RT Journal Article
SR Electronic
T1 Label-free single-cell microfluidics reveals antibiotic transport kinetics in Gram-negative bacteria
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 645507
DO 10.1101/645507
A1 Cama, Jehangir
A1 Voliotis, Margaritis
A1 Metz, Jeremy
A1 Smith, Ashley
A1 Ianucci, Jari
A1 Keyser, Ulrich F.
A1 Tsaneva-Atanasova, Krasimira
A1 Pagliara, Stefano
YR 2019
UL http://biorxiv.org/content/early/2019/05/22/645507.abstract
AB 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.