4.4.2. Efflux Pump Inhibitory Assay

The whole-cell phenotypic efflux pump inhibition assay was performed according to previously published protocols with some modification using M. smegmatis, S. aureus and P. aeruginosa [27,28,29,30]. The Gram-positive and Gram-negative cells were cultured in 10 mL nutrient broth while the Mycobacterium was cultured in Middlebrook 7H9 broth (Difco, USA) in Erlenmeyer flasks containing 10% OADC enrichment (Becton Dickinson, Franklin Lakes, NJ, USA) and 0.05% Tween 80. They were cultured at 37 °C with shaking at 150 rpm until an optical density of 600 nm (OD₆₀₀) of 0.8–1 (mid-log phase). The OD₆₀₀ of the cultures was adjusted to 0.4 after which 10 mL was centrifuged at 3000 rpm for 10 min. The supernatant was discarded, and pellets washed with sterile PBS.

The pellets were re-suspended in 10 mL of sterile PBS. The bacteria suspension (500 µL) (Test) and 500 µL of PBS (Blank) were pipetted into 2 mL Eppendorf tubes and glucose added to a final concentration of 0.4%. Different concentrations of the extracts and compounds were added at half their MIC’s. The suspension was vortexed to distribute the cells uniformly. Aliquots of 100 µL from each mixture were transferred into 96-well plates (in triplicates) and 5 µL of EtBr added to give a final concentration of 0.5 mg/L. The EtBr was added just before reading the fluorescence intensity. The fluorescence was measured using the microplate reader (Biotek Synergy H1 Hybrid Multi-Mode Reader: 271230, Vermont, VT, USA) at 530 nm (excitation) and 585 nm (emission) every 10 min over a period of 60 min at 37 °C. This measures the effect of the extracts on the accumulation of EtBr within the cells by inhibiting the efflux pumps. The known efflux pump inhibitors, verapamil and chlorpromazine, served as reference drugs and a drug-free culture was used as control.