Characterization of markedly lipid-dependent Malassezia pachydermatis isolates from healthy dogs

When 244 Malassezia colonies which had been isolated from a colony of Beagle dogs using modified Dixon's agar were sub-cultured on Sabouraud's dextrose agar to determine their lipid dependence, 30 showed poor growth resembling M. furfur , whereas the remainder were typical of M. pachydermatis . Eight of the 10 poor growing isolates selected for further study formed colonies typical of M. pachydermatis after five passages on Sabouraud's dextrose agar at 4 d intervals and two continued to show poor growth. Nine isolates had enzyme profiles identical to those of typical M. pachydermatis isolates, and one resembled M. furfur . However, seven of the poor growing isolates which were karyotyped had patterns typical of M. pachydermatis. Poor growing isolates and their non-lipid-dependent 'revertants'had identical restriction fragment length polymorphism patterns and poly(GT) hybridization profiles. These observations show that some M. pachydermatis isolates grow poorly when sub-cultured onto Sabouraud's dextrose agar and may be incorrectly identified as M. furfur if further studies are not performed.     

In vitro anti-Malassezia activity of xanthorrhizol isolated from Curcuma xanthorrhiza Roxb

AIMS: This study aimed at investigating the anti-Malassezia activity of xanthorrhizol (XTZ) isolated from Curcuma xanthorrhiza Roxb. against Malassezia furfur ATCC 14521 and Malassezia pachydermatis ATCC 14522. METHODS AND RESULTS: The in vitro susceptibility tests for XTZ were carried out in terms of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC), using broth microdilution method with endpoint after 48 h. Time-kill curves were determined at concentrations ranging from 0 to 25 microg ml(-1). The MIC values of XTZ against M. furfur and M. pachydermatis were 1.25 and 0.25 mug ml(-1), respectively. The MFC of XTZ was 5 microg ml(-1) for M. furfur and 2.5 microg ml(-1) for M. pachydermatis. Time-kill curves demonstrated that treatment with 25 microg ml(-1) of XTZ for 5 h was able to kill 100% of M. furfur, while 20 microg ml(-1) of XTZ for 15 min killed M. pachydermatis completely. CONCLUSION: XTZ shows potential as an anti-Malassezia agent for inhibiting the growth of M. furfur ATCC 14521 and M. pachydermatis ATCC 14522 in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: XTZ may be a useful alternative for treating Malassezia-associated diseases.     

Comparative study of the activity and kinetic properties of malate dehydrogenase and pyruvate decarboxylase from Candida albicans, Malassezia pachydermatis, and Saccharomyces cerevisiae

Candida albicans and Malassezia pachydermatis cause human and animal infections of the skin and internal organs. We compare the properties of two enzymes, pyruvate decarboxylase (PDC) and malate dehydrogenase (MDH), from these species and from Saccharomyces cerevisiae cultivated under aerobic and anaerobic conditions to find differences between the enzymes that adapt pathogens for virulence and help us in searching for new antifungal agents. Malassezia pachydermatis did not show any growth under anaerobic conditions, as opposed to C. albicans and S. cerevisiae. Under aerobic conditions, C. albicans showed the highest growth rate. Malassezia pachydermatis, contrary to the others, did not show any PDC activity, simultaneously showing the highest MDH activity under aerobic conditions and a Km value for oxaloacetate lower than S. cerevisiae. Candida albicans and S. cerevisiae showed a strong decrease in MDH activity under anaerobic conditions. Candida albicans shows four different isoforms of MDH, while M. pachydermatis and S. cerevisiae are characterized by two and three isoforms. Candida albicans shows about a twofold lower activity of PDC but, simultaneously, almost a threefold lower Km value for pyruvate in comparison with S. cerevisiae. The PDC apoform share under aerobic conditions in C. albicans was 47%, while in S. cerevisiae was only 26%; under anaerobic conditions, the PDC apoform decreased to 12% and 8%, respectively. The properties of enzymes from C. albicans show its high metabolic flexibility (contrary to M. pachydermatis) and cause easy switching between fermentative and oxidative metabolism. This feature allows C. albicans to cause both surface and deep infections. We take into consideration the use of thiamin antimetabolites as antifungal factors that can affect both oxidative and fermentative metabolism.     

The genus Malassezia: old facts and new concepts

Lipophilic yeasts are being considered as major opportunistic pathogens for a very long time. Most of the yeasts show an absolute requirement for long fatty acid chains and specific procedures are required for their isolation, conservation and identification. For that reason, the history of the nomenclature used for the Malassezia genus is quite complex. Before 1996, only 3 species were recognized: Malassezia furfur, M. pachydermatis and M. sympodialis. To date, the genus is composed of one non lipid-dependent species (M. pachydermatis) and 12 lipid-dependent species. No doubt that additional new taxa will be described in close future. Very recently the genome and secretory proteome of two Malassezia species was described. This analysis demonstrated the presence of multiple secreted lipases to aid in harvesting host lipids. It also revealed the presence of mating-type genes, providing an indication that Malassezia yeasts may be capable of sex.     

Chitin synthase 2 gene sequence of Malassezia species.

Nucleotide sequences of the chitin synthase 2 (CHS2) gene of seven species, Malassezia furfur, M. globosa, M. obtusa, M. pachydermatis, M. restricta, M. slooffiae and M. sympodialis, were analyzed for their phylogenetic relationship. About 620-bp genomic DNA fragments of the CHS2 gene were amplified from these Malassezia species by polymerase chain reaction (PCR) and sequenced. The CHS2 nucleotide sequences of these Malassezia species showed more than 95% similarity between the species. A phylogenetic analysis of the nucleotide sequences of CHS2 gene fragments of seven Malassezia species revealed that the species were genetically distinct from each other.     

马拉色菌相关人群及正常人群耳耵聍中马拉色菌带菌情况的调查

目的调查马拉色菌相关人群及正常人群耳耵聍中马拉色菌带菌情况。方法用结晶紫染色法对96例被调查人群耳耵聍进行马拉色菌检测,同时作培养,并以标准株作对照,用生理生化方法将耵聍中分离到的79株马拉色菌进行分类。结果马拉色菌相关人群耳耵聍中马拉色菌的直接检出率为91．84％（45／49）,培养阳性率为81．63％（40／49）,其中厚皮马拉色菌8株（16．33％）,合轴马拉色菌10株（20．41％）,糠秕马拉色菌22株（44．90％）。正常人群耳耵聍马拉色菌直接检出率为89．36％（42／47）,培养阳性率为78．72％（37／47）,其中厚皮马拉色菌5株（10．64％）,合轴马拉色菌8株（17．02％）,糠秕马拉色菌23株（48．93％）,斯洛菲马拉色菌1株（2．13％）。结论马拉色菌为正常人群及马拉色菌相关人群外耳道正常菌群,两组人群中马拉色菌的分离率和菌种分布无显著性差异。     

Identification of atypical strains of Malassezia spp. from cattle and dog

Yeast species in the genus Malassezia are lipophilic with the exception of Malassezia pachydermatis. During a study of the occurrence of Malassezia species in the external ear of 964 cattle and 6 dogs in Minas Gerais, Brazil, six lipid-dependent isolates could not be identified to known species. Four isolates came from healthy cows, one from a cow with otitis, and one from a healthy dog. When tested with Tweens and Cremophor EL as single sources of lipids, the strains grew on all sources except Cremophor EL. None of the six strains hydrolyzed esculin, and all produced catalase. Pigment production from tryptophan was variable. Partial large subunit rRNA sequences were obtained for two isolates that remained viable in culture. The strain from the cow with otitis was identified as a lipid-dependent variant of M. pachydermatis, and the strain from the dog was an atypical variant of Malassezia furfur.     

糠秕马拉色菌药敏试验新方法的探讨

目的 建立一种新的糠秕马拉色菌药敏试验方法.方法 在ATBF2半固体培养基中添加不同种类和含量的脂质,用ATB Fungus 3药敏板条对ATCC14521和临床分离的糠秕马拉色菌进行了MIC测试.结果 孵育时间为72 h时对照孔中糠秕马拉色菌生长充分,吐温40浓度为1％时糠秕马拉色菌生长充分,且对药敏结果的影响最小.脂质的种类和含量对实验结果有影响.结论 用改良ATB Fungus 3药敏试验方法对糠秕马拉色菌进行抗真菌药敏试验,方法操作简便、结果易观察、试验结果重复性好.临床分离菌株与糠秕马拉色菌ATCC14521在ATB Fungus 3对照孔中的生长状况相同,结果易于判断.     

Mode of cell growth of Malassezia (Pityrosporum) as revealed by using plasma membrane configurations as natural markers

The mode of cell growth of Malassezia was studied by freeze-fracture using the plasma membrane configurations of this organism as natural markers. The plasma membrane of the mature cell bodies of M. pachydermatis had a ring swelling, and on each side of the ring, one set of straight and spiral grooves and circumvallate bulgings. The cell always divided at the ring swelling (M. pachydermatis) or depression (M. furfur), soon followed by budding there. A new set of similar configurations formed on the bud. In all the 12 strains of Malassezia studied, the spiral grooves in the mother and bud parts were both left-handed but opposite in the direction of elongation. By comparing distances between the spiral grooves in short and long buds and in mothers, the bud tip was suggested as the major, and adjacent regions as the minor, sites of wall growth. Some characterizations of the plasma membrane invaginations, especially in relation to the mode of cell growth, were also described.     

Peptides of the constant region of antibodies display fungicidal activity

Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA) of antibodies (Fc-peptides) exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia furfur, including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro. An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents.     

Detection and quantification of specific IgE antibodies against eight Malassezia species in sera of patients with atopic dermatitis by using an enzyme-linked immunosorbent assay

The lipophilic yeast Malassezia, a member of the cutaneous microflora, is an exacerbating factor in atopic dermatitis (AD). Of the 11 currently recognized species, M. globosa and M. restricta are found to frequently colonize the skin of AD patients. In this study, we attempted to quantify specific IgE antibodies against eight Malassezia species, namely, M. dermatitis, M. furfur, M. globosa, M. obtusa, M. pachydermatis, M. slooffiae, M. sympodialis, and M. restricta, in sera from AD patients by using an enzyme-linked immunosorbent assay (ELISA). The specific IgE value against M. restricta was greater than those against other Malassezia species. Competitive ELISA inhibition tests revealed that M. restricta contained species specific as well as shared antigens. Therefore, M. restricta could be considered as a candidate diagnostic antigen for detecting anti-Malassezia IgE in sera from AD patients.     

Taxonomy of Malassezia furfur: state of the art

Malassezia furfur is a lipophilic yeast considered as a normal component of the human skin flora. Apart from pityriasis versicolor, M. furfur has been linked to several skin diseases such as seborrheic dermatitis, folliculitis or atopic dermatitis. Moreover, these yeasts have been reported as agent of invasive human diseases including pneumonia, catheter-associated sepsis and peritonitis. The existence of morphological, serological, metabolical, biochemical and karyotipical differences has been described among isolates of these yeasts. These observations gave arguments for a possible intraspecific division, and this hypothesis has been confirmed by the existence of six species within the formerly called M. furfur (lipid-dependent Malassezia strains): M. furfur, Malassezia sympodialis, Malassezia globosa, Malassezia obtusa, Malassezia restricta and Malassezia slooffiae.     

Subculture on potato dextrose agar as a complement to the broth microdilution assay for Malassezia pachydermatis

The main aim of this study was to verify the efficacy of subculture on potato dextrose agar (PDA) as a complement to the in vitro susceptibility test for Malassezia pachydermatis strains by a broth microdilution method, as well as to determine the MIC and MFC of azole derivatives, amphotericin B and caspofungin. The microdilution assay was performed in 96-well plates using a modified RPMI 1640 medium. The M. pachydermatis strains were resistant to caspofungin. All strains (n=50) had shown MIC values of <0.03, <0.03, 2.0, 4.0 and 4.0 mug/ml for itraconazole, ketoconazole, voriconazole, fluconazole and amphotericin B, respectively. Thus, the subculture on PDA improved the analysis of the in vitro antifungal susceptibility of M. pachydermatis.     

Concordance between phenotypical features and PCR-REA for the identification of Malassezia spp

The genus Malassezia has been recently revised and nowadays includes 11 species that cannot always be differentiated from each other by physiological and morphological tests. This study was aimed to evaluate the correlation between a molecular method and conventional phenotypic features in the identification of Malassezia spp. To achieve this aim, 92 Argentinean clinical strains isolated between 2001 and 2005 were analyzed along with three reference strains (Malassezia furfur CBS 7019, Malassezia sympodialis CBS 7222 and Malassezia slooffiae CBS 7956). By using PCR and restriction enzyme analysis with three different DNA endonucleases (PCR-REA), the molecular method consistently identified all three reference strains and all 92 clinical isolates as follows: 63 M. sympodialis, 18 M. furfur, 10 Malassezia globosa and one Malassezia obtusa. Phenotypic studies undentified 85 clinical isolates and two of the reference strains (total agreement > 91%). In particular for M. sympodialis, M. furfur and M. globosa, the species more frequently involved in human pathology, the agreement ranged between 84 and 96%. This result suggests that phenotypic studies are suitable for the presumptive identification of important Malassezia species in the clinical medical mycology laboratories where molecular methodologies are not available.     

ATB FUNGUS2法在念珠菌属和新生隐球菌抗真菌药敏试验中应用的评价

目的评价ATBFUNGUS2半固体培养基法在测定念珠菌属和新生隐球菌对4种常用抗真菌药物敏感性中的应用价值。方法利用CLSIM27．A2微量液基稀释法和ATBFUNGUS2法同时测定131株念珠菌和20株新生隐球菌对两性霉素B（AmB）、氟康唑（FLC）、氟胞嘧啶（5-Fc）和伊曲康唑（ITC）的敏感性。结果①两种方法对于AmB、5-FC、FLC和ITC的一致性分别为98％、89．4％、78．8％和78．1％;②所有受试菌株中两种方法的一致性为80％,但ATBFUNGUS2法将2／5株M27-A2法检查为FLC耐药的白念珠菌判断为敏感或剂量依赖,将8／10株M27-A2法检查为FLC剂量依赖的白念珠菌判断为敏感或耐药。③ATBFUNGUS2法中AmB的MIC值判读范围偏高,以致于实际工作中不能读出准确的值。结论ATBFUNGUS2半固体培养基法在测定念珠菌属和新生隐球菌对4种常用抗真菌药物的敏感性时不失为简单、快速而且重复性好的方法。     

In vitro modulation of human keratinocyte pro- and anti-inflammatory cytokine production by the capsule of Malassezia species

Malassezia spp. are commensal, cutaneous fungi that are implicated in seborrhoeic dermatitis. We hypothesize that the lipid-rich capsule of Malassezia spp. masks the organism from host detection, and depletion of this layer elicits an inflammatory response. To test this, preparations of capsulated or acapsular [10% (v/v) Triton X-100 treated], viable and nonviable, exponential or stationary phase Malassezia furfur, Malassezia globosa, Malassezia obtusa, Malassezia restricta, Malassezia slooffiae and Malassezia sympodialis, were incubated with normal human keratinocytes. Proinflammatory (IL-6, IL-8, IL-1alpha and tumour necrosis factor-alpha) and anti-inflammatory cytokine (IL-10) release and intracellular IL-10 concentrations were quantified using enzyme-linked immunosorbent assays. Capsulated Malassezia yeasts stimulated limited or no production of inflammatory cytokines, and increased intracellular IL-10 (P < 0.05). Removal of the capsule of many Malassezia preparations caused a significantly increased production of IL-6, IL-8 and IL-1alpha, and a decrease in intracellular IL-10. Notably, acapsular viable, stationary phase M. globosa caused a 66-fold increase in IL-8 production (P < 0.001) and acapsular nonviable, stationary phase M. furfur caused a 38-fold increase in IL-6 production (P < 0.001) and a 12-fold decrease in intracellular IL-10 (P < 0.001). These results support the hypothesis that the lipid layer of Malassezia spp. modulates cytokine production by keratinocytes. This has implications in the pathogenesis of seborrhoeic dermatitis.     

Biotyping of Malassezia pachydermatis strains using the killer system

The killer phenomenon has been used as epidemiological marker for Candida albicans, where hundreds of biotypes can be obtained. The objective of this study is to observe the behaviour of 30 strains of Malassezia pachydermatis isolated from dogs with otitis (15) or dermatitis (15) against 9 killer yeasts, which, when grouped in triplets produced a 3 digit code (biotype). The growth inhibition of the 30 strains of M. pachydermatis due to the effect of the killer yeasts used permitted the determination of the following biotypes: 888 (33.3%), 212 (26.7%), 111 (16.7%), 312 (6.7%), 512 (6.7%), 242 (3.3%), 311 (3.3%) and 411 (3.3%). Biotypes 888, 212 and 111 occurred most frequently in both ear canal and skin samples.     

正常人耵聍中马拉色菌菌种构成研究

目的研究正常人耵聍中马拉色菌菌种构成及同一宿主耵聍中菌种是否一致。方法采集45名健康志愿者双侧耵聍,0.1%曲拉通X-100溶解稀释后接种于含菜籽油培养基,生化及形态学方法鉴定到种,同时提取菌种DNA,用真菌通用引物ITS1/ITS4做PCR扩增并测序鉴定。结果有44例(97.78%)双侧耵聍中均培养出马拉色菌(共分离出88株菌),菌种构成:糠粃马拉色菌29株(32.95%)、斯洛菲马拉色菌23株(26.14%)、合轴马拉色菌18株(20.45%)、球形马拉色菌11株(12.50%)、限制性马拉色菌7株(7.95%)。44例(88株菌)中双侧耵聍菌种相同者有38例(76株菌)(一致率86.36%)。结论正常人耵聍中马拉色菌菌种分布较广,主要菌种为糠秕马拉色菌。同一宿主双侧耵聍中马拉色菌菌种具有一定的一致性。     

Isolation, characterization and molecular cloning of a lipolytic enzyme secreted from Malassezia pachydermatis

Lipophilic Malassezia species may induce catheter-associated sepsis in premature neonates and immunocompromised patients receiving parenteral lipid emulsions. To assess the participation of lipolytic enzymes in the pathogenesis of this yeast, we cloned a gene encoding the enzyme. A lipolytic enzyme in the culture supernatant of Malassezia pachydermatis was purified 210-fold to homogeneity. The enzyme showed high esterase activity toward p-nitrophenyl octanoate. The cDNA encoding the enzyme was cloned using a degenerate oligonucleotide primer constructed from the N-terminal amino acid sequence. The cDNA consisted of 1582 bp, including an open reading frame encoding 470 amino acids. The first 19 amino acids and the following 13 amino-acid sequence were predicted to be the signal peptides for secretion and prosequence, respectively. The predicted molecular mass of the 438-amino acid mature protein was 48 kDa. Analysis of the deduced amino acid sequence revealed that it contains the consensus motif (Gly-X-Ser-X-Gly), which is conserved among lipolytic enzymes. Homology investigations showed that the enzyme has similarities principally with 11 lipases produced by Candida albicans (29-34% identity) and some other yeast lipases.     

Pathological and clinical aspects of the diseases caused by Malassezia species

From veterinary point of view Malassezia pachydermatis has the greatest significance. It has been standing in the focus of interest since the early 1990s, mostly because of the frequency of otitis externa and dermatitis caused by this yeast in dogs. This is the only lipid-independent species in the genus Malassezia. It can be found in very large proportion on the skin of healthy animals, but can be isolated in much greater number from diseased dogs. It often causes illness together with other pathogens (e.g. Staphylococcus intermedius). Some breeds are predisposed. In addition to the treatment of the accidental concurrent diseases, therapy consists of systemic and/or topical antimicrobial treatment. Ketoconazole is used most frequently. Malassezia pachydermatis plays also a role in the skin disorders of other carnivores. It has little zoonotic potential, it can be dangerous to immunocompromised humans. The other Malassezia species have little veterinary importance, although M. sympodialis and M. globosa were isolated from asymptomatic animals (mostly cats) and from mixed infections.