Single‐Cell Antimicrobial Susceptibility Testing via Stimulus‐Responsive Resonance Raman Scattering Imaging

Abstract Antimicrobial resistance (AMR) has emerged as a formidable worldwide public health crisis. Antimicrobial susceptibility testing (AST), the current gold standard for AMR detection, is essential for guiding antibiotic usage but requires days of bacterial culture. We herein report a stimulus‐responsive resonance Raman scattering (SRRRS) imaging technique for real‐time monitoring of dynamic β‐lactamase expression at the single‐cell level, allowing AMR detection and antibiotic screening within 2.5 h. The detection limit of AMR bacteria can be down to 10 2 cfu mL −1 using the SRRRS‐imaging technique, which is 3–4 orders of magnitude lower than conventional optical probes. We further constructed a SRRRS‐imaging‐derived antibiotic breakpoints library for Pseudomonas aeruginosa ( P. aeruginosa, PAK ), enabling rapid susceptibility testing for 14 antibiotics within 3 h without bacterial culture. Clinical validation with 30 intensive care unit patients demonstrated 93% accuracy, offering a transformative tool for guiding timely antibiotic prescriptions and improving outcomes in PAK ‐related infections.