Molecular correlates of elevated terbinafine and fluconazole MICs in Trichophyton mentagrophytes complex isolates

Abstract The Trichophyton mentagrophytes complex is a major etiological group in dermatophytosis, within which Trichophyton indotineae has emerged as an important cause of recalcitrant infection, with elevated minimum inhibitory concentrations (MICs) to first-line antifungal agents being reported with increasing frequency. However, the molecular features accompanying elevated MICs to terbinafine and azoles in clinical isolates remain incompletely resolved. Seventy-two clinical isolates of T. mentagrophytes complex were identified by the Internal Transcribed Spacer (ITS) sequencing, and their susceptibility to eight antifungal agents was determined using the Clinical and Laboratory Standards Institute (CLSI) M38-Ed3 broth microdilution method. Isolates meeting predefined elevated-MIC criteria for terbinafine (≥0.5 µg/mL) and/or fluconazole (>16 µg/mL) were subjected to partial sequencing of squalene epoxidase (SQLE) and multidrug resistance (MDR2) genes. Relative SQLE and MDR2 transcript abundance was quantified by qPCR. T. indotineae was the predominant species (86.2%) and exhibited higher geometric mean MIC values for eight antifungal agents than the other members of the complex. Among isolates with elevated terbinafine MICs, nonsynonymous SQLE substitutions were identified, most frequently F397L, L393S, Q408L, and the combined F397L/A448T profile, these isolates also exhibited increased SQLE transcript levels (approximately 8.6 ± 2.7-fold) relative to susceptible comparators comprising two reference strains and two susceptible clinical isolates. In isolates with elevated fluconazole MICs, the MDR2 V438I substitution was detected exclusively in T. indotineae and was absent from T. mentagrophytes and T. interdigitale, favoring its interpretation as a species-associated polymorphism rather than a resistance-associated mutation. Elevated fluconazole MICs were additionally associated with significantly increased baseline MDR2 transcript levels (16.32 ± 2.1-fold relative to susceptible strains). Isolates with elevated MICs to both terbinafine and fluconazole showed greater SQLE and MDR2 transcript abundance than isolates with an elevated MIC to only one agent. Collectively, elevated terbinafine MICs were associated with canonical SQLE substitutions and increased SQLE expression, whereas elevated fluconazole MICs were associated primarily with higher baseline MDR2 expression. The MDR2 V438I variant appears to be species associated and showed no evident relationship with reduced fluconazole susceptibility. These findings define molecular associations within this clinical collection but do not establish causality.