Ultra-fast phenotypic antimicrobial susceptibility testing via dual-fluorescent staining and morphometric analysis on a microfluidic chip.

Antimicrobial susceptibility testing (AST) is critical for guiding timely antibiotic therapy; however conventional methods used in hospitals remain time-consuming. To allow ultra-fast AST, we developed a phenotypic-based, microfluidic AST system capable of generating susceptibility profiles for 10 antibiotics within 1 h. The platform integrates antibiotic-exposed bacterial cultivation, dual-fluorescent viability staining (dye SYTO9 for all, and dye propidium iodide (PI) for dead cells), and morphometric analysis in a three-layer microfluidic chip. Optimized staining conditions (2.5 μM SYTO9, 30 μM PI, 10-min incubation) enabled direct discrimination of drug-sensitive pathogens via the quantification of dead-cell ratios or elongated cell-lengths after drug-cultivation. Antibiotic exposure with 40 min is enough to yield distinct dead cells (18.5-51.4 % in total) or elongated cells (10.94-12.24 μm in length) for drug-sensitive strains; while almost no changes of dead cell ratios (<5 %), and cell lengths (1.4-3.8 μm) were observed for drug-resistant strains in comparison with the drug-free control. Clinical validation of chip-based AST of 19 E. coli-positive urine samples against 10 kinds of typical antibiotics demonstrated 98.7 % concordance for drug-resistant samples and 99.1 % for drug-sensitive samples compared to gold-standard AST. This system offers an ultra-fast, cost-effective solution for on-site AST in emergency (ICU) or in source-limited regions.