Previous Article | Next Article 
Journal of Clinical Microbiology, April 2002, p. 1363-1367, Vol. 40, No. 4
0095-1137/02/$04.00+0 DOI: 10.1128/JCM.40.4.1363-1367.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
New Yeast Species, Malassezia dermatis, Isolated from Patients with Atopic Dermatitis
Takashi Sugita,1* Masako Takashima,2 Takako Shinoda,1 Hajime Suto,3 Tetsushi Unno,3 Ryoji Tsuboi,3 Hideoki Ogawa,3 and Akemi Nishikawa4
Department of Microbiology,1
Department of Immunobiology, Meiji Pharmaceutical University, Kiyose,4
Department of Dermatology, School of Medicine, Juntendo University, Bunkyo-ku, Tokyo,3
Japan Collection of Microorganisms, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama, Japan2
Received 4 September 2001/
Returned for modification 31 December 2001/
Accepted 2 February 2002

ABSTRACT
Malassezia species are considered to be one of the exacerbating
factors in atopic dermatitis (AD). During examination of the
cutaneous colonization of
Malassezia species in AD patients,
we found a new species on the surface of the patients' skin.
Analysis of ribosomal DNA sequences suggested that the isolates
belonged to the genus
Malassezia. They did not grow in Sabouraud
dextrose agar but utilized specific concentrations of Tween
20, 40, 60, and 80 as a lipid source. Thus, we concluded that
our isolates were new members of the genus
Malassezia and propose
the name
Malassezia dermatis sp. nov. for these isolates.

INTRODUCTION
Malassezia species are known causative factors in pityriasis
versicolor, seborrheic dermatitis (SD), and atopic dermatitis
(AD) (
3). In the last decade, research has focused primarily
on isolating
Malassezia strains and detecting specific immunoglobulin
E (IgE) antibodies from patients (
9,
13,
14,
26). A comparison
of the isolation rates of
Malassezia species from the skin of
AD patients and healthy control subjects detected a significantly
higher rate for patients than for healthy subjects (
8). AD patients
had specific IgE antibodies against
Malassezia, while healthy
subjects did not. In recent years, studies have increasingly
been directed towards analyzing how the cutaneous microflora
at the species level are related to each disease type (pityriasis
versicolor, SD, and AD) (
1,
6,
7,
12,
16). We previously compared
the distribution of
Malassezia species in skin lesions of AD
patients and in healthy subjects using a nonculture method (nested
PCR) that is not affected by the isolating medium (
21). Of the
seven members of the genus
Malassezia,
Malassezia globosa and
M. restricta were the species most commonly associated with
AD, while
M. obtusa and
M. pachydermatis were not detected in
AD. In our survey of cutaneous
Malassezia microflora, we isolated
new
Malassezia species from several patients with AD. In this
paper, we propose a new species,
M. dermatis, for these isolates.

MATERIALS AND METHODS
Malassezia isolates.
Nineteen AD outpatients at Juntendo University Hospital were
included in the study. To obtain samples, OpSite transparent
dressings (3 by 7 cm; Smith and Nephew Medical Ltd., Hull, United
Kingdom) were applied to skin lesions (erosive, erythematous,
and lichenoid) on the scalp, back, and nape of the neck of AD
patients. Samples were then transferred onto Leeming and Notman
agar (LNA) (10.0 g of polypeptone, 5.0 g of glucose, 0.1 g of
yeast extract, 8.0 g of ox gall, 1.0 mg of glycerol, 0.5 g of
glycerol stearate, 0.5 mg of Tween 60, 10 ml of cow's milk [whole
fat], and 12.0 g of agar per liter) plates containing 50 µl
of chloramphenicol (Sankyo, Tokyo, Japan) and incubated at 32°C
for 2 weeks.
Direct DNA sequencing of rRNA genes of the isolates.
Yeast isolates recovered from LNA medium were identified by analysis of rRNA gene sequences. Nuclear DNA of the isolates was extracted by the method of Makimura et al. (15). The D1 and D2 regions of 26S ribosomal DNA (rDNA) and internal transcribed spacer (ITS) regions in the rRNA gene were sequenced directly from PCR products using the primer pairs NL-1 (forward; GCATATCAATAAGCGGAGGAAAAG) and NL-4 (reverse; GGTCCGTGTTTCAAGACGG) (11) and pITS-F (forward; GTCGTAACAAGGTTAACCTGCGG) and pITS-R (reverse; TCCTCCGCTTATTGATATGC) (22), respectively. The PCR products were sequenced using an ABI 310 DNA sequencer and an ABI PRISM BigDye Terminator Cycle Sequencing kit (Perkin-Elmer Applied Biosystems, Foster, Calif.) according to the manufacturer's instructions. Strains with 99% or more similarity of the D1 and D2 regions of 26S rDNA and the overall ITS sequences were defined as conspecific (18, 22). The sequence data were searched using the BLAST system (http://www.ncbi.nlm.nih.gov/BLAST/) at the National Center for Biotechnology Information, Bethesda, Md.
Molecular phylogenetic analysis.
The sequences were aligned using ClustalW software (25). For neighbor-joining analysis (20), the distances between sequences were calculated using Kimura's two-parameter model (10). A bootstrap analysis was conducted with 100 replications (2).
Morphological, physiological, and chemotaxonomic characteristics.
Morphology was examined on LNA after incubation at 32°C for 7 days. Tween 20, 40, 60, and 80 utilization, catalase reactions, and diazonium blue B reactions were performed as described by Guého et al. (4). Identification of ubiquinone was carried out using methods described by Nakase and Suzuki (17). The nuclear DNA base composition (moles percent G+C) was determined by high-pressure liquid chromatography after enzymatic digestion of DNA into deoxyribonucleosides (23, 24). The DNA-GC kit (Yamasa Shouyu, Chiba, Japan) was used according to the manufacturer's instructions.
Nucleotide sequence accession numbers.
The nucleotide sequences determined in this study have been deposited with DDBJ (DNA Data Bank of Japan) under the accession numbers shown in Fig. 1.

RESULTS
Molecular phylogenetic analysis.
All of the yeast isolates obtained from the 19 AD patients were
identified by analysis of rRNA gene sequences (D1 and D2 regions
of 26S rDNA and ITS). Of these, the DNA sequences of five isolates
did not match sequences in the DNA sequence database. These
isolates were designated M 9927, M 9928, M 9929, M 9930, and
M 9931. The first three strains were isolated from a single
patient, while the last two strains were isolated from one patient
each. Sequence analysis of their 26S rDNA indicated that these
isolates belonged to the genus
Malassezia (Fig.
1A). The sequences
of five isolates were completely identical in both the 26S rDNA
and ITS regions and clustered with
M. sympodialis with high
bootstrap values (100%) (Fig.
1). Dissimilarities between these
isolates and the
M. sympodialis strain in their D1 and D2 regions
of 26S, ITS1, and ITS2 were 1.2% (7 of 578), 10.5% (17 of 162),
and 10.3% (24 of 233), respectively. The overall dissimilarity
of ITS regions was 10.4% (41 of 395).
Taxonomic characteristics.
The characteristics that differentiate the new species, M. dermatis, from the other seven known Malassezia species are summarized in Table 1. The physiological characteristics of M. dermatis are identical to those of M. furfur. However, the moles percent G+C of M. dermatis nuclear DNA is 60.4% while that of M. furfur is 66.0 to 66.7%. A difference of more than 1 mol% G+C between two strains is considered taxonomically significant (19, 24).
Latin description of Malassezia dermatis Sugita, Takashima, Nishikawa et Shinoda sp. nov.
In LNA, post dies 7 ad 32°C, cellulae vegetativae sphaericae,
ovoideae vel ellipsoideae (2-8)
x (2-10) µm. Cultura xanthoalba,
semi-nitida aut hebetata, butyracea et margo glabra aut lobulata.
In agaro glucoso-peptonico Tween 20, 40, 60, 80 (0.1-1%) addito
crecit. H
2O
2 hydrolysatur. Commutatio colori per diazonium caeruleum
B positiva. Proportio molaris guanini+cytosini in acido deoxyribonucleico:
60.4 mol%. Ubiquinonum majus: Q-9. Telemorphis ignota. Typus:
JCM 11348
T, ex cute morbosa, Tokyo, Japonia, 30.9.1999, T. Sugita
(originaliter ut M 9927), conservatur in collectionibus culturarum
quas Japan Collection of Microorganisms, Saitama,
Japan sustentat.
Description of M. dermatis Sugita, Takashima, Nishikawa et Shinoda sp. nov.
After 7 days on LNA at 32°C, the vegetative cells were spherical, oval, or ellipsoidal (2 to 8 by 2 to 10 µm), and they reproduced by budding (Fig. 2). The colony was yellowish white, semishiny to dull, convex, and butyrous and had an entire or lobed margin. Filaments sometimes formed at the area of the origin of the bud. Growth was seen on glucose-peptone agar with either 0.1, 0.5, 1.0, 5.0, or 10% Tween 20, 40, 60, or 80 as sole source of lipid. Catalase reaction was positive. The diazonium blue B reaction was positive. The G+C content of nuclear DNA was 60.4 mol%, and the major ubiquinone was Q-9. Teleomorph is unknown. JCM 11348T (originally M 9927) isolated from skin lesions of an AD patient in Tokyo, Japan, by T. Sugita on 30 September 1999 is maintained in the Japan Collection of Microorganisms (JCM), Saitama, Japan. The other strains, M 9929 and M 9931 have also been deposited in the JCM, as JCM 11469 and JCM 11470, respectively. Etymology is from the Latin name for skin, from which this strain of the species was obtained.

DISCUSSION
The genus
Malassezia is phylogenetically monophyletic with a
high bootstrap value (100%) and is positioned in the class
Ustilaginomycetes (Fig.
1A). Our isolates clustered with
M. sympodialis on the
tree. We did not perform a nuclear DNA-DNA hybridization study
between
M. sympodialis and our isolates, but sequence analysis
of rDNA (26S and ITS) strongly suggests that our five isolates
represent a new species in the genus
Malassezia. At present,
D1 and D2 26S rDNA sequences are used from almost all yeasts
for species identification or phylogenetic analysis. Peterson
and Kurtzman (
18) correlated the biological species concept
with the phylogenetic species concept by the extent of nucleotide
substitutions in 26S rDNA sequences. Their study demonstrated
that strains of a single biological species show less than 1%
substitution in this region. Subsequently, Sugita et al. (
22)
also found that conspecific strains have less than 1% dissimilarity
in the ITS1 and -2 regions after comparing the nuclear DNA-DNA
hybridization values and similarity in the sequences of the
entire ITS1 and -2 regions. According to the species concept
used in the two reports cited above, the divergence between
M. sympodialis and our isolates is sufficient to resolve them
as individual species. In addition, the approximately 1 mol%
difference in the G+C content of nuclear DNA that was found
between these two species also has taxonomic significance (
19,
24). Guillot et al. (
5) described a practical approach (combination
of Tween utilization and catalase tests) for easy and simple
identification of
Malassezia species. Unfortunately,
M. dermatis cannot be identified by this system, despite having similar
characteristics to
M. furfur. At present, sequence analysis
of the D1 and D2 regions of the 26S rDNA or the ITS regions
is the most reliable and simplest method for
M. dermatis identification.
We previously demonstrated cutaneous Malassezia colonization in 32 AD patients by a nonculture method (nested PCR) and compared anti-Malassezia antibody levels with those in healthy subjects. The detection of M. globosa and M. restricta in more than 90% of AD patients suggested that these two species play an important role in AD. Since we were not aware of the existence of M. dermatis at that time, we did not detect the DNA of this species in our previous study. Although the present study examined a limited number of patients, M. dermatis was found in 3 of 19 patients. However, the degree of M. dermatis colonization of the skin surface in the AD patients was not as great as that of M. globosa and M. restricta.
In conclusion, we described a novel species, M. dermatis, isolated from skin lesions of AD patients. Further studies should examine whether this microorganism is responsible for AD or other skin diseases and whether it is specific to Japan.

ACKNOWLEDGMENTS
This study was supported in part by a Grant for the Promotion
of the Advancement of Education and Research in Graduate Schools
from the Ministry of Education, Culture, Sports, Science, and
Technology of Japan.

FOOTNOTES
* Corresponding author. Mailing address: Department of Microbiology, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan. Phone: 81-424-95-8762. Fax: 81-424-95-8762. E-mail:
sugita{at}my-pharm.ac.jp.


REFERENCES
1
- Aspiroz, C., L. A. Moreno, A. Rezusta, and C. Rubio. 1999. Differentiation of three biotypes of Malassezia species on human normal skin. Correspondence with M. globosa, M. sympodialis and M. restricta. Mycopathologia 145:69-74.[CrossRef][Medline]
2
- Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783-791.[CrossRef]
3
- Guého, E., T. Boekhout, H. R. Ashbee, J. Guillot, A. van Belkum, and J. Faergemann. 1998. The role of Malassezia species in the ecology of human skin and as pathogens. Med. Mycol. 36(Suppl. 1):220-229.
4
- Guého, E., G. Midgley, and J. Guillot. 1996. The genus Malassezia with description of four new species. Antonie van Leeuwenhoek 69:337-355.[CrossRef][Medline]
5
- Guillot, J., E. Guého, M. Lesourd, G. Midgley, G. Chevrier, and B. Dupont. 1996. Identification of Malassezia species. J. Mycol. Med. 6:103-110.
6
- Gupta, A. K., Y. Kohli, J. Faergemann, and R. C. Summerbell. 2001. Epidemiology of Malassezia yeasts associated with pityriasis versicolor in Ontario, Canada. Med. Mycol. 39:199-206.[Medline]
7
- Gupta, A. K., Y. Kohli, R. C. Summerbell, and J. Faergemann. 2001. Quantitative culture of Malassezia species from different body sites of individuals with or without dermatoses. Med. Mycol. 39:243-251.[Medline]
8
- Hiruma, M., D. J. Maeng, M. Kobayashi, H. Suto, and H. Ogawa. 1999. Fungi and atopic dermatitis. Jpn. J. Med. Mycol. 40:79-83.
9
- Kim, T. Y., I. G. Jang, Y. M. Park, H. O. Kim, and C. W. Kim. 1999. Head and neck dermatitis: the role of Malassezia furfur, topical steroid use and environmental factors in its causation. Clin. Exp. Dermatol. 24:226-231.[CrossRef][Medline]
10
- Kimura, M. 1980. A simple method for estimation evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J. Mol. E 16:111-120.
11
- Kurtzman, C. P., and C. J. Robnett. 1997. Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA gene. J. Clin. Microbiol. 35:1216-1223.[Abstract]
12
- Leeming, J. P., J. E. Sansom, and J. L. Burton. 1997. Susceptibility of Malassezia furfur subgroups to terbinafine. Br. J. Dermatol. 137:764-767.[CrossRef][Medline]
13
- Leung, D. Y. 1995. Atopic dermatitis: the skin as a window into the pathogenesis of chronic allergic diseases. J. Allergy Clin. Immunol. 96:302-318.[CrossRef][Medline]
14
- Lintu, P., J. Savolainen, and K. Kalimo. 1997. IgE antibodies to protein and mannan antigens of Pityrosporum ovale in atopic dermatitis patients. Clin. Exp. Allergy 27:87-95.[CrossRef][Medline]
15
- Makimura, K., Y. S. Murayama, and H. Yamaguchi. 1994. Detection of a wide range of medically important fungal species by polymerase chain reaction (PCR). J. Med. Microbiol. 40:358-364.[Abstract/Free Full Text]
16
- Nakabayashi, A., Y. Sei, and J. Guillot. 2000. Identification of Malassezia species isolated from patients with seborrhoeic dermatitis, atopic dermatitis, pityriasis versicolor and normal subjects. Med. Mycol. 38:337-341.[Medline]
17
- Nakase, T., and M. Suzuki. 1986. Bullera megalospora, a new species of yeast forming large ballistospores isolated from dead leaves of Oryza sativa, Miscanthus sinensis, and Sasa sp. in Japan. J. Gen. Appl. Microbiol. 32:225-240.
18
- Peterson, S. W., and C. P. Kurtzman. 1991. Ribosomal RNA sequence divergence among sibling species of yeasts. Syst. Appl. Microbiol. 14:124-129.
19
- Price, C. W., G. B. Fuson, and H. J. Phaff. 1978. Genome comparison in yeast systematics: delimitation of species within the genera Schwanniomyces, Saccharomyces, Debaryomyces, and Pichia. Microbiol. Rev. 42:161-193.[Free Full Text]
20
- Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. E 4:406-425.
21
- Sugita, T., H. Suto, T. Unno, R. Tsuboi, H. Ogawa, T. Shinoda, and A. Nishikawa. 2001. Molecular analysis of Malassezia microflora on the skin of atopic dermatitis patients and healthy subjects. J. Clin. Microbiol. 39:3486-3490.[Abstract/Free Full Text]
22
- Sugita, T., A. Nishikawa, R. Ikeda, and T. Shinoda. 1999. Identification of medically relevant Trichosporon species based on sequences of internal transcribed spacer regions and construction of a database for Trichosporon identification. J. Clin. Microbiol. 37:1985-1993.[Abstract/Free Full Text]
23
- Takashima, M., and T. Nakase. 2000. Four new species of the genus Sporobolomyces isolated from leaves in Thailand. Mycoscience 41:65-77.
24
- Tamaoka, J., and K. Komagata. 1984. Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol. Lett. 25:125-128.
25
- Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.[Abstract/Free Full Text]
26
- Wessels, M. W., G. Doekes, A. G. Van Ieperen-Van Kijk, W. J. Koers, and E. Young. 1991. IgE antibodies to Pityrosporum ovale in atopic dermatitis. Br. J. Dermatol. 125:227-232.[CrossRef][Medline]
Journal of Clinical Microbiology, April 2002, p. 1363-1367, Vol. 40, No. 4
0095-1137/02/$04.00+0 DOI: 10.1128/JCM.40.4.1363-1367.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
This article has been cited by other articles:
-
Gonzalez, A., Sierra, R., Cardenas, M. E., Grajales, A., Restrepo, S., Cepero de Garcia, M. C., Celis, A.
(2009). Physiological and Molecular Characterization of Atypical Isolates of Malassezia furfur. J. Clin. Microbiol.
47: 48-53
[Abstract]
[Full Text]
-
Kaneko, T., Makimura, K., Abe, M., Shiota, R., Nakamura, Y., Kano, R., Hasegawa, A., Sugita, T., Shibuya, S., Watanabe, S., Yamaguchi, H., Abe, S., Okamura, N.
(2007). Revised Culture-Based System for Identification of Malassezia Species. J. Clin. Microbiol.
45: 3737-3742
[Abstract]
[Full Text]
-
Diaz, M. R., Boekhout, T., Theelen, B., Bovers, M., Cabanes, F. J., Fell, J. W.
(2006). Microcoding and flow cytometry as a high-throughput fungal identification system for Malassezia species.. J Med Microbiol
55: 1197-1209
[Abstract]
[Full Text]
-
Paulino, L. C., Tseng, C.-H., Strober, B. E., Blaser, M. J.
(2006). Molecular analysis of fungal microbiota in samples from healthy human skin and psoriatic lesions.. J. Clin. Microbiol.
44: 2933-2941
[Abstract]
[Full Text]
-
Gaitanis, G., Chasapi, V., Velegraki, A.
(2005). Novel Application of the Masson-Fontana Stain for Demonstrating Malassezia Species Melanin-Like Pigment Production In Vitro and in Clinical Specimens. J. Clin. Microbiol.
43: 4147-4151
[Abstract]
[Full Text]
-
Sugita, T., Tajima, M., Ito, T., Saito, M., Tsuboi, R., Nishikawa, A.
(2005). Antifungal Activities of Tacrolimus and Azole Agents against the Eleven Currently Accepted Malassezia Species. J. Clin. Microbiol.
43: 2824-2829
[Abstract]
[Full Text]
-
Cabanes, F. J., Hernandez, J. J., Castella, G.
(2005). Molecular Analysis of Malassezia sympodialis-Related Strains from Domestic Animals. J. Clin. Microbiol.
43: 277-283
[Abstract]
[Full Text]
-
Gupta, A. K., Boekhout, T., Theelen, B., Summerbell, R., Batra, R.
(2004). Identification and Typing of Malassezia Species by Amplified Fragment Length Polymorphism and Sequence Analyses of the Internal Transcribed Spacer and Large-Subunit Regions of Ribosomal DNA. J. Clin. Microbiol.
42: 4253-4260
[Abstract]
[Full Text]
-
Velegraki, A., Alexopoulos, E. C., Kritikou, S., Gaitanis, G.
(2004). Use of Fatty Acid RPMI 1640 Media for Testing Susceptibilities of Eight Malassezia Species to the New Triazole Posaconazole and to Six Established Antifungal Agents by a Modified NCCLS M27-A2 Microdilution Method and Etest. J. Clin. Microbiol.
42: 3589-3593
[Abstract]
[Full Text]
-
Hirai, A., Kano, R., Makimura, K., Duarte, E. R., Hamdan, J. S., Lachance, M.-A., Yamaguchi, H., Hasegawa, A.
(2004). Malassezia nana sp. nov., a novel lipid-dependent yeast species isolated from animals. Int. J. Syst. Evol. Microbiol.
54: 623-627
[Abstract]
[Full Text]
-
Odds, F., Ausma, J., Van Gerven, F., Woestenborghs, F., Meerpoel, L., Heeres, J., Vanden Bossche, H., Borgers, M.
(2004). In Vitro and In Vivo Activities of the Novel Azole Antifungal Agent R126638. Antimicrob. Agents Chemother.
48: 388-391
[Abstract]
[Full Text]
-
Sugita, T., Takashima, M., Kodama, M., Tsuboi, R., Nishikawa, A.
(2003). Description of a New Yeast Species, Malassezia japonica, and Its Detection in Patients with Atopic Dermatitis and Healthy Subjects. J. Clin. Microbiol.
41: 4695-4699
[Abstract]
[Full Text]
-
Sugita, T., Kodama, M., Saito, M., Ito, T., Kato, Y., Tsuboi, R., Nishikawa, A.
(2003). Sequence Diversity of the Intergenic Spacer Region of the rRNA Gene of Malassezia globosa Colonizing the Skin of Patients with Atopic Dermatitis and Healthy Individuals. J. Clin. Microbiol.
41: 3022-3027
[Abstract]
[Full Text]