Rapid Bacteria Identification and Antimicrobial Susceptibility Testing via 5‐Min Fluorescence In Situ Hybridization

Abstract Accurate and timely diagnosis of infections, including pathogen detection and antibiotic susceptibility testing (AST), is essential to effective disease management and combating antibiotic resistance. Here, SWIFT‐FISH (Sensitive, Wash‐free, Immediate, microFluidic, Thermal permeabilization‐facilitated Fluorescence In Situ Hybridization), a novel diagnostic platform enabling bacterial identification (ID) in ≈5 min is reported, complemented by rapid AST. SWIFT‐FISH significantly accelerates the conventional FISH workflow through transient thermal permeabilization, allowing intracellular hybridization of fluorescent peptide nucleic acid (PNA) probes to bacterial 16S ribosomal RNA (rRNA) without the conventional fixation step. SWIFT‐FISH is then directly detected in solution within a continuous‐flow microfluidic platform equipped with in‐line single‐cell resolution multi‐color laser‐induced fluorescence (LIF) detection. This approach boosts the cell‐to‐background ratio, which obviates the conventional washing step, while also enabling continuous cell delivery as a means for improving detection sensitivity. Using 3 broad‐based probes and incorporating machine learning–based analysis, SWIFT‐FISH demonstrates scalable identification of 6 bacterial species with ≈99% accuracy. Finally, by quantifying bacterial proliferation, SWIFT‐FISH readily enables rapid phenotypic AST with ≈1 h turnaround time. SWIFT‐FISH thus represents a simple, rapid, sensitive, and versatile diagnostic technology with significant potential to enhance clinical microbiology and antibiotic stewardship.