In vitro and in vivo antibacterial efficacy of bacteriophage combined with tigecycline against carbapenem-resistant Klebsiella pneumoniae and characterization of phage resistant mutants

Carbapenem-resistant <i>Klebsiella pneumoniae</i> (CRKP) has emerged as a critical global public health threat, characterized by high infection rates, elevated mortality, and limited therapeutic options. In this study, we isolated and characterized a novel bacteriophage (phage), designated as HZJ31, which exhibited potent lytic activity against CRKP strains. Phylogenetic and genomic analyses revealed that phage HZJ31 belongs to the order <i>Caudovirales</i> and lacks virulence factors, antibiotic resistance genes, and lysogeny-related elements, supporting its suitability for therapeutic applications. Phage HZJ31 exhibits remarkable anti-biofilm activity by preventing biofilm formation and disrupting established biofilms, with bacterial reduction rates exceeding 70% (<i>P</i><0.05). In combination with Tigecycline, it significantly enhanced bactericidal efficacy, delayed the emergence of phage resistant mutants, and improved survival rates in <i>Galleria mellonella</i> larvae infection models. Compared to the bacterial-infected group, which had 80% larval mortality at 96 h, treatment with HZJ31 or TGC alone led to 50% and 60% survival, while their combination improved survival to 70% (<i>P</i> < 0.05). Notably, the phage-resistant mutant, which emerged due to capsule loss, resulted in reduced growth and virulence, while regaining sensitivity to certain antibiotics (such as gentamicin), indicating a fitness cost associated with phage resistance. Collectively, these findings provide valuable insights into phage-antibiotic synergy and underscore the promising clinical potential of phage HZJ31 as a therapeutic agent against CRKP infections.