Characterisation of four novel bacteriophages targeting carbapenem-resistant Klebsiella pneumoniae and their lytic activity alone and in combination

Klebsiella pneumoniae is a nosocomial pathogen with rising levels of antibiotic resistance, increasing interest in bacteriophage therapy as an adjunct or alternative treatment. This study aimed to isolate and characterise bacteriophages active against multidrug-resistant K. pneumoniae and evaluate their efficacy. Four lytic phages, Kilian, Trimon, Jurek, and Olmo were isolated from water sources using K. pneumoniae ATCC BAA-2146 as host. Genome analysis revealed no known lysogeny, antibiotic resistance, or virulence-associated genes, supporting their suitability for therapeutic use. Electron microscopy classified Kilian, Trimon, and Jurek as siphoviruses, and Olmo as a myovirus. All phages showed rapid adsorption (2.5–5 min), short latent periods (5–15 min), and variable burst sizes (22–474 PFU/cell). The phages exhibited a narrow host range, collectively infecting 13 out of 90 tested K. pneumoniae clinical isolates, including carbapenemase producers and high-risk clones (ST11, ST512, ST147, ST307, ST37). The phages were then formulated into a cocktail and tested in vitro , showing enhanced and sustained bacterial growth suppression compared to single phages, with strain- and multiplicity of infection-dependent effects. Antibiofilm activity was assessed on preformed biofilms grown on porous glass beads. Three hours after treatment, both individual phages and the cocktail reduced viable biofilm-associated cells >4-log10 compared to untreated controls. Finally, high titers of purified phages obtained by cesium chloride gradient ultracentrifugation showed no adverse effects on Galleria mellonella viability, indicating a safe profile. These findings support the potential of phage combinations to target multidrug-resistant and biofilm-associated K. pneumoniae infections.