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Home , Malassezia, Malassezia furfur, Malassezia pachydermatis, Trichosporon

Cent. Eur. J. Biol. • 7(2) • 2012 • 241-249 DOI: 10.2478/s11535-012-0018-3

Central European Journal of Biology

Atypical non--dependent strains of 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 Malassezia, including atypical strains, are lipophilic except for . New physiological features that characterize atypical Malassezia strains are mainly associated with alteration in Tween assimilation pattern – such isolates still require 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 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 . 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 [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, , 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; 10g 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. The main physiological characteristics of the known Malassezia species and our atypical isolates are summarized in Table 1.

3.2 Molecular phylogenetic analysis The atypical yeast isolates (M47, M54 and M235) obtained from the AD patients showed nearly identical Figure 2. Colonies appearance of atypical strain of M. furfur (M47) D1/D2 and ITS-5.8S-ITS2 sequences indicating that on mLNA after incubation at 32oC for five days. they are conspecific strains. The isolates formed a cluster with the reference M. furfur CBS 1878T sequences, with high bootstrap support values of 96% and 99% for the 26S and ITS-5.8S phylogeny, respectively (Figures 4 and 5). Similarities between M47, M54 and M235 isolates and the reference M. furfur strain CBS 1878T in their D1/D2 regions were 99.3%, 98.9%, and 99.5%, respectively. Similarities between M47, M54 and M235 isolates and the M. furfur CBS 1878T strain in their ITS-5.8S regions were 97.9%, 98.2%, and 98.5%, respectively. Phylogenetic analysis of sequences from these atypical strains indicated that they didn‘t exceed the variation generally observed to occur between species [32]. These isolates were identified as non- Figure 3. Colonies appearance of atypical strain of M. furfur (M47) lipid-dependent variants of M. furfur. on SGA after incubation at 32oC for five days.

243 Atypical non-lipid-dependent strains of Malassezia furfur

Figure 4. Phylogenetic tree constructed by using the D1/D2 26S rRNA sequences of atypical isolates of M. furfur and other members of the genus Malassezia. Outgroup: CBS 132T. GenBank accession numbers are indicated in parentheses. The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 0.75632576 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 472 positions in the final dataset.

Tween Tween Tween Tween Growth Cremophor ß-Gluco- Pigment Growth Growth Species 20 40 60 80 Catalase on SGA EL sidase production at 37oC at 40oC 10% 0∙5% 0∙5% 0∙1%

M. capraea - - W W W - + + ? - - M. cuniculi ------+ + ? + + M. dermatisb - + + + + w + - ? + + M. equinaa - - w w w - + - ? w - M. furfurc - + + + + + + w + + + M. globosac ------+ - - - - M. japonicad - - w + - - + + ? + - M. nanae - + + + w - + - ? + v M. obtusac ------+ + - w - M. pachyder.c + + + + + + +, w + w + + M. restrictac ------v - M. slooffiaec - + + + - - + - - + + M. sympod.c - - + + + -, w + + - + + M. yamatoen.f - + + + + ? + ? ? + - M.47 + + + + + + + w + + + M.54 + + + + + + + w + + + M.235 + + + + + + + w + + +

Table 1. Main characteristics of Malassezia species.

aCabañes et al. 2007 [7]; bSugita et al. 2002 [3]; cGuého et al. 1996 [2]; dSugita et al. 2003 [4]; eHirai et al. 2004 [6]; fSugita et al. 2004 [5].

244 A. Zinkeviciene et al.

Figure 5. Phylogenetic tree constructed by using the ITS1-5.8S-ITS2 rRNA gene sequences of atypical isolates of M. furfur and other members of the genus Malassezia. Outgroup: Cryptococcus neoformans CBS 132T. GenBank accession numbers are indicated in parentheses. The evolutionary history was inferred using the Neighbor-Joining method. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 406 positions in the final dataset.

4. Discussion robust lipid dependent species, as any lipid supplement is sufficient for its growth [33]. M. furfur was grown of All Malassezia species are recognized as lipid- samples from various hosts and body sites. The species dependent. Even M. pachydermatis, designated as has been also detected from dogs, horses, cows, non-lipid-dependent, requires growth media enriched bats, and even from a hospital floor [20,21,40-42]. with peptone (i.e. Sabouraud medium), which contains M. furfur has been described as showing high genotypic short chain fatty acids [33]. New physiological features variability, and these specific genotypes could be related that characterize atypical Malassezia strains are to geographic origin of the isolates, skin disease origin, mainly associated with alteration in Tween assimilation age groups, body sites from which they come, or the pattern - such isolates still require lipids for growth. host (human or animal associated subtypes) [43-46]. This feature seems to be related to the distribution of In Lithuania, cutaneous colonization with Malassezia these yeasts on sebum-rich areas of the body [11] and species is very low as compared with studies from other to virulence factors such as the Malassezia and countries [9,47-49]. The cutaneous microenvironment phospholipases (there are opinions that these enzymes is generally considered to be important for Malassezia are involved in pathogenicity mechanisms) [34-37]. populations. Skin diseases associated with Malassezia Besides, some isolates of M. pachydermatis are lipid- species seem to be more common in the tropics, dependent [16,21,38]. This species usually occurs on suggesting that an increased cutaneous temperature animals but could appear as a commensal yeast on and humidity favours yeast growth [50-52]. the skin of dog owners [39]. Some authors suggested There is a theory that phenotypic and genotypic that these yeasts may be in a state of adaptation to a variability in some yeast species can act as the specific host, associated with an increasing dependency determining factor in the infection strategy used by on exogenous lipid supplementation [21]. the microorganism, which is associated with switching M. furfur essentially requires olive oil or oleic acid various metabolic pathways in order to accommodate for growth on Sabouraud agar. But it is one of the most nutrient availability in some circumstances [53]. This

245 Atypical non-lipid-dependent strains of Malassezia furfur

theory was developed for Candida spp. but, in our opinion, that genetic exchange occurs within and between it could also be applicable for Malassezia spp. These two the various genetic groups of M. pachydermatis [57]. genera are phylogenetically distant human pathogenic A similar conclusion was also proposed for M. furfur and commensal fungi, but whole-genome shotgun [58]. Increasing number of Malassezia isolates, which sequencing revealed that M. globosa and C. albicans were identified as a lipid-dependent M. pachydermatis or secrete a similar set of extracellular hydrolases. It was pleomorphic variants of M. furfur, lead to the suggestion proposed that the prevalence of similar gene families about the possibility of genetic material exchange in these two yeast species may point to similar roles between these two species [19,21]. in colonization and virulence [15]. Malassezia spp. Most epidemiological studies involve only incorporate fatty acids directly into cellular lipids without molecular-based identifications of Malassezia species further metabolism [54], and there is a suggestion [59,60]. The observation of Malassezia strains with that the lipids present on the skin surface may affect atypical physiological characteristics may indicate Malassezia resulting in either immunosuppressive that the biology of the species is an essential criterion (or commensal) or immunostimulatory (or pathogen) in yeast systematics. A sequence, per se, does not phenotype [10]. describe a species [32]. There is a possibility that The assessment that M. globosa genome contains a species with atypical physiological features could mating locus indicates that the may be capable be more frequently detected if traditional culturing of sexual development, although mating has never been methods and molecular techniques both are used for observed in Malassezia species. The possibility of a sexual identification. cycle can be important in the distribution of virulence- In conclusion, the sequence analysis of the related genes among populations [15,55]. Genes at the ribosomal genes confirmed that our isolates belong mating locus are highly conserved, even in asexual fungi. to M. furfur. Discovery of the non-lipid-dependence The presence of a coding sequence does not mean that of our strains may be a new physiological pattern the gene is correctly transcribed and translated [56]. allowing better understanding of these unique Nevertheless, the results of one investigation suggested yeasts.

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Appendix

Information (GenBank), htpp://www.ncbi.nlm.nih.gov/. Malassezia furfur strain M54 (18S-ITS1-5.8S-ITS2-28S GenBank accession numbers are: region): HM014469; Malassezia furfur strain M47 (D1/D2 26S rDNA region): Malassezia furfur strain M235 (D1/D2 26S rDNA HM014475; region): HM014473; Malassezia furfur strain M47 (18S-ITS1-5.8S-ITS2-28S Malassezia furfur strain M235 (18S-ITS1-5.8S-ITS2- region): HM014470; 28S region): HM014468. Malassezia furfur strain M54 (D1/D2 26S rDNA region): HM014474;

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