Paper Title
Antifungal Susceptibility and Molecular Mechanisms of Azole Resistance Among Malassezia Isolates

Abstract
Introduction: The constant increase in the incidence of malassezioses is accompanied by the appearance or selection of species with antifungal resistance. Owing to the slow growth and lipid requirements of these yeasts, convenient and reliable antifungal drug susceptibility testing assays for Malassezia spp. are not widely available. It is therefore essential to be able to test the sensitivity of yeasts of the genus Malassezia to detect possible resistance and optimize the therapeutic choice. Material and methods: We studied using Etest strips the antifungal susceptibility profiles of 59 Malassezia strains (30 M. globosa and 29 M. restricta) isolated from patients and identified by real-time PCR. We then investigated the relative contribution of the number of copies of drug resistance genes (CDR1 and MDR1) and their overexpressions to the azole resistance of the 2 Malassezia species. Results: For M. globosa, 60% of the strains were sensitive, 30% were dose-dependent sensitive and 10% were resistant. Regarding M. restricta, 48.27% studied strains were sensitive to fluconazole, 24.13% were dose-dependent and 27.58% were resistant. The expression level of CDR1 (M. globosa) ranged from 0.23 to 15.89 times and that of MDR1 (M. restricta) ranged from 0.16 to 18.00. Hyper-expression of the CDR1 gene was observed in 6 M. globosa clinical isolates: 1 susceptible (10.78 times), 3 dose-dependent susceptible (12.13, 13.93 and 15.89 times) and 2 resistant (3.39 and 3.92 times). For M. restricta, hyper-regulation of the MDR1 gene was noted in 37.5% (3/8) resistant strains (11.08, 11.55 and 2.79 times) and 42.85% (3/7) dose-dependent sensitive strains (12.30, 18.00 and 3.14 times). Conclusion: Etest using Dixon modified medium was optimized for the testing of Malassezia antifungal drug susceptibility. It was demonstrated that overexpression of genes encoding efflux pumps is involved in azole resistance in Malassezia. However, it is likely that the presence of point mutations in the ERG11 gene or additional mutations in transcription factors, or other unknown mechanisms, likely exist. Keywords - Malassezia, Etest, Antifungal Susceptibility, Mechanisms of Resistance