Two novel lytic bacteriophages with antibiofilm activity against carbapenem-resistant Klebsiella pneumoniae infections.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a major challenge in clinical settings due to high morbidity, mortality, and limited treatment options. In response, phage therapy has reemerged as a promising alternative to conventional antibiotics. In this study, two lytic bacteriophages, Kpn_PImp2 and Kpn_PImp3, were isolated from urban sewage, a rich source of phages owing to its diverse microbial community. These phages demonstrated remarkable pH stability and thermostability, ensuring their activity under a variety of environmental and physiological conditions. Genomic analysis suggests that both phages likely belong to the Webervirus genus within the Caudoviricetes class, characterized by tailed phages with double-stranded DNA. Importantly, neither phage harbors lysogenic, toxin, nor antimicrobial resistance genes, affirming their safety for therapeutic applications. Comparative studies of tail fiber proteins, which play a crucial role in host specificity, indicate that structural variations may account for the distinct host ranges of Kpn_PImp2 and Kpn_PImp3. Moreover, both phages exhibited the ability to inhibit and disrupt biofilm formation, a key factor in CRKP persistence and resistance. Their biofilm-disrupting properties could potentially enhance the penetration and efficacy of antibiotics in combination therapies. The in vivo efficacy of these phages was further validated using the Galleria mellonella infection model, where treatment led to a significant reduction in larval mortality. However, a cocktail combining both phages did not show synergistic benefits over monophage therapy, likely due to shared host-cell receptors. These findings highlight Kpn_PImp2 and Kpn_PImp3 as promising candidates for phage therapy against CRKP, warranting further research into resistance mechanisms, delivery methods, and combination therapies to fully realize their therapeutic potential. This study also expands the bacteriophage resources against K. pneumoniae and provides valuable insights for phage-based treatments.