Distinguishing between long-term-stored and fresh chili pepper powder through fingerprinting of volatiles by headspace capillary-gas chromatography-ion mobility spectrometry.

Long-term storage of chili pepper powder results in physicochemical and microbiological changes that decrease its commercial value; these changes occur owing to fungal growth and production of off-flavor compounds. Herein, long-term-stored chili pepper powder (LSCPP) and fresh chili pepper powder (FCPP) were analyzed using internal transcribed spacer sequencing and volatile organic compound fingerprinting by headspace capillary-gas chromatography-ion mobility spectrometry. Fungal analysis detected only Xeromyces bisporus with high accuracy in all the analyzed LSCPP samples. However, the proliferation of X. bisporus on nonspecific spots complicated the distinguishing process between the two groups based solely on fungal analysis. Therefore, nine compounds (three ketones, one alcohol, two aldehydes, one ester, one furan, and one sulfur compound) obtained by autoxidation and fungal metabolism were selected as potential markers for distinguishing LSCPP and FCPP. These above-mentioned substances, which were confirmed as off-flavor species owing to "stale" odor, emitted lipid fragrance and were used to successfully distinguish LSCPP from FCPP using principal component analysis and linear discriminant analysis. PRACTICAL APPLICATION: According to the research results, it was possible to discriminate between long-term stored and fresh chili pepper powders using nine VOC markers for quality control in industry. In addition, the fungus generated from long-term storage of chili pepper powder was Xeromyces bisporus, which was confirmed to be safe for intake because it does not form secondary toxic metabolites.

In vitro activity of olorofim (F901318) against fungi of the genus, Scedosporium and Rasamsonia as well as against Lomentospora prolificans, Exophiala dermatitidis and azole-resistant Aspergillus fumigatus.

In recent decades, invasive infections caused by fungal pathogens have been reported with increasing frequency. Concurrently, the rates of detected resistance mechanisms against commonly used antifungal agents in fungi are increasing. The need for novel antifungal drugs is thus imminent. In this study, the novel drug olorofim (F901318) was tested for its antifungal activity against the human fungal pathogens Lomentospora prolificans (n = 20), Scedosporium aurantiacum (n = 2), Scedosporium apiospermum (n = 6), Rasamsonia argillacea species complex (n = 23), Exophiala dermatitidis (n = 10) and azole-resistant Aspergillus fumigatus (ARAF) (n = 25) in an in vitro broth microdilution assay according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations. Whilst olorofim was ascertained to be effective against R. argillacea species complex [minimum inhibitory concentrations (MICs) of ≤0.008 mg/L], Scedosporium spp. (MICs of 0.032-0.5 mg/L), L. prolificans (MICs of 0.032-0.5 mg/L) and ARAF (MICs of ≤0.008-0.032 mg/L), the drug had an MIC of >4 mg/L against E. dermatitidis. These data demonstrate the antifungal activity of olorofim against a broad range of filamentous fungal pathogens.

Airway persistence by the emerging multi-azole-resistant Rasamsonia argillacea complex in cystic fibrosis.

Infections caused by Rasamsonia argillacea complex have been reported in various clinical settings. Cystic fibrosis (CF) is one of the main underlying conditions. An observational cohort study of CF patients with Rasamsonia in respiratory samples was conducted. Eight isolates from 6 patients were identified as R. argillacea complex and tested for antifungal susceptibility. All isolates had high MICs to voriconazole and posaconazole and low MECs to echinocandins. Four patients experienced lung function decline in the year preceding first Rasamsonia isolation. This continued in the year following first isolation in 3 out of 4 cases. Antifungal therapy was initiated in 2 patients, to which only one exhibited a clinical response. Three out of 6 patients died within 3 years of isolating Rasamsonia. Genotyping suggests that similar genotypes of Rasamsonia can persist in CF airways. Consistent with other fungi in CF, the clinical impact of airway colonisation by Rasamsonia is variable. In certain patients, Rasamsonia may be able to drive clinical decline. In others, though a clear impact on lung function may be difficult to determine, the appearance of Rasamsonia acts as a marker of disease severity. In others it does not appear to have an obvious clinical impact on disease progression.

Long-Term Rasamsonia argillacea Complex Species Colonization Revealed by PCR Amplification of Repetitive DNA Sequences in Cystic Fibrosis Patients.

The aim of this work was to document molecular epidemiology of Rasamsonia argillacea species complex isolates from cystic fibrosis (CF) patients. In this work, 116 isolates belonging to this species complex and collected from 26 CF patients and one patient with chronic granulomatous disease were characterized using PCR amplification assays of repetitive DNA sequences and electrophoretic separation of amplicons (rep-PCR). Data revealed a clustering consistent with molecular species identification. A single species was recovered from most patients. Rasamsonia aegroticola was the most common species, followed by R. argillacea sensu stricto and R. piperina, while R. eburnea was not identified. Of 29 genotypes, 7 were shared by distinct patients while 22 were patient specific. In each clinical sample, most isolates exhibited an identical genotype. Genotyping of isolates recovered from sequential samples from the same patient confirmed the capability of R. aegroticola and R. argillacea isolates to chronically colonize the airways. A unique genotype was recovered from two siblings during a 6-month period. In the other cases, a largely dominant genotype was detected. Present results which support the use of rep-PCR for both identification and genotyping for the R. argillacea species complex provide the first molecular evidence of chronic airway colonization by these fungi in CF patients.

In Vitro Activity of Isavuconazole against Rasamsonia Species.

The in vitro susceptibilities to the novel triazole isavuconazole and six other antifungal agents of a large collection of Rasamsonia isolates (n = 47) belonging to seven species were determined. Isavuconazole and voriconazole had no in vitro activity (MIC, >32 mg/liter) against isolates of the Rasamsonia argillacea species complex. The echinocandins were the most potent antifungal drugs against all of the isolates tested (minimum effective concentration, ≤0.19 mg/liter).

Endophytic Fungi from Frankincense Tree Improves Host Growth and Produces Extracellular Enzymes and Indole Acetic Acid.

Boswellia sacra, an economically important frankincense-producing tree found in the desert woodlands of Oman, is least known for its endophytic fungal diversity and the potential of these fungi to produce extracellular enzymes and auxins. We isolated various fungal endophytes belonging to Eurotiales (11.8%), Chaetomiaceae (17.6%), Incertae sadis (29.5%), Aureobasidiaceae (17.6%), Nectriaceae (5.9%) and Sporomiaceae (17.6%) from the phylloplane (leaf) and caulosphere (stem) of the tree. Endophytes were identified using genomic DNA extraction, PCR amplification and sequencing the internal transcribed spacer regions, whereas a detailed phylogenetic analysis of the same gene fragment was made with homologous sequences. The endophytic colonization rate was significantly higher in the leaf (5.33%) than the stem (0.262%). The Shannon-Weiner diversity index was H' 0.8729, while Simpson index was higher in the leaf (0.583) than in the stem (0.416). Regarding the endophytic fungi's potential for extracellular enzyme production, fluorogenic 4-methylumbelliferone standards and substrates were used to determine the presence of cellulases, phosphatases and glucosidases in the pure culture. Among fungal strains, Penicillum citrinum BSL17 showed significantly higher amounts of glucosidases (62.15±1.8 µM-1min-1mL) and cellulases (62.11±1.6 µM-1min-1mL), whereas Preussia sp. BSL10 showed significantly higher secretion of glucosidases (69.4±0.79 µM-1min-1mL) and phosphatases (3.46±0.31µM-1min-1mL) compared to other strains. Aureobasidium sp. BSS6 and Preussia sp. BSL10 showed significantly higher potential for indole acetic acid production (tryptophan-dependent and independent pathways). Preussia sp. BSL10 was applied to the host B. sacra tree saplings, which exhibited significant improvements in plant growth parameters and accumulation of photosynthetic pigments. The current study concluded that endophytic microbial resources producing extracellular enzymes and auxin could establish a unique niche for ecological adaptation during symbiosis with the host Frankincense tree.

Identification and Antifungal Susceptibility of Penicillium-Like Fungi from Clinical Samples in the United States.

Penicillium species are some of the most common fungi observed worldwide and have an important economic impact as well as being occasional agents of human and animal mycoses. A total of 118 isolates thought to belong to the genus Penicillium based on morphological features were obtained from the Fungus Testing Laboratory at the University of Texas Health Science Center in San Antonio (United States). The isolates were studied phenotypically using standard growth conditions. Molecular identification was made using two genetic markers, the internal transcribed spacer (ITS) and a fragment of the ß-tubulin gene. In order to assess phylogenetic relationships, maximum likelihood and Bayesian inference assessments were used. Antifungal susceptibility testing was performed according to CLSI document M38-A2 for nine antifungal drugs. The isolates were identified within three genera, i.e., Penicillium, Talaromyces, and Rasamsonia The most frequent species in our study were Penicillium rubens, P. citrinum, and Talaromyces amestolkiae The potent in vitro activity of amphotericin B (AMB) and terbinafine (TRB) and of the echinocandins against Penicillium and Talaromyces species might offer a good therapeutic alternative for the treatment of infections caused by these fungi.

First recovery of Rasamsonia argillacea species complex isolated in adolescent patient with cystic fibrosis in Slovenia--case report and review of literature.

We report the isolation of the emerging fungal pathogen Rasamsonia aegroticola, which belongs Rasamsonia argillacea species complex, from a respiratory sample of a patient with cystic fibrosis. This filamentous fungus, resembling members of a Penicillium and Paecilomyces spp., was identified by morphology and confirmed by DNA sequence analysis. Susceptibility pattern showed high minimal inhibitory concentration of voriconazole and amphotericin B but low minimal inhibitory concentration of caspofungin, micafungin and itraconazole.

Dynamics and diversity of microbial community succession in traditional fermentation of Shanxi aged vinegar.

The traditional fermentation of Shanxi aged vinegar (SAV), a well-known traditional Chinese vinegar, generally involves the preparation of starter daqu, starch saccharification, alcoholic fermentation (AF) and acetic acid fermentation (AAF). Dynamics and diversity of microbial community succession in daqu and other fermentation stages were investigated by denaturing gradient gel electrophoresis (DGGE). Results showed that eight bacterial genera and four fungal genera were found in daqu. However, Staphylococcus, Saccharopolyspora, Bacillus, Oceanobacillus, Enterobacter, Streptomyces, Eurotium, Monascus and Pichia in daqu were eradicated during AF. Four bacterial genera and three fungal genera were found in this stage. Weissella, Lactobacillus, Streptococcus, Saccharomyces, and Saccharomycopsis were the dominant microorganisms in the late stage of AF. During AAF, four bacterial genera and four fungal genera were found. Weissella, Streptococcus, Klebsiella, Escherichia, and Cladosporium gradually disappeared; the dominant microorganisms were Acetobacter, Lactobacillus, Saccharomycopsis, and Alternaria in the late stage of AAF. Alpha diversity metrics showed that fungal diversity in daqu was greater than that in AF and AAF. By contrast, bacterial diversity decreased from daqu to AF and increased in the first three days of AAF and then decreased. Hence, these results could help understand dynamics of microbial community succession in continuous fermentation of traditional Chinese vinegars.

Leiothecium cristatum sp. nov. and Aspergillus posadasensis sp. nov., two species of Eurotiales from rainforest soils in South America.

We describe two novel fungi isolated from soil samples collected in Northern Argentina and belonging to the family Aspergillaceae of the order Eurotiales: Leiothecium cristatum sp. nov. and Aspergillus posadasensis sp. nov. Leiothecium cristatum sp. nov., represented by the ex-type strain FMR 11998(T) ( = CBS 134260(T) = NBRC 109843(T)), is distinguishable morphologically from the type species of the genus, Leiothecium ellipsoideum, by the presence of irregular reticulate ascospores with two prominent equatorial crests, and Aspergillus posadasensis sp. nov., represented by the ex-type strain FMR 12168(T) ( = CBS 134259(T) = NBRC 109845(T)), is differentiated from Aspergillus acanthosporus, the nearest species phylogenetically, by its non-sclerotioid ascomata and a lack of an asexual stage on all culture media tested. The taxonomic proposals are supported by the analysis of the sequences of the internal transcribed spacer region, the D1-D2 domains of the 28S rRNA gene, the fragments of the RNA polymerase II largest subunit, and the putative chaperonin complex related to TCP-1, ß-tubulin and calmodulin genes.

Heat-resistance of Hamigera avellanea and Thermoascus crustaceus isolated from pasteurized acid products.

Products containing sugar or fruit derivatives are usually subjected to a pasteurization process that can anyway be ineffective to kill ascospores from heat-resistant molds. Although the most occurring and economically relevant heat-resistant species belong to Byssochlamys, Neosartorya, Talaromyces, and Eupenicillium genera, an increasing number of uncommon heat-resistant isolates have been recently detected as spoiling microorganisms in such products. Since Hamigera spp. and Thermoascus spp. were those more frequently isolated at SSICA, heat resistance of Hamigera avellanea and Thermoascus crustaceus strains from pasteurized acid products was studied in apple juice, in blueberry and grape juice and in a buffered glucose solution. Data obtained from thermal death curves and statistical elaboration of raw data showed that D values of H. avellanea may vary between 11.11 and 66.67 min at 87°C, between 4.67 and 13.51 at 90°C, and between 0.43 and 1.52 min at 95°C. Similarly, D values of T. crustaceus may vary between 18.52 and 90.91 min at 90°C, between 2.79 and 19.23 at 93°C, and between 1.11 and 2.53 min at 95°C. For both strains studied, the z-values calculated from the decimal reduction time curves did not prove to be significantly influenced by the heating medium, that being 4.35°C, 5.39°C or 5.27°C for H. avellanea and 4.42°C, 3.69°C or 3.37°C for T. crustaceus, respectively in apple juice, in blueberry and grape juice or in the buffered glucose solution. Considering the pasteurization treatments industrially applied to fruit-based foods, the variation of thermal parameters does not seem to be a possible way to avoid product spoilage by these two species and only good practices applied to reduce the original load of heat-resistant fungi can help producers to prevent losses in contaminated finished products, as usually happens for other heat resistant molds.

Rasamsonia argillacea species complex: taxonomy, pathogenesis and clinical relevance.

Since 2010, colonizations/infections by Rasamsonia argillacea species complex, previously known as Geosmithia argillacea, have been regularly reported in literature. We reviewed all available cases focusing on pathogenesis and clinical relevance. The number of cases may be underestimated, as these fungi are frequently misidentified as Penicillium or Paecilomyces species. Major underlying conditions that predispose for infections by the R. argillacea species complex include cystic fibrosis (CF) and chronic granulomatous disease (CGD). While the pathogenic role of the colonization of CF lungs is still under debate, these molds are the causative agent of pneumonia and/or invasive infections in CGD patients. Given their thermotolerance and their resistance to various antifungals, especially the azole drugs, a special attention should be paid to the chronic colonization of the airways by these fungi in CF and CGD patients.

[Isolation of Geosmithia argillacea in a cystic fibrosis patient]. / Isolement de Geosmithia argillacea chez un enfant atteint de mucoviscidose.

We report the case of an 11-year-old child with cystic fibrosis where Geosmithia argillacea has been isolated from sputum. This is a filamentous fungus (mold) recently described as emergent infectious agent in cystic fibrosis patients. In our case, the presence of G. argillacea was not associated with clinical disorder. However, recent evidence shows that it can be responsible for very serious invasive infection, especially in chronic granulomatous disease and may be, after lung transplantation.

Isolation and identification of Geosmithia argillacea from a fungal ball in the lung of a tuberculosis patient.

Geosmithia argillacea, an anamorph of Talaromyces eburneus, is a thermophilic filamentous fungus that has a phenotype similar to that of the Penicillium species, except for the creamy-white colonies and cylindrical conidia. Recently, a new genus called Rasamsonia has been proposed, which is to accommodate the Talaromyces and Geosmithia species. Here, we report the first Korean case of G. argillacea isolated from a patient with a fungal ball. The patient was a 44-yr-old Korean man with a history of pulmonary tuberculosis and aspergilloma. The newly developed fungal ball in his lung was removed and cultured to identify the fungus. The fungal colonies were white and slow-growing, and the filaments resembled those of Penicillium. Molecular identification was carried out by sequencing the internal transcribed spacer (ITS) region of the 28S rDNA and the ß-tubulin genes. A comparative sequence analysis using the GenBank (http://blast.ncbi.nlm.nih.gov/) database was performed with the basic local alignment search tool (BLAST) algorithm. The results revealed a 97-100% similarity with the G. argillacea ITS sequence. This case should increase awareness among physicians about the pathogenic potential of G. argillacea in humans and help them accurately identify this fungus, because it can be easily confused with Penicillium and Paecilomyces species owing to their similar phenotypic and microscopic characteristics. A molecular approach should be employed to enable accurate identification of G. argillacea.

Taxonomy and antifungal susceptibility of clinically important Rasamsonia species.

In recent years, Geosmithia argillacea has been increasingly reported in humans and animals and can be considered an emerging pathogen. The taxonomy of Geosmithia was recently studied, and Geosmithia argillacea and related species were transferred to the new genus Rasamsonia. The diversity among a set of Rasamsonia argillacea strains, including 28 clinical strains, was studied, and antifungal susceptibility profiles were generated. Data obtained from morphological studies and from phylogenetic analyses of internal transcribed spacer (ITS) and partial ß-tubulin and calmodulin sequences revealed the presence of four species in the Rasamsonia argillacea complex, two of which are newly described here: R. piperina sp. nov. and R. aegroticola sp. nov. In contrast to other related genera, all Rasamsonia species can be identified with ITS sequences. A retrospective identification was performed on recently reported clinical isolates from animal or human patients. Susceptibility tests showed that the antifungal susceptibility profiles of the four members of the R. argillacea complex are similar, and caspofungin showed significant activity in vitro, followed by amphotericin B and posaconazole. Voriconazole was the least active of the antifungals tested. The phenotypically similar species R. brevistipitata and R. cylindrospora had different antifungal susceptibility profiles, and this indicates that correct species identification is important to help guide appropriate antifungal therapy.

Thermophilic fungi in an aridland ecosystem.

We report a comprehensive multi-year study of thermophilic fungi at the Sevilleta National Wildlife Refuge in central New Mexico. Recovery of thermophilic fungi from soils showed seasonal fluctuations, with greater abundance correlating with spring and summer precipitation peaks. In addition to grassland soils, we obtained and characterized isolates from grassland and riparian litter, herbivore dung and biological soil crusts. All strains belonged to either the Eurotiales or Sordariales (Chaetomiaceae). No particular substrate or microhabitat associations were detected. Molecular typing of strains revealed substantial phylogenetic diversity, eight ad hoc phylogroups across the two orders were identified and genetic diversity was present within each phylogroup. Growth tests over a range of temperatures showed substantial variation in maximum growth rates among strains and across phylogroups but consistency within phylogroups. Results demonstrated that 45-50 C represents the optimal temperature for growth of most isolates, with a dramatic decline at 60 C. Most strains grew at 60 C, albeit slowly, whereas none grew at 65 C, providing empirical confirmation that 60 C presents an evolutionary threshold for fungal growth. Our results support the hypothesis that fungal thermophily is an adaptation to transient seasonal and diurnal high temperatures, rather than simply an adaptation to specialized high-temperature environments. We note that the diversity observed among strains and the frequently confused taxonomy within these groups highlight the need for comprehensive biosystematic revision of thermophilic taxa in both orders.

Rasamsonia, a new genus comprising thermotolerant and thermophilic Talaromyces and Geosmithia species.

The phylogenetic relationship among Geosmithia argillacea, Talaromyces emersonii, Talaromyces byssochlamydoides and other members of the Trichocomaceae was studied using partial RPB2 (RNA polymerase II gene, encoding the second largest protein subunit), Tsr1 (putative ribosome biogenesis protein) and Cct8 (putative chaperonin complex component TCP-1) gene sequences. The results showed that these species form a distinct clade within the Trichocomaceae and Trichocoma paradoxa is phylogenetically most closely related. Based on phenotypic and physiological characters and molecular data, we propose Rasamsonia gen. nov. to accommodate these species. This new genus is distinct from other genera of the Trichocomaceae in being thermotolerant or thermophilic and having conidiophores with distinctly rough walled stipes, olive-brown conidia and ascomata, if present, with a scanty covering. Species within the genus Rasamsonia were distinguished using a combination of phenotypic characters, extrolite patterns, ITS and partial calmodulin and ß-tubulin sequences. Rasamsonia brevistipitata sp. nov. is described and five new combinations are proposed.

Fungal myelitis caused by Phialosimplex caninus in an immunosuppressed dog.

A bone marrow infection caused by Phialosimplex caninus was diagnosed in a seven-year-old female spayed Cocker Spaniel that was receiving prednisone for autoimmune hemolytic anemia. Histopathologic examination of a bone marrow core biopsy revealed clusters of oval to round yeast-like cells of varying shape and size and occasional irregular hyphae. Culture of a bone marrow aspirate sample yielded a mould initially suggestive of Paecilomyces inflatus or Sagenomella species but later determined to be P. caninus. The dog was treated with itraconazole and amphotericin B, and prednisone was continued at the lowest dose needed to control the hemolytic anemia. The patient died after 18 months of treatment. This is the first detailed clinical report of infection caused by P. caninus, a newly described fungus associated with disseminated disease in dogs.

Geosmithia argillacea: an emerging cause of invasive mycosis in human chronic granulomatous disease.

BACKGROUND: Chronic granulomatous disease (CGD) is an inherited disorder of the nicotinamide adenine dinucleotide phosphate oxidase that leads to defective production of microbicidal superoxide and other oxidative radicals, resulting in increased susceptibility to invasive infections, especially those due to fungi. METHODS: Geosmithia argillacea was identified from cultured isolates by genomic sequencing of the internal transcribed spacer region. Isolates previously identified as Paecilomyces variotii, a filamentous fungus closely resembling G. argillacea, were also examined. RESULTS: We identified G. argillacea as the cause of invasive mycosis in 7 CGD patients. In 5 cases, the fungus had been previously identified morphologically as P. variotii. All patients had pulmonary lesions; 1 had disseminated lesions following inhalational pneumonia. Infections involved the chest wall and contiguous ribs in 2 patients and disseminated to the brain in 1 patient. Four patients with pneumonia underwent surgical intervention. All patients responded poorly to medical treatment, and 3 died. CONCLUSIONS: We report the first cases of invasive mycosis caused by G. argillacea in CGD patients. G. argillacea infections in CGD are often refractory and severe with a high fatality rate. Surgical intervention has been effective in some cases. G. argillacea is a previously underappreciated and frequently misidentified pathogen in CGD that should be excluded when P. variotii is identified morphologically.

Emergence of disseminated infections due to Geosmithia argillacea in patients with chronic granulomatous disease receiving long-term azole antifungal prophylaxis.

We report two cases of invasive infections due to Geosmithia argillacea, an emerging mold, in patients with chronic granulomatous disease receiving prolonged azole antifungal prophylaxis. One patient died despite receiving a combination of four antifungals, and the other developed cerebral and medullary lesions under a combination of caspofungin, posaconazole, terbinafine, and gamma interferon.