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.     

Genetic variants of Malassezia pachydermatis from canine skin: body distribution and phospholipase activity

Malassezia pachydermatis isolates (n=185) from skin sites from dogs (n=30) were characterized genetically and biochemically following in vitro culture. Two regions in the chitin synthase-2 gene (chs-2) and the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA were sequenced, and the phospholipase activity of each isolate was assessed. Three chs-2 (i.e. Ac, Bc and Cc) and eight ITS-1 (i.e. AI1, AI2, AI3, AI4, BI1, CI1, CI2 and CI3) sequence types were defined for all 185 samples. The findings revealed that multiple M. pachydermatis genotypes/subgenotypes could be cultured from healthy dogs or from dogs with single or multiple, generalized skin lesions. Subgenotypes AI1 and BI1 were associated with all skin sites of dogs sampled, whereas subgenotype CI2 was mostly linked to a particular location. Isolates derived from skin lesions showed a significantly higher phospholipase activity compared with those from skin sites with no detectable lesions. Genotype B was mainly cultured from healthy skin; only four isolates (9.3%) had low phospholipase activity, whereas other genotypes/subgenotypes were predominantly associated with skin lesions and had a high phospholipase activity. The results of the present study suggest that the distribution pattern of particular genotypes or subgenotypes of M. pachydermatis on the skin of dogs relates to the affinity of the yeast to the host and to particular skin sites.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Potential therapeutic effect of yeast killer toxin

Experimental infections were produced in guinea pigs, rabbits and dogs with lesions similar to those seen in human seborrheic dermatitis and otitis externa by cutaneous application of cultures of Malassezia furfur and M. pachydermatis. Infected animals were treated by topical application of a concentrated yeast killer toxin (Hansenula anomala UCSC 25F). Clinical recovery as well as negative mycological test cultures of infected animals proved to the clearly associated with the treatment by the killer toxin.     

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.     

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.     

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。     

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.     

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.     

东南亚青年人群中马拉色菌菌种构成分析

目的：研究部分东南亚国家青年人群中马拉色菌菌种构成。方法采集285名青年人（分别来源于中国、尼泊尔、印度、巴基斯坦）面部正常皮肤及皮损区标本,接种于 Leeming ＆amp; Notman 培养基后进行培养分离,生化及形态学方法鉴定到种。结果共分离出8个菌种,共计501株马拉色菌,以球形马拉色菌为主,占40.5℅（203/501）,其次为合轴马拉色菌19.0℅（95/501）、糠秕马拉色菌16.0℅（80/501）、限制马拉色菌11.0℅（55/501）、斯洛菲马拉色菌6.2℅（31/501）等。各国家间未见明显的地理学差异。结论东南亚地区青年正常人群及马拉色菌相关疾病患者中的主要菌种为球形马拉色菌。     

Two new lipid-dependent Malassezia species from domestic animals

During a study on the occurrence of lipid-dependent Malassezia spp. in domestic animals, some atypical strains, phylogenetically related to Malassezia sympodialis Simmons et Guého, were shown to represent novel species. In this study, we describe two new taxa, Malassezia caprae sp. nov. (type strain MA383=CBS 10434), isolated mainly from goats, and Malassezia equina sp. nov. (type strain MA146=CBS 9969), isolated mainly from horses, including their morphological and physiological characteristics. The validation of these new taxa is further supported by analysis of the D1/D2 regions of the 26S rRNA gene, the ITS1-5.8S-ITS2 rRNA, the RNA polymerase subunit 1 and chitin synthase nucleotide sequences, and the amplified fragment length polymorphism patterns, which were all consistent in separating these new species from the other species of the genus, and those of the M. sympodialis species cluster, specifically.     

A new yeast, Malassezia yamatoensis, isolated from a patient with seborrheic dermatitis, and its distribution in patients and healthy subjects

Over the last few years, new Malassezia species have been found regularly in Japanese subjects. We isolated another new Malassezia species from a Japanese patient with seborrheic dermatitis (SD), and named it M. yamatoensis. In its physiological characteristics and the utilization of Tween by M. yamatoensis is similar to that of M. furfur and M. dermatis. It is distinguished by its growth temperature. To examine the distribution of the microorganism in the skin of patients with SD and atopic dermatitis (AD), and healthy subjects, we applied transparent dressings to the skin, and detected M. yamatoensis DNA using a non-culture-based method that consisted of nested PCR with specific primers. M. yamatoensis DNA was detected from 3 of 31 SD patients (9.7%), 5 of 36 AD patients (13.9%), and 1 of 22 healthy subjects (4.6%). Therefore, M. yamatoensis is a rare member of the cutaneous microflora.