Engineered reporter phages for rapid detection ofEscherichia coli, Klebsiellaspp., andEnterococcusspp. in urine

The rapid detection and species-level differentiation of bacterial pathogens facilitates antibiotic stewardship and improves disease management. Here, we develop a rapid bacteriophage-based diagnostic assay to detect the most prevalent pathogens causing urinary tract infections: Escherichia coli, Klebsiella spp., and Enterococcus spp. For each uropathogen, two virulent phages were genetically engineered to express a nanoluciferase reporter gene upon host infection. Using 206 patient urine samples, reporter phage-induced bioluminescence was quantified to identify bacteriuria and the assay was benchmarked against conventional urinalysis. Overall, E. coli, Klebsiella spp., and Enterococcus spp. were each detected with high sensitivity (68%, 78%, 85%), specificity (99%, 99%, 99%), and accuracy (90%, 94%, 96%) at a resolution of ⩾10 3 CFU/ml within 5 h. We further demonstrate how bioluminescence in urine can be used to predict phage antibacterial activity, demonstrating the future potential of reporter phages as companion diagnostics that guide patient-phage matching prior to therapeutic phage application.