A novel bacteriophage capable of efficiently lysing multi-drug-resistant Klebsiella pneumoniae

Abstract

Introduction The increasing prevalence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has posed a major challenge to clinical infection management. Phage therapy represents a promising alternative against multidrug-resistant bacterial infections; however, its application is limited by the scarcity of effective therapeutic phages. Therefore, the identification and characterization of novel phages are urgently needed. Methods A novel Klebsiella pneumoniae phage was isolated and purified from hospital wastewater using the double-layer agar plate method with K. pneumoniae ATCC 700603 as the host strain. A total of 95 bacterial strains were used to evaluate the host range and lytic activity of the phage. The biological characteristics of the phage, including optimal multiplicity of infection (MOI), one-step growth curve, and stability under different environmental conditions (temperature, pH, ethanol, and ultraviolet exposure), were systematically investigated. Morphological characterization was performed by transmission electron microscopy. Whole-genome sequencing and bioinformatics analyses were conducted to determine genomic characteristics, taxonomic classification, and the presence of antibiotic resistance genes, virulence factors, or lysogeny-related genes to assess the therapeutic potential and biosafety of the phage. Results The novel phage vB_KpnD_A2 exhibited potent lytic activity against multidrug-resistant Klebsiella pneumoniae strains. Host range analysis showed that vB_KpnD_A2 lysed 57.1% of carbapenem-resistant K. pneumoniae isolates and 63.6% of extended-spectrum β-lactamase-producing strains, while displaying specificity toward K. pneumoniae. The phage showed excellent biological properties, with an optimal MOI of 10-⁶, a latent period of 40 min, and a burst size of 8.6×10⁴PFU/cell. In addition, vB_KpnD_A2 maintained infectivity over a broad temperature range (4-70°C) and pH range (pH 2-13). Whole-genome analysis identified vB_KpnD_A2 as a strictly lytic Webervirus phage within the family Drexlerviridae, without detectable antibiotic resistance genes, virulence factors, or lysogeny-associated genes, supporting its therapeutic potential and biosafety. Discussion The newly characterized phage vB_KpnD_A2 exhibited potent lytic activity against selected multidrug-resistant Klebsiella pneumoniae isolates, favorable genomic safety, and good environmental stability. These findings support its potential as a therapeutic phage candidate for CRKP control and expand anti-Klebsiella phage resources for future phage-based interventions against antimicrobial-resistant bacterial infections.