Spectrofluorimetric quantification of antibiotic drug concentration in bacterial cells for the characterization of translocation across bacterial membranes

Nature Protocols
Julia VergalliJean-Marie Pagès

Abstract

The efficacy of antibacterial molecules depends on their capacity to reach inhibitory concentrations in the vicinity of their target. This is particularly challenging for drugs directed against Gram-negative bacteria, which have a complex envelope comprising two membranes and efflux pumps. Precise determination of the bacterial drug content is an essential prerequisite for drug development. Here we describe three approaches that have been developed in our laboratories to quantify drugs accumulated in intact cells by spectrofluorimetry, microspectrofluorimetry, and kinetics microspectrofluorimetry (KMSF). These different procedures provide complementary results that highlight the contribution of membrane-associated mechanisms, including influx through the outer membrane (OM) and efflux, and the importance of the physicochemical properties of the transported drugs for the intracellular concentration of a given antibiotic in a given bacterial population. The three key stages of this protocol are preparation of the bacterial strains in the presence of the antibiotic; preparation of the whole-cell lysates (WCLs) and fluorescence readings; and data analysis, including normalization and quantitation of the intracellular antibiotic flu...Continue Reading

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Citations

Jun 21, 2019·Nature·Eachan O JohnsonDeborah T Hung
Jul 19, 2019·Clinical Microbiology Reviews·Anne Davin-RegliJean-Marie Pagès
Dec 4, 2019·Chemistry : a European Journal·Angelo FreiMark A T Blaskovich
May 10, 2020·Journal of Synchrotron Radiation·Frédéric JammeMarine Froissard
Jan 9, 2019·Life Science Alliance·Estelle DumontJean-Marie Pagès
Dec 4, 2019·Nature Reviews. Microbiology·Julia VergalliJean-Marie Pagès
Jul 16, 2019·MedChemComm·M Rhia L StoneMark A T Blaskovich
Nov 10, 2020·Expert Opinion on Drug Delivery·Mathias Winterhalter
Nov 18, 2020·Nature Chemical Biology·Shibin ZhaoDerek S Tan
Feb 21, 2021·Scientific Reports·Erika BoulantJean Michel Bolla
Mar 12, 2021·Advanced Drug Delivery Reviews·Henni-Karoliina RopponenClaus-Michael Lehr
Mar 17, 2021·Chemical Reviews·Jigneshkumar Dahyabhai PrajapatiMathias Winterhalter
Apr 16, 2021·Advanced Drug Delivery Reviews·Robert Richter, Claus-Michael Lehr
Dec 18, 2020·ACS Medicinal Chemistry Letters·Žiga SkokJanez Ilaš
Mar 21, 2020·ACS Infectious Diseases·Mahnaz Sabeti AzadDominique Fourmy
Aug 31, 2021·RSC Chemical Biology·M Rhia L StoneMark A T Blaskovich
Oct 7, 2021·Advanced Healthcare Materials·Robert RichterClaus-Michael Lehr

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