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%.
This work demonstrates a rapid platform that can determine the antimicrobial susceptibility testing (AST) in cation-adjusted Mueller-Hinton Broth medium, urine and blood by stimulated Raman scattering (SRS) imaging of deuterium oxide (D2O) incorporation at a single bacterium level. The total AST assay time with the value of the single-cell metabolism inactivation concentration (SC-MIC) obtained is less than 2.5 h from colony to results. The SC-MIC results of 37 sets of bacterial isolate samples were systematically validated by MIC determined by the Clinical and Laboratory Standards Institute criteria, with a category agreement of 94.6% and 5.4% minor error. Furthermore, SRS imaging of D2O metabolic incorporation can rapidly determine SC-MIC directly in clinical samples for urinary tract infection or septicemia blood infection.
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