Strains
Aspergillus species isolates (11 Aspergillus fumigatus, 5 A. flavus, 3 A. terreus, and 1 A. niger) and Candida parapsilosis were stored at the Center of Chinese Medical Fungal Collection. All strains were maintained at 4 °C on sabouraud dextrose agar (SDA) slopes and grown on an SDA plate at 37 °C for 72 h to ensure purity and viability. C. parapsilosis ATCC 22019 and A. flavus ATCC 204304 were used as quality control strains.
Antifungal drug preparations
ITR (Sigma-Aldrich Co., St. Louis, MO, USA), VRC (Sigma-Aldrich Co., St. Louis, MO, USA), POS (Selleck chemicals, Houston, TX, USA), and AMD (Sigma-Aldrich Co., St. Louis, MO, USA) were obtained as pure powder. Each drug was dissolved in 100% dimethyl sulfoxide at a final concentration of 1280μg/ml. The ranges of working concentrations of ITR, VRC, POS and AMD were 0.03μg/ml to 16μg/ml, 0.03μg/ml to 16μg/ml, 0.03μg/ml to 16μg/ml, and 0.5μg/ml to 256μg/ml in drug susceptibility testing for planktonic cells, respectively. The ranges of working concentrations of ITR, VRC, POS and AMD were 0.5μg/ml to 256μg/ml, 0.5μg/ml to 256μg/ml, 0.5μg/ml to 256μg/ml, and 0.5μg/ml to 256μg/ml in drug susceptibility testing for biofilms, respectively.
The minimal inhibitory concentrations (MICs) tests
MICs for planktonic cells were tested according to the guidelines of the Clinical and Laboratory Standards Institute document M38/A2 and defined as the lowest drug concentrations that caused 80% growth inhibition compared with that of the drug-free growth control [16]. The prepared planktonic cells were seeded into 96-well plate (100μL/well). The working concentration ranges for ITR, VRC and POS are shown above. MICs are defined as the lowest concentration that achieves complete inhibition of growth.
As described previously [17], Aspergillus biofilm in vitro could be formed in 96-well plate, and the sessile MIC50 (SMIC50) and sessile MIC80 (SMIC80) were determined by the optical density values from the XTT reduction assay [18]. SMIC50 and SMIC80 mean the antifungal concentrations at which a 50% or 80% decrease in absorbance are tested in comparison to the biofilms formed by the same fungal isolate in the absence of antifungal drug.
The interaction of AMD with ITR, VRC or POS against planktonic cells and biofilms of all strains of Aspergillus spp. was evaluated with a checkerboard microdilution assay. The working concentration ranges for ITR, VRC, POS and AMD are shown above. 50μl of ITR, VRC or POS was inoculated horizontally, while another 50μl of AMD was inoculated vertically. Results were evaluated after 48 hours of incubation at 35°C. Drug combination interactions were classified according to the Fractional Inhibitory Concentration Index (FICI). Based on these results, FICI values were interpreted as: FICI≤0.5, synergy; 0.5<FICI≤4.0, indifferent; FICI>4.0, antagonism [19].
CLSM analysis of biofilms
CLSM (Olympus FV1000) was used to record the images of the biofilms. The biofilms were stained with FUN-1 (Molecular Probes, Eugene, Oregon), which can bind the intravacuolar structures of the fungal cells, according to the manufacturer’s instructions. FUN-1 solution was added at a concentration of 25μmol/L (200μL) to the surface of biofilms, followed by incubation at 37 ° C for 20min in the dark. Subsequently, the surface of the biofilms was washed with PBS and observed by CLSM. A laser with an excitation wavelength of 488nm was used, and the biofilms observed at a magnification of ×200. Spore concentrations were determined at the early stage of biofilm formation (10h) and the biofilm thickness was determined at the late stage (24h). Biofilm thickness was were scanned layer by layer from top to bottom at 1μm intervals by CLSM. 3D photos of biofilms were built up by 3D Olympus Fluoview software.
XTT reduction assay
The viability of the A. fumigatus was evaluated by XTT reduction assay. The XTT/menadione reagent (Sigma-Aldrich, USA) was freshly prepared for each experiment. The XTT/menadione solution was prepared by dissolving 2mg XTT in 10mL of PBS, and then supplemented it with 100μL of a 10mM menadione stock solution (0.4mM in acetone). After incubating the A. fumigatus spore suspension or biofilms for test, each well of the 96 multi-well plate was filled with 100μL XTT/menadione solution and incubated at 37°C for 3 hours in the dark. Following incubation, the absorbance was measured at a wavelength of 490nm using a microplate reader.
Statistics
All assays were performed on at least three independent occasions. GraphPad Prism 7 was used for statistical analyses and graphs. Significance was defined as P < 0.05.