2.5 Efflux pump inhibition assay

The test was performed as previously described by Tintino et al., 2016. Stock solutions at a concentration of 1024 μg/ml were prepared by dissolving ciprofloxacin and norfloxacin (Sigma-Aldrich) in deionized water with 0.1M hydrochloric acid solution (5%, v/v); cyanide m-chlorophenyl hydrazone (CCCP) in dilute 96% ethanol (1:1, v/v); and ethidium bromide (EtBr) in deionized water. Reserpine and kuwanon C were dissolved in DMSO. Chlorpromazine (CPZ) 1 mg/ml solution and verapamil 1 mg/ml solution were diluted to 512 μg/ml. All efflux pump inhibitors (EPIs) were purchased from Sigma-Aldrich. Briefly, the MIC was determined as previously described. Efflux pump inhibition of MRSA 7109 was investigated using a subinhibitory concentration (1/8 of the MIC) of standard EPIs and kuwanon C. The bacterial suspension in MHB was adjusted to 0.5 McFarland and diluted to 10⁷ CFU/ml 150 µL of the bacterial suspension, 1/8 of the MIC of EPI and kuwanon C were made up to 1500 µL in Eppendorf tubes with MHB, mixed well, and transferred vertically into a 96-well microtiter plate. A bacterial suspension in sterile MHB (1:10, v/v) served as a control. 100 µL of antibiotic or EtBr was added to the first column (1:1, v/v) and serially diluted so that the concentration of antibiotic and EtBr was between 512 and 0.5 μg/ml. After 18 ± 2 h of incubation at 35°C, the MIC of EtBr or the antibiotic in combination was determined after the addition of 10 µL of 1% TTC solution.