Characterization of two novel lytic bacteriophages for Klebsiella pneumoniae: molecular studies

Introduction: Multidrug-resistant Klebsiella pneumoniae (Kpn) poses a growing global health threat due to its role in severe hospital-acquired infections and increasing resistance to antibiotics. The objective of this study was to isolate, characterize, and evaluate the therapeutic potential of two novel lytic bacteriophages targeting multidrug-resistant K. pneumoniae, aiming for the development of alternatives to conventional antibiotics. Materials and methods: Phages KpnS01BRG and KpnS02SCE were analyzed using transmission electron microscopy (TEM) and whole-genome sequencing (WGS). Replication kinetics were determined through single-step growth curves (latency period and burst size). Antibacterial efficacy was tested in vitro using a phage cocktail at a multiplicity of infection (MOI) of 10,000 over 12 h. Additionally, a genome mechanics analysis was conducted to evaluate viral DNA cyclizability and flexibility. Results: TEM revealed that both phages belong to the class Caudoviricetes with a siphovirus-like morphology, and WGS classified them within the genus Webervirus (family Drexlerviridae). Phage KpnS01BRG exhibited a latency period of 60 min and a burst size of 17.7 virions/cell, while KpnS02SCE showed a latency of 5.8 min and a burst size of 18.6 virions/cell. The phage cocktail reduced the bacterial load by approximately 97.2% after 12 h, relative to the untreated control. Genomic analysis indicated that the higher DNA flexibility in KpnS02SCE correlated with a slightly higher virion production, corroborating previous findings on genome mechanics. Conclusions: The novel phages demonstrated potent antibacterial activity (i.e., strongly reduced bacterial counts after 6 h) and favorable genomic characteristics, establishing themselves as promising candidates for phage-based therapeutic strategies against multidrug-resistant K. pneumoniae infections.