Spectrofluorimetric quantification of antibiotic drug concentration in bacterial cells for the characterization of translocation across bacterial membranes
| Autor: | Laure Maigre, Jelena Pajovic, Bertrand Cinquin, Jean-Marie Pagès, Muriel Masi, Julia Vergalli, Matthieu Réfrégiers, Estelle Dumont |
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| Přispěvatelé: | Membranes et cibles thérapeutiques (MCT), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Biomédicale des Armées (IRBA)-Aix Marseille Université (AMU), University of Belgrade [Belgrade], Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Biomédicale des Armées [Brétigny-sur-Orge] (IRBA), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Biomédicale des Armées (IRBA) |
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Membranes biology Bacteria medicine.drug_class Chemistry [SDV]Life Sciences [q-bio] 030106 microbiology Antibiotics biology.organism_classification General Biochemistry Genetics and Molecular Biology Fluorescence spectroscopy Anti-Bacterial Agents Standard curve 03 medical and health sciences Membrane Spectrometry Fluorescence Biochemistry medicine Efflux Single-Cell Analysis Bacterial outer membrane Intracellular |
| Zdroj: | Nature Protocols Nature Protocols, Nature Publishing Group, 2018, 13 (6), pp.1348-1361. ⟨10.1038/nprot.2018.036⟩ Nature Protocols, 2018, 13 (6), pp.1348-1361. ⟨10.1038/nprot.2018.036⟩ |
| ISSN: | 1750-2799 |
| DOI: | 10.1038/nprot.2018.036⟩ |
| Popis: | International audience; 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 fluorescence relative to the internal standard and the antibiotic standard curve, respectively. Fluorimetry is limited to naturally fluorescent or labeled compounds, but in contrast to existing alternative methods such as mass spectrometry, it uniquely allows single-cell analysis. From culture growth to data analysis, the protocol described here takes 5 d. |
| Databáze: | OpenAIRE |
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