Novel DiOC3 96-well real-time efflux assay for discovery of NorA efflux pump inhibitors in Staphylococcus aureus

Najoua Elhidar; Ahmed Nafis; André Goehler; Abdelaziz Abbad; Lahcen Hassani; Nour-Eddine Mezrioui; Jürgen A Bohnert

doi:10.1016/j.mimet.2020.106128

Novel DiOC₃ 96-well real-time efflux assay for discovery of NorA efflux pump inhibitors in Staphylococcus aureus

J Microbiol Methods. 2021 Feb:181:106128. doi: 10.1016/j.mimet.2020.106128. Epub 2020 Dec 18.

Authors

Najoua Elhidar¹, Ahmed Nafis², André Goehler³, Abdelaziz Abbad², Lahcen Hassani², Nour-Eddine Mezrioui⁴, Jürgen A Bohnert³

Affiliations

¹ Friedrich Loeffler Institute of Medical Microbiology, University of Medicine Greifswald, Germany; Laboratory of Microbial Biotechnologies, Agrosciences and Environment, Faculty of Sciences, Semlalia, Cadi Ayyad University, Marrakech, Morocco.
² Laboratory of Microbial Biotechnologies, Agrosciences and Environment, Faculty of Sciences, Semlalia, Cadi Ayyad University, Marrakech, Morocco.
³ Friedrich Loeffler Institute of Medical Microbiology, University of Medicine Greifswald, Germany.
⁴ Laboratory of Microbial Biotechnologies, Agrosciences and Environment, Faculty of Sciences, Semlalia, Cadi Ayyad University, Marrakech, Morocco. Electronic address: mezrioui@uca.ac.ma.

PMID: 33347918
DOI: 10.1016/j.mimet.2020.106128

Abstract

The NorA efflux pump is one of the most studied efflux systems in Staphylococcus aureus and confers multidrug resistance to a variety of dyes and antimicrobial compounds. Hence, inhibition of the NorA efflux pump might be a viable option for restoring susceptibility to antibiotics like fluoroquinolones. Fluorescent real-time efflux assays are important tools to identify putative efflux pump inhibitors. Nevertheless, the number of available compounds for usage in Staphylococcus aureus is limited. Previously, a 3-dipropyloxacarbocyanine iodide (DiOC₃) efflux assay was published that circumvented problems associated with the usage of ethidium bromide, namely slow efflux and suggested mutagenicity. However, the DiOC₃ assay protocol was cuvette - based and therefore needs to be adapted to the 96-well plate format. Hence, we optimized this assay for usage with 96-well plates. The new assay allows for rapid high-throughput efflux pump inhibitor screening.

Keywords: 96-well plate; DiOC(3); Efflux assay; Multidrug resistance; NorA efflux pump; Staphylococcus aureus.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Anti-Bacterial Agents / chemistry*
Bacterial Proteins / antagonists & inhibitors*
Drug Discovery / methods*
Drug Evaluation, Preclinical / methods*
Multidrug Resistance-Associated Proteins / antagonists & inhibitors*
Staphylococcal Infections* / drug therapy
Staphylococcal Infections* / microbiology
Staphylococcus aureus / metabolism*

Substances

Anti-Bacterial Agents
Bacterial Proteins
Multidrug Resistance-Associated Proteins
NorA protein, Staphylococcus