Abstract
Aspergillus biofilms were prepared from 22 strains of Aspergillus spp. via a 96-well plate-based method. Using a broth microdilution checkerboard technique with the XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] colorimetric assay, we demonstrated a synergistic antifungal activity against 18 of 22 Aspergillus biofilm strains with a combination of caspofungin and amphotericin B and against 13 of 22 strains with a combination of caspofungin and voriconazole. We did not observe antagonism.
TEXT
Aspergilloma is increasing in incidence (2, 9). The infection is most often seen in tuberculosis cavities or in the maxillary sinus. Histopathological examination reveals that aspergilloma is an organized structure of fungal filaments, which is enveloped by an extracellular matrix without any infiltration of host cells (9). This is similar to the structure of a biofilm (4, 16, 17), and thus, it is believed to be a type of biofilm (9). In vitro studies (5, 11, 12) showed that Aspergillus spp. can form biofilms, which exhibited a significantly decreased susceptibility to antifungal drugs compared to the planktonic cells of Aspergillus spp. Therefore, we asked whether the combination of caspofungin (CAS) with amphotericin B (AMB) or voriconazole (VRC), which are also known to have synergistic inhibitory activity against planktonic cells of Aspergillus spp. (1, 6, 7, 10), could have a synergistic inhibitory activity against Aspergillus biofilms.
(This work was presented in part at the 18th Congress of the International Society for Human and Animal Mycology, Berlin, Germany, 11 to 15 June 2012 [8a].)
In the present study, Aspergillus biofilms were prepared from 22 strains of Aspergillus spp., including 11 strains of Aspergillus fumigatus, 5 strains of Aspergillus flavus, 3 strains of Aspergillus terreus, and 3 strains of Aspergillus niger, by using a 96-well plate-based method (14). All strains were stored at the Research Center for Medical Mycology of Beijing University and were subcultured on Sabouraud dextrose agar at 35°C for 3 days to ensure purity and viability. Using a broth microdilution checkerboard assay and XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] colorimetric measurement (14, 18), we tested the interaction of CAS with VRC or AMB against the Aspergillus biofilms of these 22 strains. All antifungal drugs were provided as standard powders. VRC (Shouguang Pharmaceutical, Shan Dong, China) and AMB (Sigma Chemical Co., St. Louis, MO) were dissolved in 100% dimethyl sulfoxide (DMSO), while CAS (Merck & Co., Inc., Rahway, NJ) was dissolved in sterile water. The ranges of working concentrations of CAS, VRC, and AMB were 2 to 256 μg/ml, 0.5 to 256 μg/ml, and 0.06 to 32 μg/ml, respectively. XTT powder (Sigma Chemical Co., St. Louis, MO) was prepared as a saturated solution at 500 μg/ml in phosphate-buffered saline (PBS), and menadione (vitamin K3, Sigma Chemical Co., St. Louis, MO) was dissolved in 100% acetone at a concentration of 10 mmol/liter. Both solutions were stored at −70°C. When used, 1 μl of the stock solution of menadione was added to 10 ml XTT solution to achieve a final menadione concentration of 1 μmol/liter. As described previously (11, 14), the sessile MIC50 (SMIC50) was assayed to evaluate the antifungal activity of the drugs against Aspergillus biofilms. The interaction of CAS with AMB or VRC was determined and was classified on the basis of the fractional inhibitory concentration index (FICI), defined as follows (13, 18): FICI ≤ 0.5, synergy; 0.5 < FICI ≤ 4, no interaction; FICI > 4, antagonism. At the same time, the interaction of CAS with AMB or VRC against planktonic cells of all strains of Aspergillus spp. was also evaluated as described previously (1, 3). The minimum effective concentration (MEC) of CAS, when combined with VRC and AMB, and the corresponding drug concentration of VRC and AMB were also recorded. We observed that the combination of CAS with AMB had synergistic inhibitory activity against 8 of 11 A. fumigatus biofilm strains with FICI values of 0.19 to 0.5, and 10 of 11 non-fumigatus Aspergillus biofilm strains, with FICI values of 0.13 to 0.38 (Table 1). As for the combination of CAS and VRC, synergistic inhibitory activity was also observed in 7 of 11 A. fumigatus biofilms, with FICI values of 0.188 to 0.5, and in 6 of 11 non-fumigatus Aspergillus biofilms, with FICI values of 0.27 to 0.5 (Table 1). No antagonistic effects were observed. In addition, the percentage of metabolic activity in A. fumigatus AF293 biofilm treated with a combination of CAS and AMB or VRC was calculated (XTT assay). We demonstrate that the combination of CAS and AMB or VRC significantly reduced the metabolic activity of A. fumigatus AF293 biofilm in comparison to CAS, AMB, or VRC alone at the same concentrations (Fig. 1). However, no interaction was observed in these combinations of antifungal drugs against planktonic cells (Table 1).
Table 1.
Interaction of CAS with AMB or VRC against Aspergillus biofilms and planktonic cells of 22 strains of Aspergillus spp.
| Strain | SMIC50 (μg/ml)a |
FICI | SMIC50 (μg/ml) |
FICI | MIC (μg/ml)b |
FICI | MIC (μg/ml) |
FICI | MEC (μg/ml)c |
||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CAS | VRC | CAS/VRC | AMB | CAS/AMB | CAS | VRC | CAS/VRC | AMB | CAS/AMB | CAS | CAS/VRCd | CAS/AMBd | |||||
| A. fumigatus | |||||||||||||||||
| AF293 | 32 | 32 | 8/4 | 0.375 | 4 | 4/0.25 | 0.188 | ≥16 | 0.25 | 0.03/0.25 | 1.001 | 0.25 | 0.03/0.25 | 1.001 | 0.125 | 0.03/0.125 | 0.06/0.06 |
| AFBMU01340 | 64 | 8 | 4/1 | 0.188 | 8 | 16/2 | 0.5 | ≥16 | 0.25 | 0.03/0.25 | 1.001 | 0.25 | 0.03/0.25 | 1.001 | 0.125 | 0.03/0.06 | 0.03/0.125 |
| AFBMU04718 | 32 | 8 | 4/1 | 0.25 | 2 | 4/0.25 | 0.25 | ≥16 | 0.25 | 0.03/0.25 | 1.001 | 0.5 | 2/0.25 | 0.563 | 0.125 | 0.03/0.06 | 0.06/0.25 |
| AFBMU04728 | 32 | 4 | 4/0.25 | 0.188 | 4 | 4/1 | 0.375 | ≥16 | 0.25 | 0.03/0.25 | 1.001 | 1 | 0.03/1 | 1.001 | 0.125 | 0.03/0.06 | 0.03/0.25 |
| AFBMU04736 | 64 | 4 | 32/2 | 1 | 8 | 4/0.25 | 0.094 | ≥16 | 0.25 | 0.03/0.25 | 1.001 | 1 | 0.03/1 | 1.001 | 0.125 | 0.03/0.125 | 0.06/0.25 |
| AFBMU04737 | 64 | 8 | 64/4 | 1.5 | 2 | 32/1 | 1 | ≥16 | 0.25 | 8/0.125 | 0.75 | 1 | 0.125/0.5 | 0.504 | 0.125 | ≤0.03/0.125 | 0.03/0.25 |
| AFBMU04748 | 32 | 8 | 4/2 | 0.375 | 4 | 4/1 | 0.375 | ≥16 | 0.5 | 16/0.25 | 1 | 0.5 | 0.03/0.5 | 1.001 | 0.125 | 0.03/0.125 | 0.03/0.25 |
| AFBMU01200 | 64 | 8 | 64/2 | 1.25 | 2 | 8/0.5 | 0.375 | ≥16 | 0.25 | 16/0.25 | 1.5 | 0.5 | 0.03/0.5 | 1.001 | 0.125 | 0.03/0.06 | 0.06/0.125 |
| AFBMU02731 | 64 | 8 | 16/2 | 0.5 | 2 | 16/0.25 | 0.375 | ≥16 | 0.25 | 0.03/0.25 | 1.001 | 0.25 | 16/0.125 | 1 | 0.125 | 0.125/0.125 | 0.06/0.125 |
| AFBMU02810 | 64 | 8 | 16/4 | 0.75 | 4 | 4/4 | 1.063 | ≥16 | 0.25 | 0.03/0.25 | 1.001 | 0.25 | 16/0.125 | 1 | 0.06 | 0.03/0.125 | 0.03/0.125 |
| AFBMU04787 | 32 | 8 | 2/2 | 0.313 | 2 | 32/0.25 | 1.13 | ≥16 | 4 | 16/2 | 1 | 0.5 | 16/0.125 | 0.75 | 0.125 | 0.03/1 | 0.03/0.25 |
| A. flavus | |||||||||||||||||
| AFLBMU03932 | 256 | 8 | 4/2 | 0.266 | 4 | 8/0.5 | 0.156 | ≥16 | 0.25 | 0.03/0.25 | 1.001 | 1 | 0.03/1 | 1.001 | 0.125 | 0.03/0.125 | 0.06/0.125 |
| AFLBMU03035 | 64 | 2 | 4/0.5 | 0.266 | 2 | 2/0.25 | 0.156 | ≥16 | 0.25 | 0.03/0.25 | 1.001 | 0.5 | 0.03/0.5 | 1.001 | 0.125 | 0.06/0.06 | 0.03/0.125 |
| AFLBMU00969 | >256 | >256 | 128/128 | 0.5 | >32 | 4/4 | 0.07 | ≥16 | 0.25 | 0.03/0.25 | 1.001 | 0.5 | 0.03/0.5 | 1.001 | 0.125 | 0.03/0.125 | 0.03/0.25 |
| AFLBMU03142 | 64 | 256 | 64/256 | 2 | 16 | 16/1 | 0.313 | ≥16 | 0.25 | 0.03/0.25 | 1.001 | 0.5 | 0.03/0.5 | 1.001 | 0.125 | 0.06/0.125 | 0.03/0.25 |
| AFLBMU29791 | 64 | 64 | 16/16 | 0.5 | >32 | 2/4 | 0.094 | ≥16 | 4 | 0.03/4 | 1.001 | 0.5 | 0.03/0.5 | 1.001 | 0.125 | 0.03/0.5 | 0.03/0.25 |
| A. terreus | |||||||||||||||||
| ATBMU00802 | 64 | 256 | 32/16 | 0.563 | >32 | 8/1 | 0.141 | ≥16 | 0.25 | 0.03/0.25 | 1.001 | 1 | 0.03/1 | 1.001 | 0.125 | 0.03/0.125 | 0.125/0.06 |
| ATBMU01017 | 32 | 64 | 32/8 | 1.125 | 8 | 4/1 | 0.25 | ≥16 | 0.125 | 0.03/0.125 | 1.001 | 1 | 0.03/1 | 1.001 | 0.06 | 0.06/0.06 | 0.03/0.25 |
| ATBMU04033 | 256 | 128 | 256/0.5 | 1.004 | 32 | 128/32 | 1.5 | ≥16 | 0.5 | 0.03/0.5 | 1.001 | 1 | 0.03/1 | 1.001 | 0.125 | 0.03/0.125 | 0.03/0.5 |
| A. niger | |||||||||||||||||
| ANBMU04689 | 64 | 256 | 4/128 | 0.563 | 2 | 4/0.5 | 0.313 | ≥16 | 0.125 | 0.03/0.125 | 1.001 | 0.5 | 0.03/0.5 | 1.001 | 0.125 | 0.03/0.25 | 0.125/0.06 |
| ANBMU04778 | 64 | 32 | 16/8 | 0.5 | 2 | 8/0.25 | 0.25 | ≥16 | 0.25 | 0.03/0.25 | 1.001 | 0.5 | 0.03/0.25 | 0.501 | 0.125 | ≤0.03/0.25 | 0.03/0.125 |
| ANBMU04646 | 64 | 32 | 8/8 | 0.375 | 4 | 4/1 | 0.313 | ≥16 | 0.25 | 0.03/0.25 | 1.001 | 0.25 | 0.03/0.125 | 0.501 | 0.125 | 0.03/0.25 | ≤0.03/0.125 |
SMIC50, the sessile MIC, the concentration at which a 50% decrease in absorbance was detected in comparison to the absorbance of an untreated biofilm formed by the same fungal strains.
MIC at which no fungal growth was observed with the naked eye.
MEC, the minimum effective concentration at which the stubby, aberrant fungal filaments was observed, compared to the untreated organism.
The MEC of CAS and the corresponding concentration of VRC or AMB at which stubby, aberrant growth of fungal filaments was observed when CAS was combined with VRC or AMB. No FICI could be calculated because the MIC, instead of the MEC, is used to describe the antifungal activity of VRC or AMB alone against Aspergillus spp.
Fig 1.
Inhibitory activity (XTT assays) of CAS with AMB or VRC against A. fumigatus AF293 biofilm. The metabolic activity of A. fumigatus AF293 biofilm was 97% when treated with CAS1 (4 μg/ml) or AMB (0.25 μg/ml) alone, but it was 49% when treated with CAS1 (4 μg/ml) and AMB (0.25 μg/ml). The metabolic activities of A. fumigatus AF293 biofilm were 76% and 68% when treated with CAS2 (8 μg/ml) or VRC (4 μg/ml) alone, but the activity was 48% when treated with a combination of CAS2 (8 μg/ml) and VRC (4 μg/ml).
CAS at 4 μg/ml (CAS1) was synergistic with AMB (0.25 μg/ml), and CAS at 8 μg/ml (CAS2) was synergistic with VRC (4 μg/ml) against a biofilm of A. fumigatus AF293 (Table 1). Therefore, the combination of CAS1 (4 μg/ml) with AMB (0.25 μg/ml), as well as that of CAS2 (8 μg/ml) with VRC (4 μg/ml) was used to inhibit the biofilm of A. fumigatus AF293. This combination was used as a model to observe the synergistic activity against Aspergillus biofilm by FUN-1 staining (8, 15). With an Olympus FV-500 laser scanning confocal microscope, we observed that the amount of viable organisms in the A. fumigatus AF293 biofilm was significantly less when being treated with these drugs—CAS1 (4 μg/ml) with AMB (0.25 μg/ml) or CAS2 (8 μg/ml) with VRC (4 μg/ml) (see Fig. S1 and S2 in the supplemental material). Again, these results suggest a synergistic activity of CAS with AMB or VRC against Aspergillus biofilm.
Supplementary Material
ACKNOWLEDGMENTS
This work was supported by the Program for New Century Excellent Talents in University NCET-10-0198 (Wei Liu) and BMU20110158 (Wei Liu) and by grants from National Natural Science Foundation of China 30970131 (Wei Liu) and 30930006 (Ruoyu Li). This study was also supported by grant 7102149 from the Beijing Natural Science Foundation (Wei Liu). The funders had no role in study design, data analysis, decision to publish, or preparation of the manuscript.
We thank Richard Calderone, Georgetown University, Washington, DC, for critical reading of the manuscript.
This study does not present any conflicts of interest for us.
Footnotes
Published ahead of print 1 October 2012
Supplemental material for this article may be found at http://aac.asm.org/.
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