A phage-based luminescent reporter platform for rapid typing of multiple capsular types of Klebsiella pneumoniae

Klebsiella pneumoniae is an important opportunistic pathogen, and its capsular polysaccharides are key virulence factors. Capsular typing is important for epidemiological surveillance, clinical diagnosis, and therapy development; however, methods for capsule-targeted detection remain limited. Here, we engineered a modular bioluminescent reporter phage platform leveraging bacteriophage receptor-binding proteins for specific K. pneumoniae capsular typing. Initially, the K2-specific bacteriophage ΦRCIP0109 was engineered with the nluc reporter gene to generate ΦRCIP0109:: nluc for K2 detection. Using ΦRCIP0109:: nluc as the chassis, we engineered reporter phages by swapping receptor-binding proteins (RBPs), thereby expanding the detection range to four additional clinically important K types (K64, K47, K1, and K57). Reporter phage specificity and sensitivity were evaluated against clinical isolates and in simulated polymicrobial environments. Directed evolution was employed to introduce RBP mutations for enhanced phage adsorption. These five reporter phages achieved 100% specificity against clinical isolates, detecting concentrations as low as 10 CFU/mL within 3.5–5.5 h and successfully differentiating host strains in polymicrobial synthetic urine. Directed evolution of an RBP yielded a 10- to 100-fold increase in luminescence compared to that of the wild-type phage. Together, these advances establish a scalable platform that can be expanded to additional capsular types and the detection of other pathogens, representing a platform with clear potential for integration into point-of-care diagnostics, guiding targeted antimicrobial therapy and precision phage therapy.