Journal
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume 137, Issue 43, Pages 13836-13843Publisher
AMER CHEMICAL SOC
DOI: 10.1021/jacs.5b08960
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Funding
- European Research Council (ERC) Grant [261101]
- Internal Graduate Studentship, Trinity College, Cambridge
- Cambridge Philosophical Society
- Leverhulme Trust through an Early Career Fellowship
- Konrad-Adenauer Foundation
- German National Merit Foundation
- Innovative Medicines Joint Undertaking [115525]
- European Union's seventh framework program (FP7)
- European Federation of Pharmaceutical Industries and Associates companies
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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 semi-quantitative 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 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.
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