Amylase synthesis inAspergillus flavus andAspergillus niger grown on cassava peel

Summary Aspergillus flavus andAspergillus niger produce extracellular amylase into the culture medium when grown on basal medium containing 2% (w/v) soluble starch or cassava peel as the sole carbon source. On soluble tarch the highest amylase activities were 1.6 and 5.2 mg of starch hydrolyzed/min per mg protein forA. flavus andA. niger, respectively. When grown on cassava peel, the highest amylase activity in the culture filtrate ofA. flavus was 170-times higher than that on soluble starch, while that ofA. niger was 16-times higher. The mycelial dry weight for both organisms was not significantly affected by the carbon sources. Maximum enzyme activity was obtained at the growth temperature of 29.0±1°C and pH 7 for both organisms. It is concluded that cassava peel might be a better substrate for the production of amylase byA. flavus andA. niger than commercial soluble starch.     

Discrepancies between MIC and MLC values of amphotericin B against isolates ofAspergillus species

There is little information addressing the phenomena of discrepancy between minimal inhibitory concentrations (MIC) and minimal lethal concentrations (MLC) values of amphotericin B (AMB) to clinical isolates of fungi. This study assessed in vitro activity of AMB against 70 clinical isolates of aspergilli: 30 strains ofAspergillus fumigatus, 20 strains ofAspergillus flavus and 20 strains ofAspergillus niger. Susceptibility tests were accomplished using a macro broth dilution procedure, with special emphasis on ascertainment of MLCs. AMB exhibited low MIC values against all clinical isolates. While we did not identify any AMB resistant isolates among 70Aspergillus spp. studied as judged by MIC levels, analysis of the data demonstrated a clear discrepancy between the MIC and MLC levels of AMB obtained against clinical isolates ofAspergillus spp. The MLC values of AMB were significantly higher than the MIC values with MIC 50 and MIC 90 of 0.29 and 0.5 µg/ml, respectively, at the second reading time, and MLC 50 and MLC 90 of 2.31 and 9.24 µg/ml, respectively (p<0.001). Additionally, minimal lethal concentrations in 36/70 (51%) of aspergillal isolates studied produced drug concentrations above those which can usually be sustained in patient plasma or tissue.     

Exposure to indoor fungi in different working environments: A comparative study

In this study an attempt was made to evaluate the qualitative and quantitative fungal burden (load) in five different working environments of South Assam (India) and the possible risks of indoor fungi to employees and stored products. Fungal concentrations in different working environments were studied using a Burkard personal petriplate sampler. The survey was done in five different working environments for one year. A total of 76 fungal types were recorded in the indoor air of South Assam during the survey period. The maximum fungal concentration (5,437.6 ± 145.3 CFU m⁻³ air) was recorded in the indoor air of medical wards, followed by the paper-processing industry (3,871.7 ± 93.4 CFU m⁻³ air). However the lowest concentration was observed in the indoor air of a bakery (1,796.8 ± 54.4 CFU m⁻³ air). The most dominant fungal genera were Aspergillus (34.2%) followed by Penicillium (17.8%), Geotrichum (7.0%) and the most dominant fungal species were Aspergillus fumigatus (2,650.4 CFU m⁻³ air) followed by Aspergillus flavus (1,388.2 CFU m⁻³ air), Geotrichum candidum (1,280.3 CFU m⁻³ air), Aspergillus niger (783.3 CFU m⁻³ air), and Penicillium aurantiovirens (774.0 CFU m⁻³ air). The fungal species viz., Aspergillus fumigatus, Penicillium aurantiovirens, Aspergillus flavus, Aspergillus niger, Geotrichum candidum, and Penicillium thomii, which were recorded well above threshold levels, may lead to adverse health hazards to indoor workers. Setting occupational exposure limits for indoor fungal spores as reference values is obligatory for prevention and control of adverse effects of indoor fungal exposure.     

Effects of sugar and amino acid supplementation on Aureobasidium pullulans NRRL 58536 antifungal activity against four Aspergillus species

Cultured cell extracts from ten tropical strains of Aureobasidium pullulans were screened for antifungal activity against four pathogenic Aspergillus species (Aspergillus flavus, Aspergillus niger, Aspergillus fumigatus, and Aspergillus terreus) using the well diffusion and conidial germination inhibition assays. The crude cell extract from A. pullulans NRRL 58536 resulted in the greatest fungicidal activity against all four Aspergillus species and so was selected for further investigation into enhancing the production of antifungal activity through optimization of the culture medium, carbon source (sucrose and glucose) and amino acid (phenylalanine, proline, and leucine) supplementation. Sucrose did not support the production of any detectable antifungal activity, while glucose did with the greatest antifungal activity against all four Aspergillus species being produced in cells grown in medium containing 2.5 % (w/v) glucose. With respect to the amino acid supplements, variable trends between the different Aspergillus species and amino acid combinations were observed, with the greatest antifungal activities being obtained when grown with phenylalanine plus leucine supplementation for activity against A. flavus, proline plus leucine for A. terreus, and phenylalanine plus proline and leucine for A. niger and A. fumigatus. Thin layer chromatography, spectrophotometry, high-performance liquid chromatography, ¹H-nuclear magnetic resonance, and MALDI-TOF mass spectrometry analyses were all consistent with the main component of the A. pullulans NRRL 58536 extracts being aureobasidins.     

In vitro determination of phagocytosis and intracellular killing of Aspergillus species by mononuclear phagocytes

We investigated phagocytosis and intracellular killing of clinical and environmental isolates of Aspergillus spp. by human monocyte-derived macrophages (MDMs). Serial pathogens such as Aspergillus fumigatus, Aspergillus flavus and Aspergillus terreus were examined with a microbiological assay. Phagocytosis for resting conidia of Aspergillus spp. was similar for all isolates tested. During 30 min of incubation phagocytosis ranged from 49.9% to 85.5% for clinical isolates and from 40.3% to 87.1% for environmental isolates. MDMs killed A. fumigatus, A. flavus and A. terreus conidia after ingestion for 120 min, as shown by a decrease in colony forming units (cfu) count of intracellular fungi. The killing index for all isolates of Aspergillus spp., ranged from 12.1 ± 1.1% to 90.3 ± 10.4%; isolate-dependent (P < 0.01) differences against the fungicidal action of MDMs were observed. In conclusion, significant differences were noted for killing indices between several strains of Aspergillus spp. whereas phagocytosis was similar for all isolates tested in vitro. No differences were observed within environmental and clinical isolates.     

Comparative efficacy of amphotericin B,clotrimazole and itraconazole againstAspergillus spp.

The susceptibilities of two isolates ofAspergillus flavus, one from a human case of recalcitrant mycotic keratitis, and an environmental isolate ofA. fumigatus, to itraconazole, clotrimazole and amphotericin B were measured. Observations of macroscopic growth and microscopic evaluations of conidia germination both indicated that the two isolates ofA. flavus were markedly more resistant to amphotericin B than to itraconazole and clotrimazole. Itraconazole was more effective than clotrimazole for all isolates. Ourin vitro susceptibility results suggest the use of itraconazole should be a primary consideration in the treatment ofAspergillus keratitis.     

Effects of Conidia of Various Aspergillus Species on Apoptosis of Human Pneumocytes and Bronchial Epithelial Cells

Aspergillus species can cause mycoses in human and animals. Previously, we demonstrated that A. fumigatus conidia from a human isolate inhibited apoptosis in human pneumocytes and bronchial epithelial cells. In the current study, we studied the effects of A. fumigatus conidia non-human origin and A. flavus, A. nidulans, A. niger and A. oryzae conidia on human cells apoptosis. Human pneumocytes or bronchial epithelial cells were simultaneously exposed to apoptotic inductors and aspergilli conidia. The cell cultures were analyzed by flow cytometry, immunoblotting, and examination of nuclear morphology. Similar to A. fumigatus conidia, A. flavus conidia inhibited cellular apoptosis while A. nidulans, A. niger and A. oryzae conidia did not affect apoptosis. We further studied the species specificity of conidia: there were no differences in the inhibition of apoptosis by A. fumigatus conidia from either human or bird isolates. In order to determine whether the inhibition of apoptosis by conidia is limited to certain strains, the effect on human cell apoptosis of different A. fumigatus human clinical isolates and A. fumigatus of environmental origin was evaluated. All A. fumigatus isolates inhibited apoptosis; an anti-apoptotic factor was released by conidia. For TNF-induced apoptosis, the anti-apoptotic effect of conidia of all isolates was found to be associated with a reduction of caspase-3 in human cells. The results suggest that suppression of apoptosis may play a role in reducing the efficacy of host defense mechanisms during infection with Aspergillus species. F. Féménia and D. Huet made an equal contribution to this work.     

Bio-activity of fungal culture filtrates against root-knot nematode egg hatch and juvenile motility and their effects on growth of mung bean (Vigna radiata L. Wilczek) infected with the root-knot nematode,Meloidogyne incognita

Culture filtrates of selected soil fungi, namely Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Fusarium oxysporum, Penicillium vermiculatum and Rhizopus nigricans exhibited variable response to egg hatching and mortality of the root-knot nematode, Meloidogyne incognita. Higher concentrations of the culture filtrates of all the fungi inhibited egg hatching and proved to be toxic to the juveniles of M. incognita. In addition, development of the gall and multiplication of M. incognita were also found adversely affected in varying degrees on all the plants of Vigna radiata treated with the filtrates. The culture filtrate of A. niger showed highest toxicity to the nematode than those of any other fungus tested. Soil drench application of the culture filtrates gave better seedling growth and least nematode multiplication in comparison to seed soaking treatment.     

Adherence of Candida albicans to buccal epithelial cells of neonates

Twelve commercially-prepared potting soils were screened for the presense of pathogenic Aspergillus species. Pathogenic Aspergillus species were isolated from 67% of the soils. A. fumigatus was isolated from 42% and A. flavus and A. niger from 33%.     

Air-borne fungi in the air of Barcelona (Spain). III. The genus Aspergillus link

During a survey on the presence of species of the genusAspergillus in the air of the city of Barcelona (Spain), the following species were identified:Aspergillus flavus Link,A. niger van Tieghem,A. fumigatus Fresenius,A. clavatus Desmazières,A. terreus Thorn,A. chevalieri (Mang.) Thomet Church,A. niveus Bloch, emend. Thomet Church,A. ochraceus Wilhelm,A. versicolor (Vuillemin) Tiraboschi, andA. amstelodami (Mang.) Churchet Thom.     

Isolation of fluconazole-tolerant micromycetes onto different cultivation media

On studying the dependence of quality and/or quantity of environmental fluconazole-tolerant micromycetes on culture mediumin vitro, the highest number of isolates was obtained in Sabouraud agar (SA) without fluconazole (59 CFU/mg of soil sample at 25°C and 31 CFU/mg at 37°C). In the case of SA, fluconazole inhibited the growth of micromycetes to 83%. Significant differences were found between the average concentration of CFU/mg for six cultivation media without and with fluconazole (70 mg/L) at 25 and 37°C. The genusAspergillus (A. flavus, A. fumigatus, aspergilli fromA. glaucus andA. niger group,A. nidulans, A. versicolor, A. ustus), Scopulariopsis brevicaulis andPenicillium sp. were the most common isolates. Sabouraud agar combined with malt extract agar, dichloran-18% glycerol agar and potato-dextrose agar were the most suitable for isolation of fluconazole-tolerant fungi.     

A Review Molecular Typing Methods for <Emphasis Type="Italic">Aspergillus flavus</Emphasis> Isolates

Aspergillus flavus is the second most important Aspergillus species causing human infections. The importance of this fungus increases in regions with a dry and hot climate. Small phylogenetic studies in Aspergillus flavus indicate that the morphological species contains several genetically isolated species. Different genotyping methods have been developed and employed in order to better understand the genetic and epidemiological relationships between environmental and clinical isolates. Understanding pathogen distribution and relatedness is essential for determining the epidemiology of nosocomial infections and aiding in the design of rational pathogen control methods. Typing techniques can also give us a deeper understanding of the colonization pattern in patients. Most of these studies focused on Aspergillus fumigatus because it is medically the most isolated species. To date, there has not been any publication exclusively reviewing the molecular typing techniques for Aspergillus flavus in the literature. This article reviews all these different available methods for this organism.     

Aflatoxin-producing Aspergillus species from saffron field soils in the South Khorasan province of Iran

The aim of the present study was to isolate and identify Aspergillus species associated with saffron plants in the city of Birjand (South Khorasan Province, Iran) as well as to assess their aflatoxin B1 production. Sampling was performed during 2013–2014 crop season. Aspergillus species were isolated and purified using general and specific culture media. Growth rates and macroscopic and microscopic characteristics of the isolates were determined using yeast extract, Czapek yeast extract, malt extract and creatine sucrose agar media at 25 and 37 °C. DNA was extracted by the modified CTAB method and beta-tubulin, calmodulin and internal transcribed spacer genes were amplified and sequenced. Phylogenetic position of the isolates was determined against other Aspergillus species. Thin layer chromatography was used to investigate the production of aflatoxin B1 by Aspergillus isolates. Based on the morphological characteristics, shape and colour of the colonies, and sequencing results, the isolates belonged to Aspergillus terreus, A. flavus, A. flavipes and A. niger species. Only A. flavus isolates were aflatoxin B1 producers. We concluded that the soil of the studied saffron fields contained several species of Aspergillus, with A. flavus significantly affecting crop production through contamination of the crop by aflatoxin.     

Diverse and bioactive endophytic Aspergilli inhabit Cupressaceae plant family

Aspergilli are filamentous, cosmopolitan and ubiquitous fungi which have significant impact on human, animal and plant welfare worldwide. Due to their extraordinary metabolic diversity, Aspergillus species are used in biotechnology for the production of a vast array of biomolecules. However, little is known about Aspergillus species that are able to adapt an endophytic lifestyle in Cupressaceae plant family and are capable of producing cytotoxic, antifungal and antibacterial metabolites. In this work, we report a possible ecological niche for pathogenic fungi such as Aspergillus fumigatus and Aspergillus flavus. Indeed, our findings indicate that A. fumigatus, A. flavus, Aspergillus niger var. niger and A. niger var. awamori adapt an endophytic lifestyle inside the Cupressaceous plants including Cupressus arizonica, Cupressus sempervirens var. fastigiata, Cupressus semipervirens var. cereiformis, and Thuja orientalis. In addition, we found that extracts of endophytic Aspergilli showed significant growth inhibition and cytotoxicity against the model fungus Pyricularia oryzae and bacteria such as Bacillus sp., Erwinia amylovora and Pseudomonas syringae. These endophytic Aspergilli also showed in vitro antifungal effects on the cypress fungal phytopathogens including Diplodia seriata, Phaeobotryon cupressi and Spencermartinsia viticola. In conclusion, our findings clearly support the endophytic association of Aspergilli with Cupressaceae plants and their possible role in protection of host plants against biotic stresses. Observed bioactivities of such endophytic Aspergilli may represent a significant potential for bioindustry and biocontrol applications.     

Behaviour of Aspergillus flavus in presence of Aspergillus niger during biosynthesis of aflatoxin B1

Aspergillus niger or Aspergillus tamarii when grown as mixed cultures with toxigenic A. flavus inhibits biosynthesis of aflatoxin by A. flavus, owing primarily to its ability to produce inhibitors of aflatoxin biosynthesis and to their ability to degrade aflatoxin. Gluconic acid partly prevents aflatoxin production. The other factors such as changes in pH of the medium and the effect on the growth of A. flavus have no role in imparting capabilities to these cultures to inhibit aflatoxin production by A. flavus.     

Biodegradation optimization and metabolite elucidation of Reactive Red 120 by four different <Emphasis Type="Italic">Aspergillus</Emphasis> species isolated from soil contaminated with industrial effluent

Azo dyes are recalcitrant owing to their xenobiotic nature and exhibit high resistance to degradation processes. In the present study, different Aspergillus species (A. flavus, A. fumigatus, A. niger, and A. terreus) isolated from soil samples contaminated with industrial effluent, collected from Jeddah, Saudi Arabia, were analyzed for azo dye, Reactive Red 120 (RR120) biodegradation. The physicochemical parameters such as carbon (sucrose) and nitrogen (ammonium sulfate) sources, pH, and temperature affecting the biodegradation of RR120 were optimized using central composite design–response surface methodology (CCD-RSM). The maximum RR120 degradation was found to be 84% (predicted) at the optimum level of sucrose (11.73 g/L), ammonium sulfate (1.26 g/L), pH (5.71), and temperature (28.26 °C). Further, the validation results confirmed that the predicted values are in good agreement with the experimental results for RR120 degradation by A. flavus (86%), A. fumigatus (84%), A. niger (85%), and A. terreus (86%). The metabolic product of RR120 after biodegradation by different Aspergillus species was identified as sodium 2-aminobenzenesulfonate. The present study suggests that Aspergillus species are good candidates for azo dye-loaded effluent treatment.     

Incidence of allergenically significant fungal aerosol in a rural bakery of West Bengal,India

The frequency of fungal spores in the air of three different sections of a rural bakery was analyzed using a Burkard personal slide sampler and Andersen two stage viable sampler. In average concentration of spores (No./m³) was 228–26770/m³ and concentration of viable colony forming units (CFU/m³) was 65-2061 CFU/m³. Dominant fungus species both culturable and nonculturable, were species of Aspergillus and Penicillium, Cladosporiumsp., Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Cladosporium cladosporioides, Penicillium citrinum and Alternaria alternata. Seasonal variations in the spore concentrations were clearly observed in case of some fungi. Total culturable mould concentration of different bakery sections sometimes exceeded the acceptable limit for a healthy indoor environment. Antigenic extracts prepared from some dominant culturable fungi showed high level of allergenicity in skin prick tests indicating that they could be responsible for allergic respiratory dysfunction of bakery workers.This revised version was published online in October 2005 with corrections to the Cover Date.     

Saccharification of cassava peels waste for microbial protein enrichment

Cassava waste peels may constitute up to 55% of the original tuber. These waste peels were found to contain 41.8% carbohydrate, 1.1% protein, 12.5% ether extract and 4.9%, 4.9% total ash. and 20.8% crude fibre. Studies were conducted to formulate a fermentation medium to convert the waste peels to reducing sugars and to enrich the peels with microbial protein. Amylase producing microorganisms were isolated from rotten cassava tuber discs buried in the soil at different locations. The microorganisms isolated were Aspergillus fumigatus, A. flavus, A. niger, and a Pseudomonas sp. and A. niger; the level of reducing sugar was 20.5 mg/ml. The lowest was by B. subtilis an isolate from fermenting locust bean. Generally the levels of saccharification were higher when the waste media were supplemented with different nitrogen sourses. The crude protein yield in the cassava peel waste media by different microorganisms varied from 5.6% to 17.5%. The highest protein yield was in the waste medium fermented by A. fumigatus followed by A. niger, B. subtilis, Pseudomonas sp. in decreasing order.     

Fungi that cause mouldiness of processed sheet rubber in Western Nigeria

The fung isolated from mouldy processed sheet rubber in Western Nigeria wereAspergillus fumigatus Fres,Aspergillus flavus Link andAspergillus aculeatus Iisuka. When inoculated on sterilized, uninfected sheet rubber, bothA. fumigatus andA. flavus produced symptoms which were similar to those originally observed on mouldy processed sheet from the rubber estate.

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Zusammenfassung Die folgenden Pilze sind vom verschimmelten, bearbeiteten Rubber isoliert worden:Aspergillus fumigatus Fres,A. flavus Link andA. aculeatus Iisuka. WennA. fumigatus undA. flavus an sterilisiertem, nicht infiziertem Rubber verimpft worden sind, haben sie identische Läsionen am sterilen Rubber verursacht.

     

FT-IR Spectroscopy for Rapid Differentiation of Aspergillus flavus, Aspergillus fumigatus, Aspergillus parasiticus and Characterization of Aflatoxigenic Isolates Collected from Agricultural Environments

In agricultural areas, Aspergillus flavus, Aspergillus fumigatus and Aspergillus parasiticus are commonly identified in various feedstuffs and bioaerosols originated from feed handling. Some isolates belonging to these fungal species could produce mycotoxins and constitute a risk factor for human and animal health. In this study, Fourier-transform infrared spectroscopy was used for a rapid detection and characterization of 99 isolates collected from agricultural areas. The results showed a first cluster corresponding to strains previously attributed to the A. fumigatus group according to current taxonomic concepts, and a second cluster divided in 2 groups around reference strains of A. flavus and A. parasiticus species. The toxigenic capacity of isolates was evaluated by high performance liquid chromatography coupled to mass spectrometry. In the A. flavus group, only 6 strains of A. parasiticus and 4 strains of A. flavus were able to produce aflatoxins on culture media. FT-IR spectroscopy, respectively, allowed the differentiation of non-toxigenic and toxigenic A. flavus and A. parasiticus isolates at 75 and 100%. Discrimination between toxigenic and non-toxigenic A. fumigatus was not possible because all of the isolates produced at least one mycotoxin.