| Summary: | <i>Malassezia</i> species are fastidious and slow-growing yeasts in which isolation from polymicrobial samples is hampered by fast-growing microorganisms. <i>Malassezia</i> selective culture media are needed. Although cycloheximide is often used, some fungi, including the chief human commensal <i>Candida albicans</i>, are resistant to this compound. This study aimed to test whether the macrolide rapamycin could be used in combination with cycloheximide to develop a <i>Malassezia</i>-selective culture medium. Rapamycin susceptibility testing was performed via microdilution assays in modified Dixon against two <i>M. furfur</i> and five <i>Candida</i> spp. The MIC was the lowest concentration that reduced growth by a minimum of 90%. Rapamycin ± cycloheximide 500 mg/L was also added to FastFung solid, and yeast suspensions were inoculated and incubated for 72 h. Rapamycin MICs for <i>Candida</i> spp. ranged from 0.5 to 2 mg/L, except for <i>C. krusei</i>, for which the MIC was >32 mg/L. <i>M. furfur</i> stains were rapamycin-resistant. Rapamycin and cycloheximide supplementation of the FastFung medium effectively inhibited the growth of non-<i>Malassezia</i> yeast, including cycloheximide-resistant <i>C. albicans</i> and <i>C. tropicalis</i>. Based on our findings, this “<i>MalaSelect</i>” medium should be further evaluated on polymicrobial samples for <i>Malassezia</i> isolation and culture.
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