Bacterial Metabolism-Driven Dual-Enzyme Cascade Colorimetric Sensor for Antimicrobial Susceptibility Testing with Readily Interpretable Signals

The global antimicrobial resistance (AMR) crisis urgently necessitates rapid phenotypic antimicrobial susceptibility testing (AST) to reduce empirical antibiotic prescribing and enable targeted antimicrobial therapy. Conventional phenotypic AST methods require an incubation period of 18-24 h, delaying evidence-based decisions and forcing empirical treatment, which compromises efficacy and fuels AMR emergence. Herein, we reported a simple, practical phenotypic AST assay that transduced bacterial glucose metabolic activity into a quantitative colorimetric signal via a glucose oxidase (GOD)-horseradish peroxidase (HRP) dual-enzyme cascade colorimetric sensor (DECCS) system. The assay enabled intuitive visual readout of color development by the naked eye, with quantitative signal validation via two complementary approaches: smartphone-based RGB (red-green-blue) analysis and microplate reader absorbance measurement, thus balancing operational accessibility with analytical precision. Rigorous performance validation was conducted using 155 clinical isolates and 5 reference strains, covering major clinically relevant Gram-positive and Gram-negative pathogens (<i>Staphylococcus aureus</i>, <i><i>Escherichia coli</i></i>, <i><i>Pseudomonas aeruginosa</i></i>, <i><i>Klebsiella pneumoniae</i></i>, and <i><i>Acinetobacter baumannii</i></i>). Determined minimum inhibitory concentration (MIC) values exhibited high concordance with the Clinical and Laboratory Standards Institute (CLSI)-recommended broth microdilution method (gold standard), achieving >96% essential agreement and >98% categorical agreement. This AST platform featured a ∼5 h workflow, simple operation, low cost, and multimodal readout capabilities, rendering it highly suitable for resource-limited clinical settings. By enabling same-day evidence-based antimicrobial prescribing, this method provided a practical alternative tool to strengthen antimicrobial stewardship efforts and improve the clinical outcomes of bacterial infection treatment.