Rapid Antibiotic\nSusceptibility Determination by Fluorescence\nLifetime Tracking of Bacterial Metabolism

To combat the rise of antibiotic-resistance in bacteria\nand the\nresulting effects on healthcare worldwide, new technologies are needed\nthat can perform rapid antibiotic susceptibility testing (AST). Conventional\nclinical methods for AST rely on growth-based assays, which typically\nrequire long incubation times to obtain quantitative results, representing\na major bottleneck in the determination of the optimal antibiotic\nregimen to treat patients. Here, we demonstrate a rapid AST method\nbased on the metabolic activity measured by fluorescence lifetime\nimaging microscopy (FLIM). Using lab strains and clinical isolates\nof <i>Escherichia coli</i> with tetracycline-susceptible\nand resistant phenotypes as models, we demonstrate that changes in\nmetabolic state associated with antibiotic susceptibility can be quantitatively\ntracked by FLIM. Our results show that the magnitude of metabolic\nperturbation resulting from antibiotic activity correlates with susceptibility\nevaluated by conventional metrics. Moreover, susceptible and resistant\nphenotypes can be differentiated in as short as 10 min after antibiotic\nexposure. This FLIM-AST (FAST) method can be applied to other antibiotics\nand provides insights into the nature of metabolic perturbations inside\nbacterial cells resulting from antibiotic exposure with single cell\nresolution.