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.

Lesional skin of seborrheic dermatitis patients is characterized by skin barrier dysfunction and correlating alterations in the stratum corneum ceramide composition.

Seborrheic dermatitis (SD) is a chronic inflammatory skin disease characterized by erythematous papulosquamous lesions in sebum rich areas such as the face and scalp. Its pathogenesis appears multifactorial with a disbalanced immune system, Malassezia driven microbial involvement and skin barrier perturbations. Microbial involvement has been well described in SD, but skin barrier involvement remains to be properly elucidated. To determine whether barrier impairment is a critical factor of inflammation in SD alongside microbial dysbiosis, a cross-sectional study was performed in 37 patients with mild-to-moderate facial SD. Their lesional and non-lesional skin was comprehensively and non-invasively assessed with standardized 2D-photography, optical coherence tomography (OCT), microbial profiling including Malassezia species identification, functional skin barrier assessments and ceramide profiling. The presence of inflammation was established through significant increases in erythema, epidermal thickness, vascularization and superficial roughness in lesional skin compared to non-lesional skin. Lesional skin showed a perturbed skin barrier with an underlying skewed ceramide subclass composition, impaired chain elongation and increased chain unsaturation. Changes in ceramide composition correlated with barrier impairment indicating interdependency of the functional barrier and ceramide composition. Lesional skin showed significantly increased Staphylococcus and decreased Cutibacterium abundances but similar Malassezia abundances and mycobial composition compared to non-lesional skin. Principal component analysis highlighted barrier properties as main discriminating features. To conclude, SD is associated with skin barrier dysfunction and changes in the ceramide composition. No significant differences in the abundance of Malassezia were observed. Restoring the cutaneous barrier might be a valid therapeutic approach in the treatment of facial SD.

Treatment with the Topical Antimicrobial Peptide Omiganan in Mild-to-Moderate Facial Seborrheic Dermatitis versus Ketoconazole and Placebo: Results of a Randomized Controlled Proof-of-Concept Trial.

Facial seborrheic dermatitis (SD) is an inflammatory skin disease characterized by erythematous and scaly lesions on the skin with high sebaceous gland activity. The yeast Malassezia is regarded as a key pathogenic driver in this disease, but increased Staphylococcus abundances and barrier dysfunction are implicated as well. Here, we evaluated the antimicrobial peptide omiganan as a treatment for SD since it has shown both antifungal and antibacterial activity. A randomized, patient- and evaluator-blinded trial was performed comparing the four-week, twice daily topical administration of omiganan 1.75%, the comparator ketoconazole 2.00%, and placebo in patients with mild-to-moderate facial SD. Safety was monitored, and efficacy was determined by clinical scoring complemented with imaging. Microbial profiling was performed, and barrier integrity was assessed by trans-epidermal water loss and ceramide lipidomics. Omiganan was safe and well tolerated but did not result in a significant clinical improvement of SD, nor did it affect other biomarkers, compared to the placebo. Ketoconazole significantly reduced the disease severity compared to the placebo, with reduced Malassezia abundances, increased microbial diversity, restored skin barrier function, and decreased short-chain ceramide Cer[NSc34]. No significant decreases in Staphylococcus abundances were observed compared to the placebo. Omiganan is well tolerated but not efficacious in the treatment of facial SD. Previously established antimicrobial and antifungal properties of omiganan could not be demonstrated. Our multimodal characterization of the response to ketoconazole has reaffirmed previous insights into its mechanism of action.

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.

Antimicrobial Susceptibility Testing for Three Malassezia Species.

The Malassezia genus comprises lipid-dependent yeasts that have long been associated with common skin diseases, and have recently been linked with Crohn's disease and certain cancers. Understanding Malassezia susceptibility to diverse antimicrobial agents is crucial for identifying effective antifungal therapies. Here, we tested the efficacy of isavuconazole, itraconazole, terbinafine, and artemisinin against three Malassezia species: M. restricta, M. slooffiae, and M. sympodialis. Using broth microdilution, we found antifungal properties for the two previously unstudied antimicrobials: isavuconazole and artemisinin. Overall, all Malassezia species were particularly susceptible to itraconazole, with a MIC range from 0.007 to 0.110 µg/mL. IMPORTANCE The Malassezia genus is known to be involved in a variety of skin conditions and has recently been associated with diseases such as Crohn's disease, pancreatic ductal carcinoma, and breast cancer. This work was completed to assess susceptibility to a variety of antimicrobial drugs on three Malassezia species, in particular Malassezia restricta, which is an abundant Malassezia species both on human skin and internal organs and has been implicated in Crohn's disease. We tested two previously unstudied drugs and developed a new testing method to overcome current limitations for measuring growth inhibition of slow-growing Malassezia strains.

Co-evolution of large inverted repeats and G-quadruplex DNA in fungal mitochondria may facilitate mitogenome stability: the case of Malassezia.

Mitogenomes are essential due to their contribution to cell respiration. Recently they have also been implicated in fungal pathogenicity mechanisms. Members of the basidiomycetous yeast genus Malassezia are an important fungal component of the human skin microbiome, linked to various skin diseases, bloodstream infections, and they are increasingly implicated in gut diseases and certain cancers. In this study, the comparative analysis of Malassezia mitogenomes contributed to phylogenetic tree construction for all species. The mitogenomes presented significant size and gene order diversity which correlates to their phylogeny. Most importantly, they showed the inclusion of large inverted repeats (LIRs) and G-quadruplex (G4) DNA elements, rendering Malassezia mitogenomes a valuable test case for elucidating the evolutionary mechanisms responsible for this genome diversity. Both LIRs and G4s coexist and convergently evolved to provide genome stability through recombination. This mechanism is common in chloroplasts but, hitherto, rarely found in mitogenomes.

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 incorporating newly generated 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 towards the discovery of a sexual cycle in Malassezia , with possible implications for pathogenicity. Significance Statement: Malassezia , the dominant fungal group of the mammalian skin microbiome, is associated with numerous skin disorders. Sexual development and yeast-to-hyphae transitions, governed by genes at two mating-type ( MAT ) loci, are thought to be important for fungal pathogenicity. However, Malassezia sexual reproduction has never been observed. Here, we used chromosome-level assemblies and comparative genomics to uncover unforeseen transitions in MAT loci organization within Malassezia , possibly related with fragility of centromeric-associated regions. Additionally, by expressing different MAT alleles in the same cell, we show that Malassezia can undergo hyphal development and this phenotype is associated with increased expression of key mating genes along with other genes known to be virulence factors, providing a possible connection between hyphal development, sexual reproduction, and pathogenicity.

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.

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.

Mitogenomics and mitochondrial gene phylogeny decipher the evolution of Saccharomycotina yeasts.

Saccharomycotina yeasts belong to diverse clades within the kingdom of fungi and are important to human everyday life. This work investigates the evolutionary relationships among these yeasts from a mitochondrial (mt) genomic perspective. A comparative study of 155 yeast mt genomes representing all major phylogenetic lineages of Saccharomycotina was performed, including genome size and content variability, intron and intergenic regions' diversity, genetic code alterations, and syntenic variation. Findings from this study suggest that mt genome size diversity is the result of a ceaseless random process, mainly based on genetic recombination and intron mobility. Gene order analysis revealed conserved syntenic units and many occurring rearrangements, which can be correlated with major evolutionary events as shown by the phylogenetic analysis of the concatenated mt protein matrix. For the first time, molecular dating indicated a slower mt genome divergence rate in the early stages of yeast evolution, in contrast with a faster rate in the late evolutionary stages, compared to their nuclear time divergence. Genetic code reassignments of mt genomes are a perpetual process happening in many different parallel evolutionary steps throughout the evolution of Saccharomycotina. Overall, this work shows that phylogenetic studies based on the mt genome of yeasts highlight major evolutionary events.

Multiple Hybridization Events Punctuate the Evolutionary Trajectory of Malassezia furfur.

Malassezia species are important fungal skin commensals and are part of the normal microbiota of humans and other animals. However, under certain circumstances these fungi can also display a pathogenic behavior. For example, Malassezia furfur is a common commensal of human skin and yet is often responsible for skin disorders but also systemic infections. Comparative genomics analysis of M. furfur revealed that some isolates have a hybrid origin, similar to several other recently described hybrid fungal pathogens. Because hybrid species exhibit genomic plasticity that can impact phenotypes, we sought to elucidate the genomic evolution and phenotypic characteristics of M. furfur hybrids in comparison to their parental lineages. To this end, we performed a comparative genomics analysis between hybrid strains and their presumptive parental lineages and assessed phenotypic characteristics. Our results provide evidence that at least two distinct hybridization events occurred between the same parental lineages and that the parental strains may have originally been hybrids themselves. Analysis of the mating-type locus reveals that M. furfur has a pseudobipolar mating system and provides evidence that after sexual liaisons of mating compatible cells, hybridization involved cell-cell fusion leading to a diploid/aneuploid state. This study provides new insights into the evolutionary trajectory of M. furfur and contributes with valuable genomic resources for future pathogenicity studies. IMPORTANCEMalassezia furfur is a common commensal member of human/animal microbiota that is also associated with several pathogenic states. Recent studies report involvement of Malassezia species in Crohn's disease, a type of inflammatory bowel disease, pancreatic cancer progression, and exacerbation of cystic fibrosis. A recent genomics analysis of M. furfur revealed the existence of hybrid isolates and identified their putative parental lineages. In this study, we explored the genomic and phenotypic features of these hybrids in comparison to their putative parental lineages. Our results revealed the existence of a pseudobipolar mating system in this species and showed evidence for the occurrence of multiple hybridization events in the evolutionary trajectory of M. furfur. These findings significantly advance our understanding of the evolution of this commensal microbe and are relevant for future studies exploring the role of hybridization in the adaptation to new niches or environments, including the emergence of pathogenicity.

Genetic and phenotypic diversity of fecal Candida albicans strains in irritable bowel syndrome.

Irritable bowel syndrome (IBS) is a common disorder characterized by chronic abdominal pain and changes in bowel movements. Visceral hypersensitivity is thought to be responsible for pain complaints in a subset of patients. In an IBS-like animal model, visceral hypersensitivity was triggered by intestinal fungi, and lower mycobiota α-diversity in IBS patients was accompanied by a shift toward increased presence of Candida albicans and Saccharomyces cerevisiae. Yet, this shift was observed in hypersensitive as well as normosensitive patients and diversity did not differ between IBS subgroups. The latter suggests that, when a patient changes from hyper- to normosensitivity, the relevance of intestinal fungi is not necessarily reflected in compositional mycobiota changes. We now confirmed this notion by performing ITS1 sequencing on an existing longitudinal set of fecal samples. Since ITS1 methodology does not recognize variations within species, we next focused on heterogeneity within cultured healthy volunteer and IBS-derived C. albicans strains. We observed inter- and intra-individual genomic variation and partial clustering of strains from hypersensitive patients. Phenotyping showed differences related to growth, yeast-to-hyphae morphogenesis and gene expression, specifically of the gene encoding fungal toxin candidalysin. Our investigations emphasize the need for strain-specific cause-and-effect studies within the realm of IBS research.

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.

Malassezia spp. Yeasts of Emerging Concern in Fungemia.

Malassezia spp. are lipid-dependent yeasts, inhabiting the skin and mucosa of humans and animals. They are involved in a variety of skin disorders in humans and animals and may cause bloodstream infections in severely immunocompromised patients. Despite a tremendous increase in scientific knowledge of these yeasts during the last two decades, the epidemiology of Malassezia spp. related to fungemia remains largely underestimated most likely due to the difficulty in the isolation of these yeasts species due to their lipid-dependence. This review summarizes and discusses the most recent literature on Malassezia spp. infection and fungemia, its occurrence, pathogenicity mechanisms, diagnostic methods, in vitro susceptibility testing and therapeutic approaches.

Evaluation of Molecular Epidemiology, Clinical Characteristics, Antifungal Susceptibility Profiles, and Molecular Mechanisms of Antifungal Resistance of Iranian Candida parapsilosis Species Complex Blood Isolates.

Clonal expansion of fluconazole resistant (FLZ-R) Candida parapsilosis isolates is increasingly being identified in many countries, while there is no study exploring the antifungal susceptibility pattern, genetic diversity, and clinical information for Iranian C. parapsilosis blood isolates. Candida parapsilosis species complex blood isolates (n = 98) were recovered from nine hospitals located in three major cities, identified by MALDI-TOF MS, and their genetic relatedness was examined by AFLP fingerprinting. Antifungal susceptibility testing followed CLSI-M27-A3 and ERG11, MRR1 and hotspots 1/2 (HS1/2) of FKS1 were sequenced to assess the azole and echinocandin resistance mechanisms, respectively. Ninety-four C. parapsilosis and four Candida orthopsilosis isolates were identified from 90 patients. Only 43 patients received systemic antifungal drugs with fluconazole as the main antifungal used. The overall mortality rate was 46.6% (42/90) and death mostly occurred for those receiving systemic antifungals (25/43) relative to those not treated (17/47). Although, antifungal-resistance was rare, one isolate was multidrug-resistant (FLZ = 16 µg/ml and micafungin = 8 µg/ml) and the infected patient showed therapeutic failure to FLZ prophylaxis. Mutations causing azole and echinocandin resistance were not found in the genes studied. AFLP revealed five genotypes (G) and G1 was the main one (59/94; 62.7%). Clinical outcome was significantly associated with city (P = 0.02, α <0.05) and Mashhad was significantly associated with mortality (P = 0.03, α <0.05). Overall, we found a low level of antifungal resistance for Iranian C. parapsilosis blood isolates, but the noted MDR strain can potentially become the source of future infections and challenge the antifungal therapy in antifungal-naïve patients. AFLP typing results warrants confirmation using other resolutive typing methods.

The mitochondrial intergenic regions nad1-cob and cob-rps3 as molecular identification tools for pathogenic members of the genus Cryptococcus.

Cryptococcus spp. are fungal species belonging to Tremellomycetes, Agaricomycotina, Basidiomycota, and several members are responsible for cryptococcosis, one of the most ubiquitous human mycoses. Affecting mainly immunosuppressed patients, but also immunocompetent ones, the members of this genus present a high level of genetic diversity. In this study, two mitochondrial intergenic regions, i.e. nad1-cob and cob-rps3, were tested for the intra- or interspecies discrimination and identification of strains and species of the genus Cryptococcus. Phylogenetic trees were constructed based on individual and concatenated sequences from representative pathogenic strains of the Cryptococcus neoformans/Cryptococcus gattii complex, representing serotypes and AFLP genotypes of all newly introduced species of this complex. Using both intergenic regions, as well as the concatenated dataset, the strains clustered in accordance with the new taxonomy. These results suggest that identification of Cryptococcus strains is possible by employing these mitochondrial intergenic regions using PCR amplification as a quick and effective method to elucidate genotypic and taxonomic differences. Thus, these regions may be applicable to a broad range of clinical studies, leading to a rapid recognition of the clinical profiles of patients.

The Emergence of Rare Clinical Aspergillus Species in Qatar: Molecular Characterization and Antifungal Susceptibility Profiles.

Aspergillus are ubiquitous mold species that infect immunocompetent and immunocompromised patients. The symptoms are diverse and range from allergic reactions, bronchopulmonary infection, and bronchitis, to invasive aspergillosis. The aim of this study was to characterize 70 Aspergillus isolates recovered from clinical specimens of patients with various clinical conditions presented at Hamad general hospital in Doha, Qatar, by using molecular methods and to determine their in vitro antifungal susceptibility patterns using the Clinical and Laboratory Standards Institute (CLSI) M38-A2 reference method. Fourteen Aspergillus species were identified by sequencing ß-tubulin and calmodulin genes, including 10 rare and cryptic species not commonly recovered from human clinical specimens. Aspergillus welwitschiae is reported in this study for the first time in patients with fungal rhinosinusitis (n = 6) and one patient with a lower respiratory infection. Moreover, Aspergillus pseudonomius is reported in a patient with fungal rhinosinusitis which is considered as the first report ever from clinical specimens. In addition, Aspergillus sublatus is reported for the first time in a patient with cystic fibrosis. In general, our Aspergillus strains exhibited low MIC values for most of the antifungal drugs tested. One strain of Aspergillus fumigatus showed high MECs for echinocandins and low MICs for the rest of the drugs tested. Another strain of A. fumigatus exhibited high MIC for itraconazole and categorized as non-wild type. These findings require further analysis of their molecular basis of resistance. In conclusion, reliable identification of Aspergillus species is achieved by using molecular sequencing, especially for the emerging rare and cryptic species. They are mostly indistinguishable by conventional methods and might exhibit variable antifungal susceptibility profiles. Moreover, investigation of the antifungal susceptibility patterns is necessary for improved antifungal therapy against aspergillosis.

Genotypes and population genetics of cryptococcus neoformans and cryptococcus gattii species complexes in Europe and the mediterranean area.

A total of 476 European isolates (310 Cryptococcus neoformans var. grubii, 150 C. neoformans var. neoformans, and 16 C. gattii species complex) from both clinical and environmental sources were analyzed by multi-locus sequence typing. Phylogenetic and population genetic analyses were performed. Sequence analysis identified 74 sequence types among C. neoformans var. neoformans (VNIV), 65 among C. neoformans var. grubii (56 VNI, 8 VNII, 1 VNB), and 5 among the C. gattii species complex (4 VGI and 1 VGIV) isolates. ST23 was the most frequent genotype (22%) among VNI isolates which were mostly grouped in a large clonal cluster including 50% of isolates. Among VNIV isolates, a predominant genotype was not identified. A high percentage of autochthonous STs were identified in both VNI (71%) and VNIV (96%) group of isolates. The 16 European C. gattii species complex isolates analyzed in the present study originated all from the environment and all belonged to a large cluster endemic in the Mediterranean area. Population genetic analysis confirmed that VNI group of isolates were characterized by low variability and clonal expansion while VNIV by a higher variability and a number of recombination events. However, when VNI and VNIV environmental isolates were compared, they showed a similar population structure with a high percentage of shared mutations and the absence of fixed mutations. Also linkage disequilibrium analysis reveals differences between clinical and environmental isolates showing a key role of PLB1 allele combinations in host infection as well as the key role of LAC1 allele combinations for survival of the fungus in the environment. The present study shows that genetic comparison of clinical and environmental isolates represents a first step to understand the genetic characteristics that cause the shift of some genotypes from a saprophytic to a parasitic life style.