Antifungal Susceptibility of Malassezia pachydermatis Isolates from Companion Animals and Genomic Insights into Resistance Mechanisms

<b>Background/Objectives</b>: <i>Malassezia pachydermatis</i> is a lipophilic yeast frequently associated with otitis externa and dermatological disorders in companion animals. This study aimed to evaluate the antifungal susceptibility of <i>M. pachydermatis</i> isolates from dogs and cats and to investigate the genomic determinants of reduced susceptibility. <b>Methods</b>: Susceptibility testing of 87 clinical isolates was performed using a modified CLSI broth microdilution method in Sabouraud dextrose broth supplemented with 1% Tween 80. The whole genome of ten representative isolates was sequenced and the genetic factors that are involved in drug resistance were investigated. <b>Results</b>: Ketoconazole, itraconazole, and terbinafine exhibited the highest efficacy, while miconazole and clotrimazole showed reduced activity. Whole genome sequencing revealed single nucleotide polymorphisms (SNPs) in genes that play a key role in the ergosterol biosynthesis pathway, particularly in <i>ERG11</i> and <i>ERG1</i>. While some specific amino acid substitutions (e.g., K446R in <i>ERG11</i>) were found only in isolates with elevated MIC values, no direct correlation with resistance could be unequivocally established. <b>Conclusions</b>: Genomic analyses also uncovered chromosomal mutations and the heterozygosity of certain isolates, suggesting that complex, multifactorial mechanisms may drive the development of drug resistance. These findings highlight the importance of standardized susceptibility testing and further genomic investigations to promote effective antifungal therapy in veterinary medicine.