Engineered reporter phages for detection of Escherichia coli, Enterococcus, and Klebsiella in urine

Abstract

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, Enterococcus spp., and Klebsiella 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, Enterococcus spp., and Klebsiella spp. were each detected with high sensitivity (68%, 78%, 87%), specificity (99%, 99%, 99%), and accuracy (90%, 94%, 98%) at a resolution of ≥10³ 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.

Fig. 1. Isolation and characterization of strictly lytic E. coli, Enterococcus spp. and Klebsiella spp. phage scaffolds.

a Workflow depicts the sequential multiple host method for the isolation of phages with broad host-range. Wastewater samples from different wastewater treatment plants across Switzerland were mixed with the first clinical isolate (Host A) of the respective target species selected from the Zurich Uropathogen Collection and briefly enriched in synthetic human urine medium (SHU) before performing plaque assays. Emerging plaques were pooled, extracted, and propagated on 4 subsequent hosts (B, P, C, D) before individual phages were purified on a propagation Host P. b Summary of the genomic and morphological characterization of six selected phage scaffolds infecting E. coli (phages E2 and E4), Enterococcus spp. (phages EfS3 and EfS7) and Klebsiella spp. (phages K1 and K4), respectively. Some elements of the figure were created using biorender.com. Source data are provided as a Source Data file.

Fig. 2. Engineering of virulent E. coli, Enterococcus and Klebsiella reporter phages.

a Schematic representation of the nanoluciferase gene (nluc) insertion sites in the genomes of all reporter phages. For E. coli and Enterococcus phages, codon optimized nluc (516 bp + RBS) was incorporated immediately downstream of the major capsid gene (cps), whereas for Klebsiella phages, nluc was inserted downstream of the prohead assembly protein (gp167 for K1 and gp168 for K4). b–d Engineering method for virulent Enterococcus phages combining homologous recombination with CRISPR-Cas9-assisted counterselection. b Left: Schematic representation of the pSelect CRISPR locus used to restrict wildtype phage genomes by targeting two sequences flanking the nluc integration site. Right: Spot-on-the-lawn assays of 10-fold phage dilutions show plaquing efficiency on hosts with (+) or without (scr, -) CRISPR targeting. scr = non-targeting crRNA; - = no pSelect. c Schematic representation of the editing plasmids (pEdit_EfS3, pEdit_EfS7) used to incorporate mutated protospacer-adjacent motifs (PAMs) and nluc gene sequences downstream of the major capsid protein (cps) regions of EfS3 and EfS7 by homologous recombination and subsequent CRISPR escape of EfSs::nluc and EfS7::nluc candidates (counterselection) shown by spot-on-the-lawn assays of serial dilutions of phages after recombination. d 10 individual plaques were isolated from one replicate of data shown in Fig. S2b, c. Plaques were validated genetically and functionally using PCRs with primers P19/20 and P21/22 and RLU determination from individual plaques, respectively. HAL = homology arm left; HAR = homology arm right; put. cds = putative coding sequence; mtp = major tail protein; RLU = relative light unit. Source data are provided as a Source Data file.

Fig. 3. In vitro functional assessment of the reporter phages E2::nluc, E4::nluc, EfS3::nluc, EfS7::nluc, K1::nluc and K4::nluc.

a Plaque phenotypes of wildtype and recombinant reporter phages were compared using the soft-agar overlay method. b Infection kinetics of engineered vs. wildtype phages were compared by quantifying the optical density (OD₆₀₀) of infected bacterial cultures over time (liquid infection assay). Data are mean ± SD (n = 3). c The kinetics of reporter phage-mediated NLuc signal production were determined using bioluminescence time course assays. Fold-change (FC) RLU equals RLU (sample) divided by RLU (phage only control). Data and mean±standard error of the mean (SEM) from biological triplicates. d Assay sensitivity was determined in vitro for each reporter phage by infecting serial host cell dilutions and quantifying luminescence at 3 h (E. coli and Klebsiella phages) or 4 h (Enterococcus phages) post infection. Detection limits (vertical dotted lines) were calculated as the cell number required to produce a signal that is three standard deviations (3σ) above the background luminescence (phage in medium, indicated as horizontal dotted lines). Datapoints for linear regression are from biological triplicates. All infections were performed on the phage propagation hosts. p.i. post infection; RLU relative light unit. Source data are provided as a Source Data file.

Fig. 4. Reporter phage host range analysis and infectivity in human urine and whole blood.

a Plaquing host ranges and bioluminescence detection ranges were determined in growth medium on 52 urological E. coli and E. faecalis isolates and 50 urological Klebsiella spp. isolates from the Zurich Uropathogen Collection (listed in Table S2). Strains were infected with reporter phages and luminescence quantified at 3 h (E. coli and Klebsiella spp.) or 4 h (Enterococcus spp.) post infection. Heat-map shows fold change RLU (FC-RLU). Threshold (TH): ≥ 100 FC-RLU (E. coli and Klebsiella spp.) and 50 FC-RLU (Enterococcus spp.). Clinical isolates that allow for plaque formation are indicated with a green dot. Plaquing host ranges were determined from single spot on the lawn infection assays and bioluminescence detection ranges were from biological quadruplicates (E. coli and K. pneumoniae) or biological triplicates (E. faecalis). b Summary of individual and combined host- and detection ranges. Each reporter phage was able to transduce bioluminescence into strains that were not permissive to plaque formation, effectively leading to an increased detection range compared to the plaquing host range. c Heat maps showing reporter phage-induced luminescence in human urine spiked with 6 selected strains of each bacterial target species. d, e Dose response assay with Enterococcus phages EfS3::nluc and EfS7::nluc in human urine d or blood e spiked with serial dilutions of the clinical isolate E. faecalis Ef24. After 1/10 dilution of the spiked urine/blood with buffered media and a 1 h enrichment, reactions were mixed with 2 × 10⁷ PFU/ml reporter phages and FC-RLU was determined at 2 h and 4 h p.i. FC RLU equals RLU (sample) divided by RLU (phage only control). All data in c–e is shown as mean (±SEM) from biological triplicates. Individual datapoints are shown as black dots. Source data are provided as a Source Data file.

Fig. 5. Field evaluation demonstrates rapid, sensitive, and reliable detection of E. coli, Enterococcus spp. and Klebsiella spp. in patient urine.

Workflow of the urine diagnostic assay is shown in a. Fresh patient urine was collected and bacterial metabolic activity activated by a short 1 h enrichment in growth media followed by addition of reporter phages. Luminescence was quantified at 3 h (E. coli, Klebsiella spp.) and 4 h (Enterococcus spp.) post infection. b Luminescence quantification (RLU) was correlated to bacterial CFU/ml counts from differential/selective plating of 206 patient urine samples on UriSelect and KFS agar plates. In parallel, all results were confirmed by the diagnostic laboratory of the Institute of Medical Microbiology (IMM). Urine samples were derived from two different hospitals (Site 1 and Site 2). Thresholds (TH) for luminescence were ≥ 100 FC-RLU (E. coli, green and Klebsiella spp., purple) and 7.5 FC-RLU (Enterococcus spp., blue). The test was considered positive if at least one phage per target pathogen had activity >TH. False negatives (FN, red) are samples with no luminescence but bacterial growth ( ≥ 10³ CFU/ml), false positives (FP, yellow) exhibit luminescence but no bacterial growth. c Summary of the test performance showing calculated sensitivity, specificity, accuracy for all target pathogens at both sampling sites. Some elements of the figure were created using biorender.com. Strains from the field evaluation are listed in Table S3. Source data are provided as a Source Data file.

Fig. 6. Bioluminescence in patient urine predicts antimicrobial effect of the parental phage scaffold.

a Process of phage-patient matching based on reporter phage-mediated luminescence in urine samples. b E. coli, c Enterococcus, and d Klebsiella strains from the field evaluation were isolated and tested in vitro against each wildtype phage using turbidity reduction assays over 12 h in SHU. Activity was quantified as the ratio of the area under the curve (AUC ratio: AUC_HOST+PHAGE/AUC_HOST) where a value of one corresponds to no activity and a value of zero corresponds to maximal activity. For each phage–host pair, the AUC ratio was plotted against the raw luminescence value from the field evaluation and the thresholds for optimal activity prediction were applied to these dotplots. Optimal RLU and AUC ratio thresholds values were determined as detailed in Fig. S5. For each pathogen, true positives (TP) are highlighted in yellow. Turbidity-reduction curves of selected correctly identified phage-patient matches are displayed on the left to demonstrate host killing. All in vitro data is shown as mean ± SD (n = 3). TN true negatives, FP false positives, FN false negatives. Some elements of the figure were created using biorender.com. Source data are provided as a Source Data file.