Genetic analysis of common triazole resistance mechanisms in a collection of <i>Aspergillus lentulus</i> clinical isolates from the United States

<i>Aspergillus fumigatus</i> continues to be the leading cause of invasive aspergillosis. However, the number of cases by drug-resistant cryptic species has increased in recent years. <i>Aspergillus lentulus</i> is a sibling species of <i>Aspergillus</i> section <i>Fumigati</i> that can only be distinguished from <i>A. fumigatus</i> by molecular methods. The clinical importance of this species resides in its low susceptibility to triazoles and intrinsic resistance to amphotericin B, making invasive aspergillosis treatments extremely challenging and producing high mortality rates. In this study, we investigate known molecular mechanisms important for triazole resistance in <i>A. fumigatus</i> in a collection of 25 clinical <i>A. lentulus</i> isolates from the United States. Using CRISPR-Cas9 gene editing technology, we performed <i>cyp51A</i> and <i>hmg1</i> allele replacements between susceptible and resistant isolates. Phenotypic characterization of the resulting mutants, together with mRNA expression analyzes of <i>cyp51A</i>, <i>cyp51B,</i> and the putative ABC efflux pump, <i>abcC</i>, suggests that triazole resistance in our <i>A. lentulus</i> isolates is independent of the mechanisms studied.