Culture-and amplification free nanopore sequencing for rapid detection of pathogens and antimicrobial resistance genes from urine

Urinary Tract Infections (UTIs) are among the most prevalent infections globally. Every year, approximately 150 million people are diagnosed with UTIs worldwide. The current state-of-the-art routine method diagnostic methods are culture-based and have a turnaround time of 2-4 days for pathogen identification and susceptibility testing. This study first establishes an optical density culture-based method for spiking healthy urine samples with the six most prevalent uropathogens. Urine samples were spiked at clinically significant concentrations of 103-105 CFU/ml. Three DNA extraction kits (BioStic, PowerFood, and Blood and Tissue) were investigated based on the DNA yield, average processing time, elution volume, and the average cost incurred per extraction. Results: The Blood and Tissue kit outperformed the other kits based on the DNA yield, average processing time, elution volume, and the average cost incurred per extraction. Using nanopore sequencing, we demonstrated that identifying some pathogens and antibiotic-resistance genes (ARG) was possible at as low as 103 colony formation units (CFU/mL). However, all the pathogens and corresponding genes were only identified at a spike concentration of 105 CFU/ml, achieved only after 10 min and 3 hours of sequencing, respectively. The overall turnaround time, from sample preparation to the sequencing information about pathogen ID and antimicrobial resistance (AMR) genes, was five hours. This study shows great promise in reducing the time required for informed antibiotic administration from approximately 48 hours to five hours, thereby reducing the number of empirical doses and saving lives.