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

Nature Protocols volume 13, pages 1348–1361 (2018)Cite this article

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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 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.

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Figure 1: Schematic representation of the three options described in Step 6 of the Procedure.
Figure 2: Spectrofluorimetry (Step 6A).
Figure 3: Microspectrofluorimetry (Step 6B).
Figure 4: An example of a KMSF experiment and collected data (Step 6C).

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Acknowledgements

We are especially grateful to I. Artaud for her commitment and motivation during the development of this original approach. We also thank A. Davin-Regli, R. Stavenger and M. Winterhalter for their fruitful discussions, and A.-M. Tran, V. Rouam and B. Pineau for technical assistance. The research leading to these results was conducted as part of the TRANSLOCATION consortium, and it has received support from the Innovative Medicines Initiatives Joint Undertaking under Grant Agreement no. 115525, resources that are composed of financial contribution from the European Union's seventh framework program (FP7/2007–2013), and EFPIA companies in kind contribution. J.V., E.D., J.P., B.C., L.M., and M.M. are funded by IMI-Translocation (grant 115525). This work was also supported by Aix-Marseille University and Service de Santé des Armées, and by Soleil program (project nos. 20130061, 20130949, 20140047, 20141262, 20150318, 20151274, and 20160173).

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Author notes

  1. Bertrand Cinquin

    Present address: Present address: Bertrand Cinquin, LBPA, ENS CACHAN, Cachan, France.,

  2. Julia Vergalli, Estelle Dumont and Jelena Pajović: These authors contributed equally to this work.

Authors and Affiliations

  1. UMR_MD1, U-1261, Aix-Marseille Univ., INSERM, SSA,

    Julia Vergalli, Estelle Dumont, Laure Maigre, Muriel Masi & Jean-Marie Pagés

  2. IRBA, MCT, Marseille, France

    Julia Vergalli, Estelle Dumont, Laure Maigre, Muriel Masi & Jean-Marie Pagés

  3. DISCO Beamline, Synchrotron Soleil,

    Jelena Pajović, Bertrand Cinquin & Matthieu Réfrégiers

  4. , L'Orme des Merisiers, Gif-sur-Yvette, France

    Jelena Pajović, Bertrand Cinquin & Matthieu Réfrégiers

  5. Faculty of Physics,

    Jelena Pajović

  6. University of Belgrade, Belgrade, Serbia

    Jelena Pajović

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J.V., E.D., J.P., B.C., L.M., M.M., M.R. and J.-M.P. all contributed equally to manuscript writing.

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Vergalli, J., Dumont, E., Pajović, J. et al. Spectrofluorimetric quantification of antibiotic drug concentration in bacterial cells for the characterization of translocation across bacterial membranes. Nat Protoc 13, 1348–1361 (2018). https://doi.org/10.1038/nprot.2018.036

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