Cent. Eur. J. Biol. • 7(2) • 2012 • 241-249 DOI: 10.2478/s11535-012-0018-3 Central European Journal of Biology Atypical non-lipid-dependent strains of Malassezia furfur Research Article Aukse Zinkeviciene1,2,*, Vilmantas Norkunas1, Donaldas Citavicius1 1Department of Microbiology and Biotechnology, Faculty of Natural Sciences, Vilnius University, LT-03101 Vilnius, Lithuania 2Centre of Diagnosis and Treatment of Allergic Diseases, LT-08109 Vilnius, Lithuania Received 15 August 2011; Accepted 26 January 2012 Abstract: Members of the yeast genus Malassezia, including atypical strains, are lipophilic except for Malassezia pachydermatis. New physiological features that characterize atypical Malassezia strains are mainly associated with alteration in Tween assimilation pattern – such isolates still require lipids for growth. We isolated three non-lipid-dependent strains of Malassezia from patients with diagnosed atopic dermatitis (AD). These isolates could not be identified to the species levelvia their physiological properties. Phylogenetic trees, based on the D1/D2 regions of the 26S rDNA gene sequences and nucleotide sequences of the ITS1-5.8S-ITS2 rRNA region, showed the isolates to belong to Malassezia furfur. Three non-lipid dependent isolates from AD skin were conspecific, and sequences analysis proved them to beM. furfur. Keywords: Malassezia furfur • Atypical isolates • Atopic dermatitis © Versita Sp. z o.o. 1. Introduction Physiological systems based on lipid assimilation, b-glucosidase activity, catalase reaction and pigment The genus Malassezia belongs to anamorphic production employing tryptophan as the single nitrogen basidiomycetous yeasts [1]. At present, fourteen source are widely used for identifying Malassezia Malassezia species are reported (M. furfur, M. globosa, species. In practice some individual strains can show M. restricta, M. sympodialis, M. slooffiae, M. obtusa, unrepresentative physiological features, and such M. pachydermatis [2], M. dermatis [3], M. japonica [4], atypical isolates of Malassezia species are reported with M. yamatoensis [5], M. nana [6], M. equina, M. caprae increasing frequency. These strains differ from the most [7], and M. cuniculi [8]). Malassezia yeasts comprise typical Malassezia strains in their expression of one or a part of the microbial flora of normal skin, but under more phenotypes, and they are often difficult to identify certain conditions they can cause superficial skin to the species level by routine methods [16-21]. infections, such as seborrheic dermatitis, pityriasis Atopic dermatitis skin is very susceptible to versicolor, folliculitis, atopic dermatitis, and other cutaneous bacterial, viral, and fungal infections. disorders [9-14]. The role of these opportunistic Evidence that the Malassezia species represent microorganisms in the development of diseases, and a contributing factor in AD is increasing. Because the mechanism triggering these commensal yeasts to Malassezia is a fastidious microorganism to culture, become pathogenic and induce infections in the skin, no reliable method to confirm its overgrowth during is not clear. Adaptation to the skin environment and clinical practice outside the research setting exists the associated pathogenicity may be due to metabolic [13,14]. In this study the isolates were obtained then limitations and capabilities, as these species are samples were taken from patients by physicians recognized as lipid-dependent [15]. during clinical treatments. We isolated three * E-mail: [email protected] 241 Atypical non-lipid-dependent strains of Malassezia furfur Malassezia strains that could not be identified by 2.3 Molecular analysis of Malassezia routine methods. This paper presents a biochemical Colonies grown on mLNA agar at 32oC for five days were and physiological characterization of these isolates collected by scraping them from the surface of the agar as well as their taxonomic identification by rDNA and plate. DNA was extracted by the modified glass beads ITS1-5.8S-ITS2 rRNA sequences analysis. protocol of Vogelstein and Gillespie (DNA Extraction Kit, Fermentas, Lithuania). The samples were dissolved in sterile distilled water and used for PCR amplification. 2. Experimental Procedures Primer pair for D1/D2 26S rDNA amplification was based on the previously reported sequences [26]. 2.1 Collection and cultivation of the samples Forward primer F63: 5’-GCA TAT CAA TAA GCG GAG Atypical M. furfur strains were isolated from the face GAA AAG-3’; reverse primer LR3: 5’-GGT CCG TGT and trunk of patients with a clinical diagnosis of atopic TTC AAG ACG-3’. Primer pair for internal transcribed dermatitis (from one patient each). The samples were spacer (ITS) regions of the rRNA gene was based on collected by scraping affected areas with a disposable the previously reported sequences [27]. Forward primer scalpel and collecting the scrapings in between sterile pITS-F: 5’-GTC GTA ACA AGG TTA ACC TGC GG-3’; slides sealed with tape. After transportation to the reverse primer pITS-R: 5’-TCC TCC GCT TAT TGA laboratory the specimens were cultured qualitatively TAT GC-3’. Amplification reactions were performed in on modified Leeming and Notman agar (mLNA; 10 g an Eppendorf PCR system using the following cycling glucose, 10 g peptone, 8 g bile salts, 2 g yeast extract, parameters: 95oC for two minutes; followed by 29 cycles 0.5 g glycerol monostearate, 10 ml glycerol, 5 ml Tween at 95oC for one minute, 50oC for two minutes, and 60, 20 ml olive oil, 15 g agar, 50 mg chloramphenicol and 72oC for three minutes; followed by a final extension 50 mg cycloheximide per liter), as recommended by the at 72oC for seven minutes. To confirm the absence of Centraalbureau voor Schimmelcultures (Utrecht, The false positive reactions the PCR products were inserted Netherland). Incubation was performed at 32oC for two into the plasmid vector pTZ57R/T (InsTAcloneTMPCR weeks. Written consent was obtained from each patient. Cloning Kit, Fermentas, Lithuania) according to the manufacturer’s instructions. Plasmids were extracted 2.2 Morphological and physiological from an overnight culture of Escherichia coli using the identification lysis by alkali method, dissolved in sterile distilled water, Morphology of the colonies was examined on mLNA and used for PCR amplification. The PCR products were after incubation at 32oC for five days. Isolated colonies purified. The protocol BigDye Terminator v3.1 Cycle were used for the identification. The ability to grow on Sequencing kit (Applied Biosystems, Nieuwerkerk aan Tween 20, 40, 60, and 80 utilization tests, diazonium de IJsel, The Netherlands) was used for sequencing blue B reactions, and catalase reactions [2,22] were with genetic analyzer 3130xl (Applied Biosystems). performed in order to confirm the Malassezia genus Phylogenetic and molecular evolutionary analyses were and species. Tween assimilation tests were performed conducted using MEGA version 4 program [28]. The following the Tween dilution test protocol [2] and the genetic distances were calculated using the Maximum Tween diffusion test protocol [22]. Selective growth Composite Likelihood method [29], and the phylogenetic with cremophor EL, the presence of β-glucosidase as trees were constructed using the neighbor-joining revealed by the splitting of esculin [23], the ability to method [30]. Bootstrap analysis of the data (using 1000 produce pigments on p-medium [24,25] and temperature replicates) was carried out to evaluate the validity and requirements were also tested. The ability to grow on reliability of the tree topology [31]. Sabouraud agar (SGA; 20 g glucose, 10 g peptone, 15 g agar per liter) was verified by multiple (four) transfers on SGA with intervals of five days incubations at 32oC. The 3. Results ability to grow in Sabouraud broth (SGB; 20 g glucose, 10 g peptone) was analysed by incubating the flasks We studied physiological features of three Malassezia of inoculated media (50 ml of SGB in 150 ml flask isolates. The sequencing of the 26S and ITS-5.8S rRNA was inoculated with 2 ml of a suspension containing regions supported our findings that these were atypical 105 cells/ml) on a rotary shaker at 32oC. After five days, variants of M. furfur with new physiological properties. suspensions of each strain were smeared on the SGA by means of a swab. The plates were incubated for five 3.1 Taxonomic characteristics days at 32oC and inspected for growth daily. All tests After five days of incubation on mLNA at 32oC, colonies were performed four times. of three atypical M. furfur isolates developed white 242 A. Zinkeviciene et al. to cream in color, smooth, butyrous and umbonate. Cells were globose, 2.2–4 μm in diameter, with monopolar budding on a more or less broad base (Figure 1). Growth was recorded in the presence of cremophor EL and Tweens 20, 40, 60, 80 as sole lipid sources. Catalase reaction was positive. The isolates showed β-glucosidase activity, which was revealed by the splitting of esculin. The atypical strains were characterized by active synthesis of fluorochromes and pigments from tryptophan. M. furfur is the only member of the genus able to produce brown to greenish yellow pigments from tryptophan as a sole nitrogen source [24,25]. Active growth was recorded at 37oC and 40oC. Although the atypical isolates were in many ways similar to M. furfur, biochemical identification was not possible because of their ability to grow on Sabouraud glucose agar without lipid supplementation. The growth on SGA differed from the growth on mLNA (Figures 2 and 3): colonies were very small (~0.3 mm in diameter Figure 1. Vegetative cells of M. furfur strain M47 grown on mLNA for compared with 2-3 mm colonies developing on five days at 32oC. Bar, 2 μm. mLNA), but growth was recorded on the second day of cultivation. In order to check the ability of the yeasts to survive without lipid supplementation for longer periods of time, four repeated transfers from SGA on SGA with five days intervals were performed. In addition, the growth in Sabouraud glucose broth (SGB) without any lipid supplementation was also recorded: after five days incubation in SGB at 32oC and transfer on SGA these isolates developed small (~0.3 mm in diameter) colonies.