Comparison of three culture media for testing susceptibility of <i>Malassezia pachydermatis</i> to four antifungal agents by a microdilution method and gradient diffusion strips

The yeast Malassezia pachydermatis frequently causes otitis externa and dermatitis in dogs and cats. This often requires prolonged antifungal therapy, raising concerns about reduced susceptibility. Antifungal susceptibility testing (AFST) for Malassezia spp. remains challenging because many protocols require complex lipid supplementation and are difficult to implement in routine veterinary settings. Here, we compared three culture media for AFST of M. pachydermatis: (i) a complex, lipid-enriched RPMI formulation (RPMI1); (ii) a simplified RPMI formulation supplemented with Tween 40 and Tween 80 (RPMI2); and (iii) a Sabouraud dextrose medium supplemented with Tween 80 (SD). Forty-nine canine clinical isolates were tested against ketoconazole, itraconazole, posaconazole and terbinafine using a colorimetric broth microdilution approach and a gradient diffusion method on solid media (EzyMIC®). RPMI1 broth was unstable after freezing, resulting in weak or ambiguous resazurin readings and frequent data exclusions, and the solid formulation of RPMI1 was unsuitable for reliable gradient diffusion interpretation. By contrast, RPMI2 medium provided consistent minimal inhibitory concentration (MIC) determinations for azoles in both solid and liquid media, with comparable MIC values. Conversely, terbinafine MICs were frequently elevated in RPMI-based liquid media, often exceeding the upper testing range, whereas lower and more readily interpretable MIC values were obtained on SD agar, suggesting medium-dependent effects on terbinafine assessment. The results obtained with SD media on azoles were more heterogeneous. Overall, RPMI2 offered a practical and reproducible option for azole susceptibility testing of M. pachydermatis, while terbinafine results may require cautious interpretation depending on the testing medium.