Abstract
Urinary tract infections (UTIs) are widespread bacterial infections, affecting over 400 million people globally and causing approximately 237,000 deaths. UTIs account for 15-20% of all antibiotics prescribed in primary healthcare. Uncomplicated UTIs are common in healthy individuals and outpatient settings, while complicated UTIs occur in individuals with health problems or a weakened immune system. E. coli is the primary causative agent, responsible for 75% of uncomplicated and 65% of complicated UTIs. Untreated UTIs can escalate to kidney infections, bacteremia, and urosepsis. Current culture-based diagnostic methods have a turnaround time (TAT) of 2-5 days, forcing physicians towards empirical treatments, leading to imprudent antibiotic use and eventually to antimicrobial resistance (AMR) dissemination. Culture-independent rapid detection of UTIs and AMR profiles using emerging platforms such as real-time whole genome sequencing (WGS), combined with bioinformatics, can address the time-consuming nature of conventional methods.
Five commercial kits and in-house depletion methods were evaluated in healthy urine samples spiked with two uropathogens, E. coli and E. faecalis, at 108 and 105 CFU/mL to identify the most effective strategies for enriching bacterial DNA and depleting human DNA. PCR was used to confirm the presence of target bacterial DNA and human DNA before nanopore sequencing. Whole genome amplification (WGA) was tested for samples with DNA yield lower than suggested for nanopore sequencing. Interestingly, WGA drastically amplified low DNA concentration samples within 3 hours of incubation. Therefore, the overall turnaround time for pathogen and antimicrobial resistance genes (ARGs) detection, including DNA extraction, library preparation, sequencing, and bioinformatic analysis, was approximately 5 to 14 hours.
Lastly, as urine can be stored in the clinical routine for follow-up analysis, we tested different durations, temperatures, and preserving agents to study their effect on short and long-term urine storage. Zymo condition buffer demonstrated superior efficacy for long-term urine storage among the various preserving agents tested. It effectively preserved DNA at 4 °C for up to 15 days and at -20 °C and -80 °C for up to 30 days.
The findings of this study support the future application of culture-independent nanopore sequencing for rapid UTI diagnostics in healthcare settings. This would identify the causative pathogens and their AMR profile, contributing to better clinical management of UTIs and preventing antibiotic misuse and subsequent AMR development.
Keywords: UTI, AMR, rapid detection, DNA extraction, nanopore sequencing, Whole genome amplification, long-term urine storage