In Vitro Interactions of Antifungal agents and Tacrolimus against Aspergillus Biofilms

Transcription

1 AAC Accepted Manuscript Posted Online 24 August 2015 Antimicrob. Agents Chemother. doi: /aac Copyright 2015, American Society for Microbiology. All Rights Reserved. 1 In Vitro Interactions of Antifungal agents and Tacrolimus against Aspergillus Biofilms 2 3 Lujuan Gao a, Yi Sun b # Department of Dermatology, Zhongshan Hospital Fudan University, Shanghai, People s Republic of China a ; Department of Dermatology, Jingzhou Central Hospital, Jingzhou City, Hubei Province, People s Republic of China b. 8 9 Running Head: Combination interactions against Aspergillus Biofilms #Address correspondence to Yi Sun, @qq.com 1

2 Abstract Aspergillus biofilms were prepared from Aspergillus fumigatus, Aspergillus flavus, and Aspergillus terreus, respectively, via a 96-well plate-based method and the combined antifungal activity of tacrolimus with azoles or amphotericin B against Aspergillus biofilms was investigated via 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. 2

3 Invasive 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 minimum inhibitory concentrations (MICs) of antifungal agents required to kill biofilm structures of Aspergillus spp. are much higher than that required to kill the planktonic forms of the fungus (4, 5). It has been reported that combinations of tacrolimus, which targets the calcineurin, a Ca 2+ -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 synergistic inhibitory effect against Aspergillus biofilm 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 morphologic methods. The individual MICs of tested drugs, including tacrolimus, itraconazole (ITC), posaconazole (POC), VRC and AMB, for planktonic cells of Aspergillus spp. were determined according to 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 microtitre plates and an XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)- 3

4 H-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 μg/ml, μg/ml, μg/ml, μg/ml and μg/ml, respectively. The sessile minimum inhibitory concentration (SMIC 50 and SMIC 80 ) was defined as the concentration at which a 50% or 80% decrease in optical density (OD) would be detected in comparison to the controls (9). The interaction of tacrolimus with AMB or azoles referred the fractional inhibitory concentration index (FICI), which was classified as FICI 0.5, synergy; 0.5<FICI 4, no interaction; FICI>4, antagonism (11). All experiments were conducted in triplicate The ranges of MICs of individual tested drugs for planktonic Aspergillus spp. isolates were 256μg/ml for tacrolimus, 1-4μg/ml for ITC, 0.5-2μg/ml for VRC, μg/ml for POC and μg/ml for AMB, respectively (Table 1). The ranges of SMIC 50 for Aspergillus biofilms were 256μg/ml for tacrolimus, μg/ml for ITC, 8-256μg/ml for VRC, 2-256μg/ml for POC and 2-32μg/ml for AMB, respectively, while the ranges of SMIC 80 were 256μg/ml for tacrolimus, μg/ml for ITC, μg/ml for VRC, μg/ml for POC and 2-32 μg/ml for AMB, respectively (Table 2, 3). As described in previous studies, the MICs required to kill biofilm structures of Aspergillus spp. are much higher than that required to kill the planktonic forms (4, 5). However, when tacrolimus was combined with AMB against Aspergillus biofilms, the 4

5 SMIC 50 ranges of AMB and tacrolimus decreased to μg/ml and 2-64μg/ml, respectively, while the SMIC 80 ranges decreased to μg/ml and 2-32μg/ml, respectively (Table 2). Based on the FICIs calculated from SMIC 50, favorable synergistic effects were shown against biofilms of 12 Aspergillus spp. isolates, including 5 strains of A. fumigatus (5/10) and 7 strains of A. flavus (7/8). Based on the FICIs calculated from SMIC 80, favorable synergistic effects were shown against biofilms of 14 Aspergillus spp. isolates, including 6 strains of A. fumigatus (6/10) and all strains of A. flavus. However, no synergistic effect against A. terreus biofilms was observed. As for the combination of tacrolimus and VRC, the SMIC 50 of VRC and tacrolimus decreased to 1-128μg/ml and 2-16μg/ml, respectively. The FICIs based on SMIC 50 revealed synergistic effects against biofilms of 10 Aspergillus spp. isolates, including 5 strains of A. fumigatus (5/10), 4 strains of A. flavus (4/8), and 1 strain of A. terreus (1/2) (Table 2). The SMIC 80 of VRC and tacrolimus in this combination also decreased to 1-128μg/ml and 2-16μg/ml, respectively. The FICIs based on SMIC 80 revealed synergistic effects against biofilms of 13 strains, including 7 strains of A. fumigatus (7/10), 5 strains of A. flavus (5/8), and 1 strain of A. terreus (1/2). No synergistic effect was observed when tacrolimus combined with ITC or POC (Table 3). No antagonistic effect was observed in these combinations against Aspergillus biofilms Although our results revealed unfavorable antifugal effect of tacrolimus alone against planktonic cells and biofilm form of all Aspergillus spp. isolates studied, in vitro synergism against most of the biofilms generate from A. fumigatus and non-fumigatus Aspergillus isolates tested were observed when tacrolimus was combined with AMB or 5

6 VRC, in agreement with the findings for other fungal species (7, 8, 12). This drug synergism may be attributable to the following effects: tacrolimus, the calcineurin inhibitor, renders 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 Hsp90- activated calcineurin signalling pathway has been reported to be associated with antifungal resistance in Aspergillus biofilms (13, 14). 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 AMB and VRC may result from the inhibition of the Hsp90-calcineurin pathway. In summary, our results suggest that calcineurin pathway inhibition in combination with classical antifungal agents have 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 (Lujuan Gao) and (Yi Sun) from National Natural Science Foundation of China. The funders had no role in study design, data analysis, decision to publish, or preparation of the manuscript. This study does not present any conflicts of interest for us. 6

7 112 Table 1 MICs for planktonic Aspergillus spp Strain MICs (μg/ml) FK506 ITC VRC POC AMB A. fumigatus AF AF AF AF AF AF AF AF AF AF A. flavus AFL AFL AFL AFL AFL AFL AFL AFL A. terreus AT AT

8 113 Table 2 Combinations of tacrolimus with AMB or VRC against Aspergillus biofilms Strain SMIC 50 (μg/ml) a SMIC 50 (μg/ml) SMIC 80 (μg/ml) b SMIC 80 (μg/ml) FK506 VRC FK506/VRC FICI c AMB FK506/AMB FICI FK506 VRC FK506/VRC FICI AMB FK506/AMB FICI A. fumigatus AF /8 N 2 4/0.25 S /8 S 4 8/0.25 S AF /1 S 4 2/2 N /4 S 8 8/2 S AF /1 S 2 16/2 N /1 S 2 32/2 N AF /1 S 4 8/4 N /1 S 4 16/4 N AF /16 N 2 8/2 S /16 S 2 8/2 S AF /4 N 2 16/2 S /32 N 4 16/2 S AF /4 N 4 64/1 S /16 N 4 64/1 S AF /2 S 8 2/1 S /2 S 16 64/1 S AF /2 S 2 4/2 N /4 S 2 16/2 N AF /4 N 8 4/4 N /16 N 8 64/4 N A. flavus AFL /2 S 16 32/2 S /4 S 16 32/2 S AFL /4 N 2 8/0.5 S /8 S 4 8/0.5 S AFL /4 N 2 32/0.25 S /64 N 2 32/0.25 S AFL /128 N 32 16/4 S /128 N 32 64/4 S AFL /64 N 8 64/1 S /128 N 16 64/1 S AFL /8 S 16 8/4 S /16 S 16 8/4 S AFL /16 S 8 16/1 S /16 S 16 16/1 S 8

9 AFL /8 S 4 16/2 N /16 S 8 16/2 S A. terreus AT /2 S 2 16/1 N /8 S 4 16/2 N AT /128 N 2 64/1 N /128 N 4 64/2 N a,b SMIC 50 and SMIC 80, the sessile minimum inhibitory concentration, the concentration at which a 50%/80% decrease in absorbance would be detected in comparison to the control biofilm formed by the same fungal strain in the absence of antifungal drug; c FICI: fractional inhibitory concentration index; S: FICI 0.5; N: 0.5<FICI 4. 9

10 118 Table 3 Combinations of tacrolimus with ITC or POC against Aspergillus biofilm Strain SMIC 50 (μg/ml) a SMIC 50 (μg/ml) SMIC 80 (μg/ml) b SMIC 80 (μg/ml) FK506 ITC FK506/ITC FICI POC FK506/POC FICI FK506 ITC FK506/ITC FICI POC FK506/POC FICI A. fumigatus AF /64 N 8 4/4 N /128 N 32 8/16 N AF /32 N 4 8/4 N /32 N 32 8/16 N AF /8 N 16 16/8 N /64 N 32 32/16 N AF /64 N 4 8/4 N /128 N 64 64/64 N AF /32 N 8 8/4 N /64 N 32 16/16 N AF /64 N 4 16/4 N /64 N 32 16/16 N AF /16 N 2 64/2 N /64 N 32 64/32 N AF /16 N 16 16/8 N /32 N 32 32/16 N AF /64 N 64 4/32 N /128 N /64 N AF /16 N 4 4/4 N /64 N 32 64/16 N A. flavus AFL /128 N /128 N /64 N /128 N AFL /32 N 4 8/2 N /64 N 32 8/32 N AFL /16 N 4 32/2 N /32 N 32 32/16 N AFL /128 N /128 N /64 N /32 N AFL /64 N 8 64/4 N /128 N /16 N AFL /32 N 32 8/16 N /128 N /32 N AFL /64 N /64 N /64 N /128 N 10

11 AFL /64 N 32 16/16 N /128 N 32 16/16 N A. terreus AT /64 N 64 16/16 N /64 N /64 N AT /128 N /128 N /128 N /128 N a,b SMIC 50 and SMIC 80, the sessile minimum inhibitory concentration, the concentration at which a 50%/80% decrease in absorbance would be detected in comparison to the control biofilm formed by the same fungal strain in the absence of antifungal drug; c FICI: fractional inhibitory concentration index; S: FICI 0.5; N: 0.5<FICI 4. 11

12 REFERENCE 1. Kontoyiannis DP, Bodey GP Invasive aspergillosis in 2002: an update. Eur J Clin Microbiol Infect Dis 21: Loussert C, Schmitt C, Prevost MC, Balloy V, Fadel E, Philippe B, Kauffmann-Lacroix C, Latge JP, Beauvais A In vivo biofilm composition of Aspergillus fumigatus. Cell Microbiol 12: Muller FM, Seidler M, Beauvais A Aspergillus fumigatus biofilms in the clinical setting. Med Mycol 49 Suppl 1:S96-S Seidler MJ, Salvenmoser S, Muller FM Aspergillus fumigatus forms biofilms with reduced antifungal drug susceptibility on bronchial epithelial cells. Antimicrob Agents Chemother 52: Singhal D, Baker L, Wormald PJ, Tan L Aspergillus fumigatus biofilm on primary human sinonasal epithelial culture. Am J Rhinol Allergy 25: Steinbach WJ, Schell WA, Blankenship JR, Onyewu C, Heitman J, Perfect JR In vitro interactions between antifungals and immunosuppressants against Aspergillus fumigatus. Antimicrob Agents Chemother 48: Shirazi F, Kontoyiannis DP The calcineurin pathway inhibitor tacrolimus enhances the in vitro activity of azoles against Mucorales via apoptosis. Eukaryot Cell 12: Uppuluri P, Nett J, Heitman J, Andes D Synergistic effect of calcineurin inhibitors and fluconazole against Candida albicans biofilms. Antimicrob Agents Chemother 52:

13 Pierce CG, Uppuluri P, Tristan AR, Wormley FL, Jr., Mowat E, Ramage G, Lopez-Ribot JL 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: Institute CaLS Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. Approved standard M38-A2. CLSI, Wayne, PA. 11. Ramage G, Vande Walle K, Wickes BL, Lopez-Ribot JL Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrob Agents Chemother 45: Narreddy S, Manavathu E, Chandrasekar PH, Alangaden GJ, Revankar SG In vitro interaction of posaconazole with calcineurin inhibitors and sirolimus against zygomycetes. J Antimicrob Chemother 65: Robbins N, Uppuluri P, Nett J, Rajendran R, Ramage G, Lopez-Ribot JL, Andes D, Cowen LE Hsp90 governs dispersion and drug resistance of fungal biofilms. PLoS Pathog 7:e Rajendran R, Mowat E, Jones B, Williams C, Ramage G Prior in vitro exposure to voriconazole confers resistance to amphotericin B in Aspergillus fumigatus biofilms. Int J Antimicrob Agents doi: /j.ijantimicag