In vitro susceptibility of environmental isolates of Exophiala dermatitidis to five antifungal drugs.

Several dematiaceous fungi frequently isolated from nature are involved in cases of superficial lesions to lethal cerebral infections. Antifungal susceptibility data on environmental and clinical isolates are still sparse despite the advances in testing methods. The objective of this study was to examine the activities of 5-flucytosine, amphotericin B, itraconazole, voriconazole and terbinafine against environmental isolates of Exophiala strains by minimum inhibition concentration (MIC) determination. The strains were obtained from hydrocarbon-contaminated soil, ant cuticle and fungal pellets from the infrabuccal pocket of attine gynes. Broth microdilution assay using M38-A2 reference methodology for the five antifungal drugs and DNA sequencing for fungal identification were applied. Terbinafine was the most active drug against the tested strains. It was observed that amphotericin B was less effective, notably against Exophiala spinifera, also studied. High MICs of 5-flucytosine against Exophiala dermatitidis occurred. This finding highlights the relevance of studies on the antifungal resistance of these potential opportunistic species. Our results also contribute to a future improvement of the standard methods to access the drug efficacy currently applied to black fungi.

Fungal Screening for Potential PET Depolymerization.

Approximately 400 billion PET bottles are produced annually in the world, of which from 8 to 9 million tons are discarded in oceans. This requires developing strategies to urgently recycle them. PET recycling can be carried out using the microbial hydrolysis of polymers when monomers and oligomers are released. Exploring the metabolic activity of fungi is an environmentally friendly way to treat harmful polymeric waste and obtain the production of monomers. The present study addressed: (i) the investigation of potential of strains with the potential for the depolymerization of PET bottles from different manufacturers (crystallinity of 35.5 and 10.4%); (ii) the search for a culture medium that favors the depolymerization process; and (iii) gaining more knowledge on fungal enzymes that can be applied to PET recycling. Four strains (from 100 fungal strains) were found as promising for conversion into terephthalic acid from PET nanoparticles (npPET): Curvularia trifolii CBMAI 2111, Trichoderma sp. CBMAI 2071, Trichoderma atroviride CBMAI 2073, and Cladosporium cladosporioides CBMAI 2075. The fermentation assays in the presence of PET led to the release of terephthalic acid in concentrations above 12 ppm. Biodegradation was also confirmed using mass variation analyses (reducing mass), scanning electron microscopy (SEM) that showed evidence of material roughness, FTIR analysis that showed band modification, enzymatic activities detected for lipase, and esterase and cutinase, confirmed by monomers/oligomers quantification using high performance liquid chromatography (HPLC-UV). Based on the microbial strains PET depolymerization, the results are promising for the exploration of the selected microbial strain.

Sugarcane: an unexpected habitat for black yeasts in Chaetothyriales.

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.

Isolation and identification of black yeasts by enrichment on atmospheres of monoaromatic hydrocarbons.

Black yeast members of the Herpotrichiellaceae present a complex ecological behavior: They are often isolated from rather extreme environments polluted with aromatic hydrocarbons, while they are also regularly involved in human opportunistic infections. A selective technique to promote the in vitro growth of herpotrichiellaceous fungi was applied to investigate their ecophysiology. Samples from natural ecological niches and man-made environments that might contain black yeasts were enriched on an inert solid support at low humidity and under a controlled atmosphere rich in volatile aromatic hydrocarbons. Benzene, toluene, and xylene were provided separately as the sole carbon and energy source via the gas phase. The assayed isolation protocol was highly specific toward mesophilic Exophiala species (70 strains of this genus out of 71 isolates). Those were obtained predominantly from creosote-treated railway ties (53 strains), but isolates were also found on wild berries (11 strains) and in guano-rich soil samples (six strains). Most of the isolates were obtained on toluene (43 strains), but enrichments on xylene and benzene also yielded herpotrichiellaceous fungi (17 and 10 isolates, respectively). Based upon morphological characterizations and DNA sequences of the full internal transcriber spacers (ITS) and the 8.5S rRNA genes, the majority of the obtained isolates were affiliated to the recently described species Exophiala xenobiotica (32 strains) and Exophiala bergeri (nine strains). Members of two other phylogenetic groups (24 and two strains, respectively) somewhat related to E. bergeri were also found, and a last group (three strains) corresponded to an undescribed Exophiala species.

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.

Biodegradation of PCL and PVC: Chaetomium globosum (ATCC 16021) activity.

The increasing use of plastics in human activities has resulted in an enormous amount of residues which became a matter of great environmental concern. Scientific studies on the microbial degradation of natural and synthetic molecules show the potential of fungal application on cleaning technologies. The biodegradation of PCL (polycaprolactone) and PVC (polyvinyl chloride) films by Aspergillus brasiliensis (ATCC 9642), Penicillium funiculosum (ATCC 11797), Chaetomium globosum (ATCC 16021), Trichoderma virens (ATCC 9645), and Paecilomyces variotii (ATCC 16023) was studied. According to ISO 846-1978-"Testing of Plastics - Influence of fungi and bacteria", samples of the studied polymers were inoculated with a mix suspension of 106 fungal inoculum and maintained in moisture glass chambers in a bacteriological incubator at 28 °C for 28 days. The samples were analyzed by means of morphological and color changes, mass loss, optical microscopy (OM), and scanning electron microscopy (SEM) after 28 days of culturing. After the incubation period, visual observations of the PCL films showed many micropores and cracks, pigmentation, surface erosion and hyphal adhesion on the sample surfaces, and a mass loss of up to 75%. On the contrary, there was no evidence of PVC biodegradation, such as changes in color and significant mass loss. Chaetomium globosum ATCC 16021 was a pioneer in the colonization and attack of PCL, resulting in significant mass losses. Although PVC was less attacked by the ascomycete, the polymer supported the adhesion and growth of its fertile structures (perithecia), suggesting the fungal potential to degrade both plastics.

Comparison of specific endophytic bacterial communities in different developmental stages of Passiflora incarnata using culture-dependent and culture-independent analysis.

Plants and endophytic microorganisms have coevolved unique relationships over many generations. Plants show a specific physiological status in each developmental stage, which may determine the occurrence and dominance of specific endophytic populations with a predetermined ecological role. This study aimed to compare and determine the structure and composition of cultivable and uncultivable bacterial endophytic communities in vegetative and reproductive stages (RS) of Passiflora incarnata. To that end, the endophytic communities were assessed by plating and Illumina-based 16S rRNA gene amplicon sequencing. Two hundred and four cultivable bacterial strains were successfully isolated. From the plant's RS, the isolated strains were identified mainly as belonging to the genera Sphingomonas, Curtobacterium, and Methylobacterium, whereas Bacillus was the dominant genus isolated from the vegetative stage (VS). From a total of 133,399 sequences obtained from Illumina-based sequencing, a subset of 25,092 was classified in operational taxonomy units (OTUs). Four hundred and sixteen OTUs were obtained from the VS and 66 from the RS. In the VS, the most abundant families were Pseudoalteromonadaceae and Alicyclobacillaceae, while in the RS, Enterobacteriaceae and Bacillaceae were the most abundant families. The exclusive abundance of specific bacterial populations for each developmental stage suggests that plants may modulate bacterial endophytic community structure in response to different physiological statuses occurring at the different plant developmental stages.

Hydrocarbon-associated substrates reveal promising fungi for poly (ethylene terephthalate) (PET) depolymerization.

Recalcitrant characteristics and insolubility in water make the disposal of synthetic polymers a great environmental problem to be faced by modern society. Strategies towards the recycling of post-consumer polymers, like poly (ethylene terephthalate, PET) degradation/depolymerization have been studied but still need improvement. To contribute with this purpose, 100 fungal strains from hydrocarbon-associated environments were screened for lipase and esterase activities by plate assays and high-throughput screening (HTS), using short- and long-chain fluorogenic probes. Nine isolates were selected for their outstanding hydrolytic activity, comprising the genera Microsphaeropsis, Mucor, Trichoderma, Westerdykella, and Pycnidiophora. Two strains of Microsphaeropsis arundinis were able to convert 2-3% of PET nanoparticle into terephthalic acid, and when cultured with two kinds of commercial PET bottle fragments, they also promoted weight loss, surface and chemical changes, increased lipase and esterase activities, and led to PET depolymerization with release of terephthalic acid at concentrations above 20.0 ppm and other oligomers over 0.6 ppm. The results corroborate that hydrocarbon-associated areas are important source of microorganisms for application in environmental technologies, and the sources investigated revealed important strains with potential for PET depolymerization.

Microbial ex situ preservation supporting science and bioeconomy in Brazil / Preservação de microrganismos ex situ como suporte para a ciência e a bioeconomia no Brasil

Abstract One of the texts in the "Biodiversity in the State of São Paulo" series, within the FAPESP-Biota Program, was dedicated to the Infrastructure for Biodiversity Conservation, with a focus on Biological Collections and Conservation Units. From the early 1960s, when FAPESP was established, to the present day, financial resources have been invested in the preservation of the biodiversity of the national genetic heritage, besides other fields. History of years of advances in scientific knowledge was built, which can be portrayed through the projects that resulted in high-quality data of national and international impact. Microbiological collections are centers that generate technology and specialized human resources, and act (among other things) as living repositories preserving reference material and as witnesses to the history of microbial biodiversity because they preserve what may no longer exist. They have enormous potential to promote the global bioeconomy and address problems that have resulted from the misuse of natural resources. This reading brings everyone the history, advances, and future perspectives of culture collections, within the efforts of 60-year scientific activities in Brazil.

Resumo Um dos textos da série "Biodiversidade do Estado de São Paulo", dentro do Programa FAPESP-Biota, foi dedicado à Infraestrutura para Conservação da Biodiversidade, com foco nas coleções biológicas e nas unidades de conservação. Do início dos anos 60, quando a FAPESP foi criada, até os dias atuais muito foi investido em pesquisa nas mais diversas áreas, incluindo a preservação da biodiversidade do patrimônio genético nacional. Uma história de longos anos de avanços no conhecimento científico foi construída, a qual pode ser retratada através dos projetos que resultaram em dados de alta qualidade com impacto nacional e internacional. As coleções microbiológicas são centros geradores de tecnologia e recursos humanos especializados, que atuam (dentre outros) como repositórios vivos, preservando material de referência, e como testemunho da história da biodiversidade microbiana, preservando o que pode não mais existir. Possuem enorme potencial para alavancar a bioeconomia global e tratar de problemas que resultaram do mau uso dos recursos naturais. Essa leitura traz a todos o histórico, os avanços e as perspectivas futuras das coleções de microrganismos, dentro dos esforços de 60 anos de atividades científicas no Brasil.

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.

Fontes de carbono de baixo custo para a produção de xilanase termoestável por Aspergillus niger / Low-cost carbon sources for the production of a thermostable xylanase by Aspergillus niger

A strain of the filamentous fungus Aspergillus niger was isolated and shown to possess extracellular xylanolytic activity. These enzymes have biotechnological potential and can be employed in various industries. This fungus produced its highest xylanase activity in a medium made up of 0.1% CaCO3, 0.5% NaCl, 0.1% NH4Cl, 0.5% corn steep liquor and 1% carbon source, at pH 8.0. A low-cost hemicellulose residue (powdered corncob) proved to be an excellent inducer of the A. niger xylanolytic complex. Filtration of the crude culture medium with suspended kaolin was ideal for to clarify the extract and led to partial purification of the xylanolytic activity. The apparent molecular mass of the xylanase was about 32.3 kDa. Maximum enzyme activity occurred at pH 5.0 and 55-60ºC. Apparent Km was 10.41 ± 0.282 mg/mL and Vmax was 3.32 ± 0.053 U/mg protein, with birchwood xylan as the substrate. Activation energy was 4.55 kcal/mol and half-life of the crude enzyme at 60ºC was 30 minutes. Addition of 2% glucose to the culture medium supplemented with xylan repressed xylanase production, but in the presence of xylose the enzyme production was not affected.

Uma linhagem do fungo filamentoso Aspergillus niger foi isolada e apresentou atividade xilanolítica extracelular. Estas enzimas possuem grande potencial biotecnológico e podem ser aplicadas em diversas indústrias. O fungo produziu sua maior atividade de xilanase em um meio contendo CaCO3 0,1%, NaCl 0,5%, NH4Cl 0,1%, 0,5% água de maceração de milho e 1% de fonte de carbono, em pH 8,0. Um resíduo lignocelulósico de baixo custo (sabugo de milho em pó) mostrou ser um excelente indutor do complexo xilanolítico em A. niger. A filtração do extrato cru com caulim foi ideal para a clarificação do extrato e levou à purificação parcial da enzima. A massa molecular aparente da xilanase foi de 32,3 kDa. A máxima atividade da enzima ocorreu em pH 5,0 e a 55-60ºC. O Km aparente foi de 10,41 ± 0,282 mg/mL e a Vmax foi de 3,32 ± 0,053 U/mg proteína, utilizando-se xilana birchwood como substrato. A energia de ativação foi de 4,55 kcal/mol, e a meia-vida da enzima a 60ºC foi de 30 minutos. A adição de 2% de glicose ao meio de cultura suplementado com xilana reprimiu a produção de xilanase, mas em presença de xilose a produção da enzima não foi afetada.