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Accumulation of two benzoyl esters in Aspergillus ustus after feeding with alcohols was reported 30 years ago. To the best of our knowledge, the biosynthesis for these esters has not been elucidated prior to this study. Here, we demonstrate that these compounds are artifical products of the phenethyl polyketide ustethylin A biosynthestic pathway. In addition, four aditional benzoyl esters with different methylation levels were also isolated and identified as shunt products. Feeding experiments provided evidence that the enzyme-bound polyketide acyl intermediates are hijacked by externally fed MeOH or EtOH, leading to the formation of the benzoyl esters. Graphic abstract

Aspergillus sections Usti and Cavernicolarum are accommodated in the subgenus Nidulantes. In the present study, a polyphasic approach using morphology and multi-gene phylogeny was applied to investigate the taxonomy of these two sections. Based on the phylogenetic analysis, Aspergillus section Usti includes 25 species, which can be assigned to four series: Calidousti, Deflecti, Monodiorum and Usti. Aspergillus sigarelli is newly described in this section and this species was isolated from a cigarette from PR China and belongs to series Calidousti. It is clearly distinct from other members in this series based on ITS, BenA, CaM and RPB2 sequences. Aspergillus section Usti members like A. calidoustus and A. granulosus are important opportunistic pathogens, it is speculative that more pathogenetic species will be found by using polyphasic taxonomy approaches. Aspergillus section Cavernicolarum includes five species, the growth rates on agar media and size and ornamentation of conidia are important characters for differentiating species in section Cavernicolarum.

The Aspergilli of section Usti (group ustus) are represented by over 20 species, of which Aspergillus calidoustus is the most relevant human pathogen. Invasive aspergillosis (IA) caused by these fungi is rare but could represent an emerging issue among the expanding population of patients with long-term immunosuppression receiving antifungal prophylaxis. Clinicians should be aware of this unusual type of IA, which often exhibits distinct clinical features, such as an insidious and prolonged course and a high occurrence of extra-pulmonary manifestations, such as skin/soft tissue or brain lesions. Moreover, these Aspergillus spp. pose a therapeutic challenge because of their decreased susceptibility to azole drugs. In this review, we outline the microbiological and clinical characteristics of IA due to Aspergillus spp. of section Usti and discuss the therapeutic options.

Aspergillus spp. cause serious invasive lung infections, and Aspergillus fumigatus is the most commonly encountered clinically significant species. Voriconazole is considered to be the drug of choice for treating A. fumigatus infections; however, rising resistance rates have been reported. We evaluated a matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)-based method for the differentiation between wild-type and non-wild-type isolates of 20 Aspergillus spp. (including 2 isolates of Aspergillus ustus and 1 of Aspergillus calidoustus that were used as controls due their intrinsic low azole susceptibility with respect to the in vitro response to voriconazole). At 30 and 48 h of incubation, there was complete agreement between Cyp51A sequence analysis, broth microdilution, and MALDI-TOF MS classification of isolates as wild type or non-wild type. In this proof-of-concept study, we demonstrated that MALDI-TOF MS can be used to accurately detect A. fumigatus strains with reduced voriconazole susceptibility. However, rather than proving to be a rapid and simple method for antifungal susceptibility testing, this particular MS-based method showed no benefit over conventional testing methods.

Invasive aspergillosis (IA) is the most common invasive fungal infection following a hematopoietic cell transplant, with emerging cryptic species exhibiting resistance to commonly used antifungals such as azoles. These species have been increasingly found after the introduction of anti-mold prophylaxis. We report a case of a 56-year-old female with primary myelofibrosis whose allogeneic hematopoietic cell transplant was complicated by disseminated fungal infection (skin, lung) due to Aspergillus calidoustus, a cryptic specie. Treatment of Aspergillus species remains challenging as these cryptic species are usually resistant to azoles including voriconazole which is the first line of treatment of IA. Infection was successfully treated with surgical excision and combination antifungal therapy based on in vitro susceptibility and synergy testing. Therapy included isavuconazole, a drug that has been shown to be non-inferior to voriconazole in the treatment of invasive mold infections.

Interaction between indigenous fungal strains in preventing ochratoxin A (OTA) production by Penicillium verrucosum and Penicillium nordicum was studied in 100 mL of Czapek yeast autolysate (CYA) medium in a 250-mL “U” shaped culture vessel in one end for 3 days. At the end of incubation period, test fungi inoculated and incubated at 27 ± 2 °C for another 14 days to study the inhibition of OTA production was estimated by high performance liquid chromatography (HPLC). Total inhibition of OTA production was recorded with Aspergillus fumigatus , Aspergillus flavus , Aspergillus niger , Aspergillus ustus , Fusarium culmorum , Fusarium graminarium , Fusarium proliferatum , Penicillium chrysogenum , Penicillium expansum and Trichoderma viridae . A significant correlation coefficient ( r ) on growth (0.493, P ≤ 0.0003) and OTA production (0.785, P ≤ 0.0001) was observed between the tested Penicillium species and other co-existing fungi. In conclusion, the present investigation revealed that those indigenous fungi are necessary to minimize potential losses to the poultry farmer and toxicological hazards to the consumer as biological control agent in different foods and feeds.

Two new sesterterpenoids, terretonins H ( 1 ) and I ( 2 ), together with two known compounds, strobilactone A ( 3 ) and cerebroside D ( 4 ), were isolated from the lipophilic extract of the marine-derived fungus Aspergillus ustus KMM 4664. The structures of compounds 1 and 2 were determined based on spectroscopic methods and confirmed by X-ray crystallographic analysis of terretonin H. All compounds were tested for cytotoxic and embryotoxic effects using sperm and developing embryos of the sea urchin Strongylocentrotus intermedius and human cancer cells.

In the indoor environment, people are exposed to several fungal species. Evident dampness is associated with increased respiratory symptoms. To examine the immune responses associated with fungal exposure, mice are often exposed to a single species grown on an agar medium. The aim of this study was to develop an inhalation exposure system to be able to examine responses in mice exposed to mixed fungal species aerosolized from fungus-infested building materials. Indoor airborne fungi were sampled and cultivated on gypsum boards. Aerosols were characterized and compared with aerosols in homes. Aerosols containing 10 7 CFU of fungi/m 3 air were generated repeatedly from fungus-infested gypsum boards in a mouse exposure chamber. Aerosols contained Aspergillus nidulans , Aspergillus niger , Aspergillus ustus , Aspergillus versicolor , Chaetomium globosum , Cladosporium herbarum , Penicillium brevicompactum , Penicillium camemberti , Penicillium chrysogenum , Penicillium commune , Penicillium glabrum , Penicillium olsonii , Penicillium rugulosum , Stachybotrys chartarum , and Wallemia sebi . They were all among the most abundant airborne species identified in 28 homes. Nine species from gypsum boards and 11 species in the homes are associated with water damage. Most fungi were present as single spores, but chains and clusters of different species and fragments were also present. The variation in exposure level during the 60 min of aerosol generation was similar to the variation measured in homes. Through aerosolization of fungi from the indoor environment, cultured on gypsum boards, it was possible to generate realistic aerosols in terms of species composition, concentration, and particle sizes. The inhalation-exposure system can be used to study responses to indoor fungi associated with water damage and the importance of fungal species composition.

It was shown that extracellular proteinases produced by the strains of micromycetes A. ochraceus L-1 and A. ustus 1 differ by the activity at various pH as well as by the intensity of the effect on fibrillar proteins. It was revealed that the proteinases of A. ochraceus L-1 demonstrated maximum activity during the growth of the producer in the nitrate-free growth medium (the pH of enzyme reaction was 8.0), whereas those of A. ustus 1 showed maximal activity during the growth of the micromycete in the medium containing sodium nitrate (the pH of enzyme reaction was 6.0). Values of specific fibrinolytic and collagenolytic activities of A. ochraceus L-1 were 2.2 and 1.6 times higher than those of A. ustus 1. At the same time, A. ustus 1 showed very low values of total proteolytic (caseinolytic) activity and had a high ratio of fibrinolytic activity to total proteolytic (caseinolytic) activity (6.92). It makes the strain a promising producer of proteinases, which hydrolyze fibrin and collagen.

The purpose of research is increase of honey productivity bee colonies. The studies were conducted in the early spring in March, before the departure of the insect from the hive after hibernation. Bee colonies of the experimental group treated with ozone for families for 7 consecutive days at 2 hours per day when the ozone concentration of 6 mg . Bee colonies of the control group not treated with ozone. The results showed the presence of different species of microorganisms in the intestinal tract of bees in the control group, the quantitative indicators of the content of the polymorphic bacteria, staphylococci, streptococci, pseudomonas, fungi is quite large. Also, the experiments proved devastating effect of ozonation for bacteria groups, coliforms, pseudomonas, fungi of the genus aspergillus niger in the experimental group. With regard to the fungi penicillium glaucum and aspergillus ustus, ozone caused a slight decrease in their concentration in the intestinal tract of bees. Therefore, the use of ozone as a means of prevention and treatment of infectious diseases of bees can be justified as it creates the most favorable conditions for further development of bee colonies. However, the fact that the number of fungi after ozonation in the intestinal tract of bees increased, speaks about the resistance of these microorganisms to ozone, and therefore, for suppression of fungal flora, ozonation needs to be done either longer or use a higher concentration of ozone.

Objectives To investigate the in vitro activity of olorofim (F901318), a novel broad-spectrum antifungal agent, against 150 strains belonging to 16 different cryptic species of Aspergillus by EUCAST and CLSI methodologies. Methods Olorofim, amphotericin B, micafungin, posaconazole and voriconazole were tested against cryptic species belonging to Aspergillus fumigatus complex (n = 57), Aspergillus ustus complex (n = 25), Aspergillus niger complex (n = 20), Aspergillus flavus complex (n = 20), Aspergillus circumdati complex (n = 15) and Aspergillus terreus complex (n = 13) using EUCAST and CLSI methodologies for broth microdilution susceptibility testing of antifungal agents. Results Olorofim was the only drug with activity against all cryptic species of Aspergillus tested, including the multiresistant species Aspergillus lentulus, Aspergillus fumigatiaffinis and Aspergillus calidoustus. Geometric means of MICs for olorofim were lower (0.017, 0.015 and 0.098 mg/L, respectively, for EUCAST; and 0.015, 0.015 and 0.048 mg/L, respectively, for CLSI) than for amphotericin B (4.438, 12.699 and 0.554 mg/L, respectively, for EUCAST; and 0.758, 1.320 and 0.447 mg/L, respectively, for CLSI), voriconazole (2.549, 2.297 and 5.856 mg/L, respectively, for EUCAST; and 2.071, 1.741 and 5.657 mg/L, respectively, for CLSI) and posaconazole (0.307, 0.308 and 12.996 mg/L, respectively, for EUCAST; and 0.391, 0.215 and 9.514 mg/L, respectively, for CLSI). Conclusions Olorofim shows encouraging in vitro activity against cryptic species of Aspergillus that can be hard to treat with current antifungal therapies. Further studies are warranted in order to assess its efficacy.

A co-cultivation study of two fungal strains showed that Aspergillus ustus could inhibit Aspergillus repens growth. The bioactive compound responsible for the observed activity was purified and identified as a sesterterpene, ophiobolin K. Ophiobolin K exhibited marked inhibition against both fungi and bacteria, especially A. repens, A. glaucus and gram-positive bacteria including Bacillus subtilis, Staphylococcus aureus, and Micrococcus luteus.

Microscopic fungi were isolated from different materials including muscles, bones, skin and funeral clothes from the mummified human remains of three members of the Kuffner's family and from the surrounding air environments. Their hydrolytic abilities such as cellulolytic, lipolytic, and proteolytic-keratinolytic were also assessed. The most isolated fungi, from human remains, belonged mainly to the species of Aspergillus (Aspergillus candidus, Aspergillus calidoustus, Aspergillus fumigatus, Aspergillus niger, Aspergillus sydowii, Aspergillus terreus, Aspergillus ustus, Aspergillus venenatus, Aspergillus versicolor, Aspergillus westerdijkiae) and Penicillium (Penicillium chrysogenum, Penicillium commune, Penicillium crustosum, Penicillium griseofulvum, Penicillium hordei, Penicillium polonicum). Aspergilli and penicillia were the predominant actors also in the air samples, but also many strains belonging to the Rhizopus group were isolated as well. Several fungi exhibited different hydrolytic ability, the most active isolated from human remains belonged to the species A. candidus, A. westerdijkiae, Coprinellus xanthothrix, P. chrysogenum, P. commune, P. griseofulvum and Scopulariopsis brevicaulis. The species recovered from the air displayed stronger deterioration characteristics as compared to human samples. This study can be considered one of few investigations focused on mummified human remains conserved in this kind of the environment.

Objectives Invasive mould infections are associated with a high mortality rate and the emergence of MDR moulds is of particular concern. Calcineurin and its chaperone, the heat shock protein 90 (Hsp90), represent an important pathway for fungal virulence that can be targeted at different levels. We investigated the antifungal activity of compounds directly or indirectly targeting the Hsp90-calcineurin axis against different mould species. Methods The in vitro antifungal activity of the anticalcineurin drug FK506 (tacrolimus), the Hsp90 inhibitor geldanamycin, the lysine deacetylase inhibitor trichostatin A and the Hsp70 inhibitor pifithrin-μ was assessed by the standard broth dilution method against 62 clinical isolates of Aspergillus spp. and non-Aspergillus moulds (Mucoromycotina, Fusarium spp., Scedosporium spp., Purpureocillium/Paecilomyces spp. and Scopulariopsis spp.) Results FK506 had variable antifungal activity against different Aspergillus spp. and was particularly active against Mucor spp. Geldanamycin had moderate antifungal activity against Fusarium spp. and Paecilomyces variotii. Importantly, trichostatin A had good activity against the triazole-resistant Aspergillus ustus and the amphotericin B-resistant Aspergillus terreus as well as the MDR Scedosporium prolificans. Moreover, trichostatin A exhibited synergistic interactions with caspofungin against A. ustus and with geldanamycin against Rhizopus spp. for which none of the other agents showed activity. Pifithrin-μ exhibited little antifungal activity. Conclusions Targeting the Hsp90-calcineurin axis at different levels resulted in distinct patterns of susceptibility among different fungal species. Lysine deacetylase inhibition may represent a promising novel antifungal strategy against emerging resistant moulds.

Objectives Invasive fungal diseases (IFDs) are life-threatening events in patients with haematologic disorders, and the spectrum of the aetiological pathogens continues to expand. This study aimed to evaluate the clinical utility of a panfungal polymerase chain reaction (PCR) assay for the management of IFDs in such patients. Methods We prospectively analysed 273 consecutive blood samples from 64 risk episodes in 51 patients with haematologic disorders at high risk for IFD who were treated at our hospital between April 2007 and October 2010. Results PCR-positive results were obtained in 18 of 64 risk episodes (35.3%). IFD was documented in 14 episodes (21.9%, 9 probable IFDs and 5 possible IFDs) according to the revised criteria of the European Organization for Research and Treatment of Cancer/Mycoses Study Group. PCR was positive in all of these 14 episodes, and in 4 of the 50 episodes with no IFD category. Sensitivity, specificity, positive predictive value, and negative predictive value of our assay were 100%, 92%, 78% and 100% respectively. A considerable number of fungi (44.4%) that are less common than Aspergillus and Candida species were positive by PCR. Molecular diagnoses of Cunninghamella species, Aspergillus ustus, Fusarium species, Scedosporium apiospermum, Rhodotorula species and Rhizopus species were beneficial in selecting suitable treatments. Conclusions Our panfungal PCR approach allows for the highly sensitive and specific detection and identification of a wide spectrum of fungal pathogens, which provides indispensable information for managing IFDs, especially refractory or breakthrough IFDs during antifungal therapy in high-risk patients with haematologic disorders.

Summary Mycetoma is a chronic granulomatous, subcutaneous disease endemic in tropical and subtropical countries. It is currently a health problem in rural areas of Africa, Asia and South America. Nine cases of mycetoma were analysed in a retrospective study. All isolates were identified by morphological features. The level of species identification was reached by molecular tools. Definitive identification of fungi was performed using sequence analysis of the ITS of the ribosomal DNA region and the ribosomal large-subunit D1/D2. Identification of actinomycetes was accomplished by the 16S rRNA gene sequence. Six unusual clinical isolates were identified: Aspergillus ustus, Cyphellophora oxyspora, Exophiala oligosperma, Madurella pseudomycetomatis, Nocardia farcinica and Nocardia wallacei. The prevalence of mycetoma in Venezuela remains unknown. This study represents the first report in the literature of mycetoma caused by unusual pathogens identified by molecular techniques.

Two new species of Aspergillus that are clearly distinct from all known species in section Usti were revealed during a study of microfungal communities in Spanish caves. The novel species identified in this study and additional species of Aspergillus section Usti are associated with places and substrates related to human activities in caves. Novel species are described using a polyphasic approach that combines data from four loci (ITS, benA, caM and rpb2), morphology and basic chemical and physiological analyses. Aspergillus thesauricus sp. nov. was isolated from various substrates, including the decaying organic matter, cave air and cave sediment of the Cueva del Tesoro Cave (the Treasure cave). The species is represented by twelve isolates and is most closely related to the recently described A. germanicus. Aspergillus baeticus sp. nov. was isolated from the cave sediment in the Gruta de las Maravillas Cave (the Grotto of the Marvels) and is represented by two isolates. Additional isolate was found in the Cueva del Tesoro Cave and in the Demänovska Peace Cave (Slovakia), suggesting a potentially wide distribution of this microorganism. The species is related to A. ustus and A. pseudoustus. Both species are unable to grow at 37 °C, and a weakly positive, yellow Ehrlich reaction was observed in A. thesauricus. Unique morphological features alone are sufficient to distinguish both species from related taxa., This study was supported by the Research Plan of the ISB BC AS CR, v.v.i., presented results were obtained within bilateral projects between AS CR and CSIC “Fungal diversity in Spanish caves with rock art paintings” and “Early detection of fungal outbreaks in Andalusian caves”. Molecular genetics analyses were supported by projects MSM 6007665801, AV0Z50200510 and GAUK 607812.

The majority of mycotoxins produced by Aspergillus fungi are immunosuppressive agents, and their cytotoxicity may impair defense mechanisms in humans. The objective of the study was evaluation of the cytotoxicity of fungi isolated from an environment where inpatients with impaired immunity were present. The materials comprised 57 fungal strains: Aspergillus fumigatus, Aspergillus niger: Aspergillus ochraceus, Aspergillus flavus, Aspergillus versicolor and Aspergillus ustus isolated from hospital rooms in Cracow. The cytotoxicity of all the strains was evaluated using the MTT test (3-(4,5-dimethylthiazol-2-yl) 2,5 diphenyltetrazolium bromide). To emphasize the differences in cytotoxicity among the particular strains, variance analysis (ANOVA) and Tukey's difference test were used. Out of 57 Aspergillus strains tested, 48 (84%) turned out to be cytotoxic. The cytyotoxicity was high (+++) in 21 strains, mainly in A. fumigatus. The least cytotoxic were A. niger fungi, this being statistically significant (p

This review highlights recent findings of our group on bioactive marine natural products isolated from marine sponges and marine derived fungi. The activated chemical defence of the Mediterranean sponge Aplysina aerophoba is introduced as an example of a dynamic response of marine sponges to wounding. Following tissue disrupture preformed brominated isoxazoline alkaloids are enzymatically cleaved and thereby give rise to aeroplysinin-1 which is believed to protect sponges from invasion of pathogenic bacteria. A preliminary characterization of the membrane bound enzyme(s) involved in the cleavage reaction is presented. Bromotyrosine derived, oxime group bearing peptides, the so called bastadins, obtained from the sponge Ianthella basta and some of their synthetic derivatives were shown to exhibit pronounced antifouling activity against larvae of the barnacle Balanus improvisus. The antifouling activity could be traced to the oxime group as an important pharmacophore that was also found to be present in other sponge derived natural products exhibiting antifouling activity. Marine derived fungi that reside within invertebrates such as sponges or inside Mangrove plants are emerging as a new source of bioactive metabolites as demonstrated for Aspergillus ustus and Alternaria sp. that were isolated from the sponge Suberites domuncula or from the Mangrove plant Sonneratia alba, respectively. The former fungus yielded new moderately cytotoxic sesquiterpenoids of the drimane type whereas the latter was found to produce polyketides such as alternariol that exhibited strong and selective inhibitory activity against several protein kinases, for instance Aurora A and B which are targets for anticancer chemotherapy.

Aspergillus ustus is a relatively rare human pathogen causing invasive infections in immunocompromised hosts. In this study isolates originating from clinical and other sources have been examined using molecular, morphological, and physiological approaches to clarify their species assignment. Phylogenetic analysis of partial β-tubulin, calmodulin, actin, and intergenic transcribed spacer sequences indicated that none of the clinical isolates recognized previously as A. ustus belongs to this species. All but two of these isolates formed a well-defined clade related to A. pseudodeflectus based on sequence analysis of protein-coding regions. Morphological and physiological examination of these isolates indicated that they are able to grow above 37°C, in contrast with A. ustus isolates, and give a positive Ehrlich reaction, in contrast with related species including A. granulosus, A. ustus , and A. pseudodeflectus . These isolates are proposed as a new species, A. calidoustus . Antifungal susceptibility testing showed that this species has decreased susceptibilities to several antifungal drugs. The triazoles are inactive in vitro, including the new azole posaconazole.

Specimens of Suberites domuncula that had been cultured in aquaria for 4 weeks were analyzed for their associated fungi. A total of 81 fungal strains belonging to 20 different genera was isolated and identified by morphological and molecular methods. The most frequently isolated taxa were Cladosporium spp., Penicillium spp., Petriella sp., Phialophora spp. and Engyodontium album. Based on chromatographic and mass spectrometric analysis of fungal extracts, as well as on bioassay results, Aspergillus ustus, Penicillium sp., Petriella sp. and Scopulariopsis sp. were selected for in-depth analysis of their natural products. A total of 19 different fungal metabolites, including three new natural products, was isolated and structurally identified. A. ustus yielded two sesquiterpenes, a drimane derivative and deoxyuvidin, as well as a sesterterpene ophiobolin H. The drimane derivative had an ED50 value against L5178Y cells of 1.9 μg ml-1 in vitro. The crude extract of Petriella sp. was also strongly cytotoxic against the L5178Y cell line. The cyclic tetrapeptide WF-3161 was primarily responsible for the activity; the ED50 value was -1. It was identical to the known compound WF-3161 and had been previously isolated from Petriella guttulata. In addition to WF-3161, three further natural products were obtained and unequivocally identified as new derivatives of infectopyrone by one- and two-dimensional NMR spectroscopy and by mass spectroscopy. Of the new compounds, only dihydroinfectopyrone was active against L5178Y cells; the ED50 value was 0.2 μg ml-1. Penicillium sp. yielded the largest number of metabolites. Viridicatin, viridicatol, cyclopenin and cyclopenol suppressed larval growth of the polyphagous pest insect Spodoptera littoralis when incorporated into an artificial diet at an arbitrarily chosen concentration of 237 ppm. Viridicatol was the most active compound and had an ED50 value of ca. 50 ppm. Scopulariopsis sp. yielded three metabolites, including the known acetylcholinesterase inhibitors quinolactacin A1 and A2.

Aspergillus is a ubiquitous soil-dwelling organism found in organic debris, dust, compost, foods, spices, and rotted plants. The genus Aspergillus includes over 185 species. Around 20 species have so far been reported as causative agents of opportunistic infections in humans. Among these, Aspergillus fumigatus is the most commonly isolated species, followed by Aspergillus flavus and Aspergillus niger. Aspergillus oryzae, Aspergillus terreus, Aspergillus ustus, and Aspergillus versicolor are among the other species less commonly isolated as opportunistic pathogens (Rinaldi, 1983). Aspergillus spp. are well known to play a role in three different clinical settings in humans: (1) opportunistic infections, (2) allergic states, and (3) toxicoses. Immunosuppression is the major factor predisposing to development of opportunistic infections (Ho and Yuen, 2000). Aspergillus, like other lamentous fungi, is primarily acquired from an inanimate reservoir, usually by the inhalation of airborne spores. The organism grows best at 37°C, and the small spores (2-3 µm) are easily inhaled and deposited deep in the lungs, leading to a variety of clinical syndromes (Figure 14.1). These infections may present in a wide spectrum, varying from local involvement to dissemination and as a whole called aspergillosis. Among all lamentous fungi, Aspergillus is in general the most commonly isolated one in invasive infections. It is the second most commonly recovered fungus in opportunistic mycoses following Candida. Although these are distinct pulmonary entities, on rare occasions, one condition may change to another; for example, an aspergilloma may change to invasive pulmonary aspergillosis (IPA) (Tomee et al., 1995).

This is the first report of clustered Aspergillus ustus causing systemic disease in transplant patients., Aspergillus ustus is a mold that rarely infects humans; only 15 systemic cases have been reported. We report the first outbreak of invasive infection caused by A. ustus among hematopoietic stem cell transplant (HSCT) recipients. Six patients with infections were identified; 3 infections each occurred in both 2001 and 2003. Molecular typing by using randomly amplified polymorphic DNA (RAPD) and antifungal drug susceptibility testing were performed on clinical and environmental isolates recovered from our hospital from 1999 to 2003. The highest overall attack rate in HSCT patients was 1.6%. The overall death rate was 50%, and death occurred within 8 days after diagnostic culture collection. Clinical isolates exhibited decreased susceptibility to antifungal drugs, especially azoles. RAPD and phylogenetic analysis showed genetic similarity between isolates from different patients. Based on the clustering of cases in space and time and molecular data, common-source acquisition of this unusual drug-resistant species is possible.

Molecular approaches are now being developed to provide a more rapid and objective identification of fungi compared to traditional phenotypic methods. Ribosomal targets, especially the large-subunit RNA gene (D1-D2 region) and internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions), have shown particular promise for the molecular identification of some fungi. We therefore conducted an assessment of these regions for the identification of 13 medically important Aspergillus species: Aspergillus candidus , Aspergillus ( Eurotium ) chevalieri , Aspergillus ( Fennellia ) flavipes , Aspergillus flavus , Aspergillus fumigatus , Aspergillus granulosus , Aspergillus ( Emericella ) nidulans , Aspergillus niger , Aspergillus restrictus , Aspergillus sydowii , Aspergillus terreus , Aspergillus ustus , and Aspergillus versicolor . The length of ribosomal regions could not be reliably used to differentiate among all Aspergillus species examined. DNA alignment and pairwise nucleotide comparisons demonstrated 91.9 to 99.6% interspecies sequence identities in the D1-D2 region, 57.4 to 98.1% in the ITS1 region, and 75.6 to 98.3% in the ITS2 region. Comparative analysis using GenBank reference data showed that 10 of the 13 species examined exhibited a ≤1-nucleotide divergence in the D1-D2 region from closely related but different species. In contrast, only 5 of the species examined exhibited a ≤1-nucleotide divergence from sibling species in their ITS1 or ITS2 sequences. Although the GenBank database currently lacks ITS sequence entries for some species, and major improvement in the quality and accuracy of GenBank entries is needed, current identification of medically important Aspergillus species using GenBank reference data seems more reliable using ITS query sequences than D1-D2 sequences, especially for the identification of closely related species.

We developed a PCR-based assay to differentiate medically important species of Aspergillus from one another and from other opportunistic molds and yeasts by employing universal, fungus-specific primers and DNA probes in an enzyme immunoassay format (PCR-EIA). Oligonucleotide probes, directed to the internal transcribed spacer 2 region of ribosomal DNA from Aspergillus flavus , Aspergillus fumigatus , Aspergillus nidulans , Aspergillus niger , Aspergillus terreus , Aspergillus ustus , and Aspergillus versicolor , differentiated 41 isolates (3 to 9 each of the respective species; P < 0.001) in a PCR-EIA detection matrix and gave no false-positive reactions with 33 species of Acremonium , Exophiala , Candida , Fusarium , Mucor , Paecilomyces , Penicillium , Rhizopus , Scedosporium , Sporothrix , or other aspergilli tested. A single DNA probe to detect all seven of the most medically important Aspergillus species ( A. flavus , A. fumigatus , A. nidulans , A. niger , A. terreus , A. ustus , and A. versicolor ) was also designed. Identification of Aspergillus species was accomplished within a single day by the PCR-EIA, and as little as 0.5 pg of fungal DNA could be detected by this system. In addition, fungal DNA extracted from tissues of experimentally infected rabbits was successfully amplified and identified using the PCR-EIA system. This method is simple, rapid, and sensitive for the identification of medically important Aspergillus species and for their differentiation from other opportunistic fungi.

The rhizospheric soil microfungi from a native forest (undisturbed and disturbed) were studied using soil dilution plate and soil washing methods. Fungi were isolated using slightly acid and alkaline culture media. 54 taxa were isolated: 49 from undisturbed forest soil and 37 from disturbed forest soil. Acremonium sp., Aspergillus ustus, Coemansia pectinata, Doratomyces stemonitis, Fusarium solani, F. oxysporum, Gliocladium roseum, Humicola fusco-atra, Mortierella sp., Penicillium lilacinum, Trichoderma harzianum, and T. koningii, showed the highest frequency, in both, undisturbed and disturbed forests. In undisturbed soil forest the biodiversity index was 3.97 whereas in disturbed ones was 3.89., Instituto de Botánica "Dr. Carlos Spegazzini"

A colorimetric assay for antifungal susceptibility testing of Aspergillus species ( Aspergillus fumigatus , Aspergillus flavus , Aspergillus terreus , Aspergillus nidulans , and Aspergillus ustus ) is described based on the reduction of the tetrazolium salt 2,3-bis(2-methoxy-4-nitro-5-[(sulphenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) in the presence of menadione as an electron-coupling agent. The combination of 200 μg of XTT/ml with 25 μM menadione resulted in a high production of formazan within 2 h of exposure, allowing the detection of hyphae formed by low inocula of 10 2 CFU/ml after 24 h of incubation. Under these settings, the formazan production correlated linearly with the fungal biomass and less-variable concentration effect curves for amphotericin B and itraconazole were obtained.

In this study, the ability to produce mycotoxins during growth on artificially infested building materials was investigated for Penicillium chrysogenum, Pen. polonicum, Pen. brevicompactum, Chaetomium spp., Aspergillus ustus, Asp. niger, Ulocladium spp., Alternaria spp., and Paecilomyces spp., all isolated from water-damaged building materials. Spores from the different isolates of the above mentioned species were inoculated on gypsum board with and without wallpaper and on chipboard with and without wallpaper. Fungal material was scraped off the materials, extracted, and analyzed using high performance liquid chromatography-diode array detection and thin layer chromatography. All six isolates of C. globosum produced the toxic chaetoglobosins A and C, at levels of up to 50 and 7 μg/cm2 respectively. The quantities of secondary metabolites produced by Penicillia were generally low, and no toxin production was detected from any of the five isolates of Pen. chrysogenum. Both isolates of Pen. polonicum produced 3-methoxy-viridicatin, verrucosidin, and verrucofortine. Two of five isolates of Pen. brevicompactum produced mycophenolic acid. From five out of six isolates of Alternaria spp., alternariol and alternariol monomethyl ether were detected. From Ulocladium spp., Paecilomyces spp., and Asp. ustus no known mycotoxins were detected, although the latter two are known mycotoxin producers. Asp. niger produced several naphtho-γ-pyrones and tetra-cyclic compounds. All investigated species, especially Asp. ustus and Asp. niger produced many unknown secondary metabolites on the building materials. Analyses of wallpaper and glass-fibre wallpaper naturally infested with Asp. versicolor revealed sterigmatocystin and 5-methoxysterigmatocystin. Analyses of naturally infested wallpaper showed that C. globosum produced the chaetoglobosins A and C, and Pen. chrysogenum produced the antibiotic meleagrin.

Two species of filamentous fungi, AspergiUus ustus (Bain.) Thom & Church and Graphium sp., were isolated from calcareous animal shells at depths of 860 m in the Arabian Sea and 965 m in the Bay of Bengal. Laboratory experiments showed germination of conidia, growth of hyphae and microconidiation in both the fungi at 100 bar pressure at 10 and 30°C suggesting barotolerance The fungi also secreted barotolerant protease under these conditions. Protease synthesised and secreted in cultures grown at 1 bar was also active under 100 bar pressure at 10 and 30°C. At 1O0C, the fungi showed better conidial germination, growth and protease secretion when subjected to 100 bar than when grown at 1 atmosphere. The results indicate barotolerance of terrestrial species of fungl and suggest that they might be active under deep-sea conditions.

Aspergillus ustus is an uncommon clinical species which is poorly susceptible to antifungals. We report two cases of A. ustus infections that occurred in allogeneic stem cell transplant recipients while they were receiving either voriconazole or caspofungin. Prolonged use of these new antifungal agents may increase the risk of the emergence of resistant organisms.

Primary cutaneous aspergillosis is a rare disease usually caused by Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus and Aspergillus ustus. It is usually seen in immunocompromised hosts, though some cases are also reported in immunocompetent hosts. We present a case of an immunocompetent farmer who presented with generalised nodules and plaques, mimicking erythema nodosum leprosum but turned out to be cutaneous aspergillosis caused by Aspergillus tamarii. The characteristic ascospores of Aspergillus species were found in skin lesions on fungus isolated in culture. The patient showed excellent response to antifungal therapy.

Using gellan gum as a substitute for agar-agar in a mycological medium and sequencing of the ITS 1 and 2 regions resulted in an accurate identification of Aspergillus fumigatus, Aspergillus nidulans, Aspergillus terreus and Aspergillus ustus within 24 h of subculture.

Moulds constitute the largest group of bacteria, prevailing both in the indoor and outdoor air. Approximately 200 000 species of moulds have been identified so far, where only a small group of around 200 may present a threat to human health. Fungi from Aspergillus species are among the moulds considered to be most pathogenic. They also constitute the group of most pathogenic moulds most frequently isolated from the environment. Over 250 types of this species are known; about 50 of them were precisely described before the year 2000 (Klich, 2009). Pathogenicity, due to their toxicity, was also documented in other species: Aspergillus fumigatus, Aspergillus flavus, Aspergillus ochraceus, Aspergillus niger, Aspergillus versicolor, Aspergillus parasiticus. Aspergillus nidulans, Aspergillus ustus, Aspergillus glaucus, Aspergillus clavatus, Aspergillus sydowii and Aspergillus terreus. Their taxonomic identification is still an open topic because of their morphological variability and ability to produce metabolites; new species which exhibit adverse health effects to humans are constantly being detected. Aspergillus lentulus is one of the recently detected species of considerable clinical importance; it reveals similarity to Aspergillus fumigatus, one of the most pathogenic fungus for humans (Balajee et al., 2005).Table 1 presents the classification of species of fungi pathogenic to humans from the Aspergillus species. The list has been created by the authors of “Atlas Grzybow Chorobotworczych Czlowieka" ("Atlas of Fungi Pathogenic to Humans") (Krzyściak et al., 2011).

The new compounds (I)-(V) and 14 known analogues are screened for their antitumor activity against P388, HL-60, K562, and BEL-7402 cell lines.

To deal with pathogens, plants have evolved sophisticated mechanisms including constitutive and induced defense mechanisms. Phytohormones play important roles in plant growth and development, as well as in the systemic response induced by beneficial and pathogen microorganisms. In this work, we identified an Aspergillus ustus isolate that promotes growth and induces developmental changes in Solanum tuberosum and Arabidopsis thaliana. A. ustus inoculation on A. thaliana and S. tuberosum roots induced an increase in shoot and root growth, and lateral root and root hair numbers. Assays performed on Arabidopsis lines to measure reporter gene expression of auxin-induced/ repressed or cell cycle controlled genes (DR5 and CycB1, respectively) showed enhanced GUS activity, when compared with mock-inoculated seedlings. To determine the contribution of phytohormone signaling pathways in the effect elicited by A. ustus, we evaluated the response of a collection of hormone mutants of Arabidopsis defective in auxin, ethylene, cytokinin, or abscisic acid signaling to the inoculation with this fungus. All mutant lines inoculated with A. ustus showed increased biomass production, suggesting that these genes are not required to respond to this fungus. Moreover, we demonstrated that A. ustus synthesizes auxins and gibberellins in liquid cultures. In addition, A. ustus induced systemic resistance against the necrotrophic fungus Botrytis cinerea and the hemibiotrophic bacterium Pseudomonas syringae DC3000, probably through the induction of the expression of salicylic acid, jasmonic acid/ethylene, and camalexin defense-related genes in Arabidopsis.

Recent studies have changed the taxonomy of Aspergillus section Usti, and a novel species, Aspergillus calidoustus, has been erected. It was also demonstrated that clinical isolates previously identified as A. ustus actually belong to the emerging pathogen A. calidoustus. Aspergillus ustus were frequently isolated from Norwegian water systems, and due to the taxonomical progress, these waterborne strains could be identified more precisely. A MLST study including ITS, calmodulin, β-tubulin and actin sequences was conducted on 32 strains previously identified as A. ustus. All strains were identified as A. calidoustus, which was verified by physiological, biochemical and phylogenetic analyses. This is the first report of that A. calidoustus is able to colonize water distribution systems. In respect to the potential role of water systems as a source of nosocomial infections in patients with immunodeficiency, attention should be given to water systems in hospitals and other healthcare units, especially the heated-water installations.

Introduction The purpose of this study was to research the presence of filamentous fungi in the root canals of teeth with pulp necrosis and periapical lesions and to perform the taxonomic study of the isolates. Methods Samples collected from 60 root canals with sterile absorbent paper points were inoculated in Sabouraud dextrose agar supplemented with chloramphenicol. The material was cultured from 7–14 days at room temperature to observe mycelial growth. Samples with growth were seeded in media specific for microculture (CYA agar, malt extract agar, or potato dextrose agar). Taxonomy was evaluated by macroscopic examination and optical microscopy. Results Filamentous fungi were isolated in situ from 17 of 60 samples (28.3%). Considering the positive root canal isolates, the genus Aspergillus was isolated from 7 samples (41%); 4 species were identified: Aspergillus ustus, A. granulosus, A. niger, and A. sydowii. Emericella quadriluniata , sexual form of Aspergillus , was isolated from 1 sample. Penicillium species ( Penicillium implicatum, P. micsynvisk, P. lividum, and P. citrionigrum ) were isolated from 4 samples (24%). Fusarium species ( Fusarium moniliforme and F. melanochorum ) were isolated from 2 samples (12%). The species Aureobasidium pullulans , Exophiala jeanselmei , Eurotium amstelodame, and Cladosporium sphaerospermum were isolated from 1 sample each. Conclusions The root canals of teeth with pulp necrosis and periapical lesions might present positive culture for filamentous fungi.

Aspergillus ustus infections are associated with a high mortality in immunocompromised hosts, and the mold has decreased susceptibility to most antifungal drugs, especially azoles. We report primary cutaneous A. ustus infection in a patient who failed itraconazole therapy and was switched to voriconazole (VRC). During VRC therapy, the MICs of VRC, amphotericin B (AMB), caspofungin (CFG), and terbinafine (TBF) were 4, 2, 64, and 0.13 μg/mL, respectively. Because the MIC to VRC was high, TBF was added to VRC for synergy based on anecdotal data from other mycoses. After treatment with VRC and TBF for 5 months, MICs of VRC, AMB, CFG, and TBF of A. ustus were 8, 1, 64, and 4 μg/mL respectively. Although the MICs of VRC and TBF increased during antifungal therapy, the patient responded well to the combination antifungal therapy with surgical debridement. With a successful outcome despite high MICs and with limited therapeutic options currently available, we investigated the in vitro activity of posaconazole (PCZ) and VRC individually and in combination with AMB, CFG, or TBF using the fractional inhibitory concentration index (FICI) method. Combination of VRC with TBF showed synergistic activity (FICI = 0.5). Therefore, combination of VRC and TBF with surgical debridement, when appropriate, may be a viable treatment option for refractory A. ustus infections.

The antifugal potential of indoor building materials of 7 different types – without as well as with a paint or oily coating – was studied according to the ISO 846: 1997 E. Spore suspensions of micromycetes Acremonium sp., Aspergillus ustus, A. versicolor, Cladosporium sphaerospermum, Penicillium sp. and Scedosporium apiospermum were inoculated onto the materials’ surface (clean and dusty – to simulate the real state in the indoor environment). Systems were kept under defined water activities (aw) 0.94, 0.83 and 0.75 and incubated at 22– 25°C for 3 months. Microbial growth was evaluated visually after the 1st, 2nd and 3rd months of the experiment. At the end of the period, the vitality of fungal propagules on the material surfaces was estimated by the printing method onto Sabouraud agar medium. All building materials with any lime composition or oily coating showed a certain resistance to the fungal colonization, even under very moist conditions (aw = 0.94). Thus, their broader employment in the indoor environment could be emphasized. Representatives of the common airborne mycoflora – aspergilli, penicillia, and C. sphaerospermum – were able to develop visible growth on all materials tested with and without the house dust as well, although the colonization was of different degrees depending on the humidity. The fungi of Acremonium sp. and S. apiospermum favoured – as had been expected – the highest aw = 0.94 the most. No building material showed fungicidal properties – micromycetes were capable of germinating when printed from the material surface onto the agar. The dust reduced the materials’ antifungal properties only very moderately. A. versicolor – a producer of carcinogenic mycotoxin sterigmatocystin – could colonize materials under any experimental conditions. Wooden facings seemed to be the most resistant to the moulds.

The sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of water-soluble proteins and the randomly amplified polymorphic DNA (RAPD) patterns of whole-cell lysates from 21 Aspergillus ustus isolates, including 11 reference strains and 10 patient and environmental strains from one hospital, were investigated. All isolates showed identical protein patterns. The RAPD assay discriminated between all reference strains. Comparison of hospital isolates showed identical RAPD patterns in some of the patient and environmental isolates. The data indicate that the RAPD technique is useful for fingerprinting A. ustus .

Plant wastes are very important part of biomass used and investigated for energy, chemical, and fuel production. Cellulose is the major renewable form of carbohydrate in the world, about 10{sup 11} tons of which is synthesized annually. For general use, it must be hydrolyzed first, either chemically or by cellulases derived from a few specialized microorganisms. Enzymes are acceptable environmentally but expensive to produce. Certainly, induction of mutations and selection of high cellulose microbial strains with significant adaptability to degrade cellulose to glucose is promising solutions. Induction of mutations in other fungi and Aspergillus sp. rather than Aspergillus niger was reported. Aspergillus ustus and Trichoderma harzianum were induced by gamma irradiation indicating mutants that excrete higher cellulose yields, particularly exocellobiohydrolase (Avicelase) than their respective wild types. Mutants from the celluiolytic fungus Penicillium pinophilum were induced by chemical and UV-irradiation. Enhancing the production of endo-1,4-{Beta}-D-glucanase (CMCase) and particularly {Beta}-glucosidase was obtained by gamma irradiation of Altemaria alternate. To overcome the lower activity of {beta}-glucosidase in certain fungi species rather than A. niger, mixed cultures of different species were tried. Thus, Aspergillus phonicis with Trichoderma reesei Rut 30, produced a cellulose complex that improved activity twofold over cellulose from Trichoderma alone.

We describe the second case of primary cutaneous Aspergillus ustus infection in an immunocompromised patient. Cutaneous aspergillosis was confirmed both by culture and positive fluorescent antibody staining. Few species of Aspergillus are pathogenic in human beings, and fewer still cause primary cutaneous disease. The only other reported case of aspergillosis from Aspergillus ustus occurred in an immunosuppressed patient who was temporally and geographically separated from ours. (J Am Acad Dermatol 1998;38:797-8.)

Mycotoxins constitute a group of toxic secondary fungal metabolites. Fungi that produce these toxins frequently contaminate food and feed, creating a potential threat to human and animal health. Biological activities of mycotoxins include, amongst others: toxicity, mutagenicity and carcinogenicity, which can be expressed with or without metabolic activation. Austocystins are similar in structure to aflatoxin B/sup 1/ and are probably synthesized in a similar manner. The Ames Salmonella test, a widely accepted method employed for the detection of mutagenic activity of various chemical compounds was used for testing the mutagenic activity of different mycotoxins. As aflatoxin B/sup 1/ was found by the Ames test to be highly mutagenic, the same test was applied for the study of possible mutagenicity of the austocystins. The mutagenic activity of these compounds was studied with and without metabolic activation using two tester strains of S. typhimurium, one capable of detecting frame shift mutation (strain TA98) and the other capable of detecting base pair substitution (strain TA100).

The mutants Aspergillus ustus M35 and Trichoderma harzianum M5 were selected for their ability to grow faster on crystalline cellulose as sole carbon source and to excrete higher cellulase yields, particularly exo-cellobiohydrolase (1,4-β- d -glucan cellobiohydrolase EC 3.2.1.91), than the wild types. At optimum pH and temperature of enzyme activity the respective yields of exo-cellobiohydrolase, endo-1,4-β- d -glucanase [1,4-(1,3;1,4)-β- d -glucan 4-glucanohydrolase, EC 3.2.1.4] and β- d -glucosidase (β- d -glucoside glucohydrolase, EC 3.2.1.21) were 1.0, 69.0 and 19.2 U ml −1 culture fluid for A. ustus M35 and 1.0, 33.5 and 2.5 U ml −1 culture fluid for T. harzianum M5. The exo-cellobiohydrolase yields were among, if not the highest reported in the literature .

Isolement de 2 nouveaux metabolites d'A. ustus, leur structure moleculaire est precisee. On etudie leur activite biologique sur le coleophile de ble (inhibition de la croissance), sur les cultures bacteriennes (Bacillus subtilis et Escherichia coli) sur le poussin (hyperacusie)

Four attributes required by microorganisms for biocontrol of plant diseases caused by sclerotium-forming fungi, where the strategy is to reduce the number of sclerotia, are (1) an ability to adversely affect propagule germination and (or) viability, (2) growth potential through soil, (3) activity over a range of environmental conditions, and (4) survival and reproductive potential. Three methods of assessment (soil plate, SPM; soil immersion, SIM; inert carrier, ICM) were evaluated to assess biocontrol agents with regard to attributes 1 and 3. Based on the method of assessment, the fungal antagonists (Gliocladium roseum, G. catenulatum, Trichoderma harzianum, Tr. pseudokoningii, Aspergillus ustus, A. versicolor, Penicillium crustosum, Fusarium solani, Chaetomium globosum, and Stilbum sp.) were grouped by their ability to colonize sclerotia of Phymatotrichum omnivorum in nonsterile soil. Group 1 strains colonized greater than 50% of the sclerotia with any method. Group 2 strains failed to colonize greater than 18% of the sclerotia with any method. Group 3 strains effectively colonized sclerotia when assessed by the SPM but not the ICM; opposite results were obtained with group 4 isolates. With the three methods of assessment, Gliocladium roseum (strain 1620) colonized sclerotia at 19, 23, 27, and 31 °C. Colonization with the SPM or SIM was not temperature dependent, but colonization significantly decreased with increased temperature (27 and 31 °C) for the ICM. The method of assessment altered conclusions about the effectiveness of these potential biocontrol agents.

SUMMARY: In detailed studies of the growth of Aspergillus ustus, Penicillium oxalicum, P. frequentans and P. notatum in whey with fat production in view, the first two showed the highest lactose utilization and felt weights in shaken cultures while the last two gave as good or better felt yields in stationary cultures. When A. ustus was grown in shaken culture in whey with and without the addition of 1·14 g/1. of ammonium nitrate, the extra nitrogen led to the production of larger amounts of fat, but P. frequentans did not form additional fat in these circumstances. A. ustus was the best mould; in whey plus ammonium nitrate it utilized up to 96% of the lactose and formed, per litre of whey, about 17 g of mycelial felt containing 13% of protein and 28% of fat.