Characteristics of the individual SNPs and combinations thereof impact the azole phenotype in TR34-mediated resistance genotypes

Author:

Yinggain Song (CN)

Abstract:

Background:
Aspergillus fumigatus is the main cause of invasive aspergillosis and triazole antifungals represent primary treatment options. However, the effectiveness of triazole therapy is hampered by the emergence of resistance, mainly caused by TR34/L98H and TR46/Y121F/T289A mutations in the Cyp51A-gene, which correspond with signature triazole resistance phenotypes. We investigated the occurrence of variant genotypes and phenotypes over a 29-year period in the Netherlands, and selected the novel variant to explore the impact of individual SNPs and combinations thereof on the azole phenotype in TR34-mediated resistance genotypes.
Methods:
We conducted a study on the prevalence of azole resistance among clinical Aspergillus fumigatus isolates in the Netherlands from 1994 to 2022, and identified 33 variants over this period. we focus on the novel, recent mutations and explore the effects of these SNPs: L98H, T289A, I364V, G448S, and the combination of these mutations TR34/L98H, TR34/L98H/T289A/G448S, TR34/L98H/T289A/I364V/G448S, on azole affinity and susceptibility. The mutation was introduced to the wildtype-cyp51 A. fumigatus strain by the CRISPR-Cas9 gene editing technique, with Hygromycin as the selection marker. Finally, in vitro susceptibility testing of A. fumigatus strains carrying the aforementioned mutations was conducted to confirm the azole phenotypes observed in clinical isolates.
Results:
The MICs of all four azoles against the mutated cyp51A strains harbored combination mutations were higher than that of the wild type, with highly elevated MICs of itraconazole, voriconazole, and isavuconazole. The L98H mutants and G448S mutants showed high voriconazole (8mg/l) and Isavuconazole (4mg/l), and high Agricultural Triazole; T289A mutant, and I364V mutant showed susceptible phenotype to all medical azoles, and Agricultural Triazole. TR34/L98H/T289A/G448S recombination mutant showed higher voriconazole (16mg/l) and Isavuconazole (>16mg/l). TR34/L98H, and L98H/T289A strains were resistant to the highest tested itraconazole concentration (>16 mg/l). The TR34/L98H/T289A/I364V/G448S showed consistent phenotype to the clinical strains, which are highly resistant to voriconazole but susceptible to Itraconazole. All mutants are susceptible to olorofim but resistant to agricultural fungicides Fluopyram.
Conclusions:
A. fumigatus resistance variant TR34/L98H/T289A/I364V/G448S corresponds with major shifts in phenotype. The mutations L98H and G448S contribute to the resistance of Voriconazole and Isavuconazole and reverse the high Itraconazole of TR34/L98H, which represent a major challenge in triazole resistance management and our current molecular diagnostic tools will include this mutation detection to predict the resistance phenotype. Furthermore, our study underscores the urgent need to implement effective measures that prevent triazole resistance selection in the environment

Abstract Number: 9

Conference Year: 2024

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