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Sugarcane (Saccharum officinarum, Poaceae) is cultivated on a large scale in (sub)tropical regions such as Brazil and has considerable economic value for sugar and biofuel production. The plant is a rich substrate for endo- and epiphytic fungi. Black yeasts in the family Herpotrichiellaceae (Chaetothyriales) are colonizers of human-dominated habitats, particularly those rich in toxins and hydrocarbon pollutants, and may cause severe infections in susceptible human hosts. The present study assessed the diversity of Herpotrichiellaceae associated with sugarcane, using in silico identification and selective isolation. Using metagenomics, we identified 5833 fungal sequences, while 639 black yeast-like isolates were recovered in vitro. In both strategies, the latter fungi were identified as members of the genera Cladophialophora, Exophiala, and Rhinocladiella (Herpotrichiellaceae), Cyphellophora (Cyphellophoraceae), and Knufia (Trichomeriaceae). In addition, we discovered new species of Cladophialophora and Exophiala from sugarcane and its rhizosphere. The first environmental isolation of Cladophialophora bantiana is particularly noteworthy, because this species up to now is exclusively known from the human host where it mostly causes fatal brain disease in otherwise healthy patients.
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The presence of endophytes promotes the biosynthesis of secondary plant metabolites. In this study, endophytic fungi were isolated from Schinus terebinthifolius to investigate their diversity and antimicrobial activity. A total of 272 endophytic fungi was obtained. These belonged to nine different genera: Alternaria, Colletotrichum, Diaporthe, Epicoccum, Fusarium, Pestalotiopsis, Phyllosticta, Xylaria, and Cryptococcus. Notably, Diaporthe foliorum was introduced as a new species, with accompanying morphological descriptions, illustrations, and a multigene phylogenetic analysis (using ITS, TEF1, TUB, HIS, and CAL). Among the 26 fungal morphotypes evaluated for antimicrobial activity, five strains had inhibitory effects against pathogenic microorganisms. Xylaria allantoidea CMRP1424 extracts showed antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Diaporthe terebinthifolii CMRP1430 and CMRP1436 showed antimicrobial activity against E. coli, P. aeruginosa, S. aureus, and C. albicans. Meanwhile, D. foliorum CMRP1321 and D. malorum CMRP1438 extracts inhibited C. albicans alone. Three classes of chemical compounds were identified in D. foliorum CMRP1438 extracts: ferric chloride, potassium hydroxide, and vanillin-sulfuric acid. In conclusion, the endophytic isolates were able to produce bioactive agents with pharmaceutical potential as antibacterial and antifungal agents. As such, they may provide fresh leads in the search for new, biological sources of drug therapies.
Asunto(s)
Anacardiaceae , Antiinfecciosos , Antiinfecciosos/farmacología , Ascomicetos , Endófitos/genética , Escherichia coli , Hongos , Pruebas de Sensibilidad Microbiana , Filogenia , Staphylococcus aureusRESUMEN
Chromoblastomycosis (CBM) is a neglected implantation mycosis prevalent in tropical climate zones, considered an occupational disease that affects impoverished rural populations. This retrospective study described clinical aspects of CBM in a hyperendemic area in Brazil and constructed a worldwide haplotype network of Fonsecaea spp. strains. The variables were collected from medical records using a standard report form, reporting 191 patients with CBM from Maranhão, Brazil. The mean age was 56.1 years, 168 (88%) patients were male and predominantly farmers (85.8%). The mean time of evolution of the disease until diagnosis was 9.4 years. Lower limbs (81.2%) and upper limbs (14.2%) were the main sites affected. Most patients exhibited verrucous (55%) and infiltrative plaque (48.2%). Fonsecaea spp. were identified in 136 cases and a haplotype network constructed with ITS sequences of 185 global strains revealed a total of 59 haplotypes exhibiting high haplotypic and low nucleotide diversities. No correlation was observed between the different haplotypes of Fonsecaea species and dermatological patterns, severity of disease or geographic distribution inside Maranhão. Data from this area contributed to better understanding the epidemiology of CBM. For the first time, a robust haplotype network with Fonsecaea strains reveals an evolutionary history with a recent population expansion.
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Among agents of chromoblastomycosis, Fonsecaea pugnacius presents a unique type of infection because of its secondary neurotropic dissemination from a chronic cutaneous case in an immunocompetent patient. Neurotropism occurs with remarkable frequency in the fungal family Herpotrichiellaceae, possibly associated with the ability of some species to metabolize aromatic hydrocarbons. In an attempt to understand this new disease pattern, were conducted genomic analysis of Fonsecaea pugnacius (CBS 139214) performed with de novo assembly, gene prediction, annotation and mitochondrial genome assembly, supplemented with animal infection models performed with Tenebrio molitor in Mus musculus lineages BALB/c and C57BL/6. The genome draft of 34.8 Mb was assembled with a total of 12,217 protein-coding genes. Several proteins, enzymes and metabolic pathways related to extremotolerance and virulence were recognized. The enzyme profiles of black fungi involved in chromoblastomycosis and brain infection were analyzed with the Carbohydrate-Active Enzymes (CAZY) and peptidases database (MEROPS). The capacity of the fungus to survive inside Tenebrio molitor animal model was confirmed by histopathological analysis and by presence of melanin and hyphae in host tissue. Although F. pugnacius was isolated from brain in a murine model following intraperitoneal infection, cytokine levels were not statistically significant, indicating a profile of an opportunistic agent. A dual ecological ability can be concluded from presence of metabolic pathways for nutrient scavenging and extremotolerance, combined with a capacity to infect human hosts.
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Central nervous system (CNS) infection by Histoplasma capsulatum is a rare disease in immunocompromised individuals in endemic areas. About one quarter of cases result from hematogenous dissemination. A 23-year-old upholsterer with chronic occipital headache had developed intracranial hypertension and dizziness, incoordination with ataxic gait, and acute confusion 5 months prior to admission. Laboratory examinations and chest roentgenogram were normal. Postcontrast T1-weighted MRI of the brain revealed a multiple ring-enhancing cerebellar, brain stem and parietal lobe lesions, and meningeal contrast enhancement. Cerebrospinal fluid culture was positive for H. capsulatum species complex, which was confirmed by phylogenetic analysis. Thirteen years after the diagnosis and treatment, there was no H. capsulatum recurrence; sequels related to complications due to the ventriculoperitoneal shunt. This case shows a primary neurological presentation of cerebral histoplasmosis, without meningitis or disseminated disease in nonimmune-compromised patient. The authors propose a categorization of the diagnosis of CNS histoplasmosis. Routine diagnostics of sibling species within the H. capsulatum complex proved to be difficult.
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Infecciones del Sistema Nervioso Central/microbiología , Histoplasma , Histoplasmosis/diagnóstico , Adulto , Infecciones del Sistema Nervioso Central/patología , Líquido Cefalorraquídeo/microbiología , ADN Espaciador Ribosómico/genética , Genes Fúngicos , Histoplasma/genética , Histoplasma/aislamiento & purificación , Histoplasmosis/patología , Humanos , Masculino , Filogenia , Adulto JovenRESUMEN
The species belonging to the genus Fonsecaea are the main causative agents of chromoblastomycosis. The invasive potential of Fonsecaea differs significantly among its various sibling species. Moreover, the lack of clarity on the virulence and availability of precise markers to distinguish and detect Fonsecaea species is attributed to the different ways of dissemination and pathogenicity. Therefore, the present study aimed to propose new molecular tools to differentiate between sibling species causing chromoblastomycosis. We used an infection model of chromoblastomycosis in BALB/c to study species-specific molecular markers for the in vivo detection of Fonsecaea species in biological samples. Specific primers based on the CBF5 gene were developed for Fonsecaea pedrosoi, Fonsecaea monophora, Fonsecaea nubica, and Fonsecaea pugnacius. In addition, a padlock probe was designed for F. pugnacius based on ITS sequences. We also assessed the specificity of Fonsecaea species using in silico, in vitro, and in vivo assays. The results showed that markers and probes could effectively discriminate the species in both clinical and environmental samples, enabling bioprospecting of agents of chromoblastomycosis, thereby elucidating the infection route of the disease.
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Ascomicetos/clasificación , Ascomicetos/aislamiento & purificación , Cromoblastomicosis/microbiología , Marcadores Genéticos , Técnicas de Diagnóstico Molecular/métodos , Animales , Ascomicetos/genética , ADN Espaciador Ribosómico/genética , Modelos Animales de Enfermedad , Proteínas Fúngicas/genética , Masculino , Ratones Endogámicos BALB C , Sensibilidad y EspecificidadRESUMEN
Fungal peritonitis is frequent on peritoneal dialysis, with rare cases by Exophiala dermatitidis. A 25-month-old female admitted to the pediatric ICU with acute renal failure was submitted to peritoneal dialysis. After 10 days patient presented fever. Peritoneal fluid culture showed yeast colonies molecularly identified as E. dermatitidis. Patient was treated with voriconazole and hemodialysis. The literature was reviewed. Disseminated infections are frequently fatal, but appropriate diagnose and therapeutic led to cure in this case.
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This paper presents a case of disseminated sporotrichosis in a 13-year-old female, originating from a rural area in Minas Gerais state, Brazil. The patient was hospitalized in Santa Casa hospital of Belo Horizonte, with hyporexia, prostration, fever and disseminated ulcerative lesions, besides anemia, leucopenia and sepsis of probable cutaneous focus. The patient was admitted without proven immunosuppression. She was diagnosed with cutaneous-disseminated sporotrichosis. The drug therapy chosen was itraconazole during 12 months, leading to important clinical improvement and healing of cutaneous lesions.
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Fonsecaea and Cladophialophora are genera of black yeast-like fungi harboring agents of a mutilating implantation disease in humans, along with strictly environmental species. The current hypothesis suggests that those species reside in somewhat adverse microhabitats, and pathogenic siblings share virulence factors enabling survival in mammal tissue after coincidental inoculation driven by pathogenic adaptation. A comparative genomic analysis of environmental and pathogenic siblings of Fonsecaea and Cladophialophora was undertaken, including de novo assembly of F. erecta from plant material. The genome size of Fonsecaea species varied between 33.39 and 35.23 Mb, and the core genomes of those species comprises almost 70% of the genes. Expansions of protein domains such as glyoxalases and peptidases suggested ability for pathogenicity in clinical agents, while the use of nitrogen and degradation of phenolic compounds was enriched in environmental species. The similarity of carbohydrate-active vs. protein-degrading enzymes associated with the occurrence of virulence factors suggested a general tolerance to extreme conditions, which might explain the opportunistic tendency of Fonsecaea sibling species. Virulence was tested in the Galleria mellonella model and immunological assays were performed in order to support this hypothesis. Larvae infected by environmental F. erecta had a lower survival. Fungal macrophage murine co-culture showed that F. erecta induced high levels of TNF-α contributing to macrophage activation that could increase the ability to control intracellular fungal growth although hyphal death were not observed, suggesting a higher level of extremotolerance of environmental species.
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Citrus black spot disease, caused by the phytopathogen Phyllosticta citricarpa, depreciates the market value of citrus fruits and prevents their exportation to disease-free regions. It may also reduce the productivity of citrus fruit orchards. To identify an alternative to conventional disease control measures, isolates of Diaporthe terebinthifolii, active against P. citricarpa, were selected from an endophytic fungal population of Schinus terebinthifolia leaves. Different culture media were screened to identify the culture medium that afforded the most efficient production of biologically active extracts. A particular fraction (fraction VI) of the extract completely protected orange leaves by inhibiting the germination of P. citricarpa conidia with a minimum inhibitory concentration of 0.003 µg.mL-1. The active constituents in D. terebinthifolii extract fractions were identified by gas chromatography coupled to mass spectrometry as verbanol, phenylethyl alcohol, verbenyl acetate and methyl hexadecanoate. The results obtained strongly suggest the existence of a synergistic effect among the metabolites produced. Thus, these fungal metabolites could be used to control the CBS disease. As the asexual spores of P. citricarpa play an important role in fruit lesion development and disease dispersion, fungal extracts that inhibit the spore germination can be used as an effective alternative for directional disease control.
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Antifúngicos/farmacología , Ascomicetos , Hojas de la Planta/microbiología , Esporas Fúngicas/crecimiento & desarrollo , Antifúngicos/química , Antifúngicos/aislamiento & purificación , Ascomicetos/química , Ascomicetos/crecimiento & desarrollo , Ascomicetos/aislamiento & purificación , Citrus/microbiología , Medios de Cultivo/química , Endófitos , Espectrometría de Masas , Alcohol Feniletílico/farmacología , Enfermedades de las Plantas/microbiología , Hojas de la Planta/efectos de los fármacos , Metabolismo SecundarioRESUMEN
A draft genome sequence of type strain Fonsecaea multimorphosa CBS 980.96T was obtained. This species was first isolated from a cat with cerebral phaeohyphomycosis in Queensland, Australia.
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The human mutilating disease chromoblastomycosis is caused by melanized members of the order Chaetothyriales. To assess population diversity among 123 clinical strains of agents of the disease in Brazil we applied sequencing of the rDNA internal transcribed spacer region, and partial cell division cycle and ß-tubulin genes. Strains studied were limited to three clusters divided over the single family Herpotrichiellaceae known to comprise agents of the disease. A Fonsecaea cluster contained the most important agents, among which F. pedrosoi was prevalent with 80% of the total set of strains, followed by 13% for F. monophora, 3% for F. nubica, and a single isolate of F. pugnacius. Additional agents, among which two novel species, were located among members of the genus Rhinocladiella and Cyphellophora, with frequencies of 3% and 1%, respectively.
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Ascomicetos/aislamiento & purificación , Cromoblastomicosis/microbiología , Ascomicetos/clasificación , Ascomicetos/genética , Brasil/epidemiología , Cromoblastomicosis/epidemiología , ADN de Hongos/genética , ADN Espaciador Ribosómico/genética , Humanos , Epidemiología Molecular , Técnicas de Tipificación Micológica , FilogeniaRESUMEN
On the basis of multilocus phylogenetic data, Fonsecaea nubica was described in 2010 as a molecular sibling of F. monophora, an established agent of the human skin disease chomoblastomycosis in tropical zones. Genome analysis of these pathogens is mandatory to identify genes involved in the interaction with host and virulence.
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The black yeast Fonsecaea monophora is one of the main etiologic agents of chromoblastomycosis in humans. Its pathogenicity profile is more invasive than that of related Fonsecaea species, causing brain infection in addition to (sub)cutaneous infections.
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Histoplasmosis is a systemic fungal disease that occurs worldwide, causing symptomatic infection mostly in immunocompromised hosts. Etiological agent is the dimorphic fungus, Histoplasma capsulatum, which occurs in soil contaminated with bird or bat droppings. Major limitation in recognition of H. capsulatum infections is the low awareness, since other diseases may have similar symptomatology. The molecular methods have gained importance because of unambiguous diagnostic ability and efficiency. The aim of this study was to develop and evaluate a padlock probe in view of rolling circle amplification (RCA) detection method which targets ITS (Internal Transcribed Spacer) rDNA of H. capsulatum enabling rapid and specific detection of the fungus in clinical samples. Two padlock probes were designed and one of these (HcPL2) allowed specific amplification of H. capsulatum DNA while no cross-reactivity was observed with fungi used as negative controls. This method proved to be effective for H. capsulatum specific identification and demonstrated to be faster than the traditional method of microbiological identification.
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Histoplasma/genética , Histoplasma/aislamiento & purificación , Histoplasmosis/diagnóstico , Técnicas de Amplificación de Ácido Nucleico , Animales , Sondas de ADN , ADN de Hongos/análisis , ADN de Hongos/genética , ADN Espaciador Ribosómico/genética , Histoplasma/clasificación , Histoplasmosis/microbiología , Humanos , Filogenia , Sensibilidad y EspecificidadRESUMEN
In this study, we analyzed the antimicrobial activity of extracts harvested from 17 endophytic fungi isolated from the medicinal plant Schinus terebinthifolius. Morphological and molecular analyses indicated that these fungal species belonged to the genera Alternaria, Bjerkandera, Colletotrichum, Diaporthe, Penicillium, and Xylaria. Of the endophytes analyzed, 64.7 % produced antimicrobial compounds under at least one of the fermentation conditions tested. Nine isolates produced compounds that inhibited growth of Staphylococcus aureus, four produced compounds that inhibited Candida albicans, and two that inhibited Pseudomonas aeruginosa. The fermentation conditions of the following endophytes were optimized: Alternaria sp. Sect. Alternata-LGMF626, Xylaria sp.-LGMF673, and Bjerkandera sp.-LGMF713. Specifically, the carbon and nitrogen sources, initial pH, temperature, and length of incubation were varied. In general, production of antimicrobial compounds was greatest when galactose was used as a carbon source, and acidification of the growth medium enhanced the production of compounds that inhibited C. albicans. Upon large-scale fermentation, Alternaria sp. Sect. Alternata-LGMF626 produced an extract containing two fractions that were active against methicillin-resistant S. aureus. One of the extracts exhibited high activity (minimum inhibitory concentration of 18.52 µg/mL), and the other exhibited moderate activity (minimum inhibitory concentration of 55.55 µg/mL). The compounds E-2-hexyl-cinnamaldehyde and two compounds of the pyrrolopyrazine alkaloids class were identified in the active fractions by gas chromatography-mass spectrometry.
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Anacardiaceae/microbiología , Antiinfecciosos/metabolismo , Bioprospección/métodos , Endófitos/metabolismo , Hongos/metabolismo , Medios de Cultivo/química , Endófitos/clasificación , Endófitos/aislamiento & purificación , Fermentación , Hongos/clasificación , Hongos/aislamiento & purificación , Pruebas de Sensibilidad MicrobianaRESUMEN
The human mutilating disease chromoblastomycosis is caused by melanized members of the order Chaetothyriales. To assess population diversity among 123 clinical strains of agents of the disease in Brazil we applied sequencing of the rDNA internal transcribed spacer region, and partial cell division cycle and ß-tubulin genes. Strains studied were limited to three clusters divided over the single family Herpotrichiellaceae known to comprise agents of the disease. A Fonsecaea cluster contained the most important agents, among which F. pedrosoi was prevalent with 80% of the total set of strains, followed by 13% for F. monophora, 3% for F. nubica, and a single isolate of F. pugnacius. Additional agents, among which two novel species, were located among members of the genus Rhinocladiella and Cyphellophora, with frequencies of 3% and 1%, respectively.
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Humanos , Enfermedad , CromoblastomicosisRESUMEN
We report a fatal case of a chromoblastomycosis-like infection caused by a novel species of Fonsecaea in a 52-year-old immunocompetent Caucasian male from an area of chromoblastomycosis endemicity in Brazil. The patient had a 30-year history of slowly evolving, verrucous lesions on the right upper arm which gradually affected the entire arm, the left hemifacial area, and the nose. Subsequent dissemination to the brain was observed, which led to death of the patient. The internal transcribed spacer (ITS) and partial large subunit (LSU), BT2, and CDC42 genes of the isolates recovered from skin and brain were sequenced, confirming the novelty of the species. The species is clinically unique in causing brain abscesses secondary to chromoblastomycosis lesions despite the apparent intact immunity of the patient. Histopathologic appearances were very different, showing muriform cells in skin and hyphae in brain.
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Ascomicetos/clasificación , Ascomicetos/aislamiento & purificación , Cromoblastomicosis/diagnóstico , Cromoblastomicosis/patología , Ascomicetos/genética , Encéfalo/microbiología , Encéfalo/patología , Brasil , Cromoblastomicosis/microbiología , Análisis por Conglomerados , ADN de Hongos/química , ADN de Hongos/genética , ADN Ribosómico/química , ADN Ribosómico/genética , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/genética , Resultado Fatal , Cabeza/diagnóstico por imagen , Histocitoquímica , Humanos , Imagen por Resonancia Magnética , Masculino , Microscopía , Persona de Mediana Edad , Datos de Secuencia Molecular , Filogenia , ARN Ribosómico/genética , Radiografía , Análisis de Secuencia de ADN , Piel/microbiología , Piel/patología , Población BlancaRESUMEN
Fusarium oxysporum has been described as a pathogen causing onychomycosis, its incidence has been increasing in immunocompetent and disseminated infection can occur in immunosuppressed individuals. We describe the first case of congenital onychomycosis in a child caused by Fusarium oxysporum. The infection being acquired in utero was proven by molecular methods with the identification of the fungus both in the nail and placenta, most probably as an ascending contamination/infection in a HIV-positive, immunosuppressed mother.