gms | German Medical Science

10. Kongress für Infektionskrankheiten und Tropenmedizin (KIT 2010)

Deutsche Gesellschaft für Infektiologie,
Deutsche AIDS-Gesellschaft,
Deutsche Gesellschaft für Tropenmedizin und Internationale Gesundheit,
Paul-Ehrlich-Gesellschaft für Chemotherapie

23.06. - 26.06.2010, Köln

A novel real-time efflux assay reveals that selective serotonin reuptake inhibitors inhibit efflux of the near-infrared dye 1,2-dinaphthylamine in Escherichia coli strains overexpressing different RND pumps

Ein neuer Echtzeit-Effluxassay in Escherichia coli-Stämmen mit RND-Pumpen-Überexpression zeigt die inhibitorische Wirkung von selektiven Serotonin-Wiederaufnahmehemmern auf den Efflux des Naheinfrarot-Farbstoffes 1,2-dinaphthylamine

Meeting Abstract

  • J.A. Bohnert - Universitätsklinikum Freiburg, Zentrum Infektiologie und Reisemedizin, Freiburg i.Br., Germany
  • S. Schuster - Universitätsklinikum Freiburg, Zentrum Infektiologie und Reisemedizin, Freiburg i.Br., Germany
  • M. Szymaniak-Vits - Universitätsklinikum Freiburg, Zentrum Infektiologie und Reisemedizin, Freiburg i.Br., Germany
  • W.V. Kern - Universitätsklinikum Freiburg, Zentrum Infektiologie und Reisemedizin, Freiburg i.Br., Germany

10. Kongress für Infektionskrankheiten und Tropenmedizin (KIT 2010). Köln, 23.-26.06.2010. Düsseldorf: German Medical Science GMS Publishing House; 2010. DocP15

DOI: 10.3205/10kit071, URN: urn:nbn:de:0183-10kit0712

Veröffentlicht: 2. Juni 2010

© 2010 Bohnert et al.
Dieser Artikel ist ein Open Access-Artikel und steht unter den Creative Commons Lizenzbedingungen (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.de). Er darf vervielfältigt, verbreitet und öffentlich zugänglich gemacht werden, vorausgesetzt dass Autor und Quelle genannt werden.


Gliederung

Text

Background: Multidrug resistance (MDR) is increasingly limiting treatment options in human and veterinary medicine and is often caused by MDR-efflux pumps. To combat MDR, pharmacological inhibition of efflux pumps has become an attractive approach. A variety of phenothiazines and, more recently, selective serotonin reuptake inhibitors (SSRIs) were shown to have antimicrobial activity against several groups of microorganisms [1,2]. It was demonstrated that SSRIs and other psychotropic drugs could potentiate the activity of existing antibiotics presumably due to the inhibition of MDR efflux pumps. We have developed a whole-cell real-time efflux assay using the novel dye 1,2-dinaphthylamine (DINA), which is virtually non-fluorescent in aqueous solutions but becomes highly fluorescent upon binding to membrane lipids.

Methods: Deenergized E. coli cells (OD600 0.25, room temperature, presence of CCCP 5 µM), overexpressing the RND efflux pumps AcrAB-TolC (strain 3AG100) or YhiUV-TolC, were loaded with the dye (5 µM), and efflux was triggered by addition of glucose. Several dyes were examined. Interestingly, a fluorimeter emission scan revealed that DINA is the first described RND efflux pump substrate that fluoresces within the near-infrared range of the wave-length spectrum (810 nm) thus reducing interference with other compounds present in a cell suspension.

Results: Sertraline – the most powerful SSRI tested – was found to inhibit DINA efflux in a dose dependent manner (Figure 1 [Fig. 1]). 90% DINA efflux inhibition was reached at a sertraline concentration of 50 µM in the AcrAB-TolC overexpressing strain 3-AG100, whereas the YhiUV-TolC overexpressing strain DKO 20/1 was found to be about two-fold less sertraline-sensitive. A YhiUV-TolC overexpressing V610F mutant (obtained by selection with levofloxacin) regained sertraline sensitivity comparable to 3-AG100.

Conclusions: Due to the high signal-to-noise ratio and low fluorescence interference in substrate competition assays, the near-infrared dye DINA is well suited for real-time efflux pump inhibition studies in whole cells and could facilitate the high-throughput-screening for novel inhibitors.