Bacterial catalase-responsive colorimetric array driven by nanozyme cascade for pathogen identification and phenotypic susceptibility testing.

The rapid discrimination of bacterial species and assessment of their antibiotic susceptibility are critical for combating antimicrobial resistance. Herein, we report a metabolism-driven colorimetric sensor array based on bacterial catalase-responsive hydrogen peroxide (H2O2) consumption coupled with an Au@PB (gold-coated Prussian blue) nanozyme cascade, which relies on a single 3,3',5,5'-tetramethylbenzidine (TMB) probe. Bacteria with different intrinsic catalase activities consume varying amounts of H2O2, leaving distinct residuals that drive Au@PB-catalyzed TMB oxidation to produce three characteristic absorption peaks at 370, 652, and 880 nm. These three-wavelength signals serve as unique phenotypic fingerprints. Combined with linear discriminant analysis (LDA), the single-probe array successfully discriminates multiple bacterial species and mixtures, exhibiting a linear quantitative response across 104-107 CFU mL-1. Furthermore, by monitoring real-time changes in catalase activity upon antibiotic exposure, the platform enables phenotypic antimicrobial susceptibility testing (AST) with a rapid assay time of ∼75 min for standardized bacterial suspensions, expediting the evaluation process compared to culture-based methods. Validated in water samples and clinical isolates, the AST results showed good agreement with the clinical laboratory standards institute (CLSI) gold standard. Therefore, this nanozyme-driven, single-probe sensor array provides a sensitive, and cost-effective tool for integrated pathogen identification and rapid AST.