title: Quantification of Fluoroquinolone Uptake through the Outer Membrane Channel OmpF of Escherichia coli.
abstract: Decreased drug accumulation is a common cause of antibiotic resistance in microorganisms. However, there are few reliable general techniques capable of quantifying drug uptake through bacterial membranes. We present a semiquantitative optofluidic assay for studying the uptake of autofluorescent drug molecules in single liposomes. We studied the effect of the Escherichia coli outer membrane channel OmpF on the accumulation of the fluoroquinolone antibiotic, norfloxacin, in proteoliposomes. Measurements were performed at pH 5 and pH 7, corresponding to two different charge states of norfloxacin that bacteria are likely to encounter in the human gastrointestinal tract. At both pH values, the porins significantly enhance drug permeation across the proteoliposome membranes. At pH 5, where norfloxacin permeability across pure phospholipid membranes is low, the porins increase drug permeability by 50-fold on average. We estimate a flux of about 10 norfloxacin molecules per second per OmpF trimer in the presence of a 1 mM concentration gradient of norfloxacin. We also performed single channel electrophysiology measurements and found that the application of transmembrane voltages causes an electric field driven uptake in addition to concentration driven diffusion. We use our results to propose a physical mechanism for the pH mediated change in bacterial susceptibility to fluoroquinolone antibiotics.
authors.affiliation: Biological and Soft Systems, Cavendish Laboratory, Department of Physics, University of Cambridge , JJ Thomson Avenue, Cambridge CB3 0HE, United Kingdom.
authors.forename: Jehangir
authors.initials: J
authors.lastname: Cama
authors.affiliation: Jacobs University Bremen , Campus Ring 1, D-28759, Bremen, Germany.
authors.forename: Harsha
authors.initials: H
authors.lastname: Bajaj
authors.affiliation: Biological and Soft Systems, Cavendish Laboratory, Department of Physics, University of Cambridge , JJ Thomson Avenue, Cambridge CB3 0HE, United Kingdom.|Department of Biosciences, College of Life and Environmental Sciences, University of Exeter , Exeter, Devon EX4, United Kingdom.
authors.forename: Stefano
authors.initials: S
authors.lastname: Pagliara
authors.affiliation: Biological and Soft Systems, Cavendish Laboratory, Department of Physics, University of Cambridge , JJ Thomson Avenue, Cambridge CB3 0HE, United Kingdom.
authors.forename: Theresa
authors.initials: T
authors.lastname: Maier
authors.affiliation: Jacobs University Bremen , Campus Ring 1, D-28759, Bremen, Germany.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Braun
authors.affiliation: Jacobs University Bremen , Campus Ring 1, D-28759, Bremen, Germany.
authors.forename: Mathias
authors.initials: M
authors.lastname: Winterhalter
authors.affiliation: Biological and Soft Systems, Cavendish Laboratory, Department of Physics, University of Cambridge , JJ Thomson Avenue, Cambridge CB3 0HE, United Kingdom.
authors.forename: Ulrich F
authors.initials: UF
authors.lastname: Keyser
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D004926:Escherichia coli / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D005453:Fluorescence; D024841:Fluoroquinolones / Q000737:chemistry / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008826:Microbial Sensitivity Tests; D018272:Porins / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D000900:Anti-Bacterial Agents; D024841:Fluoroquinolones; C109237:OmpF protein; D018272:Porins