Classification of dermatophytes by a multilocus phylogenetic approach based on Tef-1α, beta tubulin and ITS genes

Objectives: Identification of dermatophytes to the species level is epidemiologically, ecologically and therapeutically significant. The use of phylogenetic species concepts based on rDNA internal transcribed spacer (ITS) regions have improved the taxonomy of dermatophytes. However, it has been shown that for some species, confirmation and refinement using other genes are needed. Indeed, there are problems with accurate definition and characterization of dermatophytes especially among the Trichophyton mentagrophytes series. Intra- and interspecies variations of the translation elongation factor 1-α (Tef-1α) gene were evaluated as a new identification marker in a wide range of dermatophytes. The aim of this study was to evaluate the discriminatory power of a phylogenetic tool based on concatenated sequences of three genomic regions including Tef-1α, ITS (ITS1 to ITS2) and beta-tubulin to differentiate: 1/ the anthropophilic Trichophyton interdigitale species from the zoophilic Trichophyton mentagrophytes 2/ the two closely related African anthropophilic species Trichophyton violaceum from Trichophyton soudanense. Methods: 26 well characterized strains of T. interdigitale/mentagrophytes have been selected including 3 IHEM reference strains; 30 well characterized strains of T. soudanense/T.violaceum including 2 IHEM reference strains were also included. All strains were submitted to ITS, beta-tubulin and Tef-1α PCR/sequencing. Moreover, identification was completed by real time PCR DermaGenius® (PathoNostics). Alignments were performed using MUSCLE in Seaview. The alignments for each of the three genes were then concatenated together based on the name of the sample and gaps were added between genes. Phylogenies were then inferred on the concatenated alignment using PhyML. Results: After generation of the phylogenetic tree by concatenation of the three sequence genes, the differentiation between T. interdigitale and T. mentagrophytes was clear. The 16 strains of T. interdigitale were well classified into one distinct group on the dendrogram. The 10 strains of zoophilic T. mentagrophytes were well distinct of the anthropophilic group and defined by another clade on the dendrogram. Reference strains were correctly classified into the two previously defined groups. Regarding the differentiation between T. soudanense and T. violaceum, the concatenation permitted to define two well distinct clades on the dendrogram. One clade containing 10 T. violaceum tested and the other containing 20 T. soudanense included in the study and one strain previously identified as T. violaceum . Reference strains were correctly classified into each corresponding clade. Conclusion: The concatenation of ITS, beta-tubulin and Tef-1α genes sequences allows the generation of a discriminating dendrogram between the zoophilic T. mentagrophytes and the anthropophilic T. interdigitale. This is less powerfull between the two closely related African anthropophilic species T. violaceum and T. soudanense. Anyway This multilocus phylogenetic approach allows to better define the species boundaries between these dermatophytes and facilitates the molecular characterization of these species in routine diagnostic.