Bringing Metagenomics to the Bedside may facilitate early tailored   Management of patients with complicated UTIs and improve antimicrobial stewardship
Urinary Tract Infections (UTIs) are responsible for around 405 million infections worldwide, with millions of dollars spent on related healthcare costs. The current gold-standard pathogen detection test is culture-based and requires 2-4 days, whereas nanopore-based mNGS offers a faster alternative. Yet it faces challenges, including high host DNA levels, low bacterial levels, and impurities.  A culture-independent sample preparation method called URINN, finetuned from our previously established “optimized method”, was further evaluated on a cohort of 288 patient samples with UTIs, followed by real-time nanopore sequencing and data analysis. Results were compared with culture-based clinical microbiology techniques, including MALDI-TOF and VITEK-2. Additionally, mNGS data from patient cohorts were analyzed to examine pathogen abundance in relation to catheter use and gender-related risk factors, and to explore virulence factors and patterns of pathogen co-occurrence.  The metagenomic results closely matched those obtained by routine culture-based methods. Performance was stable, with 99% pathogen ID accuracy, 97% sensitivity, 93% PPV, and 89% NPV across 206 pathogens. Antibiotic-susceptibility prediction improved, with accuracy at 92%, specificity at 99%, and PPV and NPV at 92%. The detection limit was 10³ CFU/mL. Further analysis of patient cohorts revealed a variety of virulence factors, with a higher relative abundance of adhesion and immune-modulation factors. Gender classification indicated the presence of Escherichia coli in both groups, with higher relative abundance observed in females. Leukocyte counts remained consistent across genders, whereas bacterial load was higher in female patients. The catheterized cohort demonstrated significantly increased leukocyte counts. Besides E. coli and Enterococcus faecalis, the proportional abundances of Staphylococcus aureus, Morganella morganii, and Klebsiella oxytoca were greater in catheterized urine samples, while Klebsiella pneumoniae and Pseudomonas aeruginosa were more frequently found in non-catheterized samples. Additionally, urine from catheterized patients exhibited a higher incidence of cephalosporin resistance.  This research fine-tunes and validates a rapid, cost-effective sample preparation method, URINN, for diagnosing bacterial infections in urine samples, reducing turnaround time from 4 days to 4 hours. We also access additional pathogen-associated risk factors from the metagenomic data. Furthermore, our approach provides insights into pathogen risk factors and host immune responses, thereby aiding personalized treatment, improving UTI management, and enhancing antibiotic stewardship