Catalase-mediated Fenton reaction enabled dual fluorescence sensing for broad-spectrum bacterial rapid antimicrobial susceptibility testing

Delayed antimicrobial susceptibility testing (AST) results lead to empiric broad-spectrum antibiotic use, exerting selective pressure that promotes further antimicrobial resistance (AMR). Compared to genotypic AST approaches, phenotypic AST provides clinicians with a more comprehensive antibiotic susceptibility profile. Hence, there is a pressing need to develop phenotypic methods suitable for clinical settings with quick turnaround times. Herein, we developed a comprehensive phenotypic AST platform that harnessed catalase as a marker and the Fenton reaction as a vehicle. Signal outputs were generated through the formation of methylene blue (MB)/G-quadruplex complexes and doxorubicin (DOX)/double-stranded DNA (dsDNA) complexes. The dual signal mode enhanced the signal-to-noise ratio. Five prominent pathogens associated with AMR-related deaths were selected to validate the method's feasibility. And sixty clinical isolates were tested to assess the clinical practicality, and the results yielded susceptibility profiles that closely agreed with standard AST for 55 out of 60 isolates (91.7% concordance) but were obtained in only 111-126 minutes instead of requiring over 24 hours. Furthermore, the receiver operating characteristic curve was employed to evaluate the diagnostic value, yielding an area under the curve (AUC) of 0.958. If integrated and advocated in clinical laboratories, this technique holds promise in effectively addressing AMR.