Timely and accurate identification and antimicrobial susceptibility testing (AST) is essential to slow down the emergence of antimicrobial resistance and consequently reduce deaths due to drug-resistant infections. Here, we present multi-color fluorescence in situ hybridization (FISH) and stimulated Raman scattering (SRS) metabolic imaging to enable rapid identification and AST (iAST) of clinical samples. We have successfully developed FISH probes that utilizes oligonucleotide probes targeting the ribosomal RNA (rRNA) of bacteria strains associated with urinary tract infections (UTIs), which enables rapid and direct single-cell identification of microbes. By fast tuning between C-D and C-H vibration, we are able to use CD/(CD+CH) SRS signal ratio to quantitate the intracellular deuterium oxide (D2O) metabolism at single cell level. High-throughput, high-speed AST through robotic handling of liquid specimens and a multi-well chamber design has been demonstrated. We test the efficacy of our approach on common UTI associated bacterial isolates and clinical specimens, and demonstrate a 2 h diagnostic time, with an identification and categorical susceptible/resistant accuracy over 95%.
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