Droplet-Based Single-Cell Measurements of 16S rRNA Enable Integrated Bacteria Identification and Pheno-Molecular Antimicrobial Susceptibility Testing from Clinical Samples in 30 min

Empiric broad-spectrum antimicrobial treatments of urinary tract infections (UTIs) have contributed to widespread antimicrobial resistance. Clinical adoption of evidence-based treatments necessitates rapid diagnostic methods for pathogen identification (ID) and antimicrobial susceptibility testing (AST) with minimal sample preparation. In response, a microfluidic droplet-based platform is developed for achieving both ID and AST from urine samples within 30 min. In this platform, fluorogenic hybridization probes are utilized to detect 16S rRNA from single bacterial cells encapsulated in picoliter droplets, enabling molecular identification of uropathogenic bacteria directly from urine in as little as 16 min. Moreover, in-droplet single-bacterial measurements of 16S rRNA provide a surrogate for AST, shortening the exposure time to 10 min for gentamicin and ciprofloxacin. A fully integrated device and screening workflow were developed to test urine specimens for one of seven unique diagnostic outcomes including the presence/absence of Gram-negative bacteria, molecular ID of the bacteriaas Escherichia coli, an Enterobacterales, or other organism, and assessment of bacterial susceptibility to ciprofloxacin. In a 50-specimen clinical comparison study, the platform demonstrates excellent performance compared to clinical standard methods (areas-under-curves, AUCs >0.95), within a small fraction of the turnaround time, highlighting its clinical utility.