Back to Nature: Combating Candida albicans Biofilm, Phospholipase and Hemolysin Using Plant Essential Oils

Abstract

Candida albicans is the causative agent of fatal systemic candidiasis. Due to limitations of antifungals, new drugs are needed. The anti-virulence effect of plant essential oils (EOs) was evaluated against clinical C. albicans isolates including cinnamon, clove, jasmine and rosemary oils. Biofilm, phospholipase and hemolysin were assessed phenotypically. EOs were evaluated for their anti-virulence activity using phenotypic methods as well as scanning electron microscopy (SEM) and atomic force microscopy (AFM). Among the C. albicans isolates, biofilm, phospholipase and hemolysins were detected in 40.4, 86.5 and 78.8% of isolates, respectively. Jasmine oil showed the highest anti-biofilm activity followed by cinnamon, clove and rosemary oils. SEM and AFM analysis showed reduced adherence and roughness in the presence of EOs. For phospholipase, rosemary oil was the most inhibitory, followed by jasmine, cinnamon and clove oils, and for hemolysins, cinnamon had the highest inhibition followed by jasmine, rosemary and clove oils. A molecular docking study revealed major EO constituents as promising inhibitors of the Als3 adhesive protein, with the highest binding for eugenol, followed by 1,8-cineole, 2-phenylthiolane and cinnamaldehyde. In conclusion, EOs have a promising inhibitory impact on Candida biofilm, phospholipase and hemolysin production, hence EOs could be used as potential antifungals that impact virulence factors.

Keywords: AFM; Candida albicans; SEM; biofilm; essential oils; hemolysin; jasmine oil; molecular docking; phospholipase.

Figure 1

Confirmatory PCR for identification of C. albicans isolates. Lane 1: 100 bp ladder, lanes 2, 4 showed negative results, while the rest of lanes had a PCR products of C. albicans isolates with a band of 446 bp.

Figure 2

Assessment of biofilm and adhesion inhibition in clinical C. albicans isolates in absence (control) and presence of sub-MIC of cinnamon, clove, jasmine and rosemary oils, using (A) crystal violet assay of biofilm biomass showing significant reduction in absorbance upon exposure to essential oils (EOs). (B) SEM images of control C. albicans and those exposed to EOs, scale bars are 10 µm in upper panel and 5 µm in lower panel.

Figure 3

Atomic force microscopy 3D-topography images showing changes in cell height (A) and surface roughness (B) of Candida albicans control cells and EO-treated cells. Scanning area size is 5 × 5 µm.

Figure 4

Phospholipase inhibition in C. albicans clinical isolates. (A) Graph showing changes in Pz value upon exposure to different EOs (the smaller the Pz value, the stronger the phospholipase activity and vice versa). (B) Representative images of phospholipase inhibition on egg-yolk agar containing sub-MIC of EOs.

Figure 5

Hemolysin inhibition in C. albicans clinical isolates. (A) Graph showing changes in Hz value upon exposure to sub-MIC of different EOs (the smaller the Hz value the stronger the hemolysin activity and vice versa). (B) Representative images of hemolysin inhibition on Sabouraud dextrose-blood agar containing sub-MIC of EOs.

Figure 6

The putative binding mode (2D and 3D) with the mol-dock scores of Eugenol (clove oil); Cinnamaldehyde 2-Phenylthiolane and rosemary major constituents; 1,8-cineol; α-pinene into the pocket of the C. albicans Als3 surface protein.