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Abstract Cryptococcal meningitis is a high-mortality infection. Adding 5-fluorocytosine (5-FC) to its treatment improves outcomes, but resistance to 5-FC presents a significant challenge. We conducted whole-genome sequencing on seven C. neoformans isolates with varying 5-FC susceptibility, along with proteomic and in silico analyses. Our findings indicate that mutations in genes of the pyrimidine salvage pathway are responsible for 5-FC resistance. Specifically, we identified an E64G missense mutation in the FUR1 gene, a large deletion in the FCY1 gene, and a point mutation in FCY1 leading to a truncated protein. The proteomic data indicated that these mutations resulted in the absence or reduction of crucial enzymes in resistant isolates. Genetic transformations confirmed the association between these mutations and 5-FC resistance. Resistance to 5-FC can develop during treatment and is closely tied to mutations in key metabolic enzymes. Understanding in vivo resistance development is crucial for combating resistance and enhancing patient outcomes.

ABSTRACT Dimorphism is known among the etiologic agents of endemic mycoses as well as in filamentous Mucorales. Under appropriate thermal conditions, mononuclear yeast forms alternate with multi-nucleate hyphae. Here, we describe a dimorphic mucoralean fungus obtained from the sputum of a patient with Burkitt lymphoma and ongoing graft-versus-host reactions. The fungus is described as Mucor germinans sp. nov. Laboratory studies were performed to simulate temperature-dependent dimorphism, with two environmental strains Mucor circinelloides and Mucor kunryangriensis as controls. Both strains could be induced to form multinucleate arthrospores and subsequent yeast-like cells in vitro. Multilateral yeast cells emerge in all three Mucor species at elevated temperatures. This morphological transformation appears to occur at body temperature since the yeast-like cells were observed in the lungs of our immunocompromised patient. The microscopic appearance of the yeast-like cells in the clinical samples is easily confused with that of Paracoccidioides. The ecological role of yeast forms in Mucorales is discussed.IMPORTANCEMucormycosis is a devastating disease with high morbidity and mortality in susceptible patients. Accurate diagnosis is required for timely clinical management since antifungal susceptibility differs between species. Irregular hyphal elements are usually taken as the hallmark of mucormycosis, but here, we show that some species may also produce yeast-like cells, potentially being mistaken for Candida or Paracoccidioides. We demonstrate that the dimorphic transition is common in Mucor species and can be driven by many factors. The multi-nucleate yeast-like cells provide an effective parameter to distinguish mucoralean infections from similar yeast-like species in clinical samples.

Abstract Background Chromoblastomycosis is the World Health Organization (WHO)-recognized fungal implantation disease that eventually leads to severe mutilation. Cladophialophora carrionii (C. carrionii) is one of the agents. However, the pathogenesis of C. carrionii is not fully investigated yet. Methods We investigated the pathogenic potential of the fungus in a Galleria mellonella (G. mellonella) larvae infection model. Six strains of C. carrionii, and three of its environmental relative C. yegresii were tested. The G. mellonella model was also applied to determine antifungal efficacy of amphotericin B, itraconazole, voriconazole, posaconazole, and terbinafine. Results All strains were able to infect the larvae, but virulence potentials were strain-specific and showed no correlation with clinical background of the respective isolate. Survival of larvae also varied with infection dose, and with growth speed and melanization of the fungus. Posaconazole and voriconazole exhibited best activity against Cladophialophora, followed by itraconazole and terbinafine, while limited efficacy was seen for amphotericin B. Conclusion Infection behavior deviates significantly between strains. In vitro antifungal susceptibility of tested strains only partly explained the limited treatment efficacy in vivo.

ABSTRACTThe present paper includes a meta-analysis of literature data on 318 species of fungi belonging to 34 orders in their response to 8 antifungal agents (amphotericin B, caspofungin, fluconazole, itraconazole, ketoconazole, posaconazole, terbinafine, and voriconazole). Main trends of MIC results at the ordinal level were visualized. European Committee on Antimicrobial Susceptibility Testing and Clinical & Laboratory Standards Institute (CLSI) clinical breakpoints were used as the staff gauge to evaluate MIC values ranging from resistance to susceptibility, which were subsequently compared with a phylogenetic tree of the fungal kingdom. Several orders (Hypocreales, Microascales, and Mucorales) invariably showed resistance. Also the basidiomycetous orders Agaricales, Polyporales, Sporidiales, Tremellales, and Trichosporonales showed relatively high degrees of azole multi-resistance, while elsewhere in the fungal kingdom, including orders with numerous pathogenic and opportunistic species, that is, Onygenales, Chaetothyiales, Sordariales, and Malasseziales, in general were susceptible to azoles. In most cases, resistance vs susceptibility was consistently associated with phylogenetic distance, members of the same order showing similar behavior.IMPORTANCEA kingdom-wide the largest set of published wild-type antifungal data comparison were analyzed. Trends in resistance in taxonomic groups (monophyletic clades) can be compared with the phylogeny of the fungal kingdom, eventual relationships between fungus–drug interaction and evolution can be described.

Abstract 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.

ABSTRACTDermatophytic pseudomycetoma is a rare invasive infection, involving both immunocompetent and immunocompromised individuals. Since the discovery of inherited immune disorders such as the impairment of CARD9 gene, extended dermatophyte infections are mostly ascribed to any of these host factors. This study is to present and explore the potential causes in a fatal dermatophytic pseudomycetoma patient. We present a chronic and deep pseudomycetoma caused by the common dermatophyte Microsporum canis which ultimately led to the death of the patient. Mycological examination, genetic studies and host immune responses against fungi were performed to explore the potential factors. The patient had decreased lymphocyte counts with significantly reduced CD4+ T cells, although all currently known genetic parameters proved to be normal. Through functional studies, we demonstrated that peripheral blood mononuclear cells from the patient showed severe impairment of adaptive cytokine production upon fungus-specific stimulation, whereas innate immune responses were partially defective. This is, to our knowledge, the first report of fatal dermatophytic pseudomycetoma in a patient with non-HIV CD4 lymphocytopenia, which highlights the importance of screening for immune deficiencies in patients with deep dermatophytosis.

ABSTRACT Fungal rhinosinusitis (FRS) is a common problem worldwide, with an increasing burden in arid climate regions. Aspergillus species are the most common causative agents involved. In the present study, we investigated the prevalence, molecular characterization, and antifungal susceptibility of opportunists causing FRS in Sudan on the basis of strains collected over a period of 5 years. β-Tubulin and calmodulin sequencing were used for species identification, and antifungal susceptibility profiles were evaluated by the protocol of the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Predominant species belonged to the Aspergillus flavus complex (n = 244), A. terreus complex (n = 16), A. fumigatus complex (n = 7), and other fungi (n = 17). Molecular identification of 94 strains of Aspergillus revealed the following species: A. flavus (n = 88), A. terreus (n = 1), A. citrinoterreus (n = 2), A. fumigatus (n = 1), A. caespitosus (n = 1), and A. sydowii (n = 1). Several A. flavus and an A. fumigatus isolates showed reduced susceptibility to azoles (minimum inhibitory concentrations above the clinical breakpoints or epidemiological cutoff values). Despite several mutations revealed in cyp51A of these isolates, none could be directly linked to azole resistance. Molecular identification of fungi causing FRS is useful to identify cryptic species and for epidemiologic studies. IMPORTANCE Fungal rhinosinusitis (FRS) is a significant clinical problem in arid regions. This study provides new insights into the prevalence, etiology, and antifungal susceptibility of FRS pathogens in Sudan, where the disease burden is high. Aspergillus species, particularly the A. flavus complex, were identified as the primary FRS pathogens in the region, with some evidence of antifungal resistance. The molecular identification of fungal species causing FRS is useful for detecting antifungal resistance, identifying cryptic species, and characterizing the epidemiology of the disease. The emergence of Azole resistance Aspergilli in Sudan highlights the need for continued surveillance and appropriate use of antifungal agents. These findings have important implications for clinical management, public health policy, and future research on FRS. Publishing this study in Microbiology Spectrum would enable other researchers and clinicians to build on these findings, ultimately improving the diagnosis, treatment, and prevention of FRS.

Actinomortierella wolfii (Mortierellales), formerly Mortierella wolfii, is a causative agent of bovine systemic infection and abortion. Human infections caused by this species are extremely rare. Here, we present a case of a patient with B-Cell Acute Lymphoblastic Leukemia (B-ALL) who was diagnosed with a rhinocerebral infection caused by this fungus. Amphotericin treatment of the patient proved unsuccessful. This type of disease is otherwise nearly exclusively limited to members of the order Mucorales. The taxonomy of the causative agent is discussed.

Abstract Background The genus Sporothrix belongs to the order Ophiostomatales and contains mainly saprobic soil and plant fungi, although pathogenic species capable of causing human infections are also present. The whole-genomes of disease-causing species have already been sequenced and annotated but no comprehensive genomic resources for environmental Sporothrix species are available, thus limiting our understanding of the evolutionary origin of virulence-related genes and pathogenicity. Result The genome assembly of four environmental Sporothrix species resulted in genome size of ~ 30.9 Mbp in Sporothrix phasma, ~ 35 Mbp in S. curviconia, ~ 38.7 Mbp in S. protearum, and ~ 39 Mbp in S. variecibatus, with a variable gene content, ranging from 8142 (S. phasma) to 9502 (S. variecibatus). The analysis of mobile genetic elements showed significant differences in the content of transposable elements within the sequenced genomes, with the genome of S. phasma lacking several class I and class II transposons, compared to the other Sporothrix genomes investigated. Moreover, the comparative analysis of orthologous genes shared by clinical and environmental Sporothrix genomes revealed the presence of 3622 orthogroups shared by all species, whereas over 4200 genes were species-specific single-copy gene products. Carbohydrate-active enzyme analysis revealed a total of 2608 protein-coding genes containing single and/or multiple CAZy domains, resulting in no statistically significant differences among pathogenic and environmental species. Nevertheless, some families were not found in clinical species. Furthermore, for each sequenced Sporothrix species, the mitochondrial genomes was assembled in a single circular DNA molecule, ranging from 25,765 bp (S. variecibatus) to 58,395 bp (S. phasma). Conclusion In this study, we present four annotated genome assemblies generated using PacBio SMRT sequencing data from four environmental species: S. curviconia, S. phasma, S. protearum and S. variecibatus with the aim to provide a starting point for future comparative genome evolution studies addressing species diversification, ecological/host adaptation and origin of pathogenic lineages within the genus Sporothrix.

Paracoccidioides species have always been surrounded by taxonomic uncertainties. The continuing nomenclatoral muddle was caused in part by the failure of Adolfo Lutz and Jorge Lôbo to name the etiologic agents of human paracoccidioidomycosis and Jorge Lôbo's diseases, respectively. Early in their history, it was postulated that the cultivable species causing systemic infections belonged in the genus Paracoccidioides, whereas the uncultivable species, causing skin disease, were not part of the genus. The taxonomy of these pathogens was further complicated when a similar skin disease with numerous yeast-like cells in infected dolphins was also reported. Due to its phenotypic similarities with that described by Jorge Lôbo in human and its uncultivable nature, it was assumed that the disease in dolphins was caused by the same fungus. Recent molecular and population genetic analysis, however, found the DNA extracted from the uncultivable yeast-like cells affecting dolphins shared common phylogenetic traits with cultivable Paracoccidioides species. The study revealed that the uncultivable pathogens comprised 2 different Paracoccidioides species, now known as P. ceti and P. loboi, correspondingly. To validate P. loboi binomial, a comprehensive historical critical review of Jorge Lôbo etiology was performed. This review showed the proposed binomial P. loboi was previously used, and, thus, a replacement name is introduced, Paracoccidioides lobogeorgii nom. nov. In addition, in this review, several cultivable human Paracoccidioides species are validated, and the generic type species, P. brasiliensis, is neotypified as the original material could not be traced.

Abstract Some members of Chaetothyriales, an order containing potential agents of opportunistic infections in humans, have a natural habitat in nests of tropical arboreal ants. In these black fungi, two types of ant symbiosis are known, i.e. occurrence in domatia inside living plants, or as components of carton constructions made of ant-chewed plant tissue. In order to explain differences between strains from these types of association, we sequenced and annotated genomes of two newly described carton species, Incumbomyces lentus and Incumbomyces delicatus, and compared these with genomes of four domatia species and related Chaetothyriales. General genomic characteristics, CYP genes, carbohydrate-active enzymes (CAZymes), secondary metabolism, and sex-related genes were included in the study.

Magnusiomyces capitatus is an emerging opportunistic yeast, thus far mainly reported from the Western world where fungemia is the most frequent presentation in immunocompromised patients with high mortality. We described a rare case of Magnusiomyces capitatus infection from our hospital in China and reviewed six further cases published to date in Chinese literature. It is noted that half more of the cases (4/7) presented with fungemia in younger, immunosuppressed patients, whereas the remaining cases were with pneumonia in elderly, immunocompetent patients. All seven Chinese cases had favourable outcome with antifungal therapy. Based on the limited in vitro and clinical data, a combination of amphotericin B either with 5-fluorocytosine or voriconazole for fungemia in immunocompromised patients, and although fluconazole is not recommended as first-line therapy in the guideline, in our study, fluconazole alone or with 5-fluorocytosine for local pulmonary infection in immunocompetent patients is effective with good outcome.

CARD9-related inherited immune disorders are a major risk factor for chronic disseminated fungal infection. In addition to pathogens of Candida and dermatophytes, the environmental opportunists of the black yeast-like fungi are relatively frequent in this patient cohort. Particularly the genus Phialophora is overrepresented. We investigated two isolates of a strain of P. verrucosa residing in a CARD9 patient, sampled with a period of ten years apart. Genomes, melanization and antifungal susceptibility of progenitor and derived strains were compared, and potential adaptation to the host habitat was investigated with proteomic techniques using post-translational modification as a proxy. Global lactylation analysis was performed using high accuracy nano-LC-MS/MS in combination with enrichment of lactylated peptides from digested cell lysates, and subsequent peptide identification. The genome of the derived isolate had accumulated 6945 SNPs, of which 31 were detected in CDS. A large number of identified proteins were significantly enriched, e.g. in melanin biosynthesis. A total of 636 lactylation sites on 420 lactylated proteins were identified, which contained in 26 types of modification motifs. Lysine lactylation (Kla) was found in 23 constituent proteins of the ribosome, indicating an impact of Kla in protein synthesis. Twelve lactylated proteins participated in pathogenicity. A protein-protein interaction (PPI) network analysis suggested that protein lactylations are widely distributed influencing various biological processes. Our findings reveal widespread roles for lysine lactylation in regulating metabolism and melanin biosynthesis in black fungi. Several large rearrangements and inversions were observed in the genome, but genomic changes could not be linked to adaptation or to known clinically relevant properties of progenitor to derived isolate; in vitro antifungal susceptibility had largely remained unaltered.

ABSTRACT Seborrheic dermatitis (SD) is a common, chronic, and relapsing skin disease. The roles of Malassezia spp. in the pathogenesis of SD are still not clear due to the lack of direct evidence for the existence of hyphae within affected skin tissues. We set out to elucidate if Malassezia mycelium contributes to the onset and development of SD and if Malassezia mycelium is correlated with the clinical severity of SD patients. We detected Malassezia hyphae in patients with SD using potassium hydroxide (KOH) and calcofluor white (CFW) staining. Fluorescent microscopy was performed for the analysis of fungal cell wall and morphological characteristics of Malassezia under CFW staining. Culture growth in modified Dixon agar was used for DNA extraction and sequencing, and Malassezia species were confirmed by a sequencing data BLAST search against the NCBI database. We demonstrated that Malassezia hyphae were positively correlated with the clinical severity of SD patients (P = 3.1738 × 10−11). All the patients responded well to antifungal treatment. There is no significant difference for species dominance across the variant groups. However, the exact molecular mechanisms of how Malassezia spp. affect SD need to be further explored. The results show that Malassezia spp. in the hyphal stage are restricted to SD patients compared with healthy controls, suggesting that the presence of Malassezia hyphae contributes to the pathogenesis of SD. The results highlight the importance of the antifungal therapy for the future treatment of SD patients. IMPORTANCE Our results support the proposal that the hyphal form of Malassezia could be one of the pathogenic factors that contribute to SD, which has been previously less well studied. This clinical observation paves the way for further investigations of the molecular mechanisms of Malassezia hyphal pathogenicity in SD.

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.

Background and Purpose: Trichophyton tonsurans is a widely distributed anthropophilic dermatophyte causing different diseases of skin. In the literature limited data are available about the morphogenesis of vegetative mycelium of T. tonsurans and related anthropophilic dermatophytes. The aim of present study was to describe ultrastructural patterns of development, cellular organellography and septal pore apparatus structure of in vitro growing vegetative mycelium of T. tonsurans. Materials and Methods: Trichophyton tonsurans strain RCPFF 214/898 was grown on solid Czapek’s Agar (CzA) at 28ºС. For investigation of colonies morphology we used methods of light-, scanning and transmission electron microscopy (SEM and TEM). Results: Differences in morphogenesis of aerial and substrate hyphae were revealed. Mitochondrial reticulum and fibrosinous bodies were shown in T. tonsurans for the first time. The septal pore apparatus in hyphal cells of was comprised Woronin bodies and septal pore plugs. Woronin bodies (0.18 μm), located with 1‒4 near the pore, were spherical, membrane-bound, and had a homogeneous, electron-dense content. The cells of aerial and submerged hyphal cells of T. tonsurans contain two nuclei. Conclusion: Mature cells of substrate hyphae appeared more active than comparable cells in the aerial mycelium. During the maturation process, the differences in number and morphology of mitochondria, number of vacuoles, and in the synthesis of different types of storage substances were revealed. Presence of “mitochondrial reticulum” and variable types of storage substances in submerged hyphal cells suggested higher levels of metabolic activity compared to aerial mycelium.

ABSTRACTFilamentous basidiomycetes are uncommon agents of human diseases, despite their ubiquitous presence in the environment. We present a case of symptomatic pulmonary infection in a 38-year-old male with cough and fever; a thin-walled cyst in the posterior left upper pulmonary lobe was revealed by radiography. A non-sporulating fungus was isolated from sputum and biopsy material from the cyst. ITS and LSU sequences placed the fungus phylogenetically in Agaricales, family Cyphellaceae, and identified it as a member of shelf fungi in Gloeostereum, but without identity to any known species. The new species is described as Gloeostereum cimri. The clinical strain showed high MIC to voriconazole (>8 µg/ml) but had low MIC to amphotericin B (0.5 µg/ml).

The Global Action Fund for Fungal Infections (GAFFI) estimates that fungal diseases kill around 150 people each hour, and yet they are globally overlooked and neglected. Histoplasma and Talaromyces, which are associated with wildlife, cause systemic infections that are often lethal in patients with impaired cellular immunity. Dermatophytes that cause outbreaks in human hosts are often associated with domesticated animals. Changes in human behavior have been identified as a main cause of the emergence of animal-associated fungal diseases in humans, sometimes caused by the disturbance of natural habitats. An understanding of ecology and the transmission modes of causative agents is therefore essential. Here, we focus on fungal diseases contracted from wildlife and domesticated animals, their habitats, feces and carcasses. We discuss some basic fungal lifestyles and the risk of transmission to humans and illustrate these with examples from emerging and established diseases.

The Entomophthoromycotina, a subphylum close to the root of terrestrial fungi with a bias toward insects as their primary hosts, has been notoriously difficult to categorize taxonomically for decades. Here, we reassess the phylogeny of this group based on conserved genes encoding ribosomal RNA and RNA polymerase II subunits, confirming their general monophyly, but challenging previously assumed taxonomic relationships within and between particular clades. Furthermore, for the prominent, partially human-pathogenic taxon Conidiobolus, a new type species C. coronatus is proposed in order to compensate for the unclear, presumably lost previous type species C. utriculosus Brefeld 1884. We also performed an exhaustive survey of the broad host spectrum of the Entomophthoromycotina, which is not restricted to insects alone, and investigated potential patterns of co-evolution across their megadiverse host range. Our results suggest multiple independent origins of parasitism within this subphylum and no apparent co-evolutionary events with any particular host lineage. However, Pterygota (i.e., winged insects) clearly constitute the most dominantly parasitized superordinate host group. This appears to be in accordance with an increased dispersal capacity mediated by the radiation of the Pterygota during insect evolution, which has likely greatly facilitated the spread, infection opportunities, and evolutionary divergence of the Entomophthoromycotina as well.

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.

Eumycetoma (mycotic mycetoma) is the fungal form of mycetoma, a subcutaneous infection occurring in individuals living in endemic areas of the disease. The Sudan is hyperendemic for mycetoma, with the highest incidence being reported from Gezira State, Central Sudan. The present study was conducted at the Gezira Mycetoma Center and aimed to determine the cause of black-grain eumycetoma in the state and describe its epidemiology. Black-grain specimens were collected during the surgical operation and direct detection of the causative agent was performed using M. mycetomatis species-specific PCR and ITS PCR followed by sequencing. Black-grain was reported from 93.3% of all confirmed mycetoma cases (n = 111/119), with a prevalence in young males. Of the 91 samples subjected to direct PCR, 90.1% (n = 82) gave positive results. The predominant species (88.2%) was Madurella mycetomatis. One sample was identified as M. fahalii, one as M. tropicana, and one matched the phytopathogenic species Sphaerulina rhododendricola. The highest endemic zones were Southern Gezira (76.6%) and Northern Sinnar (23.4%). The study confirmed that direct molecular detection on grains provides rapid and specific diagnosis of agents of eumycetoma.

Chromoblastomycosis is a mutilating infection of the skin and subcutaneous tissues caused by melanized fungi belonging to the order Chaetothyriales. Proven cases of the main agent, Fonsecaea pedrosoi are mainly limited to (sub)tropical, humid climates of Latin and Central America and the Caribbean. Fonsecaea monophora has a global distribution along the equator. Cases outside the (sub)tropics have thus far mostly been considered to have been imported, but here we report the first endemic case by F. monophora from Argentina. Patient was a 82-year-old rural female worker from Corrientes, a province with a dry continental climate. Keywords: Dematiaceous fungi, Chromoblastomycosis, Fonsecaea monophora, South America, Argentina

Rhinocladiella mackenziei accounts for the majority of fungal brain infections in the Middle East, and is restricted to the arid climate zone between Saudi Arabia and Pakistan. Neurotropic dissemination caused by this fungus has been reported in immunocompromised, but also immunocompetent individuals. If untreated, the infection is fatal. Outside of humans, the environmental niche of R. mackenziei is unknown, and the fungus has been only cultured from brain biopsies. In this paper, we describe the whole-genome resequencing of two R. mackenziei strains from patients in Saudi Arabia and Qatar. We assessed intraspecies variation and genetic signatures to uncover the genomic basis of the pathogenesis, and potential niche adaptations. We found that the duplicated genes (paralogs) are more susceptible to accumulating significant mutations. Comparative genomics with other filamentous ascomycetes revealed a diverse arsenal of genes likely engaged in pathogenicity, such as the degradation of aromatic compounds and iron acquisition. In addition, intracellular accumulation of trehalose and choline suggests possible adaptations to the conditions of an arid climate region. Specifically, protein family contractions were found, including short-chain dehydrogenase/reductase SDR, the cytochrome P450 (CYP) (E-class), and the G-protein β WD-40 repeat. Gene composition and metabolic potential indicate extremotolerance and hydrocarbon assimilation, suggesting a possible environmental habitat of oil-polluted desert soil.

Black fungi of the order Chaetothyriales are grown by many tropical plant-mutualistic ants as small so-called “patches” in their nests, which are located inside hollow structures provided by the host plant (“domatia”). These fungi are introduced and fostered by the ants, indicating that they are important for the colony. As several species of Chaetothyriales tolerate, adsorb, and metabolize toxic volatiles, we investigated the composition of volatile organic compounds (VOCs) of selected domatia in the Azteca/Cecropia ant-plant mutualism. Concentrations of VOCs in ant-inhabited domatia, empty domatia, and background air were compared. In total, 211 compounds belonging to 19 chemical families were identified. Ant-inhabited domatia were dominated by ketones with 2-heptanone, a well-known ant alarm semiochemical, as the most abundant volatile. Empty domatia were characterized by relatively high concentrations of the monoterpenes d-limonene, p-cymene and β-phellandrene, as well as the heterocyclic sulphur-containing compound, benzothiazole. These compounds have biocidal properties and are primarily biosynthesized by plants as a defense mechanism. Interestingly, most of the latter compounds were present at lower concentrations in ant inhabited domatia than in non-colonized ones. We suggest that Chaetothyriales may play a role in reducing the VOCs, underlining that the mutualistic nature of these fungi as VOCs accumulation might be detrimental for the ants, especially the larvae.

Effective management and treatment of fungal diseases is hampered by poor diagnosis, limited options for antifungal therapy, and the emergence of antifungal drug resistance. An understanding of molecular mechanisms contributing to resistance is essential to optimize the efficacy of currently available antifungals. In this perspective, one of the oldest antifungals, 5-fluorocytosine (5-FC), has been the focus of recent studies applying advanced genomic and transcriptomic techniques to decipher the order of events at the molecular level that lead to resistance. These studies have highlighted the complexity of resistance and provided new insights that are reviewed in the present paper.

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. Keywords: Sporotrichosis, Sporothrix brasiliensis, Diagnosis, Molecular markers, Nucleotide sequencing

Invasive fungal infections, such as aspergillosis, candidiasis, and cryptococcosis, have significantly increased among immunocompromised people. To tackle these infections the first and most decisive step is the accurate identification of the causal pathogen. Routine identification of invasive fungal infections has progressed away from culture-dependent methods toward molecular techniques, including DNA barcoding, a highly efficient and widely used diagnostic technique. Fungal DNA barcoding previously relied on a single barcoding region, the internal transcribed spacer (ITS) region. However, this allowed only for 75% of all fungi to be correctly identified. As such, the translational elongation factor 1α (TEF1α) was recently introduced as the secondary barcode region to close the gap. Both loci together form the dual fungal DNA barcoding scheme. As a result, the ISHAM Barcoding Database has been expanded to include sequences for both barcoding regions to enable practical implementation of the dual barcoding scheme into clinical practice. The present study investigates the impact of the secondary barcode on the identification of clinically important fungal taxa, that have been demonstrated to cause severe invasive disease. Analysis of the barcoding regions was performed using barcoding gap analysis based on the genetic distances generated with the Kimura 2-parameter model. The secondary barcode demonstrated an improvement in identification for all taxa that were unidentifiable with the primary barcode, and when combined with the primary barcode ensured accurate identification for all taxa analyzed, making DNA barcoding an important, efficient and reliable addition to the diagnostic toolset of invasive fungal infections.

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.

Chromoblastomycosis is a chronic severely mutilating disease caused by fungi of the order Chaetothyriales. Classically, Phialophora verrucosa has been listed among these etiologic agents. This species is known to occur in the environment and has been found to cause other infections like phaeohyphomycosis, while reported cases of chromoblastomycosis are scant. Phialophora is phylogenetically diverse, and thus retrospective confirmation of etiology is necessary. We studied ten proven cases of chromoblastomycosis from Mexico and further analyzed the population genetics and genomics of the Phialophora species to understand their pathogenicity and predilection. The clinical strains were molecularly identified as Phialophora americana (n = 4), Phialophorachinensis (n = 4), and Phialophora macrospora (n = 2). No genetic distinction between clinical and environmental strains was possible. Further analysis of strains from diverse origins are needed to address eventual differences in virulence and niche predilection between the species.

The polyphyletic group of black fungi within the Ascomycota (Arthoniomycetes, Dothideomycetes, and Eurotiomycetes) is ubiquitous in natural and anthropogenic habitats. Partly because of their dark, melanin-based pigmentation, black fungi are resistant to stresses including UV- and ionizing-radiation, heat and desiccation, toxic metals, and organic pollutants. Consequently, they are amongst the most stunning extremophiles and poly-extreme-tolerant organisms on Earth. Even though ca. 60 black fungal genomes have been sequenced to date, [mostly in the family Herpotrichiellaceae (Eurotiomycetes)], the class Dothideomycetes that hosts the largest majority of extremophiles has only been sparsely sampled. By sequencing up to 92 species that will become reference genomes, the “Shed light in The daRk lineagES of the fungal tree of life” (STRES) project will cover a broad collection of black fungal diversity spread throughout the Fungal Tree of Life. Interestingly, the STRES project will focus on mostly unsampled genera that display different ecologies and life-styles (e.g., ant- and lichen-associated fungi, rock-inhabiting fungi, etc.). With a resequencing strategy of 10- to 15-fold depth coverage of up to ~550 strains, numerous new reference genomes will be established. To identify metabolites and functional processes, these new genomic resources will be enriched with metabolomics analyses coupled with transcriptomics experiments on selected species under various stress conditions (salinity, dryness, UV radiation, oligotrophy). The data acquired will serve as a reference and foundation for establishing an encyclopedic database for fungal metagenomics as well as the biology, evolution, and ecology of the fungi in extreme environments.

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.

ABSTRACT Sporothrix brasiliensis is the prevalent agent of a large zoonotic outbreak in Brazil. With the involvement of several thousands of cases, this is the largest cohort of human and animal sporotrichosis on record in the world. Infections are characterized by local cutaneous dissemination in humans without underlying disease. S. brasiliensis has shown a high degree of virulence in a mouse model compared to the remaining Sporothrix species, including the ancestral species, Sporothrix schenckii. The present paper investigates a genomic and expressed-proteome comparison of S. brasiliensis to S. schenckii. Using bottom-up proteomics, we found 60 proteins exclusively expressed in S. brasiliensis. No significant genomic differences were found among the genes coding for this protein set. A comparison with literature data identified nine proteins that are known to be involved in virulence and immune evasion in other species, several of which had not yet been reported for the Sporothrix species analyzed. IMPORTANCE Sporotrichosis is an important disease in Brazil that is caused by fungi of the genus Sporothrix and affects cats and humans. Our work investigated the proteins differentially expressed by S. brasiliensis in order to find out why this species is more virulent and pathogenic than S. schenckii. We verified a set of proteins that may be related to immune escape and that can explain the high virulence.

Exophiala spinifera and Exophiala dermatitidis (Fungi: Chaetothyriales) are black yeast agents potentially causing disseminated infection in apparently healthy humans. They are the only Exophiala species producing extracellular polysaccharides around yeast cells. In order to gain understanding of eventual differences in intrinsic virulence of the species, their clinical profiles were compared and found to be different, suggesting pathogenic strategies rather than coincidental opportunism. Ecologically relevant factors were compared in a model set of strains of both species, and significant differences were found in clinical and environmental preferences, but virulence, tested in Galleria mellonella larvae, yielded nearly identical results. Virulence factors, i.e., melanin, capsule and muriform cells responded in opposite direction under hydrogen peroxide and temperature stress and thus were inconsistent with their hypothesized role in survival of phagocytosis. On the basis of physiological profiles, possible natural habitats of both species were extrapolated, which proved to be environmental rather than animal-associated. Using comparative genomic analyses we found differences in gene content related to lipid metabolism, cell wall modification and polysaccharide capsule production. Despite the fact that both species cause disseminated infections in apparently healthy humans, it is concluded that they are opportunists rather than pathogens.

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.

The genus Fonsecaea comprises black yeast-like fungi of clinical relevance, including etiologic agents of chromoblastomycosis and cerebral phaeohyphomycosis. Presence of melanin and assimilation of monoaromatic hydrocarbons and alkylbenzenes have been proposed as virulence factors. Multicopper oxidase (MCO) is a family of enzymes including laccases, ferroxidases and ascorbate oxidases which are able to catalyze the oxidation of various aromatic organic compounds with the reduction of molecular oxygen to water. Additionally, laccases are required for the production of fungal melanins, a cell-wall black pigment recognized as a key polymer for pathogenicity and extremotolerance in black yeast-like fungi. Although the activity of laccase enzymes has previously been reported in many wood-rotting fungi, the diversity of laccase genes in Fonsecaea has not yet been assessed. In this study, we identified and characterized laccase-coding genes and determined their genomic location in five clinical and environmental Fonsecaea species. The identification of laccases sensu stricto will provide insights into carbon acquisition strategies as well as melanin production in Fonsecaea.

Penicillium capsulatum is a rare Penicillium species used in paper manufacturing, but recently it has been reported to cause invasive infection. To research the pathogenicity of the clinical Penicillium strain, we sequenced the genomes and transcriptome of the clinical and environmental strains of P. capsulatum. Comparative analyses of these two P. capsulatum strains and close related strains belonging to Eurotiales were performed. The assembled genome sizes of P. capsulatum are approximately 34.4 Mbp in length and encode 11,080 predicted genes. The different isolates of P. capsulatum are highly similar, with the exception of several unique genes, INDELs or SNP in the genes coding for glycosyl hydrolases, amino acid transporters and circumsporozoite protein. A phylogenomic analysis was performed based on the whole genome data of 38 strains belonging to Eurotiales. By comparing the whole genome sequences and the virulence-related genes from 20 important related species, including fungal pathogens and non-human pathogens belonging to Eurotiales, we found meaningful pathogenicity characteristics between P. capsulatum and its closely related species. Our research indicated that P. capsulatum may be a neglected opportunistic pathogen. This study is beneficial for mycologists, geneticists and epidemiologists to achieve a deeper understanding of the genetic basis of the role of P. capsulatum as a newly reported fungal pathogen.

BACKGROUND:Histoplasma capsulatum comprises a worldwide complex of saprobiotic fungi mainly found in nitrogen/phosphate (often bird guano) enriched soils. The microconidia of Histoplasma species may be inhaled by mammalian hosts, and is followed by a rapid conversion to yeast that can persist in host tissues causing histoplasmosis, a deep pulmonary/systemic mycosis. Histoplasma capsulatum sensu lato is a complex of at least eight clades geographically distributed as follows: Australia, Netherlands, Eurasia, North American classes 1 and 2 (NAm 1 and NAm 2), Latin American groups A and B (LAm A and LAm B) and Africa. With the exception of the Eurasian cluster, those clades are considered phylogenetic species. METHODOLOGY/PRINCIPAL FINDINGS:Increased Histoplasma sampling (n = 234) resulted in the revision of the phylogenetic distribution and population structure using 1,563 aligned nucleotides from four protein-coding regions. The LAm B clade appears to be divided into at least two highly supported clades, which are geographically restricted to either Colombia/Argentina or Brazil respectively. Moreover, a complex population genetic structure was identified within LAm A clade supporting multiple monophylogenetic species, which could be driven by rapid host or environmental adaptation (~0.5 MYA). We found two divergent clades, which include Latin American isolates (newly named as LAm A1 and LAm A2), harboring a cryptic cluster in association with bats. CONCLUSIONS/SIGNIFICANCE:At least six new phylogenetic species are proposed in the Histoplasma species complex supported by different phylogenetic and population genetics methods, comprising LAm A1, LAm A2, LAm B1, LAm B2, RJ and BAC-1 phylogenetic species. The genetic isolation of Histoplasma could be a result of differential dispersion potential of naturally infected bats and other mammals. In addition, the present study guides isolate selection for future population genomics and genome wide association studies in this important pathogen complex.

A global set of clinical and environmental strains of the halotolerant black yeast-like fungus Hortaea werneckii are analyzed by multilocus sequencing and AFLP, and physiological parameters are determined. Partial translation elongation factor 1-α proves to be suitable for typing because of the presence/absence of introns and also the presence of several SNPs. Local clonal expansion could be established by a combination of molecular methods, while the population from the Mediterranean Sea water also responds differently to combined temperature and salt stress. The species comprises molecular populations, which in part also differ physiologically allowing further diversification, but clinical strains did not deviate significantly from their environmental counterparts.

Although the number of mucormycosis cases has increased during the last decades, little is known about the pathogenic potential of most mucoralean fungi. Lichtheimia species represent the second and third most common cause of mucormycosis in Europe and worldwide, respectively. To date only three of the five species of the genus have been found to be involved in mucormycosis, namely L. corymbifera, L. ramosa and L. ornata. However, it is not clear whether the clinical situation reflects differences in virulence between the species of Lichtheimia or whether other factors are responsible. In this study the virulence of 46 strains of all five species of Lichtheimia was investigated in chicken embryos. Additionally, strains of the closest-related genus Dichotomocladium were tested. Full virulence was restricted to the clinically relevant species while all strains of L. hyalospora, L. sphaerocystis and Dichotomocladium species were attenuated. Although virulence differences were present in the clinically relevant species, no connection between origin (environmental vs clinical) or phylogenetic position within the species was observed. Physiological studies revealed no clear connection of stress resistance and carbon source utilization with the virulence of the strains. Slower growth at 37°C might explain low virulence of L. hyalospora, L. spaherocystis and Dichotomocladium; however, similarly slow growing strains of L. ornata were fully virulent. Thus, additional factors or a complex interplay of factors determines the virulence of strains. Our data suggest that the clinical situation in fact reflects different virulence potentials in the Lichtheimiaceae.

Long-distance dispersal in microbial eukaryotes has been shown to result in the establishment of populations on continental and global scales. Such "ubiquitous dispersal" has been claimed to be a general feature of microbial eukaryotes, homogenising populations over large scales. However, the unprecedented sampling of opportunistic infectious pathogens created by the global AIDS pandemic has revealed that a number of important species exhibit geographic endemicity despite long-distance migration via aerially dispersed spores. One mechanism that might tend to drive such endemicity in the face of aerial dispersal is the evolution of niche-adapted genotypes when sexual reproduction is rare. Dispersal of such asexual physiological "species" will be restricted when natural habitats are heterogeneous, as a consequence of reduced adaptive variation. Using the HIV-associated endemic fungus Penicillium marneffei as our model, we measured the distribution of genetic variation over a variety of spatial scales in two host species, humans and bamboo rats. Our results show that, despite widespread aerial dispersal, isolates of P. marneffei show extensive spatial genetic structure in both host species at local and country-wide scales. We show that the evolution of the P. marneffei genome is overwhelmingly clonal, and that this is perhaps the most asexual fungus yet found. We show that clusters of genotypes are specific to discrete ecological zones and argue that asexuality has led to the evolution of niche-adapted genotypes, and is driving endemicity, by reducing this pathogen's potential to diversify in nature.