Background The incidence of carbapenem-resistant and highly virulent Klebsiella pneumoniae infections has been increasing annually, posing significant clinical challenges and high mortality rates. Phage therapy, utilizing highly lytic bacteriophages, holds great promise as a potential therapeutic approach.

Objective To isolate a novel lytic T7 phage specifically targeting carbapenem-resistant and highly virulent K. pneumoniae strains and conduct biological characterization and genomic sequencing analysis of the isolated phage, PCCM_KpP1172, so as to provide applicable phage resources for clinical phage therapy.

Methods K. pneumoniae was isolated from sputum samples of patients with pulmonary infections. Bacterial identification, antibiotic susceptibility testing, whole-genome sequencing, and PCR detection were used to validate the bacterial strain's virulence and antibiotic resistance genes. A lytic bacteriophage was isolated from sewage using bacterial strain as the host and named PCCM_KpP1172. The biological characteristics of the phage were determined, and its genome sequence was analyzed. The therapeutic efficacy of phage PCCM_KpP1172 was assessed using the Galleria mellonella infection model.

Results The genomic analysis of the K. pneumoniae strain revealed the presence of antibiotic resistance genes and virulence genes, including rmpA2, rmpA, iroN, and icu. A novel K. pneumoniae phage, PCCM_KpP1172, was isolated using this bacterial strain as the host. The phage formed clear plaques with a halo on the host lawn, and under transmission electron microscopy, it exhibited typical characteristics of the Siphoviridae family within the Caudovirales order. The one-step growth curve showed a latent period of 15 minutes and an optimal multiplicity of infection (MOI) of 0.0001, indicating a broad lytic range against ST11KL64-type K. pneumoniae. Genome analysis revealed that the phage contained a double-stranded DNA with a total length of 40 222 bp and a G + C content of 53%. It consisted of 49 open reading frames (ORFs) without any genes related to virulence or antibiotic resistance. Based on phylogenetic analysis, the phage was classified as a novel species within the Studiervirinae subfamily of the Podoviridae family. Additionally, phage PCCM_KpP1172 effectively inhibited the growth of carbapenem-resistant Klebsiella pneumoniae in vitro within 3 hours and significantly increased the survival rate of G. mellonella larvae infected with K. pneumoniae (P<0.01).

Conclusion This study has successfully isolated and identified a novel T7 phage, PCCM_KpP1172, specifically targeting carbapenem-resistant and highly virulent K. pneumoniae strains. The phage exhibits high lytic activity and lacked genes associated with antibiotic resistance or virulence. Furthermore, it demonstrates significant therapeutic effects in the G. mellonella infection model. These findings provide valuable evidence for the potential clinical application of phage PCCM_KpP1172 in the treatment of hypervirulent carbapenem-resistant K. pneumoniae infections.