Rapid Determination of Antimicrobial Susceptibility by Stimulated Raman Scattering Imaging of D₂O Metabolic Incorporation in a Single Bacterium

Rapid antimicrobial susceptibility testing (AST) is urgently needed for treating infections with appropriate antibiotics and slowing down the emergence of antibiotic-resistant bacteria. Here, a phenotypic platform that rapidly produces AST results by femtosecond stimulated Raman scattering imaging of deuterium oxide (D₂O) metabolism is reported. Metabolic incorporation of D₂O into biomass in a single bacterium and the metabolic response to antibiotics are probed in as short as 10 min after culture in 70% D₂O medium, the fastest among current technologies. Single-cell metabolism inactivation concentration (SC-MIC) is obtained in less than 2.5 h from colony to results. The SC-MIC results of 37 sets of bacterial isolate samples, which include 8 major bacterial species and 14 different antibiotics often encountered in clinic, are validated by standard minimal inhibitory concentration blindly measured via broth microdilution. Toward clinical translation, stimulated Raman scattering imaging of D₂O metabolic incorporation and SC-MIC determination after 1 h antibiotic treatment and 30 min mixture of D₂O and antibiotics incubation of bacteria in urine or whole blood is demonstrated.