Abstract

Introduction The global dissemination of multidrug-resistant Klebsiella pneumoniae ( Kpn ) underscores the critical demand for alternative therapeutics such as bacteriophages. This study characterizes a novel bacteriophage, vB_KpnP-6K2 (6K2), isolated against a clinically relevant ST11-KL64 Kpn strain, and evaluates its potential for therapeutic application. Methods Phage 6K2 was morphologically examined by transmission electron microscopy and genomically analyzed via whole-genome sequencing. Its stability across pH and temperature ranges, adsorption kinetics, and burst size were determined in vitro . The inflammatory response to Kpn infection was assessed in HEK293T, A549, Hela, and THP-1 monocytic cells by measuring cytokine and chemokine expression, while cell death was evaluated in A549 lung epithelial cells. The therapeutic efficacy of 6K2 was tested in a lethal murine systemic infection model, where a single intraperitoneal dose was administered one-hour post-bacterial challenge. Survival, bacterial clearance, and phage kinetics in blood were monitored. Results Phage 6K2 exhibits a polyhedral head and short tail, classifying it within the Podoviridae family (Autographiviridae family, Przondovirus genus). Its double-stranded DNA genome comprises 40,147 bp. The phage demonstrated stability across a broad pH (4-12) and temperature (4-50°C) range, rapid adsorption, and a burst size of 13.6 PFU/cell. In vitro , Kpn infection significantly upregulated inflammatory mediators in THP-1 cells and induced death in A549 cells; both responses were potently inhibited by 6K2 treatment. In the murine infection model, a single dose of 6K2 achieved 100% survival, accompanied by rapid clearance of bacteremia and high initial phage titers in the blood. Discussion These findings highlight the promising potential of bacteriophage 6K2 as an effective therapeutic agent against multidrug-resistant Kpn infections. The phage not only suppresses bacterial load but also m