CoERG11 A395T mutation confers azole resistance in Candida orthopsilosis clinical isolates

SYNOPSIS Background: Candida orthopsilosis is a human fungal pathogen responsible for a wide spectrum of symptomatic infections. Evidence suggests that C. orthopsilosis is mainly susceptible to azoles, the most extensively used antifungals for treatment of these infections. However, fluconazole-resistant clinical isolates are reported. Objectives: This study evaluated the contribution of a single amino acid substitution in the azole target CoErg11 to the development of azole resistance in C. orthopsilosis. Methods: C. orthopsilosis clinical isolates (n = 40) were tested for their susceptibility to azoles, and their CoERG11 genes were sequenced. We used a SAT1 flipper driven transformation to integrate a mutated CoERG11 allele in the genetic background of a fluconazole-susceptible isolate. Results: Susceptibility testing revealed that 16 of 40 C. orthopsilosis clinical isolates were resistant to fluconazole and to at least another azole. We identified an A395T mutation in the CoERG11 coding sequence of azole-resistant isolates only that resulted in the non-synonymous amino acid substitution Y132F. The SAT1 flipper cassette strategy led to the creation of C. orthopsilosis mutants, which carried the A395T mutation in one or both CoERG11 alleles (heterozygous or homozygous mutant, respectively) in an azole susceptible genetic background. We tested mutant strains for azole susceptibility and for hot-spot locus heterozygosity. Either heterozygous or homozygous mutant strains exhibited an azole-resistant phenotype. Conclusions: These findings provide the first evidence that CoErg11 Y132F substitution confers multi-azole resistance in C. orthopsilosis.