Molecular Identification and Antimicrobial Activity of Foliar Endophytic Fungi on the Brazilian Pepper Tree (Schinus terebinthifolius) Reveal New Species of Diaporthe.

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.

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.

Selective isolation of agents of chromoblastomycosis from insect-associated environmental sources.

Chromoblastomycosis is a neglected disease characterized by cutaneous, subcutaneous or disseminated lesions. It is considered an occupational infectious disease that affects mostly rural workers exposed to contaminated soil and vegetal matter. Lesions mostly arise after a traumatic inoculation of herpotrichiellaceous fungi from the Chaetothyriales order. However, the environmental niche of the agents of the disease remains obscure. Its association with insects has been predicted in a few studies. Therefore, the present work aimed to analyze if social insects, specifically ants, bees, and termites, provide a suitable habitat for the fungi concerned. The mineral oil flotation method was used to isolate the microorganisms. Nine isolates were recovered and phylogenetic analysis identified two strains as potential agents of chromoblastomycosis, i.e., Fonsecaea pedrosoi CMRP 3076, obtained from a termite nest (n = 1) and Rhinocladiella similis CMRP 3079 from an ant exoskeleton (n = 1). In addition, we also identified Fonsecaea brasiliensis CMRP 3445 from termites (n = 1), Exophiala xenobiotica CMRP 3077 from ant exoskeleton (n = 1), Cyphellophoraceae CMRP 3103 from bees (n = 1), Cladosporium sp. CMRP 3119 from bees (n = 1), Hawksworthiomyces sp. CMRP 3102 from termites (n = 1), and Cryptendoxyla sp. from termites (n = 2). The environmental isolate of F. pedrosoi CMRP 3076 was tested in two animal models, Tenebrio molitor and Wistar rat, for its pathogenic potential with fungal retention in T. molitor tissue. In the Wistar rat, the cells resembling muriform cells were observed 30 d after inoculation.

A Model for Trans-Kingdom Pathogenicity in Fonsecaea Agents of Human Chromoblastomycosis.

The fungal genus Fonsecaea comprises etiological agents of human chromoblastomycosis, a chronic implantation skin disease. The current hypothesis is that patients acquire the infection through an injury from plant material. The present study aimed to evaluate a model of infection in plant and animal hosts to understand the parameters of trans-kingdom pathogenicity. Clinical strains of causative agents of chromoblastomycosis (Fonsecaea pedrosoi and Fonsecaea monophora) were compared with a strain of Fonsecaea erecta isolated from a living plant. The clinical strains of F. monophora and F. pedrosoi remained concentrated near the epidermis, whereas F. erecta colonized deeper plant tissues, resembling an endophytic behavior. In an invertebrate infection model with larvae of a beetle, Tenebrio molitor, F. erecta exhibited the lowest survival rates. However, F. pedrosoi produced dark, spherical to ovoidal cells that resembled muriform cells, the invasive form of human chromoblastomycosis confirming the role of muriform cells as a pathogenic adaptation in animal tissues. An immunologic assay in BALB/c mice demonstrated the high virulence of saprobic species in animal models was subsequently controlled via host higher immune response.

Comparative Genomics of Sibling Species of Fonsecaea Associated with Human Chromoblastomycosis.

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.

Diversity of opportunistic black fungi on babassu coconut shells, a rich source of esters and hydrocarbons.

The present study assessed the diversity of black yeast-like fungi present on babassu coconut shells, a substrate rich in lipids and several volatile organic compounds (VOCs) including aromatic hydrocarbons. Using different isolation methods, one-hundred-six isolates were obtained and were identified by ITS sequencing as members of the genera Exophiala, Cladophialophora, Veronaea, and Rhinocladiella. Two novel species were discovered. Eight strains were selected for assessing their ability to grow on toluene and phenyl acetate as the sole carbon and energy source. All strains tested were able to assimilate phenyl acetate, while two out of eight were able to use toluene. VOCs profiling in babassu samples was also investigated by GC-ToF MS, revealing that a complex mixture of VOCs was emitted, which included alkylbenzenes such as toluene. Assimilation of alkylbenzenes by the black yeasts might therefore be the result of evolutionary adaptation to symbiotic interactions with higher plants. The potential relationship between lipid/aromatic hydrocarbon metabolism and pathogenicity is also discussed.

Molecular Epidemiology of Agents of Human Chromoblastomycosis in Brazil with the Description of Two Novel Species.

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.

Susceptibility and molecular characterization of Candida species from patients with vulvovaginitis

Abstract Vulvovaginal candidiasis affects women of reproductive age, which represents approximately 15–25% of vaginitis cases. The present study aimed to isolate and characterize yeast from the patients irrespective of the presentation of clinical symptoms. The isolates were subjected to in vitro susceptibility profile and characterization by molecular markers, which intended to assess the distribution of species. A total of 40 isolates were obtained and identified through the CHROMagar, API20aux and by ITS and D1/D2 regions sequencing of DNAr gene. Candida albicans strains were genotyped by the ABC system and the isolates were divided into two genotypic groups. The identity of the C. albicans, C. glabrata, C. guilliermondii, C. kefyr and Saccharomyces cerevisiae isolates was confirmed by the multilocus analysis. The strains of Candida, isolated from patients with complications, were found to be resistant to nystatin but sensitive to fluconazole, amphotericin B and ketoconazole, as observed by in vitro sensitivity profile. The isolates from asymptomatic patients, i.e., the colonized group, showed a dose-dependent sensitivity to the anti-fungal agents, fluconazole and amphotericin B. However, the isolates of C. albicans that belong to distinct genotypic groups showed the same in vitro susceptibility profile.

Susceptibility and molecular characterization of Candida species from patients with vulvovaginitis.

Vulvovaginal candidiasis affects women of reproductive age, which represents approximately 15-25% of vaginitis cases. The present study aimed to isolate and characterize yeast from the patients irrespective of the presentation of clinical symptoms. The isolates were subjected to in vitro susceptibility profile and characterization by molecular markers, which intended to assess the distribution of species. A total of 40 isolates were obtained and identified through the CHROMagar, API20aux and by ITS and D1/D2 regions sequencing of DNAr gene. Candida albicans strains were genotyped by the ABC system and the isolates were divided into two genotypic groups. The identity of the C. albicans, C. glabrata, C. guilliermondii, C. kefyr and Saccharomyces cerevisiae isolates was confirmed by the multilocus analysis. The strains of Candida, isolated from patients with complications, were found to be resistant to nystatin but sensitive to fluconazole, amphotericin B and ketoconazole, as observed by in vitro sensitivity profile. The isolates from asymptomatic patients, i.e., the colonized group, showed a dose-dependent sensitivity to the anti-fungal agents, fluconazole and amphotericin B. However, the isolates of C. albicans that belong to distinct genotypic groups showed the same in vitro susceptibility profile.

In vitro susceptibility and molecular characterization of Candida spp. from candidemic patients / Sensibilidad in vitro y caracterización molecular de aislamientos de Candida procedentes de pacientes con candidemia

Background. Candida species are the main cause of hospital acquired fungal bloodstream infections. The main risk factors for candidemia include parenteral nutrition, long-term intensive care, neutropenia, diabetes, abdominal surgery and the use of central venous catheters. The antifungal drugs used to treat candidemia are mainly the echinocandins, however some isolates may be resistant to these drugs. Aims. This work aims to evaluate the in vitro susceptibility patterns of various Candida species isolated from blood samples and provide their identification by molecular characterization. Methods. Antifungal susceptibility testing was performed using the broth microdilution method. The sequencing of the ITS and D1/D2 regions of rDNA was used for molecular characterization. Results. Seventy-four of the 80 isolates were susceptible to anidulafungin, 5 were intermediate, and 1 was resistant. For micafungin 67 were susceptible, 8 were intermediate and 5 were resistant. All isolates were susceptible to amphotericin B. Lastly, 65 isolates were susceptible to fluconazole, 8 were dose-dependent and 4 were resistant. The molecular identification corroborated the phenotypic data in 91.3% of the isolates. Conclusions. Antifungal susceptibility data has an important role in the treatment of candidemia episodes. It was also concluded that the molecular analysis of isolates provides an accurate identification and identifies genetic variability within Candida species isolated from patients with candidemia (AU)

Antecedentes. Los hongos del género Candida son la causa principal de infección micótica del torrente sanguíneo adquirida en el hospital. Entre los factores de riesgo asociados a la candidemia destacan la nutrición parenteral, la estancia prolongada en una unidad de cuidados intensivos, la neutropenia, la diabetes, la cirugía abdominal y la utilización de catéter venoso central. Los agentes antifúngicos más utilizados para tratarla son las equinocandinas, pero determinados aislamientos son resistentes a dichos componentes, por lo que algunos pacientes no responden al tratamiento. Objetivos. Este trabajo tiene como objetivo evaluar la sensibilidad in vitro de varios aislamientos de Candida procedentes de muestras de sangre y realizar su caracterización molecular. Métodos. Se hicieron pruebas de sensibilidad a los antifúngicos mediante el método de microdilución en caldo. Para la caracterización molecular se utilizó la secuenciación de las regiones ITS y D1/D2 del DNAr. Resultados. De los 80 aislamientos evaluados, 74 fueron sensibles a la anidulafungina, 5 mostraron sensibilidad intermedia y solo uno era resistente. Cuando se utilizó la micafungina, 67 aislamientos resultaron sensibles, 8 presentaron sensibilidad intermedia y 5 fueron resistentes. Los 80 aislamientos fueron sensibles a la anfotericina B. Al menos 65 aislamientos eran sensibles al fluconazol, 8 presentaron sensibilidad dependiente de la dosis y 4 se mostraron resistentes. La identificación molecular confirmó la identificación fenotípica en un 91,3% de los aislamientos. Conclusiones. Teniendo en cuenta los resultados obtenidos con las pruebas de sensibilidad a los antifúngicos, estas resultan indispensables para el tratamiento adecuado de la candidemia. Se concluye además que la identificación molecular proporciona una identificación precisa y consigue identificar la variabilidad genética de las especies del género Candida aisladas en pacientes con candidemia (AU)

In vitro susceptibility and molecular characterization of Candida spp. from candidemic patients.

BACKGROUND: Candida species are the main cause of hospital acquired fungal bloodstream infections. The main risk factors for candidemia include parenteral nutrition, long-term intensive care, neutropenia, diabetes, abdominal surgery and the use of central venous catheters. The antifungal drugs used to treat candidemia are mainly the echinocandins, however some isolates may be resistant to these drugs. AIMS: This work aims to evaluate the in vitro susceptibility patterns of various Candida species isolated from blood samples and provide their identification by molecular characterization. METHODS: Antifungal susceptibility testing was performed using the broth microdilution method. The sequencing of the ITS and D1/D2 regions of rDNA was used for molecular characterization. RESULTS: Seventy-four of the 80 isolates were susceptible to anidulafungin, 5 were intermediate, and 1 was resistant. For micafungin 67 were susceptible, 8 were intermediate and 5 were resistant. All isolates were susceptible to amphotericin B. Lastly, 65 isolates were susceptible to fluconazole, 8 were dose-dependent and 4 were resistant. The molecular identification corroborated the phenotypic data in 91.3% of the isolates. CONCLUSIONS: Antifungal susceptibility data has an important role in the treatment of candidemia episodes. It was also concluded that the molecular analysis of isolates provides an accurate identification and identifies genetic variability within Candida species isolated from patients with candidemia.

Onychomycosis by Fusarium oxysporum probably acquired in utero.

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.

Avalia ao da susceptibilidade a antifungicos de diferentes espécies de leveduras candida isoladas de mucosa bucal e pele / Evaluation of antifungal susceptibility of different species of candida yeast isolated from buccal mucosa and skin

A variabilidade das diferentes especies de Candida sp, juntamente com as distintas respostas as formas de tratamento, desenvolveram a necessidade da utiliza tao de diferentes metodos diagn6sticos e esquemas terapeuticos. Entre as leveduras deste grupo, a Candida albicans e urn dos pat6genos mais comuns envolvidos nas candidiases mucocutaneas e da orofaringe, porem as especies nao albicans tern aumentado em numero e em importancia devido ao acrescimo do perfil de resistencia aos antifUngicos. A resistencia da Candida albicans e das especies nao albicans ao fluconazole outros derivados az61icos e descrito na literatura com freqOencia, o que torna importante a realiza tao de testes de susceptibilidade. Neste contexte esta pesquisa tern como objetivo determinar o perfil de susceptibilidade das leveduras Candida em diferentes sftios anatomicos. As leveduras isoladas da mucosa bucal e de pele foram semeadas em CHROMagar Candida, incubadas por 48 horas, a 35°C sendo posteriormente identificadas e avaliado in vitro o perfil de susceptibilidade utilizando o metodo da macrodilui tao. Entre as 25 amostras analisadas, verificou-se urn perfil de resistencia maior ao fluconazol em compara tao ao cetoconazol, sendo que 44% dos isolados de boca e 50% de pele mostraram-se resistentes ao fluconazol.

The variability of the different species of Candida sp, together with the distinct responses to treatment, elicited the need of using different diagnostic methods and therapeutic programs. Among the yeast of this group, Candida albicans is one of the most common pathogens involved in muco-cutaneous and oropharingeal candidiases, but the non-albicans species have increased in number and importance due to antifungal resistance. The resistance of Candida albicans and the non-albicans species to fluconazole and other azolic derivatives is frequently reported, which makes susceptibility tests important. Within this scope, this research has the purpose of determining the susceptibility profile of Candida yeast from different anatomical sites. The yeast cells isolated from buccal mucosa and skin were seeded in CHROMagar Candida, incubated for 48 hours at 35°C; later, they were identified and the susceptibility profile was assessed in vitro using themacrodilution method. Of the 25 samples analyzed, a higher profile of resistance to fluconazole compared to ketoconazole was noticed, where 44% ofthe buccal and 50% ofthe skin isolates were resistant to fluconazole.