Comparative Transcriptomic and Lipidomic Analysis of Amphotericin B-Susceptible and Resistant Clinical Isolates of Malassezia spp

Abstract Malassezia spp. are lipid-dependent skin commensal yeasts associated with superficial infections and fungemia. Amphotericin B remains a key treatment for systemic infections despite its toxicity and decreased susceptibility. This study characterized amphotericin B susceptibility profiles and identified transcriptomic and lipidomic signatures of polyene resistance in clinical Malassezia isolates. Clinical isolates of M. sympodialis and M. furfur, and reference strains (CBS 7222 and CBS 1878), were evaluated using the modified M27-A3 CLSI protocol for MICs and subinhibitory concentrations. One isolate per species was susceptible (MIC 0.5–2.0 μg/mL), while another was resistant (MIC ≥ 16 μg/mL). Transcriptomes generated by Oxford Nanopore sequencing were analyzed using DESeq2 (|log2FC|>2, p < 0.05). Comparisons between susceptible and resistant isolates revealed few Differentially Expressed Genes (DEGs) that were not enriched for ergosterol biosynthesis. Interaction hubs indicated roles in nucleotide homeostasis, translation, chromatin regulation, and stress pathways. Lipidomic analysis by HPLC-QTOF-MS detected approximately 200 lipid species, including 55 Differential Lipid Molecules (DLMs) for M. sympodialis and 63 for M. furfur. Although global class abundances remained unchanged, resistant clinical isolates showed differences in the relative abundance of selected lipid species, with increased levels of long-chain fatty acids, diacylglycerols, and ceramides. Resistant M. sympodialis showed higher cholesterol levels. Integrated analysis separated susceptible and resistant groups, with DEGs–DLMs correlations highlighting networks in membrane organization, stress tolerance, and metabolism. These findings suggest that amphotericin B resistance may be associated with metabolic alterations and changes in membrane lipid composition, supporting the use of multiomics markers for detection and therapeutic strategies.