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Natural products have high structural diversity with various pharmacological or biological activities, which are of great significance to our life and drug research. Millions of natural products with versatile structural diversity have been found in nature. In recent years, a large number of microbial genome sequences have been released in public databases and revealed many silent or cryptic secondary metabolite gene clusters hidden in their genomes. This shows the great potential for discovering new metabolites. Advances in sequencing technology and bioinformatics analysis also provide great advantages for studying the biosynthesis and structural diversity of these metabolites. Structural differentiation of natural products begins with the formation of basic scaffolds using basic building blocks derived from primary metabolism catalyzed by different backbone enzymes. The structural complexity of natural products mainly arises from tailoring enzymes to highly functionalize the skeletons with a set of chemical transformations. The well-studied modification enzymes range from different types of oxidoreductases, cytochrome P450 enzymes, to various prenyltransferases (PTs) and methyltransferases (MTs). In addition, nonenzymatic events have also contributed to the formation of final products with vast diversity and complexity. Therefore, fully exploring these unexplored gene clusters and the substrate promiscuity of enzymatic and non-enzymatic reactions for the natural product formation may be a promising way and a new strategy to explore the metabolite diversity. In a cooperation study with Dr. Liujuan Zheng, the biosynthesis of a highly oxygenated phenethyl derivative ustethylin A, isolated from Aspergillus ustus, was elucidated. Due to the instability of ustethylin A, it was acetylated before isolation and structure elucidation. Gene deletion and heterologous expression proved that the phenethyl core structure is assembled by a polyketide synthase (UttA) harboring a methyltransferase domain. Isotopic labelling experiments proved that the backbone of ustethylin A is derived from malonyl-CoA and the methyl groups, also in the phenethyl residue, are from L-methionine. Modifications on the core structure by an aryl acid reductase (UttJ), a putative nonheme FeII/2-oxoglutarate dependent oxygenase (UttH), a cytochrome P450 enzyme (UttC) and a O-methyltransferase (UttF) led to the final product ustethylin A. This study is the first report on the biosynthetic pathway of a phenethyl-containing natural product. In cooperation with Dr. Jing Liu, the biosynthesis of streptoazine C and guanitrypmycin D1 was elucidated. Firstly, a three-gene cluster coding for a cyclodipeptide synthase, a prenyltransferase, and a methyltransferase was identified in Streptomyces aurantiacus by genome mining. Heterologous expression and precursor incubation experiments led to the elucidation of the biosynthetic steps of streptoazine C. In vivo biotransformation experiments proved the high flexibility of the prenyltransferase SasB toward tryptophan-containing cyclodipeptides and their dehydroderivatives for regular C-3-prenylation. This study provides an enzyme with a high substrate promiscuity from the less explored prenyltransferase group in cyclodipeptide synthase-related pathways. Afterwards, a two-gene cluster coding for a CDPS and a cytochrome P450 was identified in Streptomyces sp. NRRL S-1521 by phylogenetic analysis. Heterologous expression and structural elucidation of the isolated products proved that the cytochrome P450 GutD1521 catalyzes the regiospecific transfer of guanine to C-2 of the indole ring of cyclo-(L-Trp-L-Tyr) via a C−C linkage, which represents a new chemical transformation within this enzyme class. Precursor incubation experiments revealed that GutD1521 efficiently accepts several other tryptophan-containing cyclodipeptides or derivatives for regiospecific coupling with guanine, thus generating different guanitrypmycin analogs. This study provides a biocatalyst for a new linkage pattern between a indole ring and a guanine moiety and expands the functional spectrum of P450s as tailoring enzymes.

The use of microorganisms for remediation and restoration of hydrocarbons contaminated soils is an effective and economic solution. The current study aims to find out efficient telluric filamentous fungi to degrade petroleum hydrocarbons pollutants. Six fungal strains were isolated from used engine (UE) oil contaminated soil. Fungi were screened for their ability to degrade crude oil, diesel and UE oil using 2.6-dichlorophenol indophenol (DCPIP). Two isolates were selected, identified and registered at NCBI as Aspergillus ustus HM3.aaa and Purpureocillium lilacinum HM4.aaa. Fungi were tested for their tolerance to different concentration of petroleum oils using radial growth diameter assay. Hydrocarbons removal percentage was evaluated gravimetrically. The degradation kinetic of crude oil was studied at a time interval of 10 days. A.ustus was the most tolerant fungi to high concentration of petroleum oils in solid medium. Quantitative analysis showed that crude oil was the most degraded oil by both isolate; P. lilacinium and A. ustus removed 44.55% and 30.43% of crude oil, respectively. The two fungi were able to degrade, respectively, 27.66 and 21.27% of diesel and 14.39 and 16.00% of UE oil. As compared to the controls, these fungi accumulated high biomass in liquid medium with all petroleum oils. Likewise, crude oil removal rate constant (K) and half-lives (t1/2) were 0.02 day−1, 34.66 day and 0.015 day−1, 46.21 day for P. lilacinium and A. ustus, respectively. The selected fungi appear interesting for petroleum oils biodegradation and their application for soil bioremediation require scale-up studies.

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

New antifungal agrochemicals, derived from 2-(benzylsulfonyl)benzothiazole were synthesized by an environmentally friendly method, using water as reaction medium. These compounds were prepared by a one-pot, two-step synthesis, starting from 2-mercaptobenzothiazole and benzyl halides. The potential fungicides were tested against a panel of phytopathogenic fungi, with many of them showing a significant improvement compared to the non-oxidized analogues and the commercial antifungal Captan. The new derivatives 2-((2-chlorobenzyl)sulfonyl)benzo[d]thiazole (4f) and 2-((4-methylbenzyl)sulfonyl)benzo[d]thiazole (4k) presented remarkable properties, being able to inhibit the growth of two resistant moulds (Aspergillus fumigatus and Aspergillus ustus). Both 4f and 4k could be classified as broad-spectrum fungicides, emerging as possible candidates for the control of these moulds, which have negative impact in food production. Fil: Ballari, María Sol. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina Fil: Herrera Cano, Natividad Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Ciencia y Tecnología de Alimentos Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Ciencia y Tecnología de Alimentos Córdoba; Argentina. Universidad Nacional de San Juan. Facultad de Ingeniería. Instituto de Biotecnología; Argentina Fil: Wunderlin, Daniel Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Ciencia y Tecnología de Alimentos Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Ciencia y Tecnología de Alimentos Córdoba; Argentina Fil: Feresin, Gabriela Egly. Universidad Nacional de San Juan. Facultad de Ingeniería. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Santiago, Ana Noemi. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina

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.

Highlights • Vermiculite is a new promising carrier for solid-state fermentation of micromycetes. • Aspergillus have higher protease production on vermiculite in comparison with on other carriers. • The mycelium productivity of Aspergillus was higher not only in comparison with SmF, but also in comparison with other carriers under SSF conditions (from 3 to 18 times)., A new method has been developed to increase the productivity of aspergilli - producers of extracellular proteinases based on their cultivation on vermiculite under solid-state fermentation conditions. The productivity of the mycelium Aspergillus ochraceus L-1 and Aspergillus ustus 1 was 3-18 times higher not only in comparison with submerged cultivation, but also in comparison with growth on other carriers studied under solid-state fermentation conditions. Vermiculite can be considered as a new promising carrier for solid-state fermentation of micromycetes.

Background Caffeine, theobromine and theophylline are main purine alkaloid in tea. Theophylline is the downstream metabolite and it remains at a very low level in Camellia sinensis. In our previous study, Aspergillus sydowii could convert caffeine into theophylline in solid-state fermentation of pu-erh tea through N-demethylation. In this study, tea-derived fungi caused theophylline degradation in the solid-state fermentation. The purpose of this study is identify and isolate theophylline-degrading fungi and investigate their application in production of methylxanthines with theophylline as feedstock through microbial conversion. Results Seven tea-derived fungi were collected and identified by ITS, β-tubulin and calmodulin gene sequences, Aspergillus ustus, Aspergillus tamarii, Aspergillus niger and A. sydowii associated with solid-state fermentation of pu-erh tea have shown ability to degrade theophylline in liquid culture. Particularly, A. ustus and A. tamarii could degrade theophylline highly significantly (p A. ustus and A. tamarii, respectively. The data from absolute quantification analysis suggested that 3-methylxanthine and xanthine were the main degraded metabolites in A. ustus and A. tamarii, respectively. 129.48 ± 5.81 mg/L of 3-methylxanthine and 159.11 ± 10.8 mg/L of xanthine were produced by A. ustus and A. tamarii in 300 mg/L of theophylline liquid medium, respectively. Conclusions For the first time, we confirmed that isolated A. ustus, A. tamarii degrade theophylline through N-demethylation and oxidation. We were able to biologically produce 3-methylxanthine and xanthine efficiently from theophylline through a new microbial synthesis platform with A. ustus and A. tamarii as appropriate starter strains.

The aim of research was to characterize epiphyte micromycetes observed on variable cotton fibers accessions, to estimate the range of fiber destruction and select cotton, which were the most resistant to fungus damage. The accessions of differently colored Upland Cotton varieties (Gossypium hirsutum L.) evaluated: eleven cotton of natural green, twelve – of brown and eleven of conventional white color. Cotton plants have been grown in Sothern Federal District, RF. The fiber samples for the study were placed into a thermostat in sterile Petri dishes on moistened filter paper in order to stimulate the development of mycelium or sporulation of fungi naturally occurred on fibers. Incubation carried out in a thermostat at a +24-28 °C, humidity of 90-100% and exposed for 28 days. The samples examined with a microscope or binocular magnifier. Aspergillus ustus (Bainier), A. fumigatus Fresen., A. niger v. Tiegh., A. flavus Link, Penicillium aurantiogriseum Dierckx, P. notatum Westling, Rhizopus nigricans Ehrenb. and Alternaria alternata (Fuier) Keissler were detected. Compared with exposed white, accession of green and brown colors were significantly resistant to fungus. The mean of destruction (K) of white cotton varied up to 0.95, but colored accessions not exceeded 0.3 (initial destruction of the surface, not affecting internal fiber’s structure).

Background. Aspergillus spp. of section Usti (A. ustus) represent a rare cause of invasive aspergillosis (IA). This multicenter study describes the epidemiology and outcome of A. ustus infections. Methods. Patients with A. ustus isolated from any clinical specimen were retrospectively identified in 22 hospitals from 8 coun- tries. When available, isolates were sent for species identification (BenA/CaM sequencing) and antifungal susceptibility testing. Additional cases were identified by review of the literature. Cases were classified as proven/probable IA or no infection, according to standard international criteria. Results. Clinical report forms were obtained for 90 patients, of whom 27 had proven/probable IA. An additional 45 cases were identified from literature review for a total of 72 cases of proven/probable IA. Hematopoietic cell and solid-organ transplant recipi- ents accounted for 47% and 33% cases, respectively. Only 8% patients were neutropenic at time of diagnosis. Ongoing antimold prophylaxis was present in 47% of cases. Pulmonary IA represented 67% of cases. Primary or secondary extrapulmonary sites of infection were observed in 46% of cases, with skin being affected in 28% of cases. Multiple antifungal drugs were used (consecu- tively or in combination) in 67% of cases. The 24-week mortality rate was 58%. A. calidoustus was the most frequent causal agent. Minimal inhibitory concentrations encompassing 90% isolates (MIC90) were 1, 8, >16, and 4 µg/mL for amphotericin B, voriconazole, posaconazole, and isavuconazole, respectively. Conclusions. Aspergillus ustus IA mainly occurred in nonneutropenic transplant patients and was frequently associated with extrapulmonary sites of infection. Mortality rate was high and optimal antifungal therapy remains to be defined.

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.

Background Aspergillus spp. of section Usti (A. ustus) represent a rare cause of invasive aspergillosis (IA). This multicenter study describes the epidemiology and outcome of A. ustus infections. Methods Patients with A. ustus isolated from any clinical specimen were retrospectively identified in 22 hospitals from 8 countries. When available, isolates were sent for species identification (BenA/CaM sequencing) and antifungal susceptibility testing. Additional cases were identified by review of the literature. Cases were classified as proven/probable IA or no infection, according to standard international criteria. Results Clinical report forms were obtained for 90 patients, of whom 27 had proven/probable IA. An additional 45 cases were identified from literature review for a total of 72 cases of proven/probable IA. Hematopoietic cell and solid-organ transplant recipients accounted for 47% and 33% cases, respectively. Only 8% patients were neutropenic at time of diagnosis. Ongoing antimold prophylaxis was present in 47% of cases. Pulmonary IA represented 67% of cases. Primary or secondary extrapulmonary sites of infection were observed in 46% of cases, with skin being affected in 28% of cases. Multiple antifungal drugs were used (consecutively or in combination) in 67% of cases. The 24-week mortality rate was 58%. A. calidoustus was the most frequent causal agent. Minimal inhibitory concentrations encompassing 90% isolates (MIC90) were 1, 8, >16, and 4 µg/mL for amphotericin B, voriconazole, posaconazole, and isavuconazole, respectively. Conclusions Aspergillus ustus IA mainly occurred in nonneutropenic transplant patients and was frequently associated with extrapulmonary sites of infection. Mortality rate was high and optimal antifungal therapy remains to be defined.

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The contamination of agricultural lands by pesticides, their overuse and toxicity to many non-target organisms cause serious environmental problems. Therefore, the development of bioremediation methods for distinct active ingredients, such as pyrethroids, is primordial. In this work, the biodegradation of gamma-cyhalothrin (GCH) by fungi from the cave Gruta do Catão was investigated. Experiments were conducted with different fungi strains (Aspergillus ustus CBMAI 1894, Talaromyces brunneus CBMAI 1895 and Aspergillus sp. CBMAI 1926) in 2% Malt liquid medium with 300 mg L-1 of GCH in triplicate employing a validated method. All strains biodegraded this insecticide and the most efficient was Aspergillus ustus with a degradation rate of approximately 50% of the initial GCH concentration in 21 days of experiment. We also conducted an experimental design testing three variables: temperature (25-35 degrees), pH (5,5– 8,5) and pesticide concentration (50-550 mg L-1) being the temperature and pesticide concentration were those that most influenced the biodegradation. So, we concluded that cave fungi biodegraded GCH and can be employed in future studies for enzymatic characterization and bioremediation of contaminated environments. A contaminação de áreas agriculturáveis por pesticidas, seu uso intensivo e toxicidade para muitos organismos não alvo causam sérios problemas ambientais. Portanto, o desenvolvimento de metodologias de biorremediação para ingredientes ativos distintos, tais como piretróides, é primordial. Nesse trabalho, a biodegradação de gama cialotrina (GCH) por fungos da Gruta do Catão foi investigada. Os experimentos foram realizados com três diferentes linhagens fúngicas (Aspergillus ustus CBMAI 1894, Talaromyces brunneus CBMAI 1895 e Aspergillus sp. CBMAI 1926) em meio líquido malte 2% com 300 mg L-1 de GCH em experimentos em triplicata utilizando um método validado. Todas as linhagens biodegradaram esse inseticida e a espécie mais eficiente foi Aspergillus ustus com uma taxa de degradação de aproximadamente 50% da concentração inicial de GCH em 21 dias de experimento. Também conduzimos um planejamento experimental testando três variáveis: temperatura (25-35 graus), pH (5,5 - 8,5) e concentração de pesticida (50-550 mg L-1), sendo que a temperatura e a concentração influenciaram no experimento. Concluímos que fungos de caverna biodegradaram GCH e podem ser empregados em estudos futuros para caracterização enzimática e biorremediação de ambientes contaminados. CNPq: 144586/2017-7

Made available in DSpace on 2020-12-10T19:39:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01. Added 1 bitstream(s) on 2021-07-15T14:37:10Z : No. of bitstreams: 1 S0001-37652019000500601.pdf: 975738 bytes, checksum: 4ec2cf4858b8200fba6761772dcd422f (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Isolation and screening of new fungal strains from extreme and understudied environments. such as caves, is a promising approach to find higher yields enzyme producers. Cellulolytic fungal strains isolated from a Brazilian cave were evaluated for their enzymatic production after submerged (SmF) and solid-state fermentation (SSF). After SmF, three strains were selected for their high enzymatic activities: Aspergillus ustus for endoglucanase (4.76 U/mg), Talaromyces bruneus for beta-glucosidase (11.71 U/mg) and Aspergillus sp. (CBMAI 1926) for total cellulase (1.70 U/mg). After SSF, these strains, showed better yields compared to the reference strain Aspergillus niger 3T5B8. Aspergillus sp. (CBMAI 1926) stood out as a new species that expressed activity of total cellulases (0.10 U/mg) and low protein concentration (0.44 mg/mL). In conclusion, these isolated strains have a more efficient and promising cellulolytic enzyme complex that can be used in fermentation and saccharification processes with a lower protein concentration and a higher enzymatic activity than the reference strain. Therefore, beside the new genetic material characterized, our study highlights the benefits of cave extreme environments exploitation to find new potentially valuable strains. Univ Fed Sao Carlos, Dept Ecol & Biol Evolut, Programa Posgrad Ecol & Recursos Nat, Rodovia Washington Luiz,Km 235, BR-13565905 Sao Carlos, SP, Brazil Univ Estado Sao Paulo Julio de Mesquita Filho, Inst Biociencias Rio Clara, Dept Bioquim & Microbiol, Av 24A,1515, BR-13506900 Rio Claro, SP, Brazil Empresa Brasileira Pesquisa Agr, Embrapa Instrumentacao, Rua 15 Novetnbro 1452, BR-13560970 Sao Carlos, SP, Brazil Univ Estado Sao Paulo Julio de Mesquita Filho, Inst Biociencias Rio Clara, Dept Bioquim & Microbiol, Av 24A,1515, BR-13506900 Rio Claro, SP, Brazil FAPESP: FAPESP 2015/24763-9

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.

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.

Breakthrough invasive mold infections (IMIs) that occur during posaconazole or voriconazole prophylaxis are rare complications for which epidemiological data are lacking. This retrospective analysis comparing 24 microbiologically documented breakthrough with 66 nonbreakthrough IMIs shows a shift towards non-Aspergillus molds with a significantly increased proportion of rare multidrug-resistant molds.

Invasive fungal infections due to Aspergillus calidoustus with decreased azole susceptibility are emerging in the setting of azole prophylaxis and are associated with poor outcomes. We assessed the in vitro activity of antifungal drugs used alone or in combinations against A. calidoustus and found a synergistic effect between voriconazole and terbinafine at concentrations within the therapeutic range.

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.

In the present study, we presented the complete mitochondrial genome of Aspergillus sp. The complete mitochondrial genome of Aspergillus sp. was composed of circular DNA molecules, with a total length of 31,374 bp. The base composition of this mitochondrial genome is as follows: A (36.25%), T (38.09%), G (14.55%), and C (11.10%). The mitochondrial genome contains 18 protein-coding genes, 2 ribosomal RNA genes (rRNA), and 31 transfer RNA genes. The taxonomic status of the Aspergillus sp. mitochondrial genome exhibits a closest relationship with Aspergillus ustus and A. flavus.

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.

Aeromycoflora in relation to occurrence and prevalence, their seasonal variation and distribution of fungal spores in air was studied from October, 2010 to September, 2011. During the study a total of 31 fungal species belonging to 18 genera of fungi were isolated and identified. Total fungal colony count was 2308. The highest occurrence and prevalence of fungi was contributed by Aspergillus niger 1075 colonies, Penicillium spp. 582 colonies, Aspergillus fumigatus 167, A. flavus 112 colonies, Rhizopus sp. 121 colonies, Candida spp. 39 colonies, Chladosporium spp. 36 colonies, Syncephalestrum sp. 25 colonies, and Fusarium spp. 14 colonies. Lowest colony countwas recorded one, in Aspergillus ustus, Drechslera sp., Nigrospora sp. and Ulocladium sp. Maximum fungal pollutants were found 339 in July, 2010 at temperature 27.12°C and moisture 84.2% during the wet and rainy days and declined in January, 2011 when temperature and moisture label decreased to 20.26°C and 59.5%, respectively. Highest colony density was recorded in July, 2011, it was moderate in February and March, 2011. Lowest colony density was recorded in January and May, 2011 DOI: http://dx.doi.org/10.3329/jbas.v38i1.20211 Journal of Bangladesh Academy of Sciences, Vol. 38, No. 1, 49-59, 2014

One new ergosteroid derivative, isocyathisterol (1), and eight known compounds (2–9) were isolated from the culture of an algicolous strain (cf-42) of Aspergillus ustus obtained from the fresh tissue of marine green alga Codium fragile. The structure and absolute configuration of 1 were unequivocally identified by using NMR and mass spectroscopic methods as well as quantum chemical calculations. Compound 1 exhibited weak antibacterial activity.

One new meroterpene, 1,2-dihydroterretonin F (2), five new sesterterpenes, (6α)-21-deoxyophiobolin G (3), (6α)-16,17-dihydro-21-deoxyophiobolin G (4), ophiobolin U (5), ophiobolin V (6), and ophiobolin W (7), two new sesquiterpenes, (6-strobilactone-B) esters of (E,E)-6,7-epoxy-2,4-octadienoic acids (13 and 14), and twelve known terpenes (1, 8–12, and 15–20) were isolated from Aspergillus ustus, a fungus from the fresh tissue of marine green alga Codium fragile. Their structures and absolute configurations were identified by NMR and mass spectroscopic methods as well as quantum chemical calculations. Some of the isolates exhibited antibacterial activity and brine shrimp toxicity.

Terpenoids are the most diverse and abundant natural products among which sesterterpenes account for less than 2%, with very few reports on their biosynthesis. Ophiobolins are tricyclic 5–8–5 ring sesterterpenes with potential pharmaceutical application. Aspergillus ustus 094102 from mangrove rizhosphere produces ophiobolin and other terpenes. We obtained five gene cluster knockout mutants, with altered ophiobolin yield using genome sequencing and in silico analysis, combined with in vivo genetic manipulation. Involvement of the five gene clusters in ophiobolin synthesis was confirmed by investigation of the five key terpene synthesis relevant enzymes in each gene cluster, either by gene deletion and complementation or in vitro verification of protein function. The results demonstrate that ophiobolin skeleton biosynthesis involves five gene clusters, which are responsible for C15, C20, C25, and C30 terpenoid biosynthesis.

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

Five new drimane sesquiterpenes (1-5) together with 14 known analogues (6-19) were isolated from laboratory cultures of a mangrove-derived fungus Aspergillus ustus. Their structures were established by spectroscopic methods and antitumor activities were evaluated by sulforhodamine B (SRB) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) methods.

Marine endosymbiotic fungi Aspergillus ustus (MSF3) which produce high yield of biosurfactant was isolated from the marine sponge Fasciospongia cavernosa collected from the peninsular coast of India. Maximum production of biosurfactant was obtained in Sabouraud dextrose broth. The optimized bioprocess conditions for the maximum production was pH 7.0, temperature 20 degrees C, salt concentration 3%, glucose and yeast extract as carbon source and nitrogen sources respectively. The response surface methodology based analysis of carbon and nitrogen ratio revealed that the carbon source can increase the biosurfactant yield. The biosurfactant produced by MSF3 was partially characterized as glycolipoprotein based on the estimation of macromolecules and TLC analysis. The partially purified biosurfactant showed broad spectrum of antimicrobial activity. The strain MSF3 can be used for the microbially enhanced oil recovery process.

The essential oils are well known for their antibacteric, antomycotic and insecticide effects. In this research the antomycotic activity of Rosemary essential oil has been tested in vitro versus different moulds, common contaminants of food and feed, as Aspergillus flavus, Aspergillus ochraceus, Aspergillus niger, Aspergillus ustus, Penicillium expansum, Penicillium aurantio-griseum, Fusarium moniliforme. The Rosemary essential oil tested (produced in Sardegna, Italy) shows a different efficacy against various moulds and his activity seems to be produced by borneol, α-pinene, eucalyptol, camphor and limonene. Aspergillus and Penicillium are the genus showing an important inhibition of their development in vitro. Rosemary essential oil for these natural properties can be used as antimycotic additive to extended shelf-life of many foodstuffs.

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.

Meadow chernozemic soil was studied in an idle land-shelterbelt-cropland system. It was found that the long-term agricultural use of chernozems leads to a decrease in the total nitrogen content and to changes in the soil microbial community. The portion of phytopathogenic Fusarium, Aspergillus ustus, Penicillium daleae, and Penicillium rubrum microorganisms in the complex of soil fungi increases, and the phytosanitary state of the soil worsens. The use of biotests made it possible to estimate the role of particular microorganisms in the soil phytotoxicity.