Genomic and functional characterization of a lytic Klebsiella phage UHKP with antibiofilm activity

<i>Klebsiella pneumoniae</i> is an opportunistic pathogen causing severe hospital-acquired infections, and it rapidly acquires multidrug resistance. Its robust biofilm formation further complicates treatment and drives interest in phage therapy. A phage UHKP was isolated from hospital sewage using an MDR <i>K. pneumoniae</i> strain (<i>KP-</i>03). UHKP formed clear plaques and having phage titer 2.3 × 10⁹ PFU/mL. Host-range testing on 19 clinical isolates showed a narrow spectrum. Only four MDR strains (<i>KP-</i>03, <i>KP-</i>05, <i>KP-</i>08, <i>KP-</i>11) and one K-17 serotype were lysed, with no activity on other strains or species. One-step growth analysis yielded a 30 min latent period and 85 PFU burst size. In planktonic culture, UHKP at MOI 1 stopped bacterial growth by 4 h and cleared cultures by 8 h, whereas at MOI 0.1 killing was delayed and incomplete. In static biofilm assays, UHKP eradicated 98% of 24-h and 96% of 48-h biofilm biomass by 24 h (MOI 1). Clearance of 72 - 96 h biofilms was limited (≤ 87% by 24 h). UHKP possesses an icosahedral head of 56 ± 3 nm and a short, non-contractile tail measuring around 15 ± 2 nm. Genome sequencing revealed a 62,542 bp dsDNA genome (56.6% GC) encoding 77 ORFs, and phylogenetic analysis placed UHKP in the genus <i>Lastavirus</i>. UHKP carries no lysogeny, toxin or antibiotic-resistance genes.