Hazard of agricultural triazole fungicide: Does cyproconazole induce voriconazole resistance in Aspergillus fumigatus isolates?

Abstract

Background and purpose: The present study aimed to evaluate the effect of cyproconazole, the most used fungicide in Iranian wheat farms, on the induction of voriconazole resistance in Aspergillus fumigatus isolates.

Materials and methods: A collection of 20 clinical and environmental isolates were selected for investigation of the in vitro activity of fungicides. The minimum inhibitory concentrations (MICs) were determined by the documented broth microdilution method M38-A2 (CLSI, 2008). Induction experiments were performed and the possibly induced isolate(s) were subjected to antifungal susceptibility testing, sequencing of the CYP51A promoter, and full coding gene. Furthermore, CYP51-protein homology modeling and docking modes were evaluated using SWISS-MODEL (https://swissmodel.expasy.org/) and SEESAR software (version 9.1).

Results: Among 10 susceptible isolates, only one strain showed a high MIC value against voriconazole (MIC=4µg/ml) after 25 passages. Nevertheless, sequencing of the CYP51A promoter and full coding gene did not reveal any mutations. Cyproconazole, which has 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. fumigates Cyp51 homology model.

Conclusion: Cyproconazole is being applied extensively in wheat farms in Iran. According to the results, cyproconazole may not play a key role in the induction of azole resistance in the isolates through the environmental route. However, the potential ability of the fungicide to induce medically triazole-resistant strains over a long period of application should not be neglected.

Keywords: Cyproconazole; Fungicide; Homology modeling; Wheat; Aspergillus fumigatus.

Figure 1

Effect of cyproconazole exposure on Aspergillus fumigatus macroscopic and microscopic morphology. Color of the colony turned to white and the narrow-ended mycelia revealed no asexual reproduction structures after 25 passages with 10 µg/ml of cyproconazole (B). Control (A).

Figure 2

Mapping of amino acid residues which have a key role in hydrophobic and hydrogen bonds regarding the SWISS-MODEL results.

Figure 3

Results of ExPASy modeling server which made the obtained 3D structure reliable. The red star indicates the high |Z-score| for the predicted model (A) and the red line shows high local similarity to the target Aspergillus fumigatus CYP51B (B).

Figure 4

The 2D structure of voriconazole and cyproconazole. Binding modes of cyproconazole (A) compared to the medical triazoles (B) located in the active site of human and Aspergillus fumigatus Cyp51 (https://pubchem.ncbi.nlm.nih.gov).

Figure 5

The 3D representation of cyproconazole aligned structures of CYP51 with the ligands in their active site, constructed by using the SeeSAR software (version 9.1). The ligands are represented in balls and sticks and the colored coronas depict the contributions of each atom to the estimated binding affinity. In the red estimated binding affinity, red indicates unfavorable contribution. However, green refers to a favorable contribution and the bigger the sphere is, the stronger is the effect. No sphere means that the atom is not estimated to have a significant impact on the binding affinity. Cyproconazole binds to the Fe atom of HEM indirectly through Lys147.