Malassezia colonisation on a reconstructed human epidermis: Imaging studies.

BACKGROUND: Biofilm formation represents a major microbial virulence attribute especially at epithelial surfaces such as the skin. Malassezia biofilm formation at the skin surface has not yet been addressed. OBJECTIVE: The present study aimed to evaluate Malassezia colonisation pattern on a reconstructed human epidermis (RhE) by imaging techniques. METHODS: Malassezia clinical isolates were previously isolated from volunteers with pityriasis versicolor and seborrhoeic dermatitis. Yeast of two strains of M furfur and M sympodialis were inoculated onto the SkinEthic™ RHE. The tissues were processed for light microscopy, wide-field fluorescence microscopy and scanning electron microscopy. RESULTS: Colonisation of the RhE surface with aggregates of Malassezia yeast entrapped in a multilayer sheet with variable amount of extracellular matrix was unveiled by imaging techniques following 24, 48, 72 and 96 hours of incubation. Whenever yeast were suspended in RPMI medium supplemented with lipids, the biofilm substantially increased with a dense extracellular matrix in which the yeast cells were embedded. Slight differences were found in the biofilm architectural structure between the two tested species with an apparently higher entrapment and viscosity in M furfur biofilm. CONCLUSION: Skin isolates of M furfur and M sympodialis were capable of forming biofilm in vitro at the epidermal surface simulating in vivo conditions. Following 24 hours of incubation, without added lipids, rudimental matrix was barely visible, conversely to the reported at plastic surfaces. The amount of biofilm apparently increased progressively from 48 to 96 hours. A structural heterogeneity of biofilm between species was found.

The evaluation of local oxidative/nitrosative stress in patients with pityriasis versicolor: a preliminary study.

Yeast of Malassezia, members of the microbiologic flora of the skin, cause pityriasis versicolor and have also been implicated in the pathogenesis of other superficial dermatoses. The mechanisms by which the yeast cause these dermatoses, however, are not yet clear, and there have been no studies on the interaction between fungi and keratinocytes, especially the effects of fungi on the production of reactive oxygen/nitrogen species by human keratinocytes. This is the first study we aimed to evaluate the role of oxidative/nitrosative stress in affected skin areas in patients with pityriasis versicolor. Sixty pityriasis versicolor patients and 60 healthy subjects as control group were enrolled. Scraping samples were obtained from the lesional skin areas in patients and from the normal skin areas in controls. The distribution of lesions of pityriasis versicolor was mild in all patients. We have isolated two Malassezia species: M. furfur being isolated in 75% of patients, followed by M. globosa (25%). The levels of oxidative/nitrosative stress biomarkers were significantly higher in patients than the controls (P < 0.001). Our results demonstrated that pityriasis versicolor is associated with significant alteration in oxidant/antioxidant balance. It might be secondary consequence of pityriasis versicolor infection or contributing factor in its pathogenesis.

Is transmission electron microscopy (TEM) a promising approach for qualitative and quantitative investigations of polymyxin B and miconazole interactions with cellular and subcellular structures of Staphylococcus pseudintermedius, Escherichia coli, Pseudomonas aeruginosa and Malassezia pachydermatis?

Antimicrobial therapy using a combination of polymyxin B and miconazole is effective against the main bacterial pathogens associated with otitis externa in dogs, and a synergistic effect of both drugs has been shown previously. The objective of the present investigation was to visualize ultrastructural changes after exposure of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus pseudintermedius and Malassezia pachydermatis to polymyxin B and miconazole by transmission electron microscopic (TEM). For this, cultures of E. coli, P. aeruginosa, S. pseudintermedius and M. pachydermatis were exposed to polymyxin B and miconazole, alone or in combination for 24 h. Ultrastructural changes were observed most frequently in the cell envelope of the four microorganisms. Exposure to polymyxin B seemed to cause more damage than miconazole within the range of concentrations applied. Treatment resulted in changes of the cell size: in E. coli, cell size increased significantly after treatment with either compound alone; in P. aeruginosa, cell size decreased significantly after treatment with polymyxin B and with miconazole; exposure of S. pseudintermedius to miconazole caused a decrease in cell size; in M. pachydermatis, cell size increased significantly after treatment with polymyxin B.; in E.coli, S. pseudintermedius and M. pachydermatis, cell size changed highly significant, in P. aeruginosa significantly after exposure to the combination of both compounds. In conclusion, by using a different approach than previous investigations, this study confirmed a clear combinatory effect of polymyxin B and miconazole against the tested microorganisms involved in canine otitis externa. It is the first time that visualization technologies were applied to compare the effect of single drugs to their combinatory effects on cellular and subcellular entities of selected bacterial and yeast species.

[Cutaneous Malassezia infections and Malassezia associated dermatoses: An update]. / Kutane Malassezia-Infektionen und Malassezia-assoziierte Dermatosen : Ein Update.

The lipophilic yeast fungus Malassezia (M.) spp. is the only fungal genus or species which is part of the physiological human microbiome. Today, at least 14 different Malassezia species are known; most of them can only be identified using molecular biological techniques. As a facultative pathogenic microorganism, Malassezia represents the causative agent both of superficial cutaneous infections and of blood stream infections. Pityriasis versicolor is the probably most frequent infection caused by Malassezia. Less common, Malassezia folliculitis occurs. There is only an episodic report on Malassezia-induced onychomycosis. Seborrhoeic dermatitis represents a Malassezia-associated inflammatory dermatosis. In addition, Malassezia allergenes should be considered as the trigger of "Head-Neck"-type atopic dermatitis. Ketoconazole possesses the strongest in vitro activity against Malassezia, and represents the treatment of choice for topical therapy of pityriasis versicolor. Alternatives include other azole antifungals but also the allylamine terbinafine and the hydroxypyridone antifungal agent ciclopirox olamine. "Antiseborrhoeic" agents, e.g. zinc pyrithione, selenium disulfide, and salicylic acid, are also effective in pityriasis versicolor. The drug of choice for oral treatment of pityriasis versicolor is itraconazole; an effective alternative represents fluconazole. Seborrhoeic dermatitis is best treated with topical medication, including topical corticosteroids and antifungal agents like ketoconazole or sertaconazole. Calcineurin inhibitors, e.g. pimecrolimus and tacrolimus, are reliable in seborrhoeic dermatitis, however are used off-label.

The spectrum of Malassezia species isolated from students with pityriasis vesicolor in Nigeria.

Pityriasis versicolor (PV) is a common superficial fungal infection of the skin caused by Malassezia. Initially M. furfur was suggested as its main aetiological agent; however, more recent studies suggest M. globosa as the dominant species. The possibility of a variance in predominant species based on geographical basis has not been fully evaluated. The objective of this study was to identify the Malassezia species on affected and non-affected skin of students with PV who reside in a tropical environment (Abuja, Nigeria) and correlate them to clinical characteristics. In this study, the literature on prevalence of Malassezia genus in PV was also reviewed. Samples were taken from 304 PV lesions and 110 normal appearing skin. Microscopy, culture and identification of Malassezia species utilising polymerase chain reaction-restriction fragment length polymorphism analysis were performed. Three Malassezia species were detected in PV with the major species being M. furfur. On normal appearing skin, M. furfur (77.6%) and M. restricta (10.4%) were both detected. No case of M. globosa was identified in this study. There was no significant difference between species identified and clinical features of PV. M. furfur is probably still the most predominant species causing PV in the tropical environment.

Identification of (1-->6)-beta-D-glucan as the major carbohydrate component of the Malassezia sympodialis cell wall.

Members of the genus Malassezia are commensal fungi found on the skin of both human and domestic animals and are associated with skin diseases including dandruff/seborrheic dermatitis, pityriasis versicolor, and atopic eczema (AE) in humans. In this study we have characterized the cell-wall carbohydrates of Malassezia sympodialis, one of the species most frequently isolated from both AE patients and healthy individuals. Cells were grown in liquid Dixon media at 32 degrees C, harvested, and processed using a standard Fehling's precipitation methodology for the isolation of mannan and a standard base/acid extraction for (1-->3)-beta-D-glucans. Using these classic extraction methods we were unable to isolate precipitable mannan or insoluble (1-->3)-beta-D-glucan. However, acidification and addition of methanol to the remaining Fehling's-treated sample resulted in a very clean precipitate. This material was characterized by GPC-MALLS, 1D and 2D NMR, and GC-MS for monomer-type and linkage-type composition. We determined that trace amounts of both mannan and branched (1-->3, 1-->6)-beta-D-glucan were present in the recovered precipitate, but not linear (1-->3)-beta-D-glucan. Surprisingly, NMR analysis indicated that (1-->6)-beta-D-glucan was the major carbohydrate component isolated from M. sympodialis cell wall. GC-MS linkage analysis confirmed the (1-->6)-beta-D-glucan structure. Based on these studies we have determined that the M. sympodialis cell wall contains (1-->6)-beta-D-glucan as the major carbohydrate component along with trace amounts of mannan and (1-->3, 1-->6)-beta-d-glucan. In addition, these data indicate that modification of the classic mannan isolation methodology may be useful in the simultaneous isolation of both mannan and (1-->6)-beta-D-glucan from other fungi.

[Pityriasis versicolor and the yeasts of genus Malassezia]. / La pitiriasis versicolor y las levaduras del género Malassezia.

Although pityriasis versicolor is the only human disease for which Malassezia yeasts have been fully established as pathogens, it is still not clear which species are implicated. Most studies carried out in recent years support our hypothesis - proposed in 1999 - that Malassezia globosa is the predominant species in pityriasis versicolor lesions, at least in temperate climates. Confirmation of this hypothesis could help us understand the conditions, as yet unclear, that induce transformation of this yeast from the saprophytic form present in healthy skin to the parasitic form, characterized by the formation of pseudomycelium, and could also guide therapy. In addition, isolation of another species, Malassezia furfur, which seems to be predominant in the tropics, raises the possibility of a second etiologic agent confined to certain areas, as occurs with some other human mycoses.

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.

In vitro activity of R126638 and ketoconazole against Malassezia species.

The in vitro activity of a new triazole R126638 against Malassezia yeasts was compared with that of ketoconazole. With the agar dilution technique, minimal inhibitory concentrations were lower for R126638 compared with ketoconazole against Malassezia globosa, M. obtusa, M. slooffiae, M. restricta and two strains of M. sympodialis. On human stratum corneum in vitro, both R126638 and ketoconazole were very effective in reducing the production of hyphae from 15% to 1% with R126638 and to 2% with ketoconazole. Scanning electron microscopy did not reveal obvious surface differences between untreated cultures and cultures exposed to ketoconazole or R126638 in the concentration range 0.01-1 microg/ml. However, transmission electron microscopy showed partial to complete necrosis of the cytoplasmic organelles of Malassezia yeasts. The combined scanning electron microscopy and transmission electron microscopy findings confirm earlier observations of the "mummifying" effect of azoles against Malassezia spp. In conclusion, R126638 is an interesting new triazole with high activity against the Malassezia yeasts, which are involved in pityriasis versicolor and seborrhoeic dermatitis.

[The use of calcofluor white for studying Malassezia species by direct microscopy]. / Empleo de blanco de calcoflúor para el estudio de las especies de Malassezia por microscopía directa.

Fungi of Malassezia genus are known as normal flora in human beings. However, different pathologies due to Malassezia, have been described. Traditionally, early diagnosis was delayed because of the difficulties in culture isolation of these organisms. The aim of this work, is to evaluate the technique of observation microscopy with calcofluor, for identification of Malassezia in both, clinical samples and isolates. In comparison to traditional method of direct examination with lactophenol-blue, calcofluor method offers an advantage because it turns easier the observation of fungal elements and its budding pattern. This technique contributes then, to identify species of Malassezia. The analysis of clinical specimens with calcofluor followed by observation under fluorescence microscopy is a simple and rapid method for the identification, and contribute therefore to the early diagnosis.

[Malassezia. Current knowledge and study perspectives]. / Malassezia. Estado del conocimiento y perspectivas en su estudio.

The study of Malassezia has been postponed for many years due to its nutritional requirements and its morphological variability. Molecular biology methods led to a taxonomic revision of this genus and to a new interest for its clinical importance. Nowadays, 11 Malassezia species are known, 10 are lipophilic, having similar morphological, physiological and biochemical characteristics, therefore, conventional techniques are not useful to differentiating them. Molecular methods are an accurate tool in the identification and they lead to a better knowledge of the ecology and epidemiology of this genus. Noteworthy antifungal susceptibility variations have been observed in some species, although there is not a standard method for these yeasts. There are few data about their biochemical characteristics, and the enzymes they produce might be important virulence factors, favouring host tissue invasion. Malassezia has been recognised as a member of the normal human and animal skin. Its implication in pathologic processes, including skin diseases to systemic infections, is the main issue in current investigations in order to determine the real pathogenic role of these yeasts.

Effect of circumcision on genital colonization of Malassezia spp. in a pediatric population.

This study aimed to determine the prevalence of yeast fungi in samples from the glans penis and prepuce of circumcised and uncircumcised children. Impression preparations were made on modified Dixon and Leeming-Notman agar without cycloheximide. The isolates were identified by morphological and physiological characteristics. The yeasts were detected in 32 (28.6%) of 112 children, 12 (18.2%) being among 66 circumcised and 20 (43.5%) among 46 uncircumcised children. The most common species was Malassezia globosa (40.6%) followed by, Malassezia furfur (31.3%), Malassezia slooffiae (15.6%), Candida albicans (6.3%), Candida tropicalis (3.1%) and Candida parapsilosis (3.1%). The colonization with yeasts, and especially lipophilic species was more frequently observed among uncircumcised versus circumcised children. While age was not found to be a factor (P > 0.05), circumcision was responsible for increasing the colonization rate by 4.8 times (95% CI: 1.6-14.5) (P < 0.01). The circumcision status was not found to effect the distribution of Malassezia spp.

The key role of corneocytes in pityrosporoses.

Pityrosporoses encompass various Malassezia-driven conditions. Dandruff and seborrheic dermatitis are members of this family of disorders. Their precise pathomechanisms have not been completely elucidated so far. This review focuses on the role of corneocytes in these disorders. Malassezia yeasts are not evenly distributed at the surface of the stratum corneum. Rather, they are clumped on some corneocytes while other corneocytes in their vicinity are almost free of these microorganisms. The corneocytes heavily coated by yeasts suggest a cell-related defect in the mechanisms controlling the skin biocene including the natural antimicrobial peptides and nitric oxide. Most environmental factors influencing pityrosporoses indeed affect the natural human defenses against certain microorganisms. We frame as a hypothesis that the primary defect in some Malassezia-driven disorders resides in the corneocytes and their precursor keratinocytes.

Attachment of Malassezia pachydermatis to the ear dermal cells in canine otitis externa.

To investigate the predominance of Malassezia pachydermatis (M. pachydermatis) as a causative agent of canine otitis externa, ear cerumen samples were observed for adhesion of M. pachydermatis to the cornified epithelial cells by light and electron microscopes. The yeasts appeared not to adhere to the cornified epithelial cells directly, but they seemed to exist in the proximity of the epithelial cells with an electron opaque halo-like space around them. Polysaccharide and lipid staining techniques were conducted to identify the substances existing in that space. Lipid substances, not saccharides, were observed around the yeasts and the cornified epithelial cells. These results suggested that in the canine ear canal malassezia yeast attachment to the cornified epithelial cells is mediated by lipids.

Malassezia pachydermatis isolated from a South American sea lion (Otaria byronia) with dermatitis.

A fungus was isolated from the skin of an Otaria byronia and from the water of the pool in which the animal was kept. It formed creamy colonies with soft texture on Dixon agar and grew well without supplements of long-chain fatty acids. Cells were ovoid to cylindrical in shape, budded from a broad base, and budded and divided at the same location. Thus, the isolate was identified as M. pachydermatis. We compared this very rare isolate from a marine mammal with four strains of M. pachydermatis using the freeze-etching electron-microscopy technique. The cells showed the same characteristic ring-swellings on the protoplasmic membrane on the neck site between the mother and the daughter parts, and the same accumulation of circumvallate bulgings in a small area near the straight sections of spiral grooves as four reference strains. Thus, in terms of morphology and ultrastructure, the isolate could be regarded as a typical M. pachydermatis.