Daqu is a fermentative saccharification agent that is used to initiate fermentation in the production of Chinese liquor and vinegar. Different types of Daqu can be distinguished based on the maximum fermentation temperature, location of production, and raw materials used. We aimed to characterize and distinguish the different types of Daqu using a culture-independent cloning method. The lowest microbial diversity was found in Daqu produced at high-temperature. Principal component analysis (PCA) was used to compare the bacterial composition of Daqu from different regions (i.e., northern Daqu and southern Daqu). Staphylococcus gallinarum and Staphylococcus saprophyticus were found in southern Daqu, and were absent in northern Daqu. The fungi Saccharomycopsis fibuligera and Lichtheimia ramosa dominated in low/medium-temperature Daqu, whereas Thermomyces lanuginosus occurred in high-temperature Daqu. Our study identified potential biomarkers for the different types of Daqu, which can be useful for quality control and technology development of liquor or vinegar production.
Several human skin diseases and disorders are associated with two groups of fungi, the dermatophytes and Malassezia. Although these skin-related problems are not generally life threatening, they are among the most common diseases and disorders of mankind. These fungi are phylogenetically divergent, with the dermatophytes within the Ascomycota and Malassezia within Basidiomycota. Genome analysis indicates that the adaptations to the skin environment are different in these two groups of fungi. Malassezia are dependent on host lipids and secrete lipases and phospholipases that likely release host fatty acids. The dermatophytes encode multiple enzymes with potential roles in modulating host interactions: polyketide synthases, nonribosomal peptide synthetases, LysM, proteases, kinases, and pseudokinases. These two fungal groups have maximized their interactions with the host using two very different mechanisms.
Eleven uncommon yeast species that are associated with high mortality rates irrespective of antifungal therapy were isolated from 17/187 (201 episodes) pediatric and elderly patients with fungemia from Qatar. The samples were taken over a 6-year period (January 2004-December 2010). Isolated species included Kluyveromyces marxianus, Lodderomyces elongisporus, Lindnera fabianii, Candida dubliniensis, Meyerozyma guilliermondii, Candida intermedia, Pichia kudriavzevii, Yarrowia lipolytica, Clavispora lusitaniae, Candida pararugosa, and Wickerhamomyces anomalus. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry provided correct identifications compared with molecular analysis testing of the same isolates. Low minimal inhibitory concentrations were found when isavuconazole and voriconazole were used for all uncommon yeast species evaluated in this study. Resistance to antifungal drugs was low and remained restricted to a few species.
An interlaboratory study using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to determine the identification of clinically important yeasts (n = 35) was performed at 11 clinical centers, one company, and one reference center using the Bruker Daltonics MALDI Biotyper system. The optimal cutoff for the MALDI-TOF MS score was investigated using receiver operating characteristic (ROC) curve analyses. The percentages of correct identifications were compared for different sample preparation methods and different databases. Logistic regression analysis was performed to analyze the association between the number of spectra in the database and the percentage of strains that were correctly identified. A total of 5,460 MALDI-TOF MS results were obtained. Using all results, the area under the ROC curve was 0.95 (95% confidence interval [CI], 0.94 to 0.96). With a sensitivity of 0.84 and a specificity of 0.97, a cutoff value of 1.7 was considered optimal. The overall percentage of correct identifications (formic acid-ethanol extraction method, score ? 1.7) was 61.5% when the commercial Bruker Daltonics database (BDAL) was used, and it increased to 86.8% by using an extended BDAL supplemented with a Centraalbureau voor Schimmelcultures (CBS)-KNAW Fungal Biodiversity Centre in-house database (BDAL+CBS in-house). A greater number of main spectra (MSP) in the database was associated with a higher percentage of correct identifications (odds ratio [OR], 1.10; 95% CI, 1.05 to 1.15; P < 0.01). The results from the direct transfer method ranged from 0% to 82.9% correct identifications, with the results of the top four centers ranging from 71.4% to 82.9% correct identifications. This study supports the use of a cutoff value of 1.7 for the identification of yeasts using MALDI-TOF MS. The inclusion of enough isolates of the same species in the database can enhance the proportion of correctly identified strains. Further optimization of the preparation methods, especially of the direct transfer method, may contribute to improved diagnosis of yeast-related infections.
Two boletes that frequently form fruiting bodies in Pseudomonotes tropenbosii forests are described from Colombian Amazonia. One is a new species of Austroboletus here described as A. amazonicus and the other one is Fistulinella campinaranae var. scrobiculata Singer, which is a new record for Colombia. Macromorphological, micromorphological and habitat data for these species are provided as well as DNA sequence data of the internal transcribed spacer (ITS) regions and the D1/D2 domains of the large subunit (LSU) ribosomal DNA.
Chinese Daqu is used as a starter for liquor and vinegar fermentations. It is produced by solid state fermentation of cereal-pulse mixtures. A succession of fungi, lactic acid bacteria and Bacillus spp. was observed during the production of Daqu. Mesophilic bacteria followed by fungi, dominated the first phase of fermentation. Next, lactic acid bacteria increased in relative abundance, resulting in an increase of the acidity of Daqu. At the final stages of fermentation, Bacillus spp. and thermophilic fungi became the dominant groups, possibly due to their tolerance to low water activity and high temperature. Both culture-dependent and culture-independent analyses confirmed that Bacillus spp. were ubiquitous throughout the process. Yeast species such as Wickerhamomyces anomalus, Saccharomycopsis fibuligera and Pichia kudriavzevii were present throughout almost the entire fermentation process, but the zygomycetous fungus Lichtheimia corymbifera proliferated only during the final stages of fermentation. Canonical correspondence analysis (CCA) revealed the significance of acidity, moisture content and temperature in correlation with the composition of the microbial communities at different stages.
Despite being considered an emerging yeast related to immunocompromised individuals, severe infections by Malassezia furfur have not been evaluated. During a one-year survey on yeasts fungemia, 290 neonatal and 17 pediatric patients with intravascular catheters, lipid parenteral nutrition, prolonged ward stay, and surgery were enrolled. In addition, the origin of the infection was investigated by swabbing hand skin of patients, parents, and healthcare workers and medical devices. All biological specimens and swabs were cultured on Sabouraud dextrose agar and Dixon agar. The yeasts identification was based on morphological and biochemical features and by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and confirmed by sequencing the internal transcribed spacer of nuclear ribosomal DNA. A higher prevalence of M. furfur (2.1%) over Candida spp. (1.4%) caused bloodstream infections (BSIs). Twelve fungemia episodes were recorded: 2 by M. furfur in a pediatric ward and 10 in a neonatal intensive care unit (6 caused by M. furfur and 4 by Candida spp.). M. furfur was also isolated from the skin of all patients with BSIs, from the hand skin of a parent, and from an incubator surface and sheet. Patients with Candida spp. and M. furfur BSIs were successfully treated with intravenous liposomal Amphotericin B. These findings highlight the need for a more accurate etiological diagnosis in high-risk patients by adding lipid-supplemented culture media for Malassezia in the current mycological routine as the clinical features, patient management, and outcomes in both Candida and Malassezia fungemia do not differ.
A fungal culture (FCAS11) was isolated from coastal sediments of the Arabian Sea during the anoxic season. Multigene phylogenetic analyses confidentially place the organism as a novel species within the recently defined class Tritirachiomycetes, subphylum Pucciniomycotina, phylum Basidiomycota. We named the new species Tritirachium candoliense and provide the first description of a member of this class from a marine environment. DNA sequences and morphological characters distinguish T. candoliense from previously described Tritirachium species. Its growth characteristics, morphology, and ultrastructural features showed that under anoxic conditions the species grows slowly and produces mainly hyphae with only few blastoconidia. Electron microscopy revealed differences when the culture was exposed to anoxic stress. Notable ultrastructural changes occur for example in mitochondrial cristae, irregularly shaped fat globules and the presence of intracellular membrane invaginations. We assume that the growth characteristics and substrate utilization patterns are an adaptation to its source location, the seasonally anoxic environment of the Arabian Sea.
Recognition of fungal pathogens by C-type lectin receptor (CLR) dectin-1 on human dendritic cells is essential for triggering protective antifungal TH1 and TH17 immune responses. We show that Fonsecaea monophora, a causative agent of chromoblastomycosis, a chronic fungal skin infection, evades these antifungal responses by engaging CLR mincle and suppressing IL-12, which drives TH1 differentiation. Dectin-1 triggering by F. monophora activates transcription factor IRF1, which is crucial for IL12A transcription via nucleosome remodeling. However, simultaneous F. monophora binding to mincle induces an E3 ubiquitin ligase Mdm2-dependent degradation pathway, via Syk-CARD9-mediated PKB signaling, that leads to loss of nuclear IRF1 activity, hence blocking IL12A transcription. The absence of IL-12 leads to impaired TH1 responses and promotes TH2 polarization. Notably, mincle is similarly exploited by other chromoblastomycosis-associated fungi to redirect TH responses. Thus, mincle is a fungal receptor that can suppress antifungal immunity and, as such, is a potential therapeutic target.
A wealth of microbial eukaryotes is adapted to life in oxygen-deficient marine environments. Evidence is accumulating that some of these eukaryotes survive anoxia by employing dissimilatory nitrate reduction, a strategy that otherwise is widespread in prokaryotes. Here, we report on the anaerobic nitrate metabolism of the fungus Aspergillus terreus (isolate An-4) that was obtained from sediment in the seasonal oxygen minimum zone in the Arabian Sea, a globally important site of oceanic nitrogen loss and nitrous oxide emission.
We identified Candida spp isolated from lower respiratory tract secretions obtained from cystic fibrosis (CF) patients, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), with the aim of determining the most prevalent causative agent. We also sought to determine their adhesive properties in order to understand their biology related to CF.
DNA phylogenetic comparisons have shown that morphology-based species recognition often underestimates fungal diversity. Therefore, the need for accurate DNA sequence data, tied to both correct taxonomic names and clearly annotated specimen data, has never been greater. Furthermore, the growing number of molecular ecology and microbiome projects using high-throughput sequencing require fast and effective methods for en masse species assignments. In this article, we focus on selecting and re-annotating a set of marker reference sequences that represent each currently accepted order of Fungi. The particular focus is on sequences from the internal transcribed spacer region in the nuclear ribosomal cistron, derived from type specimens and/or ex-type cultures. Re-annotated and verified sequences were deposited in a curated public database at the National Center for Biotechnology Information (NCBI), namely the RefSeq Targeted Loci (RTL) database, and will be visible during routine sequence similarity searches with NR_prefixed accession numbers. A set of standards and protocols is proposed to improve the data quality of new sequences, and we suggest how type and other reference sequences can be used to improve identification of Fungi. Database URL: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA177353.
The numerous yeast genome sequences presently available provide a rich source of information for functional as well as evolutionary genomics but unequally cover the large phylogenetic diversity of extant yeasts. We present here the complete sequence of the nuclear genome of the haploid-type strain of Kuraishia capsulata (CBS1993(T)), a nitrate-assimilating Saccharomycetales of uncertain taxonomy, isolated from tunnels of insect larvae underneath coniferous barks and characterized by its copious production of extracellular polysaccharides. The sequence is composed of seven scaffolds, one per chromosome, totaling 11.4 Mb and containing 6,029 protein-coding genes, ?13.5% of which being interrupted by introns. This GC-rich yeast genome (45.7%) appears phylogenetically related with the few other nitrate-assimilating yeasts sequenced so far, Ogataea polymorpha, O. parapolymorpha, and Dekkera bruxellensis, with which it shares a very reduced number of tRNA genes, a novel tRNA sparing strategy, and a common nitrate assimilation cluster, three specific features to this group of yeasts. Centromeres were recognized in GC-poor troughs of each scaffold. The strain bears MAT alpha genes at a single MAT locus and presents a significant degree of conservation with Saccharomyces cerevisiae genes, suggesting that it can perform sexual cycles in nature, although genes involved in meiosis were not all recognized. The complete absence of conservation of synteny between K. capsulata and any other yeast genome described so far, including the three other nitrate-assimilating species, validates the interest of this species for long-range evolutionary genomic studies among Saccharomycotina yeasts.
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was used for an extensive identification study of arthroconidial yeasts, using 85 reference strains from the CBS-KNAW yeast collection and 134 clinical isolates collected from medical centers in Qatar, Greece, and Romania. The test set included 72 strains of ascomycetous yeasts (Galactomyces, Geotrichum, Saprochaete, and Magnusiomyces spp.) and 147 strains of basidiomycetous yeasts (Trichosporon and Guehomyces spp.). With minimal preparation time, MALDI-TOF MS proved to be an excellent diagnostic tool that provided reliable identification of most (98%) of the tested strains to the species level, with good discriminatory power. The majority of strains were correctly identified at the species level with good scores (>2.0) and seven of the tested strains with log score values between 1.7 and 2.0. The MALDI-TOF MS results obtained were consistent with validated internal transcribed spacer (ITS) and/or large subunit (LSU) ribosomal DNA sequencing results. Expanding the mass spectrum database by increasing the number of reference strains for closely related species, including those of nonclinical origin, should enhance the usefulness of MALDI-TOF MS-based diagnostic analysis of these arthroconidial fungi in medical and other laboratories.
Abstract Yarrowia lipolytica has been developed as a production host for a large variety of biotechnological applications. Efficacy and safety studies have demonstrated the safe use of Yarrowia-derived products containing significant proportions of Yarrowia biomass (as for DuPonts eicosapentaenoic acid-rich oil) or with the yeast itself as the final product (as for British Petroleums single-cell protein product). The natural occurrence of the species in food, particularly cheese, other dairy products and meat, is a further argument supporting its safety. The species causes rare opportunistic infections in severely immunocompromised or otherwise seriously ill people with other underlying diseases or conditions. The infections can be treated effectively by the use of regular antifungal drugs, and in some cases even disappeared spontaneously. Based on our assessment, we conclude that Y. lipolytica is a "safe-to-use" organism.
Yeast strains were characterized to select potential starter cultures for the production of masau fermented beverages. The yeast species originally isolated from Ziziphus mauritiana (masau) fruits and their traditionally fermented fruit pulp in Zimbabwe were examined for their ability to ferment glucose and fructose using standard broth under aerated and non-aerated conditions. Most Saccharomyces cerevisiae strains were superior to other species in ethanol production. The best ethanol producing S. cerevisiae strains, and strains of the species Pichia kudriavzevii, Pichia fabianii and Saccharomycopsis fibuligera were tested for production of flavor compounds during fermentation of masau fruit juice. Significant differences in the production of ethanol and other volatile compounds during fermentation of masau juice were observed among and within the four tested species. Alcohols and esters were the major volatiles detected in the fermented juice. Trace amounts of organic acids and carbonyl compounds were detected. Ethyl hexanoate and ethyl octanoate were produced in highest amounts as compared to the other volatile compounds. S. cerevisiae strains produced higher amounts of ethanol and flavor compounds as compared to the other species, especially fatty acid ethyl esters that provide the major aroma impact of freshly fermented wines. The developed library of characteristics can help in the design of mixtures of strains to obtain a specific melange of product functionalities.
Malassezia commensal yeasts are associated with a number of skin disorders, such as atopic eczema/dermatitis and dandruff, and they also can cause systemic infections. Here we describe the 7.67-Mbp genome of Malassezia sympodialis, a species associated with atopic eczema, and contrast its genome repertoire with that of Malassezia globosa, associated with dandruff, as well as those of other closely related fungi. Ninety percent of the predicted M. sympodialis protein coding genes were experimentally verified by mass spectrometry at the protein level. We identified a relatively limited number of genes related to lipid biosynthesis, and both species lack the fatty acid synthase gene, in line with the known requirement of these yeasts to assimilate lipids from the host. Malassezia species do not appear to have many cell wall-localized glycosylphosphatidylinositol (GPI) proteins and lack other cell wall proteins previously identified in other fungi. This is surprising given that in other fungi these proteins have been shown to mediate interactions (e.g., adhesion and biofilm formation) with the host. The genome revealed a complex evolutionary history for an allergen of unknown function, Mala s 7, shown to be encoded by a member of an amplified gene family of secreted proteins. Based on genetic and biochemical studies with the basidiomycete human fungal pathogen Cryptococcus neoformans, we characterized the allergen Mala s 6 as the cytoplasmic cyclophilin A. We further present evidence that M. sympodialis may have the capacity to undergo sexual reproduction and present a model for a pseudobipolar mating system that allows limited recombination between two linked MAT loci.
The diversity of Trichoderma (Hypocreales, Ascomycota) colonizing leaf litter as well as the rhizosphere of Garcinia macrophylla (Clusiaceae) was investigated in primary and secondary rain forests in Colombian Amazonia. DNA barcoding of 107 strains based on the internal transcribed spacers 1 and 2 (ITS1 and 2) of the ribosomal RNA gene cluster and the partial sequence of the translation elongation factor 1 alpha (tef1) gene revealed that the diversity of Trichoderma was dominated (71 %) by three common cosmopolitan species, namely Trichoderma harzianum sensu lato (41 %), Trichoderma spirale (17 %) and Trichoderma koningiopsis (13 %). Four ITS 1 and 2 phylotypes (13 strains) could not be identified with certainty. Multigene phylogenetic analysis and phenotype profiling of four strains with an ITS1 and 2 phylotype similar to Trichoderma strigosum revealed a new sister species of the latter that is described here as Trichoderma strigosellum sp. nov. Sequence similarity searches revealed that this species also occurs in soils of Malaysia and Cameroon, suggesting a pantropical distribution.
Understanding more about the hosts immune response to different Cryptococcus spp. will provide additional insight into the pathogenesis of cryptocococcis. We hypothesized that the ability of C. gattii to cause disease in immunocompetent humans depends on a distinct innate cytokine response of the host to this emerging pathogen. In the current study we assessed the cytokine profile of human peripheral blood mononuclear cells (PBMCs) of healthy individuals, after in vitro stimulation with 40 different well-defined heat-killed isolates of C. gattii, C. neoformans and several hybrid strains. In addition, we investigated the involvement of TLR2, TLR4 and TLR9 in the pro-inflammatory cytokine response to C. gattii. Isolates of C. gattii induced higher concentrations of the pro-inflammatory cytokines IL-1?, TNF-? and IL-6 and the Th17/22 cytokine IL-17 and IL-22 compared to C. neoformans var neoformans and C. neoformans var grubii. In addition, clinical C. gattii isolates induced higher amounts of cytokines than environmental isolates. This difference was not observed in C. neoformans var. grubii isolates. Furthermore, we demonstrated a likely contribution of TLR4 and TLR9, but no role for TLR2, in the hosts cytokine response to C. gattii. In conclusion, clinical heat-killed C. gattii isolates induced a more pronounced inflammatory response compared to other Cryptococcus species and non-clinical C. gattii. This is dependent on TLR4 and TLR9 as cellular receptors.
Cryptococcosis is an important fungal disease in Asia with an estimated 140,000 new infections annually the majority of which occurs in patients suffering from HIV/AIDS. Cryptococcus neoformans variety grubii (serotype A) is the major causative agent of this disease. In the present study, multilocus sequence typing (MLST) using the ISHAM MLST consensus scheme for the C. neoformans/C. gattii species complex was used to analyse nucleotide polymorphisms among 476 isolates of this pathogen obtained from 8 Asian countries. Population genetic analysis showed that the Asian C. neoformans var. grubii population shows limited genetic diversity and demonstrates a largely clonal mode of reproduction when compared with the global MLST dataset. HIV-status, sequence types and geography were found to be confounded. However, a correlation between sequence types and isolates from HIV-negative patients was observed among the Asian isolates. Observations of high gene flow between the Middle Eastern and the Southeastern Asian populations suggest that immigrant workers in the Middle East were originally infected in Southeastern Asia.
Over the past two decades, several fungal outbreaks have occurred, including the high-profile Vancouver Island and Pacific Northwest outbreaks, caused by Cryptococcus gattii, which has affected hundreds of otherwise healthy humans and animals. Over the same time period, C. gattii was the cause of several additional case clusters at localities outside of the tropical and subtropical climate zones where the species normally occurs. In every case, the causative agent belongs to a previously rare genotype of C. gattii called AFLP6/VGII, but the origin of the outbreak clades remains enigmatic. Here we used phylogenetic and recombination analyses, based on AFLP and multiple MLST datasets, and coalescence gene genealogy to demonstrate that these outbreaks have arisen from a highly-recombining C. gattii population in the native rainforest of Northern Brazil. Thus the modern virulent C. gattii AFLP6/VGII outbreak lineages derived from mating events in South America and then dispersed to temperate regions where they cause serious infections in humans and animals.
A molecular taxonomic investigation performed on basidiomycetous yeast strains isolated from plant leaves collected in two areas of China revealed two novel species, Cryptococcus foliicola sp. nov. (type strain HS 23.3(T) = AS 2.2471(T) = CBS 9920(T)) and Cryptococcus taibaiensis sp. nov. (type strain ST 7.9(T) = AS 2.2444(T) = CBS 9919(T)), among the non ballistoconidium-forming strains producing cream-colored colonies. These new species differed markedly from closely related species in the internal transcribed spacer (ITS) and 26S rRNA D1/D2 region sequences. They clustered in a strongly supported clade represented by Cryptococcus victoriae in the Tremellales group in the phylogenetic trees drawn from ITS and D1/D2 sequences.
Five yeast strains isolated from plant leaves collected in south-east Tibet formed cream to brownish colonies and produced asymmetrical ballistoconidia and CoQ-9 as the major ubiquinone. Sequence analysis of the 26S rRNA D1/D2 domain and the internal transcribed spacer region indicated that these strains represented two novel species of the genus Bensingtonia. The names Bensingtonia rectispora sp. nov. (type strain XZ 4C5(T)?=?CGMCC 2.02635(T)?=?CBS 10710(T)) and Bensingtonia bomiensis sp. nov. (type strain XZ 33D1(T)?=?CGMCC 2.02670(T)?=?CBS 10713(T)) are proposed for the two novel species, which are phylogenetically closely related to Bensingtonia naganoensis, Bensingtonia pseudonaganoensis and the type species of the genus, Bensingtonia ciliata.
Cryptococcal meningitis is mainly caused by Cryptococcus neoformans and Cryptococcus gattii, but occasionally other Cryptococcus species and phylogenetically related species are involved. Herein, we present a case of cryptococcal meningitis from China, which was caused by an azole and flucytosine resistant Filobasidium uniguttulatum. In addition, we present an overview of the literature of meningitis caused by Cryptococcus species other than C. neoformans and C. gattii. Eight cases were related to infections of the central nervous system. Leukaemia and cancer were important risk factors in HIV-negative patients. Molecular identification and susceptibility testing are important for proper management of patients because the species involved may differ in susceptibility to antifungal drugs.
Cryptococcus gattii has becoming more prevalent in temperate climate zones, during the past decades. We describe a C. gattii serotype B infection in an immunocompetent Italian patient with sclerosing cholangitis. The patient traveled once to Eastern Canada and otherwise no other countries than Italy were visited. Molecular analysis revealed that the C. gattii isolate belong to genotype AFLP4/VGI and has mating-type ? which is the most common genotype in the Mediterranean environment. The C. gattii strain was found to be closely related, but not identical, to other C. gattii strains from the Mediterranean area.
Candida parapsilosis, which was previously considered to be a complex of three genetically distinct groups, has emerged as a significant agent of nosocomial infections. Recently, this complex was separated into three species: C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis. In China, data pertaining to these fungi are limited. In this study, we examined 57 isolates of members of the C. parapsilosis complex collected from four cities in East China, i.e., Nanjing (n = 22), Nanchang (n = 20), Shanghai (n = 12) and Jinan (n = 3). C. parapsilosis sensu stricto represented 71.9% of all isolates, while C. metapsilosis accounted for the remaining 28.1%. C. orthopsilosis could not be identified. A significantly high prevalence of C. metapsilosis was observed in strains recovered from Nanchang, 60% (12/20) of the isolates were C. metapsilosis. Sequence analysis of internal transcribed spacer region revealed two unevenly distributed genotypes among the C. metapsilosis strains. A PCR-restriction fragment length polymorphism assay was described for rapid identification. The strains were susceptible to fluconazole, voriconazole, amphoterincin B and micafungin. Six (15%) isolates of C. parapsilosis sensu stricto and three (18.8%) of C. metapsilosis were found to be dose-dependent susceptible to itraconazole. C. parapsilosis sensu stricto strains were less susceptible to micafungin than C. metapsilosis.
A collection of 48 clinical Cryptococcus neoformans isolates from Croatia was investigated retrospectively using in vitro antifungal susceptibility testing and molecular biological techniques to determine mating type and serotype by PCR and amplified fragment length polymorphism (AFLP) genotyping. These isolates were obtained from 15 patients: ten were human immunodeficiency virus (HIV)-negative (66.7?%) and five were HIV-positive (33.3?%). From five patients, only one isolate was available, whilst from the other ten patients, two to 11 isolates were isolated sequentially. Antifungal susceptibility was tested by a broth microdilution method. Serotype A (genotype AFLP1) and serotype D (genotype AFLP2) were both found in six patients (40?% each), and serotype AD (genotype AFLP3) in three (20.0?%) patients. Mating type ? (n?=?12; 80.0?%) predominated and ?/a hybrids were identified in 20.0?% of patients diagnosed with cryptococcosis. Two AFLP genotypes of C. neoformans were isolated during a single episode from one patient. The in vitro antifungal MIC(90) and susceptibility ranges for C. neoformans isolates were 0.5 µg ml(-1) (range 0.031-0.5 µg ml(-1)) for amphotericin B, 4 µg ml(-1) (range 1-4 µg ml(-1)) for flucytosine and fluconazole, 0.25 µg ml(-1) (range 0.031-0.5 µg ml(-1)) for itraconazole and 0.062 µg ml(-1) (range 0.031-0.25 µg ml(-1)) for voriconazole.
The Amsterdam Declaration on Fungal Nomenclature was agreed at an international symposium convened in Amsterdam on 19-20 April 2011 under the auspices of the International Commission on the Taxonomy of Fungi (ICTF). The purpose of the symposium was to address the issue of whether or how the current system of naming pleomorphic fungi should be maintained or changed now that molecular data are routinely available. The issue is urgent as mycologists currently follow different practices, and no consensus was achieved by a Special Committee appointed in 2005 by the International Botanical Congress to advise on the problem. The Declaration recognizes the need for an orderly transitition to a single-name nomenclatural system for all fungi, and to provide mechanisms to protect names that otherwise then become endangered. That is, meaning that priority should be given to the first described name, except where that is a younger name in general use when the first author to select a name of a pleomorphic monophyletic genus is to be followed, and suggests controversial cases are referred to a body, such as the ICTF, which will report to the Committee for Fungi. If appropriate, the ICTF could be mandated to promote the implementation of the Declaration. In addition, but not forming part of the Declaration, are reports of discussions held during the symposium on the governance of the nomenclature of fungi, and the naming of fungi known only from an environmental nucleic acid sequence in particular. Possible amendments to the Draft BioCode (2011) to allow for the needs of mycologists are suggested for further consideration, and a possible example of how a fungus only known from the environment might be described is presented.
Among ballistoconidium-forming yeast strains isolated from various plant leaves collected from subtropical forests in eastern and central China, four strains forming cream to yellowish coloured colonies were revealed to represent three novel Derxomyces species by conventional and molecular characterization. Phylogenetic analysis based on combined sequences of the internal transcribed spacer (ITS) and 26S rRNA gene D1/D2 domain showed that strains GT-753 and ZJJ-890T were conspecific and closely related to Derxomyces boninensis, Derxomyces mrakii and Derxomyces qinlingensis. Strain ZJJ-394T was basal to the branch formed by Derxomyces komagatae, Derxomyces pseudoschimicola and Derxomyces schimicola with strong bootstrap support. Strain GT-475T was closely related to Derxomyces linzhiensis. The strains differed significantly from their close relatives in D1/D2 and ITS sequences and in physiological criteria. Three novel species are proposed: Derxomyces amylogenes sp. nov. (type strain ZJJ-890T=CGMCC 2.4407T=CBS 12233T), Derxomyces bambusicola sp. nov. (type strain GT-475T=CGMCC 2.4411T=CBS 12234T) and Derxomyces corylopsis sp. nov. (type strain ZJJ-394T=CGMCC 2.4409T=CBS 12259T).
Recent Cryptococcus gattii infections in humans and animals, including several outbreaks in goats, were the basis of this environmental survey in six provinces of Spain. A total of 479 samples from 20 tree species were studied. Cryptococcus gattii was found for the first time in autochthonous Mediterranean trees in Spain. Fourteen isolates of this pathogen were obtained from seven trees of three different species: 12 from carob (Ceratonia siliqua), one from Mediterranean stone pine (Pinus halepensis) and another from eucalyptus (Eucalyptus camaldulensis). All C. gattii isolates were genotype AFLP4/VGI and mating type alpha, and were found to be genetically identical with C. gattii strains isolated from humans and animals in Spain. This supports the hypothesis that these trees may be a natural source for infection of humans and mammals in the Mediterranean area.
The global burden of HIV-associated cryptococcal meningitis is estimated at nearly one million cases per year, causing up to a third of all AIDS-related deaths. Molecular epidemiology constitutes the main methodology for understanding the factors underpinning the emergence of this understudied, yet increasingly important, group of pathogenic fungi. Cryptococcus species are notable in the degree that virulence differs amongst lineages, and highly-virulent emerging lineages are changing patterns of human disease both temporally and spatially. Cryptococcus neoformans variety grubii (Cng, serotype A) constitutes the most ubiquitous cause of cryptococcal meningitis worldwide, however patterns of molecular diversity are understudied across some regions experiencing significant burdens of disease. We compared 183 clinical and environmental isolates of Cng from one such region, Thailand, Southeast Asia, against a global MLST database of 77 Cng isolates. Population genetic analyses showed that Thailand isolates from 11 provinces were highly homogenous, consisting of the same genetic background (globally known as VNI) and exhibiting only ten nearly identical sequence types (STs), with three (STs 44, 45 and 46) dominating our sample. This population contains significantly less diversity when compared against the global population of Cng, specifically Africa. Genetic diversity in Cng was significantly subdivided at the continental level with nearly half (47%) of the global STs unique to a genetically diverse and recombining population in Botswana. These patterns of diversity, when combined with evidence from haplotypic networks and coalescent analyses of global populations, are highly suggestive of an expansion of the Cng VNI clade out of Africa, leading to a limited number of genotypes founding the Asian populations. Divergence time testing estimates the time to the most common ancestor between the African and Asian populations to be 6,920 years ago (95% HPD 122.96 - 27,177.76). Further high-density sampling of global Cng STs is now necessary to resolve the temporal sequence underlying the global emergence of this human pathogen.
Mucor circinelloides is a zygomycete fungus and an emerging opportunistic pathogen in immunocompromised patients, especially transplant recipients and in some cases otherwise healthy individuals. We have discovered a novel example of size dimorphism linked to virulence. M. circinelloides is a heterothallic fungus: (+) sex allele encodes SexP and (-) sex allele SexM, both of which are HMG domain protein sex determinants. M. circinelloides f. lusitanicus (Mcl) (-) mating type isolates produce larger asexual sporangiospores that are more virulent in the wax moth host compared to (+) isolates that produce smaller less virulent sporangiospores. The larger sporangiospores germinate inside and lyse macrophages, whereas the smaller sporangiospores do not. sexM? mutants are sterile and still produce larger virulent sporangiospores, suggesting that either the sex locus is not involved in virulence/spore size or the sexP allele plays an inhibitory role. Phylogenetic analysis supports that at least three extant subspecies populate the M. circinelloides complex in nature: Mcl, M. circinelloides f. griseocyanus, and M. circinelloides f. circinelloides (Mcc). Mcc was found to be more prevalent among clinical Mucor isolates, and more virulent than Mcl in a diabetic murine model in contrast to the wax moth host. The M. circinelloides sex locus encodes an HMG domain protein (SexP for plus and SexM for minus mating types) flanked by genes encoding triose phosphate transporter (TPT) and RNA helicase homologs. The borders of the sex locus between the three subspecies differ: the Mcg sex locus includes the promoters of both the TPT and the RNA helicase genes, whereas the Mcl and Mcc sex locus includes only the TPT gene promoter. Mating between subspecies was restricted compared to mating within subspecies. These findings demonstrate that spore size dimorphism is linked to virulence of M. circinelloides species and that plasticity of the sex locus and adaptations in pathogenicity have occurred during speciation of the M. circinelloides complex.
C-type lectins dectin-1 and dectin-2 on dendritic cells elicit protective immunity against fungal infections through induction of T(H)1 and T(H)-17 cellular responses. Fungal recognition by dectin-1 on human dendritic cells engages the CARD9-Bcl10-Malt1 module to activate NF-?B. Here we demonstrate that Malt1 recruitment is pivotal to T(H)-17 immunity by selective activation of NF-?B subunit c-Rel, which induces expression of T(H)-17-polarizing cytokines IL-1? and IL-23p19. Malt1 inhibition abrogates c-Rel activation and T(H)-17 immunity to Candida species. We found that Malt1-mediated activation of c-Rel is similarly essential to induction of T(H)-17-polarizing cytokines by dectin-2. Whereas dectin-1 activates all NF-?B subunits, dectin-2 selectively activates c-Rel, signifying a specialized T(H)-17-enhancing function for dectin-2 in anti-fungal immunity by human dendritic cells. Thus, dectin-1 and dectin-2 control adaptive T(H)-17 immunity to fungi via Malt1-dependent activation of c-Rel.
Field campaigns in Antarctica, Greenland and the Italian glaciers aiming to explore the biodiversity of these disappearing environments identified several undescribed yeast strains unable to grow at temperature above 20°C and belonging to unknown species. Fourteen of these strains were selected and grouped based on their morphological and physiological characteristics. Sequences of the D1/D2 and ITS regions of the ribosomal RNA demonstrated that the strains belong to unknown species related to Leucosporidium antarcticum. The new genus Glaciozyma is proposed and two new species are described, namely Glaciozyma martinii sp. nov. and Glaciozyma watsonii sp. nov. Additionally, re-classification of Leucosporidium antarcticum as Glaciozyma antarctica is proposed. Strains of Glaciozyma form a monophyletic clade and a well separated lineage within class Microbotryomycetes (Pucciniomycotina, Basidiomycota). The description of Glaciozyma genus and the re-classification of L. antarcticum reduce the polyphyletic nature of the genus Leucosporidium.
The in vitro susceptibilities of a worldwide collection of 350 Cryptococcus gattii isolates to seven antifungal drugs, including the new triazole isavuconazole, were tested. With amplified fragment length polymorphism (AFLP) fingerprinting, human, veterinary, and environmental C. gattii isolates were subdivided into seven AFLP genotypes, including the interspecies hybrids AFLP8 and AFLP9. The majority of clinical isolates (n = 215) comprised genotypes AFLP4 (n = 76) and AFLP6 (n = 103). The clinical AFLP6 isolates had significantly higher geometric mean MICs for flucytosine and fluconazole than the clinical AFLP4 isolates. Of the seven antifungal compounds examined in this study, isavuconazole had the lowest MIC(90) (0.125 ?g/ml) for all C. gattii isolates, followed by a 1 log(2) dilution step increase (MIC(90), 0.25 ?g/ml) for itraconazole, voriconazole, and posaconazole. Amphotericin B had an acceptable MIC(90) of 0.5 ?g/ml, but fluconazole and flucytosine had relatively high MIC(90)s of 8 ?g/ml.
Several species of the genus Penicillium were isolated during a survey of the mycobiota of leaf litter and soil in Colombian Amazon forest. Five species, Penicillium penarojense sp. nov. (type strain CBS 113178(T)?=?IBT 23262(T)), Penicillium wotroi sp. nov. (type strain CBS 118171(T)?=?IBT 23253(T)), Penicillium araracuarense sp. nov. (type strain CBS 113149(T)?=?IBT 23247(T)), Penicillium elleniae sp. nov. (type strain CBS 118135(T)?=?IBT 23229(T)) and Penicillium vanderhammenii sp. nov. (type strain CBS 126216(T)?=?IBT 23203(T)) are described here as novel species. Their taxonomic novelty was determined using a polyphasic approach, combining phenotypic, molecular (ITS and partial ?-tubulin sequences) and extrolite data. Phylogenetic analyses showed that each novel species formed a unique clade for both loci analysed and that they were most closely related to Penicillium simplicissimum, Penicillium janthinellum, Penicillium daleae and Penicillium brasilianum. An overview of the phylogeny of this taxonomically difficult group is presented, and 33 species are accepted. Each of the five novel species had a unique extrolite profile of known and uncharacterized metabolites and various compounds, such as penicillic acid, andrastin A, pulvilloric acid, paxillin, paspaline and janthitrem, were commonly produced by these phylogenetically related species. The novel species had a high growth rate on agar media, but could be distinguished from each other by several macro- and microscopical characteristics.
Cryptococcus neoformans is commonly associated with meningoencephalitis in immunocompromised patients and occasionally in apparently healthy individuals. Recurrence of infection after initial treatment is not uncommon. We studied C. neoformans isolates from 7 Cuban patients with recurrent cryptococcal meningitis. Antifungal susceptibility and genotyping with microsatellite molecular typing were carried out.
The novel genus Holtermanniella is proposed here to accommodate four Cryptococcus species closely related to Holtermannia corniformis that are included in the Holtermannia clade (Basidiomycota, Agaricomycotina). Thus, four novel combinations are proposed: Holtermanniella nyarrowii comb. nov., Holtermanniella festucosa comb. nov., Holtermanniella mycelialis comb. nov. and Holtermanniella wattica comb. nov. In addition, a novel anamorphic yeast species was studied with 15 isolates obtained from different habitats around the world. Analysis of the sequences of the D1/D2 region of their large subunit rDNA showed that the novel species is placed phylogenetically within the Holtermannia clade of the Tremellomycetes (Agaricomycotina, Basidiomycota). PCR fingerprinting and sequencing of ITS1-5.8S-ITS2 showed genetic intraspecific variability among the strains: three groups were formed, which did not correlate with geographical origin or substrate. This novel species, designated the type species of Holtermanniella gen. nov., is described as Holtermanniella takashimae sp. nov.; the type strain is CBS 11174(T) (=HB 982(T) =DBVPG 8012(T)). The order Holtermanniales ord. nov. is proposed here to include Holtermannia (the type genus) and Holtermanniella.
We studied the serotypes, mating-types, AFLP genotypes, and antifungal susceptibility of 58 Cryptococcus neoformans strains causing 56 episodes of cryptococcosis in 55 patients over an 18-year period in a single institution. The underlying conditions of the patients were classified as HIV infection (n = 48) or non-HIV-related immunodeficiency (n = 7). Serotype A (n = 34; 58.9%) predominated, but serotype AD was involved in 23.2% of episodes. Most of the episodes were caused by mating-type ? (n = 41; 73.2%) or ?/a strains (n = 12; 21.5%). The most common genotype was AFLP1 (n = 26; 44.8%), followed by AFLP3 (n = 21; 36.2%), and AFLP2 (n = 11; 19.0%). In two different patients, we showed the coexistence of different serotypes and/or genotypes in the same episode (AFLP1 and 3). The new triazoles voriconazole, posaconazole and isavuconazole showed high and similar antifungal activity (MICs ? 0.125 ?g/ml). Fluconazole also had good antifungal activity, but two strains from patients with HIV-infections had an MIC of 16 ?g/ml (3.4%). However, these two isolates remained very susceptible to the new triazoles (MICs ? 0.062 ?g/ml). The remaining strains always showed MICs ? 8 ?g/ml.
The distinction and monophyletic property of the basidiomycetous yeast species in the Bulleribasidium clade of the order Tremellales was resolved by molecular phylogenetic analysis based on the combined sequences of the 18S rRNA gene, internal transcribed spacer (ITS) region including 5.8S rRNA gene and 26S rRNA gene D1/D2 domain. The addition to the clade of new anamorphic species identified among ballistoconidium-forming yeasts isolated from China confirmed and strengthened the separation of this clade from other clades or lineages in the order Tremellales. A new anamorphic genus, Mingxiaea gen. nov. (type species Mingxiaea variabilis comb. nov.) is therefore proposed to accommodate the anamorphic species in the Bulleribasidium clade. Six new combinations are proposed for the described species of this clade which were formerly assigned to the genus Bullera. Four novel species in the new genus were identified among 16 ballistoconidium-forming yeast strains isolated from plant leaves collected in Hainan province, southern China, by D1/D2 and ITS sequence analyses. The novel species are described as Mingxiaea sanyaensis sp. nov. (type strain SY-3.23(T) =AS 2. 3623(T) =CBS 11408(T)), Mingxiaea hainanensis (type strain WZS-8.13(T) =AS 2.4161(T) =CBS 11409(T)), Mingxiaea foliicola (type strain WZS-8.14(T) =AS 2.3518(T) =CBS 11407(T)) and Mingxiaea wuzhishanensis (type strain WZS-29.8(T) =AS 2.4163(T) =CBS 11411(T)).
Langerhans cells (LCs) lining the stratified epithelia and mucosal tissues are the first antigen presenting cells to encounter invading pathogens, such as viruses, bacteria and fungi. Fungal infections form a health threat especially in immuno-compromised individuals. LCs express C-type lectin Langerin that has specificity for mannose, fucose and GlcNAc structures. Little is known about the role of human Langerin in fungal infections. Our data show that Langerin interacts with both mannan and beta-glucan structures, common cell-wall carbohydrate structures of fungi. We have screened a large panel of fungi for recognition by human Langerin and, strikingly, we observed strong binding of Langerin to a variety of Candida and Saccharomyces species and Malassezia furfur, but very weak binding was observed to Cryptococcus gattii and Cryptococcus neoformans. Notably, Langerin is the primary fungal receptor on LCs, since the interaction of LCs with the different fungi was blocked by antibodies against Langerin. Langerin recognizes both mannose and beta-glucans present on fungal cell walls and our data demonstrate that Langerin is the major fungal pathogen receptor on human LCs that recognizes pathogenic and commensal fungi. Together these data may provide more insight in the role of LCs in fungal infections.
Human cryptococcal infections have been associated with bird droppings as a likely source of infection. Studies toward the local and global epidemiology of Cryptococcus spp. have been hampered by the lack of rapid, discriminatory, and exchangeable molecular typing methods.
Mushroom-forming basidiomycetes colonize large areas in nature. Their hyphae are compartmentalized by perforated septa, which are usually covered by a septal pore cap (SPC). Here, we describe, for the first time, the composition and function of SPCs using the model system Schizophyllum commune. The SPC of S. commune was shown to consist of a proteinaceous matrix covered by a lipid membrane. The matrix was demonstrated to define the ultrastructure of the SPC and to consist of two main proteins, Spc14 and Spc33. Gene spc14 encodes a protein of 86 amino acids, which lacks known domain, signal or localization sequences. Gene spc33 encodes a 239 and a 340 amino acid variant. Both forms contain a predicted signal anchor that targets them to the ER. Immuno-localization showed the presence of Spc33 in the SPC but not in ER. From this and previous reports it is concluded that the SPC is derived from this organelle. Inactivation of spc33 resulted in loss of SPCs and the inability to close septa. The latter may well explain why vegetative growth and mushroom formation were severely reduced in strains in which spc33 was inactivated.
Kodamaea (Pichia) ohmeri is a yeast species that has not been reported to be a frequent cause of human infections. The current report describes a case of fungemia caused by K. ohmeri in a 3-year-old female patient hospitalized in the public hospital Maria Alice Fernandes, Natal, RN, Brazil. The patient had previously received antimicrobial therapy due to a peritoneal infection and nosocomial pneumonia, and had a central venous catheter implanted. Kodamaea ohmeri was isolated from blood and the tip of the catheter, 48 h after its implantation. The yeast was identified by standard microbiological methods and sequence analysis of the D1/D2 domains and the ITS 1 + 2 spacer regions of the ribosomal DNA. On CHROMagar Candida medium, the isolate showed a color change from pink to blue. The yeast was susceptible to amphotericin B, and liposomal AmB was used successfully to clear the infection.
Jansson and Sung showed that, given a dense set of input triplets T (representing hypotheses about the local evolutionary relationships of triplets of taxa), it is possible to determine in polynomial time whether there exists a level-1 network consistent with T, and if so, to construct such a network . Here, we extend this work by showing that this problem is even polynomial time solvable for the construction of level-2 networks. This shows that, assuming density, it is tractable to construct plausible evolutionary histories from input triplets even when such histories are heavily nontree-like. This further strengthens the case for the use of triplet-based methods in the construction of phylogenetic networks. We also implemented the algorithm and applied it to yeast data.
A novel anamorphic Cryptococcus species is described, which was isolated in New Delhi (India) from decaying wood of a tree trunk hollow of Ficus religiosa. On the basis of sequence analysis of the D1/D2 domains of the 26S rRNA gene and the internally transcribed spacer (ITS)-1 and ITS-2 region sequences, the isolate belonged to the Cryptococcus albidus cluster (Filobasidiales, Tremellomycetes) and was closely related to Cryptococcus saitoi, Cryptococcus cerealis and Cryptococcus friedmannii with 98% sequence identity. Phenotypically, the species differed from C. saitoi with respect to growth temperature (up to 37degrees C), presence of a thin capsule, ability to grow in the absence of vitamins, and inability to assimilate citrate and ethylamine. With respect to C. friedmannii, it differed in growth temperature, ability to assimilate lactose, raffinose, L: -rhamnose, myo-inositol, and inability to utilize citrate. Furthermore, our isolate also differed from C. cerealis in growth temperature, presence of capsule and inability to assimilate L: -sorbose. In view of the above phenotypic differences and unique rDNA sequences, we consider that our isolate represents a new species of Cryptococcus, and therefore, a new species, Cryptococcus randhawai is proposed for this taxon. The type strain J11/2002 has been deposited in the culture collection of the Centraalbureau voor Schimmelcultures (CBS10160) and CABI Biosciences (IMI 393306).
Two yeast strains isolated in 2007 from fermented pig feed were studied, including the analysis of sequences of the D1/D2 and ITS-regions of the rDNA-repeats, their morphology and nutritional physiology. Sequence comparison of the D1/D2 and ITS regions demonstrated that the strains do not belong to any known species. Therefore, a new species, Cryptococcus cerealis with the type strain CBS 10505, is proposed. The species belongs to Filobasidiales (Agaricomycetes, Basidiomycota), and has Cryptococcus saitoi as the closest related species. The new species is psychrophilic, showing significant growth at 4 and 10 degrees C.
Seven strains representing a novel yeast species belonging to the genus Cryptococcus were isolated from different substrates from Patagonia, Argentina, and The Netherlands. Three strains were isolated from a meltwater river draining from the Frias glacier at Mount Tronador situated in Nahuel Huapi National Park (Patagonia) and four were isolated from apple surfaces in Randwijk, The Netherlands. Analysis of the D1/D2 large-subunit rRNA gene and ITS region sequences indicated that these strains represent a single species that is distinct from other species of the Tremellales clade. The name Cryptococcus spencermartinsiae sp. nov. is proposed to accommodate these strains. The type strain is CRUB 1230(T) (=CBS 10760(T) =DBVPG 8010(T)).
Worldwide glaciers are annually retreating due to global overheating and this phenomenon determines the potential lost of microbial diversity represented by psychrophilic microbial population sharing these peculiar habitats. In this context, yeast strains, all unable to grow above 20 degrees C, consisting of 42 strains from Antarctic soil and 14 strains isolated from Alpine Glacier, were isolated and grouped together based on similar morphological and physiological characteristics. Sequences of the D1/D2 and ITS regions of the ribosomal DNA confirmed the previous analyses and demonstrated that the strains belong to unknown species. Three new species are proposed: Mrakia robertii sp. nov. (type strain CBS 8912), Mrakia blollopis sp. nov. (type strain CBS 8921) and a related anamorphic species Mrakiella niccombsii sp. nov. (type strain CBS 8917). Phylogenetic analysis of the ITS region revealed that the new proposed species were closely related to each other within the Mrakia clade in the order Cystofilobasidiales, class Tremellomycetes. The Mrakia clade now contains 8 sub-clades. Teliospores were observed in all strains except CBS 8918 and for the Mrakiella niccombsii strains.
In 1999, the population of Vancouver Island, Canada, began to experience an outbreak of a fatal fungal disease caused by a highly virulent lineage of Cryptococcus gattii. This organism has recently spread to the Canadian mainland and Pacific Northwest, but the molecular cause of the outbreak remains unknown. Here we show that the Vancouver Island outbreak (VIO) isolates have dramatically increased their ability to replicate within macrophages of the mammalian immune system in comparison with other C. gattii strains. We further demonstrate that such enhanced intracellular parasitism is directly linked to virulence in a murine model of cryptococcosis, suggesting that this phenotype may be the cause of the outbreak. Finally, microarray studies on 24 C. gattii strains reveals that the hypervirulence of the VIO isolates is characterized by the up-regulation of a large group of genes, many of which are encoded by mitochondrial genome or associated with mitochondrial activities. This expression profile correlates with an unusual mitochondrial morphology exhibited by the VIO strains after phagocytosis. Our data thus demonstrate that the intracellular parasitism of macrophages is a key driver of a human disease outbreak, a finding that has significant implications for a wide range of other human pathogens.
Cryptococcus diffluens is a recently re-established species that shares several phenotypic features with Cryptococcus neoformans. We evaluated the application of the Clinical Laboratory Standards Institute (CLSI, formerly NCCLS) macro- and microbroth dilution methods and the E-test agar diffusion method to determine the in vitro susceptibilities of known strains of C. diffluens against amphotericin B (AMB), flucytosine (5-FC), fluconazole (FLC), itraconazole (ITC) and the novel triazoles, voriconazole (VRC) and posaconazole (PSC). Seven strains were found to be resistant in vitro to AMB (MICs >/=2 microg/ml), five were resistant to 5-FC (MICs of >/=32 microg/ml), four were resistant to FLC (MICs of FLC >/=32 microg/ml) and nine were resistant to ITC (MICs of ITC >1 microg/ml). In contrast, VRC and PSC showed good in vitro activity against C.diffluens strains, even those with elevated MICs to amphotericin B and/or established azoles. Most of the isolates were inhibited by 0.5 microg/ml of both VRC and PSC. A clinical isolate showing phenotypic switching exhibited elevated MICs to both agents, i.e., VRC (>16 microg/ml) and PSC (>8 microg/ml).
This communication describes the consensus multi-locus typing scheme established by the Cryptococcal Working Group I (Genotyping of Cryptococcus neoformans and C. gattii) of the International Society for Human and Animal Mycology (ISHAM) using seven unlinked genetic loci for global strain genotyping. These genetic loci include the housekeeping genes CAP59,GPD1, LAC1, PLB1, SOD1, URA5 and the IGS1 region. Allele and sequence type information are accessible at http://www.mlst.net/ .
Trichosporon species have been reported as emerging pathogens and usually occur in severely immunocompromised patients. In the present work, 27 clinical isolates of Trichosporon species were recovered from 27 patients. The patients were not immunocompromised, except for one with acute myeloid leukemia. Sequence analysis revealed the isolation of Trichosporon dohaense Taj-Aldeen, Meis & Boekhout sp. nov., with CBS 10761(T) as the holotype strain, belonging to the Ovoides clade. In the D1-D2 large-subunit rRNA gene analysis, T. dohaense is a sister species to T. coremiiforme, and in the internal transcribed spacer analysis, the species is basal to the other species of this clade. Molecular identification of the strains yielded 17 T. asahii, 3 T. inkin, 2 T. japonicum, 2 T. faecale, and 3 T. dohaense isolates. The former four species exhibited low MICs for five antifungal azoles but showed high MICs for amphotericin B. T. dohaense demonstrated the lowest amphotericin B MIC (1 mg/liter). For the majority of T. asahii isolates, amphotericin B MICs were high (MIC at which 90% of isolates were inhibited [MIC(90)], > or = 16 mg/liter), and except for fluconazole (MIC(90), 8 mg/liter), the azole MICs were low: MIC(90)s were 0.5 mg/liter for itraconazole, 0.25 mg/liter for voriconazole, 0.25 mg/liter for posaconazole, and 0.125 mg/liter for isavuconazole. The echinocandins, caspofungin and anidulafungin, demonstrated no activity against Trichosporon species.
Cryptococcus gattii is a primary pathogenic basidiomycetous yeast comprising four genotypic groups. Here we present data on two mitochondrial loci (MtLrRNA and ATP6). Two of the genotypic groups, namely amplified fragment length polymorphism (AFLP)5/VGIII and AFLP6/VGII, formed monophyletic lineages. The AFLP4/VGI genotypic group, however, possessed five different mitochondrial genotypes that did not form a monophyletic lineage. The majority of these isolates contained mitochondrial genomes that are partially identical to those found in isolates belonging to AFLP6/VGII, which is causing the ongoing and expanding Vancouver Island outbreak. Two out of four AFLP7/VGIV isolates contained an AFLP4/VGI allele of MtLrRNA. These observations are best explained by assuming a process of mitochondrial recombination. If this is true, mitochondrial recombination seems possible between cells belonging to different genotypic groups of C. gattii, especially between AFLP6/VGII or AFLP7/VGIV and AFLP4/VGI. We also have to assume that mitochondria, most likely, were transferred from cells belonging to AFLP6/VGII to AFLP4/VGI. As such a process of mitochondrial recombination is only possible after cell-cell conjugation, this may also allow the further exchange of genetic material, for example nuclear or plasmid in nature, between different genotypes of C. gattii. This may be relevant as it may provide a possible mechanism contributing to the modulation of virulence attributes of isolates, such as has been observed in the ongoing Vancouver Island outbreak of C. gattii.
The ultrastructure of septa and septum-associated septal pore caps are important taxonomic markers in the Agaricomycotina (Basidiomycota, Fungi). The septal pore caps covering the typical basidiomycetous dolipore septum are divided into three main phenotypically recognized morphotypes: vesicular-tubular (including the vesicular, sacculate, tubular, ampulliform, and globular morphotypes), imperforate, and perforate. Until recently, the septal pore cap-type reflected the higher-order relationships within the Agaricomycotina. However, the new classification of Fungi resulted in many changes including revision of existing and addition of new orders. Therefore, the septal pore cap ultrastructure of more than 325 species as reported in literature was related to this new classification. In addition, the septal pore cap ultrastructures of Rickenella fibula and Cantharellus formosus were examined by transmission electron microscopy. Both fungi have dolipore septa associated with perforate septal pore caps. These results combined with data from the literature show that the septal pore cap-type within orders of the Agaricomycotina is generally monomorphic, except for the Cantharellales and Hymenochaetales. It appears from the fungal phylogeny combined with the septal pore cap ultrastructure that the vesicular-tubular and the imperforate type both may have arisen from endoplasmic reticulum. Thereafter, the imperforate type eventually gave rise to the perforate septal pore cap-type.
Ziziphus mauritiana (masau) fruits are consumed by many people in Zimbabwe. The fruits contribute significantly to peoples diet when they are in season. The objective of this study was to determine the nutritional content of the fruits and, hence, quantify their contribution to the diet. Samples of masau were collected in two seasons (August 2006 and August 2007). Both macronutrients and micronutrients were determined using standard AOAC methods of analysis. Dry matter content ranged from 21.1±0.2 to 24.1±0.3 g 100 g(-1) of edible portion of the sweet and sour fruits, and 84.8±0.2 to 87.2±0.2 g 100 g(-1) for the dried fruit. Crude protein per 100g edible portion of dry weight ranged between 7.9±0.0 and 8.7±0.0 g, crude fat from 0.8±0.0 to 1.5±0.0 g, crude fibre from 4.9±0.0 to 7.3±0.0 g, ash between 3.0±0.0 and 4.3±0.0 g and carbohydrate between 79.5±0.0 and 83.2±0.0 g. The fruits were rich in vitamin C (15.0±0.0-43.8±0.02 mg 100 g(-1)) and the energy values ranged between 1516.0±1.73 and 1575.0±2.3 kJ 100 g(-1). Furthermore, the fruits contained (mg 100 g(-1) of dry weight) potassium from 1865.0±1.3 to 2441.0±1.1, calcium from 160.0±0.3 to 254.0±0.1, sodium between 185.0±0.1 and 223.0±0.2, magnesium between 83.0±0.0 and 150.0±0.13 and phosphorous from 87.0±0.1 to 148.0±0.5. Manganese and copper contents ranged between 0.7±0.03 and 1.6±0.03, while iron and zinc ranged between 2.1±0.43 and 4.3±0.1, and 0.6±0.0-0.9±0.0 mg 100 g(-1) of dry weight, respectively. The masau fruit is therefore a good potential source of carbohydrates, proteins and micronutrients, such as calcium, potassium, sodium, phosphorous, copper, iron, Vitamin C and zinc.
Rapid identification of clinically important yeasts can facilitate the initiation of anti-fungal therapy, since susceptibility is largely species-dependent. We evaluated melting peak and melting curve analysis of the internally transcribed spacer region 2 fragment (ITS2-MCA) as an identification tool for distinguishing between 16 Candida spp., i.e. Candida albicans, Candida bracarensis, Candida dubliniensis, Candida famata, Candida glabrata, Candida guilliermondii, Candida inconspicua, Candida kefyr, Candida krusei, Candida lipolytica, Candida lusitaniae, Candida nivariensis, Candida norvegensis, Candida parapsilosis, Candida tropicalis and Candida sojae, and Saccharomyces cerevisiae and one species pair, i.e. Candida metapsilosis/Candida orthopsilosis. Starting from a cultured isolate, ITS2-MCA led to differentiation of these species within 6 h. According to our findings, ITS2-MCA offers a simple, rapid and cost-effective method for identification of cultured isolates of the clinically most relevant and prevalent Candida species. Further studies will be necessary to evaluate how it performs on mixed samples and clinical samples.
Until recently, Cryptococcus gattii infections occurred mainly in tropical and subtropical climate zones. However, during the past decade, C. gattii infections in humans and animals in Europe have increased. To determine whether the infections in Europe were acquired from an autochthonous source or associated with travel, we used multilocus sequence typing to compare 100 isolates from Europe (57 from 40 human patients, 22 from the environment, and 21 from animals) with 191 isolates from around the world. Of the 57 human patient isolates, 47 (83%) were obtained since 1995. Among the 40 patients, 24 (60%) probably acquired the C. gattii infection outside Europe; the remaining 16 (40%) probably acquired the infection within Europe. Human patient isolates from Mediterranean Europe clustered into a distinct genotype with animal and environmental isolates. These results indicate that reactivation of dormant C. gattii infections can occur many years after the infectious agent was acquired elsewhere.
The aim of the study was to compare the effect of two low-cost, low technology traditional methods for drying starter cultures with standard lyophilisation. Lyophilised yeast cultures and yeast cultures preserved in dry rice cakes and dry plant fibre strands were examined for viable cell counts during 6 months storage at 4 and 25 °C. None of the yeast cultures showed a significant loss in viable cell count during 6 months of storage at 4 °C upon lyophilisation and preservation in dry rice cakes. During storage at 25 °C in the dark, yeast cultures preserved in dry rice cakes, and lyophilised cultures of Saccharomyces cerevisiae and Issatchenkia orientalis showed no significant loss of viable cells up to 4 months of storage. Yeast cultures preserved in dry plant fibre strands had the greatest loss of viable count during the 6 months of storage at 25 °C. Preservation of yeasts cultures in dry rice cakes provided better survival during storage at 4 °C than lyophilisation. The current study demonstrated that traditional methods can be useful and effective for starter culture preservation in small-scale, low-tech applications.
Daqu is a traditional fermentation starter that is used for Chinese liquor production. Although partly mechanized, its manufacturing process has remained traditional. We investigated the microbial diversity of Fen-Daqu, a starter for light-flavour liquor, using combined culture-dependent and culture-independent approaches (PCR-DGGE). A total of 190 microbial strains, comprising 109 bacteria and 81 yeasts and moulds, were isolated and identified on the basis of the sequences of their 16S rDNA (bacteria) and 26S rDNA and ITS regions (fungi). DGGE of DNA extracted from Daqu was used to complement the culture-dependent method in order to include non-culturable microbes. Both approaches revealed that Bacillus licheniformis was an abundant bacterial species, and Saccharomycopsis fibuligera, Wickerhamomyces anomalus, and Pichia kudriavzevii were the most common yeasts encountered in Fen-Daqu. Six genera of moulds (Absidia, Aspergillus, Mucor, Rhizopus, Rhizomucor and Penicillium) were found. The potential function of these microorganisms in starters for alcoholic fermentation is discussed. In general the culture-based findings overlapped with those obtained by DGGE by a large extent. However, Weissella cibaria, Weissella confusa, Staphylococcus saprophyticus, Enterobacter aerogenes, Lactobacillus sanfranciscensis, Lactobacillus lactis, and Bacillus megaterium were only revealed by DGGE.
A set of 300 Dutch Cryptococcus neoformans isolates, obtained from 237 patients during 1977 to 2007, was investigated by determining the mating type, serotype, and AFLP and microsatellite genotype and susceptibility to seven antifungal compounds. Almost half of the studied cases were from HIV-infected patients, followed by a patient group of individuals with other underlying diseases and immunocompetent individuals. The majority of the isolates were mating type ? and serotype A, followed by ?D isolates and other minor categories. The most frequently observed genotype was AFLP1, distantly followed by AFLP2 and AFLP3. Microsatellite typing revealed a high genetic diversity among serotype A isolates but a lower diversity within the serotype D set of isolates. One patient was infected by multiple AFLP genotypes. Fluconazole and flucytosine had the highest geometric mean MICs of 2.9 and 3.5 ?g/ml, respectively, while amphotericin B (0.24 ?g/ml), itraconazole (0.08 ?g/ml), voriconazole (0.07 ?g/ml), posaconazole (0.06 ?g/ml), and isavuconazole (0.03 ?g/ml) had much lower geometric mean MICs. One isolate had a high flucytosine MIC (>64 ?g/ml), while decreased susceptibility (?16 ?g/ml) for flucytosine and fluconazole was found in 9 and 10 C. neoformans isolates, respectively.
Cryptococcus neoformans is a pathogenic yeast that causes cryptococcosis, a life threatening disease. The prevalence of cryptococcosis in Asia has been rising after the onset of the AIDS epidemic and estimates indicate more than 120 cases per 1,000 HIV-infected individuals per year. Almost all cryptococcal disease cases in both immunocompromised and immunocompetent patients in Asia are caused by C. neoformans var. grubii. Epidemiological studies on C. neoformans in pan-Asia have not been reported. The present work studies the genetic diversity of the fungus by microsatellite typing and susceptibility analysis of approximately 500 isolates from seven Asian countries.
Production of the proinflammatory cytokine interleukin 1? (IL-1?) 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-1? 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-1? 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-1? production and maturation through a noncanonical caspase-8-dependent inflammasome for protective immunity.
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