ABSTRACT
In vitro interactions of tacrolimus, a calcineurin inhibitor, and azoles, including itraconazole, voriconazole, and posaconazole, against planktonic cells and biofilms of Exophiala dermatitidis were assessed via a broth microdilution checkerboard technique. A total of 16 clinical isolates were studied. The results revealed favorable synergistic inhibitory activity between tacrolimus and itraconazole, voriconazole, or posaconazole against 68.8%, 87.5%, and 100% of tested strains of planktonic E. dermatitidis, respectively.However, limited synergism was observed against biofilms of E. dermatitidis. No antagonism was observed in all combinations.
KEYWORDS: Exophiala dermatitidis, FK506, azoles, biofilms, calcineurin inhibitor, synergy, tacrolimus
TEXT
The opportunistic black yeast-like species Exophiala dermatitidis is an increasingly recognized pathogen and a leading cause of severe phaeohyphomycosis in both immunocompetent and immunocompromised humans worldwide (1). It has been abundantly isolated from human-made environments, such as sauna facilities and dishwashers in tropical and temperate regions (2). Clinical forms of the infection range from localized cutaneous and subcutaneous phaeohyphomycosis to more severe systemic forms, such as neurotropic infections, whose prevalence is effectively growing and has the worst outcome, with mortality of more than 90% (3, 4). In addition, this species is frequently found as a pulmonary colonizer in patients with cystic fibrosis and appears to be associated with more advanced disease (5). Its colonization in the airway tracts and presence in such proximity to humans generate risks for biofilm formation, which is a prerequisite event toward the development of invasive disease and has been reported to be involved in about 80% of nonacute infections in humans (6). Unlike the planktonic form, fungal biofilms are relatively resistant to conventional antifungal agents (6). E. dermatitidis biofilms have been demonstrated to exhibit a higher resistance to the tested compounds than the organism's planktonic cells (7) and biofilms of Candida albicans (8). It has been demonstrated that tacrolimus (also known as FK506), which targets calcineurin, a Ca2+-calmodulin-dependent protein phosphatase that is important in various process in fungi, synergizes with azoles against planktonic cells and biofilms of Aspergillus spp. and C. albicans (9, 10). Thus, it is reasonable to suspect that the combination of FK506 and azoles would also generate synergy against planktonic cells and biofilms of E. dermatitidis.
In the present study, a total of 16 strains of E. dermatitidis and one control strain of Candida parapsilosis (ATCC 22019) were studied. All E. dermatitidis strains were all clinical isolates and identified by microscopic morphology and by molecular sequencing of the internal transcribed spacer (ITS) ribosomal DNA (rDNA), as required. All tested agents, including FK506, itraconazole (ITC), posaconazole (POS), and voriconazole (VRC), were purchased in powder form from Sigma Chemical Co., St. Louis, MO, and prepared as outlined in CLSI document M38-A2 (11). The interactions between FK506 and azoles against planktonic cells and biofilms of E. dermatitidis were tested via the microdilution checkerboard technique, adapted from the CLSI broth microdilution method M38-A2 (11). The working concentration ranges of azoles and FK506 were 0.015 to 8 μg/ml and 0.25 to 16 μg/ml against planktonic cells and 0.5 to 64 μg/ml and 1 to 64 μg/ml against biofilm cells, respectively. The MICs applied for the evaluation of effects against planktonic cells were determined as the lowest concentration resulting in complete (100%) inhibition of growth (11). An XTT {2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide}-based colorimetric assay was applied for the evaluation of effects on biofilms (12). E. dermatitidis biofilms were prepared via a 96-well plate-based method (13). Briefly, conidia were collected from Sabouraud's dextrose agar (SDA) and resuspended in 20 ml RPMI 1640. The suspension was then adjusted to the final concentration of 1 × 107 spores/ml. Subsequently, the suspension was added into the 96-well plate with 200 μl in each cell and incubated at 37°C for 72 h. The media were then carefully extracted without disturbing the biofilm. The 96-well plate was washed with sterile phosphate-buffered saline (PBS) three times to remove detached spores (13). Subsequently, the 96-well plate containing prepared biofilm was inoculated with 100 μl serially double-diluted FK506 in the horizontal direction and another 100 μl serially double-diluted azoles in the vertical direction. After incubation at 37°C for 48 h, 50 μl XTT-menadione solution was added to each well, achieving a working concentration of 1 μg/ml, and then incubated for another 4 h. Subsequently, 80 μl of the colored supernatant from each well was removed, transferred into a new plate, and read at 490 nm. The sessile MIC50 and MIC80 (SMIC50 and SMIC80) were defined as the concentrations at which a 50% or 80% decrease in optical density (OD) would be detected in comparison to the controls (12). The interactions between FK506 and azoles were classified on the basis of the fractional inhibitory concentration index (FICI). The FICI as calculated by the formula FICI = (Ac/Aa) + (Bc/Ba), where Ac and Bc are the MICs/SMICs of antifungal drugs in combination, and Aa and Ba are the MICs/SMICs of antifungal drugs A and B alone. The FICI results are classified as follows: FICI of ≤0.5, synergy; FICI of >0.5 to ≤4, no interaction (indifference); and FICI of >4, antagonism (14). All experiments were conducted in triplicate.
The MIC ranges of individual tested agents against planktonic E. dermatitidis isolates were >16 μg/ml for FK506, 0.5 to 1 μg/ml for ITC, 0.25 to 0.5 μg/ml for VRC, and 0.25 to 1 μg/ml for POS (Table 1). FK506 individually did not show any significant antifungal activity against planktonic E. dermatitidis. However, when FK506 was combined with ITC, VRC, or POS, favorable synergistic effects were demonstrated in 11 (68.8%), 14 (87.5%), and 16 (100%) strains of planktonic E. dermatitidis. The effective MIC ranges of FK506 and ITC in the FK506-ITC combination were 1 to 2 μg/ml and 0.03 to 0.5 μg/ml, respectively. In the FK506-VRC combination, the effective working ranges of FK506 and VRC were 1 to 4 μg/ml and 0.03 to 0.125 μg/ml, respectively, while in the FK506-POS combination, the effective MIC ranges of FK506 and POS were 1 to 2 μg/ml and 0.015 to 0.03 μg/ml, respectively. With respect to biofilms, the ranges of SMIC50 and SMIC80 of individual tested agents were all >64 μg/ml (Table 2). When FK506 was combined with azoles, the SMIC50 ranges of FK506, ITC, POS, and VRC decreased to 4 to 32, 8 to 32, 4 to 32, and 4 to 16 μg/ml, respectively. Therefore, based on the FICIs calculated from the SMIC50, there would be favorable synergistic effects against almost all biofilms. However, the SMIC80s in combinations were all >64 μg/ml, suggesting that even in these combinations, the inhibitory activity never reached 80% inhibition levels. Thus, limited synergism was observed against E. dermatitidis biofilms. No antagonism was observed in all combinations.
TABLE 1.
MICs and FICI results with combinations of FK506 with triazoles against planktonic E. dermatitidis
| Strain | MIC (μg/ml) for: |
||||||
|---|---|---|---|---|---|---|---|
| Agent alone |
Combinationa |
||||||
| FK506 | ITC | VRC | POS | FK506/ITC | FK506/VRC | FK506/POS | |
| BMU00028 | >16 | 1 | 0.25 | 0.5 | 2/0.03 (S) | 4/0.03 (S) | 2/0.03 (S) |
| BMU00029 | >16 | 1 | 0.5 | 0.5 | 1/0.03 (S) | 1/0.5 (I) | 2/0.015 (S) |
| BMU00030 | >16 | 0.5 | 0.25 | 0.5 | 1/0.25 (I) | 1/0.06 (S) | 2/0.015 (S) |
| BMU00031 | >16 | 1 | 0.5 | 0.5 | 1/0.06 (S) | 2/0.125 (S) | 2/0.03 (S) |
| BMU00034 | >16 | 0.5 | 0.5 | 0.5 | 1/0.06 (S) | 2/0.125 (S) | 1/0.03 (S) |
| BMU00035 | >16 | 1 | 0.5 | 0.5 | 1/0.25 (S) | 2/0.03 (S) | 2/0.015 (S) |
| BMU00037 | >16 | 1 | 0.5 | 1 | 2/0.5 (I) | 0.25/0.5 (I) | 1/0.015 (S) |
| BMU00038 | >16 | 0.5 | 0.25 | 0.25 | 4/0.25 (I) | 2/0.03 (S) | 2/0.015 (S) |
| BMU00039 | >16 | 0.5 | 0.25 | 0.5 | 2/0.125 (S) | 2/0.06 (S) | 2/0.015 (S) |
| BMU00040 | >16 | 1 | 0.5 | 1 | 2/0.125 (S) | 2/0.125 (S) | 1/0.03 (S) |
| BMU00041 | >16 | 1 | 0.25 | 0.5 | 2/0.06 (S) | 2/0.06 (S) | 1/0.03 (S) |
| 109140 | >16 | 0.5 | 0.5 | 1 | 1/0.125 (S) | 2/0.125 (S) | 1/0.03 (S) |
| 109145 | >16 | 0.5 | 0.25 | 1 | 2/0.25 (I) | 2/0.06 (S) | 1/0.03 (S) |
| 109148 | >16 | 1 | 0.25 | 0.5 | 1/0.125 (S) | 2/0.06 (S) | 2/0.03 (S) |
| 109149 | >16 | 1 | 0.5 | 0.5 | 1/0.06 (S) | 4/0.06 (S) | 2/0.03 (S) |
| 109152 | >16 | 0.5 | 0.25 | 0.5 | 2/0.25 (I) | 1/0.03 (S) | 1/0.015 (S) |
FICI results are shown in parentheses. S, synergy (FICI of ≤0.5); I, indifference (no interaction [FICI of >0.5 to ≤4]).
TABLE 2.
SMICs and FICI results with combinations of FK506 with triazoles against E. dermatitidis biofilms
| Strain | SMIC50 (μg/ml) for: |
SMIC80 (μg/ml) for: |
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Agent alone |
Combinationa |
Agent alone |
Combinationa |
||||||||||
| FK506 | ITC | VRC | POS | FK506/ITC | FK506/VRC | FK506/POS | FK506 | ITC | VRC | POS | FK506/VRC | FK506/POS | |
| BMU00028 | >64 | >64 | >64 | >64 | 16/8 (S) | 16/16 (S) | 16/8 (S) | >64 | >64 | >64 | >64 | >64/>64 (I) | >64/>64 (I) |
| BMU00029 | >64 | >64 | >64 | >64 | 16/8 (S) | 16/8 (S) | 8/8 (S) | >64 | >64 | >64 | >64 | >64/>64 (I) | >64/>64 (I) |
| BMU00031 | >64 | >64 | >64 | >64 | 16/8 (S) | 16/4 (S) | 8/4 (S) | >64 | >64 | >64 | >64 | >64/>64 (I) | >64/>64 (I) |
| BMU00030 | >64 | >64 | >64 | >64 | 8/8 (S) | 8/8 (S) | 16/8 (S) | >64 | >64 | >64 | >64 | >64/>64 (I) | >64/>64 (I) |
| BMU00034 | >64 | >64 | >64 | >64 | 16/8 (S) | 16/4 (S) | 16/4 (S) | >64 | >64 | >64 | >64 | >64/>64 (I) | >64/>64 (I) |
| BMU00035 | >64 | >64 | >64 | >64 | 8/8 (S) | 8/8 (S) | 8/8 (S) | >64 | >64 | >64 | >64 | >64/>64 (I) | >64/>64 (I) |
| BMU00037 | >64 | >64 | >64 | >64 | 8/16 (S) | 8/8 (S) | 8/8 (S) | >64 | >64 | >64 | >64 | >64/>64 (I) | >64/>64 (I) |
| BMU00038 | >64 | >64 | >64 | >64 | 16/8 (S) | 8/8 (S) | 8/4 (S) | >64 | >64 | >64 | >64 | >64/>64 (I) | >64/>64 (I) |
| BMU00039 | >64 | >64 | >64 | >64 | 32/32 (I) | 16/16 (S) | 8/8 (S) | >64 | >64 | >64 | >64 | >64/>64 (I) | >64/>64 (I) |
| BMU00040 | >64 | >64 | >64 | >64 | 16/8 (S) | 16/32 (I) | 8/8 (S) | >64 | >64 | >64 | >64 | >64/>64 (I) | >64/>64 (I) |
| BMU00041 | >64 | >64 | >64 | >64 | 16/8 (S) | 8/8 (S) | 8/4 (S) | >64 | >64 | >64 | >64 | >64/>64 (I) | >64/>64 (I) |
| 109140 | >64 | >64 | >64 | >64 | 16/16 (S) | 16/4 (S) | 16/4 (S) | >64 | >64 | >64 | >64 | >64/>64 (I) | >64/>64 (I) |
| 109145 | >64 | >64 | >64 | >64 | 16/8 (S) | 16/4 (S) | 16/4 (S) | >64 | >64 | >64 | >64 | >64/>64 (I) | >64/>64 (I) |
| 109148 | >64 | >64 | >64 | >64 | 16/16 (S) | 8/8 (S) | 16/16 (S) | >64 | >64 | >64 | >64 | >64/>64 (I) | >64/>64 (I) |
| 109149 | >64 | >64 | >64 | >64 | 16/16 (S) | 8/8 (S) | 16/16 (S) | >64 | >64 | >64 | >64 | >64/>64 (I) | >64/>64 (I) |
| 109152 | >64 | >64 | >64 | >64 | 8/8 (S) | 8/4 (S) | 16/8 (S) | >64 | >64 | >64 | >64 | >64/>64 (I) | >64/>64 (I) |
FICI results are shown in parentheses. S, synergy (FICI of ≤0.5); I, indifference (no interaction [FICI of >0.5 to ≤4]).
The calcineurin pathway governs multiple important process of fungal physiology, which include morphogenesis, antifungal drug susceptibility, and virulence (15). Calcineurin inhibitors have been demonstrated to render azoles fungicidal instead of simply fungistatic (16). In another aspect, azoles could increase the intracellular concentration of calcineurin inhibitor (17). FK506 has been demonstrated to synergize with multiple classical antifungals against planktonic cells of several pathogenic fungi, including Candida spp., Aspergillus spp., Cryptococcus neoformans, and Mucorales, and biofilms of C. albicans and Aspergillus spp. (10, 18–21). In the present study, FK506 also exhibits favorable synergistic effects with ITC, VRC, or POS against 68.8%, 87.5%, and 100% of tested strains of E. dermatitidis under the planktonic condition. It is notable that the FK506-POS combination was highly synergistic in action against all planktonic E. dermatitidis tests. Although Exophiala biofilms were more than 1,000-fold resistant to azoles than planktonic cells, FK506 also potentiated the antibiofilm activity of azoles against the biofilm cells when reading the SMIC50. However, since all combinations were unable to achieve more than 80% inhibitory effect, there was limited synergistic effect against Exophiala biofilms. It is well known that Hsp90, the molecular chaperone of calcineurin, has been implicated as a key regulator of biofilm dispersion and drug resistance (22). However, under planktonic conditions of C. albicans, Hsp90 pathway potentiates the emergence and maintenance of resistance to antifungals, at least in part via calcineurin (23), while in biofilms, Hsp90 regulates drug resistance through distinct mechanisms independent of calcineurin (22). Therefore, we suspected that this may explain the discrepancy of the combination effect between planktonic cells and biofilms in this study. However, further studies are warranted to elucidate the mechanism in E. dermatitidis.
In conclusion, FK506 could enhance the in vitro antifungal activity of itraconazole, voriconazole, and posaconazole against E. dermatitidis, suggesting that the combination of azoles with a calcineurin inhibitor that selectively targets fungal calcineurin pathways without having collateral effects on human cells might prove a potential antifungal regimen.
ACKNOWLEDGMENTS
We want to thank Ruoyu Li and Wei Liu from Peking University First Hospital, Research Center for Medical Mycology, Peking University, Beijing, China, and G. Sybren de Hoog from CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands, for kindly providing us with the E. dermatitidis isolates studied.
This work was supported by grants 31400131 (Lujuan Gao) and 81401677 (Yi Sun) from the National Natural Science Foundation of China and grant WJ2015MB281 from the Hubei Province Health and Family Planning Scientific Research Project (Yi Sun). The funders had no role in study design, data analysis, decision to publish, or preparation of the manuscript.
We declare no conflicts of interest.
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