Environmental Circulation of Aspergillus fumigatus With Reduced Susceptibility to Agricultural Triazole in Brazil: Clonal Dissemination of Potentially Resistant Genotypes.

Abstract

Background: Aspergillus fumigatus resistance to triazole antifungals poses an increasing global health concern. Moreover, the cross-resistance between azole antifungal agents used in clinical settings and those applied in agriculture has become an important emerging issue.

Objectives: In this study, we investigated the five environmental A. fumigatus strains showing reduced susceptibility to tebuconazole.

Methods: Fungal strains were recovered from air samples collected around the homes of two patients with suspected aspergillosis caused by resistant isolates. Species identification was performed by sequencing the β-tubulin gene, and minimum inhibitory concentrations were determined by broth microdilution. The cyp51A gene was sequenced to detect mutations, and CRISPR-Cas9 genome editing was employed to investigate their influence on susceptibility patterns. Microsatellite genotyping was performed to assess genetic variability, followed by whole genome sequencing and single nucleotide polymorphism analysis.

Results: The environmental strains presented the same cyp51A genotype characterised by the M172V substitution and silent mutations. Microsatellite genotyping and whole genome sequencing confirmed that the strains were clonal. Functional validation demonstrated that the M172V and silent mutations partially contribute to reduced susceptibility to tebuconazole but are not the main mechanism of resistance involved. Analysis of polymorphisms in genes other than cyp51A revealed no resistance-conferring mutations.

Conclusions: The findings described herein suggest the possibility of local clonal dissemination of environmental strains under selective pressure from agricultural azoles in a major agribusiness region of the Midwest of Brazil. This study highlights the silent spread of potentially resistant genotypes in urban areas and reinforces the need for environmental surveillance and expanded genomic monitoring in South America.