Isolamento e caracterização de leveduras negras em formigas, abelhas e cupins

Orientadora: Profª Drª Vânia Aparecida VicenteCoorientadora: Profª Drª Débora do Rocio KlisiowiczDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Microbiologia, Parasitologia e Patologia. Defesa : Curitiba, 23/03/2018Inclui referênciasResumo: A cromoblastomicose é uma doença negligenciada caracterizada pela ocorrência de lesões cutâneas, subcutâneas ou disseminadas, de desenvolvimento lento, que podem comprometer a qualidade de vida e o desenvolvimento econômico do indivíduo afetado. Trata-se de uma doença ocupacional que afeta principalmente trabalhadores rurais e aqueles expostos a solo e material vegetal contaminados, homens (numa relação 4:1), com idades entre 25 e 85 anos. O desenvolvimento da doença se dá principalmente a partir da inoculação de leveduras negras por lesão traumática com material contaminado. No Brasil, Fonsecaea pedrosoi é considerado o principal causador de cromoblastomicose, porém outras espécies como por exemplo, F. monophora, F. nubica, F. pugnacius, Rhinocladiella tropicalis e Cypehllophora ludoviensis também são relacionadas com a doença. Leveduras negras têm sido encontradas em substratos vegetais, madeira, solo e material vegetal em decomposição. Além disso, são capazes de se desenvolver em diversos microambientes considerados extremos, como solo contaminado com hidrocarbonetos, material contaminado com petróleo e gasolina, madeira tratada com creosoto, entre outros. O desenvolvimento dessas leveduras em condições extremas, como presença de radiação ultravioleta, alta osmolaridade, altas temperaturas, escassez de nutrientes, além da capacidade de assimilação de compostos aromáticos de carbono as caracteriza como seres oligotróficos. Embora se conheça os agentes causais da cromoblastomicose, o nicho ambiental e a forma com que essas leveduras se dispersam no ambiente ainda não estão totalmente esclarecidos, sendo uma das hipóteses sugeridas a de disseminação ambiental mediada por insetos. Dessa forma, o objetivo deste estudo foi determinar se insetos sociais, especificamente formigas, abelhas e cupins, podem atuar como vetores na disseminação de leveduras negras no ambiente. Para isso foram realizados isolamentos utilizando duas técnicas seletivas para leveduras negras, flotação em óleo mineral e enriquecimento seletivo em compostos aromáticos voláteis, sendo obtidos oito isolados de fungos dematiáceos, posteriormente identificados por micromorfologia e sequenciamento como Fonsecaea pedrosoi, Rhinocladiella similis, Exophiala xenobiotica, Cyphellophora sp., Cladosporium sp., Microascus murinus e Hawksworthiomyces sp. Dentre os isolados, Fonsecaea pedrosoi, Rhinocladiella similis, Exophiala xenobiotica e Cyphellophora sp. são relacionadas com o desenvolvimento de cromoblastomicose ou outras micoses de implantação. Assim sendo, formigas, abelhas e cupins podem ser considerados vetores e agentes de dispersão de leveduras negras patogênicas, visto que tais leveduras são encontradas no exoesqueleto destes insetos e em material diretamente associado a estes hospedeiros. Palavras-chave: Cromoblastomicose; Leveduras negras; Atta laevigata; Melipona flavolineata; Scaptotrigona postica; Nasutitermes sp.Abstract: Chromoblastomycosis is a neglected disease characterized by cutaneous, subcutaneous or disseminated lesions with slow progression that affects mostly farm workers and individuals exposed to contaminated soil and plant debris, impairing life's quality and affecting individual's economic status. The onset of this disease is mostly by traumatic inoculation of black yeasts from plant material contaminated. Fonsecaea pedrosoi is the major agent of chromoblastomycosis in Brazil, but other species are related to the disease, as F. monophora, F. nubica, F. pugnacius, Rhinocladiella tropicalis and Cypehllophora ludoviensis. Black yeasts have been isolated from decaying plant material, soil, wood and other environments. They are capable of growing in harmful environments, like soil contaminated with hydrocarbons, contaminated sites with petroleum and gas, creosote treated wood and others. Black yeasts are known to be oligotrophic organisms because of their capacity for development in extreme conditions, such as UV presence, high osmolarity and temperature, low availability of nutrients, besides their capacity to assimilate hydrocarbons. It is well known which species are agents of chromoblastomycosis, but until now little is known about the environmental niche and mode of dispersion in nature. Because this, is hypothesized that insects can act as vectors mediating fungal dispersion. In this way, the aim of this study was determinate if social insects, being them ants, bees and termites, can act as vectors disseminating black yeasts in nature. To elucidate this, two selective methods of isolation were realized, oil flotation and selective enrichment on volatile aromatics. Eight isolates of dematiaceous fungi were obtained and were identified by micromorphology and sequencing as Fonsecaea pedrosoi, Rhinocladiella similis, Exophiala xenobiotica, Cyphellophora sp., Cladosporium sp., Microascus murinus and Hawksworthiomyces sp. Four of these are known as the agents of chromoblastomycosis or another implantation mycosis, being them Fonsecaea pedrosoi, Rhinocladiella similis, Exophiala xenobiotica and Cyphellophora sp. These results demonstrated that ants, bees and termites could be considered vectors and pathogenic black yeasts' dispersion agents since these fungi were found in insects' exoskeleton and in material associated with them. Keywords: Chromoblastomycosis; Black yeast; Atta laevigata; Melipona flavolineata; Scaptotrigona postica; Nasutitermes sp

The origin of human pathogenicity and biological interactions in Chaetothyriales

Fungi in the order Chaetothyriales are renowned for their ability to cause human infections. Nevertheless, they are not regarded as primary pathogens, but rather as opportunists with a natural habitat in the environment. Extremotolerance is a major trend in the order, but quite diferent from black yeasts in Capnodiales which focus on endurance, an important additional parameter is advancing toxin management. In the ancestral ecology of rock colonization, the association with metabolite-producing lichens is signifcant. Ant-association, dealing with pheromones and repellents, is another mainstay in the order. The phylogenetically derived family, Herpotrichiellaceae, shows dual ecology in monoaromatic hydrocarbon assimilation and the ability to cause disease in humans and cold-blooded vertebrates. In this study, data on ecology, phylogeny, and genomics were collected and analyzed in order to support this hypothesis on the evolutionary route of the species of Chaetothyriales. Comparing the ribosomal tree with that of enzymes involved in toluene degradation, a signifcant expansion of cytochromes is observed and the toluene catabolism is found to be complete in some of the Herpotrichiellaceae. This might enhance human systemic infection. However, since most species have to be traumatically inoculated in order to cause disease, their invasive potential is categorized as opportunism. Only in chromoblastomycosis, true pathogenicity might be surmised. The criterion would be the possible escape of agents of vertebrate disease from the host, enabling dispersal of adapted genotypes to subsequent generations.info:eu-repo/semantics/publishedVersio

Isolamento e caracterização de leveduras negras em formigas, abelhas e cupins

Orientadora: Profª Drª Vânia Aparecida VicenteCoorientadora: Profª Drª Débora do Rocio KlisiowiczDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Microbiologia, Parasitologia e Patologia. Defesa : Curitiba, 23/03/2018Inclui referênciasResumo: A cromoblastomicose é uma doença negligenciada caracterizada pela ocorrência de lesões cutâneas, subcutâneas ou disseminadas, de desenvolvimento lento, que podem comprometer a qualidade de vida e o desenvolvimento econômico do indivíduo afetado. Trata-se de uma doença ocupacional que afeta principalmente trabalhadores rurais e aqueles expostos a solo e material vegetal contaminados, homens (numa relação 4:1), com idades entre 25 e 85 anos. O desenvolvimento da doença se dá principalmente a partir da inoculação de leveduras negras por lesão traumática com material contaminado. No Brasil, Fonsecaea pedrosoi é considerado o principal causador de cromoblastomicose, porém outras espécies como por exemplo, F. monophora, F. nubica, F. pugnacius, Rhinocladiella tropicalis e Cypehllophora ludoviensis também são relacionadas com a doença. Leveduras negras têm sido encontradas em substratos vegetais, madeira, solo e material vegetal em decomposição. Além disso, são capazes de se desenvolver em diversos microambientes considerados extremos, como solo contaminado com hidrocarbonetos, material contaminado com petróleo e gasolina, madeira tratada com creosoto, entre outros. O desenvolvimento dessas leveduras em condições extremas, como presença de radiação ultravioleta, alta osmolaridade, altas temperaturas, escassez de nutrientes, além da capacidade de assimilação de compostos aromáticos de carbono as caracteriza como seres oligotróficos. Embora se conheça os agentes causais da cromoblastomicose, o nicho ambiental e a forma com que essas leveduras se dispersam no ambiente ainda não estão totalmente esclarecidos, sendo uma das hipóteses sugeridas a de disseminação ambiental mediada por insetos. Dessa forma, o objetivo deste estudo foi determinar se insetos sociais, especificamente formigas, abelhas e cupins, podem atuar como vetores na disseminação de leveduras negras no ambiente. Para isso foram realizados isolamentos utilizando duas técnicas seletivas para leveduras negras, flotação em óleo mineral e enriquecimento seletivo em compostos aromáticos voláteis, sendo obtidos oito isolados de fungos dematiáceos, posteriormente identificados por micromorfologia e sequenciamento como Fonsecaea pedrosoi, Rhinocladiella similis, Exophiala xenobiotica, Cyphellophora sp., Cladosporium sp., Microascus murinus e Hawksworthiomyces sp. Dentre os isolados, Fonsecaea pedrosoi, Rhinocladiella similis, Exophiala xenobiotica e Cyphellophora sp. são relacionadas com o desenvolvimento de cromoblastomicose ou outras micoses de implantação. Assim sendo, formigas, abelhas e cupins podem ser considerados vetores e agentes de dispersão de leveduras negras patogênicas, visto que tais leveduras são encontradas no exoesqueleto destes insetos e em material diretamente associado a estes hospedeiros. Palavras-chave: Cromoblastomicose; Leveduras negras; Atta laevigata; Melipona flavolineata; Scaptotrigona postica; Nasutitermes sp.Abstract: Chromoblastomycosis is a neglected disease characterized by cutaneous, subcutaneous or disseminated lesions with slow progression that affects mostly farm workers and individuals exposed to contaminated soil and plant debris, impairing life's quality and affecting individual's economic status. The onset of this disease is mostly by traumatic inoculation of black yeasts from plant material contaminated. Fonsecaea pedrosoi is the major agent of chromoblastomycosis in Brazil, but other species are related to the disease, as F. monophora, F. nubica, F. pugnacius, Rhinocladiella tropicalis and Cypehllophora ludoviensis. Black yeasts have been isolated from decaying plant material, soil, wood and other environments. They are capable of growing in harmful environments, like soil contaminated with hydrocarbons, contaminated sites with petroleum and gas, creosote treated wood and others. Black yeasts are known to be oligotrophic organisms because of their capacity for development in extreme conditions, such as UV presence, high osmolarity and temperature, low availability of nutrients, besides their capacity to assimilate hydrocarbons. It is well known which species are agents of chromoblastomycosis, but until now little is known about the environmental niche and mode of dispersion in nature. Because this, is hypothesized that insects can act as vectors mediating fungal dispersion. In this way, the aim of this study was determinate if social insects, being them ants, bees and termites, can act as vectors disseminating black yeasts in nature. To elucidate this, two selective methods of isolation were realized, oil flotation and selective enrichment on volatile aromatics. Eight isolates of dematiaceous fungi were obtained and were identified by micromorphology and sequencing as Fonsecaea pedrosoi, Rhinocladiella similis, Exophiala xenobiotica, Cyphellophora sp., Cladosporium sp., Microascus murinus and Hawksworthiomyces sp. Four of these are known as the agents of chromoblastomycosis or another implantation mycosis, being them Fonsecaea pedrosoi, Rhinocladiella similis, Exophiala xenobiotica and Cyphellophora sp. These results demonstrated that ants, bees and termites could be considered vectors and pathogenic black yeasts' dispersion agents since these fungi were found in insects' exoskeleton and in material associated with them. Keywords: Chromoblastomycosis; Black yeast; Atta laevigata; Melipona flavolineata; Scaptotrigona postica; Nasutitermes sp

Black fungi and ants: a genomic comparison of species inhabiting carton nests versus domatia

Some members of Chaetothyriales, an order containing potential agents of opportunistic infections in humans, have a natural habitat in nests of tropical arboreal ants. In these black fungi, two types of ant symbiosis are known, i.e. occurrence in domatia inside living plants, or as components of carton constructions made of ant-chewed plant tissue. In order to explain differences between strains from these types of association, we sequenced and annotated genomes of two newly described carton species, Incumbomyces lentus and Incumbomyces delicatus, and compared these with genomes of four domatia species and related Chaetothyriales. General genomic characteristics, CYP genes, carbohydrate-active enzymes (CAZymes), secondary metabolism, and sex-related genes were included in the study. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s43008-022-00091-5

Environmental Screening of Fonsecaea Agents of Chromoblastomycosis Using Rolling Circle Amplification

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Environmental Screening of Fonsecaea Agents of Chromoblastomycosis Using Rolling Circle Amplification

Chromoblastomycosis is a chronic, cutaneous or subcutaneous mycosis characterized by the presence of muriform cells in host tissue. Implantation disease is caused by melanized fungi related to black yeasts, which, in humid tropical climates, are mainly members of the genus Fonsecaea. In endemic areas of Brazil, F. pedrosoi and F. monophora are the prevalent species. The current hypothesis of infection is traumatic introduction via plant materials, especially by plant thorns. However, isolation studies have demonstrated a low frequency of the agents in environmental substrates. The present study aimed to detect F. pedrosoi and F. monophora in shells of babassu coconuts, soil, plant debris, and thorns from endemic areas of chromoblastomycosis in Maranhão state, northern Brazil, using Rolling Circle Amplification (RCA) with padlock probes as a new environmental screening tool for agents of chromoblastomycosis. In addition to molecular screening, the environmental samples were analyzed by fungal isolation using mineral oil flotation. The limit of detection of the RCA method was 2.88 × 107 copies of DNA per sample for the used padlock probes, indicating that this represents an efficient and sensitive molecular tool for the environmental screening of Fonsecaea agents. In contrast, with isolation from the same samples using several selective methods, no agents of chromoblastomycosis were recovered