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J Clin Microbiol. Apr 1999; 37(4): 920–924.
PMCID: PMC88625

Phylogenetic Classification and Species Identification of Dermatophyte Strains Based on DNA Sequences of Nuclear Ribosomal Internal Transcribed Spacer 1 Regions

Abstract

The mutual phylogenetic relationships of dermatophytes of the genera Trichophyton, Microsporum, and Epidermophyton were demonstrated by using internal transcribed spacer 1 (ITS1) region ribosomal DNA sequences. Trichophyton spp. and Microsporum spp. form a cluster in the phylogenetic tree with Epidermophyton floccosum as an outgroup, and within this cluster, all Trichophyton spp. except Trichophyton terrestre form a nested cluster (100% bootstrap support). Members of dermatophytes in the cluster of Trichophyton spp. were classified into three groups with ITS1 homologies, with each of them being a monophyletic cluster (100% bootstrap support). The Arthroderma vanbreuseghemii-Arthroderma simii group consists of A. vanbreuseghemii, A. simii, Trichophyton mentagrophytes isolates from humans, T. mentagrophytes var. quinckeanum, Trichophyton tonsurans, and Trichophyton schoenleinii. Arthroderma benhamiae, T. mentagrophytes var. erinacei, and Trichophyton verrucosum are members of the Arthroderma benhamiae group. Trichophyton rubrum and Trichophyton violaceum form the T. rubrum group. This suggests that these “species” of dermatophytes have been overclassified. The ITS1 sequences of 11 clinical isolates were also determined to identify the species, and all strains were successfully identified by comparison of their base sequences with those in the ITS1 DNA sequence database.

Dermatophytes (dermatomycetes) have the capacity to invade keratinized tissues of humans and other animals to produce an infection, dermatophytosis (dermatomycosis) (28). The phylogeny of dermatophytes, however, remains unclear because their members are phylogenetically and taxonomically very closely related, their phenotypic features are sometimes poor, and many isolates from medical and veterinary samples have lost their sexual activity (25). From a clinical point of view, for definition of species or for performance of an epidemiological study, it is important to have a reliable method for the identification of dermatophyte species. Molecular biological studies of the phylogeny of the fungi have been performed, primarily by using the G+C content of chromosomal DNA (4), total DNA homology (5), restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA) (6, 14, 15, 21, 23), random amplification of polymorphic DNA (10, 13, 17, 22), and determination of the base sequence of 18S (11) or 28S (16) rRNA or ribosomal DNA (rDNA). For dermatophytes, however, the phylogenetic relationships of species or species-specific sequences cannot be fully defined by these methods.

Specific DNA sequences of internal transcribed spacer (ITS) 1 (ITS1) of rDNA in the dermatophytes were therefore determined and were analyzed phylogenetically. ITS1 is located between the 18S and the 5.8S rDNAs. As reported previously, the variable ITS regions have been proven to be useful in resolving relationships between close taxonomic relatives (2, 3, 18), and in the field of medical mycology, several phylogenetic studies in which the ITS1 region and the primer system designed by Makimura et al. (20) or White et al. (29) were used were reported on recently (1, 20, 26, 27). We have reported that it is feasible to successfully differentiate between members of the Trichophyton mentagrophytes complex, the major dermatophytes, which are hard to identify by their morphological features, by demonstrating their phylogenetic relationship by comparing the base pair sequences of the ITS1 regions (20). In the present study we determined the phylogeny of the group of dermatophytes, including the genera Trichophyton, Microsporum, and Epidermophyton, and identified the species using the base pair sequences of ITS1.

MATERIALS AND METHODS

Fungal strains.

The 12 standard strains and 11 clinical isolates of dermatophytes used in this study are described in Tables Tables11 and and2.2. The clinical strains were isolated in Japan and were identified by their morphological features, but the species of two of them could not be specified because they did not have typical microscopic structures.

TABLE 1
Standard strains of dermatophytes used in this study
TABLE 2
Clinical isolates used in this study

Preparation of DNA from fungal cells.

All fungal strains were grown on Sabouraud dextrose agar (peptone, 1% [wt/vol]; glucose, 1% [wt/vol]; agar, 1.5% [wt/vol]) at 27°C for 5 days. Rapid preparation of DNA from strains was performed by the method described by the authors (20). A small amount of mycelium grown on Sabouraud dextrose agar was placed in lysis buffer (200 mM Tris-HCl [pH 8.0], 0.5% [wt/vol] sodium dodecyl sulfate, 250 mM NaCl, 25 mM EDTA) and crushed with a conical grinder. It was then incubated at 100°C for 15 min and mixed with 150 μl of 3.0 M sodium acetate, kept at −20°C for 10 min, and then centrifuged at 10,000 × g for 5 min. The supernatant was extracted once with phenol-chloroform-isoamyl alcohol (25:24:1 [vol/vol]) and was subsequently extracted once with chloroform. The DNA was precipitated with an equal volume of isopropanol at −20°C for 10 min, washed with 0.5 ml of 99% ethanol, dried, and suspended in 50 μl of ultrapure water (Milli-Q Synthesis A10; Millipore). One microliter of solution was used as the template for PCR. The total time required to prepare the DNA was 80 min.

Oligonucleotides.

The oligonucleotide primers, designed by the authors (20), (18SF1, 5′-AGGTTTCCGTAGGTGAACCT-3′; 58SR1, 5′-TTCGCTGCGTTCTTCATCGA-3′) were made by Amersham Pharmacia Biotech Co., Ltd. (Tokyo, Japan).

PCR.

Each PCR mixture contained 10 μl of 10× reaction buffer (Pharmacia), 100 μM (each) dATP, dCTP, dGTP, and dTTP (Pharmacia), 2.5 U of Taq polymerase (Pharmacia), 30 pmol of each primer, and DNA template solution. Ultrapure water was added to increase the volume to 100 μl. Each reaction mixture was heated to 94°C for 5 min, and PCR was performed under the following conditions: 94°C for 1 min, 60°C for 15 s, and 72°C for 15 s for 25 cycles. The thermal cycles were terminated by polymerization at 72°C for 10 min. The products were detected as a single band of 0.3 kbp by agarose gel electrophoresis and UV irradiation.

ITS1 DNA sequencing and phylogenetic analysis.

Both strands of the PCR products were directly sequenced with a DNA Sequencing Kit (Perkin-Elmer) with primers 18SF1 and 58SR1 and an automatic sequencer (Genetic Analyzer 310; Perkin-Elmer), according to the manufacturer’s instructions.

The ITS1 sequences of the standard strains used in this study and of members of the T. mentagrophytes complex (Arthroderma vanbreuseghemii, DDBJ/EMBL/GenBank accession no. AB011488; Arthroderma benhamiae [Americano-European race], accession no. AB011457; Arthroderma benhamiae [African race], accession no. AB011454; Arthroderma simii, accession no. AB011461; T. mentagrophytes isolates from humans, accession no. AB011463; T. mentagrophytes var. erinacei, accession no. AB011455; Trichophyton rubrum, accession no. AB011453), as reported by the authors (20), were aligned by using the Clustal W computer program (12) and GENETYX-MAC 10.1 software (Software Development Co., Ltd., Tokyo, Japan). Phylogenetic trees were then constructed by the DNA maximum-likelihood (ML) method in the PHYLIP program (Phylogeny Inference Package), version 3.5c (8), and the neighbor-joining (NJ) (24) method in the NJPLOT program (9). Bootstrap (7) analysis with the Clustal W program was performed by taking 1,000 random samples from the multiple alignment. This provided a measure of how well supported parts of the tree are, given the data set and the method used to construct the tree. The tree was rooted with Epidermophyton floccosum as an outgroup, because it was shown that this species is phylogenetically distant from other dermatophytes by the RFLP analysis of mtDNA (15). The evolutionary distance between organisms is indicated by the horizontal branch length, which reflects the number of nucleotide substitutions per site along that branch from the node to the endpoint. In the NJ tree, the percentage of bootstrap samplings that support the interior branches is noted.

Nucleotide sequence accession numbers.

The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence database with the accession numbers presented in Table Table11.

RESULTS

Each strain of the dermatophytes tested was shown to have unique ITS1 base sequences, and the two strains of T. mentagrophytes var. quinckeanum were found to be identical. Phylogenetic trees were prepared by the NJ (Fig. (Fig.1)1) and ML (Fig. (Fig.2)2) methods and were constructed from data for 18 species of dermatophytes. The sequences of their ITS1 regions are presented in Fig. Fig.3;3; the sizes of these regions ranged from 175 to 293 bp. In the NJ tree, Trichophyton spp. and Microsporum spp. form cluster A, and all Trichophyton spp. except Trichophyton terrestre form cluster B (100% bootstrap support). The members of the dermatophytes in cluster B were classified into three groups with ITS1 homology (groups a, b, and c) according to their ITS1 DNA sequences, and each of them is a monophyletic cluster (100% bootstrap support). ITS1 homology group a (A. vanbreuseghemii-A. simii group) consists of A. vanbreuseghemii, A. simii, T. mentagrophytes isolates from humans, T. mentagrophytes var. quinckeanum, Trichophyton tonsurans, and Trichophyton schoenleinii. Both races of A. benhamiae, T. mentagrophytes var. erinacei, and Trichophyton verrucosum are members of ITS1 homology group b (A. benhamiae group). T. rubrum and Trichophyton violaceum form a cluster of ITS1 homology group c (T. rubrum group). The phylogenetic relationships mentioned above were also supported by the ML tree.

FIG. 1
NJ tree of dermatophytes on the basis of their ITS1 sequences. The NJ tree was constructed with data for standard strains of dermatophytes (see Table Table11 and the text). The numbers above the branches indicate the percentage of bootstrap samplings. ...
FIG. 2
ML tree of dermatophytes on the basis of their ITS1 sequences. The ML tree was constructed with data for standard strains of dermatophytes (see Table Table11 and the text). A, cluster of Trichophyton spp. and Microsporum spp.; B, cluster of all ...
FIG. 3
Alignment of ITS1 sequences of dermatophytes. The sequences of 18 species of dermatophytes (see Table Table11 and the text) were aligned by using the Clustal W (12) and GENETYX-MAC 10.1 (Software Development Co., Ltd.) computer programs. Hyphens ...

The ITS1 sequences of 11 clinical isolates were also determined. Each of the morphologically identified strains of dermatophytes (three strains of T. rubrum, four strains of T. mentagrophytes, one strain of T. violaceum, and one strain of Microsporum canis) was shown to have ITS1 base pair sequences identical to that of the respective standard strain tested (Table (Table2).2). Two morphologically unidentifiable strains were then subjected to ITS1 sequencing; one was revealed to be T. rubrum, and the other was shown to be T. violaceum on the basis of the ITS1 DNA sequence database constructed by the authors.

DISCUSSION

Using ITS1 rDNA sequences from 12 newly sequenced and 7 previously reported strains of fungi, we have described the phylogeny of members of the dermatophytes. The phylogenetic relationship based on the ITS1 DNA sequence alignment of meiosporic (perfect) and mitosporic (imperfect) states of the strains agreed with the proposed taxonomic connection in their sexual compatibility (25), RFLP analysis of mtDNA (14, 15, 21, 23), and a phylogenetic study based on 18S rDNA (11) or 28S rDNA (16) sequences. In particular, the last two papers dealt with the base sequences of the rDNA region, but they omitted A. benhamiae, T. verrucosum, and T. mentagrophytes var. erinacei, which together formed a unique cluster, cluster B-b, in the phylogenetic trees that appear in Fig. Fig.11 and and2.2. Thus, because of their highly variable ITS1 sequences, the phylogenetic analysis of the members of the dermatophytes was achieved in more accurate detail.

We stated earlier that there were three ITS1 homology groups (groups a, b, and c) in the cluster of Trichophyton spp. The clusters of ITS1 homology group a (A. vanbreuseghemii-A. simii group), group b (A. benhamiae group), and group c (T. rubrum group) were 100% supported by bootstrap analysis. A. vanbreuseghemii, A. simii, A. benhamiae, and anamorphic species of T. mentagrophytes constitute the T. mentagrophytes complex (20, 25) because they are difficult to distinguish from each other by their morphological features. By using sexual compatibility (25), RFLP analysis of mtDNA (21), or DNA sequence analysis of ITS1 (20), the T. mentagrophytes complex was shown to be monophyletic, and each of the members was identified. The present study showed that the species of dermatophytes pathogenic for humans or animals, T. tonsurans, T. schoenleinii, and T. verrucosum, are members of the A. vanbreuseghemii-A. simii group (Fig. (Fig.1,1, cluster a) or the A. benhamiae group (Fig. (Fig.1,1, cluster b). This suggests that these medically important dermatophytes have been overclassified. However, since each of the “species” has a unique phenotype, pathogenicity, and host-specific affinity, it is reasonable to retain their “species” identifications in order to identify the pathogen.

In addition to establishing the significance of the ITS1 region from a taxonomic standpoint, we also identified these clinically important species using the ITS1 DNA sequence database. With this system, not only the morphologically identified strains of T. mentagrophytes, T. rubrum, T. violaceum, and M. canis but also the two strains of morphologically unidentifiable strains, T. rubrum and T. violaceum, were successfully identified. This ITS1-based identification system saves time (it takes 2 to 3 days) and is accurate and applicable even to strains with atypical morphological features.

Further evaluation of the phylogenetic analysis and identification system, both of which are based on ITS1 rDNA sequences, is under way in our laboratory with other species and strains. Moreover, a new approach to the detection and identification of pathogenic fungi from clinical specimens, i.e., skin, nail, or hair samples, with this database, along with previously reported molecular diagnostic systems (19), is also in progress in our laboratory.

ACKNOWLEDGMENTS

We thank Takashi Sugita, Department of Microbiology, Meiji College of Pharmacy, for technical advice.

This study was partly supported by a research grant for bioscience from the Sapporo Bioscience Foundation of Japan and the Proposal-Based Advanced Industrial Technology R & D Program (grant B-276) of the New Energy and Industrial Technology Development Organization (NEDO) of Japan.

REFERENCES

1. Attili D S, de Hoog G S, Pizzirani-Kleiner A A. rDNA-RFLP and ITS1 sequencing of species of the genus Fonsecaea, agents of chromoblastomycosis. Med Mycol. 1998;36:219–225. [PubMed]
2. Berbee M L, Yoshimura A, Sugiyama J, Taylor J W. Is Penicillium monophyletic? An evaluation of phylogeny in the family Trichocomaceae from 18S, 5.8S and ITS ribosomal DNA sequence data. Mycologia. 1995;87:210–222.
3. Carbone I, Kohn L M. Ribosomal DNA sequence divergence within internal transcribed spacer 1 of the Sclerotiniaceae. Mycologia. 1993;85:415–427.
4. Davidson F D, Mackenzie D W R, Owen R J. Deoxyribonucleic acid base compositions of dermatophytes. J Gen Microbiol. 1980;118:465–470. [PubMed]
5. Davidson F D, Mackenzie D W R. DNA homology studies in the taxonomy of dermatophytes. Sabouraudia. 1984;2:117–123. [PubMed]
6. de Bièvre C, Dauguet C, Nguyen V H, Iburahim-Granet O. Polymorphism in mitochondria DNA of several Trichophyton rubrum isolates from clinical specimens. Ann Inst Pasteur Microbiol. 1987;138:719–727. [PubMed]
7. Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 1985;39:783–791.
8. Felsenstein J. PHYLIP (Phylogeny Inference Package), version 3.5c. Seattle: University of Washington; 1995.
9. Gouy M. NJPLOT. Lyon, France: University of Lyon 1; 1995.
10. Graser Y, el Fari M, Presber W, Sterry W, Tietz H J. Identification of common dermatophytes (Trichophyton, Microsporum, Epidermophyton) using polymerase chain reactions. Br J Dermatol. 1998;138:576–582. [PubMed]
11. Harmsen D, Schwinn A, Weig M, Bröcker E-B, Heesemann J. Phylogeny and dating of some pathogenic keratinophilic fungi using small subunit ribosomal RNA. J Med Vet Mycol. 1995;33:299–303. [PubMed]
12. Higgins D. Clustal W, version 1.6. Cambridge, United Kingdom: The European Bioinformatics Institute; 1996.
13. Kano R, Nakamura Y, Watari T, Watanabe S, Takahashi H, Tsujimoto H, Hasegawa A. Identification of clinical isolates of Microsporum canis and M. gypseum by random amplification of polymorphic DNA (RAPD) and Southern hybridization analysis. Mycoses. 1998;41:139–143. [PubMed]
14. Kawasaki M, Aoki M, Ishizaki H, Nishio K, Mochizuki T, Watanabe S. Phylogenetic relationships of the genera Arthroderma and Nanizzia referred from mitochondrial DNA analysis. Mycopathologia. 1992;118:95–102. [PubMed]
15. Kawasaki M, Aoki M, Ishizaki H, Nishimura K, Miyaji M. Phylogeny of Epidermophyton floccosum and other dermatophytes. Mycopathologia. 1996;134:121–128. [PubMed]
16. Leclerc M C, Philippe H, Guého E. Phylogeny of dermatophytes and dimorphic fungi based on large subunit ribosomal RNA sequence comparisons. J Med Vet Mycol. 1994;32:331–341. [PubMed]
17. Liu D, Coloe S, Baird R, Pedersen J. Molecular determination of dermatophyte fungi using the arbitrarily primed polymerase chain reaction. Br J Dermatol. 1997;137:351–355. [PubMed]
18. Lobuglio K, Pitt J I, Taylor J W. Phylogenetic analysis of two ribosomal DNA regions indicates multiple independent losses of a sexual Talaromyces state among asexual Penicillium species in subgenus Biverticillium. Mycologia. 1993;85:592–604.
19. Makimura K, Murayama S Y, Yamaguchi H. Detection of a wide range of medically important fungi by the polymerase chain reaction. J Med Microbiol. 1994;40:358–364. [PubMed]
20. Makimura K, Mochizuki T, Hasagawa A, Uchida K, Yamaguchi H. Phylogenetic classification of Trichophyton mentagrophytes complex strains based on DNA sequences of nuclear ribosomal internal transcribed spacer 1 regions. J Clin Microbiol. 1998;36:2629–2633. [PMC free article] [PubMed]
21. Mochizuki T, Takada K, Watanabe S, Kawasaki W, Ishizaki H. Taxonomy of Trichophyton interdigitale (Trichophyton mentagrophytes var. interdigitale) by restriction enzyme analysis of mitochondrial DNA. J Med Vet Mycol. 1990;28:191–196. [PubMed]
22. Mochizuki T, Sugie N, Uehara M. Random amplification of polymorphic DNA is useful for the differentiation of several anthropophilic dermatophytes. Mycoses. 1997;40:405–409. [PubMed]
23. Nishio K, Kawasaki M, Ishizaki H. Phylogeny of the genera Trichophyton using mitochondrial DNA analysis. Mycopathologia. 1992;117:127–132. [PubMed]
24. Saito N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4:406–425. [PubMed]
25. Takashio M. The Trichophyton mentagrophytes complex. In: Iwata K, editor. Recent advances in medical and veterinary mycology. Tokyo, Japan: University of Tokyo Press; 1977. pp. 271–276.
26. Uijthof J M J, van Belkum A, de Hoog G S, Haase G. Exophiala deimatitidis and Sarcinomyces phaeomuriformis: ITS1-sequencing and nutritional physiology. Med Mycol. 1998;36:143–151. [PubMed]
27. Wedde M, Müller D, Tintelnot K, de Hoog G S, Stahl U. PCR-based identification of clinically relevant Pseudallescheria/Scedosporium strains. Med Mycol. 1998;36:61–67. [PubMed]
28. Weitzman I, Summerbell R C. The dermatophytes. Clin Microbiol Rev. 1995;8:240–259. [PMC free article] [PubMed]
29. White T J, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis M A, Gelfand D H, Sninsky J J, White T J, editors. PCR protocols: a guide to methods and applications. San Diego, Calif: Academic Press; 1990.

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