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Previously, 1,8-dihydroxynaphthalene (DHN)-melanin was described to protect Aspergillus fumigatus against hydrogen peroxide (H2O2), thereby protecting this opportunistic human pathogen from reactive oxygen species generated by the immune system. This was based on the finding that the ATCC 46645 mutant with mutations in the pksP gene of the DHN-melanin synthesis pathway showed increased sensitivity to reactive oxygen species compared to the wild type. Here, it is shown that deletion of the pksP gene in A. fumigatus strain CEA10 did not affect sensitivity for H2O2 and superoxide in a plate stress assay. In addition, direct exposure of the dormant white conidia of the pksP deletion strains to H2O2 did not result in increased sensitivity. Moreover, complementation of the ATCC 46645 pksP mutant strain with the wild-type pksP gene did result in pigmented conidia but did not rescue the H2O2-sensitive phenotype observed in the plate stress assay. Genome sequencing of the ATCC 46645 pksP mutant strain and its complemented strain revealed a mutation in the cat1 gene, likely due to the UV mutagenesis procedure used previously, which could explain the increased sensitivity toward H2O2. In summary, DHN-melanin is not involved in protection against H2O2 or superoxide and, thus, has no role in survival of conidia when attacked by these reactive oxygen species.

Objective: The form of human fungal sinusitis that most closely approximates the disease occurring in the dog is chronic erosive non-invasive fungal sinusitis. This disease is characterized by final destruction of bone in the absence of tissue invasion by the fungus and requires both removal of fungal plaques, necrotic tissue and medical therapy with antifungals. Remarkably, these fungal plaques are white indicated that asexual development does not proceed in the patients. Immune response in SNA infections has been studied via biopsy and cytokine profiling as well as transcriptomic analysis of the host tissue. However, a transcriptomic study of this fungal pathogen growing in patients causing a non-invasive infection has never being performed. We obtained fungal plaques directly from canine patients suffering from SNA and characterize the transcriptome of the causative fungus A. fumigatus in order understand gene expression in the context of the host and particularly in the field of in-host adaptation Methods: Four different fungal plaques were isolated from dogs suffering from SNA. After surgical removal using endoscope or trephination part of fungal plaques were immediately frozen in liquid nitrogen and stored at -80°C for RNA isolation and sequencing. RNA isolation was performed using RNeasy Mini Kit® from Qiagen and sequencing was performed by ServiceXS (Leiden, The Netherlands). RNA-seq analysis involved quality check with fastQC. Cleaning and trimming of reads with Fastx-toolkit. Kallisto was used for transcript quantification(TPM) with A. fumigatus Af293 (AspGD) as reference. For functional characterization of the transcriptome, highly variable expressed genes between samples were removed, and 3 subjective levels of expression were established: low (1 to 39.8 TPM), median (39.8 to 1584.8 TPM) and high (1584.8 to 79432.8 TPM), for each group an enrichment analysis was performed. Additionally a more targeted categorization of the transcriptome was done using pu

Objective: Malassezia species are lipid-dependent due to the lack of cytosolic fatty acid synthase required for De novo synthesis of fatty acids (FAs) and these yeasts are part of the skin mycobiome. Pathogenicity of Malassezia has been related to several factors including the ability to produce enzymes such as esterases, lipases, lipoxygenases and proteases which enable growth on the host skin and lead to changes in sebum (skin fat) composition. The skin functions in the innate defense against pathogens due to its low water content, acidic pH, its microbiota, and antimicrobial compounds like free fatty acids. Understanding lipid dependency of Malassezia will help to understand how these yeasts establish themselves as part of the skin microbiota, which adaptation mechanisms are involved, and how, and whether, lipid metabolism impacts the shift to pathogenicity. The complex nutritional requirements of Malassezia have delayed the full comprehension of its lipid metabolism. Reconstruction of the lipid-synthesis pathways of Malassezia species in silico predicted amongst others a defect in the assimilation of palmitic acid in M. globosa, M. sympodialis, M. pachydermatis and an atypical isolate of M. furfur, but not in M. furfur. This prediction was validated by physiological characterization in chemically defined media (MM) using different lipid sources. Methods: Growth on FAs in liquid MM: Strains were first grown for 7 days at 33°C in lipid-rich mDixon medium. To prevent subsequent growth in MM due to the presence of residual lipids we performed a two-phase growth in MM. First, cells were diluted into MM containing specific lipids. After 3 days, these cells were diluted again in fresh MM with the same lipids. Growth was monitored for 7 days by determining OD600 nm and CFU by plating on mDixon plates. Results: M. furfur could assimilate palmitic acid or oleic acid as well as all Tween variants tested. The atypical M. fufur strain could assimilate only Tween 80, Tween 20

Objective: We explored the phenotypical and genetic variability among isolates of the ubiquitous and saprophytic fungus Aspergillus fumigatus which has the remarkable ability to adapt and grow in many different niches. Due to this ability it can also cause invasive and non-invasive infections in humans and animals, for example invasive pulmonary aspergillosis (IPA) in humans and sinonasal aspergillosis (SNA) in dogs. Our main objective is to understand how this fungus adapts to different niches and to find the factors and genetic traits that influences host adaptation and development in the context of fungal infections. To address this question we have compared a set of clinical and environmental isolates at a genetic and phenotypic level. Isolates were derived from sputum or bronchoalveolar lavage from human patients at the intensive care unit who were suspected to developed IPA. In addition we cultured isolates from fungal plaques isolated from the sinus of dogs suffering with SNA using endoscopy or trephination. Methods: We have compared a set of isolates of A. fumigatus from A) humans (29 isolates from a preselected set of 9 patients), B) dogs with SNA (27 isolates form 9 patients) C) environmental isolates (27 isolates) with reference strains. Azole resistance was determined by microdilution assay antifungal susceptibility testing and tandem repeats in the promotor region of the cyp51A gene. Sequencing of calmodulin (CaM), beta-tubulin(benA) and mating type genes (MAT1-1 and 1-2) and microsatellite (STRAf) analysis were performed to detect genetic differences between isolates. Plating on different media was performed to observe differences in macro and micromorphology Results: Genotyping of the different isolates showed that each human patient carried multiple fungal genotypes. In contrast, each dog suffering from SNA appeared to be infected by only one single genotype. Remarkably, different isolates from each dogs, and having the same genotype, showed a large

Volatile organic compounds (VOCs) in exhaled breath may identify the presence of invasive pulmonary aspergillosis. We aimed to detect VOC profiles emitted by in vitro cultured, clinical Aspergillus isolates using gas chromatography-mass spectrometry (GC-MS). Three clinical Aspergillus isolates and a reference strain were cultured while conidiation was prevented. Headspace samples were analyzed using a standardized method. Breath samples of patients from which the cultures were obtained were checked for the presence of the VOCs found in vitro. Each Aspergillus isolate produced a distinct VOC profile. These profiles could not be confirmed in exhaled breath in vivo.

Background: Aspergillus tubingensis is a black Aspergillus belonging to the Aspergillus section Nigri, which includes species that morphologically resemble Aspergillus niger. Recent developments in species determination have resulted in clinical isolates presumed to be Aspergillus niger being reclassified as Aspergillus tubingensis by sequencing. We present a report of a patient with an osteomyelitis of the maxillary bone with a probable invasive Aspergillus tubingensis infection.Case presentation: We describe an immune compromised patient suffering from osteomyelitis of the maxillary bone after tooth extraction. The osteomyelitis probably resulted in dentogenic pansinusitis presenting as an acute ethmoiditis. Histologic examination of biopsy samples showed osteomyelitis, and inflammation of the surrounding connective tissue. Cultures of the alveolar wound grew Aspergillus tubingensis. The patient was treated with liposomal amphoterocin B, which was changed to oral treatment with voriconazole based on susceptibility testing (MIC for voriconazole was 1 μg/ml).Conclusion: This case shows that Aspergillus tubingensis may have the potential to cause severe invasive infections in immunocompromised hosts. A larger proportion of Aspergillus tubingensis isolates are less susceptible to azoles compared to Aspergillus niger. Therefore, correct species identification and susceptibility testing is crucial for the choice of anti-fungal treatment, screening of azole resistance, and characterization of the pathogenic potential of the various species within Aspergillus section Nigri. © 2013 Bathoorn et al.; licensee BioMed Central Ltd., Background: Aspergillus tubingensis is a black Aspergillus belonging to the Aspergillus section Nigri, which includes species that morphologically resemble Aspergillus niger. Recent developments in species determination have resulted in clinical isolates presumed to be Aspergillus niger being reclassified as Aspergillus tubingensis by sequencing. We present a report of a patient with an osteomyelitis of the maxillary bone with a probable invasive Aspergillus tubingensis infection.Case presentation: We describe an immune compromised patient suffering from osteomyelitis of the maxillary bone after tooth extraction. The osteomyelitis probably resulted in dentogenic pansinusitis presenting as an acute ethmoiditis. Histologic examination of biopsy samples showed osteomyelitis, and inflammation of the surrounding connective tissue. Cultures of the alveolar wound grew Aspergillus tubingensis. The patient was treated with liposomal amphoterocin B, which was changed to oral treatment with voriconazole based on susceptibility testing (MIC for voriconazole was 1 μg/ml).Conclusion: This case shows that Aspergillus tubingensis may have the potential to cause severe invasive infections in immunocompromised hosts. A larger proportion of Aspergillus tubingensis isolates are less susceptible to azoles compared to Aspergillus niger. Therefore, correct species identification and susceptibility testing is crucial for the choice of anti-fungal treatment, screening of azole resistance, and characterization of the pathogenic potential of the various species within Aspergillus section Nigri. © 2013 Bathoorn et al.; licensee BioMed Central Ltd.