Malassezia Pachydermatis Strains 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 Antimycotic Activity and Structural Damage against Canine Malassezia pachydermatis Strains Caused by Mexican Stingless Bee Propolis.

This work describes the antimycotic activity of propolis from the stingless bees Scaptotrigona mexicana and Tetragonisca angustula, collected from two Mexican regions (Veracruz and Chiapas, respectively), against three clinical isolates and the reference strain ATCC 14522 of Malassezia pachydermatis, the causative agent of canine otitis. The chemical components of the ethanolic extracts of propolis were determined by gas chromatography coupled with mass spectrometry (GC-MS), and sesquiterpenes were the predominant compounds. The antimycotic activity was evaluated by plate microdilution. The induced changes in the yeasts were evaluated by fluorescence microscopy and staining with calcofluor white and propidium iodide. The minimum inhibitory concentration (MIC) was 7.11 mg/mL, and the minimum fungicidal concentration was 21.33 mg/mL for both extracts. The EPPs of Scaptotrigona mexicana and Tetragonisca angustula caused substantial damage to yeast morphology, where the propidium iodide staining of the yeasts treated with both EEPs revealed the penetration of this marker, which indicates the destruction of the cell wall and plasma membrane of the fungi. This result suggests that these types of propolis could be used as alternative treatments for canine external otitis. To the best of our knowledge, this seems to be the first scientific report that has demonstrated structural damage in Malassezia pachydermatis by Mexican stingless bee propolis.

Azole and terbinafine susceptibility testing of Malassezia pachydermatis in Japan.

Canine Malassezia dermatitis and otitis externa are generally treated by antifungal drugs. However, multi-drug-resistant strains of Malassezia pachydermatis have been reported worldwide. Given the presence of these multi-drug-resistant strains, it is unclear which antifungal agent is the most effective for canine Malassezia dermatitis and canine otitis. In this study, we attempted to determine the most effective drug against azole-resistant M. pachydermatis. Susceptibility to azoles and terbinafine (TBF) was assessed using a modified broth microdilution method. In all tested isolates, the minimum inhibitory concentration at 90% of organisms (MIC90) were 16 to >32 μg/mL for the azoles, and 2 μg/mL for TBF. All of the strains that showed low susceptibility to both itraconazole and miconazole were also relatively susceptible to TBF.

In Vitro Antimicrobial Activity of Thymus vulgaris, Origanum vulgare, Satureja montana and Their Mixture against Clinical Isolates Responsible for Canine Otitis Externa.

Otitis externa is a frequent inflammation among dogs, mainly caused by bacteria and yeasts that are often resistant to conventional drugs. The aim of the present study was to evaluate the in vitro antibacterial and antifungal activities of commercial essential oils (EOs) from Origanum vulgare, Satureja montana, and Thymus vulgaris, as well as a mixture of these three components, against 47 clinical bacterial strains (Staphylococcus sp., Streptococcus sp., Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens) and 5 Malassezia pachydermatis strains, previously cultured from the ears of dogs affected by otitis externa. The tested Gram-positive bacteria were sensible to the analysed EOs with MICs ranging from 1.25% (v/v) to <0.0195% (v/v); Gram-negative isolates, mainly P. aeruginosa, were less sensitive with MICs from >10% (v/v) to 0.039% (v/v). M. pachydermatis isolates were sensitive to all EOs with MICs from 4.25% (v/v) to 2% (v/v). However, the mixture was active against all bacterial (except one P. aeruginosa strain) and fungal tested isolates. The three EOs and their mixture seem to be an interesting alternative for treating canine otitis externa when conventional antimicrobials are not active.

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

ABSTRACTMalassezia 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.

Oxythiamine improves antifungal activity of ketoconazole evaluated in canine Malassezia pachydermatis strains.

Malassezia pachydermatis is an opportunistic yeast involved in skin and ear canal infections of dogs and cats. Reports suggest that strains of M.pachydermatis resistant to commonly used antifungal agents may be emerging. Therefore, new therapeutic strategies should be explored. The synergistic effect of oxythiamine (OT) and ketoconazole (KTC) was analysed using a reference strain and field isolates (n=66) of M.pachydermatis. Hydrogel formulations containing these components also were evaluated. The minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) of OT, KTC and their mixtures were determined by a broth macrodilution method. The antifungal effects of hydrogel formulations were determined by a plate diffusion method. The MIC and MFC values of OT were in the range 0.08×103 to 10×103 mg/L. All M.pachydermatis strains showed higher susceptibility to KTC (MICs and MFCs in the range 0.04-0.32mg/L). Formulations that combined OT and KTC showed a synergistic effect for all tested isolates (n=66). Hydrogels that contained OT at a concentration of 10×103 or 20×103 mg/L and KTC at the concentration of 0.1×103 mg/L showed a stronger effect than a commercially available product with KTC alone (20×103 mg/L). Synergy of these drugs may allow for successful topical treatment which utilizes lower doses of KTC without changing its therapeutic effectiveness. Hydrogel formulations proved to be attractive drug carriers for potential topical use.

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.

Malassezia pachydermatis up-regulates AhR related CYP1A1 gene and epidermal barrier markers in human keratinocytes.

Cytochrome P450 CYP1A1 and CYP1B1 enzymes are regulated by the aryl hydrocarbon receptor (AhR), a transcription factor activated by a variety of ligands among which Malassezia metabolites. In this study, we analyzed the modulation of CYP1A1, CYP1B1, and AhR in human keratinocytes infected with different strains of Malassezia pachydermatis, as well as the upregulation of some genes involved in the epidermal homeostasis. We demonstrated that all the strains induced AhR activation and its nuclear translocation in HaCaT cells infected for 24h, compared to untreated cells. The expression of CYP1A1 and CYP1B1, prototypical markers of the AhR signaling pathway, were upregulated with the level of CYP1A1 mRNA approximately 100-fold greater than that for CYP1B1. Filaggrin, involucrin, and TGaseI, proteins involved in epidermal differentiation, were all modulated by Malassezia pachydermatis strains, with the strongest induction observed for filaggrin. By contrast, quinone oxidoreductase 1 (NQO1), which is part of the antioxidant defense system involved in detoxification, was not modulated in our experimental model. In conclusions, our findings suggest that Malassezia pachydermatis infection of human keratinocytes induces activation of the AhR, and increases the expression of its responsive genes and markers of epidermal differentiation, paving the way for occurrence/exacerbation of pathological skin conditions.

Preservation of Malassezia pachydermatis using skimmed milk as cryoprotective agent

This study evaluated the effect of skimmed milk as a cryoprotectant for the maintenance and long-term preservation of 70 Malassezia pachydermatis strains. An initial suspension of each strain was prepared in sterile distilled water with skimmed milk. The most effective method was cryopreservation that ensured 100% viability for 2years.

Biofilm formation in Malassezia pachydermatis strains isolated from dogs decreases susceptibility to ketoconazole and itraconazole.

Malassezia pachydermatis is a commonly isolated yeast in veterinary dermatology that can produce biofilms in vitro and in vivo, lowering its susceptibility to antimicrobial drugs. The aim of this study was to determine and compare the in vitro susceptibility of planktonic cells and biofilms of M. pachydermatis isolates to ketoconazole and itraconazole. The presence of biofilm formation was confirmed by crystal violet staining and absorbance measurement at 595 nm wavelength, and by a scanning electron microscopy method. Cell viability was determined by the Celltiter 96 Aqueous One solution assay containing a water-soluble tetrazolium compound (MTS) with absorbance measurement at 490 nm. Planktonic cell minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) of ketoconazole and itraconazole were very low: MIC90 and MFC90 were 0.032 and 0.125 μg/ml for ketoconazole, while 0.063 and 0.25 μg/ml for itraconazole, respectively. Also, the half maximal effective concentrations (EC50) of itraconazole were higher for planktonic cells and biofilms compared to ketoconazole. The EC50 values of ketoconazole were 18-169 times higher and those of itraconazole 13-124 times higher for biofilms than for planktonic cells. Biofilm EC50 levels exceeded MICs 103-2060 times for ketoconazole and 84-1400 times for itraconazole. No significant difference was found between these values of the two substances. In conclusion, biofilms of all examined M. pachydermatis strains were much less susceptible to ketoconazole and itraconazole than their planktonic forms.

Comparative Analysis of Protein Profiles of Malassezia Pachydermatis Strains Isolated from Healthy Dogs and Dogs with Otitis Externa

Abstract The study included 40 strains of Malassezia pachydermatis obtained in pure culture from external ear canal of dogs and the reference strain. Protein fractions were received by mechanical disruption of the fungal cells. Denaturing protein electrophoresis was performed according to Laemmli method. More than 90% of the all tested strains were characterised by the presence of the following protein fractions: 83.0; 77.0; 59.0; 55.0; 48.0; 38.0; 35.0; 28.0, and 27.0 kDa. In other regions of electrophoregrams, a relatively high differentiation was observed. The strains isolated from animals with otitis externa were characterised by the presence of the following protein fractions: 75.0; 61.0; 52.0; 36.0; 19.5; 16.0; 13.8, and 13.0 kDa. These fractions were absent in the commensal strains. The use of statistical analysis confirmed the obtained results and allowed to generate dendrogram grouping potentially pathogenic and commensal strains on two different branches. Such results may indicate significant differences between protein profiles of strains of M. pachydermatis isolated from healthy and diseased animals.

Evaluation of In Vitro Synergistic Interaction of Miconazole and Polymyxin B Against Clinical Strains of Malassezia pachydermatis

Malassezia pachydermatis is a yeast that is frequently involved as a secondary/perpetuating factor in canine otitis externa, along with numerous species of bacteria. As a result, otitis is generally treated with topical therapies using combinations of antifungal, antimicrobial and anti-inflammatory agents. The evaluation of the combined effect of drugs included in commercially available products is therefore important. In vitro synergy of polymyxin B and miconazole has been demonstrated against one strain type of M. pachydermatis. This study was aimed at investigating the in vitro interactions of these two agents against five clinical strains of this yeast. A chequerboard broth microdilution method was employed. We observed a synergistic action against all strains tested (fractional inhibitory concentration index comprised between 0.2 and 0.4). Our results indicate a strong therapeutic value for the association of these antimicrobial agents in the treatment of canine otitis externa caused by M. pachydermatis.

Evaluation of Antifungal Effects of a Traditional Medicine Containing 17 Components on Trichophyton verrucosum and Malassezia pachydermatis by Microdilution

The minimum inhibitory concentration(MIC)of a traditional medicine containing 17 components against 9 strains of Trichophyton verrucosum and 13 strains of Malassezia pachydermatis was determined using a method recommended by the Clinical and Laboratory Standards Institute(CLSI). We also measured the MIC of each of the 17 components using the same method, and identified the main antifungal components.In order to evaluate MIC as a parameter of the antifungal effects using the microdilution method, we prepared 10% working solutions from 10% (w/v)medicines. The geometric mean MIC of the medicinal extract against T. verrucosum was 2.51%, and that against M. pachydermatis was 2.25%. The components that exhibited antifungal effects were Rheum palmatum, Glycyrrhiza uralensis, Magnolia obovata, Phellodendron amurense, and Geranium thunbergii.

Subculture on potato dextrose agar as a complement to the broth microdilution assay for Malassezia pachydermatis

The main aim of this study was to verify the efficacy of subculture on potato dextrose agar (PDA) as a complement to the in vitro susceptibility test for Malassezia pachydermatis strains by a broth microdilution method, as well as to determine the MIC and MFC of azole derivatives, amphotericin B and caspofungin. The microdilution assay was performed in 96-well plates using a modified RPMI 1640 medium. The M. pachydermatis strains were resistant to caspofungin. All strains ( n = 50) had shown MIC values of < 0.03, < 0.03, 2.0, 4.0 and 4.0 μg/ml for itraconazole, ketoconazole, voriconazole, fluconazole and amphotericin B, respectively. Thus, the subculture on PDA improved the analysis of the in vitro antifungal susceptibility of M. pachydermatis.

Molecular characterization of Malassezia isolates from dogs using three distinct genetic markers in nuclear DNA

Little precise information is available on the systematics, genetics, ecology and epidemiology of yeasts of the genus Malassezia from different animal species. In the present study, one hundred and four isolates of Malassezia (lipid dependent or non-lipid dependent) from dogs were characterized by their chitin synthase 2 gene (CHS2), and the large subunit (LSU) and the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA sequences, and compared genetically with well-defined reference strains of Malassezia pachydermatis and heterologous species, including Malassezia furfur and Candida albicans. For each locus examined, three main sequence types (i.e. A, B and C) represented all of the 104 isolates, which were designated as genotypes A, B and C, respectively. A fourth, minor sequence type was also defined for the ITS-1. The nucleotide differences among genotypes was consistent with the magnitudes of intraspecific variability reported in previous studies. The genetic analysis of the sequence data sets (for individual loci) showed that all Malassezia genotypes clustered (with moderate to strong support) with the reference sequences of M. pachydermatis to the exclusion of the outgroups M. furfur and C. albicans. The present study reveals that multiple genetic variants of M. pachydermatis occur on dogs. The multilocus approach employed herein provides a foundation for future investigations of M. pachydermatis from other animals and humans, and their ecology and epidemiology.

THE ANTIMYCOTIC EFFECT OF SOME BEE PRODUCTS AND VEGETAL EXTRACTS ON GENUS MALASSEZIA FUNGI

For both antimicrobial and antimycotic therapy, th e researches of the latest years are mentioning an increased resistance of the bacterial and fungal strains to these substances. A natural alternative to classical therapy was aimed, so 14 Malassezia pachydermatis strains isolated from dogs suffering with dermatiti s and otitis Cluj-Napoca were tested regarding the capacity of some bee products to inhibit their in vitro development. From the products tested, the best res ults were registered for propolis tincture, polioel 3 and royal jelly. The media of the diameter inhibit area mm , was of 22,21 mm for propolis tincture, 14,92 mm f or polioel 3 and respectively of 8,78 mm for royal jelly.

Phenotypic characterization and in vitro antifungal sensitivity of Candida spp. and Malassezia pachydermatis strains from dogs

Yeasts of the genera Candida and Malassezia can be found as commensal microorganisms in animals. The main species of importance in veterinary medicine are Malassezia pachydermatis and Candida albicans. The objectives of this study were to conduct a phenotypic characterization and to evaluate the in vitro antifungal sensitivity of strains of C. albicans ( n = 5), C. tropicalis ( n = 3) and M. pachydermatis ( n = 32) isolated from dogs. The phenotyping was based on macro and micromorphological features as well as biochemical analysis. The techniques of microdilution in broth and dilution in agar were used to evaluate the in vitro sensitivity of Candida spp. and M. pachydermatis, respectively. The tested drugs were ketoconazole (KTC), itraconazole (ITC), fluconazole (FLC) and amphotericin B (AMB). The morphological analysis of the strains of Candida spp. and M. pachydermatis did not show any noteworthy alterations when compared to standard strains. On the other hand, in the biochemical tests, 34.4% of the strains of M. pachydermatis were negative for the urease test. Four strains of C. albicans were resistant to FLC with a minimum inhibitory concentration (MIC) >64 μg/mL and all were resistant to KTC and ITC (MIC > 16 μg/mL). The MIC for two strains of C. tropicalis were >16 μg/mL for KTC and ITC, and >64 μg/mL for FLC. It is worth highlighting that all of the strains tested were sensitive to AMB with the MIC varying from 0.25–1.0 μg/mL. All strains of M. pachydermatis were sensitive to ITC with a minimum fungistatic concentration (MFC) ⩽0.0075 μg/mL. The MIC for 29 strains was the same (MFC ⩽ 0.0075 μg/mL) for KTC. The MFCs for FLC varied from ⩽1 to ⩽16 μg/mL, and for AMB, the MFC interval was 0.125–8 μg/mL. There were no alterations in the classic phenotypic features of the strains of Candida spp. and M. pachydermatis isolated from dogs but, unlike M. pachydermatis, Candida spp. were much more resistant to azole antifungal agents.

Effects of .BETA.-Thujaplicin on Anti-Malassezia pachydermatis Remedy for Canine Otitis Externa

The antifungal activity of beta-thujaplicin was evaluated against 51 Malassezia pachydermatis strains isolated from canine ear canals with or without otitis externa. For comparison, sensitivity tests were performed on M. pachydermatis isolates for nystatin, ketoconazole, and terbinafine HCl, all clinically available antifungal agents. The minimal inhibition concentrations over 50% of the tested isolates (MIC50) were 3.13 microg/ml for beta-thujaplicin and nystatin, 0.016 microg/ml for ketoconazole, and 1.56 microg/ml for terbinafine HCl. The antifungal effect for M. pachydermatis of beta-thujaplicin compared favorably with commercial antifungal agents. None of the 51 M. pachydermatis isolates showed resistance against any of the tested antibiotics investigated in this study. Ten representative isolates of M. pachydermatis were subcultured for 30 generations at concentrations close to the MIC levels of beta-thujaplicin, nystatin, ketoconazole, and terbinafine HCl, and examined to determine whether they had acquired resistance to each drug. As a result, M. pachydermatis was found to achieve resistance more easily for ketoconazole and terbinafine HCl than for beta-thujaplicin or nystatin. The MIC50 of beta-thujaplicin did not change during the course of subculture, and it is thought that the potential development of a resistant strain is low, even with continuous infusion for otitis externa therapy. beta-Thujaplicin is an inexpensive and safe treatment with anti-inflammatory and deodorant effects that can be recommended as an effective remedy for canine otitis externa.

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.

Proteinase, phospholipase, hyaluronidase and chondroitin-sulphatase production by Malassezia pachydermatis.

The production of four functional enzyme categories was investigated in 30 strains of Malassezia pachydermatis isolated from dogs with otitis or dermatitis. The most appropriate reading intervals for these assays were determined with the aid of statistical comparisons. All strains produced proteinase and chondroitin-sulphatase; hyaluronidase and phospholipase were produced by all skin isolates (15/15) and 14 out of 15 ear canal isolates. Strains from ear canals did not differ significantly as a group from skin strains in quantitative production of any of the four enzymes; production of proteinase and chondroitin-sulphatase in particular was markedly uniform.