JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1999, p. 920–924 Vol. 37, No.

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Phylogenetic Classification and Species Identification of Dermatophyte

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Strains Based on DNA Sequences of Nuclear Ribosomal
Internal Transcribed Spacer 1 Regions
KOICHI MAKIMURA,1* YOSHIKO TAMURA,1 TAKASHI MOCHIZUKI,2 ATSUHIKO HASEGAWA,3
YOSHITO TAJIRI,1 RYO HANAZAWA,1 KATSUHISA UCHIDA,1 HIUGA SAITO,1
1
AND HIDEYO YAMAGUCHI

Teikyo University Institute of Medical Mycology, Tokyo,1 Department of Dermatology, Kanazawa Medical University,
Ishikawa,2 and Department of Veterinary Medicine, Nihon University, Kanagawa,3 Japan
Received 4 September 1998/Returned for modification 5 November 1998/Accepted 24 December 1998

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 Tricho-
phyton 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 overclas-
sified. 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 in-
vade keratinized tissues of humans and other animals to pro-
duce 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 epide-
miological study, it is important to have a reliable method for
the identification of dermatophyte species. Molecular biologi-
cal studies of the phylogeny of the fungi have been performed,
primarily by using the G1C content of chromosomal DNA (4),
total DNA homology (5), restriction fragment length polymor-
phism (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 dermato-
phytes, 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 deter-
mined 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,
* Corresponding author. Mailing address: Teikyo University Insti-
tute of Medical Mycology, Otsuka, Hachioji, Tokyo 192-0395 Japan.
Phone: 81-426-78-3256. Fax: 81-426-74-9190. E-mail: makimura@main
.teikyo-u.ac.jp.
18), and in the field of medical mycology, several phylogenetic
studies in which the ITS1 region and the primer system de-
signed 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 der-
matophytes, 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, Microspo-
rum, 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 dermato-
phytes used in this study are described in Tables 1 and 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.
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 per-
formed by the method described by the authors (20). A small amount of myce-
lium 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 ml of 3.0 M sodium acetate, kept at 220°C for 10 min,
and then centrifuged at 10,000 3 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 vol-
ume of isopropanol at 220°C for 10 min, washed with 0.5 ml of 99% ethanol,
dried, and suspended in 50 ml of ultrapure water (Milli-Q Synthesis A10; Milli-
pore). 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, 59-AGGTTTCCGTAGGTGAACCT-39; 58SR1, 59-TTCGCTGCGTTC

920
VOL. 37, 1999 PHYLOGENY OF DERMATOPHYTES FROM ITS1 921

TABLE 1. Standard strains of dermatophytes used in this study set and the method used to construct the tree. The tree was rooted with Epider-
mophyton floccosum as an outgroup, because it was shown that this species is
ITS1 phylogenetically distant from other dermatophytes by the RFLP analysis of
Species Straina

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accession no. mtDNA (15). The evolutionary distance between organisms is indicated by the
horizontal branch length, which reflects the number of nucleotide substitutions
Trichophyton mentagrophytes SM72834IMI140690 AB017171 per site along that branch from the node to the endpoint. In the NJ tree, the
var. quinckeanum percentage of bootstrap samplings that support the interior branches is noted.
Trichophyton mentagrophytes SM72844IMI163337 Nucleotide sequence accession numbers. The nucleotide sequence data re-
var. quinckeanum ported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide se-
Trichophyton tonsurans TIMM12544IP133-73 AB017172 quence database with the accession numbers presented in Table 1.
Trichophyton schoenleinii TIMM3395 AB017173
Trichophyton violaceum TIMM3396 AB017174
Trichophyton terrestre TIMM33944CBS307.65 AB017175 RESULTS
Trichophyton verrucosum TIMM3328 AB017176
Microsporum gypseum TIMM33974CBS161.69 AB017177
Each strain of the dermatophytes tested was shown to have
Microsporum audouinii TIMM07574IP941 AB017178
Microsporum canis TIMM1502 AB017179 unique ITS1 base sequences, and the two strains of T. menta-
Microsporum cookei TIMM3398 AB017180 grophytes var. quinckeanum were found to be identical. Phylo-
Epidermophyton floccosum TIMM0431 AB017181 genetic trees were prepared by the NJ (Fig. 1) and ML (Fig. 2)
a
methods and were constructed from data for 18 species of
Arrows are used to indicate the history of the strain. CBS, Centraalbureau
voor Schimmelcultures, Barn, The Netherlands; IMI, International Mycological dermatophytes. The sequences of their ITS1 regions are pre-
Institute, Surrey, United Kingdom; Institut Pasteur, Paris, France; RV, Institute sented in Fig. 3; the sizes of these regions ranged from 175 to
de Medicine Tropicale, Antwerp, Belgium; SM, Department of Dermatology, 293 bp. In the NJ tree, Trichophyton spp. and Microsporum spp.
Shiga University of Medical Science, Otsu, Japan; TIMM, Teikyo University
Institute of Medical Mycology, Tokyo, Japan.
form cluster A, and all Trichophyton spp. except Trichophyton
terrestre form cluster B (100% bootstrap support). The mem-
bers of the dermatophytes in cluster B were classified into
three groups with ITS1 homology (groups a, b, and c) accord-
TTCATCGA-39) were made by Amersham Pharmacia Biotech Co., Ltd. (Tokyo,
Japan). ing to their ITS1 DNA sequences, and each of them is a
PCR. Each PCR mixture contained 10 ml of 103 reaction buffer (Pharmacia), monophyletic cluster (100% bootstrap support). ITS1 homol-
100 mM (each) dATP, dCTP, dGTP, and dTTP (Pharmacia), 2.5 U of Taq ogy group a (A. vanbreuseghemii-A. simii group) consists of
polymerase (Pharmacia), 30 pmol of each primer, and DNA template solution.
Ultrapure water was added to increase the volume to 100 ml. Each reaction
A. vanbreuseghemii, A. simii, T. mentagrophytes isolates from
mixture was heated to 94°C for 5 min, and PCR was performed under the humans, T. mentagrophytes var. quinckeanum, Trichophyton ton-
following conditions: 94°C for 1 min, 60°C for 15 s, and 72°C for 15 s for 25 cycles. surans, and Trichophyton schoenleinii. Both races of A. benha-
The thermal cycles were terminated by polymerization at 72°C for 10 min. The miae, T. mentagrophytes var. erinacei, and Trichophyton verru-
products were detected as a single band of 0.3 kbp by agarose gel electrophoresis
and UV irradiation. cosum are members of ITS1 homology group b (A. benhamiae
ITS1 DNA sequencing and phylogenetic analysis. Both strands of the PCR group). T. rubrum and Trichophyton violaceum form a cluster
products were directly sequenced with a DNA Sequencing Kit (Perkin-Elmer) of ITS1 homology group c (T. rubrum group). The phyloge-
with primers 18SF1 and 58SR1 and an automatic sequencer (Genetic Analyzer
310; Perkin-Elmer), according to the manufacturer’s instructions.
netic relationships mentioned above were also supported by
The ITS1 sequences of the standard strains used in this study and of members the ML tree.
of the T. mentagrophytes complex (Arthroderma vanbreuseghemii, DDBJ/EMBL/ The ITS1 sequences of 11 clinical isolates were also deter-
GenBank accession no. AB011488; Arthroderma benhamiae [Americano-Euro- mined. Each of the morphologically identified strains of der-
pean race], accession no. AB011457; Arthroderma benhamiae [African race],
accession no. AB011454; Arthroderma simii, accession no. AB011461; T. menta- matophytes (three strains of T. rubrum, four strains of T. men-
grophytes isolates from humans, accession no. AB011463; T. mentagrophytes var. tagrophytes, one strain of T. violaceum, and one strain of
erinacei, accession no. AB011455; Trichophyton rubrum, accession no. AB011453), Microsporum canis) was shown to have ITS1 base pair se-
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.,
quences identical to that of the respective standard strain
Ltd., Tokyo, Japan). Phylogenetic trees were then constructed by the DNA tested (Table 2). Two morphologically unidentifiable strains
maximum-likelihood (ML) method in the PHYLIP program (Phylogeny Infer- were then subjected to ITS1 sequencing; one was revealed to
ence Package), version 3.5c (8), and the neighbor-joining (NJ) (24) method in be T. rubrum, and the other was shown to be T. violaceum on
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 the basis of the ITS1 DNA sequence database constructed by
provided a measure of how well supported parts of the tree are, given the data the authors.

TABLE 2. Clinical isolates used in this study

Identification according to
Strain Source Morphological identification
ITS1 sequence

CN9708001 Tinea pedis Trichophyton rubrum Trichophyton rubrum

CN9708002 Tinea pedis Trichophyton rubrum Trichophyton rubrum
CN9708003 Tinea pedis Trichophyton rubrum Trichophyton rubrum
CN9708004 Tinea pedis Trichophyton mentagrophytes Trichophyton mentagrophytes
CN9708005 Tinea pedis Trichophyton mentagrophytes Trichophyton mentagrophytes
CN9708006 Tinea pedis Trichophyton mentagrophytes Trichophyton mentagrophytes
CN9708007 Tinea pedis Trichophyton mentagrophytes Trichophyton mentagrophytes
CN9710001 Tinea capitis Trichophyton violaceum Trichophyton violaceum
CN9703001 Tinea corporis Microsporum canis Microsporum canis
CN9708001 Tinea corporis Unidentifiablea Trichophyton rubrum
CN9802001 Kerion celsi Unidentifiable Trichophyton violaceum
a
Unidentifiable, strains in which the species could not be specified because they did not show the typical microscopic structures.
922 MAKIMURA ET AL. J. CLIN. MICROBIOL.

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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 1
and the text). The numbers above the branches indicate the percentage of bootstrap samplings. Branches without numbers have frequencies of less than 70%. A, cluster
of Trichophyton spp. and Microsporum spp.; B, cluster of all Trichophyton spp. except T. terrestre; a, Arthroderma vanbreuseghemii-A. simii group; b, A. benhamiae group;
c, T. rubrum group. K nuc, thousands of nucleotides.

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. erina-
cei, which together formed a unique cluster, cluster B-b, in the
phylogenetic trees that appear in Fig. 1 and 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

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
1 and the text). A, cluster of Trichophyton spp. and Microsporum spp.; B, cluster of all Trichophyton spp. except T. terrestre; a, A. vanbreuseghemii-A. simii group; b, A.
benhamiae group; c, T. rubrum group. K nuc, thousands of nucleotides.
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FIG. 3. Alignment of ITS1 sequences of dermatophytes. The sequences of 18 species of dermatophytes (see Table 1 and the text) were aligned by using the Clustal
W (12) and GENETYX-MAC 10.1 (Software Development Co., Ltd.) computer programs. Hyphens designate gaps that were added to permit alignment. T. men.
quinckeanum, T. mentagrophytes var. quinckeanum; T. men. erinacei, T. mentagrophytes var. erinacei; A. benhamiae A/E and Af, A. benhamiae Americano-European race
and African race, respectively.

923
924 MAKIMURA ET AL.
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 spe-
cies of T. mentagrophytes constitute the T. mentagrophytes com-
plex (20, 25) because they are difficult to distinguish from each
other by their morphological features. By using sexual compat-
ibility (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. 1, cluster a) or the A. benhamiae group (Fig.
1, cluster b). This suggests that these medically important der-
matophytes 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” iden-
tifications in order to identify the pathogen.
In addition to establishing the significance of the ITS1 re-
gion from a taxonomic standpoint, we also identified these
clinically important species using the ITS1 DNA sequence
database. With this system, not only the morphologically iden-
tified strains of T. mentagrophytes, T. rubrum, T. violaceum, and
M. canis but also the two strains of morphologically unidenti-
fiable 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 identi-
fication system, both of which are based on ITS1 rDNA se-
quences, is under way in our laboratory with other species and
strains. Moreover, a new approach to the detection and iden-
tification 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 Col-
lege 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 Organi-
zation (NEDO) of Japan.
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