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

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.

Keywords: 26S rDNA PCR-RFLP; Malassezia.

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.