The pathogenesis of Malassezia yeasts

The genus Malassezia includes twelve species of yeast, many of which have been mainly associated with human and canine diseases. Malassezia pachydermatis colonizes the skin and mucosal sites of healthy dogs and cats. Despite being part of the normal cutaneous microflora, Malassezia spp. yeast may become pathogenic under certain circumstances. This article reviews the factors related to both host and yeast which affect the pathogenical or commensal phenotypes of Malassezia yeasts.     

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.     

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.     

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.     

Epidemiology and variability of Malassezia spp

A short review on Malassezia spp., completed with our experience, is made. The main epidemiological characteristics with particular regard to the diffusion in several animal species, the characteristics of skin colonization (in particular of the dog) and the distribution of the different Malassezia spp. in some hosts are discussed. Lastly the main phenotypic and genotypic characteristics, referred to M. pachydermatis especially, were described, showing their high variability and differentiation.     

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.     

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.     

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.     

A survey of mycotic otitis externa of dogs in Lisbon

One hundred and thirteen dogs of different breeds and with different clinical forms of external otitis were mycologically and bacteriologically examined. Forty six of those dogs showed abnormal cerumen with a high yeasts contamination. These yeasts belong to four species: Malassezia pachydermatis (80.4%), Cryptococcus laurentii (13.1%), Candida parapsilosis (4.3%) and Trichosporon cutaneum (2.1%). All strains, excepting C. laurentii were highly lipolytic. Most of the clinical cases associated with those yeasts were chronic, with hyperkeratosis and lichenification, and most of them were relapsed otitis (91.3%). The most affected dogs were a pendulous ears breeding (65.7%) and males (86.8%). Some dogs had other cutaneous disorders (seborrhoeic dermatitis, pemphigus). In vitro tests, using seven different antifungal drugs were systematically performed. All strains revealed to be 5-fluorocytosine-resistant and 32% of them were also resistant to nystatin. One M. pachydermatis isolated was resistant to all of the tested antifungal drugs.     

Malassezia pityrosporum pachydermatis (Weidman) Dodge 1935

Priority of the name Malassezia pachydermatis (Weidman) Dodge 1935 is indicated for the microorganism which has been called Pityrosporum pachydermatis Weidman 1925 and P. canis Gustafson 1955. M. pachydermatis is here further characterized in culture with information drawn from 2 recent isolates, in particular the presence of spiral grooves on the inner surface of the cell wall, good growth on Mycosel agar, rapid production of urease, and assimilation of glucose by the Wickerham method.     

Occurrence of Malassezia species in healthy and dermatologically diseased dogs

The presence of Malassezia spp. yeasts was investigated in dermatological specimens of 224 dogs, 164 dermatologically diseased and 60 normal dogs. Subjects included in the study were of different breed, age, sex and habitat. Malassezia spp. positive cultures were obtained in 142 (63.4%) specimens: 67.6% from dermatologically diseased subjects and 51.6% from healthy dogs. Malassezia pachydermatis, either as a pure culture or in association with lipid-dependent species, was identified in 138 (97%) specimens. Malassezia furfur was identified in 69 (48.6%) specimens and was associated with other Malassezia species in 68 dogs, as a pure culture in one subject: at the best of our knowledge, this species was identified before as the sole species from canine dermatitis. Malassezia sympodialis was identified in 11 (7.7%) specimens, always in association with other species: it was never isolated from kennel dogs. Statistical analysis of data showed a very significant difference (P < 0.01) in the prevalence of isolation of Malassezia spp. between animals with and without dermatological signs, and in the distribution of cultural burden between diseased and healthy dogs. A statistically significant difference (P<0.05) was also detected in the group of animals between 1- and 5-years of age. No significant difference was found between male and female dogs.     

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.     

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.     

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.     

The influence of newly synthesised fenporpimorph derivatives on some pathogen yeasts

The effect of minimum inhibitory concentrations (MICs) of six novel fenpropimorph derivatives on lipid and sterol composition of Candida albicans, Cryptococcus neoformans, Malassezia pachydermatis and Malassezia furfur was investigated. The MICs for the most effective derivatives were found in the range from 3.7 to 56.7 microM and were 2-3 times lower compared to the commercial fungicide bifonazol. The more efficient fenpropimorph derivatives were the piperidine derivative for C. albicans and the allylamine derivative for Cr. neoformans, M. pachydermatis and M. furfur. The inhibitor in the growth medium reduced the unsaturation index of the total lipid content in M. furfur and C. albicans.     

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.     

Skin diseases associated with Malassezia species in humans. Clinical features and diagnostic criteria

Malassezia yeasts not only cause the well known pityriasis versicolor and folliculitis, but also play an important role in other skin diseases, including seborrheic dermatitis and atopic dermatitis. The presence of Malassezia yeasts may be confirmed by direct microscopic examination and cultures of skin scrapings. In pityriasis versicolor the direct microscopic examination is the rapidest and surest test for confirming the clinical diagnosis. The preparation will show a cluster of globose budding spores with thick or double wall and short hyphae. For detecting Malassezia in the other diseases the cultures is preferable. Culture is useful both for confirming the clinical diagnosis and for epidemiological investigations. The identification of the Malassezia species is not easy. The microscopic observation of the colony direct towards the identification of Malassezia species, but it is not enough to identify the colonies definitely. Several biochemical tests are necessary for a precise identification, such as catalase reaction, growth on media without lipid sources, ability to utilize hydrophilic emulsifiers as sole lipid source, esculin test, tryptophan test.     

Microtubular and actin cytoskeletons and ultrastructural characteristics of the potentially pathogenic basidiomycetous yeast Malassezia pachydermatis

Microtubular and actin cytoskeletons were investigated in the lipophilic yeast Malassezia pachydermatis by fluorescence and electron microscopy. To detect microtubules by indirect immunofluorescence using monoclonal anti-tubulin antibody, a prolonged incubation with lysing enzymes was necessary due to its very thick cell wall. Cytoplasmic microtubules were detected in interphase and a spindle with astral microtubules was seen in M-phase. The disintegration of cytoplasmic microtubules and migration of the nucleus to the bud before mitosis were characteristic features of the basidiomycetous yeast Malassezia pachydermatis. The visualisation of F-actin structures (patches, cables and cytokinetic rings) by fluorescence microscopy using both monoclonal anti-actin antibody and rhodamine-phalloidin failed, but actin was detected by electron microscopy with immunogold labelling. Clusters of gold particles indicating actin structures were detected at the plasma membrane of cells with unique cortical ultrastructural features characteristic of the genus Malassezia. A possible association of these with the actin cytoskeleton is suggested.     

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。     

Genotype analysis of Malassezia restricta as the major cutaneous flora in patients with atopic dermatitis and healthy subjects

Lipophilic yeasts of the genus Malassezia colonize the skin surface of humans and are an exacerbating factor in atopic dermatitis (AD). Two species, M. restricta and M. globosa are major cutaneous microflora in both AD patients and healthy subjects. We compared the DNA sequences of the intergenic spacer (IGS) region, located between the 26S and 5S rRNA genes of M. restricta colonizing the skin surfaces of 13 AD patients and 12 healthy subjects, and of three CBS stock strains as references. The IGS 1 sequences were divided into two major groups, corresponding to AD patients and healthy subjects. These findings suggest that a specific genotype of M. restricta plays a significant role in AD, although M. restricta commonly colonizes both AD patients and healthy subjects.