Protein profiles of Malassezia pachydermatis isolated from dogs

Chronic dermatophytosis was observed in 2276 (10.02%) of 22 692 patients with dermatophytosis during a period of 5.5 years. Males were affected at least 3 times more frequently than females. The age group most commonly affected was between 20 and 40 years of age. Females were affected more between the ages of 30 to 40 years. Tinea cruris and tinea corporis were the most common clinical types and tinea pedis was the least common type observed. The most frequent isolate was Trichophyton rubrum followed by T. mentagrophytes and T. violaceum. Ichthyosis vulgaris was the most common cutaneous association whereas atopy and diabetes mellitus were the most common systemic associations. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterization of markedly lipid-dependent Malassezia pachydermatis isolates from healthy dogs

When 244 Malassezia colonies which had been isolated from a colony of Beagle dogs using modified Dixon's agar were sub-cultured on Sabouraud's dextrose agar to determine their lipid dependence, 30 showed poor growth resembling M. furfur , whereas the remainder were typical of M. pachydermatis . Eight of the 10 poor growing isolates selected for further study formed colonies typical of M. pachydermatis after five passages on Sabouraud's dextrose agar at 4 d intervals and two continued to show poor growth. Nine isolates had enzyme profiles identical to those of typical M. pachydermatis isolates, and one resembled M. furfur . However, seven of the poor growing isolates which were karyotyped had patterns typical of M. pachydermatis. Poor growing isolates and their non-lipid-dependent 'revertants'had identical restriction fragment length polymorphism patterns and poly(GT) hybridization profiles. These observations show that some M. pachydermatis isolates grow poorly when sub-cultured onto Sabouraud's dextrose agar and may be incorrectly identified as M. furfur if further studies are not performed.     

Gentamycin inhibits the growth of Malassezia pachydermatis in culture

BackgroundMalassezia pachydermatis is a yeast of importance in both veterinary and human medicine.AimsTo know if M. pachydermatis grow on micological media with high concentrations of gentamycin.MethodsTwenty M. pachydermatis strains were streaked on Sabouraud Dextrose Agar plates with different concentrations of gentamycin.ResultsAll isolates were inhibited when high concentrations of gentamycin were added.ConclusionsThe use of plates with high concentrations of gentamycin can lead to some important misdiagnoses: firstly, false-negative cultures, and secondly, an erroneous classification of M. pachydermatis as a lipid-dependent species. Morever, all of this could be useful in two therapeutic fields: i) in animals, topical gentamycin could be an efficacious treatment for a disease such as external otitis in dogs; ii) in humans, we hypothesize that gentamycin could be regarded as a possible therapy (“antibiotic-lock”) for catheter-associated Malassezia spp. infections.     

Isolation, characterization and molecular cloning of a lipolytic enzyme secreted from Malassezia pachydermatis

Lipophilic Malassezia species may induce catheter-associated sepsis in premature neonates and immunocompromised patients receiving parenteral lipid emulsions. To assess the participation of lipolytic enzymes in the pathogenesis of this yeast, we cloned a gene encoding the enzyme. A lipolytic enzyme in the culture supernatant of Malassezia pachydermatis was purified 210-fold to homogeneity. The enzyme showed high esterase activity toward p-nitrophenyl octanoate. The cDNA encoding the enzyme was cloned using a degenerate oligonucleotide primer constructed from the N-terminal amino acid sequence. The cDNA consisted of 1582 bp, including an open reading frame encoding 470 amino acids. The first 19 amino acids and the following 13 amino-acid sequence were predicted to be the signal peptides for secretion and prosequence, respectively. The predicted molecular mass of the 438-amino acid mature protein was 48 kDa. Analysis of the deduced amino acid sequence revealed that it contains the consensus motif (Gly-X-Ser-X-Gly), which is conserved among lipolytic enzymes. Homology investigations showed that the enzyme has similarities principally with 11 lipases produced by Candida albicans (29-34% identity) and some other yeast lipases.     

Biotyping of Malassezia pachydermatis strains using the killer system

The killer phenomenon has been used as epidemiological marker for Candida albicans, where hundreds of biotypes can be obtained. The objective of this study is to observe the behaviour of 30 strains of Malassezia pachydermatis isolated from dogs with otitis (15) or dermatitis (15) against 9 killer yeasts, which, when grouped in triplets produced a 3 digit code (biotype). The growth inhibition of the 30 strains of M. pachydermatis due to the effect of the killer yeasts used permitted the determination of the following biotypes: 888 (33.3%), 212 (26.7%), 111 (16.7%), 312 (6.7%), 512 (6.7%), 242 (3.3%), 311 (3.3%) and 411 (3.3%). Biotypes 888, 212 and 111 occurred most frequently in both ear canal and skin samples.     

Genetic variants of Malassezia pachydermatis from canine skin: body distribution and phospholipase activity

Malassezia pachydermatis isolates (n=185) from skin sites from dogs (n=30) were characterized genetically and biochemically following in vitro culture. Two regions in the chitin synthase-2 gene (chs-2) and the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA were sequenced, and the phospholipase activity of each isolate was assessed. Three chs-2 (i.e. Ac, Bc and Cc) and eight ITS-1 (i.e. AI1, AI2, AI3, AI4, BI1, CI1, CI2 and CI3) sequence types were defined for all 185 samples. The findings revealed that multiple M. pachydermatis genotypes/subgenotypes could be cultured from healthy dogs or from dogs with single or multiple, generalized skin lesions. Subgenotypes AI1 and BI1 were associated with all skin sites of dogs sampled, whereas subgenotype CI2 was mostly linked to a particular location. Isolates derived from skin lesions showed a significantly higher phospholipase activity compared with those from skin sites with no detectable lesions. Genotype B was mainly cultured from healthy skin; only four isolates (9.3%) had low phospholipase activity, whereas other genotypes/subgenotypes were predominantly associated with skin lesions and had a high phospholipase activity. The results of the present study suggest that the distribution pattern of particular genotypes or subgenotypes of M. pachydermatis on the skin of dogs relates to the affinity of the yeast to the host and to particular skin sites.     

Studies on the isolation,growth and maintenance of Malassezia pachydermatis

Results related to the isolation, cultivation, culture and maintenance of the opportunistic pathogen Malassezia pachydermatis are reported. A dextrose nutrient medium with 1.5% yeast extract turned out to be the most favourable medium for its development. It permitted identification in 24 hours and maintenance of isolates for three months without subculturing. Addition of Tween 80 (1%) significantly enhanced the isolation of this yeast from clinical materials.     

A survey of 120 isolates of Malassezia (Pityrosporum) pachydermatis

The morphological, cultural and biochemical characteristics of 120 isolates of Malassezia (Pityrosporum) pachydermatis, isolated from chronic otitis externa in the dog, are discussed.     

Malassezia Intra-Specific Diversity and Potentially New Species in the Skin Microbiota from Brazilian Healthy Subjects and Seborrheic Dermatitis Patients

Malassezia yeasts are part of the resident cutaneous microbiota, and are also associated with skin diseases such as seborrheic dermatitis (SD). The role these fungi play in skin diseases and why they are pathogenic for only some individuals remain unclear. This study aimed to characterize Malassezia microbiota from different body sites in healthy and SD subjects from Brazil. Scalp and forehead samples from healthy, mild SD and severe SD subjects were collected. Non-scalp lesions from severe SD patients were also sampled. 5.8S rDNA/ITS2 amplicons from Malassezia sp. were analyzed by RFLP and sequencing. Results indicate that Malassezia microbiota did not group according to health condition or body area. Phylogenetic analysis revealed that three groups of sequences did not cluster together with any formally described species, suggesting that they might belong to potential new species. One of them was found in high proportions in scalp samples. A large variety of Malassezia subtypes were detected, indicating intra-specific diversity. Higher M. globosa proportions were found in non-scalp lesions from severe SD subjects compared with other areas, suggesting closer association of this species with SD lesions from areas other than scalp. Our results show the first panorama of Malassezia microbiota in Brazilian subjects using molecular techniques and provide new perspectives for further studies to elucidate the association between Malassezia microbiota and skin diseases.     

In Vitro Antifungal Susceptibility of Malassezia pachydermatis Strains Isolated from Dogs with Chronic and Acute Otitis Externa

Malassezia pachydermatis is a yeast that is frequently involved as a secondary/perpetuating factor in canine otitis externa. Topical therapies with different antifungal agents, mainly azole compounds, are generally successful in controlling the yeast overgrowth, but treatment failure and rapid recurrences are common. This study compared the in vitro antifungal susceptibility of M. pachydermatis isolates obtained from chronic and acute cases of otitis externa. The aim was to assess the possible onset of resistance mechanisms in isolates involved in long-lasting episodes with poor response to treatment. We evaluated the in vitro susceptibility to miconazole (MCZ) and clotrimazole (CTZ) of 42 isolates of M. pachydermatis obtained from dogs with chronic (group A, n = 25) and acute otitis (group B, n = 17), using a modified CLSI M27-A3 microdilution method. All isolates were inhibited by the antifungal agents employed, but Malassezia isolates from group A were significantly associated with higher minimum inhibitory concentration (MIC) values for both agents (Median MIC values: MCZ group A 2 µg/ml, group B 1 µg/ml; CTZ group A 8 µg/ml, group B 4 µg/ml). These findings prove that these isolates had a reduced in vitro susceptibility to the antifungal agents tested. However, it is unlikely that this could have any influence on the outcome of a topical treatment. Indeed, marketed products include concentrations of the tested agents that largely exceed even the highest MICs found in this study (in most cases at least 1,000 × the MIC, or greater). In conclusion, this study suggests that isolates of M. pachydermatis involved in chronic cases of canine external otitis and exposed to repeated antifungal treatments are unlikely to develop mechanisms of resistance of clinical relevance.     

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.     

Microbiota Communities of Healthy and Bacterial Pustule Diseased Soybean

Soybean is an important source of protein and for a wide range of agricultural, food, and industrial applications. Soybean is being affected by Xanthomonas citri pv. glycines, a causal pathogen of bacterial pustule disease, result in a reduction in yield and quality. Diverse microbial communities of plants are involved in various plant stresses is known. Therefore, we designed to investigate the microbial community differentiation depending on the infection of X. citri pv. glycines. The microbial community’s abundance, diversity, and similarity showed a difference between infected and non-infected soybean. Microbiota community analysis, excluding X. citri pv. glycines, revealed that Pseudomonas spp. would increase the population of the infected soybean. Results of DESeq analyses suggested that energy metabolism, secondary metabolite, and TCA cycle metabolism were actively diverse in the non-infected soybeans. Additionally, Streptomyces bacillaris S8, an endophyte microbiota member, was nominated as a key microbe in the healthy soybeans. Genome analysis of S. bacillaris S8 presented that salinomycin may be the critical antibacterial metabolite. Our findings on the composition of soybean microbiota communities and the key strain information will contribute to developing biological control strategies against X. citri pv. glycines.     

Bacterial Community Associated with Healthy and Diseased Reef Coral Mussismilia hispida from Eastern Brazil

In order to characterize the bacterial community diversity associated to mucus of the coral Mussismilia hispida, four 16S rDNA libraries were constructed and 400 clones from each library were analyzed from two healthy colonies, one diseased colony and the surrounding water. Nine bacterial phyla were identified in healthy M. hispida, with a dominance of Proteobacteria, Actinobacteria, Acidobacteria, Lentisphaerae, and Nitrospira. The most commonly found species were related to the genera Azospirillum, Hirschia, Fabibacter, Blastochloris, Stella, Vibrio, Flavobacterium, Ochrobactrum, Terasakiella, Alkalibacter, Staphylococcus, Azospirillum, Propionibacterium, Arcobacter, and Paenibacillus. In contrast, diseased M. hispida had a predominance of one single species of Bacteroidetes, corresponding to more than 70% of the sequences. Rarefaction curves using evolutionary distance of 1% showed a greater decrease in bacterial diversity in the diseased M. hispida, with a reduction of almost 85% in OTUs in comparison to healthy colonies. ∫-Libshuff analyses show that significant p values obtained were <0.0001, demonstrating that the four libraries are significantly different. Furthermore, the sympatric corals M. hispida and Mussismilia braziliensis appear to have different bacterial community compositions according to Principal Component Analysis and Lineage-specific Analysis. Moreover, lineages that contribute to those differences were identified as α-Proteobacteria, Bacteroidetes, and Firmicutes. The results obtained in this study suggest host–microbe co-evolution in Mussismilia, and it was the first study on the diversity of the microbiota of the endemic and endangered of extinction Brazilian coral M. hispida from Abrolhos bank.     

Preliminary studies on the mechanism of infection and characterization of Malassezia pachydermatis in association with canine otitis externa

A study on the mechanism of infection and the characterization of Malassezia pachydermatis in connection with canine otitis externa was conducted.The ability of this yeast to grow uninhibited in intimate contact with the diversity of other microbial isolants of the canine aural canal was demonstrated. Canine cerumen seemed to promote the growth of this yeast.Although the source of infection is still obscure, M. pachydermatis managed to survive in soil and dust at different temperatures for four weeks.Among the commonly used diagnostic media, Sabouraud's dextrose agar was shown to be the most supportive for the growth of this yeast.The study was presented in parts on the 65th and 66th Conference of Research Worker in Animal disease CRWAD in Chicago, Illinois, 1984 and 1985.     

Comparative study of the activity and kinetic properties of malate dehydrogenase and pyruvate decarboxylase from Candida albicans, Malassezia pachydermatis, and Saccharomyces cerevisiae

Candida albicans and Malassezia pachydermatis cause human and animal infections of the skin and internal organs. We compare the properties of two enzymes, pyruvate decarboxylase (PDC) and malate dehydrogenase (MDH), from these species and from Saccharomyces cerevisiae cultivated under aerobic and anaerobic conditions to find differences between the enzymes that adapt pathogens for virulence and help us in searching for new antifungal agents. Malassezia pachydermatis did not show any growth under anaerobic conditions, as opposed to C. albicans and S. cerevisiae. Under aerobic conditions, C. albicans showed the highest growth rate. Malassezia pachydermatis, contrary to the others, did not show any PDC activity, simultaneously showing the highest MDH activity under aerobic conditions and a Km value for oxaloacetate lower than S. cerevisiae. Candida albicans and S. cerevisiae showed a strong decrease in MDH activity under anaerobic conditions. Candida albicans shows four different isoforms of MDH, while M. pachydermatis and S. cerevisiae are characterized by two and three isoforms. Candida albicans shows about a twofold lower activity of PDC but, simultaneously, almost a threefold lower Km value for pyruvate in comparison with S. cerevisiae. The PDC apoform share under aerobic conditions in C. albicans was 47%, while in S. cerevisiae was only 26%; under anaerobic conditions, the PDC apoform decreased to 12% and 8%, respectively. The properties of enzymes from C. albicans show its high metabolic flexibility (contrary to M. pachydermatis) and cause easy switching between fermentative and oxidative metabolism. This feature allows C. albicans to cause both surface and deep infections. We take into consideration the use of thiamin antimetabolites as antifungal factors that can affect both oxidative and fermentative metabolism.     

Isolation and Characterization of Mycoplasma and Acholeplasma from Apparently Healthy and Diseased (Infectious Sinusitis) Turkeys

Investigation of 136 turkeys (24 manifesting infra-orbital sinusitis, 112 apparently healthy) resulted in isolation of 79 strains of Mycoplasma and 4 of Acholeplasma. By the disc growth inhibition test with 16 reference antisera of avian serogroups, 55 strains were identified serologically and 28 remained unidentified. Thirteen strains of Mycoplasma gallisepticum, 1 of M. meleagridis, and 2 of Acholeplasma laidlawii were isolated from turkey sinusitis whereas serogroups C (2), D (19), F (8), M. meleagridis (4), M. anatis (4), A. laidlawii (2), and 28 unidentified strains were isolated from apparently healthy turkeys. Three patterns were recognized on the basis of glucose, maltose, and sucrose, fermentation. The most frequent, pattern I, included 13 M. gallisepticum strains whereas 5 M. meleagridis strains belonged to fermentation pattern III. Isolates were also studied for reduction of tetrazolium, methylene blue, potassium tellurite, resistance to methylene blue and sodium taurocholate, and production of arginine deiminase and “film and spots.” Inoculation of selected isolates into developing chick embryos revealed that 2 A. laidlawii strains were nonpathogenic and 13 M. gallisepticum, 1 serogroup D and 2 serogroup F strains were pathogenic, causing 50–100% mortality. In vitro antibiotic disc sensitivity tests indicated that rovamycin (solubilized spiramycin) may be recommended for turkey mycoplasmosis. Isolation of 2 A. laidlawii strains from turkey sinusitis and 4 M. anatis strains from apparently healthy turkeys appears interesting.