Endolysin potentiates depolymerase efficacy against Klebsiella pneumoniae under low-serum conditions

Klebsiella pneumoniae is a Gram-negative opportunistic pathogen responsible for severe healthcare-associated infections. The increasing prevalence of multidrug-resistant (MDR) strains, particularly those resistant to carbapenems, poses a significant clinical challenge. In this study, we isolated Jasmi2, a lytic bacteriophage targeting K2-type K. pneumoniae strains. Jasmi2 was classified within the genus Webervirus and contained a linear double-stranded DNA genome of 45,811 bp with 67 predicted open reading frames. ORF67 was identified as a K2-targeting tail depolymerase and designated DpoJ2. Recombinant DpoJ2 efficiently degraded the surface polysaccharide of K2-type K. pneumoniae and reduced biofilm biomass without significantly affecting viable cell counts. While DpoJ2 sensitized K. pneumoniae to serum-mediated killing, achieving an approximately 3.7-log reduction in bacterial counts in 25% human serum, its bactericidal activity diminished at lower serum concentrations. We successfully overcame this threshold by developing a novel combinatorial approach with the endolysin LysTS6. The DpoJ2-LysTS6 combination demonstrated superior efficacy compared to individual treatment, significantly enhancing bacterial eradication under low-serum conditions. DpoJ2 and LysTS6 exhibited no detectable cytotoxicity or hemolytic activity at the tested concentrations. The enhanced efficacy of the combination was further validated in vivo Galleria mellonella infection models, where the combination of DpoJ2 and LysTS6 increased larval survival to 20% at 72 h, whereas no larvae survived following either individual treatment. These findings establish a novel antibiotic-independent strategy combining a phage-derived depolymerase with an endolysin to enhance antibacterial activity against MDR K. pneumoniae, particularly under conditions where serum-mediated killing is limited.