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

Highlights

• Fluorescent efflux assays are important tools to identify efflux pump inhibitors.

• DiOC₃ acts as a fluorescent substrate in the S. aureus NorA efflux pump.

• The DiOC₃ circumvent problems associated with the usage of ethidum bromide.

• DiOC₃ cuvette assay doesn't allow rapid screening for efflux pumps inhibitors (EPI).

• The novel 96-well plate DiOC₃ method is suitable for high-throughput EPI screening.

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.

Introduction

In the 20th century, the introduction of antibiotics has significantly contributed to the control of infectious diseases that were once the leading causes of human mortality and morbidity worldwide (Walsh and Wright, 2005). However, the emergence of multidrug resistant (MDR) bacteria has contributed to the decline of the antibacterial effect of these compounds, leading to worse treatment options in the health-care setting (Costa et al., 2013). Nowadays, MDR has become a major concern in both hospitals and the community. Among these resistant bacteria, Staphylococcus aureus (S. aureus) constitutes one of the most common human pathogens responsible for a wide variety of infections – some of them are life-threatening by presenting a remarkable diversity of resistance mechanisms towards antibiotic agents, notably production of beta-lactamases and modification of penicillin-binding proteins. Moreover, the overproduction of efflux pumps, most of which belong to the major facilitator superfamily (MFS) is an important cause of resistance to antibiotics, disinfectants and antiseptics in S. aureus (Chambers and Deleo, 2009).The MFS transporter NorA is produced in roughly half of the clinical isolates and remains the most studied one (Buonerba et al., 2017). This pump confers MDR to a broad spectrum of compounds including fluoroquinolones, quaternary ammonium compounds, reserpine, verapamil, and dyes like ethidium bromide, rhodamine and acridines (Kaatz et al., 1993; Neyfakh et al., 1993; Yoshida et al., 1990).

Since overproduction of the NorA efflux pump in S. aureus contributes to fluoroquinolone resistance in the clinical setting, restoration of antibiotic susceptibility by usage of efflux pumps inhibitors (EPIs) seems to be an attractive route. To test the potency and specificity of putative EPIs, real-time efflux assays are typically used in which extrusion of a fluorescent dye is triggered by addition of an energy source like glucose to whole cells.

One of the classic compounds used in these assays is ethidium bromide (EtBr) in S. aureus NorA overproducers (Pal et al., 2020; Tintino et al., 2020). However, its use comes with some drawbacks. In fact, EtBr is a DNA intercalator and concerns about its mutagenicity have been raised (Andre et al., 1977; Kuypers et al., 2013). For this, EtBr must be handled with extreme caution, significant amount of time and requires expensive techniques for managing its wastes (Singh and Singh, 2018). Furthermore, in S. aureus its efflux is very slow and might take up to 1 h (de Sousa et al., 2020; Sun et al., 2016). To circumvent these drawbacks, Zimmermann and collaborators optimized an efflux assays using 3-dipropyloxacarbocyanine iodide (DiOC₃) as S. aureus NorA substrate (Zimmermann et al., 2017) which is considered as nontoxic (for humans or the environment) according to EU-Regulation (EC) No. 1272/2008 [CLP]. Despite its advantages, the DiOC₃ assay was optimized for usage with glass cuvettes and we discovered in preliminary experiments that its usage with 96-well plates did not lead to comparable results. In fact, when we tested the cuvette-based protocol previously published by Zimmermann et al. (2017) in 96 well plates (Following the same reagents and protocols) we did not get any important efflux of the DiOC₃ in SA119B, this is probably due to the usage of small volumes of the cultures and reagents in 96 well plates which gave low limit of detection in 96 well plates in comparison with cuvettes and shortens the way the light passes through the samples and since high-throughput screening for new S. aureus EPIs is not practical with a cuvette-based assay, we set out to optimize the previously described DiOC₃ assay for usage of 96-well plates.