The hazard of triazole fungicide: Cyproconazole can induce voriconazole-resistant in Aspergillusfumigatus isolates

Introduction: Increasing trends in resistance to azole is a global problem especially among Aspergillus species. This phenomenon would be appeared through two distinct pathways amongst the widespread use of azole fungicides in agriculture have been considered to be notable. In the present study, the effect of cyproconazole, the most used fungicide in Iranian wheat farms, was evaluated on inducing medical-triazole resistance in Aspergillusfumigatus isolates. Methods: A collection of 3 clinical azole-resistant TR34/L98H A. fumigatus isolates, 10 environmental wild type isolates, and 7 environmental TR34/L98H isolates were selected for investigation of the in vitro activity of fungicides.The Minimum Inhibition Concentrations (MICs) for the isolates were determined bythe broth microdilution method according to the Clinical and Laboratory Standards Institute document M38-A2 (CLSI, 2008) as previously described. Induction experiments were performed with cyproconazoleon GYEP agar plates containing cyproconazole. After each 5 passages, the possible-induced isolate(s) were subjected to AFST andsequencing of the CYP51A promoter and full coding gene was performed to detect mutations. Moreover, CYP51-protein homology modeling and molecule alignment studies were performed to identify similarity in molecule structure and docking modes using SWISS-MODEL and SEESAR softwares. Results: The MIC range was obtained as 0.05-0.1 mg/ml (50-100 μg/ml) for all investigated isolates.Induction experiments were performed with the maximum concentration of cyproconazole, by which the susceptible isolates were able to grow. Hence, a solution of 8.8μg/ml of cyproconazole was selected for the experiment. Among 10 susceptible isolates, only one strain showed high MIC value against voriconazole (MIC= 4μg/ml) after 25 passages. Nevertheless, sequencing of the CYP51A promoter and full coding gene didn’t revealed any mutations. The 3-D structure of voriconazole and cyproconazole showed similar structures harboring an azole ring by which they can bind to their ligand, heme.Cyproconazole , which have three nitrogen atoms in the aromatic ring, coordinated to the iron atom of heme through a hydrogen bond contact to residue Lys147, present in the active site of the A. fumigatusCYP51 homology model. Conclusion: Cyproconazole is being applied extensively in wheat farm in IRAN. Fortunately, the fungicide was not able to induce voriconazole-resistant A. fumigatus strains except for one strains. The low rate of induced-resistant isolate, lack of common mutations in both promoter and the whole CYP51A gene; and the results for molecular docking, demonstrate that cyproconazole may not play a key role in inducing azole-resistant isolates through environment rout.However, the potential ability of the fungicide to induce medically triazole resistant strains would not be neglectedover a long time of application.