Isolation and genomic characterization of a lytic bacteriophage vB_KpBD_211 with strong antibiofilm activity against a multidrug-resistant Klebsiella pneumoniae isolate

The rapid emergence of multidrug-resistant (MDR) Klebsiella pneumoniae has become a major global health concern, particularly in resource-limited settings such as Bangladesh. Bacteriophages offer a promising alternative to conventional antibiotics due to their specificity and ability to lyse resistant bacterial strains. A lytic bacteriophage, vB_KpBD_211, was isolated from sewage samples using the double-layer agar method against a multidrug-resistant K. pneumoniae CP4 host strain. The phage was characterized through plaque morphology, one-step growth curve, multiplicity of infection (MOI) optimization, host range determination, and physicochemical stability assays. Biofilm inhibition and disruption assays were performed using a microtiter plate method. Whole-genome sequencing and bioinformatic analyses were conducted to determine genomic features, functional annotation, comparative genomics, phylogeny, pangenome structure, and lifestyle prediction. Structural and functional characterization of the endolysin was performed using InterProScan, DeepTMHMM, and AlphaFold2. The phage formed clear plaques (~ 4 mm) and exhibited strong lytic activity with a short latent period (~ 25 min) and a high burst size (295 PFU/cell). The optimal MOI was 0.1, and the phage demonstrated stability across a broad pH range (4–9) and moderate thermal tolerance. The phage significantly inhibited biofilm formation (up to 86.1%) and moderately disrupted preformed biofilms (58.33%). The genome size was 47,938 bp with 57.05% GC content and lacked tRNA genes. Comparative genomics revealed close similarity (95.8–96.7% identity) to KP_NC20/RCIP-like phages. Pangenome analysis identified 174 orthogroups, including 40 core genes. Phylogenetic and synteny analyses confirmed its placement within a highly conserved lineage. Structural analysis of the endolysin revealed a conserved glycoside hydrolase family 24 domain and a well-folded globular structure. Lifestyle prediction classified the phage as virulent with high confidence. Bacteriophage vB_KpBD_211 is a strictly lytic phage with strong antibacterial and antibiofilm activity against MDR K. pneumoniae . Its genomic features, evolutionary placement, and functional properties highlight its potential as a candidate for phage-based therapeutic applications, particularly in regions with a high burden of antimicrobial resistance. Not applicable.