Novel Phage Exhibits Effective <i>In Vitro</i> and <i>In Vivo</i> Activity Against Bovine-Derived Multidrug-Resistant <i>Klebsiella pneumoniae</i>

AIMS: Multidrug-resistant (MDR) Klebsiella pneumoniae (K. pneumoniae) poses a serious threat to cattle health. This study aimed to isolate phages against bovine-origin MDR K. pneumoniae and evaluate their therapeutic potential. METHODS AND RESULTS: We successfully isolated five lytic bacteriophages (phage) (vB_Kpn_Y1-Y5) from hospital sewage samples. They showed favorable temperature and pH stability, efficient lytic cycles, and narrow host ranges. Whole-genome analysis further classified these phages into three categories belonging to the families Siphoviridae and Podoviridae within the class of Caudoviricetes. This genetic diversity provides a foundation for constructing a phage cocktail. A phage cocktail constructed from these phages exhibited synergistic antibacterial effects in vitro, with superior inhibition and duration compared to single phages. In a mouse bacteremia model, the phage cocktail therapy significantly increased the survival rate of mice from 50% to 91.7% (p < 0.05), effectively reduced bacterial loads in multiple organs (p < 0.0001), alleviated tissue inflammatory damage, and showed no obvious toxicity. DISCUSSION: The genetic diversity of the phages underpinned their synergy. The phage cocktail demonstrated better efficacy than monotherapy both in vitro and in vivo.