Stepwise phage resistance and collateral phage susceptibility in Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a difficult-to-treat pathogen. Phages recovered by a "stepwise" approach may constitute a cocktail able to prolong retardation of bacterial regrowth. We stepwise recovered three lytic phages (namely P04, P40, and P49) from different families against ST11KL64 CRKP and created a cocktail that restrained CRKP growth for 15 h. Phage P04 recognized bacterial capsular polysaccharide (CPS). P04/P40-resistant mutants lost the large fragments containing CPS gene due to homologous recombination between two insertion sequences. Through gene cloning and complementation experiments, CPS and lipopolysaccharide (LPS) quantification, and untargeted lipid metabolism assays, deletion of the ugd/wbgU genes alters lipid A modification and outer membrane environment. This change affects the accessibility of an unidentified membrane protein or lipid A itself, which serves as the receptor for P40. P49 recognized bacterial transmembrane protein involved in vitamin B12 transportation. Synergistic antibacterial activity was observed because the three phages recognize different receptors. Notably, P49 also lysed Salmonella enterica, Escherichia coli, Enterobacter ludwigii, and Kluyvera tianfuensis, suggesting new receptors would be exposed when the synthesis of CPS was inhibited, thereby allowing efficient attack by phages originally targeting other species (the "close-one-door-but-open-another" phenomenon). The conserved structure of BtuB provides a molecular basis for the rare cross-genus activity of P49. Insertion sequences provide a generalized anti-phage defence in encapsulated bacteria. Our findings provide critical insights into the versatile mechanisms underpinning bacteria-phages interactions. Specifically, the collateral susceptibility to phages targeting other bacterial species may provide a novel and highly promising approach for creating clinically viable phage cocktails.