A NOVEL PROPIDIUM MONOAZIDE-BASED PCR ASSAY CAN DETECT VIABLE BUT NON-CULTURABLE BACTERIA IN URINE AFTER ANTIBIOTIC TREATMENT OF UTI

INTRODUCTION AND OBJECTIVE: Bacterial urinary tract infections (UTIs) are among the most common microbial diseases in all age groups and major contributors to the injudicious use of antibiotics. Rapid and accurate pathogen identification directly from urine samples can drastically improve the clinical management of UTIs, alleviating their socio-economic impacts and facilitating antibiotic stewardship. METHODS: Luciferase reporter phages are genetically engineered viruses capable of rapidly detecting their target bacterial hosts with exquisite specificity and sensitivity. As signal production requires phage genome injection and subsequent host-dependent expression of phage-encoded reporter proteins, this diagnostic method does not produce false-positive results in the presence of dead target cells. Here, using a set of six genetically distinct luciferase reporter phages, we report the development and evaluation of a novel phage-based bioluminescence assay for rapid detection of the three predominant UTI pathogens, Escherichia coli, Klebsiella spp. and Enterococcus spp. RESULTS: In vitro, with pure cultures, the assay positively identified 89% of 155 relevant clinical isolates, showing the highest inclusivity for Enterococcus spp. (50/52), followed by Klebsiella spp. (45/51) and E. coli (43/52). Cross-reactivity of the phage cocktails with other closely related genus including Raoultella and Staphylococcus was observed, but only at minimal rate (4.3%; 3/70). The assay performance was further assessed alongside routine clinical testing on 145 patient specimens collected from a local hospital in Switzerland over 5 months. Following short pre-enrichment steps and with simple substrate addition, the assay reliably detected the three target pathogens with 73% sensitivity and 96% specificity at a resolution of 10 CFU/ml in less than 6 h. CONCLUSIONS: This phage-based diagnostic platform offers opportunities for prompt bacterial UTI diagnosis in point-of-care settings and could be harnessed as companion diagnostics to assist rapid identification of potential responder patients during phage therapy.