Phylogenetic classification of Trichophyton mentagrophytes complex strains based on DNA sequences of nuclear ribosomal internal transcribed spacer 1 regions

Abstract

Using internal transcribed spacer 1 (ITS1) region ribosomal DNA sequences from 37 stock strains and clinical isolates provisionally termed Trichophyton mentagrophytes complex in Japan, we demonstrated the mutual phylogenetic relationships of these strains. Members of this complex were classified into 3 ITS1-homologous groups and 13 ITS1-identical groups by their sequences. ITS1-homologous group I consists of Arthroderma vanbreuseghemii, T. mentagrophytes human isolates, and several strains of T. mentagrophytes animal isolates. Five strains of Arthroderma simii form a cluster comprising ITS1-homologous group II. The Americano-European and African races of Arthroderma benhamiae, T. mentagrophytes var. erinacei, and one strain of a T. mentagrophytes animal isolate constitute ITS1-homologous group III. According to the phylogenetic tree constructed with Trichophyton rubrum as an outgroup, ITS1-homologous groups I and II comprised a monophyletic cluster and ITS1-homologous group III constituted another cluster which was rather distant from the others in the complex. This system was applicable to the phylogenetic analysis of closely related strains. Using this technique, human and animal isolates of T. mentagrophytes were also clearly distinguishable from each other.

FIG. 1

Gel image of PCR products prepared with primer pair 18SF1 and 58SR1 from DNA of various species of fungi. To determine the DNA sequence of the ITS1 region, a specific primer pair, 18SF1 and 58SR1, was used to amplify by PCR a DNA fragment from genomic DNA isolated from fungal cells. The PCR products were electrophoresed, and after being stained, the gel was visualized under UV irradiation. Lanes: M, HindIII-digested lambda phage DNA; 1, A. vanbreuseghemii VUT77008; 2, T. mentagrophytes human clinical isolate TIMM3295; 3, A. simii VUT77010; 4, A. benhamiae, Americano-European race, SM103; 5, T. rubrum TIMM3231; 6, Microsporum canis TIMM0765; 7, P. marneffei IFM41707; 8, C. albicans ATCC10231; 9, C. glabrata TIMM1062; 10, Trichosporon beigelii TIMM3140; 11, Mucor circinelloides TIMM1325.

FIG. 2

T. mentagrophytes complex and T. rubrum phylogenetic tree, based on ITS1 rDNA sequences. The NJ tree was constructed by using ITS1 sequence data for strains T. mentagrophytes complex and 11 T. rubrum (Table 1). The evolutionary distances between organisms are indicated by the horizontal branch lengths, which reflect the numbers of nucleotide substitutions per site along the branches from node to endpoint. The percentages of bootstrap samplings, derived from 1,000 samples which were supporting the interior branches, are noted. Members of the T. mentagrophytes complex were classified into 3 ITS1-homologous groups and 13 ITS1-identical groups (Table 2), and all strains of T. rubrum had identical ITS1 sequences. I, ITS1-homologous group I; II, ITS1-homologous group II; III, ITS1-homologous group III.

FIG. 3

Alignment of ITS1 sequences of T. mentagrophytes complex and T. rubrum. The ITS1 rDNA sequences of 13 ITS1-identical individual groups of the T. mentagrophytes complex (Table 2) and of T. rubrum were aligned by using the Clustal W computer program (11). Hyphens designate gaps added to permit alignment; asterisks indicate conserved bases. A. vanbreu., A. vanbreuseghemii; T.men. (animal1) to T.men. (animal4), T. mentagrophytes animal type 1 to 4 isolates, respectively; T.men.(human), T. mentagrophytes human isolate; A.simii(1) to A.simii(4), A. simii type 1s to 4, respectively; A.ben.(A/E) and A.ben.(Af), A. benhamiae Americano-European and African races, respectively; T.men.eri, T. mentagrophytes var. erinacei.