Diagnostic characterization of Malassezia pachydermatis isolates from canine otitis externa

Canine otitis externa (OE) is a common cause of dog visits to the veterinary clinic and can be complicated by Malassezia pachydermatis overgrowth. Anecdotal evidence from a referral hospital suggested that treating M. pachydermatis OE with miconazole was no longer successful in a number of cases. This finding raised the question of whether the treatment failure was due to intrinsic resistance of a cryptic species or if azole resistance was emerging in M. pachydermatis clinical isolates. To identify the isolates and further characterize them, conserved loci were sequenced, phylogenetic analysis was performed, matrix-assisted laser/desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS) identification was used, and common biochemical assays were conducted. A subset of isolates could not be identified using the MALDI-ToF MS standard library and had different colony morphologies than the M. pachydermatis type strain, ATCC 14522. DNA sequencing the ITS region and portions of the LSU, SSU, and ACT1 genes revealed that the atypical isolates were M. pachydermatis. The unidentifiable isolates formed their own clade, Clade II, and the other isolates belonged to Clades I and III. A new MALDI-ToF reference spectrum was created that could be used to identify the atypical isolates to the species level. MALDI-ToF MS identification revealed that the clinical isolates identified to specific reference spectra, and never others. Identifications and which reference spectra the isolates matched did not change when using different growth media or protein extraction methods. A prominent peak between m/z 8700-8800 was identified in the mass spectra of the isolates, and the m/z value of the peak identified the phylogenetic clade. The isolates clustered into their phylogenetic clade using the mass spectra, and the prominent peak contributed to their clustering. Common biochemical assays did not differentiate the isolates but could be distinguished using common growth media. Sequencing of the miconazole drug target, ERG11, and a miconazole disk diffusion assay were used to assess susceptibility compared to the type strain and investigate substitutions to the drug target as a mechanism of reduced susceptibility. Multiple isolates had a reduced susceptibility to miconazole compared to the type strain. The Erg11 sequences revealed multiple substitutions compared to the type strain that did not affect miconazole susceptibility and were associated with the phylogenetic clade. Amino acid 302 substitutions were present in isolates with reduced susceptibility to miconazole. These results will allow for more-accurate species-level identification of M. pachydermatis and can be used by the diagnostic field. Identification of isolates with a reduced susceptibility to miconazole will help inform about antifungal usage and the emergence of reduced susceptibility to first-line azole antifungals. Future studies can identify the interaction of these amino acid substitutions with azole antifungals and the genetic determinants of the atypical phenotypes observed for the clinical isolates.

Name thesis:degree_name

M.S.

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

VMS - Pathobiology

Grantor

University of Illinois Urbana-Champaign

Year dc:date

2025

Author dc:creator

• Belcher, Cole Michael

Contributors dc:contributor

• Hoyer, Lois L
• Hung, Chien-Che
• Souza, Clarissa P
• Witola, William H

• Malassezia pachydermatis
• otitis externa
• dog

Statement dc:rights

• Copyright 2025 Cole M Belcher

Language dc:language

en

Handle dc:identifier

https://hdl.handle.net/2142/132694

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/132694