Effect of Escin Alone or in Combination with Antifungal Agents on Resistant Candida glabrata Biofilms: Mechanisms of Action

Abstract

Nowadays, the increase in antimicrobial-resistant fungi (AMR) is certainly a major health concern, and the development of alternative therapeutic strategies has become crucial. Natural products have been used to treat various infections, and their chemical properties contribute to the performance of their biological activities, such as antifungal action. The various virulence factors and mechanisms of resistance to antifungals contribute to making Candida glabrata one of the most frequent agents of candidiasis. Here we investigate the in vitro and in vivo activity of β-escin against Candida glabrata. The β-escin MICs were determined for a reference strain and two clinical isolates of C. glabrata. Furthermore, growth kinetics assays and biofilm inhibition/eradication assays (crystal violet) were performed. The differences in the expression of some anti-biofilm-associated genes were analyzed during biofilm inhibition treatment so that reactive oxygen species could be detected. The efficacy of β-escin was evaluated in combination with fluconazole, ketoconazole, and itraconazole. In addition, a Galleria mellonella infection model was used for in vivo treatment assays. Results have shown that β-escin had no toxicity in vitro or in vivo and was able to inhibit or destroy biofilm formation by downregulating some important genes, inducing ROS activity and affecting the membrane integrity of C. glabrata cells. Furthermore, our study suggests that the combination with azoles can have synergistic effects against C. glabrata biofilm. In summary, the discovery of new antifungal drugs against these resistant fungi is crucial and could potentially lead to the development of future treatment strategies.

Keywords: Candida glabrata; antifungal therapy; azole resistance; biofilm; β-escin.

Figure 1

Determination of cytotoxic activity of β-escin shown as the percent cell viability of the HaCaT cell line treated with different concentrations for 24 h. The assays were performed in three independent experiments. Standard deviations are always less than 10%.

Figure 2

Growth curves of C. glabrata DSM 1226, C18, and C27 strains (A,C,E) treated with β-escin at concentrations 1/2 × MIC and 1 × MIC and 2 × MIC compared to non-treated cells. Time to kill curves (B,D,F) of the same strains treated with β-escin at the same concentrations and compared to non-treated. Data are reported as mean of three independent experiments ± SD.

Figure 3

The effect of β-escin on the biofilms of C. glabrata DSM11226 and C. glabrata clinical isolates C18 and C27. Inhibition (panel (A)) and eradication (panel (B)) quantified with crystal violet after 24 h of treatment with different doses of β-escin.

Figure 4

Expression analysis of selected genes (ALS3, CDR1, ERG11, FKS1, and HOG1) of C. glabrata DSM 1226, C18, and C27 strains using real-time qPCR in response to β-escin. Histograms represent the fold differences in the expression levels of the genes selected during inhibition of biofilm with β-escin at concentration of 5 µg mL^-1. Red lines show fold change thresholds of -1 and +1, respectively. * p < 0.05 indicate fold changes significantly different from untreated samples (t-test).

Figure 5

Intracellular ROS overproduction detected using DCFH-DA (panel (A)) and mitochondrial ROS detected using MitoSOX Red (panel (B)) during inhibition of C. glabrata biofilms of DSM 1226, C18, and C27 strains by 5 mg mL⁻¹ β-escin. * p < 0.05, **** p < 0.0001 (Tukey’s test).

Figure 6

FACS analysis revealed an increase in cell membrane permeability induced by β-escin. C. glabrata cells were treated with β-escin at the concentrations of 5 µg mL^-1, 10 µg mL^-1, and 20 µg mL^-1 for 18 h. Fungal cells without β-escin were used as negative control. Dot plot images (A) were analyzed by FlowJo 8.7 software. The histograms (B) represent the % of cell positive to PI staining compared to control.

Figure 7

Synergistic effect of β-escin/azole combination against the biofilms of C. glabrata DSM 1226, C18, and C27 strains. Heat-maps show the synergistic effect of Esc/FLC (panel (A)), Esc/KET (panel (B)), Esc/ITZ (panel (C)); * indicate the synergistic effect (FICI ≤ 0.5). The corresponding percentage of biofilm inhibition for Esc/FLC (panel (D)), Esc/KET (panel (E)), and Esc/ITZ (panel (F)) are shown. Data are means of three independent experiments. *** p < 0.001, **** p < 0.0001 (Tukey’s test).

Figure 8

In vivo experiments in Galleria mellonella. Toxicity of β-escin and azole drugs alone or in combination (panel (A)). Determination of a suitable concentration of C18 per larva (panel (B)). Prevention/treatment of larvae inoculated with the three combinations Esc/FLC, Esc/KET, and Esc/ITZ (panel (C)). Control larvae were inoculated with 10 μL of PBS or intact. (N = 20 for each experiment and condition).