Ann Dermatol.  2009 Feb;21(1):18-26. 10.5021/ad.2009.21.1.18.

The Investigation on the Distribution of Malassezia Yeasts on the Normal Korean Skin by 26S rDNA PCR-RFLP

• Jang SJ
• Lim SH
• Ko JH
• Oh BH
• Kim SM
• Song YC
• Yim SM
• Lee YW
• Choe YB
• Ahn KJ

• KMID: 2172060
• DOI: http://doi.org/10.5021/ad.2009.21.1.18

Abstract

BACKGROUND
Malassezia yeasts are normal flora of the skin that are discovered in 75~98% of health subjects, but since its association with various skin disorders have been known, many studies have been conducted in the distribution of the yeasts.
OBJECTIVE
To isolate, identify, and classify Malassezia yeasts from the normal human skin of Koreans by using the rapid and accurate molecular biology method (26S rDNA PCR-RFLP) which overcome the limits of morphological and biochemical methods, and to gather a basic database that will show its relation to various skin diseases.
METHODS
Malassezia yeasts were cultured from clinically healthy human skin using scrub-wash technique at five sites (forehead, cheek, chest, upper arm, and thigh) and swabbing technique at scalp in 160 participants comprised of 80 males and 80 females aged from 0 to 80. Identification of obtained strains were placed into the one of the 11 species by 26S rDNA PCR-RFLP.
RESULTS
An overall positive culture rate was 62.4% (599/960). As shown in the experiment groups by their age, the positive culture rate was the highest (74.2%) in the age 21~30 and 31~40 (89/120). In the experiment groups by different body areas, the scalp showed the highest positive culture rate of 90% (144/160). On analysis of 26S rDNA PCR-RFLP, M. globosa was the most predominant species in the age 0~10 (32.8%), 11~20 (28.9%), 21~30 (32.3%). M. restricta was identified as predominant species in the age 41~50 (27.9%), 61~70 (31.5%) and 71~80 (24.0%). In the age 31~40 years, M. sympodialis was found to be the most common species (24.6%). According to body site, M. restricta was more frequently recovered in the scalp (56.8%), forehead (39.8%) and cheek (24.0%) and while M. globosa was more frequently recovered in the chest (36.8%). Higher positive culture rates of Malassezia yeasts were shown in male subjects than female counterparts in all body areas except scalp (p<0.05). Especially in this study, M. dermatis, newly isolated Malassezia species from atopic dermatitis patient in Japan, was isolated and identified in 19 cases (1.9%) in healthy subjects.
CONCLUSION
The key is to recognize the existence of a difference in the type of Malassezia species in different ages as well as body areas, which reflects differing skin lipid levels in various ages and different body areas. Moreover, 26S rDNA PCR-RFLP analysis which was opted in this study could provide a sensitive and rapid identification system for Malassezia species, which may be applied to epidemiological surveys and clinical practice.

Keyword

26S rDNA PCR-RFLP; Malassezia

MeSH Terms

Aged
Arm
Cheek
Dermatitis, Atopic
DNA, Ribosomal
Female
Forehead
Humans
Japan
Malassezia
Male
Molecular Biology
Scalp
Skin
Skin Diseases
Thorax
Yeasts
DNA, Ribosomal

Figure

• 

  Fig. 1 26S rDNA PCR products of 11 Malassezia standard strains. In PCR to amplify the 26S rDNA, approximately 580 bp PCR band was identified in all of 11 standard colonies (Lane M: 100 bp DNA ladder, Lane 1: M. furfur, Lane 2: M. sympodialis, Lane 3: M. globosa, Lane 4: M. restricta, Lane 5: M. slooffiae, Lane 6: M. pachydermatis, Lane 7: M. japonica, Lane 8: M. nana, Lane 9: M. dermatis, Lane 10: M. obtusa, Lane 11: M. yamatoensis, Lane 12: negative control).

• 

  Fig. 2 26S rDNA PCR-RFLP patterns that digested with Hha1 (A), BstF51 (B) of Malassezia standard strains. On analysis of PCR-RFLP of 26S rDNA using restriction enzymes Hha1, 9 different species, including M. furfur, M. globosa, M. restricta, M. slooffiae, M. pachydermatis, M. japonica, M. nana, M. obtusa, and M. yamatoensis showed interspecifically distinguishable restriction pattern but M. sympodialis and M. dermatis produced same pattern (A). In order to differentiate the two species, we used an additional restriction enzyme BstF51 to prove those two are indeed different species (B) (Lane M: 100 bp DNA ladder, Lane 1: M. furfur, Lane 2: M. sympodialis, Lane 3: M. globosa, Lane 4: M. restricta, Lane 5: M. slooffiae, Lane 6: M. pachydermatis, Lane 7: M. japonica, Lane 8: M. nana, Lane 9: M. dermatis, Lane 10: M. obtusa, Lane 11: M. yamatoensis).

• 

  Fig. 3 The frequency of co-culture of different Malassezia species in one media.

Reference

1. Janik MP, Heffernan MP. Wolff K, Goldsmith LA, Katz SI, Gilchrest BA, Paller AS, Leffell DJ, editors. Yeasts infection: Candidiasis, Pityriasis (Tinea) versicolor. Fitzpatrick's dermatology in general medicine. 2008. 7th ed. New York: McGraw-Hill;1822–1830.

2. Ahn KJ. Taxonomy of the genus Malassezia. Korean J Med Mycol. 1998. 3:81–88.

3. Ljubojević S, Skerlev M, Lipozencić J, Basta-Juzbasic A. The role of Malassezia furfur in dermatology. Clin Dermatol. 2002. 20:179–182.

4. Kanda N, Tani K, Enomoto U, Nakai K, Watanabe S. The skin fungus-induced Th1- and Th2-related cytokine, chemokine and prostaglandin E2 production in peripheral blood mononuclear cells from patients with atopic dermatitis and psoriasis vulgaris. Clin Exp Allergy. 2002. 32:1243–1250.

5. Ginarte M, Fabeiro JM, Toribio J. Confluent and reticulated papillomatosis (Gougerot-Carteaud) successfully treated with tacalcitol. J Dermatolog Treat. 2002. 13:27–30.

6. Yesudian P, Kamalam S, Razack A. Confluent and reticulated papillomatosis (Gougerot-Carteaud). An abnormal host reaction to Malassezia furfur. Acta Derm Venereol. 1973. 53:381–384.

7. Chowdhary A, Randhawa HS, Sharma S, Brandt ME, Kumar S. Malassezia furfur in a case of onychomycosis: colonizer or etiologic agent? Med Mycol. 2005. 43:87–90.

8. Gupta AK, Batra R, Bluhm R, Boekhout T, Dawson TL Jr. Skin diseases associated with Malassezia species. J Am Acad Dermatol. 2004. 51:785–798.

9. Devlin RK. Invasive fungal infections caused by Candida and Malassezia species in the neonatal intensive care unit. Adv Neonatal Care. 2006. 6:68–77.

10. Curvale-Fauchet N, Botterel F, Legrand P, Guillot J, Bretagne S. Frequency of intravascular catheter colonization by Malassezia spp. in adult patients. Mycoses. 2004. 47:491–494.

11. Guého E, Midgley G, Guillot J. The genus Malassezia with description of four new species. Antonie Van Leeuwenhoek. 1996. 69:337–355.

12. Lee YW, Yim SM, Lim SH, Choe YB, Ahn KJ. Quantitative investigation on the distribution of Malassezia species on healthy human skin in Korea. Mycoses. 2006. 49:405–410.

13. Guillot J, Gueho E, Lesourd M, Midgley G, Chevrier G, Dupont B. Identification of Malassezia species: a practical approach. J Mycol Med. 1996. 6:103–110.

14. Rodriguez-Valero S, Mesa LM, Gonzalez-Moran E, Delmonte ML, Robertiz S, Valero A. Phenotypic characterization of species of Malassezia in healthy skin of an university student population. Invest Clin. 2005. 46:329–335.

15. Bernier V, Weill FX, Hirigoyen V, Elleau C, Feyler A, Labrèze C, et al. Skin colonization by Malassezia species in neonates: a prospective study and relationship with neonatal cephalic pustulosis. Arch Dermatol. 2002. 138:215–218.

16. Morishita N, Sei Y, Sugita T. Molecular analysis of Malassezia microflora from patients with pityriasis versicolor. Mycopathologia. 2006. 161:61–65.

17. Sugita T, Tajima M, Tsubuku H, Tsuboi R, Nishikawa A. Quantitative analysis of cutaneous Malassezia in atopic dermatitis patients using real-time PCR. Microbiol Immunol. 2006. 50:549–552.

18. Senczek D, Siesenop U, Bohm KH. Characterization of Malassezia species by means of phenotypic characteristics and detection of electrophoretic karyotypes by pulsed-field gel electrophoresis (PFGE). Mycoses. 1999. 42:409–414.

19. Gupta AK, Boekhout T, Theelen B, Summerbell R, Batra R. 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. 2004. 42:4253–4260.

20. Theelen B, Silvestri M, Guého E, van Belkum A, Boekhout T. Identification and typing of Malassezia yeasts using amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE). FEMS Yeast Res. 2001. 1:79–86.

21. Gandra RF, Simao RC, Matsumoto FE, da Silva BC, Ruiz LS, da Silva EG, et al. Genotyping by RAPD-PCR analyses of Malassezia furfur strains from pityriasis versicolor and seborrhoeic dermatitis patients. Mycopathologia. 2006. 162:273–280.

22. Gaitanis G, Velegraki A, Alexopoulos EC, Chasapi V, Tsigonia A, Katsambas A. Distribution of Malassezia species in pityriasis versicolor and seborrhoeic dermatitis in Greece. Typing of the major pityriasis versicolor isolate M. globosa. Br J Dermatol. 2006. 154:854–859.

23. Gemmer CM, DeAngelis YM, Theelen B, Boekhout T, Dawson TL Jr. Fast, noninvasive method for molecular detection and differentiation of Malassezia yeast species on human skin and application of the method to dandruff microbiology. J Clin Microbiol. 2002. 40:3350–3357.

24. Guillot J, Deville M, Berthelemy M, Provost F, Gueho E. A single PCR-restriction endonuclease analysis for rapid identification of Malassezia species. Lett Appl Microbiol. 2000. 31:400–403.

25. Makimura K, Tamura Y, Kudo M, Uchida K, Saito H, Yamaguchi H. Species identification and strain typing of Malassezia species stock strains and clinical isolates based on the DNA sequences of nuclear ribosomal internal transcribed spacer 1 regions. J Med Microbiol. 2000. 49:29–35.

26. Gaitanis G, Velegraki A, Frangoulis E, Mitroussia A, Tsigonia A, Tzimogianni A, et al. Identification of Malassezia species from patient skin scales by PCR-RFLP. Clin Microbiol Infect. 2002. 8:162–173.

27. Mirhendi H, Makimura K, Zomorodian K, Yamada T, Sugita T, Yamaguchi H. A simple PCR-RFLP method for identification and differentiation of 11 Malassezia species. J Microbiol Methods. 2005. 61:281–284.

28. Lee YW, Lim SH, Ahn KJ. The application of 26S rDNA PCR-RFLP in the identification and classification of Malassezia Yeast. Korean J Med Mycol. 2006. 11:141–153.

29. Sugita T, Kodama M, Saito M, Ito T, Kato Y, Tsuboi R, et al. 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. 2003. 41:3022–3027.

30. Sugita T, Takashima M, Shinoda T, Suto H, Unno T, Tsuboi R, et al. New yeast species, Malassezia dermatis, isolated from patients with atopic dermatitis. J Clin Microbiol. 2002. 40:1363–1367.

31. Sugita T, Takashima M, Kodama M, Tsuboi R, Nishikawa A. Description of a new yeast species, Malassezia japonica, and its detection in patients with atopic dermatitis and healthy subjects. J Clin Microbiol. 2003. 41:4695–4699.

32. Hirai A, Kano R, Makimura K, Duarte ER, Hamdan JS, Lachance MA, et al. Malassezia nana sp. nov., a novel lipid-dependent yeast species isolated from animals. Int J Syst Evol Microbiol. 2004. 54:623–627.

33. Williamson P, Kligman AM. A new method for the quantitative investigation of cutaneous bacteria. J Invest Dermatol. 1965. 45:498–503.

34. Leeming JP, Notman FH, Holland KT. The distribution and ecology of Malassezia furfur and cutaneous bacteria on human skin. J Appl Bacteriol. 1989. 67:47–52.

35. Leeming JP, Notman FH. Improved methods for isolation and enumeration of Malassezia furfur from human skin. J Clin Microbiol. 1987. 25:2017–2019.

36. Sugita T, Tajima M, Takashima M, Amaya M, Saito M, Tsuboi R, et al. A new yeast, Malassezia yamatoensis, isolated from a patient with seborrheic dermatitis, and its distribution in patients and healthy subjects. Microbiol Immunol. 2004. 48:579–583.

37. Faergemann J, Fredriksson T. Age incidence of Pityrosporum orbiculare on human skin. Acta Derm Venereol. 1980. 60:531–533.

38. Bergbrant IM, Faergemann J. Variations of Pityrosporum orbiculare in middle-aged and elderly individuals. Acta Derm Venereol. 1988. 68:537–540.

39. Kwon HC, Kang SH, Kim HU. The distribution of Malassezia yeasts on normal human skin by culture study using the scrub-wash technique. Korean J Dermatol. 1999. 37:38–45.

40. Gupta AK, Kohli Y. Prevalence of Malassezia species on various body sites in clinically healthy subjects representing different age groups. Med Mycol. 2004. 42:35–42.

41. Nakabayashi A, Sei Y, Guillot J. Identification of Malassezia species isolated from patients with seborrhoeic dermatitis, atopic dermatitis, pityriasis versicolor and normal subjects. Med Mycol. 2000. 38:337–341.

42. Leeming JP, Sansom JE, Burton JL. Susceptibility of Malassezia furfur subgroups to terbinafine. Br J Dermatol. 1997. 137:764–767.

43. Ashbee HR, Ingham E, Holland KT, Cunliffe WJ. The carriage of Malassezia furfur serovars A, B and C in patients with pityriasis versicolor, seborrhoeic dermatitis and controls. Br J Dermatol. 1993. 129:533–540.