Johansen, T. E. B. | bioRxiv Science

bioRxiv2026

Rapid clinical metagenomics enables early tailored therapy in complicated urinary tract infections and strengthens antimicrobial stewardship

Rapid and accurate diagnosis of UTIs remains difficult because culture-based methods are slow and less sensitive. This study evaluates URINN, a metagenomic workflow that detects uropathogens, antibiotic resistance genes, and virulence factors directly from patient urine samples. The optimized protocol was tested on a combined set of 349 clinical urine samples. URINN demonstrated 99% accuracy across all samples and 97% sensitivity for identifying 294 pathogens, including both bacteria and fungi. It predicted antibiotic susceptibility with 91% accuracy across 2099 antibiotics. The method detected pathogens at concentrations as low as 9.3 x 103 CFU/mL and provided results within approximately four hours. Flow cytometry and DNA yield analyses helped establish thresholds to differentiate culture-positive from culture-negative samples, with genome coverage linked to the accuracy of susceptibility predictions for certain species. Virulence profiling revealed that adherence and nutritional factors are crucial for colonization and persistence. Leukocyte counts were comparable between genders, but bacterial loads were higher in females. The catheterized group had significantly higher leukocyte counts, and their urine showed increased cephalosporin resistance. This approach could enhance clinical decision-making, support personalized treatment, and improve the management of complicated UTIs, thereby contributing to better UTI care and antibiotic stewardship.

genomics↗

bioRxiv2024

Detection of pathogens and antimicrobial resistant genes from urine within 5 hours using Nanopore sequencing

PurposeUrinary Tract Infection (UTI) is a prevalent global health concern accounting for 1-3% of primary healthcare visits. The current methods for UTI diagnosis have a high turnaround time of 3-5 days for pathogen identification and susceptibility testing. This work is a proof-of-concept study aimed at determining the detection limit by establishing a culture and amplification-free DNA extraction methodology from spiked urine samples followed by real-time Nanopore sequencing and data analysis. MethodsThis study first establishes an optical density culture-based method for spiking healthy urine samples with the six most prevalent uropathogens. Pathogens were spiked at two clinically significant concentrations of 103 and 105 CFU/ml. Three commercial DNA extraction kits were investigated based on the quantity of isolated DNA, average processing time, elution volume and the average cost incurred per extraction. The outperforming kit was used for direct DNA extraction and subsequent sequencing on MinION and Flongle flowcells. ResultsThe Blood and Tissue kit outperformed the other kits. All pathogens were identified at a concentration of 105 CFU/ml within ten minutes, and the corresponding AMR genes were detected within three hours of the sequencing start. The overall turnaround time including the DNA extraction and sequencing steps was five hours. Moreover, we also demonstrate that the identification of some pathogens and antibiotic-resistance genes was possible at a spike concentration of 103 CFU/mL. ConclusionThis study shows great promise toward reducing the time required for making an informed antibiotic administration from approximately 48 hours to five hours thereby reducing the number of empirical doses and saving lives.

microbiology↗