Journal of Medical Microbiology (2012), 61, 57–63 DOI 10.1099/jmm.0.

036541-0

Multilocus differentiation of the related

dermatophytes Microsporum canis, Microsporum
ferrugineum and Microsporum audouinii
Ali Rezaei-Matehkolaei,1 Koichi Makimura,2 G. Sybren de Hoog,3,4
Mohammad Reza Shidfar,1 Kazuo Satoh,2 Mohammad Javad Najafzadeh5
and Hossein Mirhendi1
Correspondence 1
Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of
Hossein Mirhendi Medical Sciences, Tehran, Iran
mirhendi@tums.ac.ir 2
Teikyo University Institute of Medical Mycology, Tokyo, Japan
3
CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands
4
Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, The Netherlands
5
Department of Parasitology and Mycology, Faculty of Medicine, Mashhad University of Medical
Sciences, Mashhad, Iran

Microsporum ferrugineum, an uncommon causative agent of dermatophytosis, has restricted

endemicity. Iranian strains suspected to be M. ferrugineum from two patients with tinea were
analysed using the rDNA internal transcribed spacer (ITS) region and the partial b-tubulin (BT2)
and translation elongation factor 1-a (TEF1) genes. Strains were compared to reference strains to
differentiate M. ferrugineum from its relatives Microsporum canis and Microsporum audouinii.
Inter-species differences for TEF1 and BT2 were found to be higher than for the ITS region, which
is the current molecular standard for species identification in dermatophytes. Intra-species
variation was zero for each of the markers. In silico analysis showed that the restriction enzymes
BanI and BshNI were together sufficient to differentiate the three species based on TEF1,
whereas a two-step digestion was needed with BT2 or the ITS region. The prevalence of M.
Received 26 July 2011 ferrugineum in clinical samples in Iran appeared to be higher than suspected on the basis of
Accepted 4 September 2011 routine phenotypic identification.

INTRODUCTION 1996). In Iran, this species is rare (Mahmoudabadi, 2006).

Routine identification of this species is based on the
Microsporum canis, Microsporum ferrugineum and
macroscopic features of colonies on Sabouraud’s glucose
Microsporum audouinii are three phylogenetically closely
agar with cycloheximide and chloramphenicol (SCC) or on
related dermatophytes in the Arthroderma otae complex
potato dextrose agar. Physiological criteria involve urease
(Gräser et al., 2008). They have different ecological niches:
production, hair perforation and the response to bromocre-
the former two are anthropophilic, whilst the latter is a sol purple/milk solids/glucose agar (Ellis et al., 2007; Kane
zoophilic species. However, all are involved in human et al., 1997; Weitzman & Summerbell, 1995). Microscopy is
infections, and can cause similar clinical manifestations and limited because microconidia are absent. The most import-
be transmitted via human-to-human or animal-to-human ant feature is the production of hyphae with prominent
routes. Identification/differentiation of these related species cross-walls (‘bamboo hyphae’; Kane et al., 1997; Ishizaki et al.,
is important from an epidemiological point of view. 2003). Chlamydospores and racket hyphae are commonly
M. ferrugineum has limited endemicity in the Balkans, observed, and occasionally macroconidia are present. How-
the Middle East, East Asia and Nigeria (Kane et al., 1997; ever, phenotypic identification remains difficult because
Weitzman & Summerbell, 1995; Wisuthsarewong et al., colonies may be similar to atypical (dysgonic) variants of M.
canis (Ellis et al., 2007; Gräser et al., 2008). In addition, long
incubation times are needed (7214 days) for characteristics
The GenBank/EMBL/DDBJ accession numbers for the sequences
determined in this study for M. canis, M. ferrugineum and M. audouinii,
to appear, and these characteristics may also be unstable
respectively, are JN134129, JN134137 and JN134145 for the ITS (Ates et al., 2008; Ninet et al., 2003). Moreover, identification
region; JF731103, JF731106 and JF731113 for BT2; and JN662936, of uncommon dermatophytes outside endemic areas may be
JN662934 and JN662935 for TEF1. difficult due to a lack of experience among microbiologists.

036541 G 2012 SGM Printed in Great Britain 57

A. Rezaei-Matehkolaei and others

Recently, rapid molecular diagnostics tools have become et al., 1999; Woodgyer, 2004) and PCR-RFLP (De Baere
available for dermatophytes, particularly sequencing of the et al., 2010; Jackson et al., 1999). The present paper reports
rDNA internal transcribed spacer (ITS) region (Makimura on an 18-month screening study of dermatophytoses in

58 Journal of Medical Microbiology 61

 Differentiation of M. canis and its close relatives

Fig. 1. DNA sequence alignment of the TEF1 and BT2 gene and the ITS region in M. canis, M. ferrugineum and M. audouinii.

Tehran, Iran, where .700 isolates were identified using acetate, kept at 220 uC for 10 min and centrifuged at 12 000 g for
ITS-RFLP with MvaI. Two dermatophyte strains were 10 min. Supernatants were extracted once with phenol : chloroform :
isoamyl alcohol (25 : 24 : 1) and subsequently with chloroform. The
encountered that had the RFLP profile characteristics of
DNA was precipitated with an equal volume of 2-propanol, washed
M. canis and M. ferrugineum but phenotypically were with 300 ml 70 % ethanol, dried and suspended in 50 ml ultrapure
suspected to be M. ferrugineum. This prompted a study of water. One microlitre of the final suspension was used as template in
the identification of M. ferrugineum and its close relatives, each PCR.
M. canis and M. audouinii. We analysed sequences of the
rDNA ITS region and partial sequences of the b-tubulin Primers and amplification conditions. The following primer sets
were used: the universal fungal primers ITS1 (59-TCCGTAGGTGA-
(BT2) and translation elongation factor 1-a (TEF1) genes
ACCTGCGG-39) and ITS4 (59-TCCTCCGCTTATTGATATGC-39)
with a view to reliable discrimination of these species. (White et al., 1990) for amplification of the ITS region; the universal
fungal primers T1 (59-AACATGCGTGAGATTGTAAGT-39) (O’Donnell
& Cigelnik, 1997) and Bt2b (59-ACCCTCAGTGTAGTGACCCTTGGC-
METHODS 39) (Glass & Donaldson, 1995) for partial amplification of the BT2 gene;
and the pan-dermatophyte primers EF-DermF (59-CACATTAACTT-
Strains. Two dermatophytes suspected to be M. ferrugineum were GGTCGTTATCG-39) and EF-DermR (59-CATCCTTGGAGATACC-
isolated from patients with tinea pedis and ectothrix-type tinea AGC-39), designed in this study, for partial amplification of the TEF1
capitis. The strains were compared with 30 clinical strains identified gene. All primers were synthesized by Sigma-Aldrich. The PCR mixture
morphologically as M. canis and originating from tinea capitis (n55), was prepared using a commercial kit (Takara Bio) and contained 2.5 ml
tinea corporis (n521), tinea cruris (n52) and tinea faciei (n52) 106 reaction buffer, 200 mM dNTPs, 1 U Taq DNA polymerase,
infections, and with 10 reference strains (M. canis: NBRC 9182, CBS 30 pmol each primer and 1 ml DNA template in a final volume of 25 ml.
132.88 and CBS 277.62; M. ferrugineum: CBS 457.80, NBRC 6081 and Reaction mixtures were pre-heated to 94 uC for 6 min and PCR was
NBRC 5831; M. audouinii: NBRC 6074, CBS 280.63, CBS 119448 and performed as follows: 35 cycles of 30 s at 94 uC, 30 s at 58262 uC and
CBS 332.68). Strains were cultured on SCC (Oxoid) and incubated at 1 min at 72 uC, with a final extension at 72 uC for 10 min and cooling at
28 uC for 2 weeks. 4 uC. The thermal conditions, except for annealing temperatures, were
the same for all three markers.
DNA extraction. Genomic DNA was prepared using methods
described previously (Makimura et al., 1999). Briefly, small amounts Sequencing. PCR products were purified using a QIAquick
of young colonies were placed in 1.5 ml tubes containing 200 ml lysis purification kit (Qiagen) and an ABI PRISM BigDye Terminator
buffer [200 mM Tris/HCl (pH 7.5), 25 mM EDTA, 0.5 % SDS, Cycle Sequencing Ready Reaction kit (Applied Biosystems), and
250 mM NaCl] and crushed with a conical grinder. Samples were sequenced using an automated DNA Sequencer (ABI Prism 3730
incubated for 20 min at 100 uC and mixed with 150 ml 3.0 M sodium Genetic Analyzer; Applied Biosystems).

http://jmm.sgmjournals.org 59
A. Rezaei-Matehkolaei and others

Sequence analysis. Forward and reverse sequences were edited identical restriction fragments of 441, 165, 103 and 28 bp.
using MEGA4 (Tamura et al., 2007) and Geneious (www.geneious. An appropriate restriction enzyme was then selected for
com) software. Trimmed ITS sequences were compared with similar an RFLP assay for identification of M. canis and M.
sequences in the open access validated CBS database of dermato-
ferrugineum by analysing the ITS sequences of 38 strains
phytes (www.cbs.knaw.nl/dermatophytes). Inter- and intra-species
differences were evaluated using BioEdit (www.mbio.ncsu.edu/ that were identified as M. canis and M. ferrugineum,
bioedit) or Geneious software. Levels of sequence diversity (D) in supplemented by sequences from GenBank. Sequences
each target were calculated pairwise using the formula D512(M/L) were subjected in silico to 610 restriction enzymes included
(Chilton et al., 1995), in which M is the number of shared positions in the DNASIS software. Only MaeIII proved to be suitable
and L is the total number of alignment positions compared. for differentiation of the two species. This enzyme had a
Restriction sites and fragment sizes were predicted in silico and single cutting site in M. canis, yielding bands of 527 and
appropriate restriction enzymes for RFLP analysis were selected using
210 bp, whereas restriction sites were absent from M.
DNASIS software (Hitachi DNASIS MAX version 3.0).
ferrugineum and M. audouinii and their profile was
RFLP. ITS region PCR products of all standard and clinical strains, identical to the original PCR product. Following digestion,
except for M. audouinii strains that were identified during an initial the band patterns exactly matched the expectations from in
ITS RFLP screening with MvaI, were digested in 15 ml reaction volumes silico analysis. Comparison of ITS sequences with the ITS
containing 0.5 ml (5 U) of the enzyme MaeIII (Roche), 1.5 ml buffer RFLP with MaeIII confirmed the identities of the clinical
(supplied by the manufacturer), 5 ml PCR product and 8 ml molecular- isolates from Iran as M. canis and M. ferrugineum (Fig. 2).
grade water. Reactions were incubated at 55 uC for 120 min. Clinical isolates and reference strains of M. canis produced
Electrophoresis. A total of 5 ml amplicon and 8 ml digested DNA
white to cream-coloured colonies with a dense cottony
were separated by electrophoresis on a 1.5 and 2 % agarose gel, surface and yellow to yellow/orange pigments exuding into
respectively. TAE buffer [40 mM Tris/acetate (pH 8.0), 2 mM EDTA] the SCC medium after incubation for 1 week at 28 uC.
was used for preparing the gel and for electrophoresis. A 100 bp DNA In microscopic preparations, spindle-shaped, thick-walled
ladder was used as a molecular size marker. The DNA bands were macroconidia with terminal knobs characteristic of M. canis
stained with 0.5 mg ethidium bromide ml21, visualized by UV were observed. The reference strains of M. ferrugineum and
transillumination and photographed. the two clinical isolates were waxy to glabrous in texture,
slightly raised and folded at the centre, with a cream- to buff-
coloured surface and without pigments on the reverse side of
RESULTS AND DISCUSSION the colony (Fig. 3a, b). Microscopic examination revealed
PCR amplification of the three target regions yielded single bamboo-like and racket hyphae, whilst macro- and micro-
bands of 7202770 bp in all clinical and reference strains. conidia were absent (Fig. 3c, d). The reference strains of M.
The band sizes for the three species were almost the same audouinii produced flat, spreading, cream-coloured colonies
for each of the ITS regions and the BT2 and TEF1 genes. with a densely cottony surface. The reverse side of the colony
Sequence analysis indicated that the DNA products was brownish yellow. Macroconidia were absent, but a few
amplified by primers ITS1/ITS4, T1/Bt2b and EF-DermF/ pyriform microconidia were observed.
EF-DermR were 737, 770 and 720 bp for M. canis and M. Computer analysis of the TEF1 sequences indicated that
ferrugineum, respectively, and 734, 771 and 721 bp for M. restriction enzymes BanI and BshNI could differentiate the
audouinii (Fig. 1). Inter-species differences for TEF1 and three species in a single reaction. These enzymes yielded
BT2 were higher than for the ITS region. Whilst the mean electrophoretic patterns comprising bands of 610, 78 and
nucleotide difference between the three tested species was 32 bp for M. canis, 721 bp (no restriction site) for M.
5.33 nt for the ITS region, it was 9.33 and 10.67 nt for ferrugineum and 643 and 78 bp for M. audouinii. In silico
TEF1 and BT2, respectively (Table 1). No intra-species digestion of the BT2 gene showed that, like the ITS region,
variation among strains of each species and each marker a two-step RFLP assay was needed to differentiate the three
was seen (data not shown). species (data not shown).
In preliminary screening of the tested dermatophytes using Conventionally, dermatophytes including M. ferrugineum are
ITS region digestion by MvaI, all four reference strains of identified by the macro- and microscopic morphology of the
M. audouinii yielded a specific RFLP pattern (441, 162 and colony, supplemented with physiological tests (Kane et al.,
131 bp) and were excluded from the following process, 1997; Weitzman & Padhye, 1996; Weitzman & Summerbell,
whilst all strains of M. canis and M. ferrugineum produced 1995). However, tests are time-consuming and require

Table 1. Inter-species distances of the ITS region and the BT2 and TEF1 genes between M. canis, M. ferrugineum and M. audouinii

DNA sequence M. canis/M. ferrugineum M. canis/M. audouinii M. ferrugineum/M. audouinii

ITS region 0.3 % (2 nt) 1 % (7 nt) 1 % (7 nt)

TEF1 1.4 % (10 nt) 1.1 % (8 nt) 1.4 % (10 nt)
BT2 1.6 % (12 nt) 1.9 % (14 nt) 0.8 % (6 nt)

60 Journal of Medical Microbiology 61

 Differentiation of M. canis and its close relatives

the sequence database of the rDNA ITS region is considered

the gold standard for dermatophytes (Gräser et al., 2008;
Makimura et al., 1999).
M. canis, M. ferrugineum and M. audouinii are three
phylogenetically closely related dermatophytes in the A.
otae complex (Gräser et al., 2008). In the present study, we
focused on two genetic markers other than the ITS region
to facilitate differentiation of these species. The ITS regions
of M. ferrugineum and M. canis were highly similar,
differing by just 2 bp at one site in ITS2 (Fig. 1), whilst M.
audouinii differed from these two species in five locations
Fig. 2. Agarose gel electrophoresis of the ITS RFLP profiles using (7 bp) in both ITS1 and ITS2 (Fig. 1). With MvaI digestion
MaeIII for representative strains of M. canis and M. ferrugineum. of the ITS region, the reference strains of M. audouinii
Lanes: 1 and 2, clinical isolates suspected to be M. ferrugineum; produced a specific profile, whereas the profiles of M.
3 and 4, M. ferrugineum NBRC 6081 and NBRC 5831, respectively; ferrugineum and M. canis were identical to each other.
5–9, clinical isolates of M. canis; 10, M. canis NBRC 9182; M, These results were in accordance with those of Leon-
100 bp DNA molecular mass marker. Mateos et al. (2006), who were unable to find an enzyme
differentiating M. ferrugineum and M. canis; however, we
found that digestion of the ITS region with MaeIII enabled
simple PCR-RFLP differentiation of these two species.
special skills for reliable diagnosis (Arabatzis et al., 2007; De
Baere et al., 2010), particularly in species with a degenerate We also characterized partial sequences of the BT2 and TEF1
appearance such as M. ferrugineum. Recently, molecular genes encoding b-tubulin and translation elongation factor
techniques have become available (Kanbe, 2008). Currently, 1-a for M. canis, M. ferrugineum and M. audouinii. Several

Fig. 3. Two Iranian clinical isolates confirmed as M. ferrugineum. (a, b) Colony morphology on SCC after 2 weeks’ incubation at
28 6C. (c, d) Microscopy showing bamboo-like hyphae (c) and non-specific racket hyphae (d). Bars, 30 mm.

http://jmm.sgmjournals.org 61
A. Rezaei-Matehkolaei and others

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