Interaction of Amiodarone with Azoles Against Aspergillus Planktonic Cells and Biofilms in vitro

Aspergillus spp. is the most common clinical pathogen of invasive fungal infection with high mortality. Existing treatments for Aspergillus spp. infection are still inefficient and accompanied by drug resistance, so it is still urgent to find new treatment approaches. The antiarrhythmic drug amiodarone (AMD) has demonstrated antifungal activity against a range of fungi. This study evaluated the efficacy of AMD in combination with triazoles for Aspergillus spp. infection. We tested the combined effect of AMD and three triazole drugs, namely, itraconazole (ITR), voriconazole (VRC), and posaconazole (POS), on the planktonic cells and biofilms of 20 strains of Aspergillus spp. via a checkerboard microdilution assay derived from 96-well plate-based method. Our results reveal that the combination of AMD with ITR or POS against Aspergillus biofilms has synergistic fungicidal effects. By contrast, the combination of AMD with VRC exhibits no antagonistic and synergistic effects. In this way, the use of AMD in combination with ITR or POS could be an effective adjunctive treatment for Aspergillus spp. infection.


Introduction
Aspergillus spp. remain the most common invasive fungal infection pathogens, followed by Candida spp. Although new triazole drugs are widely used in treating aspergillosis, the mortality rate for aspergillosis remains high, ranging from 40 to 90% [1][2][3][4]. This high mortality rate is closely related to the formation of Aspergillus biofilms. Compared with planktonic morphologies, Aspergillus biofilms have increased resistance to antimicrobial agents [5,6] and enhanced pathogenicity against the host [7,8]. The control of Aspergillus spp. infection is mainly based on antifungal therapy, such as amphotericin B, itraconazole (ITR), voriconazole (VRC) and posaconazole (POS), while the optimal antifungal therapy remains uncertain and need to be explored [9].
The antiarrhythmic drug amiodarone (AMD) exhibits a growth inhibition for a broad range of several diverse fungi (Candida, Aspergillus, Cryptococcus, Saccharomyces, and Fusarium) [10,11]. Mechanisms by which AMD is involved in anti-fungal infection include the disruption of Ca 2? homeostasis, the activation of the calcineurin pathway, the accumulation of transcription factor Crz1, etc. [12,13]. The combination of AMD and triazole drugs shows a synergistic inhibitive effect on the planktonic cells of Candida albicans [14,15] and Aspergillus niger [10]. Therefore, it is reasonable and promising to study the killing and inhibitory effects of AMD combined with triazole antifungal drugs ITR, VRC or POS on Aspergillus biofilms. In this study, we tested the inhibitory effect of AMD combined with ITR, VRC or POS on 20 Aspergillus species (11 Aspergillus fumigatus, 5 Aspergillus flavus, 3 Aspergillus terreus, and 1 A. niger). Our study revealed that the use of AMD in combination with ITR or POS could be an effective adjunctive treatment for Aspergillus spp. infection. The ranges of working concentrations of ITR, VRC, POS and AMD were 0.5-256 lg/ml, 0.5-256 lg/ml, 0.5-256 lg/ml, and 0.5-256 lg/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 (100 lL/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. 50 lL of ITR, VRC or POS was inoculated horizontally, while another 50 lL of AMD was inoculated vertically. Results were evaluated after 48 h 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 B 0.5, synergy; 0.5 \ FICI B 4.0, indifferent; FICI [ 4.0, antagonism [19]. Conidia concentrations were determined at the early stage of biofilm formation (10 h) and the biofilm thickness was determined at the late stage (24 h). Biofilm thickness was were scanned layer by layer from top to bottom at 1 lm 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 2 mg XTT in 10 mL of PBS, and then supplemented it with 100 lL of a 10 mM menadione stock solution (0.4 mM in acetone). After incubating the A. fumigatus conidia suspension or biofilms for test, each well of the 96 multi-well plate was filled with 100 lL XTT/menadione solution and incubated at 37°C for 3 h in the dark. Following incubation, the absorbance was measured at a wavelength of 490 nm 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.

Effects of AMD on Morphology and Viability of Aspergillus Biofilm
CLSM was used to record the entire process of biofilm formation, and the vitality gradually increased with incubation time, as shown in Supplement Fig. 1, A. fumigatus biofilm can be effectively formed in vitro. Through XTT test and CLSM observation, it was found that AMD exhibited an inhibitory effect on biofilms at concentrations above 64 lg/ml, and showed an enhanced inhibitory effect with the increase of AMD concentration in a concentrationdependent manner (Fig. 1). The ranges of the MICs of ITR, VRC, POS and AMD for the planktonic cells of Aspergillus spp. were 0.5-4 lg/ml for ITR, 0.03-1 lg/ml for VRC, 0.25-1 lg/ml for POS, and C 256 lg/ml for AMD ( Table 1). The ranges of the SMIC50 and SMIC80 of ITR, VRC, POS and AMD for Aspergillus biofilms were 16 to C 256 lg/ml and 64 to C 256 lg/ml, 8-128 lg/ml and 32-256 lg/ml, 4 to C 256 lg/ml and 32 to C 256 lg/ml, and C 256 and C 256 lg/ml, respectively (Tables s 2, 3 and 4). As described in previous studies [17], the susceptibility of biofilms was higher than that of planktonic cells against the three antifungal drugs (ITR, VRC and POS).

Effects of AMD with ITR, VRC and POS on Morphology and Viability of Aspergillus Biofilm
The biofilms were photographed at 10 and 24 h (Fig. 2a). XTT tests revealed variations in viability during biofilm development (Fig. 2b). ITR, POS and VRC all had inhibitory effects on the adhesion (10 h) and formation (24 h) of biofilms. Combined with AMD, ITR and POS exhibited synergistically enhanced anti-biofilm formation effects. However, the antibacterial effect of VRC in combination with AMD did not change.

Discussion
Courchesne [11] found that AMD has a potent fungicidal activity against A. fumigatus by growth rate test and colony-forming assay. By contrast, the result did not show fungicidal activity alone in our antifungal susceptibility test on planktonic cells or biofilms of Aspergillus spp. Guo [14] found that the MIC range of AMD was 512 lg/ml in the in vitro susceptibility of C. albicans. The different detection methods may result to different results. However, we observed that the combination of AMD with ITR, VRC or POS against planktonic cells and biofilms of Aspergillus spp. had some encouraging results (Tables 1, 2, 3 and 4). In the interaction of AMD with ITR, VRC or POS against planktonic cells of Aspergillus, the combination of AMD with ITR displayed a synergistic inhibitory effect in 11 of 11 A. fumigatus strains, with FICI values ranging from 0.14 to 0.28, and 7 of 9 non-fumigatus Aspergillus, with FICI values ranging from 0.13 to 0.26 (Table 1). The synergistic inhibitory effect could also be found in the combination of AMD with POS in 10 of 11 A. fumigatus strains, with FICI values ranging from 0.09 to 0.32, and 8 of 9 non-fumigatus Aspergillus, with FICI values ranging from 0.04 to 0.28 (Table 1). When AMD was combined with VCR, no interaction effect against Aspergillus spp. was observed with FICI values ranging from 0.56 to 1.12 (Table 1). In the interaction of AMD with ITR, VRC or POS against biofilms of Aspergillus, some attractive results were identified. When ITR was combined with AMD, the SMIC50 ranges of ITR and AMD decreased to 8-64 lg/ml and 32-128 lg/ml, respectively, the SMIC80 ranges decreased to 16-32 lg/ml and 32-128 lg/ml, respectively (Table 2). Based on SMIC50, 5 of 11 A. fumigatus strains, with FICI values ranging from 0.375 to 0.5, and 5 of 9 nonfumigatus Aspergillus, with FICI values ranging from 0.375 to 0.5, displayed a synergistic effect. Based on SMIC80, 8 of 11 A. fumigatus strains, with FICI values ranging from 0.375 to 0.5, and 6 of 9 non-fumigatus Aspergillus, with FICI values ranging from 0.25 to 0.5, presented a synergistic effect. When POS was combined with AMD, the SMIC50 ranges of POS and AMD decreased to 1-64 lg/ml and 8-64 lg/ml, respectively, the SMIC80 ranges decreased to 2-64 lg/ml and 8-64 lg/ml, respectively (Table 3). Based on SMIC50, all strains of Aspergillus spp., with FICI values ranging from 0.16 to 0.5, exhibited a synergistic effect. Based on SMIC80, all strains of A. fumigatus, with FICI values ranging from 0.28 to 0.5, and 7 of 9 non-fumigatus Aspergillus, with FICI values ranging from 0.25 to 0.38, indicated a synergistic effect. When AMD was combined with VCR, no interaction effect against biofilms of Aspergillus spp. was determined, with FICI values ranging from 0.51 to 1.5 based on SMIC50 and FICI values ranging from 0.75 to 1.5 based on SMIC80 (Table 4). No antagonism was observed against Aspergillus planktonic cells or biofilms with these combinations.
We are the first to study the in vitro interaction effect of AMD with triazole drugs against Aspergillus biofilm. Our results revealed that AMD alone did not significantly inhibit the planktonic cells and biofilms of Aspergillus spp. However, when AMD was combined with triazole drugs (such as ITR or POS), most of the isolates exhibited a synergistic effect. This mechanism is not clear enough. AMD has been found Combined with AMD, ITR and POS exhibited synergistically enhanced anti-biofilm formation effects. However, the antibacterial effect of VRC in combination with AMD did not change to have a potent fungicidal activity against a broad range of fungi by targets calcium and pH homeostasis, which lead to slow growth and poor sporulation on fungi [10,15]. Triazoles are fungistatic by inhibiting the production of ergosterol, thereby destroying the integrity of the fungal membrane. Gamarra found that AMD combined with fluconazole can significantly dampen the compensatory response pathways for the production of ergosterol [15]. Therefore, we speculate that the synergy of these two drugs may be the mechanism that the break-up of intracellular calcium and pH homeostasis can lead to downregulation of the synthase of ergosterol, whereas the destruction of the cell membrane further results in the destruction of calcium and pH homeostasis.
Overall, our results supported the role for AMD combined with triazole antifungal agents as a novel combination therapy for Aspergillus biofilm-associated fungal infections. More animal test and clinical experience are needed to verify this effect.