Simplicity and Reliability Achieved in Antimicrobial Resistance Testing through Transformative Design of a β‐Lactamase‐Responsive Molecular Probe

The molecular probe developed in this study enables the design of next‐generation phenotypic diagnostic tests that directly assess antimicrobial resistance in pathogenic bacterial colonies on agar, that is, in a spatially heterogeneous setting. Its robust and distinctive performance arises from a unique mode of action: the generation of an intensely fluorescent precipitate from an initially silent probe (off–on mode). The probe's utility is verified across seven major human pathogens, each secreting a distinct, clinically relevant β‐lactamase. Its exquisite sensitivity and selectivity are achieved through a probe design that allows for fluorescent readout, enzymatic signal amplification, operation via an off–on mode, and crucially: the ability to generate and retain fluorescence precisely at the site of the enzyme activity. Beyond this newly established feasibility of agar‐based colony assays, the probe technology also delivers a limit of detection of 10 4 bacteria per milliliter of urine in the test tube.