Klebsiella pneumoniae is an important zoonotic opportunistic pathogen. Of particular concern is the emergence of multidrug-resistant hypervirulent K. pneumoniae (MDR-hvKP), which represents a serious public health threat. Bacteriophage (phage) therapy has emerged as a promising solution to combat antibiotic-resistant K. pneumoniae infections. Here, we characterize a novel lytic phage, vB_Kp_H122, that specifically targets K57 capsular-type MDR-hvKP. vB_Kp_H122 exhibited the characteristic morphology of a siphovirus and demonstrated efficient infection kinetics, with an optimal multiplicity of infection (MOI) of 0.001 and a latent period of ~5 min. It also demonstrated considerable stability across a pH range of 4-11 and at temperatures from 4°C to 50°C, as well as potent activity against K. pneumoniae biofilms. The phage has a linear double-stranded DNA genome of 46,077 bp with a G + C content of 47.61% and belongs to a novel species within the genus Roufvirus. Its genome contained no identifiable genes associated with lysogeny, virulence, or antibiotic resistance, supporting its therapeutic safety. In a mouse infection model, a single dose of vB_Kp_H122 at 2 × 106 PFU significantly reduced bacterial loads in organs, alleviated pathological damage, and provided complete protection against lethal challenge with K57 MDR-hvKP. These findings suggest that vB_Kp_H122 may have potential as an antibacterial candidate against K57 MDR-hvKP isolates.

Keywords: Klebsiella pneumoniae; antibiofilm activity; bacteriophage; biological characteristics; genome analysis; phage therapy.