FR207944, an antifungal antibiotic from Chaetomium sp. no. 217 I. Taxonomy, fermentation, and biological properties

An antifungal antibiotic, FR207944, was isolated from the culture broth of a fungal strain Chaetomium sp. no. 217. FR207944 is a triterpene glucoside with antifungal activity against Aspergillus fumigatus and Candida albicans. Specifically, FR207944 exhibits in vitro and in vivo antifungal activity against A. fumigatus. The effects of FR207944 on the morphology of A. fumigatus were shown to be similar to those of FR901379, a known 1,3-beta-glucan synthase inhibitor. The MECs of FR207944 against A. fumigatus FP1305 and C. albicans FP633 in micro-broth dilution test were 0.039 and 1.6 mug/ml respectively. FR207944 showed good potency by subcutaneous injection and oral administration against A. fumigatus in a murine systemic infection model, with ED(50)s of 5.7 and 17 mg/kg respectively.     

Abundant respirable ergot alkaloids from the common airborne fungus Aspergillus fumigatus

Ergot alkaloids are mycotoxins that interact with several monoamine receptors, negatively affecting cardiovascular, nervous, reproductive, and immune systems of exposed humans and animals. Aspergillus fumigatus, a common airborne fungus and opportunistic human pathogen, can produce ergot alkaloids in broth culture. The objectives of this study were to determine if A. fumigatus accumulates ergot alkaloids in a respirable form in or on its conidia, to quantify ergot alkaloids associated with conidia produced on several different substrates, and to measure relevant physical properties of the conidia. We found at least four ergot alkaloids, fumigaclavine C, festuclavine, fumigaclavine A, and fumigaclavine B (in order of abundance), associated with conidia of A. fumigatus. Under environmentally relevant conditions, the total mass of ergot alkaloids often constituted >1% of the mass of the conidium. Ergot alkaloids were extracted from conidia produced on all media tested, and the greatest quantities were observed when the fungus was cultured on latex paint or cultured maize seedlings. The values for physical properties of conidia likely to affect their respirability (i.e., diameter, mass, and specific gravity) were significantly lower for A. fumigatus than for Aspergillus nidulans, Aspergillus niger, and Stachybotrys chartarum. The demonstration of relatively high concentrations of ergot alkaloids associated with conidia of A. fumigatus presents opportunities for investigations of potential contributions of the toxins to adverse health effects associated with the fungus and to aspects of the biology of the fungus that contribute to its success.     

烟曲霉关键致病因子研究进展

烟曲霉是侵袭性真菌病常见的致病菌之一。近年来一些细胞表面蛋白（如GPI锚定蛋白）以及分泌的毒素被认为是烟曲霉的关键致病因子。现从烟曲霉的细胞壁蛋白、多糖,细胞外基质成分,烟曲霉的分泌产物几方面综述已发现的烟曲霉关键致病因子,并对未来的研究方向进行展望。     

THTA, a thermotolerance gene of Aspergillus fumigatus

Aspergillus fumigatus grows optimally from 37 to 42 degrees C but can grow at temperatures up to 55 degrees C. To study the genetic basis of thermotolerance and its role in virulence of A. fumigatus, temperature sensitive mutants were isolated. One of the mutants that grew at 42 degrees C but not at 48 degrees C was complemented and the gene, THTA, was identified. Deletion of THTA showed the same temperature sensitivity as the original mutant. THTA encodes a putative protein of 141 kDa with unknown function and the HA-tagged ThtAp accumulated to similar levels in cultures grown at either 37 or 48 degrees C. Southern blot analysis and database searches revealed the presence of THTA-related sequences in several other ascomycetous fungi. No difference in virulence was observed between the deltathtA and wild-type strains. Thus, THTA is essential for growth of A. fumigatus at high temperatures but does not contribute to the pathogenicity of the species.     

Bioinformatic and expression analysis of the putative gliotoxin biosynthetic gene cluster of Aspergillus fumigatus

Gliotoxin is a secondary metabolite produced by several fungi including the opportunistic animal pathogen Aspergillus fumigatus. It is a member of the epipolythiodioxopiperazine (ETP) class of toxins characterised by a disulphide bridged cyclic dipeptide. A putative cluster of 12 genes involved in gliotoxin biosynthesis has been identified in A. fumigatus by a comparative genomics approach based on homology to genes from the sirodesmin (another ETP) biosynthetic gene cluster of Leptosphaeria maculans. The physical limits of the cluster in A. fumigatus have been defined by bioinformatics and by identifying the genes that are co-regulated and whose timing of expression correlates with the production of gliotoxin in culture.     

Effets de la fumitoxine A sur le tissu foliaire de Pisum sativum L.

Etiolated pea seedlings are treated with fumitoxin A, a mycotoxin produced by Aspergillus fumigatus Fres. The effects observed on the greening process relate to the production of chlorophyll and fatty acid biosynthesis. The effects vary in relation to the dose of toxin used. With high doses, fumitoxin A gives rise to a diminution of chlorophyll level and an inhibition of fatty acid biosynthesis. Lower doses cause a significant augmentation of chlorophyll level and at the same time, an acceleration of polyunsaturated fatty acid biosynthesis.     

1株盾叶薯蓣植物内生烟曲霉菌的分离鉴定

从药用盾叶薯蓣地下茎组织中分离内生菌。选取盾叶薯蓣地下茎核心组织,32℃恒温孵育,分离菌株;依据形态学、分子生物学特征鉴定菌株;通过纸片扩散法检测分离株与同种植物内生Bcillus subtilis SWB8菌株的拮抗作用。结果显示,孵育48 h后,培养基表面呈现扁平、白色或深绿色绒毛状菌落,显微镜下见孢子囊和圆形分生孢子;ITS序列分析显示,分离株ITS基因序列与GenBank数据库中烟曲霉菌同源性为100%,鉴定为烟曲霉菌(A.fumigatus YHY01)。拮抗实验显示,其与B.subtilis SWB8菌没有明显相互抑制现象。首次从盾叶薯蓣植物中分离出A.fumigatus,推测其与B.subtilis SWB8菌株间存在共生关系。     

Occurrence of Aspergillus fumigatus during composting of sewage sludge.

Aspergillus fumigatus, a medically important fungal opportunist and respiratory allergen, was isolated from woodchips and sewage sludge used in the production of compost at the U.S. Department of Agriculture's composting research facility in Beltsville, Md. It was also regularly isolated as a dominant fungus during forced aeration composting and after 30 days in an unaerated stationary curing pile; in both cases, the fungus was found in pile zones with temperatures less than 60 degrees C. Compost stored outdoors in stationary unaerated piles from 1 to 4 months after screening out of woodchips contained easily detectable amounts of A. fumigatus in the exterior pile zones (0- to 25-cm depths). Semiquantitative studies of the airspora at the composting site revealed that A. fumigatus constituted 75% of the total viable mycoflora captured. At locations 320 m to 8 km from the compost site, the fungus constituted only 2% of the total viable mycoflora in the air. Of 21 samples of commercially available potting soil, one had levels of A. fumigatus nearly equivalent to those of 1-month-old storage compost; 15 others had lower but detectable levels.     

Occurrence of Aspergillus fumigatus during composting of sewage sludge.

Aspergillus fumigatus, a medically important fungal opportunist and respiratory allergen, was isolated from woodchips and sewage sludge used in the production of compost at the U.S. Department of Agriculture's composting research facility in Beltsville, Md. It was also regularly isolated as a dominant fungus during forced aeration composting and after 30 days in an unaerated stationary curing pile; in both cases, the fungus was found in pile zones with temperatures less than 60 degrees C. Compost stored outdoors in stationary unaerated piles from 1 to 4 months after screening out of woodchips contained easily detectable amounts of A. fumigatus in the exterior pile zones (0- to 25-cm depths). Semiquantitative studies of the airspora at the composting site revealed that A. fumigatus constituted 75% of the total viable mycoflora captured. At locations 320 m to 8 km from the compost site, the fungus constituted only 2% of the total viable mycoflora in the air. Of 21 samples of commercially available potting soil, one had levels of A. fumigatus nearly equivalent to those of 1-month-old storage compost; 15 others had lower but detectable levels.     

Occurrence of Clostridium difficile in fecal samples of HIV-infected children in Poland

The prevalence of Clostridium difficile and its toxins (A and B) in HIV-positive children in Poland was investigated in a group of 18 children, aged 6 months to 8 1/2 years. Stool samples were tested using an antigen detection method for toxin A/B, cytotoxicity-neutralization and culture. In 3 cases (17%) C. difficile toxins were detected in both stool samples and strains recovered from culture. The three strains isolated were shown by PCR methods to contain toxins A and B genes. All children had been treated previously with antimicrobial and antiviral agents. All three C. difficile-positive children had mild diarrhea that resolved without specific therapy. Further studies involving a large number of children and molecular analyses of isolated C. difficile strains are necessary to determine the frequency and rate of carriage of C. difficile strains among HIV-positive children in Poland.     

烟曲霉再鉴定、标准化CSP分型及体外药物敏感性

目的 了解烟曲霉复合体临床株菌种分布,经典烟曲霉临床株CSP基因型及对常见抗真菌药物敏感性状况.方法 菌株来源:北京大学真菌和真菌病研究中心保存分离自125名患者的162株烟曲霉复合体菌株.通过形态学,最高生长温度及分子生物学测序分步鉴定;对CSP基因进行扩增、测序,采用国际化命名体系进行CSP分型;采用微量液基稀释法测定经典烟曲霉对伊曲康唑(ITC)、两性霉素B(AMB)、伏立康唑(VRC)及卡泊芬净(CAS)的敏感性.结果 所有烟曲霉复合体菌株均为经典烟曲霉;共分为16个CSP基因型,最常见为t04A、t03和t01;分离自4名患者的13株菌对ITC的MICs≥4 μg/mL,其中2株菌AMB和VRC的MICa分别为4μg/mL和16 μg/mL.CAS的MECs最高为4μg/mL,仅1株.结论 未检出烟曲霉相关新种;经典烟曲霉临床株共16个CSP基因型,分布与国际研究结果基本一致,其中5个为新型.我国经典烟曲霉临床株ITC耐药率为3.2％,个别菌株AMB,VRC和CAS耐药.     

New restriction fragment length polymorphism (RFLP) markers for Aspergillus fumigatus

In this study, we isolated and tested restriction fragment length polymorphism (RFLP) markers for Aspergillus fumigatus based on PCR products amplified by the random amplified polymorphic DNA (RAPD) primer R108. Four DNA fragments, Afd, Af5, Af4, and Af4A, were amplified. Fragments Afd and Af5 were 85% and 88% identical at the DNA level to part of the Afut1 retrotransposon from A. fumigatus. Fragment Af4A is a duplication of fragment Af4 and both showed similarity at the amino acid level with endonucleases from other fungal retrotransposons. We used both RAPD with primer R108 and RFLP assays with Afut1, Afd, and Af4A, to determine the genetic relatedness of clinical isolates of A. fumigatus isolated sequentially from four patients colonized with A. fumigatus. The combination of these different methods suggested that the isolates infecting the four patients were not identical.     

Production of phospholipase C (alpha-toxin), haemolysins and lethal toxins by Clostridium perfringens types A to D.

To obtain high yields of extracellular enzymes and toxins for immunological analysis, type culture collection strains of Clostridium perfringens types A to D and 28 fresh isolates of C. perfringens type A from humans were grown in fermenters under controlled conditions in a pre-reduced proteose peptone medium. The type culture collection strains all showed different characteristics with respect to growth rates and pH optima for growth. Production of phospholipase C (alpha-toxin), haemolysin and lethal activity varied considerably between the different types. Growth and extracellular protein production in fermenters with pH control and static or stirred cultures were compared. Production of all extracellular proteins measured was markedly improved by cultivation in fermenters with pH control. Strain ATCC13124 produced five times more phospholipase C than any of 28 freshly isolated strains of C. perfringens type A, grown under identical conditions. Haemolytic and lethal activities of the ATCC strain were equal or superior to the activities of any of the freshly isolated strains. There were no differences in the bacterial yields and in the production of extracellular toxins between type A strains isolated from clinical cases of gas gangrene and abdominal wounds, and those isolated from faecal samples from healthy persons.     

Isolation and Characterization of Antigens of Aspergillus Fischeri

Dilute acid extraction of Aspergillus fischeri mycelia contained two distinct antigens that were purified and separated by ethanol fractionation, concanavalin A precipitation, and chromatography on a Sephadex G-100 column. One antigen which emerges from the column among the early fractions contained 66% hexose determined as galactose and mannose but no demonstrable protein. The second antigen contained mannose as the sole hexose, was inactivated by pronase, and had a molecular weight of approximately 8000.

Fungi of the genus Aspergillus are ubiquitous saprophytes capable of causing debilitating disease under certain conditions. Although aspergilli can attack any part of the body, they are most often encountered as respiratory pathogens. Aspergillus fumigatus is the species most frequently responsible for infection.

Aspergillus fischeri is an ascosporic species within the aspergilli group, closely related to A. fumigatus This organism grows on Czapek's agar at 25 and 37°C and at 37°C it produces abundant conidia indistinguishable from A. fumigatus. Aspergillus fischeri is worldwide in distribution and can be readily isolated from the soil. Only rarely has it been associated with disease in man and it is generally considered a nonpathogen. However, A. fischeri has been shown by Biguet et al. to have at least eight antigens in common with A_. fumigatus. Aspergillus fischeri has been neglected in serological studies of the aspergilli, probably because of its noted nonpathogenicity. However, the obvious morphological similarity of these two aspergilli led us to study the antigens shared by the two species.     

Cultivation of fungi from fecal specimens in cases of antibiotic associated diarrhea (AAD)

The aim of performed examinations was to isolate, identify and determine a drug susceptibility of fungi cultured from faecal specimens submitted for detection of Clostridium difficile in cases of antibiotic-associated diarrhoea (AAD). One hundred samples of diarrhoeic faeces were examined using routine bacteriological methods (isolation and identification of C. difficile), serological test (detection of C. difficile toxins A/B) and mycological methods (isolation, identification and drug susceptibility testing of fungi). Out of twenty seven specimens of diarrhoeic faeces fungal strains were isolated, in 20 samples C. difficile strain and/or C. difficile toxins A/B were detected, in 23 specimens fungal strains, C. difficile strains and/or toxins A/B of this species were present. The most active in vitro agent against cultured fungal strains was nystatin. In conclusion it can be stated, that fungal strains are responsible for some cases of antibiotic-associated diarrhoea. So, mycological diagnostics of faecal samples from patients with diarrhoea after antibiotic therapy is necessary. Cases of diarrhoea with mixed bacterial and fungal aetiology (C. difficile + yeast-like fungus) were observed.     

Studies on mycoflora of salt marshes in Egypt. IV=osmophilic fungi

Ninety-five species and seven varieties which belong to thirty-nine genera were isolated from 74 soil samples from salt marshes. At 28°C, on 30% sucrose Czapek's agar, ninety species and four varieties which belong to thirty-six genera were encountered, from which A. niger, A. fumigatus A. terreus and P. notatum were the most frequent. On 60% sucrose Czapek's agar, sixty-three species and three varieties were recovered which belong to twenty-five genera, from which A. niger, A. fumigatus, Cladosporium herbarum and A. terreus were the most frequent.At 45°C, on 30% sucrose, sixteen species and four varieties were identified, but on 60% sucrose, fourteen species and three varieties were isolated. A. fumigatus and A. niger were the most frequent on both sucrose concentrations.The results reveal that the soil samples poor in total osmophilic fungi (at 28°C) were significantly higher in their content of total soluble salts, Na and K and significantly lower in the average number of species per soil samples than the rich ones; the difference was nonsignificant in case of organic matter.     

Microbiological and molecular determination of mycobiota in fresh and ensiled maize silage

The mycobiota of fresh and ensiled maize was studied with culturing techniques and a DNA sequence-based approach. Freshly chopped and ensiled maize were collected for 2 y from 12 farms in Pennsylvania. Samples were plated on selective media and isolates identified by morphology and sequences of the internal transcribed spacer regions of rDNA, 800-900 bp of the 5' end of the translation elongation factor 1-alpha gene and a portion of the rodA gene (Aspergillus fumigatus only). ITS regions were amplified from total silage DNA, cloned, sequenced and compared to fungal ITS sequences in GenBank with the BLAST-N algorithm. For samples analyzed by both methods, the molecular technique detected a greater number of species than selective plating. Plating recovered several Penicillium and Fusarium species and Aspergillus fumigatus, while molecular analysis detected Alternaria, Penicillium and Fusarium species. Data from both methods found that Fusarium and Penicillium were the dominant mycotoxigenic fungi in silage, while yeast made up the majority all fungi recovered or detected. Known mycotoxigenic species often accounted for 50% or more of the total number of species isolated or detected at each site. Viable Fusaria were not isolated from or detected in ensiled maize, suggesting that Fusarium species do not survive the ensiling process. Results from this study suggest that given the numerous species of fungi present in silage with mycotoxin producing ability, there is a strong possibility that silage may be contaminated with multiple toxins simultaneously.     

Aspergillus fumigatus toxicity and gliotoxin levels in feedstuff for domestic animals and pets in Argentina

Aims:  To evaluate gliotoxin production by Aspergillus fumigatus strains isolated from feedstuff intended for domestic animals and pets, and to determine the amount of gliotoxin in these substrates.
Methods and Results:  A total of 150 feedstuff samples were collected. They were composed of 30 samples each of five different feed types (pigs, poultry, cattle, horse and pets). Aspergillus fumigatus gliotoxin production ability and gliotoxin presence in feedstuff was determined by HPLC. Aspergillus fumigatus strains were isolated from all of the tested samples. Strains from cattle, horses and pet food were able to produce gliotoxin. Corn silage samples intended for cattle did not show gliotoxin contamination. All the other tested samples had gliotoxin levels ranging from 29 to 209 μg g⁻¹. Horse and poultry feed samples had the greatest contamination frequency.
Conclusions:  Feed samples contaminated with gliotoxin are potentially toxic to animals.
Significance and Impact of the Study:  The presence of gliotoxin could affect animal productivity and health. Moreover, there are risks of contamination to farm workers handling improperly stored animal feed. Aspergillus fumigatus strains isolated from different sources should be investigated to determine prevention and control strategies.     

Analysis of Volatile Fingerprints for Monitoring Anti-Fungal Efficacy Against the Primary and Opportunistic Pathogen Aspergillus fumigatus

The aims of this study were to use qualitative volatile fingerprints obtained using a hybrid sensor array system to screen anti-fungals for controlling the important lung infecting fungus, Aspergillus fumigatus, especially in immunocompromised patients. SIFT-MS was also used to try and identify key volatiles produced by A. fumigatus. Initial studies were carried out to identify the ED(50) and ED(90) (effective dose) for inhibiting growth of A. fumigatus using three anti-fungal compounds, benomyl, tebuconazole and fluconazole. Subsequent studies involved inoculation of malt extract agar plates with spores of A. fumigatus (25 and 37°C) over periods of 24-72 h to examine the headspace volatile fingerprints generated from the sample treatments using the hybrid sensor array system to compare controls and ED(50)/ED(90) concentrations. The sensor responses showed discrimination between treatments after 48-h incubation when benomyl and tebuconazole were used against A. fumigatus at 37°C using Principal Components Analysis and Cluster Analysis. SIFT-MS analysis showed that methyl pentadiene, ethanol, isoprene and methanol were key biomarker volatiles produced by A. fumigatus in the presence of anti-fungal compounds. This may also be a good approach for the development of rapid screening of anti-microbial compounds and potentially useful for monitoring the possible build up of resistance to specific drug types. Volatile fingerprints produced by patient samples could also be used to evaluate whether lung infections are caused by bacteria or specific fungi to facilitate early diagnosis and enable the right drug treatment to be prescribed.