Isolation and characterization of novel bacteriophages with broad-spectrum lytic activity against carbapenem-resistant Klebsiella pneumoniae capsular pathotypes K1, K2, K3, K5, and K20

Abstract

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP), particularly strains associated with virulent capsular pathotypes K1, K2, K3, K5, and K20, represent a major global health burden due to limited therapeutic options and poor clinical outcomes. This study aimed to isolate and characterize lytic bacteriophages targeting clinically relevant CRKP capsular types. METHODS: Bacteriophages were isolated from hospital sewage and purified by successive plaque assays. Biological characterization included adsorption kinetics, one-step growth analysis, and host range determination. TEM was used for morphological classification. Molecular characterization of the phages was achieved using whole-genome sequencing (WGS). RESULTS: Three phages, designated UGKSKpnP1, UGKSKpnP2, and UGKSKpnP3, were isolated. The phages exhibited a high and rapid adsorption rate of ~ 95% within 9–10.5 min, short latent periods (~ 25 min), and large burst sizes exceeding 300 PFU/infected cell. All phages efficiently lysed CRKP capsular types K1, K2, K3, K5, and K20. TEM analysis revealed that the phages exhibited siphovirus-like morphology, characterized by similar capsid–tail architecture with polyhedral heads and long non-contractile tails. Based on the current ICTV taxonomy, they were classified within the class Caudoviricetes and the family Drexlerviridae. WGS showed that UGKSKpnP1, UGKSKpnP2, and UGKSKpnP3 possess nearly identical genome sizes of 51,620 bp, 51,622 bp, and 51,622 bp, respectively, with high intra-group average nucleotide similarity (> 99% ANI) and were devoid of tRNA genes, lysogenic markers, virulence factors, and antibiotic-resistance determinants. Phylogenomic and VIRIDIC analyses placed the phages within the family Drexlerviridae and genus Webervirus, and revealed that the phages represent novel species within the Webervirus genus. CONCLUSION: Therefore, UGKSKpnP1, UGKSKpnP2, and UGKSKpnP3 demonstrated strong lytic activity, favorable replication dynamics, and genomic safety, underscoring their potential as candidates for phage therapy against CRKP.