Development of a lytic bacteriophage BPK01 impregnated biopolymer (chitosan) hydrogel for combating high-risk strains of carbapenem resistant Klebsiella pneumoniae (CRKP) pathogens- in vitro and in vivo evaluation.

Alternative strategies are urgently required to combat the rise of high-risk carbapenem-resistant Klebsiella pneumoniae (CRKP), including blaNDM-positive strains that produce carbapenemase enzymes, which deactivate beta-lactam antibiotics and result in poor treatment outcomes. In this study, we isolated a bacteriophage BPK01, targeting a high-risk strain of Klebsiella pneumoniae (carbapenem-resistant, blaNDM-positive, ST147, capsular type K64, biofilm former). BPK01 demonstrated strong lytic activity (84%) against a panel of genetically characterized CRKP strains (n = 59) from clinical specimens, including pus, urine, sputum, blood, and tracheal aspirates. BPK01 was classified as a Caudoviricetes phage, exhibiting a burst size of 220 virions and a short latent period of 10 min. It demonstrated stability across a range of conditions (temperature, pH, and organic solvents) and effectively disrupted biofilms on silicone catheters. In vivo, BPK01 improved survival rates in the Galleria mellonella infection model and reduced bacterial burden in a murine bacteremia model, underscoring its therapeutic potential. Subsequently, we developed a hydrogel by incorporating BPK01 into a chitosan biopolymer, which demonstrated efficient lytic activity (spot assay, scanning electron microscopy, time kill assay) against CRKP pathogens, stability of biological activity for 6 months of storage, and controlled release kinetics, with the mathematical model Korsmeyer - Peppas being the best fit (R2 = 0.9962). The hydrogel expedited the healing of CRKP-infected lesions in a murine model, suggesting its potential as an effective topical treatment. This study highlights BPK01 as a promising biotherapeutic candidate for treating CRKP infection, with the phage hydrogel offering an ecofriendly and sustainable solution for treating infected lesions. Further research could expand its use in phage cocktails and other formulations for broader CRKP infection management.