Since conventional culture-based antibiotic susceptibility testing (AST) methods are too\ntime-consuming (typically 24-72 h), rapid AST is urgently needed for preventing the increasing\nemergence and spread of antibiotic resistant infections. Although several phenotypic antibiotic\nresistance sensing modalities are able to reduce the AST time to a few hours or less, concerning the\nbiological heterogeneity, their accuracy or limit of detection are limited by low throughput. Here,\nwe present a rapid AST method based on whole slide imaging (WSI)-enabled high-throughput\nsensing antibiotic resistance at single-bacterium level. The time for determining the minimum\ninhibitory concentration (MIC) was theoretically shortest, which ensures that the growth of each\nindividual cell present in a large population is inhibited. As a demonstration, our technique was\nable to sense the growth of at least several thousand bacteria at single-cell level. Reliable MIC of\nEnterobacter cloacae against gentamicin was obtained within 1 h, while the gold standard broth dilution\nmethod required at least 16 h for the same result. In addition, the application of our method prevails\nover other imaging-based AST approaches in allowing rapid and accurate determination of antibiotic\nsusceptibility for phenotypically heterogeneous samples, in which the number of antibiotic resistant\ncells was negligible compared to that of the susceptible cells. Hence, our method shows great promise\nfor both rapid AST determination and point-of-care testing of complex clinical bacteria isolates.
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