Genomic Characterization and Therapeutic Potential of the Lytic Bacteriophage Curly against Klebsiella pneumoniae in Human Innate Immune Cells and a Murine Pneumonia Model

Abstract

Klebsiella pneumoniae is an important cause of severe respiratory and systemic infections, and the increasing prevalence of multidrug-resistant strains has created an urgent need for alternative antibacterial strategies. In this study, nine K. pneumoniae-infecting bacteriophages isolated from diverse environmental sources were characterized genomically and functionally. Genome analyses revealed substantial genomic and proteomic diversity among the isolates. Functional screening against the clinical K. pneumoniae isolate JJD85 identified Curly as the most active phage, producing the highest plaque-forming titer and rapid suppression of bacterial growth in liquid culture. Curly was predicted to have a virulent lifestyle and encoded structural, genome-packaging, and DNA replication-associated proteins. In primary human monocyte-derived macrophage cultures, Curly markedly reduced bacterial burden in both cell-associated and cell-free fractions, while treatment of primary human neutrophil cultures produced an approximately 10^6-fold reduction in total recoverable bacterial burden. Transmission electron microscopy demonstrated phage-like particles within bacterial profiles located in both extracellular and macrophage-associated intracellular compartments. In a C57BL/6J murine pneumonia model, intranasal Curly treatment reduced pulmonary bacterial burden in a dose-associated manner, with approximately 10-fold and 100-fold reductions at the low and high doses, respectively. Curly treatment also attenuated infection-associated lung inflammation and preserved pulmonary architecture. These findings identify Curly as a promising bacteriophage candidate against K. pneumoniae and support further evaluation of its host range, resistance profile, and therapeutic potential.