A novel phage preparation capable of simultaneously inhibiting both K1 and K2 serotypes of <i>Klebsiella pneumoniae</i>

Given the high pathogenicity and escalating antimicrobial resistance of <i>Klebsiella pneumoniae</i>, the efficacy of conventional antibiotics is waning, creating an urgent need for alternative therapeutic strategies. Phage therapy has emerged as a particularly promising approach for combating highly virulent and multidrug-resistant <i>K. pneumoniae</i> due to its target specificity and potent bactericidal activity. This study isolated a lytic <i>K. pneumoniae</i> phage vB_kpnP_ZB27 (ZB27) from domestic sewage in Hefei, China. This phage demonstrated specificity against clinically relevant serotypes K1 and K2. It exhibited remarkable stability across a wide range of temperatures (20°C-50°C) and pH values (4-11). Crucially, genomic analysis confirmed the absence of antibiotic resistance or virulence genes. These findings collectively indicated that phage ZB27 is a promising and safe candidate for phage therapy. The core objective of this study was to optimize the phage fermentation process. To enhance phage yield, we optimized inoculum concentration and medium composition. Laboratory-scale fermentation determined the optimal multiplicity of infection to be 0.0001. Furthermore, response surface methodology optimized key additive concentrations: sorbitol to 46.8 mg/mL, soybean meal to 59.9 mg/mL, and calcium chloride to 1 mM. This optimized protocol increased phage yield by 2.96-fold during scale-up, establishing an effective method for industrial production of therapeutic phages.IMPORTANCETraditional phage preparations typically consist of a single phage with a narrow host range, often requiring the use of complex phage cocktails to cover target strains, which substantially increases production costs. In this study, we successfully isolated broad-spectrum phages capable of lysing both K1 and K2 serotypes, thereby significantly expanding the bactericidal spectrum of the phage preparation. Furthermore, through industrial process optimization, we achieved dual benefits: reducing host bacterial consumption while increasing phage yield. These findings demonstrate that artificially optimized production processes can improve both the economic feasibility and safety of phage biomanufacturing, thus opening new pathways for the industrialization of phage therapy.