Journal of Microbiological Methods 61 (2005) 281 – 284

www.elsevier.com/locate/jmicmeth

Note

A simple PCR-RFLP method for identification and

differentiation of 11 Malassezia species
Hossein Mirhendia,*, Koichi Makimurab, Kamiar Zomorodiana, Tsuyoshi Yamadab,
Takashi Sugitac, Hideyo Yamaguchib
a
Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences,
PO Box 14155-6446, Tehran, Iran
b
Institute of Medical Mycology and Genome Research Center, Teikyo University, Tokyo, Japan
c
Department of Microbiology, Meiji Pharmaceutical University, Kiyose, Tokyo, Japan
Received 19 July 2004; received in revised form 11 November 2004; accepted 22 November 2004
Available online 23 December 2004

Abstract

A PCR-RFLP method targeted toward 26S rDNA and with 2 restriction enzymes, CfoI and BstF51, was developed to
identify 11 Malassezia species. Not only type and standard strains but also 13 clinical isolates were identified successfully in
this study. The results of identifications were confirmed by DNA sequencing.
D 2004 Elsevier B.V. All rights reserved.

Keywords: Malassezia; Identification; PCR; RFLP; CfoI; BstF51

Malassezia species are part of the resident skin
flora of humans and other warm-blooded verte-
brates (Leeming et al., 1989). These yeasts are
associated with various superficial diseases, includ-
ing pityriasis versicolor, seborrheic dermatitis, and
folliculitis (Gupta et al., 2002; Tarazooie et al.,
2004), as well as nosocomial bloodstream infec-
tion in pediatric care units (Chryssanthou et al.,
2001).
* Corresponding author. Tel.: +98 21 89 51 583; fax: +98 21
646 22 67.
E-mail addresses: mirhendi@yahoo.com,
mirhendi@sphtums.com (H. Mirhendi).
0167-7012/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.mimet.2004.11.016
The genus Malassezia has undergone several
taxonomic revisions. In the reclassification by Gueho
et al. (1996), seven distinct species were identified
within this genus. Recently, on the basis of DNA
relatedness, four new species have been included:
Malassezia dermatis, Malassezia nana, Malassezia
japonica, and Malassezia yamatoensis (Sugita et al.,
2002, 2003, 2004; Hirai et al., 2004).
Malassezia species can be identified through their
morphological features and biochemical characte-
rization (Guillot et al., 1996). However, these pheno-
typic methods are usually time consuming, lack
sufficient discriminatory power, and are unable to
unambiguously differentiate newly identified species.
282 H. Mirhendi et al. / Journal of Microbiological Methods 61 (2005) 281–284

Although various DNA-based molecular methods PCR amplification was carried out in a final
have been described to overcome this problem volume of 50 Al. Each reaction contained 1 Al of
(Makimura et al., 2000; Senczek et al., 1999; template DNA, 0.5 AM of each primer, 0.20 mM of
Theelen et al., 2001), a simple, reliable, and cost each deoxynucleoside triphosphate (dNTPs), 5 Al of
effective method is still needed for differentiation of 10PCR buffer, and 1.25 U of Taq polymerase. An
Malassezia species. Here, we report application of a initial denaturation step at 948C for 5 min was
PCR-based technique using restriction enzyme followed by 30 cycles of denaturation at 94 8C for
digestion for discrimination of 11 Malassezia 45 s, annealing at 55 8C for 45 s, and extension at
species. 72 8C for 1 min, with a final extension step of 72 8C
Malassezia type and standard strains used in this for 7 min. Amplified products were visualized by
study are listed in Table 1. Thirteen clinical isolates of 1.5% (w/v) agarose gel electrophoresis in TBE buffer,
Malassezia were also used. All strains were charac- stained with ethidium bromide (0.5 Ag ml 1), and
terized by ITS1 sequencing. The yeasts were cultured photographed under UV transillumination.
on modified Dixon agar and incubated at 32 8C for 7– According to sequence data, the 26S rDNA
14 days. The harvested cells were preserved at 20 8C sequences of all Malassezia species were aligned
until analysis. and the digestion patterns of the species were
Genomic DNA was extracted and purified using predicted for various restriction enzymes using
glass bead preparation (Yamada et al., 2002). Primer Genetyx software (version 6.1, Gynetyx Corporation,
selection was based on alignment of published 26S Tokyo, Japan). Finally, the enzymes CfoI and BstF51
rDNA sequences of known Malassezia species (acces- were selected to achieve the best species-specific
sion numbers: AJ249950, AJ249951, AJ249952, pattern. Digestion was performed by incubating a
AJ249953, AJ249954, AJ249955, AJ249956). Pri- 21.5-Al aliquot of PCR product with 10 U of the
mers were selected to allow the amplification of target enzyme in a final reaction volume of 25 Al at 37 8C
DNA in all species and their sequences were: forward, for 3 h, followed by 2% agarose gel electrophoresis in
5V-TAACAAGGATTCCCCTAGTA and reverse, 5V- TBE buffer and staining with ethidium bromide.
ATTACGCCAGCATCCTAAG. DNA sequencing of ITS1 (forward, 18SF1: 5-
AGGTTTCCGTAGGTGAACCT-3; and reverse,
5.8SR1: 5-TTCGCTGCGTTCTTCATCGA-3 primers
designed by Makimura et al., 2000) was performed
Table 1
for Malassezia standard and clinical strains using a
Type and standard strains used for verification of the method used in
this study DNA sequencing kit and analyzed on an automated
Species Type strains Standard strains
sequencer (ABI Prim-Perkin-Elmer 310, Genetic
Analyzer, Wellesley, MA).
M. furfur CBS 1878 M9970, M9971
M. globosa CBS 7966 CBS 8744, CBS 8745,
The primers successfully amplified the target part
CBS 7989, isolate 15, of 26S rDNA from all Malassezia strains tested,
isolate 16 providing a single PCR product of the expected size
M. restricta CBS 7877 CBS 8747, CBS 7991 (approximately 580 bp) (Fig. 1A).
M. pachydermatis CBS 1879 isolate A-3, isolate B-3, The products of restriction digestion are shown
isolate C-1, and isolate D-2
M. sympodialis CBS 7222 M 9978, M9979
separately in Fig. 1B; the bands generated were of
M. obtusa CBS 7876 M 9974, M9975 the predicted sizes. Using CfoI (Roche Diagnostics,
M. slooffiae CBS 7956 M 9980, M 9981 Mannheim, Germany), we could distinguish nine
M. dermatis JCM 11348 JCM 11469, JCM 11470 different species, including Malassezia furfur, Malas-
M. nana JCM 12085 JCM 12086, JCM 12087, sezia pachydermatis, Malassezia globosa, Malasse-
JCM 12088,
JCM 12089
zia obtuse, Malassezia restricta, Malassezia
M. japonica CBS 9432 CBS 9431 slooffiae, M. nana, M. japonica, and M. yamatoen-
M. yamatoensis CBS 9725 sis, but Malassezia sympodialis and M. dermatis
CBS, Centraalbureau voor Schimmelcultures; JCM, Japanese produced the same pattern. However, these two
Collection of Microorganisms. species could be differentiated using the additional
 H. Mirhendi et al. / Journal of Microbiological Methods 61 (2005) 281–284 283

Fig. 1. 26S rDNA PCR products before (A) (Lanes 1–12: M. furfur, M. sympodialis, M. globosa, M. restricta, M. slooffiae, M. pachydermatis,
M. japonica, M. nana, M. dermatis, M. obtusa, 100 bp ladder, and M. yamatoensis) and after digestion with CfoI (B) (Lanes 1–12: M. furfur, M.
sympodialis, M. globosa, M. restricta, M. slooffiae, M. pachydermatis, M. japonica, M. nana, M. dermatis, M. obtuse, M. yamatoensis, and 100
bp ladder) and BstF51 (C) (Lanes 1 to 12: M. furfur, M. sympodialis, M. dermatis, M. globosa, M. restricta, M. obtusa, M. pachydermatis, M.
japonica, M. nana, M. slooffiae, M. yamatoensis and 100 bp ladder).

enzyme BstF51 (SibEnzyme, Novosibirsk, Russia) Identification of Malassezia isolates by PCR-RFLP

(Fig. 1C). of ITS1-1TS2 regions has been applied recently
All standard strains were identified by the (Gupta et al., 2000; Guillot et al., 2000) albeit for a
enzymes. No band variation was seen within the restricted number of species. In another study,
strains of same species examined. Malassezia yeasts Gaitanis et al. (2002) proposed the use of endonu-
isolated from clinical specimens were identified by the clease enzymes for direct identification of Malassezia
above method as well as ITS1 sequencing. The results species from patient skin scales.
of PCR-RFLP analyses of clinical isolates were in The 26S rDNA contains several regions of highly
complete agreement with those from DNA sequencing conserved sequence useful for obtaining proper
and included 5 cases of M. furfur, 5 cases of M. sequence alignments. It also contains sufficient
restricta, and 2 cases of M. globosa (Table 2). We sequence variability in other regions of the molecule
could not identify one isolate because it was a mixture that can serve as markers for species-specific restric-
of two species. tion enzyme analyses. In the present study, restriction
Accurate and reproducible methods of species digestion of the partial 26S rDNA PCR product with
identification are essential for epidemiological purpo- the enzyme CfoI produced the predicted species-
ses. Various molecular methods of characterizing
species of Malassezia have been used. Pulsed-field Table 2
gel electrophoresis (PFGE) of Malassezia species Clinical isolates used in this study
yielded seven distinct karyotypes and allowed reliable Strain No. Identification method % Homology
differentiation of these species (Senczek et al., 1999). PCR-RFLP ITS1 (to DDBJ/EMBL/GenBank
However, the method is both time consuming and labor sequencing Accession number)
intensive and therefore cannot be used as a rapid 1 6-1 M. furfur M. furfur 99% (AB105151)
diagnostic method. Application of internal transcribed 2 7-1 M. globosa M. globosa 100% (AY387134)
spacer 1 (ITS1) ribosomal DNA sequences for dis- 3 8-1 M. restricta M. restricta 100% (AY387143)
4 8-3 Mixture of NT NT
crimination of Malassezia species (Makimura et al., 2 species
2000) is too expensive for use as a routine diagnostic 5 9-1 M. restricta M. restricta 100% (AY387143)
method and is likely to be restricted to research and 6 9-3 M. restricta M. restricta 99% (AY387144)
reference laboratories. AFLP genotyping and random 7 10-1 M. restricta M. restricta 99% (AJ437695)
amplified polymorphic DNA (RAPD) have proved to 8 12-1 M. furfur M. furfur 99% (AB105154)
9 13-3 M. furfur M. furfur 99% (AY623429)
be useful methods for subtyping and discriminating 10 15-1 M. restricta M. restricta 100% (AY387143)
between Malassezia strains (Theelen et al., 2001). 11 17-1 M. furfur M. furfur 99% (AB019331)
However, the results are usually be too complicated to 12 D-1 M. furfur M. furfur 100% (AB105153)
interpret and not sufficiently reliable for identification 13 D-3-2 M. globosa M. globosa 100%(AY387134)
and discrimination of clinically isolated yeasts. NT, not tested.
284 H. Mirhendi et al. / Journal of Microbiological Methods 61 (2005) 281–284
specific patterns for all species tested. While the
above studies discriminated only seven Malassezia
species using three restriction enzymes, we identified
nine species with only a single enzyme and two
further species using an additional one enzyme. Thus,
our PCR-RFLP method was capable of determining
the species of isolates in agreement with the new
taxonomic classification. This was demonstrated with
Malassezia standard strains. This simple typical PCR-
RFLP method was applied to amplicons from cultured
clinical isolates, allowing immediate identification of
Malassezia yeasts. Moreover, the results of PCR-
RFLP analysis were comparable with those sequenc-
ing of the ITS region of the clinical isolates.
In conclusion, this method is a genotypic identi-
fication approach that can be applied for the identi-
fication of nearly all known Malassezia species and
that is flexible because patterns of newly described
species can be added directly to a database without the
need for sequence information. This PCR-RFLP
method requires only PCR and one or two enzyme(s),
and is technically less demanding than most other
molecular biological approaches. We sought to design
another PCR-RFLP method for identification of 11
Malassezia species that would be capable of identify-
ing species with high accuracy and reliability, and
would be simple, fast, and cost effective for applica-
tion even in routine laboratories.
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