Ultraviolet germicidal irradiation disinfection of Stachybotrys chartarum

The ultraviolet germicidal irradiation (UVGI) dose necessary to inactivate fungal spores on an agar surface and the efficacy of UVGI were determined for cultures of Stachybotrys chartarum (ATCC 208877). This study employed a UVGI testing unit consisting of four chambers with a 9-W, Phillips, low pressure, mercury UVGI lamp in each chamber. The testing unit's apertures were adjusted to provide 50, 100, 150, and 200 microW/cm2 of uniform flux to the Petri dish surfaces, resulting in a total UVGI surface dose ranging from 12 to 144 mJ/cm2. The UVGI dose necessary to inactivate 90% of the S. chartarum was greater than the maximum dose of 144 mJ/cm2 evaluated in this study. While UVGI has been used to inactivate several strains of culturable fungal spores, S. chartarum was not susceptible to an appropriate dose of UVGI. The results of this study may not correlate directly to the effect of UVGI on airborne fungal spores. However, they indicate that current technology may not be efficacious as a supplement to ventilation unless it can provide higher doses of UVGI to kill spores, such as S. chartarum, traveling through the irradiated zone.     

Targeting antioxidative signal transduction and stress response system: control of pathogenic Aspergillus with phenolics that inhibit mitochondrial function

AIMS: The aim of this study was to show whether antioxidative response systems are potentially useful molecular targets for control of Aspergillus fumigatus and Aspergillus flavus. Selected phenolic agents are used in target-gene-based bioassays to determine their impact on mitochondrial respiration. METHODS AND RESULTS: Vanillyl acetone, vanillic acid, vanillin, cinnamic acid, veratraldehyde, m-coumaric acid (phenolic agents to which Saccharomyces cerevisiae sod2delta mutant showed sensitivity), carboxin (inhibits complex II of the mitochondrial respiratory chain), strobilurins/antimycin A (inhibits complex III of the mitochondrial respiratory chain) and fludioxonil/fenpiclonil [antifungals potentiated by mitogen-activated protein kinase (MAPK)] were examined in A. fumigatus, A. flavus and S. cerevisiae. Individual or combined application of phenolics with inhibitors of mitochondrial respiration showed some of the phenolics effectively inhibited fungal growth. Target-gene bioassays were performed using a sakAdelta (MAPK deletion) strain of A. fumigatus and a complementation analysis using the mitochondrial superoxide dismutase (Mn-SOD) gene (sodA) of A. flavus in the ortholog mutant, sod2delta, of S. cerevisiae. The results demonstrated that mitochondrial antioxidative stress system plays important roles in fungal response to antifungal agents tested. CONCLUSIONS: Antioxidative response systems of fungi can be an efficient molecular target of phenolics for pathogen control. Combined application of phenolics with inhibitors of mitochondrial respiration can effectively suppress the growth of fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: Natural compounds that do not pose any significant medical or environmental risks could serve as useful alternatives or additives to conventional antifungals. Identifying the antioxidative response systems in other pathogens could improve methods for fungal control.     

曲霉生物膜的形成过程与结构特征

目的 研究曲霉生物膜的形成过程和结构特征.方法 我们利用一个曲霉生物膜体外模型研究其形成过程和结构特征.将200 μL浓度为1×10<'5>孢子/mL的受试曲霉(烟曲霉AF293,黄曲霉BMU03940,土曲霉BMU00802,黑曲霉BMU04689)的孢子悬液加到24孔组织培养板中的无菌塑料细胞培养盖玻片上,37℃孵育不同时间(0、2、4、8、10、12、16、18、24、48、72 h),加入25 μmol/L的FUN-1室温避光染色后,用波长488 nm激光激发,通过共聚焦激光扫描显微镜观察曲霉生物膜的形成过程;再用波长为488 am和633 am激光同时激发,将两个波长下的图像叠加后观察曲霉生物膜的活力;利用:XYZ轴成像观察其结构特征.在上述不同的时间点用钙荧光白染色后,用波长为405 nm的紫外光激发,观察曲霉生物膜细胞外基质的产生.结果 烟曲霉AF293在第4 h即开始有散在的孢子黏附于盖玻片上;8 h时孢子开始萌芽,10～12 h菌丝延长形成单细胞层;16～20 h菌丝缠绕形成多层立体结构;24 h形成一个具有复杂的三维立体结构特征的多细胞菌落,菌丝有序排列,细胞外基质弥散的分布在菌丝的周围;48～72 h生物膜逐渐成熟.成熟的烟曲霉生物膜是由细胞外基质包裹的有序排列的菌丝形成的复杂立体结构.黄曲霉BMU03940、土曲霉BMU00802、黑曲霉BMU04689与烟曲霉AF293有类似的生物膜发育阶段,包括黏附、孢子萌芽、菌丝延长、菌丝有序排列形成三维立体结构.结论 烟曲霉、黄曲霉、土曲霉和黑曲霉在体外都能形成典型的生物膜,它的形成过程和结构特征与其他真菌生物膜类似.     

Allergic potential of someAspergilli on saw mill workers in Lucknow,India

Fifty fungal types were isolated from the indoor atmosphere of saw mills by exposing Petri plates containing Czapek-dox Agar, Potato-dextrose Agar and Sabouraud Agar media for 5 min. The fungal flora of the outdoor surroundings was also studied for comparison. Species ofAspergilli dominated in the saw mills, being represented by 16 species including one ascosporic form. Other fungi were species ofCladosporium, Alternaria, Curvularia, Penicillium, Fusarium, etc. Variations in the fungal population in different months were also observed. Fungal spores recovered using the Rotorod Sampler wereAlternaria, Curvalaria lunata, Curvularia tetramera, Cladosporium, Dreschslera sp.,Epicoccum sp.,Pithomyes sp.,Nigrospora, Stemphylium sp. andTorula sp. Mycelial fragments and unidentifiable spores were also seen in abundance. Varying allergic responses of patients were also recorded by testing intradermally, the antigens of nineAspergilli, vizAspergillus flavus, A. fumigatus, A. japonicus, A. melleus, A. nidulans, A. niger, A. niveus, A. tammarii and A. terreus.     

医院病房环境真菌监测分析

目的 调查医院各病区环境中真菌含量及分布特点.方法 运用分离培养及DNA测序方法对医院不同病区、不同环境中真菌进行监测分析.结果 医院不同病区真菌含量不同,呼吸科、血液科、小儿科空气中真菌含量较高,分别为400、225和200 CFU/m3,其中以烟曲霉菌( 325 CFU/m3)、黄曲霉菌( 275 CFU/m3)、枝孢霉菌( 125 CFU/m3)、根霉( 125 CFU/m3)为主;老年病科、肿瘤科和血液科空调出气口真菌含量较高,分别为0.559、0.500和0.323 CFU/cm2,以链格孢霉(0.441 CFU/cm2)、根霉(0.412 CFU/cm2)、烟曲霉菌(0.294 CFU/cm2)为主;不同环境中真菌含量不同,其中以空气和空调出气口真菌含量最高,分别为130 CFU/m3、0.173 CFU/cm2.结论 真菌广泛存在于医院环境中,且不同病区、不同环境真菌污染程度不同,因而我们必须制订健全的消毒管理制度,预防医院真菌感染.     

Allergic potential of someAspergilli on saw mill workers in Lucknow,India

Fifty fungal types were isolated from the indoor atmosphere of saw mills by exposing Petri plates containing Czapek-dox Agar, Potato-dextrose Agar and Sabouraud Agar media for 5 min. The fungal flora of the outdoor surroundings was also studied for comparison. Species ofAspergilli dominated in the saw mills, being represented by 16 species including one ascosporic form. Other fungi were species ofCladosporium, Alternaria, Curvularia, Penicillium, Fusarium, etc. Variations in the fungal population in different months were also observed. Fungal spores recovered using the Rotorod Sampler wereAlternaria, Curvalaria lunata, Curvularia tetramera, Cladosporium, Dreschslera sp.,Epicoccum sp.,Pithomyes sp.,Nigrospora, Stemphylium sp. andTorula sp. Mycelial fragments and unidentifiable spores were also seen in abundance. Varying allergic responses of patients were also recorded by testing intradermally, the antigens of nineAspergilli, vizAspergillus flavus, A. fumigatus, A. japonicus, A. melleus, A. nidulans, A. niger, A. niveus, A. tammarii and A. terreus.     

Selective expression of a major allergen and cytotoxin, Asp f I, in Aspergillus fumigatus. Implications for the immunopathogenesis of Aspergillus-related diseases.

Asp f I is a major 18-kDa Aspergillus fumigatus allergen and a member of the mitogillin family of cytotoxins. The nucleotide sequence of the Asp f I gene was determined by sequencing polymerase chain reaction products amplified from A. fumigatus spore DNA. The entire 678-bp DNA includes an 81-bp leader sequence, preceding the N-terminal alanine codon, a 52-bp intron, and a 444-bp open reading frame, encoding a 149-amino acid protein (M(r) 16,899), which is 99% homologous to mitogillin from Aspergillus restrictus. A mAb-based ELISA was used to compare Asp f I levels in spores, mycelia, and culture filtrate, and to determine the kinetics of allergen production. Disrupted hyphae or spore extracts had a 1000-fold lower level of Asp f I than culture filtrate, suggesting that germination of spores and growth of the fungus are essential for allergen production. Asp f I levels in A. fumigatus and A. restrictus peaked at day 3 (0.87 to 12.1 micrograms/ml), however, the allergen was not detected in Aspergillus flavus, Aspergillus niger, Aspergillus terreus, and Aspergillus nidulans cultures (< 1.5 ng/ml) on either days 3 or 8. Northern analysis confirmed that Asp f I mRNA was detected only in A. fumigatus and A. restrictus, but not in the other four Aspergillus spp. Asp f I-specific DNA was generated after polymerase chain reaction amplification of genomic mycelial DNA obtained from A. fumigatus and A. restrictus, but not from the other Aspergillus spp. The results show that Asp f I is selectively expressed in A. fumigatus, and suggest that this cytotoxin could be a specific virulence factor for A. fumigatus.     

Intratracheal exposure of rats to Aspergillus fumigatus spores isolated from sawmills in Sweden.

Five strains of Aspergillus fumigatus (A, B, D, H, and K) isolated from sawmills were used to expose groups of three rats by intratracheal intubation. The dose was 10(9) spores per rat. At 48 h after administration, two rats from the D group and all rats from the K group died with symptoms of strong dyspnea and tachypnea. At 72 h postadministration and after, some animals showed mild to moderate dyspnea and tachypnea. Autopsies of all animals were performed, including a histopathological examination of the lungs. At 72 h after administration, two distinct morphological groups were identified histopathologically. Severe necrotizing pneumonia characterized by the presence of abundant fungal hyphae was seen in animals that died spontaneously within 48 h postadministration and rats with bronchopneumonia and was characterized by the presence of numerous fungal spores. There was an obvious difference in pathogenicity among the strains of A. fumigatus. Strains D and K were more pathogenic, and only the rats exposed to these strains showed the presence of fungal hyphae in the lungs. The mycotoxin gliotoxin that is produced by A. fumigatus and has antiphagocytic activity was not detected in the spores from any of the A. fumigatus strains.     

Occurrence of indole alkaloids among secondary metabolites of soil Aspergillus

The occurrence of indole alkaloids among secondary fungal metabolites was studied in species of the genus Aspergillus, isolated from soils that were sampled in various regions of Russia (a total of 102 isolates of the species A. niger, A. phoenicis, A. fumigatus, A. flavus, A. versicolor, A. ustus, A. clavatus, and A. ochraceus). Clavine alkaloids were represented by fumigaclavine, which was formed by A. fumigatus. alpha-Cyclopiazonic acid was formed by isolates of A. fumigatus, A. flavus, A. versicolor, A. phoenicis, and A. clavatus. The occurrence of indole-containing diketopiperazine alkaloids was documented for isolates of A. flavus, A. fumigatus, A. clavatus, and A. ochraceus. No indole-containing metabolites were found among the metabolites of A. ustus or A. niger.     

Intratracheal exposure of rats to Aspergillus fumigatus spores isolated from sawmills in Sweden

Five strains of Aspergillus fumigatus (A, B, D, H, and K) isolated from sawmills were used to expose groups of three rats by intratracheal intubation. The dose was 10(9) spores per rat. At 48 h after administration, two rats from the D group and all rats from the K group died with symptoms of strong dyspnea and tachypnea. At 72 h postadministration and after, some animals showed mild to moderate dyspnea and tachypnea. Autopsies of all animals were performed, including a histopathological examination of the lungs. At 72 h after administration, two distinct morphological groups were identified histopathologically. Severe necrotizing pneumonia characterized by the presence of abundant fungal hyphae was seen in animals that died spontaneously within 48 h postadministration and rats with bronchopneumonia and was characterized by the presence of numerous fungal spores. There was an obvious difference in pathogenicity among the strains of A. fumigatus. Strains D and K were more pathogenic, and only the rats exposed to these strains showed the presence of fungal hyphae in the lungs. The mycotoxin gliotoxin that is produced by A. fumigatus and has antiphagocytic activity was not detected in the spores from any of the A. fumigatus strains.     

Endoglucanase production by paper-degrading mycoflora

Fourteen fungal species, namely Aspergillus flavus, A. fumigatus, A. niger, A. ustus, Penicillium islandicum, P. wortmannii, Memnoniella echinata, Cladosporium herbarum, Stachybotrys atra, Chaetomium globosum, Fusarium oxysporum, Torula herbarum, Alternaria alternata and Curvularia uncinata were isolated from different grades of paper. They differ in their distribution on various kinds of paper and also in relative occurrence. While seasonal influence on mycoflora was observed, most of the moulds were capable of growing in all three seasons examined (summer, winter, rainy season). The moulds were cellulolytic in nature and endoglucanase activity was greatest in Aspergillus flavus, A. niger, A. fumigatus, P. wortmannii and P. islandicum.     

Effect of Antifungal Compounds on Aspergillosis in Hatching Chick Embryos

Aspergillosis was induced experimentally in hatching chick embryos by dipping them in water containing spores of Aspergillus fumigatus or A. flavus. The addition to the dip water of antifungal compounds prevented the development of this syndrome. Of the compounds studied, amphotericin B was most effective, followed by pimaricin and nystatin. Sorbic acid was without effect.     

Culture supernatants of patient-derived Aspergillus isolates have toxic and lytic activity towards neurons and glial cells

Infection of the central nervous system by the ubiquitous fungi Aspergillus spp. is a life-threatening disease. Therefore we investigated the mechanism of brain damage by fungal infection. To examine whether secretory factors of Aspergillus isolates derived from patients can induce death of different brain cells, culture supernatants of Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus and Aspergillus niger were added to different astrocytes as well as to the neuroblastoma cell line SK-N-SH, and to the microglial cell line CHME. All four fungal species were shown to secrete toxic factors with neurons being most sensitive against these factors. Very low amounts and short incubation times are sufficient to induce irreversible cell damage, indicating that secreted factors might also affect distant brain regions. Further characterization of the toxic factors revealed that A. fumigatus and A. terreus produced small, heat-stable components whereas the toxic activity of A. niger filtrates was triggered by a high molecular mass factor which could be inactivated by heat. The active component of A. flavus had a molecular mass similar to that of A. niger but was heat-stable and had a significantly lower activity. Taken together these results indicate that secretion of different necrotizing factors might contribute to brain lesions in patients with cerebral aspergillosis.     

Quantifying fungal viability in air and water samples using quantitative PCR after treatment with propidium monoazide (PMA)

A method is described to discriminate between live and dead cells of the infectious fungi Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Mucor racemosus, Rhizopus stolonifer and Paecilomyces variotii. To test the method, conidial suspensions were heat inactivated at 85 degrees C or held at 5 degrees C (controls) for 1 h. Polycarbonate filters (25 mm diameter, 0.8 microm pore size) were placed on "welled" slides (14 mm diameter) and the filters treated with either PBS or PMA. Propidium monoazide (PMA), which enters dead cells but not live cells, was incubated with cell suspensions, exposed to blue wavelength light-emitting diodes (LED) to inactivate remaining PMA and secure intercalation of PMA with DNA of dead cells. Treated cells were extracted and the live and dead cells evaluated with quantitative PCR (QPCR). After heat treatment and DNA modification with PMA, all fungal species tested showed an approximate 100- to 1000-fold difference in cell viability estimated by QPCR analysis which was consistent with estimates of viability based on culturing.     

Cytotoxicity of Aspergillus fungi isolated from hospital environment

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<0,05). To protect a patient from the adverse effects of mycotoxins, not only his or her immunity status should be evaluated but also the presence of fungi in hospital environment and their cytotoxicity should be monitored (possible exposure).     

Standardization and characterization of antigens for the diagnosis of aspergillosis

The aim of this study was to develop and characterize antigens for the diagnosis of aspergillosis. Nine strains of Aspergillus species Aspergillus fumigatus , Aspergillus flavus , and Aspergillus niger were grown in Sabouraud and Smith broth to produce exoantigens. The antigens were tested by immunodiffusion against sera from patients with aspergillosis and other systemic mycoses. The protein fraction of the antigens was detected by SDS-PAGE; Western blot and representative bands were assessed by mass spectrometry coupled to a nano Acquity UltraPerformance LC and analyzed by the Mascot search engine. Concurrently, all sera were tested with Platelia Aspergillus EIA. The most reactive antigens to sera from patients infected by A. fumigatus were produced by A. fumigatus MG2 Sabouraud and pooled A.?fumigatus Sabouraud samples, both with a sensitivity of 93% and specificity of 100% and 97%, respectively. Aspergillus niger and A. flavus antigens were reactive against A. niger and A. flavus sera, each one with a sensitivity and specificity of 100%. Two proteins, probably responsible for antigenic activity, β-glucosidase in A. fumigatus and α-amylase in A. niger were attained. The commercial kit had a specificity of 22%, sensitivity of 100%, positive predictive value of 48%, and negative predictive value of 100%. The antigens produced showed high sensitivity and specificity and can be exploited for diagnostics of aspergilloma.     

TLR9 is actively recruited to Aspergillus fumigatus phagosomes and requires the N-terminal proteolytic cleavage domain for proper intracellular trafficking

TLR9 recognizes unmethylated CpG DNA and induces innate immune responses. TLR9 activation is a multistep process requiring proteolytic cleavage and trafficking to endolysosomal compartments for ligand-induced signaling. However, the rules that govern the dynamic subcellular trafficking for TLR9 after pathogen uptake have not been established. In this study, we demonstrate that uptake of Aspergillus fumigatus conidia induced drastic spatial redistribution of TLR9 to the phagosomal membrane of A. fumigatus-containing phagosomes but not to bead-containing phagosomes in murine macrophages. Specific TLR9 recruitment to the fungal phagosome was consistent using A. fumigatus spores at different germination stages and selected mutants affecting the display of Ags on the fungal cell surface. Spatiotemporal regulation of TLR9 compartmentalization to the A. fumigatus phagosome was independent of TLR2, TLR4, and downstream TLR signaling. Our data demonstrate that the TLR9 N-terminal proteolytic cleavage domain was critical for successful intracellular trafficking and accumulation of TLR9 in CpG-containing compartments and A. fumigatus phagosomal membranes. Our study provides evidence for a model in which A. fumigatus spore phagocytosis by macrophages specifically induces TLR9 recruitment to A. fumigatus phagosomes and may thereby mediate TLR9-induced antifungal innate immune responses.     

Aspergillus fumigatus triggers inflammatory responses by stage-specific beta-glucan display

Inhalation of fungal spores (conidia) occurs commonly and, in specific circumstances, can result in invasive disease. We investigated the murine inflammatory response to conidia of Aspergillus fumigatus, the most common invasive mold in immunocompromised hosts. In contrast to dormant spores, germinating conidia induce neutrophil recruitment to the airways and TNF-alpha/MIP-2 secretion by alveolar macrophages. Fungal beta-glucans act as a trigger for the induction of these inflammatory responses through their time-dependent exposure on the surface of germinating conidia. Dectin-1, an innate immune receptor that recognizes fungal beta-glucans, is recruited in vivo to alveolar macrophage phagosomes that have internalized conidia with exposed beta-glucans. Antibody-mediated blockade of Dectin-1 partially inhibits TNF-alpha/MIP-2 induction by metabolically active conidia. TLR-2- and MyD88-mediated signals provide an additive contribution to macrophage activation by germinating conidia. Selective responsiveness to germinating conidia provides the innate immune system with a mechanism to restrict inflammatory responses to metabolically active, potentially invasive fungal spores.     

黄曲霉菌原生质体的制备和再生技术优化

黄曲霉菌的遗传转化是研究黄曲霉菌致病相关功能基因的前提和基础,而原生质体是研究和建立真菌遗传转化系统的重要工具。本文分别以黄曲霉孢子和菌丝为材料,研究不同条件下黄曲霉原生质体的形成和再生,结果表明,黄曲霉孢子在酶液浓度为纤维素酶∶蜗牛酶∶溶壁酶=1.5%∶1.5%∶1.5%,30℃酶解3 h,原生质体制备率高达97.3%,再生率达89.2%;黄曲霉菌丝在菌龄为42 h,酶液浓度为纤维素酶∶蜗牛酶∶溶壁酶=1.5%∶1.5%∶1.5%,30℃酶解1 h,可获得最高原生质体产量为2.0×10^6个/m L,再生培养基中以1 mol/L蔗糖作为渗透压稳定剂时,原生质体再生率达5.5%。故本实验条件下,黄曲霉孢子原生质体的形成和再生优于菌丝。     

Gliotoxin effects on fungal growth: mechanisms and exploitation

Although initially investigated for its antifungal properties, little is actually known about the effect of gliotoxin on Aspergillus fumigatus and other fungi. We have observed that exposure of A. fumigatus to exogenous gliotoxin (14 μg/ml), under gliotoxin-limited growth conditions, results in significant alteration of the expression of 27 proteins (up- and down-regulated >1.9-fold; p<0.05) including de novo expression of Cu, Zn superoxide dismutase, up-regulated allergen Asp f3 expression and down-regulated catalase and a peroxiredoxin levels. Significantly elevated glutathione GSH levels (p<0.05), along with concomitant resistance to diamide, were evident in A. fumigatus ΔgliT, lacking gliotoxin oxidoreductase, a gliotoxin self-protection gene. Saccharomyces cerevisiae deletents (Δsod1 and Δyap1) were hypersensitive to exogenous gliotoxin, while Δgsh1 was resistant. Significant gliotoxin-mediated (5 μg/ml) growth inhibition (p<0.001) of Aspergillus nidulans, Aspergillus terreus, Aspergillus niger, Cochliobolus heterostrophus and Neurospora crassa was also observed. Growth of Aspergillus flavus, Fusarium graminearum and Aspergillus oryzae was significantly inhibited (p<0.001) at gliotoxin (10 μg/ml), indicating differential gliotoxin sensitivity amongst fungi. Re-introduction of gliT into A. fumigatus ΔgliT, at a different locus (ctsD; AFUA_4G07040, an aspartic protease), with selection on gliotoxin, facilitated deletion of ctsD without use of additional antibiotic selection markers. Absence of ctsD expression was accompanied by restoration of gliT expression, and resistance to gliotoxin. Thus, we propose gliT/gliotoxin as a useful selection marker system for fungal transformation. Finally, we suggest incorporation of gliotoxin sensitivity assays into all future fungal functional genomic studies.