Skin Commensal Fungus Malassezia and Its Lipases.

Malassezia is the most abundant genus in the fungal microflora found on human skin, and it is associated with various skin diseases. Among the 18 different species of Malassezia that have been identified to date, M. restricta and M. globosa are the most predominant fungal species found on human skin. Several studies have suggested a possible link between Malassezia and skin disorders. However, our knowledge on the physiology and pathogenesis of Malassezia in human body is still limited. Malassezia is unable to synthesize fatty acids; hence, it uptakes external fatty acids as a nutrient source for survival, a characteristic compensated by the secretion of lipases and degradation of sebum to produce and uptake external fatty acids. Although it has been reported that the activity of secreted lipases may contribute to pathogenesis of Malassezia, majority of the data were indirect evidences; therefore, enzymes' role in the pathogenesis of Malassezia infections is still largely unknown. This review focuses on the recent advances on Malassezia in the context of an emerging interest for lipases and summarizes the existing knowledge on Malassezia, diseases associated with the fungus, and the role of the reported lipases in its physiology and pathogenesis.

Distribution of Malassezia species on the skin of patients with psoriasis.

INTRODUCTION: Malassezia species can induce the expression of interleukin-17 (IL-17), which plays an important role in the inflammatory and immune response in psoriasis (PS). The purpose of this study was to investigate the Malassezia species composition in patients with PS and healthy individuals and explore the role of Malassezia species in the pathogenesis of PS. MATERIALS AND METHODS: A total of 28 patients with PS and 10 age- and sex-matched healthy individuals participated in this study. Specimens collected from the lesional and non-lesional skin of patients with PS and the skin of healthy individuals were analyzed by using nested PCR. RESULTS: The relative abundance of Malassezia species was 84.96% in healthy subjects, more than twice that in patients with PS (P<0.01). M. restricta (43.09%) and M. globosa (41.38%) were the main Malassezia species in patients with PS followed by M. furfur (4.84%) and M. sympodialis (2.49%). M. sympodialis accounted for 18. 81% of the Malassezia species in healthy subjects, which was nearly eight times higher than in patients with PS (P<0.01). Further, M. furfur was detected both on lesional and non-lesional psoriatic skin, but it was not found on the skin of healthy individuals. CONCLUSIONS: The Malassezia species composition in patients with PS differed from that of healthy individuals. M. restricta and M. globosa were the main Malassezia species in patients with PS.

Malassezia and Staphylococcus dominate scalp microbiome for seborrheic dermatitis.

Seborrheic dermatitis (SD) is a common disease of the human scalp that causes physical damage and psychological problems for patients. Studies have indicated that dysbiosis of the scalp microbiome results in SD. However, the specific fungal and bacterial microbiome changes related to SD remain elusive. To further investigate the fungal and bacterial microbiome changes associated with SD, we recruited 57 SD patients and 53 healthy individuals and explored their scalp microbiomes using next generation sequencing and the QIIME and LEfSe bioinformatics tools. Skin pH, sebum secretion, hydration, and trans-epidermal water loss (TWEL) were also measured at the scalp. We found no statistically significant differences between the normal and lesion sites in SD patients with different subtypes of dandruff and erythema. However, the fungal and bacterial microbiome could differentiate SD patients from healthy controls. The presence of Malassezia and Aspergillus was both found to be potential fungal biomarkers for SD, while Staphylococcus and Pseudomonas were found to be potential bacterial biomarkers. The fungal and bacterial microbiome were divided into three clusters through co-abundance analysis and their correlations with host factors indicated the interactions and potential cooperation and resistance between microbe communities and host. Our research showed the skin microbe dysbiosis of SD and highlighted specific microorganisms that may serve as potential biomarkers of SD. The etiology of SD is multi-pathogenetic-dependent on the linkage of several microbes with host. Scalp microbiome homeostasis could be a promising new target in the risk assessment, prevention, and treatment of SD disease.

Sociodemographic characteristics and spectrum of Malassezia species in individuals with and without seborrhoeic dermatitis/dandruff: A comparison of residents of the urban and rural populations.

Seborrhoeic dermatitis/dandruff (SD/D) is a common, persistent, relapsing inflammatory condition affecting the areas rich in sebaceous glands. SD/D is widely prevalent in India but Malassezia species implicated are not well studied. To estimate the prevalence and spectrum of Malassezia species causing SD/D and understand the sociodemographic characteristics of SD/D in rural and urban populations, a total of 200 SD/D patients and 100 healthy controls (HC) from both rural and urban backgrounds were enrolled in this study. SD/D severity was clinically graded as mild, moderate, severe, and very severe. The isolates were identified by phenotypic characters and confirmed by ITS2 PCR-RFLP and sequencing of the ITS region of rDNA. Severe (59%) and very severe (71%) form of SD/D was higher in the rural population compared to the urban population (P = .004). The isolation rate of Malassezia was significantly higher in overall SD/D patients scalp (82%) compared to HC (67%) (P = .005). From the scalp of SD/D patients, M. globosa (36.2%) was predominantly isolated followed by M. restricta (31.3%), M. furfur (15.7%), a mixture of M. globosa and M. restricta (12%) or M. arunalokei (4.8%). Similarly, M. globosa (49.3%) was predominately isolated from the scalp of HC followed by M. restricta (22.4%). M. restricta was significantly higher in the scalp of SD/D patients compared to HC and/or nasolabial fold of both SD/D patients and HC (P = .0001). Our findings indicate that M. restricta has a high association with SD/D. More severe disease frequency was observed in the rural population. PRECIS: Dandruff is associated with Malassezia restricta and very severe cases are higher in rural population, probably due the poor hygiene. Moderate to severe hair loss and itching were strongly associated with dandruff. Use of soaps to cleanse scalp appears to be better than shampoo in preventing dandruff.

Inflammasome-mediated Inflammation by Malassezia in human keratinocytes: A comparative analysis with different strains.

Malassezia species are associated with several common dermatologic conditions including pityriasis versicolor, seborrhoeic dermatitis, folliculitis, and atopic dermatitis and dandruff. However, its causal role remains to be established. We intended to explore the role of inflammasome activation in human keratinocytes in response to three different Malassezia species. We compared the different activation patterns of inflammasomes and the expression of pro-inflammatory cytokines and antimicrobial peptides by three different Malassezia species-M. restricta, M. globosa and M. sympodialis-in human keratinocytes. We found that different Malassezia species, especially M. restricta and M. globosa could induce nucleotide-binding oligomerisation domain, leucine-rich repeat and pyrin-domain-containing protein (NLRP)3-apoptosis-associated speck-like protein containing CARD (ASC) inflammasome activation and subsequent interleukin (IL)-1ß secretion in human keratinocytes. Malassezia species variably induced thymic stromal lymphopoietin, ß-defensin 2, and LL-37. IL-8 mRNA and IL-22 protein significantly increased in the M. sympodialis-treated group, and Chemokine C-C motif ligand (CCL)17 and CCL22 mRNA were increased in response to M. globosa- and M. restricta- treated keratinocytes, respectively. Our data show that various species of Malassezia promote variable inflammatory responses in keratinocytes by activating NLRP3 inflammasomes, pro-inflammatory cytokines and chemokines, and antimicrobial peptides.

External ear canal mycobiome of some rabbit breeds.

The genus Malassezia is part of the normal skin mycobiota of a wide range of warm-blooded animals. In this genus, M. cuniculi is the only species described from rabbits. However, Malassezia species are rarely studied in lagomorphs. In the present study, the presence of Malassezia was assessed in samples from the external ear canal of healthy rabbits of different breeds. Cytological and culture techniques, Sanger sequencing, and Next-generation sequencing (NGS) were used to describe the ear mycobiota in the samples. Although no growth was observed in the cultured plates, cytological examination revealed the presence of round cells similar to those of Malassezia yeasts. For metagenomics analysis, the D1/D2 domain of the large subunit of the ribosomal DNA (LSU rDNA) was PCR amplified and the resulting reads were mapped against a custom-made cured database of 26S fungal sequences. NGS analysis revealed that Basidiomycota was the most abundant phylum in all the samples followed by Ascomycota. Malassezia was the most common genus presenting the highest abundance in the external ear canal. Malassezia phylotype 131 and M. cuniculi were the main sequences detected in the external auditory canal of rabbits. The study included both lop-eared and erect-eared rabbits and no differences were observed in the results when comparing both groups. This is the first attempt to study the external ear canal mycobiome of rabbits of different breeds using NGS. LAY SUMMARY: In the present study, the presence of Malassezia was assessed in samples from the external ear canal of healthy rabbits of different breeds. Cytological and culture techniques, Sanger sequencing, and Next-generation sequencing (NGS) were used to describe the ear mycobiota in the samples.

Effect of zinc pyrithione shampoo treatment on skin commensal Malassezia.

Malassezia restricta and Malassezia globosa are lipid dependent commensal yeasts associated with dandruff. Antifungal actives such as zinc pyrithione are commonly used in antidandruff shampoos, although their efficacy is not clearly demonstrated. In this study, we assessed the efficacy of antifungal treatments on scalp Malassezia via a combination of culturomic and genomic detection methods. Zinc pyrithione inhibited Malassezia growth at low minimum inhibitory concentrations (MICs). In a longitudinal pilot study, quantitative polymerase chain reaction (qPCR) analysis showed a decrease in M. restricta on the scalp after zinc pyrithione treatment. These findings validate the antifungal efficacy of zinc pyrithione as a dandruff treatment. LAY ABSTRACT: Malassezia yeasts are associated with dandruff and seborrheic dermatitis. Zinc pyrithione is effective against Malassezia growth in vitro and when tested on human skin as a shampoo. These findings will be useful for investigating the role of Malassezia in skin microbiome intervention studies.

Epidemiological pattern of Malassezia, its phenotypic identification and antifungal susceptibility profile to azoles by broth microdilution method.

Background: Malassezia though known for its cutaneous infections can potentially cause invasion. The skin infections caused by Malassezia have poor patient compliance due to its chronicity and recurrent nature of the disease. There is also a lack of standardised antifungal susceptibility profile for Malassezia due to its complex growth requirement. Objective: This study was performed to understand the epidemiological pattern of disease and to study the antifungal susceptibility testing (AFST) profile so as to choose the appropriate drug/drugs to treat the infections caused by Malassezia. Materials and Methods: Samples were collected and processed, species were identified by conventional method and AFST was done by broth microdilution method. Results: The epidemiological pattern showed adolescent females commonly affected in torso. The most common lesion was pityriasis versicolor. The systemic antifungal of choice was itraconazole with the lowest minimum inhibitory concentration (MIC) of 0.125-1 µg/ml. The best topical drug with the lowest MIC value was clotrimazole 0.03-0.5 µg/ml. Conclusion: AFST is important as it will help the dermatologist to choose the appropriate antifungal agents for the patient and thereby reduce the chronicity of the disease with good patient compliance.

Metagenomic next-generation sequencing of viruses, bacteria, and fungi in the epineurium of the facial nerve with Bell's palsy patients.

Bell's palsy (BP) represents a major cause leading to facial paralysis in the world. The etiology of BP is still unknown, and virology is the prevailing theory. The purpose of this study is to explore the pathogenic microorganisms that may be related to BP, and it is of great significance to study the pathogenesis and treatment of BP. Metagenomic next-generation sequencing (mNGS) detection was performed in the epineurium of the facial nerve of 30 BP patients who underwent facial nerve epineurium decompression. A total of 84 pathogenic microorganisms were detected in 30 clinical samples, including 4 viruses, 10 fungi, and 70 bacteria. The species with the highest detection frequency in virus was human betaherpesvirus 7 (HHV-7). The species with the highest detection frequency in Fungi was Malassezia restricta. The species with the highest detection frequency in Bacteria was Pseudomonas aeruginosa. In this study, mNGS method was firstly used to detect the pathogenic microorganisms in the epineurium of the facial nerve with BP patients. We have for the first time identified HHV-7 and aspergillus in the epineurium of the facial nerve of BP patients. These results suggest that these two pathogenic microorganisms should be considered in the pathogenesis of BP.

HGT in the human and skin commensal Malassezia: A bacterially derived flavohemoglobin is required for NO resistance and host interaction.

The skin of humans and animals is colonized by commensal and pathogenic fungi and bacteria that share this ecological niche and have established microbial interactions. Malassezia are the most abundant fungal skin inhabitant of warm-blooded animals and have been implicated in skin diseases and systemic disorders, including Crohn's disease and pancreatic cancer. Flavohemoglobin is a key enzyme involved in microbial nitrosative stress resistance and nitric oxide degradation. Comparative genomics and phylogenetic analyses within the Malassezia genus revealed that flavohemoglobin-encoding genes were acquired through independent horizontal gene transfer events from different donor bacteria that are part of the mammalian microbiome. Through targeted gene deletion and functional complementation in Malassezia sympodialis, we demonstrated that bacterially derived flavohemoglobins are cytoplasmic proteins required for nitric oxide detoxification and nitrosative stress resistance under aerobic conditions. RNA-sequencing analysis revealed that endogenous accumulation of nitric oxide resulted in up-regulation of genes involved in stress response and down-regulation of the MalaS7 allergen-encoding genes. Solution of the high-resolution X-ray crystal structure of Malassezia flavohemoglobin revealed features conserved with both bacterial and fungal flavohemoglobins. In vivo pathogenesis is independent of Malassezia flavohemoglobin. Lastly, we identified an additional 30 genus- and species-specific horizontal gene transfer candidates that might have contributed to the evolution of this genus as the most common inhabitants of animal skin.

Diversity and adaptation within the genus Malassezia: bats already have their species / Diversidad y adaptación en el género Malassezia: los murciélagos ya tienen su especie

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Medium-chain fatty acid esters-Optimising their efficacy as anti-Malassezia agents.

BACKGROUND: For fatty acid esters of monohydric alcohols, cleavage by exo-enzymes of Malassezia (M.) spp. and release of fatty acids with antimicrobial activity have been shown recently. On skin surface, this selective activation of antimicrobial activity might result in a 'self-kill' targeted locally at the site with the highest M. density. OBJECTIVES: As for the disadvantage of strong odour, use of these esters for topical therapy is limited to low concentrations. Therefore, cleavage was also tested for monoesters of octanoic and undec-10-enoic acid with the bihydric alcohol propane-1,3-diol or the trihydric glycerol. METHODS: In an agar dilution test, the minimal inhibitory concentrations of these compounds were determined for M. furfur, M. globosa, M. sympodialis and M. restricta, respectively. GC analysis of parent compounds and liberated fatty acids was used to reveal ester cleavage. RESULTS: Ester cleavage started immediately. MICs for the test compounds ranged between ~1000-8000 ppm after 14 days of incubation. 1,3-propanediol esters, especially 3-hydroxypropyl octanoate and 3-hydroxypropyl undecylenate were most effective, binary combinations exerted synergistic effects. CONCLUSIONS: The new substances are advantageous in terms of odour and substantivity and have also beneficial skin caring properties if not hydrolysed by M. spp. As a different panel of hydrolases of each single M. species is responsible for variation in efficacy among the test substances, tailored products to treat preferentially single species or blends with a broader effectivity can be designed. In vivo verification will be the next step for the successful development of this new therapeutical concept for M.-associated diseases.

Biofilm formation, adherence, and hydrophobicity of M. sympodialis, M. globosa, and M. slooffiae from clinical isolates and normal skinVirulence factors of M. sympodialis, M. globosa and M. slooffiae.

The genus Malassezia comprises a heterogeneous group of species that cause similar pathologies. Malassezia yeasts were considered as the most abundant skin eukaryotes of the total skin mycobiome. The ability of this fungus to colonize or infect is determined by complex interactions between the fungal cell and its virulence factors. This study aims to evaluate in vitro the hydrophobicity levels, the adherence capacity on a polystyrene surface and the ability to form biofilm of 19 isolates, including M. sympodialis, M. globosa, and M. slooffiae, from healthy subjects and from dermatological disorders. Cellular surface hydrophobicity levels were determined by two-phase system. The biofilm formation was determined by tetrazolium salt (XTT) reduction assay and by Scanning Electron Microscopy (SEM). Strain dependence was observed in all virulence factors studied. All isolates of M. sympodialis, M. globosa, and M. slooffiae demonstrated their ability to form biofilm at variable capacities. SEM observations confirmed a variable extracellular matrix after 48 hours of biofilm formation. All isolates of M. globosa were highly adherent and/or hydrophobic as well as biofilm producers. In contrast, M. slooffiae was the least biofilm producer. No significant differences between virulence factors were demonstrated for M. sympodialis, either as clinical isolate or as inhabitant of human microbiota. Results of this work together with the previous M. furfur research confirm that the most frequently Malassezia species isolated from normal subject's skin and patients with dermatosis, form biofilm with different capacities. The study of these virulence factors is important to highlight differences between Malassezia species and to determine their involvement in pathological processes.

Malassezia Yeasts in Veterinary Dermatology: An Updated Overview.

Lipophilic yeasts of the genus Malassezia are important skin commensals and opportunistic skin pathogens in a variety of animals. The species M. pachydermatis was first isolated from the skin of a captive Indian rhinoceros with an exfoliative dermatitis in 1925, recognized as an important otic pathogen of dogs in the 1950's, and finally accepted, after several years of controversy, as a common cause of canine dermatitis in the 1990's. Since then, there has been considerable research into the biology of Malassezia yeasts and their interaction with their animal hosts. In dogs and cats, M. pachydermatis is associated with ceruminous otitis externa and a "seborrhoeic" dermatitis, wherein pruritic, erythematous skin lesions, often with brown/black greasy, malodourous material matting hairs, preferentially develop in intertriginous areas. Skin disease is favored by folds, underlying hypersensitivity disorders, endocrinopathies, defects of cornification, and in cats, various visceral paraneoplastic syndromes. Diagnosis is based on detecting the yeast in compatible skin lesions, usually by cytology, and observing a clinical and mycological response to therapy. Treatment normally comprises topical or systemic azole therapy, often with miconazole-chlorhexidine shampoos or oral itraconazole or ketoconazole. Management of concurrent diseases is important to minimize relapses. Historically, wild-type Malassezia isolates from dogs and cats were typically susceptible to azoles, with the exception of fluconazole, but emerging azole resistance in field strains has recently been associated with either mutations or quadruplication of the ERG11 gene. These observations have prompted increased interest in alternative topical antifungal drugs, such as chlorhexidine, and various essential oils. Further clinical trials are awaited with interest.

Loss of centromere function drives karyotype evolution in closely related Malassezia species.

Genomic rearrangements associated with speciation often result in variation in chromosome number among closely related species. Malassezia species show variable karyotypes ranging between six and nine chromosomes. Here, we experimentally identified all eight centromeres in M. sympodialis as 3-5-kb long kinetochore-bound regions that span an AT-rich core and are depleted of the canonical histone H3. Centromeres of similar sequence features were identified as CENP-A-rich regions in Malassezia furfur, which has seven chromosomes, and histone H3 depleted regions in Malassezia slooffiae and Malassezia globosa with nine chromosomes each. Analysis of synteny conservation across centromeres with newly generated chromosome-level genome assemblies suggests two distinct mechanisms of chromosome number reduction from an inferred nine-chromosome ancestral state: (a) chromosome breakage followed by loss of centromere DNA and (b) centromere inactivation accompanied by changes in DNA sequence following chromosome-chromosome fusion. We propose that AT-rich centromeres drive karyotype diversity in the Malassezia species complex through breakage and inactivation.

Millions of yeast, bacteria and other microbes live in or on the human body. A type of yeast known as Malassezia is one of the most abundantmicrobes living on our skin. Generally, Malassezia do not cause symptoms in humans but are associated with dandruff, dermatitis and other skin conditions in susceptible individuals. They have also been found in the human gut, where they exacerbate Crohn's disease and pancreatic cancer. There are 18 closely related species of Malassezia and all have an unusually small amount of genetic material compared with other types of yeast. In yeast, like in humans, the genetic material is divided among several chromosomes. The number of chromosomes in different Malassezia species varies between six and nine. A region of each chromosome known as the centromere is responsible for ensuring that the equal numbers of chromosomes are passed on to their offspring. This means that any defects in centromeres can lead to the daughter yeast cells inheriting unequal numbers of chromosomes. Changes in chromosome number can drive the evolution of new species, but it remains unclear if and how centromere loss may have contributed to the evolution of Malassezia species. Sankaranarayanan et al. have now used biochemical, molecular genetic, and comparative genomic approaches to study the chromosomes of Malassezia species. The experiments revealed that nine Malassezia species had centromeres that shared common features such as being rich in adenine and thymine nucleotides, two of the building blocks of DNA. Sankaranarayanan et al. propose that these adenines and thymines make the centromeres more fragile leading to occasional breaks. This may have contributed to the loss of centromeres in some Malassezia cells and helped new species to evolve with fewer chromosomes. A better understanding of how Malassezia organize their genetic material should enable in-depth studies of how these yeasts interact with their human hosts and how they contribute to skin disease, cancer, Crohn's disease and other health conditions. More broadly, these findings may help scientists to better understand how changes in chromosomes cause new species to evolve.

Malassezia species dysbiosis in natural and allergen-induced atopic dermatitis in dogs.

Malassezia dermatitis and otitis are recurrent features of canine atopic dermatitis, increasing the cost of care, and contributing to a reduced quality of life for the pet. The exact pathogenesis of secondary yeast infections in allergic dogs remains unclear, but some have proposed an overgrowth of M. pachydermatis to be one of the flare factors. The distribution of Malassezia populations on healthy and allergic canine skin has not been previously investigated using culture-independent methods. Skin swabs were collected from healthy, naturally affected allergic, and experimentally sensitized atopic dogs. From the extracted DNA, fungal next-generations sequencing (NGS) targeting the ITS region with phylogenetic analysis of sequences for species level classification, and Malassezia species-specific quantitative real-time polymerase chain reaction (qPCR) were performed. M. globosa was significantly more abundant on healthy canine skin by both methods (NGS P < .0001, qPCR P < .0001). M. restricta was significantly more abundant on healthy skin by NGS (P = .0023), and M. pachydermatis was significantly more abundant on naturally-affected allergic skin by NGS (P < .0001) and on allergen-induced atopic skin lesions by qPCR (P = .0015). Shifts in Malassezia populations were not observed in correlation with the development of allergen-induced skin lesions. Differences in the lipid dependency of predominant Malassezia commensals between groups suggests a role of the skin lipid content in driving community composition and raises questions of whether targeting skin lipids with therapeutics could promote healthy Malassezia populations on canine skin.

The diversity and abundance of fungi and bacteria on the healthy and dandruff affected human scalp.

Dandruff is a skin condition that affects the scalp of up to half the world's population, it is characterised by an itchy, flaky scalp and is associated with colonisation of the skin by Malassezia spp. Management of this condition is typically via antifungal therapies, however the precise role of microbes in the aggravation of the condition are incompletely characterised. Here, a combination of 454 sequencing and qPCR techniques were used to compare the scalp microbiota of dandruff and non-dandruff affected Chinese subjects. Based on 454 sequencing of the scalp microbiome, the two most abundant bacterial genera found on the scalp surface were Cutibacterium (formerly Propionibacterium) and Staphylococcus, while Malassezia was the main fungal inhabitant. Quantitative PCR (qPCR) analysis of four scalp taxa (M. restricta, M. globosa, C. acnes and Staphylococcus spp.) believed to represent the bulk of the overall population was additionally carried out. Metataxonomic and qPCR analyses were performed on healthy and lesional buffer scrub samples to facilitate assessment of whether the scalp condition is associated with differential microbial communities on the sampled skin. Dandruff was associated with greater frequencies of M. restricta and Staphylococcus spp. compared with the healthy population (p<0.05). Analysis also revealed the presence of an unclassified fungal taxon that could represent a novel Malassezia species.

Differences in protein profiles between Malassezia pachydermatis strains obtained from healthy and infected dogs.

Malassezia pachydermatis causes infections of the skin and mucous membranes, especially in individuals with metabolic, hormonal, and immunological disorders. The search for M. pachydermatis properties that differentiate isolates from healthy and infected animals may result in the identification of typically commensal and potentially pathogenic strains within the entire species. We aimed to determine and compare protein profiles of M. pachydermatis strains isolated from 30 dogs with clinical symptoms of otitis externa and 34 dogs without symptoms of any disease. Two-dimensional gel electrophoresis was applied, and proteins distinguishing the two groups of strains were identified by liquid chromatography coupled with tandem mass spectrometry. Significant differences were found between potentially pathogenic and commensal isolates. The most significant finding was the presence of nicotinamide adenine dinucleotide phosphate (NADP)-dependent mannitol dehydrogenase and ketol-acid reductoisomerase among M. pachydermatis strains obtained from dogs with otitis externa. Nevertheless, it is not clear whether they are associated directly with the pathogenicity or they play the role of fungal allergen. On the basis of these findings, we can conclude that there may be two distinct groups of M. pachydermatis strains-one typically commensal and the other with properties that enhance the infection process. These results may be used for more precise diagnosis and identification of potentially pathogenic strains in the future.

Quantification of Malassezia pachydermatis by real-time PCR in swabs from the external ear canal of dogs.

Malassezia pachydermatis is part of the normal microbiota of canine skin and external ear canal, and is also associated with otitis externa in dogs. Laboratory detection of Malassezia otitis relies on the presence of elevated numbers of the yeast on cytologic examination of otic exudate. Although cytology has high specificity, it has low sensitivity, resulting in false-negatives and posing a challenge for clinicians to accurately diagnose Malassezia otitis. We developed a quantitative PCR (qPCR) to detect and quantify M. pachydermatis yeasts and validate the method with swabs from external ear canals of dogs. Our qPCR uses the ß-tubulin gene, a single-copy gene, as a target. The limit of quantification was established as 0.18 ng/reaction, equivalent to 2.0 × 104 genome equivalents (gEq). Swabs from healthy dogs yielded quantification values of ≤2.7 × 104 gEq in the qPCR, whereas swabs from dogs with otitis yielded quantification values of ≥2.5 × 105 gEq. Our qPCR assay provides accurate quantification of M. pachydermatis yeasts from swab samples from dogs, is more sensitive than cytology, and could be used to monitor response to treatment. Our assay could also be valuable in a research setting to better understand the pathogenesis of M. pachydermatis.