Indian Journal of Medical Microbiology, (2004) 22 (3):179-181

Brief Communication

IDENTIFICATION OF MALASSEZIA SPECIES

AJ Kindo, SKC Sophia, *J Kalyani, S Anandan

Abstract
Malassezia spp. are lipophilic unipolar yeasts recognized as commensals of skin that may be pathogenic under certain conditions. The genus Malassezia now comprises of seven species. This study was aimed at using a simple practical approach to speciate Malassezia yeasts from clinical material. Seventy skin scrapings from patients with pityriasis versicolor infection, positive in 10% potassium hydroxide (KOH), were cultured onto modified Dixons agar (mDixons agar) and Sabouraud dextrose agar (SDA) and incubated at 32C. Speciation was done on the basis of Gram stain morphology, catalase test, and utilization of Tweens. Out of 70 scrapings 48 (68.75%) showed growth on mDixons agar. The commonest isolate was M. sympodialis (28, 58%) followed by M. globosa (19, 40%) and one isolate was (2%) of M. restricta. M. sympodialis was the commonest species affecting our population and there was no isolation of M. obtusa, M. slooffiae, M. pachydermatis and M. furfur. Key words: Malassezia spp., pityriasis versicolor, lipophilic yeasts, tween assimilation

For more than a century, yeasts of the genus Malassezia have been known to be a part of the normal flora of human skin and other warm-blooded animals.1,2 Being lipid dependent, they are normally found in areas that are rich in sebaceous glands.3 Once the lipophilic nature of these yeasts was recognized and cultures were made possible, different workers observed spontaneous changes from one morphological type to the other which led to the conclusion that P.orbiculare, P. ovale and M. furfur were only variants of the same species.4 However, apart from their lipid dependence, little is known about the metabolism and nutritional requirements of Malassezia species.2 A first approach, recently presented by Gueho et al5 has been translated into a practice oriented identification system by Guillot et al.6 There was considerable confusion regarding the classification of Malassezia, with different groups tending to favour their own classification scheme. Following the reclassification of the genus Malassezia and the definition of four new species9 much of this confusion has been resolved. There are seven proposed species in the genus Malassezia based on molecular, morphological and biochemical profiles: six lipid dependent species namely M.furfur, M.sympodialis, M. globosa, M.obtusa, M. restricta and M.slooffiae and one lipid independent species, Malassezia pachydermatis. Apart from *Corresponding author Department of Microbiology (AJK, JK), and Department of Dermatology and STD (SKCS, SA), Sri Ramachandra Medical College and Research Institute, Porur, Chennai - 600 116, Tamil Nadu, India. Received : 28-04-2003 Accepted : 19-09-2003

morphological criteria, Malassezia yeasts are primarily differentiated by their ability to assimilate various polyoxyetheylene sorbitan esters (Tween) following the methodology of Guillot et al.6 The present study was aimed to identify the Malassezia spp. causing pityriasis versicolor in a south Indian population. Materials and Methods This study was conducted from August 2001 to October 2002.The samples were collected from patients with pityriasis versicolor in the out-patient department of skin and STD and processed in the mycology laboratory of the department of microbiology. Entry of observations in a detailed proforma, clinical evaluation, Woods lamp examination and mycological evaluation by microscopic examination of KOH treated skin scrapings were done. Culture Only samples which were KOH positive (n=70) were cultured. The scales were inoculated into modified Dixons agar (mDixons agar) as described by Guillot et al6 and into Sabouraud dextrose agar containing 0.05% chloramphenicol and 0.05% cycloheximide (SDA). The tubes were incubated at 32C for 3-4 days. Microscopic features The morphology of the yeast cells was studied by making Gram stained smears of the isolates from mDixons agar after one week incubation at 32C. Physiological characteristics The catalase reaction was determined by application of a drop of hydrogen peroxide (10 vol.) onto a portion

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of a colony on a glass slide, or directly on colonies on the culture media. The production of gas bubbles indicated a positive reaction. For each isolate, the ability to utilize individual Tweens was tested by the following procedure. Sterile SDA (16 mL) was melted and allowed to cool to about 50C. The yeast being identified were added (2 mL) to the medium. The suspension was obtained by inoculating 5 mL of sterile distilled water with a loopful of actively growing yeasts and the concentration was adjusted to about 105 cell/mL. The seeded agar was then vigorously mixed and poured into a 9 cm diameter petri dish. Once the medium had solidified, four wells were made by means of a 2 mm diameter punch and filled with 5 L of Tween 20, 40, 60 and 80 respectively. Since these reagents are watersoluble, a concentration gradient formed around each well. The plates were systematically incubated for one week at 32C. Utilization of Tweens was assessed by the degree of growth and/or reaction (precipitation) of the lipophilic yeasts around individual wells. Results All the 70 skin scrapings showed hyphae and spores exhibiting the characteristic spaghetti and meatball appearance in the KOH preparation (Fig. 1a). Growth was obtained on mDixons agar from 48 (68.57%) of the 70 skin scrapings (Fig. 1b). Twenty-eight (58%) of the isolates belonged to M. sympodialis, 19 (40%) isolates belonged to M. globosa and 1(2%) case belonged to M. restricta. There was no growth in SDA, ruling out the presence of M. pachydermatis the only lipidindependent species. Other species namely M.furfur, M. slooffiae, and M. obtusa, were not isolated from our population.

Figure 1b : Smooth, cream coloured colonies of Malassezia spp. in modified Dixons agar.

the narrow base (Fig.3a, 3b). The catalase reaction was positive for all except one isolate of M. restricta, which is the only lipid dependent species of Malassezia to consistently lack catalase. The Tween diffusion test allowed the differentiation of most Malassezia species in our study population. The growth of 28 isolates was inhibited by high

Figure 2 : Malassezia sympodialis with small ovoid and characteristic sympodial budding (Gram stain, x 1000).

Figure 1a : Microscopic examination of KOH treated skin scrapings showing the hyphae and spores- characteristic spaghetti and meatball appearance (KOH, x 400).

M.sympodialis had small ovoid cells with sympodial budding, which is a characteristic feature (Fig.2). M. globosa had stable spherical cells. Buds were formed on

Figure 3a : Malassezia globosa with stable, spherical cells with a broad base (Gram stain, x 1000).

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of the Tweens ( M. globosa or M. restricta or M. obtusa ). Based on the catalase reaction and the microscopic morphology in Gram stained smears, these isolates were speciated as M. globosa. Discussion The few reports that have appeared since the taxonomic revision of the genus Malassezia in 1996, suggest that M.sympodialis is the predominant species of human skin, in health or disease, and is mainly found on the trunk, M.globosa is found in scales of pitryiasis versicolor and in healthy skin, and M. restricta seems to be associated with pityriasis capitis.7
Figure 3b : Single budding cell of Malassezia globosa (Gram stain, x 1000).

concentrations of Tween 20. This phenomenon resulted in a characteristic ring of tiny colonies around the corresponding well (Fig. 4). 19 isolates did not utilize any

In our study, out of the 70 specimens that were inoculated, 68.75% yielded growth of Malassezia in culture. Out of this, the most frequently isolated species was Malassezia sympodialis (58.33%), followed by M.globosa (39.58%) and M.restricta (2.08%). In an earlier study, Crespo et al 9 reported that M.globosa was recovered from 97% of their patients, alone in 60% of them and associated with M.sympodialis in 29% and M. slooffiae in 7%. These authors concluded that M.globosa in its mycelial phase is the causative agent of pityriasis versicolor.8 Our results suggest that M. sympodialis in its mycelial phase, is the most common Malassezia spp. associated with pityriasis versicolor in the population attending our hospital in south India. The presence of this species, in its yeast phase in diseased and even in healthy skin, indicates that local factors (humidity, sweat, heat) together with some degree of idiosyncratic individual predisposition, are responsible for the transformation to the mycelial form and development of clinical lesions. 6. 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. Midley G, Gueho E, Guillot J. Diseases caused by Malassezia species. In: Medical Mycology (Hay RJ, Ajello L, eds). Topley and Wilsons Microbiology and Microbial Infections. Vol. 4, part iv (Superficial Basidiomycetous Yeasts). London: Arnold. 1997:201-211. Crespo VE, Ojedo AM, Vera AC, Crespo AE, Sanchez FF. Malassezia globosa as the causative agent of pityriasis versicolor. Br J Dermatol 2000;143:799-803. Ashbee HR, Evans EGV. Immunology of diseases associated with Malassezia species. Clin Microbiol Rev 2002;15(1):21-57.

Figure 4 : Tween assimilation test- Malassezia sympodialis: growth around all Tweens but inhibited by high concentration of Tween 20.

References
1. Yarrow D, Ahearn DG. Malassezia Baillon. The Yeasts: A Taxonomic study (Kreger- van Rij NJW, ed.). 3rd edn. Amsterdam: North Holland Publishing Company. 1984:882-885. Ingham E, Cunningham AC. Malassezia furfur. J Med Vet Mycol 1993;31:265-288. Nazarro-Porro, Passi S, Caprilli F, et al. Growth requirement and lipid metabolism of Pityriasis orbiculare. J Invest Dermatol 1976;66:178-182. Faergeman J. Pityrosporum infections. In: Topics in Clinical Dermatology Cutaneous Fungal Infections. New York: Igaku shoin, 1992:69-83. Gueho E, Midley G, Guillot J. The genus Malassezia with description of four new species. Antonie Van Leewenhoek 1996;69:337-355. 9.

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