Deciphering shared receptor usage in genomically unrelated bacteriophages infecting hypervirulent Klebsiella...

Klebsiella pneumoniae is a critical pathogen often associated with multidrug resistance and hypervirulence. We report the isolation and characterization of three distinct lytic bacteriophages-Spear, Loop, and Shorty-from sewage, using a hypervirulent, hypermucoid K. pneumoniae K1 ST23 strain as the host. Despite genomic and structural differences, all three phages exhibited a narrow host range, infecting only the K1 serotype. Transmission electron microscopy and genomic analyses confirmed their lytic lifestyle and classifications: Spear and Loop are siphovirus-like, while Shorty is podovirus-like. A key focus was phage-host interaction and receptor usage. DNA sequence analysis showed no homology between the receptor-binding proteins, yet structural modelling revealed high similarity between Loop and Shorty tail fibers, aligning within a K1-specific lyase domain, suggesting phage genetic mosaicism. All three phages rely on capsular polysaccharide (CPS) for infection. Resistance selection under phage pressure yielded non-mucoid mutants, characteristic of CPS loss. Cross-resistance and adsorption assays confirmed CPS-dependence. Loop and Shorty showed near-complete loss of binding; Spear retained partial binding, suggesting additional receptors. These results highlight that unrelated phages can target the same bacterial structure, CPS. This has important implications for rational phage cocktail design, as CPS mutations may undermine seemingly diverse phage combinations.