Fungal sensing by dectin-1 directs the non-pathogenic polarization of TH17 cells through balanced type I IFN responses in human DCs.

The non-pathogenic TH17 subset of helper T cells clears fungal infections, whereas pathogenic TH17 cells cause inflammation and tissue damage; however, the mechanisms controlling these distinct responses remain unclear. Here we found that fungi sensing by the C-type lectin dectin-1 in human dendritic cells (DCs) directed the polarization of non-pathogenic TH17 cells. Dectin-1 signaling triggered transient and intermediate expression of interferon (IFN)-ß in DCs, which was mediated by the opposed activities of transcription factors IRF1 and IRF5. IFN-ß-induced signaling led to integrin αvß8 expression directly and to the release of the active form of the cytokine transforming growth factor (TGF)-ß indirectly. Uncontrolled IFN-ß responses as a result of IRF1 deficiency induced high expression of the IFN-stimulated gene BST2 in DCs and restrained TGF-ß activation. Active TGF-ß was required for polarization of non-pathogenic TH17 cells, whereas pathogenic TH17 cells developed in the absence of active TGF-ß. Thus, dectin-1-mediated modulation of type I IFN responses allowed TGF-ß activation and non-pathogenic TH17 cell development during fungal infections in humans.

Frequent transitions in mating-type locus chromosomal organization in Malassezia and early steps in sexual reproduction.

Fungi in the basidiomycete genus Malassezia are the most prevalent eukaryotic microbes resident on the skin of human and other warm-blooded animals and have been implicated in skin diseases and systemic disorders. Analysis of Malassezia genomes revealed that key adaptations to the skin microenvironment have a direct genomic basis, and the identification of mating/meiotic genes suggests a capacity to reproduce sexually, even though no sexual cycle has yet been observed. In contrast to other bipolar or tetrapolar basidiomycetes that have either two linked mating-type-determining (MAT) loci or two MAT loci on separate chromosomes, in Malassezia species studied thus far the two MAT loci are arranged in a pseudobipolar configuration (linked on the same chromosome but capable of recombining). By generating additional chromosome-level genome assemblies, and an improved Malassezia phylogeny, we infer that the pseudobipolar arrangement was the ancestral state of this group and revealed six independent transitions to tetrapolarity, seemingly driven by centromere fission or translocations in centromere-flanking regions. Additionally, in an approach to uncover a sexual cycle, Malassezia furfur strains were engineered to express different MAT alleles in the same cell. The resulting strains produce hyphae reminiscent of early steps in sexual development and display upregulation of genes associated with sexual development as well as others encoding lipases and a protease potentially relevant for pathogenesis of the fungus. Our study reveals a previously unseen genomic relocation of mating-type loci in fungi and provides insight toward the identification of a sexual cycle in Malassezia, with possible implications for pathogenicity.

Yeast-insect interactions in southern Africa: Tapping the diversity of yeasts for modern bioprocessing.

Yeast-insect interactions are one of the most interesting long-standing relationships whose research has contributed to our understanding of yeast biodiversity and their industrial applications. Although insect-derived yeast strains are exploited for industrial fermentations, only a limited number of such applications has been documented. The search for novel yeasts from insects is attractive to augment the currently domesticated and commercialized production strains. More specifically, there is potential in tapping the insects native to southern Africa. Southern Africa is home to a disproportionately high fraction of global biodiversity with a cluster of biomes and a broad climate range. This review presents arguments on the roles of the mutualistic relationship between yeasts and insects, the presence of diverse pristine environments and a long history of spontaneous food and beverage fermentations as the potential source of novelty. The review further discusses the recent advances in novelty of industrial strains of insect origin, as well as various ancient and modern-day industries that could be improved by use yeasts from insect origin. The major focus of the review is on the relationship between insects and yeasts in southern African ecosystems as a potential source of novel industrial yeast strains for modern bioprocesses.

Dectin-1 is an extracellular pathogen sensor for the induction and processing of IL-1ß via a noncanonical caspase-8 inflammasome.

Production of the proinflammatory cytokine interleukin 1ß (IL-1ß) by dendritic cells is crucial in host defense. Here we identify a previously unknown role for dectin-1 in the activation of a noncanonical caspase-8 inflammasome in response to fungi and mycobacteria. Dectin-1 induced both the production and maturation of IL-1ß through signaling routes mediated by the kinase Syk. Whereas the CARD9-Bcl-10-MALT1 scaffold directed IL1B transcription, the recruitment of MALT1-caspase-8 and ASC into this scaffold was crucial for processing of pro-IL-1ß by caspase-8. In contrast to activation of the canonical caspase-1 inflammasome, which requires additional activation of cytosolic receptors, activation of the noncanonical caspase-8 inflammasome was independent of pathogen internalization. Thus, dectin-1 acted as an extracellular sensor for pathogens that induced both IL-1ß production and maturation through a noncanonical caspase-8-dependent inflammasome for protective immunity.

Blastobotrys nigripullensis, a new yeast species isolated from a fungal outbreak on an ancient Roman shipwreck in the Netherlands.

A new species of the yeast genus Blastobotrys was discovered on ancient ship timbers in the Netherlands. The species had developed on the wood of a river barge dating to the Roman period. The growth occurred after the preservative polyethylene glycol (PEG 4000) was washed out of some of the timbers due to an undetected leak in the storage unit. Mycological analysis of various timber samples revealed the presence of Microascus melanosporus (predominant), Microascus paisii, a member of the Acremonium chrysogenum-clade, and a new Blastrobotrys species. The new species produced sporothrix-like conidiophores with clavate blastoconidia (3-7 × 1-3.5 µm) and was found to be osmotolerant, capable of growth on low water activity media like malt yeast 50% glucose agar (MY50G). In this article we formally describe and introduce Blastrobotrys nigripullensis (CBS 17879 T) based on its morphology, physiology and phylogenetic placement.

Evolution of loss of heterozygosity patterns in hybrid genomes of Candida yeast pathogens.

BACKGROUND: Hybrids are chimeric organisms with highly plastic heterozygous genomes that may confer unique traits enabling the adaptation to new environments. However, most evolutionary theory frameworks predict that the high levels of genetic heterozygosity present in hybrids from divergent parents are likely to result in numerous deleterious epistatic interactions. Under this scenario, selection is expected to favor recombination events resulting in loss of heterozygosity (LOH) affecting genes involved in such negative interactions. Nevertheless, it is so far unknown whether this phenomenon actually drives genomic evolution in natural populations of hybrids. To determine the balance between selection and drift in the evolution of LOH patterns in natural yeast hybrids, we analyzed the genomic sequences from fifty-five hybrid strains of the pathogenic yeasts Candida orthopsilosis and Candida metapsilosis, which derived from at least six distinct natural hybridization events. RESULTS: We found that, although LOH patterns in independent hybrid clades share some level of convergence that would not be expected from random occurrence, there is an apparent lack of strong functional selection. Moreover, while mitosis is associated with a limited number of inter-homeologous chromosome recombinations in these genomes, induced DNA breaks seem to increase the LOH rate. We also found that LOH does not accumulate linearly with time in these hybrids. Furthermore, some C. orthopsilosis hybrids present LOH patterns compatible with footprints of meiotic recombination. These meiotic-like patterns are at odds with a lack of evidence of sexual recombination and with our inability to experimentally induce sporulation in these hybrids. CONCLUSIONS: Our results suggest that genetic drift is the prevailing force shaping LOH patterns in these hybrid genomes. Moreover, the observed LOH patterns suggest that these are likely not the result of continuous accumulation of sporadic events-as expected by mitotic repair of rare chromosomal breaks-but rather of acute episodes involving many LOH events in a short period of time.

Unveiling Trichosporon austroamericanum sp. nov.: A Novel Emerging Opportunistic Basidiomycetous Yeast Species.

During an epidemiological survey, a potential novel species within the basidiomycetous yeast genus Trichosporon was observed. The clinical strain was obtained from a urine sample taken from a Brazilian kidney transplant recipient. The strain was molecularly identified using the intergenic spacer (IGS1) ribosomal DNA locus and a subsequent phylogenetic analysis showed that multiple strains that were previously reported by other studies shared an identical IGS1-genotype most closely related to that of Trichosporon inkin. However, none of these studies provided an in-depth characterization of the involved strains to describe it as a new taxon. Here, we present the novel clinically relevant yeast for which we propose the name Trichosporon austroamericanum sp. nov. (holotype CBS H-24937). T. austroamericanum can be distinguished from other siblings in the genus Trichosporon using morphological, physiological, and phylogenetic characters.

Comparative genomics of smut fungi suggest the ability of meiosis and mating in asexual species of the genus Pseudozyma (Ustilaginales).

BACKGROUND: The Ustilaginales comprise hundreds of plant-parasitic fungi with a characteristic life cycle that directly links sexual reproduction and parasitism: One of the two mating-type loci codes for a transcription factor that not only facilitates mating, but also initiates the infection process. However, several species within the Ustilaginales have no described parasitic stage and were historically assigned to the genus Pseudozyma. Molecular studies have shown that the group is polyphyletic, with members being scattered in various lineages of the Ustilaginales. Together with recent findings of conserved fungal effectors in these non-parasitic species, this raises the question if parasitism has been lost recently and in multiple independent events or if there are hitherto undescribed parasitic stages of these fungi. RESULTS: In this study, we sequenced genomes of five Pseudozyma species together with six parasitic species from the Ustilaginales to compare their genomic capability to perform two central functions in sexual reproduction: mating and meiosis. While the loss of sexual capability is assumed in certain lineages and asexual species are common in Asco- and Basidiomycota, we were able to successfully annotate potentially functional mating and meiosis genes that are conserved throughout the whole group. CONCLUSION: Our data suggest that at least the key functions of a sexual lifestyle are maintained in the analyzed genomes, challenging the current understanding of the so-called asexual species with respect to their evolution and ecological role.

Diversity of dung beetle-associated yeasts from pristine environments of Botswana.

Yeast-insect interactions are increasingly becoming an attractive source of discovery for previously unknown, unique, diverse, and industrially relevant yeast species. Despite a wealth of studies that have recently focused on yeasts in symbiotic association with Hymenopteran insects, yeasts associated with Coleopteran insects, such as lignocellulosic-rich dung-dependent beetles, remain poorly studied. Trends in yeast discovery suggest that species richness and diversity can be attributed to the ecological niche of the insect. Here, we considered the potential of dung beetles inhabiting the extreme environments of Botswana, characterized by desert-like conditions (semi-arid to arid and hot) as well as protected pristine environments, as possible attribute niches that can shape the extremophilic and diverse life history strategies of yeasts. We obtained a total of 97 phylogenetically diverse yeast isolates from six species of dung beetles from Botswana's unexplored environments, representing 19 species belonging to 11 genera. The findings suggest that the guts of dung beetles are a rich niche for non-Saccharomyces yeast species. Meyerozyma and Pichia were the most dominant genera associated with dung beetles, representing 55% (53 out of 97) of the yeast isolates in our study. Trichosporon and Cutaneotrichosporon genera represented 32% (31 out of 97) of the isolates. The remaining isolates belonged to Apiotrichum, Candida, Diutina, Naganishia, Rhodotorula, and Wickerhamiella genera (12 out of 97). We found out that about 62% (60 out of 97) of the isolates were potentially new species because of their low internal transcribed spacer (ITS) sequence similarity when compared to the most recent optimal species delineation threshold. A single isolate was unidentifiable using the ITS sequences. Using an in silico polymerase chain reaction-restriction fragment length polymorphism approach, we revealed that there was genetic diversity within isolates of the same species. Our results contribute to the knowledge and understanding of the diversity of dung beetle-associated yeasts.

Da Vinci's yeast: Blastobotrys davincii f.a., sp. nov.

A new species of the yeast genus Blastobotrys was discovered during a worldwide survey of culturable xerophilic fungi in house dust. Several culture-dependent and independent studies from around the world detected the same species from a wide range of substrates including indoor air, cave wall paintings, bats, mummies, and the iconic self-portrait of Leonardo da Vinci from ca 1512. However, none of these studies identified their strains, clones, or OTUs as Blastobotrys. We introduce the new species as Blastobotrys davincii f.a., sp. nov. (holotype CBS H-24879) and delineate it from other species using morphological, phylogenetic, and physiological characters. The new species of asexually (anamorphic) budding yeast is classified in Trichomonascaceae and forms a clade along with its associated sexual state genus Trichomonascus. Despite the decade-old requirement to use a single generic name for fungi, both names are still used. Selection of the preferred name awaits a formal nomenclatural proposal. We present arguments for adopting Blastobotrys over Trichomonascus and introduce four new combinations as Blastobotrys allociferrii (≡ Candida allociferrii), B. fungorum (≡ Sporothrix fungorum), B. mucifer (≡ Candida mucifera), and Blastobotrys vanleenenianus (≡ Trichomonascus vanleenenianus). We provide a nomenclatural review and an accepted species list for the 37 accepted species in the Blastobotrys/Trichomonascus clade. Finally, we discuss the identity of the DNA clones detected on the da Vinci portrait, and the importance of using appropriate media to isolate xerophilic or halophilic fungi.

Dung beetle-associated yeasts display multiple stress tolerance: a desirable trait of potential industrial strains.

BACKGROUND: Stress-tolerant yeasts are highly desirable for cost-effective bioprocessing. Several strategies have been documented to develop robust yeasts, such as genetic and metabolic engineering, artificial selection, and natural selection strategies, among others. However, the significant drawbacks of such techniques have motivated the exploration of naturally occurring stress-tolerant yeasts. We previously explored the biodiversity of non-conventional dung beetle-associated yeasts from extremophilic and pristine environments in Botswana (Nwaefuna AE et.al., Yeast, 2023). Here, we assessed their tolerance to industrially relevant stressors individually, such as elevated concentrations of osmolytes, organic acids, ethanol, and oxidizing agents, as well as elevated temperatures. RESULTS: Our findings suggest that these dung beetle-associated yeasts tolerate various stresses comparable to those of the robust bioethanol yeast strain, Saccharomyces cerevisiae (Ethanol Red™). Fifty-six percent of the yeast isolates were tolerant of temperatures up to 42 °C, 12.4% of them could tolerate ethanol concentrations up to 9% (v/v), 43.2% of them were tolerant to formic acid concentrations up to 20 mM, 22.7% were tolerant to acetic acid concentrations up to 45 mM, 34.0% of them could tolerate hydrogen peroxide up to 7 mM, and 44.3% of the yeasts could tolerate osmotic stress up to 1.5 M. CONCLUSION: The ability to tolerate multiple stresses is a desirable trait in the selection of novel production strains for diverse biotechnological applications, such as bioethanol production. Our study shows that the exploration of natural diversity in the search for stress-tolerant yeasts is an appealing approach for the development of robust yeasts.

SeqEditor: an application for primer design and sequence analysis with or without GTF/GFF files.

MOTIVATION: Sequence analyses oriented to investigate specific features, patterns and functions of protein and DNA/RNA sequences usually require tools based on graphic interfaces whose main characteristic is their intuitiveness and interactivity with the user's expertise, especially when curation or primer design tasks are required. However, interface-based tools usually pose certain computational limitations when managing large sequences or complex datasets, such as genome and transcriptome assemblies. Having these requirments in mind we have developed SeqEditor an interactive software tool for nucleotide and protein sequences' analysis. RESULT: SeqEditor is a cross-platform desktop application for the analysis of nucleotide and protein sequences. It is managed through a Graphical User Interface and can work either as a graphical sequence browser or as a fasta task manager for multi-fasta files. SeqEditor has been optimized for the management of large sequences, such as contigs, scaffolds or even chromosomes, and includes a GTF/GFF viewer to visualize and manage annotation files. In turn, this allows for content mining from reference genomes and transcriptomes with similar efficiency to that of command line tools. SeqEditor also incorporates a set of tools for singleplex and multiplex PCR primer design and pooling that uses a newly optimized and validated search strategy for target and species-specific primers. All these features make SeqEditor a flexible application that can be used to analyses complex sequences, design primers in PCR assays oriented for diagnosis, and/or manage, edit and personalize reference sequence datasets. AVAILABILITYAND IMPLEMENTATION: SeqEditor was developed in Java using Eclipse Rich Client Platform and is publicly available at https://gpro.biotechvana.com/download/SeqEditor as binaries for Windows, Linux and Mac OS. The user manual and tutorials are available online at https://gpro.biotechvana.com/tool/seqeditor/manual. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Molecular epidemiology of clinical filamentous fungi in Qatar beyond Aspergillus and Fusarium with notes on the rare species.

Due to an increasing number of patients at risk (i.e., those with a highly compromised immune system and/or receiving aggressive chemotherapy treatment), invasive fungal infections (IFI) are increasingly being reported and associated with high mortality rates. Aspergillus spp., particularly A. fumigatus, is the major cause of IFI caused by filamentous fungi around the world followed by Fusarium spp., however, other fungi are emerging as human pathogens. The aim of this study was to explore the epidemiology and prevalence of the non-Aspergillus and non-Fusarium filamentous fungi in human clinical samples over an 11-year period in Qatar using molecular techniques. We recovered 53 filamentous fungal isolates from patients with various clinical conditions. Most patients were males (75.5%), 9.4% were immunocompromised, 20.7% had IFI, and 11.3% died within 30 days of diagnosis. The fungal isolates were recovered from a variety of clinical samples, including the nasal cavity, wounds, respiratory samples, body fluids, eye, ear, tissue, abscess, and blood specimens. Among the fungi isolated, 49% were dematiaceous fungi, followed by Mucorales (30%), with the latter group Mucorales being the major cause of IFI (5/11, 45.5%). The current study highlights the epidemiology and spectrum of filamentous fungal genera, other than Aspergillus and Fusarium, recovered from human clinical samples in Qatar, excluding superficial infections, which can aid in the surveillance of uncommon and emerging mycoses.

We recovered 53 non-Aspergillus and non-Fusarium filamentous fungal isolates from 53 patients in Qatar. Dematiaceous (black) fungi were the most isolated fungi followed by Mucorales, with the latter group Mucorales being the major cause of invasive infections in this study.

Communities of culturable yeasts and yeast-like fungi in oligotrophic hypersaline coastal waters of the Arabian Gulf surrounding Qatar.

This report is the first investigation of yeast biodiversity from the oligotrophic hypersaline coastal waters of the Arabian Gulf surrounding Qatar. Yeasts and yeast-like fungi, were cultured from seawater sampled at 13 coastal areas surrounding Qatar over a period of 2 years (December 2013-September 2015). Eight hundred and forty-two isolates belonging to 82 species representing two phyla viz., Ascomycota (23 genera) and Basidiomycota (16 genera) were identified by molecular sequencing. The results indicated that the coastal waters of the Qatari oligotrophic marine environment harbor a diverse pool of yeast species, most of which have been reported from terrestrial, clinical and aquatic sources in various parts of the world. Five species, i.e., Candida albicans, C. parapsilosis, C. tropicalis, Pichia kudriavzevii and Meyerozyma guilliermondii (n = 252/842; 30% isolates) are known as major opportunistic human pathogens. Fifteen species belonging to nine genera (n = 498/842; 59%) and 12 species belonging to seven genera (n = 459/842; 55%) are hydrocarbon degrading yeast and pollution indicator yeast species, respectively. Ascomycetous yeasts were predominant (66.38%; 559/842) as compared to their basidiomycetous counterparts (33.6%; 283/842). The most isolated yeast genera were Candida (28%; 236/842) (e.g., C. aaseri, C. boidinii, C. glabrata, C. intermedia, C. oleophila, C. orthopsilosis, C. palmioleophila, C. parapsilosis, C. pseudointermedia, C. rugopelliculosa, C. sake, C. tropicalis and C. zeylanoides), Rhodotorula (12.7%; 107/842), Naganishia (8.4%; 71/842), Aureobasidium (7.4%; 62/842), Pichia (7.3%; 62/842), and Debaryomyces (6.4%; 54/842). A total of eleven yeast species ( n = 38) isolated in this study are reported for the first time from the marine environment. Chemical testing demonstrated that seven out of the 13 sites had levels of total petroleum hydrocarbons (TPH) ranging from 200 to 900 µg/L, whereas 6 sites showed higher TPH levels (> 1000-21000 µg/L). The results suggest that the yeast community structure and density are impacted by various physico-chemical factors, namely total organic carbon, dissolved organic carbon and sulphur.

A New Filter Based Cultivation Approach for Improving Aspergillus Identification using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS).

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) is widely used in clinical laboratories for routine identification of bacteria and yeasts. However, methodological difficulties are still apparent when applied to filamentous fungi. The liquid cultivation method recommended by Bruker Daltonics GmbH for identification of filamentous fungi by MALDI-TOF MS is labour intensive and time-consuming. In this study, growth of Aspergillus species on different (porous) surfaces was investigated with the aim to develop a more reliable, quicker and less laborious identification method using MALDI-TOF MS. Mycelial growth without sporulation mimicking liquid cultivation and reliable MALDI-TOF MS spectra were obtained when A. fumigatus strains were grown on and in between a polycarbonate membrane filter on Sabouraud dextrose agar. A database of in-house reference spectra was created by growing Aspergillus reference strains (mainly focusing on sections Fumigati and Flavi) under these selected conditions. A test set of 50 molecularly identified strains grown under different conditions was used to select the best growth condition for identification and to perform an initial validation of the in-house database. Based on these results, the cultivation method on top of a polycarbonate filter proved to be most successful for species identification. This method was therefore selected for the identification of two sets of clinical isolates that mainly consisted of Aspergilli (100 strains originating from Indonesia, 70 isolates from Qatar). The results showed that this cultivation method is reliable for identification of clinically relevant Aspergillus species, with 67% and 76% correct identification of strains from Indonesia and Qatar, respectively. In conclusion, cultivation of Aspergilli on top of a polycarbonate filter showed improved results compared to the liquid cultivation protocol recommended by Bruker in terms of percentage of correct identification, ease of MSP creation, time consumption, cost and labour intensity. This method can be reliably applied for identification of clinically important Aspergilli and has potential for identification of other filamentous fungi.

Evolutionary engineering to improve Wickerhamomyces subpelliculosus and Kazachstania gamospora for baking.

The conventional baker's yeast, Saccharomyces cerevisiae, is the indispensable baking yeast of all times. Its monopoly coupled to its major drawbacks, such as streamlined carbon substrate utilisation base and a poor ability to withstand a number of baking associated stresses, prompt the need to search for alternative yeasts to leaven bread in the era of increasingly complex consumer lifestyles. Our previous work identified the inefficient baking attributes of Wickerhamomyces subpelliculosus and Kazachstania gamospora as well as preliminarily observations of improving the fermentative capacity of these potential alternative baker's yeasts using evolutionary engineering. Here we report on the characterisation and improvement in baking traits in five out of six independently evolved lines incubated for longer time and passaged for at least 60 passages relative to their parental strains as well as the conventional baker's yeast. In addition, the evolved clones produced bread with a higher loaf volume when compared to bread baked with either the ancestral strain or the control conventional baker's yeast. Remarkably, our approach improved the yeasts' ability to withstand baking associated stresses, a key baking trait exhibited poorly in both the conventional baker's yeast and their ancestral strains. W. subpelliculosus evolved the best characteristics attractive for alternative baker's yeasts as compared to the evolved K. gamospora strains. These results demonstrate the robustness of evolutionary engineering in development of alternative baker's yeasts.

Genetically related micafungin-resistant Candida parapsilosis blood isolates harbouring novel mutation R658G in hotspot 1 of Fks1p: a new challenge?

BACKGROUND: Echinocandin resistance rarely occurs in clinical Candida parapsilosis isolates and the underlying mechanism is unknown. OBJECTIVES: To determine the prevalence of echinocandin resistance and the underlying mechanism for a large collection of C. parapsilosis blood isolates and to determine whether the echinocandin-resistant isolates were clonally related. METHODS: C. parapsilosis blood isolates (n = 213) were subjected to antifungal susceptibility testing (CLSI M27), for micafungin, anidulafungin, amphotericin B and, if appropriate, caspofungin. Hotspot (HS) 1 and HS2 of FKS1 were sequenced for all isolates (n = 213) and microsatellite typing was performed for echinocandin-resistant isolates. RESULTS: All isolates were susceptible to amphotericin B and two isolates were intermediate to anidulafungin (MIC = 4 mg/L), while micafungin resistance was noted in four isolates (MIC >8 mg/L); three of which were also fluconazole resistant and therefore were MDR. Interestingly, micafungin-resistant isolates, but not those intermediate to anidulafungin, carried novel mutation R658G in HS1 of Fks1p; three of which also harboured Y132F+K143R in Erg11. The first isolate (MICR1) was recovered in November 2017 from a patient admitted to paediatric gastroenterology who showed therapeutic failure under caspofungin treatment. MICR2-MICR4 were collected during 2018-19 and were recovered from three echinocandin-naive paediatric-surgery patients; the isolates shared the same genotype. CONCLUSIONS: Herein, for the first time (to the best of our knowledge), we identified micafungin-resistant C. parapsilosis blood isolates harbouring a novel mutation in HS1 of FKS1, which was likely attributable to in vitro micafungin resistance and in vivo caspofungin therapeutic failure. The acquisition of micafungin-resistant C. parapsilosis isolates in echinocandin-naive patients likely implicates clonal expansion, as supported by the close genetic relatedness of MICR2-MICR4.

Multidrug-resistant Trichosporon species: underestimated fungal pathogens posing imminent threats in clinical settings.

Species of Trichosporon and related genera are widely used in biotechnology and, hence, many species have their genome sequenced. Importantly, yeasts of the genus Trichosporon have been increasingly identified as a cause of life-threatening invasive trichosporonosis (IT) in humans and are associated with an exceptionally high mortality rate. Trichosporon spp. are intrinsically resistant to frontline antifungal agents, which accounts for numerous reports of therapeutic failure when echinocandins are used to treat IT. Moreover, these fungi have low sensitivity to polyenes and azoles and, therefore, are potentially regarded as multidrug-resistant pathogens. However, despite the clinical importance of Trichosporon spp., our understanding of their antifungal resistance mechanisms is quite limited. Furthermore, antifungal susceptibility testing is not standardized, and there is a lack of interpretive epidemiological cut-off values for minimal inhibitory concentrations to distinguish non-wild type Trichosporon isolates. The route of infection remains obscure and detailed clinical and environmental studies are required to determine whether the Trichosporon infections are endogenous or exogenous in nature. Although our knowledge on effective IT treatments is rather limited and future randomized clinical trials are required to identify the best antifungal agent, the current paradigm advocates the use of voriconazole, removal of central venous catheters and recovery from neutropenia.

Comparative analysis of Malassezia furfur mitogenomes and the development of a mitochondria-based typing approach.

Malassezia furfur is a yeast species belonging to Malasseziomycetes, Ustilaginomycotina and Basidiomycota that is found on healthy warm-blooded animal skin, but also involved in various skin disorders like seborrheic dermatitis/dandruff and pityriasis versicolor. Moreover, Malassezia are associated with bloodstream infections, Crohn's disease and pancreatic carcinoma. Recent advances in Malassezia genomics and genetics have focused on the nuclear genome. In this work, we present the M. furfur mitochondrial (mt) genetic heterogenicity with full analysis of 14 novel and six available M. furfur mt genomes. The mitogenome analysis reveals a mt gene content typical for fungi, including identification of variable mt regions suitable for intra-species discrimination. Three of them, namely the trnK-atp6 and cox3-nad3 intergenic regions and intron 2 of the cob gene, were selected for primer design to identify strain differences. Malassezia furfur strains belonging to known genetic variable clusters, based on AFLP and nuclear loci, were assessed for their mt variation using PCR amplification and sequencing. The results suggest that these mt regions are excellent molecular markers for the typing of M. furfur strains and may provide added value to nuclear regions when assessing evolutionary relationships at the intraspecies level.

Conventional therapy and new antifungal drugs against Malassezia infections.

Malassezia yeasts are commensal microorganisms occurring on the skin of humans and animals causing dermatological disorders or systemic infections in severely immunocompromised hosts. Despite attempts to control such yeast infections with topical and systemic antifungals, recurrence of clinical signs of skin infections as well as treatment failure in preventing or treating Malassezia furfur fungemia have been reported most likely due to wrong management of these infections (e.g., due to early termination of treatment) or due to the occurrence of resistant phenomena. Standardized methods for in vitro antifungal susceptibility tests of these yeasts are still lacking, thus resulting in variable susceptibility profiles to azoles among Malassezia spp. and a lack of clinical breakpoints. The inherent limitations to the current pharmacological treatments for Malassezia infections both in humans and animals, stimulated the interest of the scientific community to discover new, effective antifungal drugs or substances to treat these infections. In this review, data about the in vivo and in vitro antifungal activity of the most commonly employed drugs (i.e., azoles, polyenes, allylamines, and echinocandins) against Malassezia yeasts, with a focus on human bloodstream infections, are summarized and their clinical implications are discussed. In addition, the usefulness of alternative compounds is discussed.