Mycobiome of the external ear canal of healthy cows.

Malassezia yeasts belong to the normal skin microbiota of a wide range of warm-blooded animals. However, their significance in cattle is still poorly understood. In the present study, the mycobiota of the external ear canal of 20 healthy dairy Holstein cows was assessed by cytology, culture, PCR, and next-generation sequencing. The presence of Malassezia was detected in 15 cows by cytology and PCR. The metagenomic analysis revealed that Ascomycota was the predominant phylum but M. pachydermatis the main species. The Malassezia phylotype 131 was detected in low abundance. Nor M. nana nor M. equina were detected in the samples.

The mycobiota of the external ear canal of healthy cows was assessed by cytology, culture, PCR, and NGS. The presence of Malassezia was detected by cytology and PCR. Ascomycota was the main phylum and M. pachydermatis the main species. The Malassezia phylotype 131 was also detected in the samples.

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.

ERG11 Gene Variability and Azole Susceptibility in Malassezia pachydermatis.

Malassezia pachydermatis is part of the normal skin microbiota of various animal species but under certain circumstances becomes an opportunistic pathogen producing otitis and dermatitis. Commonly these Malassezia diseases are effectively treated using azoles. However, some cases of treatment failure have been reported. Alterations in the ERG11 gene have been associated with in vitro azole resistance in M. pachydermatis. In the present study, in vitro antifungal susceptibility of 89 different strains of M. pachydermatis isolated from different animal species and health status was studied. The susceptibility to fluconazole (FLZ), itraconazole (ITZ), ketoconazole and amphotericin B was tested by a disk diffusion method and 17 strains were also subjected to an ITZ E-test. Mueller-Hinton supplemented with 2% glucose and methylene blue was used as culture medium in both susceptibility assays. Multilocus sequence typing was performed in 30 selected strains using D1D2, ITS, CHS2 and ß-tubulin genes. Also, ERG11 gene was sequenced. The four antifungals tested were highly effective against most of the strains. Only two strains showed no inhibition zone to antifungals and a strain showed an increased MIC to ITZ. The study of the ERG11 sequences revealed a high diversity of DNA sequences and a total of 23 amino acid substitutions, from which only two have been previously described. Also, three deleterious substitutions (A302T, G459D and G461D) previously associated with azole resistance in this yeast were recovered. A correlation between certain genotypes and ERG11 mutations was observed. Some of the ERG11 mutations recovered were correlated with a reduced susceptibility to azoles.

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.

Trichophyton erinacei: an emergent pathogen of pediatric dermatophytosis / Trichophyton erinacei: un patógeno emergente en las dermatofitosis en niños

BACKGROUND: Dermatophytoses in children are common pathologies worldwide caused mainly by Trichophyton rubrum. However, due to the globalization and the atypical pets that people nowadays own, some zoonotic species are also involved in these lesions. CASE REPORT: We present two cases of tinea faciei caused by the zoonotic mould Trichophyton erinacei in two children that owned a guinea pig and a hedgehog, respectively. Mycological diagnosis was performed inoculating skin scales on Sabouraud-glucose agar plates supplemented with chloramphenicol, with and without gentamicin, and on Sabouraud-glucose agar tubes, with and without cycloheximide. Microscopical examination in both cases and ITS region sequencing to confirm the identification (performed in one of them) were compatible with T. erinacei. Multiple treatments like corticosteroids and antibiotics were prescribed prior to the accurate diagnosis. Finally, both patients received topical and oral terbinafine, respectively, the lesions being resolved entirely. CONCLUSIONS: Zoonotic fungi must be considered in the diagnosis of skin lesions. An accurate medical record, with a guided anamnesis about possible risk factors and an ongoing and open dialogue between health professionals, are essential to improve both the management of these exotic and zoophilic dermatophytoses

ANTECEDENTES: Las dermatofitosis son patologías comunes en niños y son causadas principalmente por Trichophyton rubrum. Sin embargo, debido a la globalización y a la presencia cada vez más frecuente de animales exóticos como mascotas, algunas especies zoonóticas menos habituales pueden convertirse en agentes causales. CASO CLÍNICO: Nuestro objetivo es describir dos casos de Tinea faciei causados por Trichophyton erinacei en dos niños que poseían, respectivamente, una cobaya y un erizo como mascotas. Se tomó muestra de escamas cutáneas que fueron inoculadas en placas de agar Sabouraud-glucosa suplementado con cloranfenicol, con y sin gentamicina, y en tubos de agar Sabouraud-glucosa con y sin cicloheximida. El examen microscópico fue compatible con Trichophyton erinacei, cuya identificación pudo ser confirmada por secuenciación de la región ITS en uno de los casos. Antes del correcto diagnóstico los pacientes habían recibido múltiples tratamientos (corticosteroides, antibióticos). Finalmente, los dos pacientes recibieron terbinafina tópica y oral, respectivamente, lo que llevó a la resolución completa de las lesiones. CONCLUSIONES: Los hongos zoonóticos deben ser considerados en el diagnóstico diferencial de las lesiones cutáneas. Una historia clínica con anamnesis guiada sobre posibles factores de riesgo, junto con una comunicación multidisciplinar fluida, es indispensable para mejorar el manejo de estas dermatofitosis

Trichophyton erinacei: an emergent pathogen of pediatric dermatophytosis.

BACKGROUND: Dermatophytoses in children are common pathologies worldwide caused mainly by Trichophyton rubrum. However, due to the globalization and the atypical pets that people nowadays own, some zoonotic species are also involved in these lesions. CASE REPORT: We present two cases of tinea faciei caused by the zoonotic mould Trichophyton erinacei in two children that owned a guinea pig and a hedgehog, respectively. Mycological diagnosis was performed inoculating skin scales on Sabouraud-glucose agar plates supplemented with chloramphenicol, with and without gentamicin, and on Sabouraud-glucose agar tubes, with and without cycloheximide. Microscopical examination in both cases and ITS region sequencing to confirm the identification (performed in one of them) were compatible with T. erinacei. Multiple treatments like corticosteroids and antibiotics were prescribed prior to the accurate diagnosis. Finally, both patients received topical and oral terbinafine, respectively, the lesions being resolved entirely. CONCLUSIONS: Zoonotic fungi must be considered in the diagnosis of skin lesions. An accurate medical record, with a guided anamnesis about possible risk factors and an ongoing and open dialogue between health professionals, are essential to improve both the management of these exotic and zoophilic dermatophytoses.

Transcriptome analysis of non-ochratoxigenic Aspergillus carbonarius strains and interactions between some black aspergilli species.

Aspergillus carbonarius consistently produces large amounts of ochratoxin A (OTA), a mycotoxin with nephrotoxic effects on animals and humans. In the present study, we analyzed the transcriptional changes associated to OTA production in three atypical non-ochratoxigenic strains of A. carbonarius. In addition, in vitro interactions between ochratoxigenic strains of A. carbonarius and A. niger and non-ochratoxigenic strains of A. carbonarius and A. tubingensis were studied in order to evaluate their potential for controlling OTA production. RNA-seq analysis revealed that there are 696 differentially expressed genes identified in the three non-OTA-producing strains, including 280 up-regulated and 333 down-regulated genes. A functional and gene ontology enrichment analysis revealed that the processes related to metabolic and oxidation processes, associated with functions such as oxidoreductase and hydrolase activity were down regulated. All the genes related with OTA biosynthesis in A. carbonarius were the most down-regulated genes in non-ochratoxigenic strains. We also showed that these strains possess a deleterious mutation in the AcOTApks gene required for OTA biosynthesis. Moreover, one of these strains gave the best control of OTA production resulting in an OTA reduction of 98-100% in co-inoculation with an ochratoxigenic strain of A. niger and an OTA reduction of 79-89% with an ochratoxigenic strain of A. carbonarius. Results of this study provided novel insights into the knowledge of the OTA biosynthetic pathway in these non-ochratoxigenic wild strains, and showed the biocontrol potential of these strains.

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.

Genomic diversity in ochratoxigenic and non ochratoxigenic strains of Aspergillus carbonarius.

Ochratoxin A (OTA) is a mycotoxin with nephrotoxic effects on animals and humans. Aspergillus carbonarius is the main responsible for OTA contamination of grapes and derived products. We present the genome resequencing of four A. carbonarius strains, one OTA producer and three atypical and unique non-OTA producing strains. These strains were sequenced using Illumina technology and compared with a reference genome of this species. We performed some specific bioinformatics analyses in genes involved in OTA biosynthesis. Data obtained in this study revealed the high genomic diversity within A. carbonarius strains. Although some gaps of more than 1,000 bp were identified in non-ochratoxigenic strains, no large deletions in functional genes related with OTA production were found. Moreover, the expression of five genes of the putative OTA biosynthetic cluster was down regulated under OTA-inducing conditions in the non-ochratoxigenic strains. Knowledge of the regulatory mechanisms involved in OTA biosynthesis will provide a deeper understanding of these non-ochratoxigenic strains.

Phenotypic and genetic diversity of Malassezia furfur from domestic and zoo animals.

Malassezia furfur is traditionally associated to human skin, although more recent studies have been revealing its presence in a variety of animals. The aim of this study was to analyze phenotypically and genetically the diversity among strains isolated from animals of this species. We have examined 21 strains of M. furfur from domestic and wild animals held in captivity. On the one hand, their phenotypic characteristics were studied, by assessing its growth at different incubation temperatures, their catalase and ß-glucosidase activities and the Tween diffusion test on Sabouraud glucose agar (SGA), and on yeast nitrogen base agar (YNBA), a synthetic medium without lipids. On the other hand, the large subunit (LSU) and the internal transcribed spacer (ITS) of ribosomal RNA and the ß-tubulin gene were sequenced. Different sequence types were identified for each target gene, and fourteen genotypes were revealed. While several genotypes were obtained from the strains from domestic animals, the strains from zoo animals appeared to be genetically more stable. With ITS and ß-tubulin gene, M. furfur strains grouped in two clades. One clade included the strains from domestic animals and the other clade included the strains from zoo animals. The phenotypic tests also revealed a remarkable diversity within this species, which appeared to be more significant among strains from domestic animals. Moreover, the Tween diffusion test using YNBA was more useful to observe differences among strains, which could not be perceived using SGA.

Characterization and phylogenetic analysis of a Cunninghamella bertholletiae isolate from a bottlenose dolphin (Tursiops truncatus) / Tipificación y análisis filogenético de una cepa de Cunninghamella bertholletiae aislada de un delfín mular (Tursiops truncatus)

Background. Cunninghamella is a genus of the order Mucorales which includes saprophytic species, rarely causing mycoses. The most frequently reported in human mycoses is the thermophilic species Cunninghamella bertholletiae. However, this species does not appear to cause mucormycosis in animals, so there is scarce information about C. bertholletiae isolates from animals. Aims. In this paper we describe the phenotypic and genotypic characterization, and the phylogenetic analysis, of an isolate of C. bertholletiae involved in a central nervous system mucormycosis in a dolphin. Methods. The isolate studied in this publication was characterized using the current morphological and physiological identification system for Cunninghamella species. DNA sequencing and analysis of the D1/D2 regions of the 26S rRNA gene and the ITS-5.8S rRNA gene sequences were also performed. Results. Colonies were fast-growing, white at first, although they became tannish-gray, covering the whole plate after 7 days of incubation at 30 and 40°C. Limited growth was observed after 7 days at 45°C. The micromorphology showed characteristic erect sporangiophores. The identification of the isolate was confirmed by DNA sequencing of the D1/D2 regions of the 26S and the ITS-5.8S (ITS) rRNA gene sequencing. Conclusions. In the phylogenetic study, the isolate clustered in the same clade as C. bertholletiae neotype strain although some differences were observed in the ITS sequences. In the cetacean cases, the possible sources of infection are unclear. The reasons why this pathogen has been found only in cetaceans and not in other domestic or wild animals are at the moment unknown and need further study (AU)

Antecedentes. Cunninghamella es un género perteneciente al orden Mucorales, que incluye especies saprófitas que raramente causan micosis. De este género, Cunninghamella bertholletiae es la especie termófila más frecuentemente citada en micosis humanas. No obstante, no parece que sea una causa habitual de mucormicosis en animales, ya que es escasa la información sobre cepas de esta especie procedentes de estos. Objetivos. En esta publicación describimos la tipificación fenotípica, genotípica y el análisis filogenético de una cepa de C. bertholletiae causante de una mucormicosis del sistema nervioso central en un delfín. Métodos. La cepa fue tipificada mediante los criterios morfológicos y fisiológicos actualmente utilizados para la identificación de estas especies. También se llevó a cabo la secuenciación y el análisis de los fragmentos génicos D1/D2 26S e ITS-5.8S del ARN ribosómico. Resultados. Las colonias presentaron un crecimiento rápido; eran blanquecinas al principio y se volvieron de color marrón agrisado con el tiempo, y cubrieron totalmente las placas a los 7 días de incubación a las temperaturas de 30 y 40°C. A 45°C, después de 7 días de incubación, el crecimiento fue limitado. Al microscopio se pudieron observar los característicos esporangióforos de esta especie. La identificación de la cepa se confirmó mediante la secuenciación de los fragmentos génicos D1/D2 26S e ITS-5.8S del ARN ribosómico. Conclusiones. En el estudio filogenético, la cepa se agrupó en el mismo clado que la cepa neotipo de C. bertholletiae, aunque se detectaron algunas diferencias en las secuencias correspondientes a los ITS. En los casos causados por esta especie en cetáceos, se desconocen las posibles fuentes de infección. Tampoco se conoce por el momento por qué este patógeno ha sido aislado solo de cetáceos y no de otros animales domésticos o salvajes (AU)

Characterization and phylogenetic analysis of a Cunninghamella bertholletiae isolate from a bottlenose dolphin (Tursiops truncatus).

BACKGROUND: Cunninghamella is a genus of the order Mucorales which includes saprophytic species, rarely causing mycoses. The most frequently reported in human mycoses is the thermophilic species Cunninghamella bertholletiae. However, this species does not appear to cause mucormycosis in animals, so there is scarce information about C. bertholletiae isolates from animals. AIMS: In this paper we describe the phenotypic and genotypic characterization, and the phylogenetic analysis, of an isolate of C. bertholletiae involved in a central nervous system mucormycosis in a dolphin. METHODS: The isolate studied in this publication was characterized using the current morphological and physiological identification system for Cunninghamella species. DNA sequencing and analysis of the D1/D2 regions of the 26S rRNA gene and the ITS-5.8S rRNA gene sequences were also performed. RESULTS: Colonies were fast-growing, white at first, although they became tannish-gray, covering the whole plate after 7 days of incubation at 30 and 40°C. Limited growth was observed after 7 days at 45°C. The micromorphology showed characteristic erect sporangiophores. The identification of the isolate was confirmed by DNA sequencing of the D1/D2 regions of the 26S and the ITS-5.8S (ITS) rRNA gene sequencing. CONCLUSIONS: In the phylogenetic study, the isolate clustered in the same clade as C. bertholletiae neotype strain although some differences were observed in the ITS sequences. In the cetacean cases, the possible sources of infection are unclear. The reasons why this pathogen has been found only in cetaceans and not in other domestic or wild animals are at the moment unknown and need further study.

Characterization of the species Malassezia pachydermatis and re-evaluation of its lipid dependence using a synthetic agar medium.

The genus Malassezia includes lipophilic yeasts, which are part of the skin microbiota of various mammals and birds. Unlike the rest of Malassezia species, M. pachydermatis is described as non-lipid-dependent, as it is able to grow on Sabouraud glucose agar (SGA) without lipid supplementation. In this study we have examined the phenotypic variability within M. pachydermatis and confirmed its lipid-dependent nature using a synthetic agar medium. We used a selection of representative non-lipid-dependent strains from different animal species and three atypical lipid-dependent strains of this species, which were not able to grow after multiple passages on SGA. More than 400 lipid-dependent Malassezia isolates from animals were studied in order to detect the three lipid-dependent strains of M. pachydermatis. The identity of the atypical strains was confirmed by DNA sequencing. On the other hand, we have modified the Tween diffusion test, which is widely used in the characterization of these yeasts, by using a synthetic agar-based medium instead of SGA. This modification has proved to be useful for differentiation of M. pachydermatis strains, providing reproducible results and a straightforward interpretation. The finding of these peculiar lipid-dependent strains exemplifies the large variability within the species M. pachydermatis, which involves rare atypical strains with particular growth requirements.

Cryptic Diversity of Malassezia pachydermatis from Healthy and Diseased Domestic Animals.

Malassezia pachydermatis is part of the normal cutaneous microbiota of wild and domestic carnivores. However, under certain conditions this yeast can overproliferate and cause several diseases in its host, mainly otitis and dermatitis in dogs. The aim of this study was to conduct a molecular characterization of M. pachydermatis isolates from healthy and diseased domestic animals, in order to assess the molecular diversity and phylogenetic relationship within this species. The large subunit (LSU) and the internal transcribed spacer (ITS) of ribosomal RNA, chitin synthase 2 (CHS2) and ß-tubulin genes from sixteen strains isolated from dogs, cats, a goat, a pig and a horse were sequenced. A different number of types of sequences were identified for each target gene, including some types described for the first time. Five sequence types were characterized for the LSU, eleven for the ITS region, nine for CHS2 and eight for ß-tubulin. A multilocus analysis was performed including the four genes, and the resulting phylogenetic tree revealed fifteen genotypes. Genotypes were distributed in two well-supported clades. One clade comprised strains isolated from different domestic animals and a strongly supported cluster constituted by strains isolated from cats. The second clade included strains isolated mainly from dogs and an outlier strain isolated from a horse. No apparent association could be observed between the health status of the animal hosts and concrete strains. The multilocus phylogenetic analysis is a useful tool to assess the intraspecific variation within this species and could help understand the ecology, epidemiology and speciation process of M. pachydermatis.

New lipid-dependent Malassezia species from parrots / Nuevas especies lipodependientes del género Malassezia procedentes de loros

Background. All the currently recognized Malassezia species have been isolated from mammals. However, only a few of them have been isolated from birds. In fact, birds have been less frequently studied as carriers of Malassezia yeasts than mammals. Aim. In this study we describe two new taxa, Malassezia brasiliensis sp. nov. and Malassezia psittaci sp. nov. Methods. The isolates studied in this publication were isolated from pet parrots from Brazil. They were characterized using the current morphological and physiological identification scheme. DNA sequencing and analysis of the D1/D2 regions of the 26S rRNA gene, the ITS-5.8S rRNA gene sequences and the β-tubulin gene were also performed. Results. The strains proposed as new species did not completely fit the phenotypic profiles of any the described species. The validation of these new species was supported by analysis of the genes studied. The multilocus sequence analysis of the three loci provides robust support to delineate these species. Conclusions. These studies confirm the separation of these two new species from the other species of the genus Malassezia, as well as the presence of lipid-dependent Malassezia yeasts on parrots (AU)

Antecedentes. Todas las especies del género Malassezia actualmente identificadas se han aislado de mamíferos. Sin embargo, tan solo unas pocas de ellas se han aislado de aves. De hecho, las aves han sido estudiadas con menos frecuencia como portadoras de estas levaduras que los mamíferos. Objetivos. En este estudio describimos dos nuevas especies del género Malassezia: Malassezia brasiliensis sp. nov. y Malassezia psittaci sp. nov. Métodos. Las cepas estudiadas en esta publicación se aislaron de loros utilizados como animales de compañía en Brasil. Las cepas se caracterizaron mediante los criterios morfológicos y fisiológicos actualmente utilizados para la identificación de estas levaduras. También se llevó a cabo la secuenciación y el análisis de los fragmentos génicos D1/D2 26S e ITS-5.8S del ADN ribosómico y del gen de la β-tubulina. Resultados. Los perfiles fenotípicos de las cepas propuestas como nuevas especies no encajaron completamente con los de las especies descritas en este género. Además, el análisis de los genes estudiados respaldó la validez de las nuevas especies. El análisis multilocus de secuencias de los tres loci estudiados reforzó con mayor firmeza la definición de las nuevas especies. Conclusiones. Todos estos estudios confirman la separación de estas dos nuevas especies del resto de las especies descritas del género Malassezia, así como la existencia de especies dependientes de lípidos del género Malassezia en loros (AU)

New lipid-dependent Malassezia species from parrots.

BACKGROUND: All the currently recognized Malassezia species have been isolated from mammals. However, only a few of them have been isolated from birds. In fact, birds have been less frequently studied as carriers of Malassezia yeasts than mammals. AIM: In this study we describe two new taxa, Malassezia brasiliensis sp. nov. and Malassezia psittaci sp. nov. METHODS: The isolates studied in this publication were isolated from pet parrots from Brazil. They were characterized using the current morphological and physiological identification scheme. DNA sequencing and analysis of the D1/D2 regions of the 26S rRNA gene, the ITS-5.8S rRNA gene sequences and the ß-tubulin gene were also performed. RESULTS: The strains proposed as new species did not completely fit the phenotypic profiles of any the described species. The validation of these new species was supported by analysis of the genes studied. The multilocus sequence analysis of the three loci provides robust support to delineate these species. CONCLUSIONS: These studies confirm the separation of these two new species from the other species of the genus Malassezia, as well as the presence of lipid-dependent Malassezia yeasts on parrots.

Rapid genome resequencing of an atoxigenic strain of Aspergillus carbonarius.

In microorganisms, Ion Torrent sequencing technology has been proved to be useful in whole-genome sequencing of bacterial genomes (5 Mbp). In our study, for the first time we used this technology to perform a resequencing approach in a whole fungal genome (36 Mbp), a non-ochratoxin A producing strain of Aspergillus carbonarius. Ochratoxin A (OTA) is a potent nephrotoxin which is found mainly in cereals and their products, but it also occurs in a variety of common foods and beverages. Due to the fact that this strain does not produce OTA, we focused some of the bioinformatics analyses in genes involved in OTA biosynthesis, using a reference genome of an OTA producing strain of the same species. This study revealed that in the atoxigenic strain there is a high accumulation of nonsense and missense mutations in several genes. Importantly, a two fold increase in gene mutation ratio was observed in PKS and NRPS encoding genes which are suggested to be involved in OTA biosynthesis.

Central nervous system mucormycosis caused by Cunninghamella bertholletiae in a bottlenose dolphin (Tursiops truncatus).

In May 2012, an adult, male bottlenose dolphin (Tursiops truncatus) was found stranded and dead on the Spanish Mediterranean coast. At necropsy, several areas of malacia were macroscopically observed in the periventricular parenchyma of the cerebrum. Microscopically a severe, diffuse, pyogranulomatous, and necrotizing meningoencephalomyelitis was associated with numerous intralesional highly pleomorphic fungal structures. After culture, the fungus, Cunninghamella bertholletiae, was identified by culture and PCR. To our knowledge, this is the first reported case of central nervous system mucormycosis due to Cunninghamella bertholletiae in a cetacean.

Phylogenetic relationships of Malassezia species based on multilocus sequence analysis.

Members of the genus Malassezia are lipophilic basidiomycetous yeasts, which are part of the normal cutaneous microbiota of humans and other warm-blooded animals. Currently, this genus consists of 14 species that have been characterized by phenetic and molecular methods. Although several molecular methods have been used to identify and/or differentiate Malassezia species, the sequencing of the rRNA genes and the chitin synthase-2 gene (CHS2) are the most widely employed. There is little information about the ß-tubulin gene in the genus Malassezia, a gene has been used for the analysis of complex species groups. The aim of the present study was to sequence a fragment of the ß-tubulin gene of Malassezia species and analyze their phylogenetic relationship using a multilocus sequence approach based on two rRNA genes (ITS including 5.8S rRNA and D1/D2 region of 26S rRNA) together with two protein encoding genes (CHS2 and ß-tubulin). The phylogenetic study of the partial ß-tubulin gene sequences indicated that this molecular marker can be used to assess diversity and identify new species. The multilocus sequence analysis of the four loci provides robust support to delineate species at the terminal nodes and could help to estimate divergence times for the origin and diversification of Malassezia species.

Hortaea werneckii isolated from silicone scuba diving equipment in Spain.

During a survey of black yeasts of marine origin, some isolates of Hortaea werneckii were recovered from scuba diving equipment, such as silicone masks and snorkel mouthpieces, which had been kept under poor storage conditions. These yeasts were unambiguously identified by phenotypic and genotypic methods. Phylogenetic analysis of both the D1/D2 regions of 26S rRNA gene and ITS-5.8S rRNA gene sequences showed three distinct genetic types. This species is the agent of tinea nigra which is a rarely diagnosed superficial mycosis in Europe. In fact this mycosis is considered an imported fungal infection being much more prevalent in warm, humid parts of the world such as the Central and South Americas, Africa, and Asia. Although H. werneckii has been found in hypersaline environments in Europe, this is the first instance of the isolation of this halotolerant species from scuba diving equipment made with silicone rubber which is used in close contact with human skin and mucous membranes. The occurrence of this fungus in Spain is also an unexpected finding because cases of tinea nigra in this country are practically not seen.