3,5-Dicaffeoylquinic Acid Disperses Aspergillus Fumigatus Biofilm and Enhances Fungicidal Efficacy of Voriconazole and Amphotericin B

Abstract

BACKGROUND The aim of this study was to evaluate the dispersal effects of 3,5-dicaffeoylquinic acid (3,5-DCQA) against the preformed biofilm of Aspergillus fumigatus and to investigate its potential mechanism. MATERIAL AND METHODS Aspergillus fumigatus biofilms of laboratory strain AF293 and clinical strain GXMU04 were generated in 24- or 96-well polystyrene microtiter plates in vitro. Crystal violet assay and XTT reduction assay were performed to evaluate the effects of 3,5-DCQA on biofilm biomass, extracellular matrix, and metabolic activity alteration of cells in biofilms. Real-time PCR was performed to quantify the expression of hydrophobin genes. The cytotoxicity of 3,5-DCQA on human erythrocytes was evaluated by a hemolytic assay. RESULTS The results indicated that 3,5-DCQA in subminimum inhibitory concentrations (256 to 1024 mg/L) elicited optimal A. fumigatus biofilm dispersion activity and improved the efficacy of VRC and AMB in minimal fungicidal concentrations (MFCs) to combat fungal cells embedded in biofilms. The results of scanning electron microscope (SEM) and confocal laser scanning microscopy (CLSM) revealed 3,5-DCQA facilitated the entry of antifungal agents into the A. fumigatus biofilm through eliminating the hydrophobic extracellular matrix (ECM) without affecting fungal growth. Real-time PCR indicated that 3,5-DCQA down-regulated the expression of hydrophobin genes. Hemolytic assay confirmed that 3,5-DCQA exhibited a low cytotoxicity against human erythrocytes. CONCLUSIONS Subminimum inhibitory concentrations of 3,5-DCQA can disperse A. fumigatus biofilm and enhance fungicidal efficacy of VRC and AMB through down-regulating expression of the hydrophobin genes. The study indicated the anti-biofilm potential of 3,5-DCQA for the management of A. fumigatus biofilm-associated infection.

Figure 1

Chemical structure of 3,5-DCQA.

Figure 2

Biomass alterations of A. fumigatus GXMU04 (A) and A. fumigatus AF293 (B) using crystal violet assay. Error bars represent standard error. * P<0.05 vs. drug-free control group; ^# P<0.05 vs. the VRC group; ^@ P<0.05 vs. the AMB group. VRC – MFC of Voriconazole; AMB – MFC of amphotericin B.

Figure 3

Metabolic activity alteration of cells in A. fumigatus GXMU04 (A) and A. fumigatus AF293 (B) of preformed biofilms after exposing to the tested agents evaluated by XTT reduction assay. Error bars represent standard error. * P<0.05 vs. drug-free control group; ^# P<0.05 vs. the VRC group; ^& P<0.05 vs. the AMB group; ^@ P<0.05 vs. the AMB group. VRC – MFC of Voriconazole; AMB – MFC of amphotericin B.

Figure 4

SEM (A) images and CLSM (B) view of preformed A. fumigatus GXMU04 biofilms stained with the combination of PI and Syto-9 in control group, MFC of VRC or AMB, 3,5-DCQA (1024 mg/L), and 3,5-DCQA in combination with VRC or AMB.

Fiugre 5

(A) CLSM Images of preformed A. fumigatus GXMU04 biofilm stained with the combination of ECA and ConA in control group, VRC or AMB alone of MFC, 3,5-DCQA (1024 mg/L), and 3,5-DCQA in combination with VRC or AMB. Scale bars indicates 100 μm. Three-dimensional reconstructions were shown in the bottom views. (B) Fluorescence intensity of CLSM images in each group were also quantified by Image Pro Plus software.

Figure 6

(A–F) Expression of hydrophobin genes (RODA-F) in the biofilms of A. fumigatus GXMU04 and A. fumigatus AF293 after exposing to MFC of AMB or VRC, and 3,5-DCQA at 1024 mg/L. The 18srRNA gene served as the reference gene. * P<0.05 vs. the drug-free control group.