Diversidade genética e mecanismos de resistência antifúngica em isolados clínicos e ambientais de Aspergillusfumigatus

Abstract

Aspergillosis is a group of opportunistic diseases resulting from the inhalation of conidia from fungi of the genus Aspergillus. Therapeutic management is challenging due to both diagnostic limitations and the emergence of isolates resistant to triazole antifungals, which is frequently associated with prolonged use in hospitalized patients. Furthermore, there are reports of resistant isolates infecting patients with no prior history of treatment, suggesting that resistance is acquired in the environment as a result of excessive fungal exposure to triazole pesticides used in agriculture. Compounding this scenario, there are few studies on the epidemiology of resistance in South America, particularly in Brazil, a country with significant agricultural activity and specifically in the state of Mato Grosso do Sul (MS).This study aimed to analyze the frequency of resistant clinical and environmental isolates of Aspergillus spp. in Mato Grosso do Sul, investigate mutations, understand the influence of specific mutations on resistance phenotypes, and determine the genetic similarity of resistant A. fumigatus isolates. Between 2022 and 2024, 86 clinical isolates and 67 atmospheric air isolates of Aspergillus spp. were recovered. Molecular species identification was performed by sequencing the β-tubulin and calmodulin genes. Species delimitation was based on the monophyly of clades obtained through phylogenetic analyses. Antifungal susceptibility testing was conducted via broth microdilution, following the EUCAST E.DEF 9.3.2 method, to determine the minimum inhibitory concentration (MIC). Sequencing of the cyp51A gene was performed on resistant A. fumigatus isolates, and microsatellite genotyping was used to assess the genetic relationship between them. CRISPR-Cas9 experiments were conducted to investigate the contribution of mutations to the resistance phenotype. Of the 84 A. fumigatus isolates, seven showed MIC values above the cutoff points, indicating resistance. Among these, one clinical isolate (CMMS 154) harboring the G138S mutation was resistant to voriconazole (4 mg/L) and itraconazole (>16 mg/L), and one environmental isolate (CMMS 321) with the G54A mutation was resistant to posaconazole (8 mg/L) and itraconazole (> 16 mg/L). Five other environmental isolates (CMMS 340 to 344), resistant only to the agrochemical tebuconazole, presented the non-silent M172V mutation in addition to four silent mutations and microsatellite analysis indicated they belong to the same genetic group. Isolates CMMS 154 and CMMS 321 clustered distinctly but near wild-type isolates. Validation of the M172V and silent mutations demonstrated that they contribute, in part, to reduced susceptibility to tebuconazole, but are not the sole resistance mechanism involved. The results suggest the possible circulation and expansion of environmental isolates under selective pressure from agricultural azoles in an important agro-industrial region of the Brazilian Midwest. Furthermore, the silent dissemination of different resistant environmental genotypes in urban and rural areas was evidenced, reinforcing the need for genomic monitoring of aspergillosis agents in South America.