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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. 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 min instead of requiring over 24 h. 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. • The biosensor is broadly applicable to most aerobic bacteria, with catalase as the marker. • We employ the I--facilitated Fenton reaction as intermediary, connecting target recognition to signal outputs. • The dual signal output capability provides mutual correction and signal-to-noise ratio enhancement. • The platform is validated by testing 60 clinical isolates from five prominent pathogenic bacteria. [ABSTRACT FROM AUTHOR]

Culture-free, rapid, and ultrasensitive quantification of Escherichia coli (E. coli) is urgently needed to diagnose clinical urinary tract infections (UTIs), especially point-of-care testing (POCT). Herein, a homogeneous sensitive multi-modal (fluorometer, smartphone, and self-developed handheld fluorometer) assay was developed to detect E. coli within 30 min, and nanoscale luminescent cadmium telluride quantum dots (CdTe QDs) were employed as signal reporters. E. coli outer membrane protease T (OmpT) is a biomarker for the specific proteolytic cleavage of peptides. p -Aminophenol (PAP), generated by leucine aminopeptidase (LAP) via secondary cleavage, was used to reduce Ag+. Cascade amplifications included Ag+ and copper sulfide nanoparticles (CuS NPs) to release Cu2+, CdTe QDs, and Cu2+/CuS NPs, which can ensure detection sensitivity. The method achieved single bacterium detection with great stability and selectivity. The fluorometer, handheld fluorometer and smartphone red-green-blue (RGB) value-based modes were determined to be 50, 15, and 15 clinical urine samples, respectively. The findings were consistent with clinical culture counting, and an area under the curve (AUC) of 0.95 with 90% sensitivity and 100% specificity was achieved. Overall, this OmpT-based catalytic biosensor can provide a solution for the POCT diagnosis of UTIs based on E. coli detection. • A homogeneous rapid fluorescence one-pot assay for Escherichia coli was developed. • The non-culture, rapid, multi-modal sensor had high sensitivity and selectivity. • This system was successfully applied to the analysis of E. coli in 50 urine clinical samples. [ABSTRACT FROM AUTHOR]