CANINE MALASSEZIA PACHYDERMATIS FIELD STRAINS SUSCEPTIBILITY TO AZOLES: A SURVEY

Malassezia genus count 18 species, of which Malassezia pachydermatis (MP) is the most represented yeast in cutaneous mycobiome of animals, both as commensal and pathogen. MP is responsible of severe cutaneous infections in companion animals - particularly in dogs - causing dermatitis (MD) and otitis externa (MO). Therapeutic protocols for MP infections in companion animals involve azoles derivatives (miconazole, ketoconazole, itraconazole, fluconazole) - both topical and systemic - followed by terbinafine, chlorhexidine, and selenium disulfide (SeS2). Antifungal treatments are effective to control MP overgrowth when primary causes and predisposing factors are resolved firstly, but the onset of antifungal resistance can lead to failure. In human medicine the antifungal resistance increased, leading the WHO to draft the Fungal Priority Pathogens List (FPPL) of public health importance, which should include MP due to zoonotic and resistance potential [1]. Aim of this work was to evaluate the antifungal susceptibility of MP field strains to a small library of azole compounds, isolated from canine patients with dermatitis and/or otitis externa. Fifty samples were collected from auricular (35) or cutaneous swabs (15) taken from canine patients with a suspected yeast etiology. Each sample was firstly cultured onto Sabouraud agar then isolates were identified by CHROM-agar® Malassezia. 36 strains were isolated: 26 from auricular and 10 from cutaneous swabs. Confirmation of the ID was performed with a nested PCR for internal transcribed spacer region of rRNA gene; all the 36 strains were identified as MP. Then, each isolate was tested with MIC assay for susceptibility to ketoconazole (KTZ), miconazole (MCZ) and fluconazole (FCZ), following CLSI guidelines for yeasts, using modified RPMI 1640 medium [2]. Since no breakpoints were established for susceptibility of MP to KTZ, MCZ and FCZ, criteria suggested by other authors were followed: susceptible (S) = MIC sample £ MIC50; intermediary susceptible (I) = MIC50 < MIC sample £ MIC90; resistant (R) = MIC sample > MIC90 [3]. Lowest MIC50 and MIC90 values were found with KTZ (0,016 μg/ml and 5 μg/ml, respectively), while MIC50 of MCZ and FCZ were 16 and 24 μg/ml, respectively, and MIC90 was 64 μg/ml for both. On 36 strains, 3 (8,33%) were resistant to KTZ, 1 (2,78%) was resistant to MCZ and 16 (2,78%) was resistant to FCZ. None of the strains were resistant to more than one azole tested compounds. Intermediary susceptibilities were found for 13/36 (36,11%) strains to KTZ, 11/36 (30,55%) to MCZ and 17/36 (47,22%) to FCZ. All the resistant strains to azoles were from auricular swabs from patients with otitis externa (5/26) and none of these were previously treated with azoles (only one was treated with florfenicol + terbinafine). Limitations of the study were represented by both the low sample size, as well as the difficulty of interpreting susceptibility in absence of breakpoints. Furthermore, a molecular investigation of the resistance mechanism needs to be carried out.