Molecular analysis of Malassezia species isolated from hospitalized neonates

Abstract:  Malassezia species are a part of the skin microflora of neonates. Under certain circumstances, they can cause diseases ranging from simple pustulosis to lifethreatening fungemia in newborn infants. Little information is available about the epidemiology of Malassezia species in neonates. In the present study, we successfully isolated Malassezia yeasts from 68.7% of hospitalized neonates. Using the polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFPL), M. furfur (88.06%) was identified as the most isolated species, followed in frequency by M. globosa (10.48%), M. obtusa (0.73%), and M. slooffiae (0.73%). Among the variables studied, only a longer stay in the ward resulted in a higher colonization rate. Using multiple logistic regression, only the type of hospital and ward had some effects on the colonization rate. Our results supported the hypothesis that neonates acquire Malassezia flora through direct contact with their mothers or hospital personnel.     

Distribution of Malassezia species in pityriasis versicolor and seborrhoeic dermatitis in Greece. Typing of the major pityriasis versicolor isolate M. globosa

BACKGROUND: The expansion of the genus Malassezia has generated interest in the epidemiological investigation of the distribution of new species in a range of dermatoses, on which variable results have been reported from different geographical regions. No data are thus far available from South-east Europe (Greece). OBJECTIVES: To study the distribution of Malassezia species in pityriasis versicolor (PV) and seborrhoeic dermatitis (SD) and to investigate whether polymorphisms in the internal transcribed spacer (ITS) 1 region facilitate detection of M. globosa and M. sympodialis subtypes. METHODS: In total, 109 patients with PV and SD and positive Malassezia cultures were included in the study. Age, gender, primary/recurrent episode, disease extent and clinical form of PV were recorded. ITS 1 polymorphisms of M. globosa and M. sympodialis type and clinical strains were investigated by polymerase chain reaction (PCR)-single-strand conformational polymorphism (SSCP) analysis. RESULTS: Malassezia globosa was the prevalent species isolated from PV and SD either alone (77% and 39%, respectively) or in combination (13% and 18%, respectively) with other Malassezia species. The pigmented form of PV was strongly correlated with the female gender. PCR-SSCP differentiated five subgroups of M. globosa with one being associated with extensive clinical disease. All M. sympodialis isolates displayed a homogeneous ITS 1 PCR-SSCP profile. CONCLUSIONS: Malassezia species isolation rates were in agreement with those reported from South-west Europe. PCR-SSCP of the ITS 1 is useful for highlighting prospective clinical implications of M. globosa subtypes.     

马拉色菌在新生儿皮肤定植的研究

目的探讨马拉色菌在新生儿皮肤的定植及影响因素。方法采用含菜籽油培养基32℃培养。对拟自然分娩的母亲临产时取阴道分泌物接种于试管斜面,用无菌微孔透气胶带粘取新生儿(8处皮肤,从出生当时到出院)、母亲(胸部和手掌,分娩后第2天)及其护士(手掌,新生儿出生后第2天)皮肤标本,接种于平板,根据菌落形态及生理生化方法鉴定菌种。结果①52份母亲阴道分泌物培养阴性。②15名护士共104份手掌部皮肤标本培养阴性。③104例新生儿(自然分娩和剖宫产各52例)出生当时的皮肤标本培养阴性,但出生后第1天有28例(26.92%)培养出马拉色菌,到第8天上升到59例(56.73%),其中糠秕马拉色菌41例(69.49%)。④从104例母亲皮肤上培养出马拉色菌65例(62.50%),其中糠秕马拉色菌49例(75.38%)。⑤母亲和新生儿124株马拉色菌中菌种相同者92例,一致率为74.19%。⑥新生儿前额和面颊马拉色菌培养阳性率最高。结论马拉色菌在新生儿皮肤定植最早发生在出生后的第1天内,主要部位为额面部,主要菌种为糠秕马拉色菌,存在于母亲皮肤的马拉色菌可能是定植菌种的主要来源。     

Antigenic components of Malassezia species for immunoglobulin E antibodies in sera of patients with atopic dermatitis

Antigenic components of Malassezia furfur, M. globosa, M. restricta, M. slooffiae, and M. sympodialis were studied for immunoglobulin E antibodies in sera of patients with atopic dermatitis (AD). Antigenic components were extracted from Malassezia cells by treatment with beta-mercaptoethanol, referred to as 2-ME extract. CBB staining and lectin blots using Con A, LCA, PHA-E4, PNA or RCA120 showed that the 2-ME extracts contained several species-dependent components that differed quantitatively and qualitatively among the Malassezia species at the protein level. In the Western blot with the 2-ME extracts, of 54 sera of the patients with AD (54 patients), the patients' IgE antibodies most frequently recognized components showing molecular weights of 43-46 kDa for M. slooffiae, 12-22 kDa for M. sympodialis, 35-40 kDa for M. restricta, 45-50 kDa for M. globosa, and of 67-72 kDa for M. furfur, respectively. In the correlative study, in which the total band intensities generated for each extract in Western blot were compared among the Malassezia species, the intensity for M. globosa was well correlated with that for M. sympodialis (r=0.756). In the Western blot inhibition test, the 2-ME extract of M. globosa partially inhibited the reaction of the antigenic components of other Malassezia species with the patient's IgE antibodies. These results indicated that Malassezia species contained both species-specific and common antigenic components at the IgE antibody level.     

Effects of topical vehicles on growth of the lipophilic Malassezia species

In the present study, the abilities of major Malassezia species, M. sympodialis, M. globosa and M. furfur, to assimilate topical agents, which have been widely used as a material of ointment for skin diseases, were tested. Obvious growth of M. furfur on GYEP agar plate was noted in the presence of white petrolatum, purified white petrolatum, hydrophilic ointment and heparinoid in hydrophilic ointment, and also M. sympodialis showed similar growth when they were cultured with hydrophilic or heparinoid in hydrophilic ointment. In contrast, M. globosa did not grow on GYEP in the presence of the any topical agents tested. These results suggest that Malassezia species, especially M. furfur and M. sympodialis, assimilate several topical agents and showed the drug-depended cell growth.     

Quantitative analysis of Malassezia in the scale of patients with psoriasis using a real-time polymerase chain reaction assay

BACKGROUND: It has been suggested that Malassezia is associated with the development of skin lesions in psoriasis because of the response of the scalp lesions in psoriasis to antifungal agents. Malassezia restricta and M. globosa are the two major members of the cutaneous Malassezia flora in patients with psoriasis, although they have not been analysed quantitatively. OBJECTIVES: This study quantified the two major cutaneous Malassezia species in psoriatic scale from different body sites using a real-time polymerase chain reaction (PCR) assay. METHODS: Scale samples were collected from lesional and nonlesional skin of 20 Japanese patients with psoriasis and fungal DNA was extracted from the samples directly. All the Malassezia species, including the two major species M. globosa and M. restricta, were quantified with high accuracy, using a real-time PCR assay. RESULTS: Colonization by M. restricta was approximately five times higher at all body sites than colonization by M. globosa. Malassezia colonization was significantly lower in patients with hyperlipidaemia than in patients with normolipidaemia. CONCLUSIONS: Malassezia restricta is the predominant species in psoriatic scale.     

花斑糠疹和马拉色菌毛囊炎菌种分布特点分析

目的探讨花斑糠疹和马拉色菌毛囊炎的菌种分布特点。方法从临床诊断为花斑糠疹和马拉色菌毛囊炎的患者分离培养菌种,通过形态学和生理生化学方法鉴定菌种,并比较两组患者菌种分布情况。结果共收集花斑糠疹病例161例,培养阳性121株,其中合轴马拉色菌46株,糠秕马拉色菌13株,球形马拉色菌29株,钝性马拉色菌33株;马拉色菌毛囊炎135例,培养阳性114株,其中合轴马拉色菌52株,糠秕马拉色菌43株,球形马拉色菌13株,钝性马拉色菌6株。两种疾病菌种分布差异有显著性(P<0.005)。结论花斑糠疹和马拉色菌毛囊炎菌种分布存在差异,可能为两种疾病不同临床表现的原因之一,但结果尚需慎重解释,需要分子水平的进一步研究。     

In vitro susceptibility of the seven Malassezia species to ketoconazole, voriconazole, itraconazole and terbinafine

Fifty-five strains, either authentic or ex-type, of seven Malassezia species were investigated for in vitro susceptibility to various concentrations (0.03-64.0 microg/mL) of three azole drugs, ketoconazole, voriconazole and itraconazole, as well as the allylamine terbinafine, using the agar dilution method. All strains of the seven Malassezia species were susceptible to the three azole drugs at low concentrations. M. furfur, M. sympodialis, M. slooffiae, M. pachydermatis, M. globosa, M. obtusa and M. restricta were most sensitive to ketoconazole and itraconazole, with minimum inhibitory concentrations (MICs) ranging from < or = 0.03 to 0.125 microg/mL. The recently introduced antifungal, voriconazole, was also very effective, with MIC80 values < or = 0.03 microg/mL for 80% of strains. MICs of terbinafine against the seven Malassezia species ranged from 相似文献   

从包皮龟头炎患者皮损处分离鉴定马拉色菌

目的：了解马拉色菌属各菌种在包皮龟头炎皮损处的构成及其在发病中的作用。方法：从患处取材直接镜检查见马拉色菌孢子和(或)菌丝的包皮龟头炎患者作为研究对象。用胶带法取材后分别接种在含菜子油的培养基及无放线菌酮的沙堡培养基分离菌种。依据生理生化和形念学特点及转种到科玛嘉显色培养基和米粉吐温80琼脂培养结果鉴定出马拉色菌和(或)念珠菌。结果：81例患者中有57例(70．37％)培养并鉴定出马拉色菌(共60株)，其中糠秕马拉色菌20株(33．33％），合轴马拉色菌18株(30．00％)，钝形马拉色菌17株(28．33％)，球形马拉色菌5株(8．33％)。有37例同时分离到念珠菌(其中72．97％为白念珠菌）。44例仅检出马拉色菌的患者中有23例接受抗真菌治疗。结论：糠秕马拉色菌、合轴马拉色菌、钝形马拉色菌是包皮龟头炎患者皮损处的主要菌种；马拉色菌可能单独或与念珠菌协同引起包皮龟头炎。     

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.     

从夫妻双方的头皮屑中分离和鉴定马拉色菌

目的 探讨夫妻双方的头皮屑中马拉色菌的带菌情况，菌种构成及夫妻间共用梳子与马拉色菌菌种一致性的关系，方法 采用Leeming和Notman培养基，Dixon培养基和含菜子油培养基分别同时培养114对夫妻的头皮屑，观察培养阳性率与头皮屑严重程度的关系。根据生理生化学及形态学特点鉴定菌种，并比较在这3种培养基中菌落初长时间及对菌种有无选择性。结果 从114对夫妻（共228人0中153人的头皮屑中分离到马拉色菌，阳性率为67.11%。培养阳性率与头皮屑的严重程度有正相关关系，3种培养基共分离到马拉色菌，阳性率为67.11%。培养阳性率与头皮屑的严重程度有正相关关系。3种培养基共分离到459株马拉色菌，从中鉴定出合轴马拉色菌（33.33%)，球形马拉色菌（25.05%)，限制马拉色菌（15.47%)，糠秕马拉色菌（13.73%)和钝形马拉色菌（12.42%)共5个种，3种培养基对菌种无选择性，但在含菜子油的培养基中菌落初长时间最短，同一头皮屑在3种培养基上分离到同一菌种有129人（56.58%)，分离到2种菌的有24人（10.53%)，共用梳子的夫妻双方的头皮屑中菌种相同者（35.96%)显著多于菌种不同者（13.16%)；夫妻间共用梳子者头皮屑中菌种相同者（50.67%)显著多于非共用梳子者（7.69%)。结论 头皮屑中的优势菌种主要为合轴和球形马拉色菌，夫妻双方的头皮屑中马拉色菌菌种一致率较高，而夫妻间共用梳子组的菌种一致率更高，提示马拉色菌可在夫妻之间传播，共用梳子可能是重要的传播媒介。     

Cutaneous Malassezia microbiota of healthy subjects differ by sex, body part and season

Malassezia is a component of normal cutaneous resident microbiota. The aim of this study was to quantitatively clarify the differences in cutaneous Malassezia microbiota in healthy subjects by sex, body part and season. Samples were collected from the forehead, cheek, upper chest and upper back of 20 healthy men and 20 healthy women (average age 32 years) in summer and winter by the swab method. Malassezia DNA was analyzed using a real-time PCR system. As a result, in sex, body parts and season, men, the upper trunk and summer showed the highest total numbers of cutaneous Malassezia species on average. There were also differences depending on the analytical method. The predominant species were M. restricta on the face of men, M. globosa and M. dermatis on the upper trunk of men, and M. globosa and M. sympodialis on the upper trunk of women. This study clarified that the cutaneous Malassezia microbiota of healthy subjects differed by sex, body part and season.     

南通和南京马拉色菌毛囊炎临床调查及致病菌种分析

目的 探讨南通和南京马拉色菌毛囊炎的易感因素及致病菌种在不同地区、不同部位马拉色菌毛囊炎中的菌种分布情况。方法 对符合病例收集纳入标准的患者进行问卷调查,取毛囊内容物进行真菌涂片、培养;并根据形态学和生理生化特征进行菌种鉴定。结果 241例临床诊断为马拉色菌毛囊炎的患者,真菌涂片204例阳性,涂片阳性率84.65%;收集标本259份,共得阳性株213株,其中马拉色菌209株,念珠菌4株（占1.54%）,真菌培养阳性率82.24%。菌种鉴定：209株马拉色菌活化菌种后,可供鉴定的马拉色菌菌株186株,共检测到6个菌种的马拉色菌,其中糠秕马拉色菌111株（59.68%）、斯洛菲马拉色菌43株（23.12%）、合轴马拉色菌17株（9.14%）、球形马拉色菌9株（4.84%）、厚皮马拉色菌4株（2.15%）、钝形马拉色菌2株（1.08%）。不同个体、不同部位的菌种分布：胸部、后背、腹部和面颈部以糠秕马拉色菌为主,上肢、肩部和头顶以斯洛菲马拉色菌为主,下肢均为球形马拉色菌。同一个体、不同发病部位存在不同的菌种,主要为糠秕马拉色菌合并合轴或斯洛菲马拉色菌。 结论 南通和南京马拉色菌毛囊炎存在6种马拉色菌致病菌种,糠秕和斯洛菲马拉色菌是主要的致病菌种。     

The prevalence of Malassezia yeasts in patients with atopic dermatitis, seborrhoeic dermatitis and healthy controls

Cultures for Malassezia yeasts were taken from both normal-looking skin and lesional skin in 124 patients with atopic dermatitis, 16 patients with seborrhoeic dermatitis and from normal skin of 31 healthy controls. Positive Malassezia growth was found in fewer patients with atopic dermatitis (56%) than in patients with seborrhoeic dermatitis (88%) or in healthy controls (84%, p<0.01). In the patients with atopic dermatitis, fewer positive cultures were found in lesional (28%) than in non-lesional skin (44%, p<0.05), while positive cultures were found in 75% of both lesional and non-lesional skin of patients with seborrhoeic dermatitis (not significant). M. sympodialis dominated in patients with atopic dermatitis (46%) and in healthy controls (69%). In patients with seborrhoeic dermatitis both M. sympodialis and M. obtusa were cultured in 43%. A Malassezia species extract mixture would increase the possibility of detecting IgE sensitization to Malassezia in patients with atopic dermatitis.     

Differentiation of Malassezia species: selectivity of Cremophor EL, castor oil and ricinoleic acid for M. furfur

In recent years, the genus Malassezia has been reclassified based on molecular data. In addition to M. furfur , M. pachydermatis and M. sympodialis , four new species, M. globosa , M. obtusa , M. restricta and M. slooffiiae , have been described. However, apart from their lipid dependence, little is known about the metabolism and nutritional requirements of all the seven species. Further to recent studies, 10 hydrophilic emulsifiers (HLB > 10) were examined in an agar diffusion test to determine their growth-promoting effect on reference strains of the different Malassezia species. Polyethylene glycol (PEG) 7 glyceryl monoalcanoate (Cetiol HE), PEG–glyceryl stearate (Tagat S2) and macrogol-50 stearate (Myrj 53) were metabolized by all strains, while PEG-35 castor oil (Cremophor EL) was metabolized only by M. furfur . The latter observation is due to a different metabolism of castor oil and its main component, ricinoleic acid (12-hydroxy oleic acid), which may also give an insight into the pathogenesis of diseases that are associated with Malassezia spp. As hydroxy fatty acids are important in maintaining the epidermal structure and function, their metabolism specifically by M. furfur might clarify some clinical aspects of pityriasis versicolor. Apart from this speculation, use of Cremophor EL, with splitting of esculin as an additional key character, improves the distinction of the species M. furfur , M. slooffiae and M. sympodialis .     

Hyperkeratotic head and neck Malassezia dermatosis

BACKGROUND: Pityriasis versicolor (tinea versicolor) is a common skin disorder due to Malassezia usually affecting adolescents and young adults, more frequently in the tropics. Facial involvement, isolated or not, is not frequent in white adults. OBJECTIVE: Here, we report a possible atypical hyperkeratotic form of dermatosis of the face, in two young immunocompetent Caucasian patients, particularly recalcitrant to therapy. RESULTS: Skin scrapings grew yeasts belonging to the genus Malassezia, including both M. globosa and M. sympodialis. This unusual variant needs long-term therapy with systemic and topical imidazoles together with facial cleansing. CONCLUSION: We propose the name hyperkeratotic head and neck Malassezia dermatosis for this distinctive clinical entity. This variant of pityriasis versicolor should be considered in the differential diagnosis of seborrheic dermatitis and dermatitis neglecta.     

马拉色菌与人角质形成细胞株共同培养液对培养人黑素细胞生长及酪氨酸酶mRNA表达的影响

目的:观察不同马拉色菌与人角质形成细胞株共同培养液对培养人黑素细胞生长及酪氨酸酶mRNA表达的影响,探讨花斑癣的色素改变是否与马拉色菌的不同菌种有关。方法:将球形、钝形、糠秕、合轴马拉色菌分别与人角质形成细胞株共同培养,24 h后收集上清液,按一定比例加入黑素细胞培养体系中继续培养24 h:①用四甲基偶氮唑兰比色法观察对黑素细胞生长的影响;②用实时荧光定量PCR法测定黑素细胞酪氨酸酶mRNA的表达。结果:各共同培养液对黑素细胞生长无明显影响,但除球形马拉色菌与人角质形成细胞共同培养液可上调黑素细胞酪氨酸酶mRNA的表达(P<0.05)外,未见其它种马拉色菌与人角质形成细胞株共同培养液对黑素细胞酪氨酸酶mRNA的表达有影响。结论:球形马拉色菌与人角质形成细胞株共同培养液刺激黑素细胞后,能提高酪氨酸酶mR-NA的表达,该菌可能与花斑癣的色素沉着有关。     

Malassezia globosa as the causative agent of pityriasis versicolor

BACKGROUND: The taxonomic revision carried out in 1996 on the genus Malassezia, which now comprises seven different species, made necessary a re-evaluation of the data concerning the ecology and pathogenicity of these lipophilic yeasts. Very little has been published since then. OBJECTIVES: The aim of this work was to contribute to the knowledge of the aetiology of pityriasis versicolor (PV) with a mycological study made according to the new species. METHODS: Ninety-six patients with PV completed the study. In all of them, samples were taken from the lesions for direct microscopy with KOH+ Parker ink and culture. Samples were also taken from normal skin of the trunk and the forehead of the same patients for culture. Cultures were made in modified Dixon medium in Petri dishes, incubated at 31 degrees C and the isolates were identified by morphological and physiological characteristics. RESULTS: In the PV lesions, direct microscopy always showed the typical mixture of globose blastoconidia and pseudomycelium. Only one patient, having received previous topical antimycotic treatment, was negative. The spherical yeasts observed in vivo were morphologically identical to the globose yeasts characteristic of M. globosa. In culture, M. globosa was found in 97% of cases, alone in 60% of them and associated with M. sympodialis in 29% and M. slooffiae in 7%. These two species were also found in similar percentages on the clinically uninvolved skin of the trunk, whereas M. globosa was not isolated at these sites. However, on the forehead, a small number of colonies of M. globosa was recovered in 12% of cases. CONCLUSIONS: These results support the data previously reported and strongly suggest that M. globosa in its mycelial phase is the causative agent of PV.     

从花斑癣患者的皮损区及非皮损区分离和鉴定马拉色菌

目的 研究花斑癣患者皮损区及非皮损区马拉色菌菌种构成;不同解剖部位、皮损颜色及各菌种的分布情况;患者病情和年龄与菌种构成的关系。方法 用无菌胶带粘取113例花斑癣患者皮损区及非皮损区共629个部位的皮屑,分别接种于含菜子油培养基中分离马拉色菌,用生理生化及形态学方法鉴定菌种。结果 皮损区与相对应的非皮损区马拉色菌分离阳性率无差别,非皮损区额部和胸背部分离阳性率高于上、下肢。共分离到565株马拉色菌,鉴定出合轴马拉色菌(44.78%)、糠秕马拉色菌(32.94%)、球形马拉色菌(11.68%)、钝形马拉色菌(5.84%)及限制马拉色菌(4.76%)共5个种,有27处(5.01%)同时分离到两种菌。皮损区与非皮损区菌种构成无明显差别,限制马拉色菌主要从额部分离出。菌种构成与皮损面积无关,但与皮损颜色和患者年龄有关。皮损颜色与病程无关。结论 花斑癣患者皮损区与非皮损区马拉色菌分离阳性率和菌种构成基本一致,与病情无关,而不同解剖部位、皮损类型及年龄患者的菌种构成有一定差异。     

Malassezia globosa and restricta: breakthrough understanding of the etiology and treatment of dandruff and seborrheic dermatitis through whole-genome analysis

Dandruff and seborrheic dermatitis (D/SD) share an etiology dependent upon three factors: sebum, microbial metabolism (specifically, Malassezia yeasts), and individual susceptibility. Advances in microbiological and analytical techniques permit a more detailed understanding of these etiologic factors, especially the role of Malassezia. Malassezia are lipid-dependent and demonstrate adaptation allowing them to exploit a narrow niche on sebum-rich skin. Work in our and our collaborators' laboratories has focused on understanding these adaptations by detailed analysis of biochemistry and gene expression. We have shown that Malassezia globosa and M. restricta predominate on dandruff scalp, that oleic acid alone can initiate dandruff-like desquamation, that M. globosa is the most likely initiating organism by virtue of its high lipase activity, and that an M. globosa lipase is expressed on human scalp. Considering the importance of M. globosa in D/SD (and the overall importance of commensal fungi), we have sequenced the M. globosa and M. restricta genomes. Genomic analysis indicates key adaptations to the skin environment, several of which yield important clues to the role Malassezia play in human disease. This work offers the promise of defining new treatments to D/SD that are targeted at changing the level or activities of Malassezia genes.