In Vitro Interactions of Antifungal Agents and Tacrolimus against Aspergillus Biofilms Lujuan Gao,a Yi Sunb Department of Dermatology, Zhongshan Hospital, Fudan University, Shanghai, People’s Republic of Chinaa; Department of Dermatology, Jingzhou Central Hospital, Jingzhou City, Hubei Province, People’s Republic of Chinab
Aspergillus biofilms were prepared from Aspergillus fumigatus, Aspergillus flavus, and Aspergillus terreus via a 96-well platebased method, and the combined antifungal activity of tacrolimus with azoles or amphotericin B against Aspergillus biofilms was investigated via a broth microdilution checkerboard technique system. Our results suggest that combinations of tacrolimus with voriconazole or amphotericin B have synergistic inhibitory activity against Aspergillus biofilms. However, combinations of tacrolimus with itraconazole or posaconazole exhibit no synergistic or antagonistic effects.
I
nvasive fungal infections caused by Aspergillus spp. have already become one of the major causes of death in immunocompromised patients in recent years (1). Recent studies have suggested that biofilm formation by Aspergillus spp. may be one of the most important virulence factors in invasive pulmonary aspergillosis and aspergilloma (2, 3). The MICs of antifungal agents required to kill biofilm structures of Aspergillus spp. are much higher than those required to kill the planktonic forms of the fungus (4, 5). It has been reported that combinations of tacrolimus, which targets calcineurin, a Ca2⫹-calmodulin-dependent protein phosphatase, and antifungal agents have synergistic activity against pathogenic fungi, such as Candida albicans, Mucorales, and Aspergillus fumigatus (6–8). Therefore, it is reasonable to determine whether the combination of tacrolimus and amphotericin (AMB) or azoles would generate a synergistic inhibitory effect against Aspergillus biofilms. In the present study, Aspergillus biofilms were prepared from 20 strains of Aspergillus spp., including 10 strains of Aspergillus fumigatus, 8 strains of Aspergillus flavus, and 2 strains of Aspergillus terreus, via a 96-well plate-based method (9). The Aspergillus strains were all clinical isolates from patients with invasive aspergillosis and identified by molecular and morphological methods. The individual MICs of tested drugs, including tacrolimus, itraconazole (ITC), posaconazole (POC), voriconazole (VRC), and AMB, for planktonic cells of Aspergillus spp. were determined according to the M38-A2 method (10). The effects of tacrolimus and antifungals alone, and combinations of antifungals with tacrolimus, on Aspergillus biofilms were assessed by a checkerboard method with biofilms formed in the wells of microtiter plates and an XTT (2,3-bis-[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide)-based colorimetric assay (11). All antifungal drugs and chemical agents were purchased in powder form from Sigma Chemical Co. (St. Louis, MO) and prepared as outlined in the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method M38-A2 (10). The working concentration ranges of tacrolimus, ITC, VRC, POC, and AMB were 2 to 256 g/ml, 0.5 to 256 g/ml, 0.5 to 256 g/ml, 0.5 to 256 g/ml, and 0.06 to 32 g/ml, respectively. The sessile MICs (SMIC50 and SMIC80) were defined as the concentrations at which a 50% or 80% decrease, respectively, in optical density (OD) would be detected in comparison to the controls (11). The interaction of tacrolimus with AMB or azoles referred to the fractional inhibitory concentration index (FICI), which was classified as follows: FICI of ⱕ0.5, synergy;
November 2015 Volume 59 Number 11
FIC of ⬎0.5 to ⱕ4, no interaction; FICI of ⬎4, antagonism (12). All experiments were conducted in triplicate. The ranges of the MICs of individual tested drugs for isolates of planktonic Aspergillus spp. were ⱖ256 g/ml for tacrolimus, 1 to 4 g/ml for ITC, 0.5 to 2 g/ml for VRC, 0.125 to 2 g/ml for POC, and 0.125 to 4 g/ml for AMB (Table 1). The ranges of the SMIC50 for Aspergillus biofilms were ⱖ256 g/ml for tacrolimus, 16 to ⱖ256 g/ml for ITC, 8 to ⱖ256 g/ml for VRC, 2 to ⱖ256 g/ml for POC, and 2 to 32 g/ml for AMB, while the ranges of the SMIC80 were ⱖ256 g/ml for tacrolimus, 64 to ⱖ256 g/ml for ITC, 32 to ⱖ256 g/ml for VRC, 32 to ⱖ256 g/ml for POC, and 2 to 32 g/ml for AMB (Table 2, Table 3). As described in previous studies, the MICs required to kill biofilm structures of Aspergillus spp. are much higher than those required to kill the planktonic forms (4, 5). However, when tacrolimus was combined with AMB against Aspergillus biofilms, the SMIC50 ranges of AMB and tacrolimus decreased to 0.25 to 4 g/ml and 2 to 64 g/ml, respectively, while the SMIC80 ranges decreased to 0.25 to 4 g/ml and 2 to 32 g/ml, respectively (Table 2). Based on the FICIs calculated from the SMIC50, favorable synergistic effects were shown against biofilms of 12 isolates of Aspergillus spp., including 5 strains of A. fumigatus (5 of 10) and 7 strains of A. flavus (7 of 8). Based on the FICIs calculated from the SMIC80, favorable synergistic effects were shown against biofilms of 14 isolates of Aspergillus spp., including 6 strains of A. fumigatus (6 of 10) and all strains of A. flavus. However, no synergistic effect against A. terreus biofilms was observed. For the combination of tacrolimus and VRC, the SMIC50 of VRC and tacrolimus decreased to 1 to 128 g/ml and 2 to 16 g/ml, respectively. The FICIs based on the SMIC50 revealed syn-
Received 29 June 2015 Returned for modification 27 July 2015 Accepted 16 August 2015 Accepted manuscript posted online 24 August 2015 Citation Gao L, Sun Y. 2015. In vitro interactions of antifungal agents and tacrolimus against Aspergillus biofilms. Antimicrob Agents Chemother 59:7097–7099. doi:10.1128/AAC.01510-15. Address correspondence to Yi Sun, [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Antimicrobial Agents and Chemotherapy
aac.asm.org
7097
Gao and Sun
TABLE 1 MICs for planktonic Aspergillus spp. MIC (g/ml) Strain
FK506
ITC
VRC
POC
AMB
A. fumigatus AF1 AF2 AF3 AF4 AF5 AF6 AF7 AF8 AF9 AF10
ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256
4 1 2 2 1 4 1 1 4 2
2 1 1 1 1 2 0.5 2 2 0.5
2 2 1 1 2 1 0.5 1 1 0.125
1 2 1 2 2 1 2 2 1 0.125
A. flavus AFL1 AFL2 AFL3 AFL4 AFL5 AFL6 AFL7 AFL8
ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256
4 2 4 4 2 4 4 1
2 1 1 2 1 2 1 0.5
0.5 1 0.25 1 0.25 0.25 0.5 1
4 0.25 0.5 4 2 2 2 1
A. terreus AT1 AT2
ⱖ256 ⱖ256
2 2
0.5 1
1 0.5
1 0.5
ergistic effects against biofilms of 10 isolates of Aspergillus spp., including 5 strains of A. fumigatus (5 of 10), 4 strains of A. flavus (4 of 8), and 1 strain of A. terreus (1 of 2) (Table 2). The SMIC80 of VRC and tacrolimus in this combination also decreased to 1 to 128 g/ml and 2 to 16 g/ml, respectively. The FICIs based on the SMIC80 revealed synergistic effects against biofilms of 13 strains, including 7 strains of A. fumigatus (7 of 10), 5 strains of A. flavus (5 of 8), and 1 strain of A. terreus (1 of 2). No synergistic effect was observed when tacrolimus was combined with ITC or POC (Table 3). No antagonistic effect was observed against Aspergillus biofilms with these combinations. Although our results revealed an unfavorable antifungal effect of tacrolimus alone against planktonic cells and biofilm forms of all isolates of Aspergillus spp. studied, in vitro synergism against most of the biofilms generated from A. fumigatus isolates and from nonfumigatus Aspergillus isolates tested were observed when tacrolimus was combined with AMB or VRC, in agreement with the findings for other fungal species (7, 8, 13). This drug synergism may be attributable to the following effects: tacrolimus, the calcineurin inhibitor, renders the azole fungicidal rather than simply fungistatic, and membrane perturbation by antifungal inhibition of ergosterol biosynthesis increases intracellular calcineurin inhibitor concentrations (8). In addition, the molecular chaperone Hsp90 has been implicated as a key regulator of biofilm dispersion and drug resistance, and an Hsp90activated calcineurin signaling pathway has been reported to be associated with antifungal resistance in Aspergillus biofilms (14, 15). We speculate that the decreased SMIC50 and SMIC80 of tacrolimus may result from the membrane perturbation and stress response due to AMB or VRC, and the decreased SMIC50 and SMIC80 of
TABLE 2 SMIC and FICI results with combinations of tacrolimus with AMB or VRC against Aspergillus biofilms SMIC80 (g/ml)
SMIC50 (g/ml) Strain
FK506 VRC
FK506/ FK506/AMB FK506/ FK506/VRC AMB AMB FICIa FK506 VRC VRC FICIa
A. fumigatus AF1 AF2 AF3 AF4 AF5 AF6 AF7 AF8 AF9 AF10
ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256
16 8 16 8 32 8 8 64 128 8
4/8 2/1 4/1 8/1 8/16 16/4 4/4 4/2 16/2 8/4
N S S S N N N S S N
2 4 2 4 2 2 4 8 2 8
4/0.25 2/2 16/2 8/4 8/2 16/2 64/1 2/1 4/2 4/4
S N N N S S S S N N
ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256
64 64 32 32 64 64 32 128 ⱖ256 32
4/8 4/4 4/1 8/1 8/16 16/32 4/16 8/2 16/4 8/16
S S S S S N N S S N
4 8 2 4 2 4 4 16 2 8
8/0.25 8/2 32/2 16/4 8/2 16/2 64/1 64/1 16/2 64/4
S S N N S S S S N N
A. flavus AFL1 AFL2 AFL3 AFL4 AFL5 AFL6 AFL7 AFL8
ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256
ⱖ256 8 8 ⱖ256 128 64 ⱖ256 64
8/2 2/4 4/4 8/128 16/64 16/8 4/16 8/8
S N N N N S S S
16 2 2 32 8 16 8 4
32/2 8/0.5 32/0.25 16/4 64/1 8/4 16/1 16/2
S S S S S S S N
ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256
ⱖ256 64 64 ⱖ256 ⱖ256 128 ⱖ256 128
8/4 2/8 8/64 8/128 16/128 16/16 4/16 16/16
S S N N N S S S
16 4 2 32 16 16 16 8
32/2 8/0.5 32/0.25 64/4 64/1 8/4 16/1 16/2
S S S S S S S S
A. terreus AT1 AT2
ⱖ256 ⱖ256
128 8/2 ⱖ256 2/128
S N
2 2
16/1 64/1
N N
ⱖ256 ⱖ256
128 8/8 ⱖ256 4/128
S N
4 4
16/2 64/2
N N
a
FK506/ FK506/VRC FK506/ FK506/AMB VRC FICIa AMB AMB FICIa
S, synergy (FICI of ⱕ0.5); N, no interaction (FICI of ⬎0.5 to ⱕ4).
7098
aac.asm.org
Antimicrobial Agents and Chemotherapy
November 2015 Volume 59 Number 11
Combination Interactions against Aspergillus Biofilms
TABLE 3 SMIC and FICI results with combinations of tacrolimus with ITC or POC against Aspergillus biofilms SMIC50 (g/ml)
SMIC80 (g/ml)
Strain
FK506 ITC
FK506/ FK506/ITC POC ITC FICIa
FK506/ FK506/POC POC FICIa FK506 ITC
A. fumigatus AF1 AF2 AF3 AF4 AF5 AF6 AF7 AF8 AF9 AF10
ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256
128 16 64 128 64 128 32 32 128 32
16/64 8/32 64/8 32/64 16/32 8/64 32/16 32/16 32/64 16/16
N N N N N N N N N N
8 4 16 4 8 4 2 16 64 4
4/4 8/4 16/8 8/4 8/4 16/4 64/2 16/8 4/32 4/4
N N N N N N N N N N
ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256
A. flavus AFL1 AFL2 AFL3 AFL4 AFL5 AFL6 AFL7 AFL8
ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256
ⱖ256 64 32 ⱖ256 128 64 ⱖ256 64
16/128 8/32 32/16 32/128 32/64 32/32 64/64 16/64
N N N N N N N N
ⱖ256 4 4 ⱖ256 8 32 128 32
32/128 8/2 32/2 16/128 64/4 8/16 16/64 16/16
N N N N N N N N
A. terreus AT1 AT2
ⱖ256 ⱖ256
128 16/64 ⱖ256 32/128
N N
64 16/16 ⱖ256 32/128
N N
a
FK506/ ITC
FK506/ITC FICIa POC
FK506/ POC
FK506/POC FICIa
128 64 128 ⱖ256 128 ⱖ256 128 64 ⱖ256 64
128/128 64/32 64/64 128/128 128/64 128/64 64/64 32/32 128/128 128/64
N N N N N N N N N N
32 32 32 64 32 32 32 32 128 32
8/16 8/16 32/16 64/64 16/16 16/16 64/32 32/16 64/64 64/16
N N N N N N N N N N
ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256
ⱖ256 128 64 ⱖ256 ⱖ256 128 ⱖ256 ⱖ256
128/64 128/64 32/32 128/64 128/128 128/128 128/64 128/128
N N N N N N N N
ⱖ256 32 32 ⱖ256 32 64 ⱖ256 32
128/128 8/32 32/16 64/32 128/16 128/32 128/128 16/16
N N N N N N N N
ⱖ256 ⱖ256
128 128/64 N ⱖ256 128/128 N
128 64/64 N ⱖ256 128/128 N
N, no interaction (FICI of ⬎0.5 to ⱕ4).
AMB and VRC may result from the inhibition of the Hsp90-activated calcineurin signaling pathway. In summary, our results suggest that calcineurin pathway inhibition in combination with classical antifungal agents has therapeutic potential in Aspergillus biofilm-associated fungal infections. Due to the immunosuppressive properties of calcineurin inhibitors, the clinical use would ultimately require a selectively fungal calcineurin pathway-targeted agent without collateral effects on human cells. ACKNOWLEDGMENTS This work was supported by grants 31400131 (Lujuan Gao) and 81401677 (Yi Sun) from the National Natural Science Foundation of China. The funders had no role in study design, data analysis, decision to publish, or preparation of the manuscript. We declare no conflicts of interest.
REFERENCES 1. Kontoyiannis DP, Bodey GP. 2002. Invasive aspergillosis in 2002: an update. Eur J Clin Microbiol Infect Dis 21:161–172. http://dx.doi.org/10 .1007/s10096-002-0699-z. 2. Loussert C, Schmitt C, Prevost MC, Balloy V, Fadel E, Philippe B, Kauffmann-Lacroix C, Latge JP, Beauvais A. 2010. In vivo biofilm composition of Aspergillus fumigatus. Cell Microbiol 12:405– 410. http://dx .doi.org/10.1111/j.1462-5822.2009.01409.x. 3. Müller FM, Seidler M, Beauvais A. 2011. Aspergillus fumigatus biofilms in the clinical setting. Med Mycol 49(Suppl 1):S96 –S100. http://dx.doi .org/10.3109/13693786.2010.502190. 4. Seidler MJ, Salvenmoser S, Muller FM. 2008. Aspergillus fumigatus forms biofilms with reduced antifungal drug susceptibility on bronchial epithelial cells. Antimicrob Agents Chemother 52:4130 – 4136. http://dx.doi.org /10.1128/AAC.00234-08. 5. Singhal D, Baker L, Wormald PJ, Tan L. 2011. Aspergillus fumigatus biofilm on primary human sinonasal epithelial culture. Am J Rhinol Allergy 25:219 –225. http://dx.doi.org/10.2500/ajra.2011.25.3622.
November 2015 Volume 59 Number 11
6. Steinbach WJ, Schell WA, Blankenship JR, Onyewu C, Heitman J, Perfect JR. 2004. In vitro interactions between antifungals and immunosuppressants against Aspergillus fumigatus. Antimicrob Agents Chemother 48:1664 – 1669. http://dx.doi.org/10.1128/AAC.48.5.1664-1669.2004. 7. Shirazi F, Kontoyiannis DP. 2013. The calcineurin pathway inhibitor tacrolimus enhances the in vitro activity of azoles against Mucorales via apoptosis. Eukaryot Cell 12:1225–1234. http://dx.doi.org/10.1128/EC.00138-13. 8. Uppuluri P, Nett J, Heitman J, Andes D. 2008. Synergistic effect of calcineurin inhibitors and fluconazole against Candida albicans biofilms. Antimicrob Agents Chemother 52:1127–1132. http://dx.doi.org/10.1128/AAC.01397-07. 9. Pierce CG, Uppuluri P, Tristan AR, Wormley FL, Jr, Mowat E, Ramage G, Lopez-Ribot JL. 2008. A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing. Nat Protoc 3:1494–1500. http://dx.doi.org/10.1038/nprot.2008.141. 10. Clinical and Laboratory Standards Institute. 2008. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard. CLSI M38-A2. Clinical and Laboratory Standards Institute, Wayne, PA. 11. Ramage G, Vande Walle K, Wickes BL, Lopez-Ribot JL. 2001. Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrob Agents Chemother 45:2475–2479. http://dx.doi .org/10.1128/AAC.45.9.2475-2479.2001. 12. Tobudic S, Kratzer C, Lassnigg A, Graninger W, Presterl E. 2010. In vitro activity of antifungal combinations against Candida albicans biofilms. J Antimicrob Chemother 65:271–274. http://dx.doi.org/10.1093/jac/dkp429. 13. Narreddy S, Manavathu E, Chandrasekar PH, Alangaden GJ, Revankar SG. 2010. In vitro interaction of posaconazole with calcineurin inhibitors and sirolimus against zygomycetes. J Antimicrob Chemother 65:701–703. http://dx.doi.org/10.1093/jac/dkq020. 14. Robbins N, Uppuluri P, Nett J, Rajendran R, Ramage G, Lopez-Ribot JL, Andes D, Cowen LE. 2011. Hsp90 governs dispersion and drug resistance of fungal biofilms. PLoS Pathog 7:e1002257. http://dx.doi.org/10 .1371/journal.ppat.1002257. 15. Rajendran R, Mowat E, Jones B, Williams C, Ramage G. 24 April 2015. Prior in vitro exposure to voriconazole confers resistance to amphotericin B in Aspergillus fumigatus biofilms. Int J Antimicrob Agents http://dx.doi .org/10.1016/j.ijantimicag.2015.03.006.
Antimicrobial Agents and Chemotherapy
aac.asm.org
7099
Aspergillus biofilms were prepared from Aspergillus fumigatus, Aspergillus flavus, and Aspergillus terreus via a 96-well platebased method, and the combined antifungal activity of tacrolimus with azoles or amphotericin B against Aspergillus biofilms was investigated via a broth microdilution checkerboard technique system. Our results suggest that combinations of tacrolimus with voriconazole or amphotericin B have synergistic inhibitory activity against Aspergillus biofilms. However, combinations of tacrolimus with itraconazole or posaconazole exhibit no synergistic or antagonistic effects.
I
nvasive fungal infections caused by Aspergillus spp. have already become one of the major causes of death in immunocompromised patients in recent years (1). Recent studies have suggested that biofilm formation by Aspergillus spp. may be one of the most important virulence factors in invasive pulmonary aspergillosis and aspergilloma (2, 3). The MICs of antifungal agents required to kill biofilm structures of Aspergillus spp. are much higher than those required to kill the planktonic forms of the fungus (4, 5). It has been reported that combinations of tacrolimus, which targets calcineurin, a Ca2⫹-calmodulin-dependent protein phosphatase, and antifungal agents have synergistic activity against pathogenic fungi, such as Candida albicans, Mucorales, and Aspergillus fumigatus (6–8). Therefore, it is reasonable to determine whether the combination of tacrolimus and amphotericin (AMB) or azoles would generate a synergistic inhibitory effect against Aspergillus biofilms. In the present study, Aspergillus biofilms were prepared from 20 strains of Aspergillus spp., including 10 strains of Aspergillus fumigatus, 8 strains of Aspergillus flavus, and 2 strains of Aspergillus terreus, via a 96-well plate-based method (9). The Aspergillus strains were all clinical isolates from patients with invasive aspergillosis and identified by molecular and morphological methods. The individual MICs of tested drugs, including tacrolimus, itraconazole (ITC), posaconazole (POC), voriconazole (VRC), and AMB, for planktonic cells of Aspergillus spp. were determined according to the M38-A2 method (10). The effects of tacrolimus and antifungals alone, and combinations of antifungals with tacrolimus, on Aspergillus biofilms were assessed by a checkerboard method with biofilms formed in the wells of microtiter plates and an XTT (2,3-bis-[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide)-based colorimetric assay (11). All antifungal drugs and chemical agents were purchased in powder form from Sigma Chemical Co. (St. Louis, MO) and prepared as outlined in the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method M38-A2 (10). The working concentration ranges of tacrolimus, ITC, VRC, POC, and AMB were 2 to 256 g/ml, 0.5 to 256 g/ml, 0.5 to 256 g/ml, 0.5 to 256 g/ml, and 0.06 to 32 g/ml, respectively. The sessile MICs (SMIC50 and SMIC80) were defined as the concentrations at which a 50% or 80% decrease, respectively, in optical density (OD) would be detected in comparison to the controls (11). The interaction of tacrolimus with AMB or azoles referred to the fractional inhibitory concentration index (FICI), which was classified as follows: FICI of ⱕ0.5, synergy;
November 2015 Volume 59 Number 11
FIC of ⬎0.5 to ⱕ4, no interaction; FICI of ⬎4, antagonism (12). All experiments were conducted in triplicate. The ranges of the MICs of individual tested drugs for isolates of planktonic Aspergillus spp. were ⱖ256 g/ml for tacrolimus, 1 to 4 g/ml for ITC, 0.5 to 2 g/ml for VRC, 0.125 to 2 g/ml for POC, and 0.125 to 4 g/ml for AMB (Table 1). The ranges of the SMIC50 for Aspergillus biofilms were ⱖ256 g/ml for tacrolimus, 16 to ⱖ256 g/ml for ITC, 8 to ⱖ256 g/ml for VRC, 2 to ⱖ256 g/ml for POC, and 2 to 32 g/ml for AMB, while the ranges of the SMIC80 were ⱖ256 g/ml for tacrolimus, 64 to ⱖ256 g/ml for ITC, 32 to ⱖ256 g/ml for VRC, 32 to ⱖ256 g/ml for POC, and 2 to 32 g/ml for AMB (Table 2, Table 3). As described in previous studies, the MICs required to kill biofilm structures of Aspergillus spp. are much higher than those required to kill the planktonic forms (4, 5). However, when tacrolimus was combined with AMB against Aspergillus biofilms, the SMIC50 ranges of AMB and tacrolimus decreased to 0.25 to 4 g/ml and 2 to 64 g/ml, respectively, while the SMIC80 ranges decreased to 0.25 to 4 g/ml and 2 to 32 g/ml, respectively (Table 2). Based on the FICIs calculated from the SMIC50, favorable synergistic effects were shown against biofilms of 12 isolates of Aspergillus spp., including 5 strains of A. fumigatus (5 of 10) and 7 strains of A. flavus (7 of 8). Based on the FICIs calculated from the SMIC80, favorable synergistic effects were shown against biofilms of 14 isolates of Aspergillus spp., including 6 strains of A. fumigatus (6 of 10) and all strains of A. flavus. However, no synergistic effect against A. terreus biofilms was observed. For the combination of tacrolimus and VRC, the SMIC50 of VRC and tacrolimus decreased to 1 to 128 g/ml and 2 to 16 g/ml, respectively. The FICIs based on the SMIC50 revealed syn-
Received 29 June 2015 Returned for modification 27 July 2015 Accepted 16 August 2015 Accepted manuscript posted online 24 August 2015 Citation Gao L, Sun Y. 2015. In vitro interactions of antifungal agents and tacrolimus against Aspergillus biofilms. Antimicrob Agents Chemother 59:7097–7099. doi:10.1128/AAC.01510-15. Address correspondence to Yi Sun, [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Antimicrobial Agents and Chemotherapy
aac.asm.org
7097
Gao and Sun
TABLE 1 MICs for planktonic Aspergillus spp. MIC (g/ml) Strain
FK506
ITC
VRC
POC
AMB
A. fumigatus AF1 AF2 AF3 AF4 AF5 AF6 AF7 AF8 AF9 AF10
ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256
4 1 2 2 1 4 1 1 4 2
2 1 1 1 1 2 0.5 2 2 0.5
2 2 1 1 2 1 0.5 1 1 0.125
1 2 1 2 2 1 2 2 1 0.125
A. flavus AFL1 AFL2 AFL3 AFL4 AFL5 AFL6 AFL7 AFL8
ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256
4 2 4 4 2 4 4 1
2 1 1 2 1 2 1 0.5
0.5 1 0.25 1 0.25 0.25 0.5 1
4 0.25 0.5 4 2 2 2 1
A. terreus AT1 AT2
ⱖ256 ⱖ256
2 2
0.5 1
1 0.5
1 0.5
ergistic effects against biofilms of 10 isolates of Aspergillus spp., including 5 strains of A. fumigatus (5 of 10), 4 strains of A. flavus (4 of 8), and 1 strain of A. terreus (1 of 2) (Table 2). The SMIC80 of VRC and tacrolimus in this combination also decreased to 1 to 128 g/ml and 2 to 16 g/ml, respectively. The FICIs based on the SMIC80 revealed synergistic effects against biofilms of 13 strains, including 7 strains of A. fumigatus (7 of 10), 5 strains of A. flavus (5 of 8), and 1 strain of A. terreus (1 of 2). No synergistic effect was observed when tacrolimus was combined with ITC or POC (Table 3). No antagonistic effect was observed against Aspergillus biofilms with these combinations. Although our results revealed an unfavorable antifungal effect of tacrolimus alone against planktonic cells and biofilm forms of all isolates of Aspergillus spp. studied, in vitro synergism against most of the biofilms generated from A. fumigatus isolates and from nonfumigatus Aspergillus isolates tested were observed when tacrolimus was combined with AMB or VRC, in agreement with the findings for other fungal species (7, 8, 13). This drug synergism may be attributable to the following effects: tacrolimus, the calcineurin inhibitor, renders the azole fungicidal rather than simply fungistatic, and membrane perturbation by antifungal inhibition of ergosterol biosynthesis increases intracellular calcineurin inhibitor concentrations (8). In addition, the molecular chaperone Hsp90 has been implicated as a key regulator of biofilm dispersion and drug resistance, and an Hsp90activated calcineurin signaling pathway has been reported to be associated with antifungal resistance in Aspergillus biofilms (14, 15). We speculate that the decreased SMIC50 and SMIC80 of tacrolimus may result from the membrane perturbation and stress response due to AMB or VRC, and the decreased SMIC50 and SMIC80 of
TABLE 2 SMIC and FICI results with combinations of tacrolimus with AMB or VRC against Aspergillus biofilms SMIC80 (g/ml)
SMIC50 (g/ml) Strain
FK506 VRC
FK506/ FK506/AMB FK506/ FK506/VRC AMB AMB FICIa FK506 VRC VRC FICIa
A. fumigatus AF1 AF2 AF3 AF4 AF5 AF6 AF7 AF8 AF9 AF10
ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256
16 8 16 8 32 8 8 64 128 8
4/8 2/1 4/1 8/1 8/16 16/4 4/4 4/2 16/2 8/4
N S S S N N N S S N
2 4 2 4 2 2 4 8 2 8
4/0.25 2/2 16/2 8/4 8/2 16/2 64/1 2/1 4/2 4/4
S N N N S S S S N N
ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256
64 64 32 32 64 64 32 128 ⱖ256 32
4/8 4/4 4/1 8/1 8/16 16/32 4/16 8/2 16/4 8/16
S S S S S N N S S N
4 8 2 4 2 4 4 16 2 8
8/0.25 8/2 32/2 16/4 8/2 16/2 64/1 64/1 16/2 64/4
S S N N S S S S N N
A. flavus AFL1 AFL2 AFL3 AFL4 AFL5 AFL6 AFL7 AFL8
ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256
ⱖ256 8 8 ⱖ256 128 64 ⱖ256 64
8/2 2/4 4/4 8/128 16/64 16/8 4/16 8/8
S N N N N S S S
16 2 2 32 8 16 8 4
32/2 8/0.5 32/0.25 16/4 64/1 8/4 16/1 16/2
S S S S S S S N
ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256
ⱖ256 64 64 ⱖ256 ⱖ256 128 ⱖ256 128
8/4 2/8 8/64 8/128 16/128 16/16 4/16 16/16
S S N N N S S S
16 4 2 32 16 16 16 8
32/2 8/0.5 32/0.25 64/4 64/1 8/4 16/1 16/2
S S S S S S S S
A. terreus AT1 AT2
ⱖ256 ⱖ256
128 8/2 ⱖ256 2/128
S N
2 2
16/1 64/1
N N
ⱖ256 ⱖ256
128 8/8 ⱖ256 4/128
S N
4 4
16/2 64/2
N N
a
FK506/ FK506/VRC FK506/ FK506/AMB VRC FICIa AMB AMB FICIa
S, synergy (FICI of ⱕ0.5); N, no interaction (FICI of ⬎0.5 to ⱕ4).
7098
aac.asm.org
Antimicrobial Agents and Chemotherapy
November 2015 Volume 59 Number 11
Combination Interactions against Aspergillus Biofilms
TABLE 3 SMIC and FICI results with combinations of tacrolimus with ITC or POC against Aspergillus biofilms SMIC50 (g/ml)
SMIC80 (g/ml)
Strain
FK506 ITC
FK506/ FK506/ITC POC ITC FICIa
FK506/ FK506/POC POC FICIa FK506 ITC
A. fumigatus AF1 AF2 AF3 AF4 AF5 AF6 AF7 AF8 AF9 AF10
ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256
128 16 64 128 64 128 32 32 128 32
16/64 8/32 64/8 32/64 16/32 8/64 32/16 32/16 32/64 16/16
N N N N N N N N N N
8 4 16 4 8 4 2 16 64 4
4/4 8/4 16/8 8/4 8/4 16/4 64/2 16/8 4/32 4/4
N N N N N N N N N N
ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256
A. flavus AFL1 AFL2 AFL3 AFL4 AFL5 AFL6 AFL7 AFL8
ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256
ⱖ256 64 32 ⱖ256 128 64 ⱖ256 64
16/128 8/32 32/16 32/128 32/64 32/32 64/64 16/64
N N N N N N N N
ⱖ256 4 4 ⱖ256 8 32 128 32
32/128 8/2 32/2 16/128 64/4 8/16 16/64 16/16
N N N N N N N N
A. terreus AT1 AT2
ⱖ256 ⱖ256
128 16/64 ⱖ256 32/128
N N
64 16/16 ⱖ256 32/128
N N
a
FK506/ ITC
FK506/ITC FICIa POC
FK506/ POC
FK506/POC FICIa
128 64 128 ⱖ256 128 ⱖ256 128 64 ⱖ256 64
128/128 64/32 64/64 128/128 128/64 128/64 64/64 32/32 128/128 128/64
N N N N N N N N N N
32 32 32 64 32 32 32 32 128 32
8/16 8/16 32/16 64/64 16/16 16/16 64/32 32/16 64/64 64/16
N N N N N N N N N N
ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256 ⱖ256
ⱖ256 128 64 ⱖ256 ⱖ256 128 ⱖ256 ⱖ256
128/64 128/64 32/32 128/64 128/128 128/128 128/64 128/128
N N N N N N N N
ⱖ256 32 32 ⱖ256 32 64 ⱖ256 32
128/128 8/32 32/16 64/32 128/16 128/32 128/128 16/16
N N N N N N N N
ⱖ256 ⱖ256
128 128/64 N ⱖ256 128/128 N
128 64/64 N ⱖ256 128/128 N
N, no interaction (FICI of ⬎0.5 to ⱕ4).
AMB and VRC may result from the inhibition of the Hsp90-activated calcineurin signaling pathway. In summary, our results suggest that calcineurin pathway inhibition in combination with classical antifungal agents has therapeutic potential in Aspergillus biofilm-associated fungal infections. Due to the immunosuppressive properties of calcineurin inhibitors, the clinical use would ultimately require a selectively fungal calcineurin pathway-targeted agent without collateral effects on human cells. ACKNOWLEDGMENTS This work was supported by grants 31400131 (Lujuan Gao) and 81401677 (Yi Sun) from the National Natural Science Foundation of China. The funders had no role in study design, data analysis, decision to publish, or preparation of the manuscript. We declare no conflicts of interest.
REFERENCES 1. Kontoyiannis DP, Bodey GP. 2002. Invasive aspergillosis in 2002: an update. Eur J Clin Microbiol Infect Dis 21:161–172. http://dx.doi.org/10 .1007/s10096-002-0699-z. 2. Loussert C, Schmitt C, Prevost MC, Balloy V, Fadel E, Philippe B, Kauffmann-Lacroix C, Latge JP, Beauvais A. 2010. In vivo biofilm composition of Aspergillus fumigatus. Cell Microbiol 12:405– 410. http://dx .doi.org/10.1111/j.1462-5822.2009.01409.x. 3. Müller FM, Seidler M, Beauvais A. 2011. Aspergillus fumigatus biofilms in the clinical setting. Med Mycol 49(Suppl 1):S96 –S100. http://dx.doi .org/10.3109/13693786.2010.502190. 4. Seidler MJ, Salvenmoser S, Muller FM. 2008. Aspergillus fumigatus forms biofilms with reduced antifungal drug susceptibility on bronchial epithelial cells. Antimicrob Agents Chemother 52:4130 – 4136. http://dx.doi.org /10.1128/AAC.00234-08. 5. Singhal D, Baker L, Wormald PJ, Tan L. 2011. Aspergillus fumigatus biofilm on primary human sinonasal epithelial culture. Am J Rhinol Allergy 25:219 –225. http://dx.doi.org/10.2500/ajra.2011.25.3622.
November 2015 Volume 59 Number 11
6. Steinbach WJ, Schell WA, Blankenship JR, Onyewu C, Heitman J, Perfect JR. 2004. In vitro interactions between antifungals and immunosuppressants against Aspergillus fumigatus. Antimicrob Agents Chemother 48:1664 – 1669. http://dx.doi.org/10.1128/AAC.48.5.1664-1669.2004. 7. Shirazi F, Kontoyiannis DP. 2013. The calcineurin pathway inhibitor tacrolimus enhances the in vitro activity of azoles against Mucorales via apoptosis. Eukaryot Cell 12:1225–1234. http://dx.doi.org/10.1128/EC.00138-13. 8. Uppuluri P, Nett J, Heitman J, Andes D. 2008. Synergistic effect of calcineurin inhibitors and fluconazole against Candida albicans biofilms. Antimicrob Agents Chemother 52:1127–1132. http://dx.doi.org/10.1128/AAC.01397-07. 9. Pierce CG, Uppuluri P, Tristan AR, Wormley FL, Jr, Mowat E, Ramage G, Lopez-Ribot JL. 2008. A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing. Nat Protoc 3:1494–1500. http://dx.doi.org/10.1038/nprot.2008.141. 10. Clinical and Laboratory Standards Institute. 2008. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard. CLSI M38-A2. Clinical and Laboratory Standards Institute, Wayne, PA. 11. Ramage G, Vande Walle K, Wickes BL, Lopez-Ribot JL. 2001. Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrob Agents Chemother 45:2475–2479. http://dx.doi .org/10.1128/AAC.45.9.2475-2479.2001. 12. Tobudic S, Kratzer C, Lassnigg A, Graninger W, Presterl E. 2010. In vitro activity of antifungal combinations against Candida albicans biofilms. J Antimicrob Chemother 65:271–274. http://dx.doi.org/10.1093/jac/dkp429. 13. Narreddy S, Manavathu E, Chandrasekar PH, Alangaden GJ, Revankar SG. 2010. In vitro interaction of posaconazole with calcineurin inhibitors and sirolimus against zygomycetes. J Antimicrob Chemother 65:701–703. http://dx.doi.org/10.1093/jac/dkq020. 14. Robbins N, Uppuluri P, Nett J, Rajendran R, Ramage G, Lopez-Ribot JL, Andes D, Cowen LE. 2011. Hsp90 governs dispersion and drug resistance of fungal biofilms. PLoS Pathog 7:e1002257. http://dx.doi.org/10 .1371/journal.ppat.1002257. 15. Rajendran R, Mowat E, Jones B, Williams C, Ramage G. 24 April 2015. Prior in vitro exposure to voriconazole confers resistance to amphotericin B in Aspergillus fumigatus biofilms. Int J Antimicrob Agents http://dx.doi .org/10.1016/j.ijantimicag.2015.03.006.
Antimicrobial Agents and Chemotherapy
aac.asm.org
7099
