Increased Absorption and Inhibitory Activity against Candida spp. of Imidazole Derivatives in Synergistic Association with a Surface Active Agent

Abstract

This paper's purpose was to evaluate the interaction between three imidazole derivatives, (2-methyl-1H-imidazol-1-yl)methanol (SAM3), 1,1'-methanediylbis(1H-benzimidazole (AM5) and (1H-benzo[d]imidazol-1-yl)methanol 1-hydroxymethylbenzimidazole (SAM5) on the one hand, and sodium dodecyl sulphate (SDS) on the other, as antifungal combinations against Candida spp. Inhibitory activity was assessed using the agar diffusion method and Minimal Inhibitory Concentration (MIC) and showed moderate inhibitory activity of single imidazole derivatives against Candida spp. The mean value of MIC ranged from 200 µg/mL (SAM3) to 312.5 µg/mL (SAM3), while for SDS the MIC was around 1000 µg/mL. When used in combination with SDS, the imidazole derivatives demonstrated an improvement in their antifungal activity. Their MIC decreased over five times for AM5 and over seven times for SAM3 and SAM5, respectively, and ranged from 26.56 µg/mL (SAM3) to 53.90 µg/mL (AM5). Most combinations displayed an additive effect while a clear synergistic effect was recorded in only a few cases. Thus, the FIC Index (FICI) with values between 0.311 and 0.375 showed a synergistic effect against Candida spp. when SDS was associated with SAM3 (three strains), SAM5 (two strains) and AM5 (one strain). The association of imidazole derivatives with SDS led to the increased release of cellular material as well as the intracellular influx of crystal violet (CV), which indicated an alteration of the membrane permeability of Candida spp. cells. This favored the synergistic effect via increasing the intracellular influx of imidazoles.

Keywords: Candida spp.; SDS; imidazole derivatives; synergistic association.

Figure 1

Time-kill of Candida albicans ATCC 10231 during exposure to imidazole derivatives and SDS (SAM3 = 500 µg/mL; AM5 = 500 µg/mL; SAM5 = 500 µg/mL; SDS = 2000 µg/mL).

Figure 2

Time-kill of Candida albicans ATCC 10231 during exposure to imidazole derivatives and SDS (SAM5 = 500 µg/mL; SAM5 + SDS = 62.5 + 1250 µg/mL; SDS = 1500 µg/mL).

Figure 3

Time-kill of Candida albicans ATCC 10231 during exposure to imidazole derivatives and SDS (SAM3 = 500 µg/mL; SAM3 + SDS = 31.25 + 625 µg/mL; SDS = 1500 µg/mL).

Figure 4

Time-kill of Candida albicans ATCC 10231 during exposure to imidazole derivatives and SDS (AM5 = 500 µg/mL; AM5 + SDS = 62.5 + 1250 µg/mL; SDS = 1500 µg/mL).

Figure 5

Leakage of cellular material in C. albicans cells detected as absorbance at 260 nm. A—negative control (cultures without compounds); B—positive control (SDS 2%); C—SAM3 1 MIC + SDS; D—SAM3 2 MIC + SDS; E—SAM3 4 MIC + SDS; F—SAM3 1 MIC; G—SAM3 2 MIC; H—SAM3 4 MIC.

Figure 6

Intracellular uptake of CV in C. albicans cells after exposure to compounds and their combinations with SDS. A—control (cells without compounds); B–D—CV uptake by Candida spp. cells in the presence of compounds alone (B—SDS 1000 μg/mL; C—SAM3 1 MIC; D—SAM5 1 MIC); E–G—CV uptake by Candida spp. cells after exposure to SAM3/SDS combination at increasing concentrations of SAM3 (E—SAM3 1 MIC + SDS; F—SAM3 2 MIC + SDS; G—SAM3 4 MIC + SDS); H–J—CV uptake by Candida spp. cells after exposure to SAM5/SDS combination at increasing SAM5 concentrations (H—SAM5 1 MIC + SDS; I—SAM5 2 MIC + SDS; J—SAM5 4 MIC + SDS).

Figure 7

The putative mechanism of imidazoles/SDS synergistic interaction. (a) Imidazole molecules enter the cells, but most are removed with functional efflux pumps. (b) SDS induces a conformational change in the efflux pumps and inactivates them; therefore, azoles are trapped intracellularly and achieve a lethal concentration. (c) SDS can also induce, as a secondary mechanism, a minimal local destabilization of the membrane phospholipid layer, which leads to an additional increase in imidazole influx.