Genomic characterization of lytic bacteriophages A¥L and A¥M infecting ESBL K. pneumoniae and its therapeutic potential on biofilm dispersal and in-vivo bacterial clearance.

BACKGROUND

The emerging carbapenem resistance and extended spectrum β-lactamases (ESBL) producing strains of Klebsiella pneumoniae, are one of the critical pathogens for which novel therapeutic alternatives are required on urgent basis. Biofilm formation further aids in virulence due to impermeable nature. Bacteriophages are bacterial predators which due to their selective, and nontoxic nature can be used as an alternate to counter MDR infections. Hence, the current study was intended to isolate, characterize, and develop phage cocktail as a possible therapy against ESBL K. pneumoniae.

MATERIAL AND METHOD

The two-novel bacteriophage A¥L and A¥M were isolated from environmental samples and characterized for host specificity and physicochemical stability (i.e., temperature and pH). Isolated phages alone or together as cocktail was further evaluated for in vitro biofilm eradication and infection clearance using in vivo murine model. Whole genome sequencing was performed for identification, evolutionary relationship, and bioinformatics analysis.

RESULT

The isolated phage A¥L and A¥M belonged to Myoviridae and Siphoviridae family, respectively and showed good thermal (-20, 37, 45, and 60̊C) and pH (5, 7, 9, and 11) stability. At MOI of 0.001, both phages displayed short eclipse period of 5 and 10 min, respectively. Phages alone or together as cocktail showed 50-70% eradication of 48 h mature biofilm. Majority of the cells within biofilm was found dead as evinced from live dead staining. Atomic force and scanning electron microscopic analysis showed distorted biofilm with ruptured cells. The isolated phage or their cocktail significantly inhibited K. pneumoniae associated mortality in intraperitoneal inoculated mice model CONCLUSION: These findings imply that the phage might be a good option for eliminating K. pneumoniae infections and further studies could help in development of these phage as a bio-control product.