Fig. 4.

Felodipine enhance antibacterial efficacy of gentamicin and disrupt the ultrastructure of bacteria. (A) Synergy testing of felodipine and gentamicin against MRSA. OD600nm was measured after 18h incubation at 37 °C and fractional inhibitory concentration index (FICI) value is 0.25. Synergy, FICI ≤0.5; no interaction, 0.5 < FICI ≤4; antagonism, FICI >4. Experiments were independently repeated twice. (B) Synergy testing of felodipine and gentamicin against MRSE and the FICI value is 0.25. (C) After incubated with 1MIC felodipine, 1/8 MIC felodipine (Fd) combined with 1/8 MIC gentamicin (Gen), the Gentamicin-Texas red uptake was detected using flow cytometry. (D) After incubated with 1MIC felodipine (8 μg/mL), 1/8 MIC felodipine (Fd) and 1/8 MIC gentamicin (Gen), the morphological changes of bacteria were observed using SEM. Red arrowheads indicate bacteria with damaged structure. (E) The ultrastructure changes of bacteria were observed using TEM. The experiment was repeated three times. (F) MRSA membrane potential was examined by detecting the change of fluorescent dye, DiOC2(3), using CLSM and flow cytometer. DiOC2(3) exhibits green fluorescence in all bacterial cells, but in healthy cells its fluorescence shift to red. The proton ionophore CCCP served as positive control. (G) Bacterial membrane permeability was assessed by monitoring the uptake of SYTOX Green dye using a spectrophotometer. Data are expressed as the mean ± SD; n = 3; ***p < 0.001. (H) The membrane fluidity of MRSA was evaluated based on Laurdan generalized polarization (Laurdan GP). The benzyl alcohol (50 mM) was used as a positive control. Data are expressed as the mean ± SD; n = 3; ***p < 0.001.