Methods for rapid antimicrobial susceptibility testing (AST) are urgently needed to address the emergence and spread of antimicrobial resistance. Here, we report a new method based on stimulated Raman scattering (SRS) microscopy, which measures both the metabolic activity and the morphological deformation of bacteria to determine the antimicrobial susceptibility of β-lactam antibiotics rapidly. In this approach, we quantify single bacteria's metabolic activity by the carbon-deuterium (C−D) bond concentrations in bacteria after D2O incubation. In the meantime, bacterial morphological deformation caused by β-lactam antibiotics is also measured. With these two quantifiable markers, we develop an evaluation method to perform AST of cefotaxime on 103 E. coli strains. Our method achieved a 93.2% categorical agreement and a 93.2% essential agreement with the standard reference method.
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