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.

Primary Central Nervous System Infection by Histoplasma in an Immunocompetent Adult.

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.

New Molecular Markers Distinguishing Fonsecaea Agents of Chromoblastomycosis.

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.

Fungal infections in animals: a patchwork of different situations.

The importance of fungal infections in both human and animals has increased over the last decades. This article represents an overview of the different categories of fungal infections that can be encountered in animals originating from environmental sources without transmission to humans. In addition, the endemic infections with indirect transmission from the environment, the zoophilic fungal pathogens with near-direct transmission, the zoonotic fungi that can be directly transmitted from animals to humans, mycotoxicoses and antifungal resistance in animals will also be discussed. Opportunistic mycoses are responsible for a wide range of diseases from localized infections to fatal disseminated diseases, such as aspergillosis, mucormycosis, candidiasis, cryptococcosis and infections caused by melanized fungi. The amphibian fungal disease chytridiomycosis and the Bat White-nose syndrome are due to obligatory fungal pathogens. Zoonotic agents are naturally transmitted from vertebrate animals to humans and vice versa. The list of zoonotic fungal agents is limited but some species, like Microsporum canis and Sporothrix brasiliensis from cats, have a strong public health impact. Mycotoxins are defined as the chemicals of fungal origin being toxic for warm-blooded vertebrates. Intoxications by aflatoxins and ochratoxins represent a threat for both human and animal health. Resistance to antifungals can occur in different animal species that receive these drugs, although the true epidemiology of resistance in animals is unknown, and options to treat infections caused by resistant infections are limited.

Influence of Culturing Conditions on Bioprospecting and the Antimicrobial Potential of Endophytic Fungi from Schinus terebinthifolius.

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.

Molecular identification of Histoplasma capsulatum using rolling circle amplification.

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.

Fonsecaea pugnacius, a Novel Agent of Disseminated Chromoblastomycosis.

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.

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.

Genome Sequence of the Human Opportunistic Fungus Arthrocladium fulminans (CBS 136243).

The black yeast-like fungus Arthrocladium fulminans is known from strains that cause severe and eventually fatal disseminated infections in immunocompromised patients. Given the dramatic outcome of this clinical case, it is essential to understand the virulence potential of this species. The fungus is a member of the family Trichomeriaceae, at some phylogenetic distance from the Herpotrichiellaceae where most infectious fungi in the order Chaetothyriales are located. Main ecological preferences among Trichomeriaceae include colonization of exposed inert surfaces. Currently, black yeasts genomes that are available in public databases cover members of the families Herpotrichiellaceae and Cyphellophoraceae In the present report, we sequenced the genome of the first member and only clinical representative of the family Trichomeriaceae.