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

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

Journal of microbiological methods (2020-12-22)
Najoua Elhidar, Ahmed Nafis, André Goehler, Abdelaziz Abbad, Lahcen Hassani, Nour-Eddine Mezrioui, Jürgen A Bohnert
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Carbonyl cyanide 3-chlorophenylhydrazone, ≥97% (TLC), powder
Corning®  96 Well Black Polystyrene Microplate, flat bottom clear, black polystyrene plate (Ideal for fluorescent assays), Tissue Culture (TC)-treated surface, pkg of (individually wrapped), sterile, lid