Amesia hispanica sp. nov., Producer of the Antifungal Class of Antibiotics Dactylfungins.

During a study of the diversity of soilborne fungi from Spain, a strain belonging to the family Chaetomiaceae (Sordariales) was isolated. The multigene phylogenetic inference using five DNA loci showed that this strain represents an undescribed species of the genus Amesia, herein introduced as A. hispanica sp. nov. Investigation of its secondary metabolome led to the isolation of two new derivatives (2 and 3) of the known antifungal antibiotic dactylfungin A (1), together with the known compound cochliodinol (4). The planar structures of 1-4 were determined by ultrahigh performance liquid chromatography coupled with diode array detection and ion mobility tandem mass spectrometry (UHPLC-DAD-IM-MS/MS) and extensive 1D and 2D nuclear magnetic resonance (NMR) spectroscopy after isolation by HPLC. All isolated secondary metabolites were tested for their antimicrobial and cytotoxic activities. Dactylfungin A (1) showed selective and strong antifungal activity against some of the tested human pathogens (Aspergillus fumigatus and Cryptococcus neoformans). The additional hydroxyl group in 2 resulted in the loss of activity against C. neoformans but still retained the inhibition of As. fumigatus in a lower concentration than that of the respective control, without showing any cytotoxic effects. In contrast, 25″-dehydroxy-dactylfungin A (3) exhibited improved activity against yeasts (Schizosaccharomyces pombe and Rhodotorula glutinis) than 1 and 2, but resulted in the appearance of slight cytotoxicity. The present study exemplifies how even in a well-studied taxonomic group such as the Chaetomiaceae, the investigation of novel taxa still brings chemistry novelty, as demonstrated in this first report of this antibiotic class for chaetomiaceous and sordarialean taxa.

Higher white-nose syndrome fungal isolate yields from UV-guided wing biopsies compared with skin swabs and optimal culture media.

BACKGROUND: North American bat populations have suffered severe declines over the last decade due to the Pseudogymnoascus destructans fungus infection. The skin disease associated with this causative agent, known as white-nose syndrome (WNS), is specific to bats hibernating in temperate regions. As cultured fungal isolates are required for epidemiological and phylogeographical studies, the purpose of the present work was to compare the efficacy and reliability of different culture approaches based on either skin swabs or wing membrane tissue biopsies for obtaining viable fungal isolates of P. destructans. RESULTS: In total, we collected and analysed 69 fungal and 65 bacterial skin swabs and 51 wing membrane tissue biopsies from three bat species in the Czech Republic, Poland and the Republic of Armenia. From these, we obtained 12 viable P. destructans culture isolates. CONCLUSIONS: Our results indicated that the efficacy of cultures based on wing membrane biopsies were significantly higher. Cultivable samples tended to be based on collections from bats with lower body surface temperature and higher counts of UV-visualised lesions. While cultures based on both skin swabs and wing membrane tissue biopsies can be utilised for monitoring and surveillance of P. destructans in bat populations, wing membrane biopsies guided by UV light for skin lesions proved higher efficacy. Interactions between bacteria on the host's skin also appear to play an important role.

Ten decadal advances in fungal biology leading towards human well-being.

Fungi are an understudied resource possessing huge potential for developing products that can greatly improve human well-being. In the current paper, we highlight some important discoveries and developments in applied mycology and interdisciplinary Life Science research. These examples concern recently introduced drugs for the treatment of infections and neurological diseases; application of -OMICS techniques and genetic tools in medical mycology and the regulation of mycotoxin production; as well as some highlights of mushroom cultivaton in Asia. Examples for new diagnostic tools in medical mycology and the exploitation of new candidates for therapeutic drugs, are also given. In addition, two entries illustrating the latest developments in the use of fungi for biodegradation and fungal biomaterial production are provided. Some other areas where there have been and/or will be significant developments are also included. It is our hope that this paper will help realise the importance of fungi as a potential industrial resource and see the next two decades bring forward many new fungal and fungus-derived products.

Piperazine-Modified Ketoconazole Derivatives Show Increased Activity against Fungal and Trypanosomatid Pathogens.

Ketoconazole (KTZ) is an imidazole drug applied topically to treat numerous skin infections. However, as a systemic antifungal, KTZ' efficacy and safety no longer justify its use as a first-line treatment. Azole conjugates often display higher solubility and better antifungal activities than their parent azoles. Accordingly, we aimed at developing suitable linkers for clickable azole conjugation with a second antifungal molecule, and targeted drug delivery towards improving antifungal activity. For its low price and high availability, we selected KTZ as a molecular scaffold to introduce such chemical modifications. We prepared a series of piperazine-modified KTZ derivatives and we evaluated their in vitro antifungal and antitrypanosomal activity against fourteen strains of pathogenic fungi and two strains of Trypanosoma parasites. Several compounds were more effective against the pathogens than KTZ. Compound 5 was 24 times more potent against Aspergillus flavus and 8 times more potent against A. fumigatus than KTZ, with similarly low cytotoxicity to HEK cells up to 100 µM. Derivative 6 had 9- and 7-fold higher activity against T. brucei gambiense and T. brucei brucei than KTZ, respectively, and inhibited trypanosoma growth at single micromolar EC50 values. Combined, our findings will foster further research of piperazine-modified KTZs as promising antifungal and antiparasitic drugs towards enhancing the properties of both KTZ and other azole derivatives.

Re-discovery of Trichophyton bullosum in North Africa as a cause of severe dermatophytosis in donkeys.

This article reports the first verified cases of infection by Trichophyton bullosum in Africa since the description of the fungus, isolated in 1933 from the coat of horses in Tunisia and Mali. We found the fungus in cutaneous samples obtained from donkeys suffering from severe dermatitis with areas of alopecia and scaling in the surroundings of Cairo (Egypt). Fungal elements (arthroconidia and hyphae) were seen at the microscopy of material collected by skin scraping and digested in NaOH. Fungal colonies grown on various culture media were identified through PCR and sequencing of the ITS rDNA region. Since the original report in Africa and the Middle East, only a few cases have been reported thus far in humans in France and two cases in horses in the Czech Republic and Japan. Trichophyton bullosum seems thus an infrequent cause of dermatophytosis. However, the actual prevalence of this pathogen may be underestimated due to the similarity with T. verrucosum, a predominant cause of infection in cattle, occasionally found on horses and donkeys. Indeed, the two fungi can be distinguished only via molecular methods, which are poorly employed in epidemiological studies on equine and bovine dermatophytosis. The present study results add to our knowledge on the ecology of this poorly explored dermatophyte, supporting the concept that equines are the primary hosts of T. bullosum and confirming the presence of this pathogen in Africa. At the same time, these are the first unequivocally documented infections in donkeys due to T. bullosum.

An Outbreak of Trichophyton quinckeanum Zoonotic Infections in the Czech Republic Transmitted from Cats and Dogs.

Trichophyton quinckeanum, a zoophilic dermatophyte mostly known as the causative agent of rodent favus, is relatively rarely reported to cause human infections. Indeed, no infections were detected in Czechia between 2012 and 2015 despite routine verification of species identification by ITS rDNA sequencing. By contrast, 25 human and 11 animal cases of infection were documented from December 2016 to December 2020 and the rates tended to grow every following year. Interestingly, most of the cases were reported in the Olomouc region, suggesting a local outbreak. We bring the evidence that human T. quinckeanum infections are most commonly contracted from infected cats or, less frequently, dogs. Although rodents or contaminated soil and environment could be the source of infection to cats and dogs, the occurrence of infections in multiple animals in the same household suggests direct transmission among animals. Confirmation of the identification by molecular methods is highly recommended due to morphological similarity with T. mentagrophytes/T. interdigitale. Antifungal susceptibility testing of isolates to eight antifungals was performed using EUCAST methodology (E.Def 11.0). Among the tested antifungals, terbinafine, amorolfine, ciclopirox and efinaconazole were most potent in vitro and elevated minimum inhibitory concentrations were obtained for fluconazole and ketoconazole.

Discovery of New Trichophyton Members, T. persicum and T. spiraliforme spp. nov., as a Cause of Highly Inflammatory Tinea Cases in Iran and Czechia.

Pathogens from the Trichophyton benhamiae complex are one of the most important causes of animal mycoses with significant zoonotic potential. In light of the recently revised taxonomy of this complex, we retrospectively identified 38 Trichophyton isolates that could not be resolved into any of the existing species. These strains were isolated from Iranian and Czech patients during molecular epidemiological surveys on dermatophytosis and were predominantly associated with highly inflammatory tinea corporis cases, suggesting possible zoonotic etiology. Subsequent phylogenetic (4 markers), population genetic (10 markers), and phenotypic analyses supported recognition of two novel species. The first species, Trichophyton persicum sp. nov., was identified in 36 cases of human dermatophytosis and one case of feline dermatophytosis, mainly in Southern and Western Iran. The second species, Trichophyton spiraliforme sp. nov., is only known from a single case of tinea corporis in a Czech patient who probably contracted the infection from a dog. Although the zoonotic sources of infections summarized in this study are very likely, little is known about the host spectrum of these pathogens. Awareness of these new pathogens among clinicians should refine our knowledge about their poorly explored geographic distribution. IMPORTANCE In this study, we describe two novel agents of dermatophytosis and summarize the clinical manifestation of infections. These new pathogens were discovered thanks to long-term molecular epidemiological studies conducted in Czechia and Iran. Zoonotic origins of the human infections are highly probable, but the animal hosts of these pathogens are poorly known. Further research is needed to refine our knowledge about these new dermatophytes.

In vitro antifungal susceptibility patterns of Trichophyton benhamiae complex isolates from diverse origin.

BACKGROUND: Species from the Trichophyton benhamiae complex are mostly zoophilic dermatophytes which cause inflammatory dermatophytosis in animals and humans worldwide. OBJECTIVES: This study was purposed to (a) to identify 169 reference and clinical dermatophyte strains from the T benhamiae complex species by molecular method and adhering to the newest taxonomy in the complex (b) to evaluate the in vitro antifungal susceptibility profile of these strains against eight common and new antifungal agents that may be used for the treatment of dermatophytosis. METHODS: All isolates, mainly originated from Europe but also from Iran, Japan and USA, were subjected to ITS-rDNA sequencing. The in vitro antifungal susceptibility profiles of eight common and new antifungal drugs against the isolates were determined by CLSI M38-A2 protocol and according to microdilution method. RESULTS: Based on the ITS-rDNA sequencing, T benhamiae was the dominant species (n = 102), followed by T europaeum (n = 29), T erinacei (n = 23), T japonicum (n = 10), Trichophyton sp (n = 4) and T eriotrephon (n = 1). MIC ranges across all isolates were as follows: luliconazole: 0.0002-0.002 µg/ml, terbinafine: 0.008-0.125 µg/ml, efinaconazole: 0.008-0.125 µg/ml, ciclopirox olamine: 0.03-0.5 µg/ml, itraconazole: 0.06-2 µg/ml, griseofulvin: 0.25-4 µg/ml, amorolfine hydrochloride: 0.125-4 µg/ml and tavaborole: 1-16 µg/ml. CONCLUSION: Luliconazole, efinaconazole and terbinafine were the most potent antifungals against T benhamiae complex isolates, regardless of the geographic locations where strains were isolated. These data might help dermatologists to develop effective therapies for successful treatment of infections due to T benhamiae complex species.

Detection and Control of Dermatophytosis in Wild European Hedgehogs (Erinaceus europaeus) Admitted to a French Wildlife Rehabilitation Centre.

The rising number of European hedgehogs (Erinaceus europaeus) admitted every year to wildlife rehabilitation centres might be a source of concern to animal and public health since transmissible diseases, such as dermatophytosis, can be easily disseminated. This study seeks to evaluate the frequency of dermatophyte detection in hedgehogs admitted to a wildlife rehabilitation centre located near Paris, France, and to assess the risk of contamination in the centre in order to adapt prevention measures. A longitudinal cohort study was performed on 412 hedgehogs hosted at the Wildlife Animal Hospital of the Veterinary College of Alfort from January to December 2016. Animals were sampled once a month for fungal culture. Dermatophyte colonies were obtained from 174 out of 686 skin samples (25.4%). Besides Trichophyton erinacei, Trichophyton mentagrophytes and Nannizzia gypsea were also found. Dermatophyte detection seemed to be associated with the presence of skin lesions, while more than one-third of T. erinacei-positive animals were asymptomatic carriers. Healing required several months of treatment with topical and systemic azoles, but dermatophytosis did not seem to reduce the probability of release. Daily disinfection procedures and early detection and treatment of infected and asymptomatic carriers succeeded in limiting dermatophyte transmission between hedgehogs and humans.

Subtyping Options for Microsporum canis Using Microsatellites and MLST: A Case Study from Southern Italy.

Microsporum canis is considered one of the most common zoophilic dermatophyte species causing infections in animals and humans worldwide. However, molecular epidemiological studies on this dermatophyte are still rare. In this study, we aimed to analyse the population structure and relationships between M. canis strains (n = 66) collected in southern Italy and those isolated from symptomatic and asymptomatic animals (cats, dogs and rabbits) and humans. For subtyping purposes, using multilocus sequence typing (MLST) and multilocus microsatellite typing (MLMT), we first used a limited set of strains to screen for variability. No intraspecies variability was detected in six out of the eight reference genes tested and only the ITS and IGS regions showed two and three sequence genotypes, respectively, resulting in five MLST genotypes. All of eight genes were, however, useful for discrimination among M. canis, M. audouinii and M. ferrugineum. In total, eighteen microsatellite genotypes (A-R) were recognized using MLMT based on six loci, allowing a subdivision of strains into two clusters based on the Bayesian iterative algorithm. Six MLMT genotypes were from multiple host species, while 12 genotypes were found only in one host. There were no statistically significant differences between clusters in terms of host spectrum and the presence or absence of lesions. Our results confirmed that the MLST approach is not useful for detailed subtyping and examining the population structure of M. canis, while microsatellite analysis is a powerful tool for conducting surveillance studies and gaining insight into the epidemiology of infections due to this pathogen.

Multilocus phylogeny- and fruiting feature-assisted delimitation of European Cyclocybe aegerita from a new Asian species complex and related species.

Cyclocybe aegerita (synonym: Agrocybe aegerita) is a widely cultivated edible and reportedly almost cosmopolitan mushroom species that serves as a model fungus for basidiome formation and as producer of useful natural products and enzymes. Focusing on strains from different continents, here, we present a phylogenetic analysis of this species and some adjacent taxa that employs four phylogenetic markers. In addition, we tested the strains' capability to fructify on agar media. Our analysis reveals that "C. aegerita sensu lato" splits up into the following two well-supported monophyletic geographic lineages: a European clade and an Asian clade. The European one is closely associated with the Chinese species Cyclocybe salicaceicola. In contrast, the Asian lineage, which we preliminarily designate as Cyclocybe chaxingu agg., may comprise several species (species complex) and clusters with the Pacific species Cyclocybe parasitica (New Zealand). In addition, fruiting properties differ across C. aegerita and its Asian and Pacific relatives; however, strains from the Asian clade and C. parasitica tend to form larger basidiomes with relatively big caps and long stipes and strains from the European clade exhibit a more variable fruiting productivity with the tendency to form more basidiomes, with smaller caps and shorter stipes. Moreover, some strains showed individual fruiting patterns, such as the preference to fruit where they were exposed to injuring stimuli. In conclusion, the delimitation of the newly delimited Asian species complex from our multilocus phylogeny of "C. aegerita sensu lato", which is supported by phenotypic data, depicts an exemplary case of biogeographic diversity within a previously thought homogeneous species of near worldwide distribution.

White-nose syndrome without borders: Pseudogymnoascus destructans infection tolerated in Europe and Palearctic Asia but not in North America.

A striking feature of white-nose syndrome, a fungal infection of hibernating bats, is the difference in infection outcome between North America and Europe. Here we show high WNS prevalence both in Europe and on the West Siberian Plain in Asia. Palearctic bat communities tolerate similar fungal loads of Pseudogymnoascus destructans infection as their Nearctic counterparts and histopathology indicates equal focal skin tissue invasiveness pathognomonic for WNS lesions. Fungal load positively correlates with disease intensity and it reaches highest values at intermediate latitudes. Prevalence and fungal load dynamics in Palearctic bats remained persistent and high between 2012 and 2014. Dominant haplotypes of five genes are widespread in North America, Europe and Asia, expanding the source region of white-nose syndrome to non-European hibernacula. Our data provides evidence for both endemicity and tolerance to this persistent virulent fungus in the Palearctic, suggesting that host-pathogen interaction equilibrium has been established.

Assemblage of filamentous fungi associated with aculeate hymenopteran brood in reed galls.

Monotypic stands of common reed and the reed-gall-associated insect assemblages are distributed worldwide. However, fungi associated with these assemblages have not been characterized in detail. Here we examined 5200 individuals (12 species) of immature aculeate hymenopterans or their parasitoids collected at 34 sampling sites in Central Europe. We noticed fungal outgrowth on exoskeletons of 83 (1.60%) larvae and pupae. The most common host was eudominant Pemphredon fabricii. However, the less abundant aculeate hymenopteran reed gall inquilines were infected at higher prevalence, these included Trypoxylon deceptorium, Trypoxylon minus, Hoplitis leucomelana and Hylaeus moricei (all considered new host records). We identified three fungal species, Penicillium buchwaldii (72% of cases), Aspergillus pseudoglaucus (22%) and Penicillium quebecense (6%). When multibrooded nests were affected, only a part of individuals was infected in 62% of cases. The sampling site-specific infection rate reached up to 13%, thus fungal infections should be considered an important variable driving the abundance of gall inquilines. Infections of generalist host species were more frequent than those of reed gall specialists, suggesting that suboptimal conditions decreased the immunocompetence of non-specialized species, which only occasionally nest in reed galls and feed in reed beds.

Equine Dermatophytosis due to Trichophyton bullosum, a Poorly Known Zoophilic Dermatophyte Masquerading as T. verrucosum.

Trichophyton bullosum is a zoophilic dermatophyte from the Arthroderma benhamiae complex with a poorly known distribution. In this study, we report a case of dermatophytosis caused by T. bullosum in a 6-year-old male horse who had a skin lesion located in a saddle area. The infection spread rapidly to the upper chest and to both sides of the trunk. The dermatophyte was isolated in culture and identified by sequence analysis of the internal transcribed spacer regions (ITS rDNA). To date, this is the first verified case of animal infection due to T. bullosum in Europe following the 2012 report of human infection in France. We hypothesize that this species can be relatively common in horses and donkeys, but it is confused with other zoophilic species responsible for infections with similar clinical manifestations, and when isolated in culture, it is misidentified as the phenotypically similar T. verrucosum. Previous cases of dermatophytosis caused by T. verrucosum-like dermatophytes in horses and donkeys were reviewed together with human infections transmitted from these animals. This summary estimates possible distribution width of T. bullosum. The taxonomy of T. verrucosum-like dermatophytes is extremely difficult due to lack of original material and poor morphology of species. Molecular genetic methods are necessary to verify the identification of these fungi. ITS1 or ITS2 region of rDNA alone is sufficient for correct identification.

Discovery of a sexual stage in Trichophyton onychocola, a presumed geophilic dermatophyte isolated from toenails of patients with a history of T. rubrum onychomycosis.

Trichophyton onychocola is a recently described geophilic dermatophyte that has been isolated from a toenail of Czech patient with a history of onychomycosis due to T. rubrum and clinical suspicion of relapse. In this study, we report a similar case from Denmark in an otherwise healthy 56-year-old man. The patient had a history of great toenail infection caused by T. rubrum in 2004 and presented with suspected relapse in 2011 and 2013. Trichophyton onychocola was the only microbial agent isolated at the second visit in 2013 and the identification was confirmed by DNA sequencing. Direct microscopic nail examination was positive for hyphae, however the etiological significance of T. onychocola was not supported by repeated isolation of the fungus. This new species may be an overlooked geophilic species due to the resemblance to some common species, for example, zoophilic T. interdigitale or some species of geophilic dermatophytes. We included differential diagnosis with phenotypically similar species; however, it is recommended that molecular methods are used for correct identification. The MAT locus of Danish strain was of opposite mating type than in the previously isolated Czech strain and the two isolates were successfully mated. The mating experiments with related heterothallic species T. thuringiense and Arthroderma melis were negative. The sexual state showed all typical signs of arthroderma-morph and is described by using optical as well as scanning electron microscopy. The sexual state was induced on a set of agar media, however low cultivation temperature and the presence of keratin source were crucial for the success rather than formulation of medium.

Trichophyton onychocola sp. nov. isolated from human nail.

A previously undescribed Trichophyton species was isolated from the nail of a 33-year-old man with a history of probable distal lateral subungual onychomycosis (without confirmation by mycological examination). The infection occurred for the first time five years earlier (in 2006) and affected the right great toenail, with complete clinical remission after treatment with ciclopirox olamine. This undescribed species was isolated during probable relapse in 2011, but its etiological significance was not confirmed, that is, direct microscopy was negative and additional clinical samples were not collected. The species is probably geophilic based on phylogenetic analysis (internal transcribed spacer [ITS] rDNA) and is most closely related to the anamorphic T. thuringiense, homothallic Arthroderma ciferrii (anamorph T. georgiae), and heterothallic A. melis. The new species is characterized by yellowish colonies, red reverse on several media, positive urease test, negative hair-perforation test, absence of growth at 34°C, absence of macroconidia, formation of one-celled clavate microconidia, and spiral hyphae. The species grows well on sterilized human hairs placed on agar medium without any additional nutrients and forms gymnothecium-like structures covered by peridial hyphae. The combination of unique micro- and macromorphological features and physiological and sequence data from four unlinked loci (ITS, benA, RPB2, and act1 gene) justified the proposal of a new species T. onychocola sp. nov.