Emerging antifungal resistance in Trichophyton mentagrophytes: insights from susceptibility profiling and genetic mutation analysis

Abstract

ABSTRACTTrichophyton species, the leading cause of dermatophytosis globally, are increasingly resistant to antifungal treatments, concerns about effective management strategies. In light of the absence of established resistance criteria for terbinafine and azoles, coupled with a dearth of research on resistance mechanisms in Trichophyton, antifungal susceptibility and drug resistance gene diversity were analyzed across 64 T. mentagrophytes, 65 T. interdigitale, and 2 T. indotineae isolates collected in China between 1999 and 2024 and 101 published T. indotineae strains. Analyses of the minimum inhibitory concentrations (MICs) of terbinafine, itraconazole, voriconazole, posaconazole, and isavuconazole revealed a concerning increase in T. indotineae with terbinafine resistance, including two novel isolates from China. Compared with T. interdigitale, T. mentagrophytes presented higher terbinafine MICs but similar azole susceptibility. Notably, 27 T. interdigitale isolates were classified as non-wild-type for terbinafine. Genetic diversity was analyzed for the SQLE, CYP51A and CYP51B gene. Specifically, T. indotineae isolates presented SQLE protein changes linked to terbinafine resistance. SQLE diversity was linked to terbinafine sensitivity, whereas alterations in CYP51A were associated with itraconazole sensitivity, with notable statistical significance evident across various protein isoforms. The relationship between protein diversity and drug sensitivity is presented in detail. Together, these findings highlight a growing prevalence of antibiotic resistance among Trichophyton and identify potential target genes for new therapies, underscoring the need for ongoing monitoring and offering directions for novel therapeutics.

Keywords: CYP51A/B; SQLE; Trichophyton mentagrophytes complex; antifungal susceptibility; terbinafine.

Figure 1.

Phylogenetic trees of all strains with characteristics. (A) Maximum-likelihood tree. (B) Neighbor-joining tree. Phylogenetic trees were constructed using 101 reference strains of T. indotineae and 131 isolates from this study. ITS gene sequences were aligned with MAFFT 7.515, and phylogenetic analyses were performed using FastTree (ML) and TreeBest (NJ) with bootstrap analysis (1,000 replicates). The trees show the relationships among the strains, with bootstrap values (>70%) at branch nodes and a scale bar of 0.02 changes per nucleotide position. MT038040 was rooted at the midpoint due to inaccuracies in previous identifications. The circular phylogenetic tree presents the genetic relationships among Trichophyton species alongside the sensitivity to terbinafine by MIC values. Both the maximum-likelihood tree (A) and the neighbor-joining tree (B) accurately classified the isolates. Within the T. mentagrophytes complex, two primary clades are identified: one for T. indotineae and another comprising various ITS genotypes of T. mentagrophytes and T. interdigitale, shown in different colours. The outer layers of the tree indicate the geographical origins of the strains, mutations in the squalene epoxidase (SQLE) gene, and terbinafine sensitivity categorized as sensitive (<0.25 mg/L), moderate (0.25 mg/L ≤ MIC ≤ 1 mg/L), or resistant (>1 mg/L).

Figure 2.

Antifungal Comparison. Comparison of MICs of antifungal agents against Trichophyton mentagrophytes (T. m) and Trichophyton interdigitale (T. i) by Grouped Violin Plot. The graph displays the MIC values for terbinafine (TBF), voriconazole (VOR), posaconazole (POS), itraconazole (ITR), and isavuconazole (ISA) for both species. The abscissa represents the antifungal agents tested, grouped by class, while the ordinate indicates the log₂ MIC values. Statistical analysis indicated significant differences in MICs for terbinafine and voriconazole between T. mentagrophytes and T. interdigitale (** for p < 0.001, * for p < 0.05), while no significant differences between the two species were found for posaconazole, itraconazole, and isavuconazole.

Figure 3.

Alterations of SQLE amino acid sequence in Trichophyton spp. Amino acid diversity of 11 types of SQLE alterations, including those in the WT (wild-type) protein. Sites of differences among the protein sequences are highlighted with red boxes.

Figure 4.

Antifungal sensitivity and amino acid alterations. (A) Terbinafine sensitivity and SQLE alterations. Distribution of alterations in the SQLE protein types among various isolates with different terbinafine minimum inhibitory concentrations (MICs). The x-axis represents the MIC of terbinafine (mg/L), whereas the y-axis represents the number of isolates (n). The coloured bars indicate different SQLE genotypes, including wild-type (SQLE-WT) and various mutations (SQLE-S1 to SQLE-S9). The majority of the wild-type isolates presented low MIC values, whereas the isolates with mutations presented relatively high MICs, indicating reduced susceptibility to terbinafine. (B) Azole sensitivity and CYP51A alterations. Distribution of MICs for four azole antifungal agents (isavuconazole, voriconazole, posaconazole, and itraconazole) against various CYP51A protein alterations in 131 T. mentagrophytes complex isolates. Each bar indicates the quantity of isolates corresponding to a specific CYP51A protein type, with colours used to differentiate between the various protein types. The x-axis shows the MIC values (mg/L), whereas the y-axis indicates the number of isolates. (C) Azole sensitivity and CYP51B alterations. The correlation between various mutations and resistance levels to antifungal drugs. Four bar charts depict the distribution of isolates with different CYP51B alterations across varying MICs for isavuconazole, voriconazole, posaconazole, and itraconazole; the y-axis indicates the number of isolates, and the x-axis indicates the MICs (mg/L). Different CYP51B protein types are indicated in different colours.

Figure 5.

Alterations of CYP51A amino acid sequence in Trichophyton spp. Amino acid diversity of 10 types of CYP51A protein alterations, including those in the CYP51A protein sequence of the ATCC reference strain. Sites of differences among the protein sequences are highlighted with red boxes.

Figure 6.

Alterations of CYP51B amino acid sequence in Trichophyton spp. Sequence alignment illustrating the 10 types of CYP51B protein alterations, including those in the CYP51B protein of the TIMM20114 reference strain. Sites of differences among the protein sequences are highlighted with red boxes.