Abstract
The double-membrane cell envelope of Gram-negative bacteria is a formidable barrier to the cellular entry of antibiotics. Therefore, quantifying antibiotic accumulation in these bacteria is crucial for Gram-negative drug development. However, there is a dearth of techniques capable of studying the kinetics of drug accumulation in single bacteria while also controlling the surrounding microenvironment. By combining microfluidics and time-lapse auto-fluorescence microscopy, we quantified ofloxacin uptake label-free in hundreds of individual Escherichia coli bacteria revealing homogeneous kinetics of drug accumulation within clonal populations. By manipulating the microenvironment, we showed that ofloxacin accumulation is higher in growing versus non-growing cells. We investigated mutants lacking major transport proteins to inform a new Bayesian hierarchical model that quantifies the kinetics of ofloxacin uptake in individual bacteria. Importantly, our combined experimental-theoretical approach predicts drug accumulation in subcellular compartments, which is essential for the rational design of new Gram-negative antibiotics.
