Yeast Malassezia Pachydermatis Research Articles

Extracellular phospholipase production by Malassezia pachydermatis strains and its inhibition by selected antimycotics and plant essential oil components

Extracellular phospholipase (EPL) plays an important role in the pathogenesis of the yeast Malassezia pachydermatis. Currently, the attention of researchers is focused on studying the virulence factors involved in this process and searching solutions to reduce their activity. One of the options is the use of natural remedies as anti-virulence agents. This study is aimed at investigating the production of extracellular phospholipase in M. pachydermatis strains (18 samples) and followed by the time-dependent inhibitory effect of selected azole antifungals (itraconazole, posaconazole and voriconazole) and plant essential oil components (terpinen-4-ol, thymol, carvacrol, eugenol and geraniol), evaluated by Egg Yolk Agar plate method. Almost all strains (17 isolates, (94.4%) were found to be intense EPL producers. A significant, time-dependent inhibition of EPL was noted after 1-, 3- and 6-h exposure of Malassezia cells to itraconazole (26.4%, 47.2% and 50.9%, respectively) compared to exposure to posaconazole (26.4%, 28.3% and 28.3%, respectively) and voriconazole (18.8%, 20.8% and 35.8%, respectively). After one-hour exposure to plant essential oil components, the best inhibitory effect was recorded for eugenol (62.3%), followed by terpinen-4-ol and thymol (56.6%), geraniol (41.5%) and carvacrol (26.4%). A 3-h exposure revealed that thymol retained the best inhibitory effect (88.7%) on EPL production, followed by carvacrol (73.6%), eugenol (56.6%), terpinen-4-ol (52.8%) and geraniol (49.1%). After 6-h exposure, no growth of M. pachydermatis strains exposed to carvacrol was observed, and the inhibitory efficiency for the other tested essential oil (EO) components achieved 88.7%. The obtained results indicate the promising efficacy of plant essential oils components in the inhibition of virulence factors such as EPL production.

In Vitro Biofilm Formation by Malassezia pachydermatis Isolates and Its Susceptibility to Azole Antifungals.

The yeast Malassezia pachydermatis, an opportunistic pathogen that inhabits the skin of various domestic and wild animals, is capable of producing a biofilm that plays an important role in antifungal resistance. The aim of this research study was to find the intensity of biofilm production by M. pachydermatis strains isolated from the ear canal of healthy dogs, and to determine the susceptibility of planktonic, adhered and biofilm-forming cells to three azole antifungals-itraco-nazole, voriconazole and posaconazole-that are most commonly used to treat Malassezia infections. Out of 52 isolates, 43 M. pachydermatis strains (82.7%) were biofilm producers with varying levels of intensity. For planktonic cells, the minimum inhibitory concentration (MIC) range was 0.125-2 µg/mL for itraconazole, 0.03-1 µg/mL for voriconazole and 0.03-0.25 µg/mL for posaconazole. Only two isolates (4.7%) were resistant to itraconazole, one strain (2.3%) to voriconazole and none to posaconazole. For adhered cells and the mature biofilm, the following MIC ranges were found: 0.25-16 µg/mL and 4-16 µg/mL for itraconazole, 0.125-8 µg/mL and 0.25-26 µg/mL for voriconazole, and 0.03-4 µg/mL and 0.25-16 µg/mL for posaconazole, respectively. The least resistance for adhered cells was observed for posaconazole (55.8%), followed by voriconazole (62.8%) and itraconazole (88.4%). The mature biofilm of M. pachydermatis showed 100% resistance to itraconazole, 95.3% to posaconazole and 83.7% to voriconazole. The results of this study show that higher concentrations of commonly used antifungal agents are needed to control infections caused by biofilm-forming strains of M. pachydermatis.

Study of the variation of the Malassezia load in the interdigital fold of dogs with pododermatitis

The yeast Malassezia pachydermatis is a common inhabitant of the skin and mucosae of dogs. However, under certain circumstances this yeast can overgrow and act as an opportunistic pathogen causing otitis and dermatitis in dogs. Canine pododermatitis is a common disorder in dogs in which M. pachydermatis acts as an opportunistic pathogen. In the present study, the presence of Malassezia yeasts was assessed and quantified in samples collected from the interdigital space of dogs with pododermatitis before and after treatment, and from healthy dogs. The samples were subjected to two different cytological examinations, culture on Sabouraud glucose agar and modified Dixon’s agar and a quantitative PCR targeting the internal transcribed spacer (ITS) genomic region. A selection of samples was analyzed by next generation sequencing (NGS) using the D1D2 domain of the large subunit of the ribosomal DNA as target. The pododermatitis samples before treatment showed higher cell counts, colony-forming units and ITS copies than the rest of samples. The NGS analysis revealed that Ascomycota was the main phylum in the healthy and post-treatment samples. However, Basidiomycota and M. pachydermatis was more abundant in the pododermatitis samples before treatment. These results support M. pachydermatis as an opportunistic agent in canine pododermatitis by a variety of methods, and demonstrate the correlation between cytologic and molecular methods for quantification.

The use of essential oils in the treatment of malasseziasis in dogs and cats: Review

Malasseziasis is a common fungal skin disease of humans and animals, caused by the lipophilic yeast Malassezia pachydermatis. Treatment is based on the association between systemic and topical antifungal; however, recurrences are frequent and complicated by intercurrent dermatoses that complicate the clinical case. This recurrence is usually due to errors in dosage of the drugs, administration of the same by owners and resistance of the yeast. The mechanisms of antifungal resistance are associated with the specific or acquired characteristics of the pathogen to the drugs. As an alternative to traditional treatments, the use of herbal products, such as essential oils, in the veterinary clinic has become popular. Although the possible action of essential oils has been reported in human medicine for about 17 years, the specialized literature on its use in canine and feline malasseziasis is still scarce and studies are needed to investigate the mechanisms of action of essential oils in the metabolism of the yeast Malassezia pachydermatis. Thus, the objective of this work is to present a bibliographic review on the use of four essential oils for the treatment of primary or secondary malasseziasis in dogs and cats.

Cytological and Microbiological Analysis of Canine Otitis Externa

The ear discharge of fifteen dogs was examined for cytological and microbiological evaluation along with antibiotic sensitivity testing. The ear discharge of ten healthy dogs was also analyzed for comparative data. Cytological analysis showed that inflammatory cells and rod shaped organisms in highly infected cases. Otic microbiota in healthy cases revealed Bacillus spp. and Staphylococcus spp., while in clinical cases Pseudomonas spp., E coli, Klebsiella spp. and Alcaligenes spp., were also identified by using universal primer 16S rRNA PCR. The yeast Malassezia pachydermatis was isolated from ear discharge of all dogs. Among the anti – microbial drugs tested, Gentamicin had the most effective response against the otic microbes, followed by ciprofloxacin while neomycin had the least

Antifungal in vitro Activity of Essential Oils against Clinical Isolates of Malassezia pachydermatis from Canine Ears: A Report from a Practice Laboratory

Background: The yeast Malassezia pachydermatis is a commensal but also the most isolated pathogenic yeast in canine otitis externa. Essential oils (EOs) exhibit antimicrobial activity and thus could be an alternative for treatment of otitis. Objective: The aim of this study was to analyse the in vitro efficacy of EOs against M. pachydermatis isolates from canine ears. Materials and Methods: Twenty-two EOs were tested for their in vitro activity against 15 M. pachydermatis isolates by agar disc diffusion. In addition, diluted EOs were tested and volatile antifungal activity was analysed by vapour assay. Results: Most EOs showed in vitro efficacy against M. pachydermatis. A different susceptibility pattern of tested isolates was found. Overall, the most potential EO was lemon grass. Also, cinnamon leaf, clove, manuka, Indian melissa, oregano, palmarosa, and winter savory oil exhibited a strong antifungal activity. Volatile activity was proven by the inhibiting effect in vapour assay. Conclusion: EOs show in vitro activity against M. pachydermatis. This study gives scientific background for the use of EOs in veterinary medicine. Diverse susceptibility patterns of different M. pachydermatis strains emphasise the helpfulness of an aromatogram as one lead for the best choice of an EO for therapy.

In vitro combination of antifungal agents against Malassezia pachydermatis.

The yeast Malassezia pachydermatis is a common commensal and occasional opportunistic pathogen of theskin microbiota of animals and humans. In this study, the susceptibility of M. pachydermatis isolates to fluconazole (FLC), itraconazole (ITZ), ketoconazole (KTZ), clotrimazole (CLZ), and miconazole (MCZ) alone and in combination with terbinafine (TRB), nystatin (NYS), and caspofungin (CSP) was evaluated in vitro based on the M27-A3 technique and the checkerboard microdilution method using Sabouraud dextrose broth with 1% tween 80 (SDB). Based on the mean FICI values, the main synergies observed were combinations of ITZ+CSP and CLZ+CSP (55.17%). The most significant combinations deserve in vivo evaluations because might provide effective alternative treatments against M. pachydermatis due to their synergistic interactions.

Antifungal Effect of Selected Essential Oils on Malassezia pachydermatis Growth

Abstract The antifungal activities of 14 selected essential oils (at the concentrations of 0.5 %, 5 %, and 30 %) against the yeast Malassezia pachydermatis (18 isolates and one reference strain) were investigated. The isolates of M. pachydermatis were obtained from swabs of external ear canals of healthy dogs using sterile swabs. The determination of the efficacy was based on a modified disc diffusion method (CLSI M44-A2). The best antifungal efficacy (100 %) was shown by clove, cinnamon and oregano at the concentration of 30 %; less significant efficacy was shown at the concentration of 5 % (38 %, 33 % and 5 %, respectively). Satureja inhibited the growth of Malassezia (efficacy of 16 %) only at the concentration of 30 %. Bergamot, lavender, juniper, cedar, sage, tea-tree, grapefruit, pine, chamomile and yarrow essential oils were not able to form inhibition zones as defined in the methodology used (greater or equal to 15 mm) in all concentrations used. Therefore, according to the interpretation criterion, they were considered ineffective. In all cases, the concentration of 0.5 % was not effective against the growth of Malassezia yeasts.

Malassezia pachydermatis from animals: Planktonic and biofilm antifungal susceptibility and its virulence arsenal

The yeast Malassezia pachydermatis is a component of the microbiota of dogs and cats, however it can cause otitis and seborrheic dermatitis in these animals. The objective of this study was to determine the antifungal susceptibility, and evaluate virulence and pathogenicity of 25 M. pachydermatis strains from animals. Susceptibility to ketoconazole, fluconazole, itraconazole, voriconazole, terbinafine, and amphotericin B was evaluated by broth microdilution assay. In addition, biofilm-forming ability, protease, phospholipase, hemolysin and melanin production and adhesion to epithelial cells by this yeast species were assessed. Finally, strain pathogenicity was investigated using the nematode Caenorhabditis elegans. Concerning the planktonic susceptibility, minimum inhibitory concentrations varied from <0.03 to>64 μg/mL for azole derivatives, 1 to >16 μg/mL for amphotericin B and 0.03 to 0.25 μg/mL for terbinafine. All strains were classified as strong biofilm producers, and ketoconazole, fluconazole and amphotericin B presented the best inhibitory effect against mature biofilms. All fungal isolates produced proteases, whereas 14/25 strains were positive for phospholipase production. Hemolytic activity was not observed and 18/25 strains showed dark pigmentation in the presence of L-DOPA. Regarding adhesion to epithelial cells, a low adhesion rate was observed in 10/12 evaluated strains. C. elegans mortality rate reached 95.9% after 96 h of exposure of the worms to M. pachydermatis. This yeast species produces important virulence factors and presents high pathogenicity, corroborating its clinical importance.

In vitro activity of amphotericin B-azole combinations against Malassezia pachydermatis strains.

Combination therapy has become popular in clinical practice, but limited data on the effects of combinations of antifungal agents is still available for most fungal pathogens. We studied the in vitro response of 30 genetically diverse clinical strains of the basidiomycetous lipophilic yeast Malassezia pachydermatis obtained from cases of canine otitis to several amphotericin B (AMB)-azole combinations. Broth microdilution checkerboard tests revealed that AMB antagonized the effects of itraconazole (ITC) and voriconazole (VRC) in 50% and 6.7% of the strains, respectively, but did not interact with fluconazole or posaconazole (fractional inhibitory concentration index (FICI) values were <4 in all cases). Subsequent Etest-based assays performed for a subset of strains did not confirm the antagonism between AMB and ITC or AMB and VRC. In summary, the results of this study suggest that antagonistic combination effects between AMB and azoles might occur when tested against M. pachydermatis. Nevertheless, as observed for other fungi, different in vitro analyses yielded contrasting results, and the response to AMB-azole combinations was compound- and strain-dependant.

Pseudomonas otitis in dogs: a general practitioner's guide to treatment

Otitis externa in dogs is a very common clinical problem encountered in general practice; it is also a very frustrating one to treat, especially when cases are recurrent. Many organisms...

Molecular epidemiology of a Malassezia pachydermatis neonatal unit outbreak.

The non-lipid-dependent yeast Malassezia pachydermatis is predominantly zoophilic but occasionally colonizes the human skin. This yeast caused an outbreak in a neonatal iIntensive care unit (NICU). This study aimed to describe the molecular epidemiology of this M. pachydermatis outbreak. All the M. pachydermatis isolates collected at a French University Hospital from January 2012 to April 2013 were included in the study. M. pachydermatis isolates, sampled from various biological samples sites in 25 patients, were identified via MALDI-TOF mass spectrometry and typed using intergenic-spacer 1 (IGS1) nucleotide sequence polymorphisms analysis. By analyzing 90 IGS1 sequences (including 43 deposited in GenBank), we found that of the 186M. pachydermatis isolates, 47 were viable for typing and all of them clustered within type 3; 78.7% clustered within the 3D subtype; the remaining clustered within three newly described subtypes: 3E (4.3%), 3F (8.5%) and 3G (8.5%). No particular subtype was associated with a collection site or a particular time period. This first molecular investigation of a M. pachydermatis outbreak in neonates showed that multiple genotypes can colonize the same neonate patient by. The source of this polyclonal outbreak could not be identified. It stopped after infection control measures, including the prohibition of a lipid-rich moisturizing hand cream used by the health care staff, had been implemented.

&lt;i&gt;In Vitro&lt;/i&gt; Susceptibility of &lt;i&gt;Malassezia pachydermatis&lt;/i&gt; Isolates from Canine Skin with Atopic Dermatitis to Ketoconazole and Itraconazole in East Asia

ABSTRACTTopical or oral azole antifungals are commonly used in canine atopic dermatitis (AD), as the lipophilic yeast Malassezia pachydermatis exacerbates canine AD. To examine whether canine AD lesions harbor azole-resistant M. pachydermatis isolates in East Asia, we investigated the in vitro susceptibility of M. pachydermatis isolates to ketoconazole (KTZ) and itraconazole (ITZ) obtained from AD lesions of canines in Japan, Korea and Taiwan. The minimum inhibitory concentrations (MICs) of KTZ and ITZ were measured by the E-test using Sabouraud dextrose agar with 0.5% Tween 40. The MICs of KTZ and ITZ for isolates from canines with AD were significantly higher than the MICs for isolates from healthy canines. Our findings suggested that the clinical isolates from canine AD skin lesions were less susceptible to azoles than those from normal canine skin in East Asia.

Genotyping of &lt;i&gt;Malassezia pachydermatis&lt;/i&gt; Isolates from Canine Healthy Skin and Lesional Skin of Atopic Dermatitis in Japan, Korea and Taiwan

Isolates of the yeast Malassezia pachydermatis obtained from skin samples of healthy dogs and of dogs with atopic dermatitis in Japan, Taiwan and Korea were molecularly characterized using intergenic pacer 1 (IGS1) region analysis. The percentage of IGS1 subtype isolates detected in healthy skin was as follows: 1A (6%), 1B (27%), 1C (11%), 2A (6%), 2B (6%), 3A (11%), 3B (6%), 3C (3%) and 3D (24%). In contrast, the most prevalent isolates detected in skin lesions of atopic dermatitis were subtype 3D in Japan and Taiwan and subtype 3C in Korea. All subtype isolates grew well on acidic medium (pH 6). However, subtype 3C and 3D isolates grew better than the other subtype isolates on medium at pH 8.

Enzymatic characterization of Malassezia pachydermatis isolates from dogs

The yeast Malassezia pachydermatis is part of the normal cutaneous microflora of most homoeothermic vertebrates. However, under certain conditions as high humidity, seborrheic skin, corticotherapy and immunodeficiency, it can become pathogenic and cause dermatopathies. The pathogenic role of the genus Malassezia seems to be related to physical, chemical and immunological disturbances and to the production of enzymes, including lipases, phospholipases and hydrolases. The Api-Zym® system is a semi quantitative method designed for the study of 19 enzymatic activities of tissues, cell suspensions, biological fluids, microorganisms, soil and others. Herein, the use of the Api-Zym system in 30 M. pachydermatis isolates detected the presence of the phosphohydrolases: acid phosphatase and naphtol-AS-BI-phosphohydrolase among all isolates, independently of the clinical sign associated to the animal. The knowledge of the enzymatic profile of M. pachydermatis aims to contribute to the comprehension of the role of each enzyme in the pathogeny of this yeast. Key words: Malassezia pachydermatis, enzymes, Api-Zym®, enzymatic profile, dogs.

Prevalence of Malassezia pachydermatis in dogs with suspected Malassezia dermatitis or otitis in Slovakia

The aim of the study was to evaluate the prevalence of yeastMalassezia pachydermatisin dogs from Slovakia in relation to different predisposition factors (sex, age, body localisation, hair type, and season). Samples of ear swabs (58) and dermal swabs (131) from 147 dogs with clinical symptoms of suspected yeast dermatitis and/or otitis, were examined between June 2005 to June 2007. Relatively higher prevalence ofM. pachydermatiswas found in samples taken from males (45.2%) than in females (35.2%), and in geriatric dogs (63.6%) than in young (42.5%) or adult (38.5%) dogs.Malassezia pachydermatiswas isolated more often from ear swabs (44.8%) than from skin swabs (38.9%). Prevalence ofM. pachydermatiswas significantly higher (p&lt; 0.05) in samples from the trunk area (60.3%) than in samples from other skin areas. Significantly higher prevalence was found in samples from long-haired (51.5%) and short-haired (45.9%) dogs compared to smooth-haired (21.4%) dogs. The prevalence was relatively higher in the samples taken in autumn (52.6%), than the other seasons: spring (36.1%), summer (27.3%), winter (45.7%); those differences were not significant.Malassezia pachydermatisis one of the most frequent yeasts isolated in dogs. Knowledge of factors predisposing to development of infection is valuable attribute of the correct diagnostic approach and case management.

Malassezia and seborrhoeic dermatitis

During the last 20 years, the yeast Malassezia pachydermatis has been acknowledged as a relatively common cause of dermatitis in dogs. The organism occurs naturally on the skin, mucosal surfaces and in the ear canals of dogs, but it can proliferate in response to abnormalities, for example, associated with allergic diseases, keratinisation defects and hormone disturbances.

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.

Antifungal effect of Australian tea tree oil on Malassezia pachydermatis isolated from canines suffering from cutaneous skin disease.

The lipophilic yeast Malassezia pachydermatis is part of the normal skin flora of most warm-blooded organisms. In a number of surveys it could be demonstrated that this yeast species might be involved in different skin diseases like seborrhoeic dermatitis, especially in dogs and cats. In order to look for an alternative therapeutic agent to the commonly used antimycotic and antiseptic synthetic substances the in vitro activity of Australian tea tree oil, the essential oil of Melaleuca alternifolia, against several strains of Malassezia pachydermatis was examined. All tested strains showed remarkably high susceptibility to tea tree oil. With these results the excellent antibacterial activity of tea tree oil is extended to a new group of fungal pathogens colonizing mainly mammals' skin. During the last ten years there was an increasing popularity of tea tree oil containing human health care products. The presented data open up new horizons for this essential oil as a promising alternative agent for topical use in veterinary medicine as well.

Type-1 hypersensitivity reactions to Malassezia pachydermatis extracts in atopic dogs

SUMMARY Objective To investigate the potential allergenic role of the yeast Malassezia pachydermatis in dogs with clinical diagnosis of atopic dermatitis. Animals 5 clinically normal nonatopic dogs, 10 atopic dogs with cytologic evidence of Malassezia dermatitis, and 12 atopic dogs without cytologic evidence of Malassezia dermatitis. Procedure A crude yeast extract was produced by disrupting the cell wall of M pachydermatis. The crude extract and 8 of its fractions, which were generated by fractionation in a high-performance liquid chromatography column, were injected along with 46 commercial allergens for intradermal allergy testing of normal and atopic sample populations. Significant difference between atopic populations was evaluated, using a threshold concentration of crude yeast extract that failed to induce wheal-and-flare responses in normal nonatopic dogs. Results Atopic dogs with cytologic evidence of Malassezia dermatitis had significantly greater wheal-and-flare reactions to intradermal injection of crude extract of M pachydermatis than did atopic dogs without cytologic evidence of Malassezia dermatitis. Conclusions It is concluded that M pachydermatis is capable of promoting type-1 hypersensitivity reactions in dogs with an atopic dermatitis phenotype. Clinical Relevance Currently, Malassezia dermatitis is principally managed by use of antifungal chemotherapy. Because the yeast appears to be a contributing allergen in dogs with atopic dermatitis, hyposensitization with M pachydermatis extracts may offer a future alternative to extended or repeated episodic administration of antifungals for extended control of recurrent infections. (Am J Vet Res 1998;59:836–841)