Skin innate immune response against fungal infections and the potential role of trained immunity.

Fungal skin infections are distributed worldwide and can be associated with economic and social traits. The immune response related to skin cells is complex and its understanding is essential to the comprehension of each cell's role and the discovery of treatment alternatives. The first studies of trained immunity (TI) described the ability of monocytes, macrophages and natural killer (NK) cells to develop a memory-like response. However, the duration of TI does not reflect the shorter lifespan of these cells. These conclusions supported later studies showing that TI can be observed in stem and haematopoietic cells and, more recently, also in non-immune skin cells such as fibroblasts, highlighting the importance of resident cells in response to skin disorders. Besides, the participation of less studied proinflammatory cytokines in the skin immune response, such as IL-36γ, shed light into a new possibility of inflammatory pathway blockade by drugs. In this review, we will discuss the skin immune response associated with fungal infections, the role of TI in skin and clinical evidence supporting opportunities and challenges of TI and other inflammatory responses in the pathogenesis of fungal skin infections.

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.

Genotyping and antifungal susceptibility testing of Sporothrix brasiliensis isolates from Southern Brazil.

Sporotrichosis is an implantation mycosis caused by the dimorphic fungus Sporothrix and mostly involves cutaneous and subcutaneous tissues and the lymphatic vessels. Among more than 50 different species, only Sporothrix schenckii, Sporothrix globosa and Sporothrix brasiliensis are frequently reported to cause infections in humans. Sporothrix brasiliensis is remarkably virulent and has been spreading rapidly in Brazil and other Latin American countries. In this study, we aimed to determine the genetic relatedness and antifungal susceptibility of Sporothrix strains by analysing 89 isolates from humans and cats in Curitiba, Southern Brazil. Calmodulin sequencing identified 81 S. brasiliensis and seven S. schenckii isolates. Amplified fragment length polymorphism genotyping analysis showed feline and human isolates clustering together. In vitro susceptibility testing with seven antifungals demonstrated a broad activity against all tested S. brasiliensis isolates, with no significant differences in minimal inhibitory concentration (MIC) values between feline and human isolates. Resistance was solely observed in one human isolate against itraconazole and posaconazole, with MICs of ≥16 µg/mL against both antifungals. Whole genome sequencing (WGS) analysis on this isolate and two related susceptible isolates did not reveal any unique substitutions in resistance-associated genes, including cyp51, hmg and erg6, when compared to two related susceptible isolates. The novel antifungal olorofim exhibited excellent activity against this large isolate collection, with all isolates considered as susceptible. Altogether, we indicate zoonotic transmission based on genotyping and revealed a broad activity of seven common antifungals, including olorofim, against a large S. brasiliensis isolate collection.

New Insights on Environmental Occurrence of Pathogenic Fungi Based on Metagenomic Data from Brazilian Cerrado Biome

Abstract: Cerrado is the second largest biome in Brazil and majorly contributes to the country's grain production. Previous studies on soil metagenomics from the Cerrado revealed an outstanding microbial diversity. In this study, the abundance of pathogenic fungi was analyzed using metagenomic sequences of the Cerrado soils under native vegetation, and under agriculture with no-tillage and conventional tillage. In total, 128,627 sequences of fungi were identified, with 43,439 representing pathogenic fungi and were distributed as follows: native 17,301 (40%), no-tillage 13,780 (32%), and conventional tillage 12,358 (28%). We identified 41 pathogenic fungal species associated with human and animal infections. The data analysis revealed that the native soils had a higher relative abundance of fungal sequences, similar to pathogenic species sequences, in relation to the total eukaryotic sequences, than the conventional tillage and no-tillage treatments, which observed a reduction in fungal abundance because of anthropogenic activities.

Agrobacterium tumefaciens-Mediated Transformation of Fonsecaea monophora and Fonsecaea erecta for Host-Environment Interaction Studies.

The fungal genus Fonsecaea contains etiological agents of human chromoblastomycosis, a (sub)tropical, (sub)cutaneous implantation disease caused by plant contact. The invasive potential differs significantly between species. Infections by Fonsecaea monophora are believed to originate from the environment and the species has been reported as one of the main causative agents of the disease, but also of cases of primary brain infection. The epidemiology of the disease has not been fully elucidated and questions related to its infection route and virulence are still to be clarified. The environmental species Fonsecaea erecta was isolated from organic material and living plants in endemic areas for chromoblastomycosis in Brazil. The present paper describes Agrobacteriumtumefaciens-mediated transformation (AMT) of the environmental species F. erecta and the pathogenic species F. monophora. We propose the use of Agrobacterium transformation for future gene function studies related to Fonsecaea virulence and pathogenicity. We evaluated the co-cultivation ratios 1:1, 10:1 and 100:1 (Agrobacterium:conidia) at 28 °C during 72 h. pAD1625 and pCAMDsRed plasmids were inserted into both species. Confirmation of transformation was realized by hph gene amplification and Southern blot determined the amount of foreign DNA integrated into the genome. In order to evaluate a potential link between environmental and clinical strains, we obtained red fluorescent transformants after pCAMDsRed insertion. We observed by confocal fluorescence microscopy that both F. monophora and F. erecta were able to colonize the palm Bactris gasipaes, penetrating the epidermis. These results contribute to understanding the ability of Fonsecaea species to adapt to different environmental and host conditions.

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.

Genomics and Virulence of Fonsecaea pugnacius, Agent of Disseminated Chromoblastomycosis.

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.

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.

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.

Fusarium oxysporum is an onychomycosis etiopathogenic agent.

AIM: To evaluate and characterize the etiopathogenesis of the fusarial onychomycosis in an ex vivo study through fragments of sterile human nail, without the addition of any nutritional source. MATERIALS & METHODS: The infection and invasion of Fusarium oxysporum in the nail were evaluated by scanning electron microscopy (SEM), CFU, matrix, histopathology and Fourier Transform Infrared Spectrometer coupled to an equipment with diamond accessory (FTIR-ATR). RESULTS: F. oxysporum infected and invaded across the nail, regardless of application face. However, the dorsal nail surface was the strongest barrier, while the ventral was more vulnerable to infection and invasion process. The fungal-nail interaction resulted in the formation of a dense biofilm. CONCLUSION: F. oxysporum infect and invade the healthy human nail, resulting in biofilm formation. Therefore, F. oxysporum is likely a primary onychomycosis agent.

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.

Genome Sequence of Type Strain Fonsecaea multimorphosa CBS 980.96T, a Causal Agent of Feline Cerebral Phaeohyphomycosis.

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.

Draft Genome Sequence of Fonsecaea nubica Strain CBS 269.64, Causative Agent of Human Chromoblastomycosis.

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.

Draft Genome Sequence of Fonsecaea monophora Strain CBS 269.37, an Agent of Human Chromoblastomycosis.

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.

Resistance to extended-spectrum ß-lactamases in Salmonella from a broiler supply Chain.

The prevalence of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae varies worldwide, however, the incidence of ESBL-producing environmental Salmonella isolates is increasing. Salmonella is still one of the most important pathogens that occur in the poultry supply chain. Therefore, this study analyzed the susceptibility of Salmonella isolates collected from a poultry supply chain to ß-lactam antibiotics, and examined the phenotypes of the isolates based on enzyme-inducible AmpC ß-lactamase analysis. All analysis of the putative positive isolates in the current study confirmed that 27.02% (77/285 analysis) of all ESBL tests realized with the isolates produced a profile of resistance consistent with ß-lactamase production. All isolates of S. Minnesota serotype had ESBL phenotype. Aztreonam resistance was the least common amongst the Salmonella isolates, followed by ceftazidime. The presence of inducible chromosomal ESBL was detected in 14 different isolates of the 19 serotypes investigated. These results are very indicatives of the presence of ESBL genes in Salmonella isolates from a broiler supply chain, reaffirming the growing global problem of ESBL resistance.