability within 2 h. In a Galleria mellonella infection model, a single dose (MOI 100) rescued 80% of larvae from lethal challenge without toxicity. Genomic analysis of resistant mutant SXK7-R11 identified a single-nucleotide deletion in wzm, which encodes an ABC transporter permease essential for O-antigen assembly. This mutation impaired phage adsorption, which was restored by genetic complementation. These findings establish vB_KpnS_SXK7 as a promising preclinical candidate but highlight its vulnerability to resistance mechanisms involving O‑antigen‑associated pathways, supporting the development of phage cocktails targeting multiple receptors against MDR infections.