A Mycological and Molecular Biological Study of Malassezia dermatis Isolated from Korean / 대한피부과학회지

BACKGROUND: Malassezia (M.) yeasts are lipophilic fungi which are regarded as normal flora of the skin, and are recovered in 75~98% of healthy adults. Gueho et al reclassified the Malassezia yeasts into 7 species (M. furfur, M. obtusa, M. globosa, M. slooffiae, M. sympodialis, M. pachydermatis, M. restricta) on the basis of molecular biology and by employing an interdisciplinary approach of morphology, microstructurology and physiology. Recently novel species of the genus Malassezia have been discovered as a result of molecular analysis. But there are no additional reports in Korea regarding newly reported Malassezia species because most identification and classification of Malassezia in Korea depend on classical methods and research on molecular biologic application is insufficient. OBJECTIVE: Five clinical isolates of M. dermatis were isolated from the skin of healthy subjects without skin disease or seborrheic dermatitis patients using molecular biology techniques for the first time in Korea. Hence the present study describes mycological and molecular biological characteristics of these five isolates as a novel species of M. dermatis. METHODS: Morphological and biochemical analyses, such as colony morphologies, microscopic morphologies and physiological characteristic were done targeting 5 clinical isolates of M. dermatis. Molecular techniques, namely, 26S rDNA PCR-RFLP, 26S rDNA and internal transcribed spacer region 1 (ITS1) sequencing, were done for identification and phylogenetic systematic analysis. RESULTS: Five clinical isolates of M. dermatis showed positive in the catalase test. No growth is obtained on Sabouraud's dextrose agar (SDA) without lipid supplementation but all grew in 0.5% Tween 60 and 0.1% Tween 80 added 2% glucose/1% peptone culture medium. Round and ellipsoidal yeast cells and budding of the yeast cells were observed under microscope, resembling M. sympodialis, M. furfur, and M. nana. The 26S rDNA PCR-RFLP pattern showed the same pattern as M. dermatis (JCM 11348), the standard strain. 26S rDNA and ITS1 sequencing were performed for exact identification, showing 99% accordance with M. dermatis (AB070361), and M. dermatis (AB070356), confirming the species to be new, the first to be reported in Korea. Phylogenetic trees based on the D1/D2 domains of the 26S rDNA sequences and nucleotide sequences of the ITS 1 region showed that the isolates were conspecific and belonged to the genus Malassezia and crusted with M. sympodialis. CONCLUSION: Taking a molecular biological classification approach, we have successfully isolated 5 cases of M. dermatis-the first in Korea. Although it is not known whether M. dermatis plays a role in Malassezia-related skin disease, this species was part of the microflora in both patients with seborrheic dermatitis and healthy subjects.

The Application of Pyrosequencing Method in the Identification and Classification of Malassezia Yeasts / 대한의진균학회지

BACKGROUND: Malassezia yeasts are lipophilic fungi that are found in 75~80% of healthy adults. Recently, various molecular biological techniques are being preferred to identify and classify the Malassezia yeasts. Pyrosequencing is a real-time DNA sequencing technique. This technology has the potential advantage of accuracy, ease-of-use, high flexibility and is now emerging as a popular platform for microbial typing. OBJECTIVE: We sought to implement novel molecular biology technique, namely pyrosequencing method in identifying and classifying Malassezia yeasts, and assess its clinical applicability. METHODS: We obtained ribosomal RNA sequences of 11 Malassezia standard strains from NCBI database. Primers for the initial PCR amplification of the target region (ITS2) and sequencing primers within the regions amplified by the PCR primers were designed using Pyrosequencing Assay Design Software (Biotage AB, Uppsala, Sweden). We obtained PCR amplifying fragments of genomic DNA isolated from the Malassezia yeasts. And pyrosequence reactions were performed using reagents provided with the PSQ 96 Sample Preparation kit. RESULTS: In the PCR analysis, all of 11 standard strains are shown at the 130 bp levels. In the pyrosequencing analysis, M. obtusa and M. furfur sequences were corresponded among 11 Malassezia standard strains. But, in 4 cases, Malassezia strains mismatched with expected Malassezia strain and in rest of 5 Malassezia strains, pyrosequencing was failed. CONCLUSION: As evidenced above, pyrosequencing analysis could provide a sensitive and rapid identification system for Malassezia species. But it still has many limitation to be applied to epidemiological surveys and clinical practice.

The Application of Colony PCR in the Molecular Biological Analysis of Malassezia Yeasts / 대한의진균학회지

BACKGROUND: Malassezia yeasts are lipophilic fungi that are found in 75~80% of healthy adults. The yeasts are known to be associated with pityriasis versicolor, seborrheic dermatitis, Malassezia folliculitis, and recently its pathogenicity is being expanded to other various skin disorders, such as atopic dermatitis and acne vulgaris. Recently, various molecular biological techniques are being preferred over morphological analysis. In order to perform a DNA-based diagnostic test, availability of a simple, rapid, and reliable DNA extraction protocol is essential. OBJECTIVE: We sought to implement novel molecular biology technique, namely colony PCR method using microwave as the easiest way to amplification of Malassezia target DNA, and assess its clinical applicability. METHODS: Instead of using templates of purified genomic DNA, we performed the PCR directly from Malassezia colonies. A fresh yeast colony transferred to the bottom of a microcentrifuge tube and microwaved for 1 min three times in the presence of a pyrex beaker containing 50 ml of sterile water to dissipate excess heat. Following this microwave lysis, PCR-reaction mixture was added directly to the microcentrifuge tube. Two DNA extraction methods (boiling method, glass beads method) were used for comparing the sensitivity and effectiveness with the colony PCR method. All reactions were performed using the primers 26S and ITS1 complementary to the rDNA region. Results 1. As a result of gel electrophoresis, we recognized expected PCR products (approximately 580 bp for 26S rDNA and 250~320 bp for ITS1) from both colony PCR method and two DNA extraction methods (boiling method, glass beads method). 2. As a result of measuring nucleic acid level with the spectrophotometer, colony PCR disregarding DNA extraction process shows relatively similar PCR efficacy compared with the boiling and glass beads method. And there is no significant difference among those methods statistically (p>0.001). 3. In conducting the PCR method, boiling method required approximately 400 minutes, and glass beads method required approximately 360 minutes, respectively. As contrasted with two methods, colony PCR method required approximately 150 minutes, and could be capable of saving time. In addithion, colony PCR had an economic efficiency comparing with boiling method and glass beads methods. CONCLUSIONS: All these findings suggest that directly application of the Malassezia yeasts obtained from culture colony for PCR reaction is a fast, reliable, cost-effective and simple method for performing any PCR-based protocol including diagnostic tests.