Leishmania donovani: Metabolite mapping of promastigotes using proton nuclear magnetic resonance spectroscopy

Proton nuclear magnetic resonance spectroscopy was used for studying the intracellular metabolite profile of promastigotes of Leishmania donovani. The major intracellular metabolites observed in the promastigotes were acetate, alanine, succinate, glycine, -glycerophosphorylcholine, acetoacetate, arginine and ethanol. A comparative study of the intracellular metabolite profile of promastigotes of different strains of L. donovani showed that, all the major intracellular metabolites were present in promastigotes of different strains. A quantitative estimation of metabolites showed a strain specific (Finger print) metabolite profile which can be used for strain/species identification/differentiation.     

Mites and fungi in heavily infested stores in the Czech Republic

Toxigenic and allergen-producing fungi represent a serious hazard to human food and animal feed safety. Ninety-four fungal species were isolated from mite-infested samples of seeds taken from Czech seed stores. Fungi were isolated from the surface of four kinds of seeds (wheat, poppy, lettuce, and mustard) and from the gut and external surface of five species of mites (i.e., Acarus siro L., 1758, Caloglyphus rhizoglyphoides (Zachvatkin, 1973), Lepidoglyphus destructor (Schrank, 1781), Tyrophagus putrescentnae (Schrank, 1781) and Cheyletus malaccensis Oudemans 1903) separately. Multivariate analysis of fungi complex composition showed that the frequency of fungal was species significantly influenced by the kind of seed. Fungal frequencies differed between mites gut and exoskeleton surface and between the surfaces of mites and seeds. Three groups of fungal species were recognized: 1) mite surface-associated fungi: Penicillium brevicompactum, Alternaria alternata, and Aspergillus versicolor; 2) mite surface- and seed-associated fungi: Aspergillus niger, Penicillium crustosum, Penicillium aurantiogriseum, Penicillium chrysogenum, and Aspergillus flavus; and 3) seed-associated fungi: Cladosporium herbarum, Mucor dimorphosporus f. dimorphosporus, Botrytis cinerea, Penicillium griseofulvum, and Eurotium repens. Mite-carried species of microfungi are known to produce serious mycotoxins (e.g., aflatoxin B1, cyclopiazonic acid, sterigmatocystin, ochratoxin A, and nephrotoxic glycopeptides) as well as allergen producers (e.g., A. alternata and P. brevicompactum). Storage mites may play an important role in the spread of some medically hazardous micromycetes. In addition, these mite-fungi associations may heighten the risk of occurrence of mycotoxins in food and feed stuffs and cause mixed contamination by fungal and mite allergens.     

The system approach to the identification of aflatoxigenic fungi in foodstuffs and feedstuffs

Aspergillus flavus, A parasiticus, A nomius, A tamarii andA pseudotamarii are important microorganisms capable of producing aflatoxins and further mycotoxins. Aflatoxigenic Aspergillus species are morphologically similar species belonging to the Aspergillus section Flavi. The aflatoxigenic fungal strains were isolated from foods (cereals, pulses, oilseeds, dried fruit, spices), soil, air and water. Mycological analyses are based on valid standards and recommendations of the International Commission for Food Mycology (ICFM). The identification of isolated aflatoxigenic fungi in foodstuffs and feedstuffs can be proved by using classical mycological cultivation methods, diagnostic nutrient media, chemotaxonomy and molecular biological methods (PCR). The system approach to the identification of aflatoxigenic fungi combines these four methods. Thirty strains of the aflatoxigenic fungi were tested.     

Primer R108 performs best in the RAPD strain typing of threeAspergillus species frequently isolated from patients

We evaluated the suitability of various primers for the RAPD (random amplified polymorphic DNA) accurate species identification and strain typing of Aspergillus clinical isolates. Five primers described previously were tested for their discriminatory power in three Aspergillus species (A. fumigatus, A. niger agg. and A. flavus - 23 clinical isolates and 2 reference strains). Clustering of RAPD fingerprints corresponded well with the identification based on morphological features. All isolates were resolved as different strains using the primer R108 and the RAPD protocol optimized for a Robocycler thermal cycler. RAPD with the primer R108 thus can be considered to be a valuable, simple and powerful tool for identification and strain delineation of Aspergillus spp.     

Production of fungal biomass protein using microfungi from winery wastewater treatment

This study was carried out to investigate the production of fungal biomass protein (FBP) in treatment of winery wastewater using microfungi. Three fungal strains, Trichoderma viride WEBL0702, Aspergillus niger WEBL0901 and Aspergillus oryzae WEBL0401, were selected in terms of microbial capability for FBP production and COD reduction. T. viride appeared to be the best strain for FBP production due to high productivity and less nitrogen requirement. More than 5 g/L of fungal biomass was produced in shake fermentation using T. viride without nitrogen addition, and by A. oryzae and A. niger with addition of 0.5-1.0 g/L (NH4)2SO4. The FBP production process corresponded to 84-90% COD reduction of winery wastewater. Fungal biomass contained approximately 36% protein produced by two Aspergillus strains, while biomass produced by T. viride consisted of 19.8% protein. Kinetic study indicated that maximum fungal cell growth could be achieved in 24h for T. viride and 48 h for A. oryzae and A. niger. Current results indicated that it could be feasible to develop a biotechnological treatment process integrated with FBP production from the winery waste streams.     

Raman microspectroscopy as an identification tool within the phylogenetically homogeneous 'Bacillus subtilis' group

Vibrational methods have multiple advantages compared to more classic, chemotaxonomic and even molecular microbial tools for the identification of bacteria. Nevertheless, their definite breakthrough in diagnostic microbiology laboratories is determined by their identification potential. This paper reports on the profound evaluation of Raman spectroscopy to identify closely related species by means of 68 Bacillus strains that are assigned or closely related to the phylogenetically homogeneous ‘Bacillus subtilis’-group (sensu stricto). These strains were chosen to represent biological variation within the selected species and to create a realistic view on the possibilities of this technique

The evaluation resulted in 49/54 correct identifications at the species level for intern and 15/19 for extern testing. The correct identification of strains, which were not represented in the training set, supports the potential as an identification tool within the ‘B. subtilis group’. Considering the vague borderline between the species studied, Raman spectroscopy can be regarded here as a promising application for identifications at the species level.     

Genetic characterization of Brazilian strains of Aspergillus flavus using DNA markers

The Aspergillus genus belongs to a filamentous fungal group characterized by wide dispersion in the environment. Some species are associated with diseases, especially in immunocompromised patients, while others are of economical importance due to aflatoxin production or biotechnological applications. Its species identification is nowadays performed by traditional techniques combined with molecular markers, resulting in a higher efficiency of isolate characterization. In the present study, internal transcribed spacer, inter-simple sequence repeats (ISSR), and random amplified polymorphic DNA (RAPD) molecular markers were used, with the aim of genetically characterizing strains of Aspergillus flavus and strains of other species of the A. flavus group. High genetic diversity was revealed by RAPD and by ISSR, in which the use of the (GACA)4 primer yielded a higher diversity than with the (GTG)5 primer, although the latter showed a characteristic banding profile for each species. These data were used to create a similarity matrix for the construction of dendrograms by means of the UPGMA method. The ISSR and RAPD profiles showed that among the strains previously identificated as A. flavus, one should be A. oryzae, one A. parasiticus and two A. tamarii. On the other hand, a strain previously identified as A. parasiticus should be A. flavus. All these strains were retested by traditional methods and their new species identification was confirmed. These results strongly support the need for using molecular markers as an auxiliary tool in differentiating fungal species and strains.     

Discrimination of enterobacterial repetitive intergenic consensus PCR types of Campylobacter coli and Campylobacter jejuni by Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy (FT-IR) has been used together with pattern recognition methodology to study isolates belonging to the species Campylobacter coli and Campylobacter jejuni and to compare FT-IR typing schemes with established genomic profiles based on enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Seventeen isolates were cultivated under standardized conditions for 2, 3, and 4 days to study variability and improve reproducibility. ERIC-PCR profiles and FT-IR spectra were obtained from strains belonging to the species Campylobacter coli and C. jejuni, normalized, and explored by hierarchical clustering and stepwise discriminant analysis. Strains could be differentiated by using mainly the first-derivative FT-IR spectral range, 1,200 to 900 cm(-1) (described as the carbohydrate region). The reproducibility index varied depending on the ages of the cultures and on the spectral ranges investigated. Classification obtained by FT-IR spectroscopy provided valuable taxonomic information and was mostly in agreement with data from the genotypic method, ERIC-PCR. The classification functions obtained from the discriminant analysis allowed the identification of 98.72% of isolates from the validation set. FT-IR can serve as a valuable tool in the classification, identification, and typing of thermophilic Campylobacter isolates, and a number of types can be differentiated by means of FT-IR spectroscopy.     

FT-IR spectroscopy for identification of closely related lactobacilli

Fourier Transform Infrared (FT-IR) spectroscopy was used to analyse 56 strains from four closely related species of Lactobacillus, L. sakei, L. plantarum, L. curvatus and L. paracasei. Hierarchical Cluster Analysis (HCA) was used to study the clusters in the data, but in the dendrogram, the spectra were not differentiated into four separate clusters corresponding to species. When the data were analysed with Partial Least Squares Regression (PLSR), the strains were differentiated into four clusters according to species. It was also possible to recognise strains that were incorrectly identified by conventional methods prior to the FT-IR analysis. PLSR was used to identify strains from three of the species, and the results were compared to two other multivariate methods, Soft Independent Modelling of Class Analogy (SIMCA) and K-Nearest Neighbour (KNN). The three methods gave equally good identification results. The results show that FT-IR spectroscopy in combination with PLSR, or other multivariate methods, is well suited for identification of Lactobacillus at the species level, even in quite large data sets.     

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.     

Fourier-transform infrared microspectroscopy,a novel and rapid tool for identification of yeasts

Fourier-transform infrared (FT-IR) microspectroscopy was used in this study to identify yeasts. Cells were grown to microcolonies of 70 to 250 micro m in diameter and transferred from the agar plate by replica stamping to an IR-transparent ZnSe carrier. IR spectra of the replicas on the carrier were recorded using an IR microscope coupled to an IR spectrometer, and identification was performed by comparison to reference spectra. The method was tested by using small model libraries comprising reference spectra of 45 strains from 9 genera and 13 species, recorded with both FT-IR microspectroscopy and FT-IR macrospectroscopy. The results show that identification by FT-IR microspectroscopy is equivalent to that achieved by FT-IR macrospectroscopy but the time-consuming isolation of the organisms prior to identification is not necessary. Therefore, this method also provides a rapid tool to analyze mixed populations. Furthermore, identification of 21 Debaryomyces hansenii and 9 Saccharomyces cerevisiae strains resulted in 92% correct identification at the strain level for S. cerevisiae and 91% for D. hansenii, which demonstrates that the resolution power of FT-IR microspectroscopy may also be used for yeast typing at the strain level.     

Tremorgenic mycotoxins from Aspergillus fumigatus as a possible occupational health problem in sawmills

Wood-trimmers' disease, generally called extrinsic allergic alveolitis, which affects workers in sawmills, is thought to be caused by fungal diaspores. The importance of Aspergillus fumigatus on the surface of wood dried in kilns is accentuated by its ability to produce tremorgenic mycotoxins. Eight strains of A. fumigatus from five different sawmills were isolated and cultivated on liquid media, and one of the strains was also cultivated on wood blocks. Extracts were prepared, and the tremorgenic reactions were induced by oral administration of extracts to rats. Extracts of the strain grown in liquid medium and on wood blocks induced very strong tremorgenic reactions when administered orally to rats. Four other strains induced mild tremorgenic reactions. High-performance liquid chromatography analysis revealed two tremorgenic mycotoxins, verruculogen and fumitremorgen C, in the five toxic strains. One nontoxic strain produced detectable levels of verruculogen. These results, coupled with the known resemblance of the acutely toxic phase of wood-trimmers' disease to the symptoms produced by these tremorgens, imply that wood-trimmers' disease and similar occupational diseases are, at least in part, mycotoxicoses.     

Tremorgenic mycotoxins from Aspergillus fumigatus as a possible occupational health problem in sawmills.

Wood-trimmers' disease, generally called extrinsic allergic alveolitis, which affects workers in sawmills, is thought to be caused by fungal diaspores. The importance of Aspergillus fumigatus on the surface of wood dried in kilns is accentuated by its ability to produce tremorgenic mycotoxins. Eight strains of A. fumigatus from five different sawmills were isolated and cultivated on liquid media, and one of the strains was also cultivated on wood blocks. Extracts were prepared, and the tremorgenic reactions were induced by oral administration of extracts to rats. Extracts of the strain grown in liquid medium and on wood blocks induced very strong tremorgenic reactions when administered orally to rats. Four other strains induced mild tremorgenic reactions. High-performance liquid chromatography analysis revealed two tremorgenic mycotoxins, verruculogen and fumitremorgen C, in the five toxic strains. One nontoxic strain produced detectable levels of verruculogen. These results, coupled with the known resemblance of the acutely toxic phase of wood-trimmers' disease to the symptoms produced by these tremorgens, imply that wood-trimmers' disease and similar occupational diseases are, at least in part, mycotoxicoses.     

Aspergillus niger contains the cryptic phylogenetic species A. awamori

Aspergillus section Nigri is an important group of species for food and medical mycology, and biotechnology. The Aspergillus niger 'aggregate' represents its most complicated taxonomic subgroup containing eight morphologically indistinguishable taxa: A. niger, Aspergillus tubingensis, Aspergillus acidus, Aspergillus brasiliensis, Aspergillus costaricaensis, Aspergillus lacticoffeatus, Aspergillus piperis, and Aspergillus vadensis. Aspergillus awamori, first described by Nakazawa, has been compared taxonomically with other black aspergilli and recently it has been treated as a synonym of A. niger. Phylogenetic analyses of sequences generated from portions of three genes coding for the proteins β-tubulin (benA), calmodulin (CaM), and the translation elongation factor-1 alpha (TEF-1α) of a population of A. niger strains isolated from grapes in Europe revealed the presence of a cryptic phylogenetic species within this population, A. awamori. Morphological, physiological, ecological and chemical data overlap occurred between A. niger and the cryptic A. awamori, however the splitting of these two species was also supported by AFLP analysis of the full genome. Isolates in both phylospecies can produce the mycotoxins ochratoxin A and fumonisin B?, and they also share the production of pyranonigrin A, tensidol B, funalenone, malformins, and naphtho-γ-pyrones. In addition, sequence analysis of four putative A. awamori strains from Japan, used in the koji industrial fermentation, revealed that none of these strains belong to the A. awamori phylospecies.     

Rapid species and strain differentiation of non-tubercoulous mycobacteria by Fourier-Transform Infrared microspectroscopy

Rapid identification of non-tuberculous mycobacteria (NTM) species is important in clinical laboratories to stipulate the appropriate therapy and to offer a comprehensive infection control. We applied Fourier-Transform Infrared microspectroscopy to evaluate, whether the most frequent species of NTM can be rapidly and uniformly identified by this method using microcolonies of NTM growing on solid nutrient agar plates. To establish a standardized protocol, the heterogeneity of cell growth within the microcolonies and the reproducibility of measuring the IR spectra from whole mycobacterial microcolonies were first studied. Hierarchical cluster analysis applied to spectra obtained by linear mapping across microcolony imprints from fast- and slow-growing NTM revealed only little spectral variance between the various microcolony zones. In parallel, when repetitive measurements were performed on independently grown whole single microcolonies with diameters of 80 and 140 mum, excellent reproducibility could be achieved, verifying that mycobacterial microcolonies are well suited for FT-IR-based identification. Twenty-eight different and well-defined strains, comprising the most frequent species of NTM isolated in clinical laboratories, were used to create a classification system based on FT-IR spectra from single microcolonies. Hierarchical cluster analysis allowed the assignment of all isolates measured in replicates to their correct species-specific clusters. Additionally, a clear separation of all strains into strain-specific sub-clusters was observed. These results demonstrate the potential of FT-IR microspectroscopy to rapidly differentiate NTM at the species and strain level. The data so far obtained suggest that an extended spectral database, containing more NTM strains and covering a broader biological variance, may provide a practical solution to rapidly identify unknown NTM isolates in routine clinical-microbiological laboratories with the additional possibility to type these microorganisms at the sub-species level.     

Fourier transform infrared spectroscopy as a new tool for characterization of mollicutes

Fourier transform infrared (FT-IR) spectroscopy is a convenient physico-chemical technique to investigate various cell materials. Bacteria of class Mollicutes, identified by conventional methods, as Mycoplasma, Acholeplasma and Ureaplasma genera were characterized using this method. A data set of 74 independent experiments corresponding to fourteen reference strains of Mollicutes was examined by FT-IR spectroscopy to attempt a spectral characterization based on the biomolecular structures. In addition to the separation of Mollicutes within the lipidic region into five main clusters corresponding to the three phylogenetic groups tested, FT-IR spectroscopy allowed a fine discrimination between strains belonging to the same species by using selective spectral windows, particularly in the 1200-900 cm(-1) saccharide range. The results obtained by FT-IR were in good agreement with both taxonomic and phylogenetic classifications of tested strains. Thus, this technique appears to be a useful tool and an accurate mean for a rapid characterization of Mollicutes observed in humans.     

Rapid strain classification and taxa delimitation within the edible mushroom genus Pleurotus through the use of diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy

Fourier transform infrared (FT-IR) spectroscopy has been successfully applied for the identification of bacteria and yeasts, but only to a limited extent for discriminating specific groups of filamentous fungi. In the frame of this study, 73 strains - from different associated hosts/substrates and geographic regions - representing 16 taxa of the edible mushroom genus Pleurotus (Basidiomycota, Agaricales) were examined through the use of diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy. A binary matrix, elaborated on the basis of presence/absence of specific absorbance peaks combined with cluster analysis, demonstrated that the spectral region 1800-600 cm(-1) permitted clear delimitation of individual strains into Pleurotus species. In addition, closely related species (e.g., Pleurotus ostreatus and Pleurotus pulmonarius) or taxa of the subgenus Coremiopleurotus demonstrated high similarity in their absorbance patterns, whereas genetically distinct entities such as Pleurotus dryinus, Pleurotus djamor, and Pleurotus eryngii provided spectra with noteworthy differences. When specific regions (1800-1700, 1360-1285, 1125-1068, and 950-650 cm(-1)) were evaluated in respect to the absorbance values demonstrated by individual strains, it was evidenced that this methodology could be eventually exploited for the identification of unknown Pleurotus specimens with a stepwise process and with the aid of a dichotomous key developed for this purpose. Moreover, it was shown that the nature of original fungal material examined (mycelium, basidiomata, and basidiospores) had an effect on the outcome of such analyses, and so did the use of different mycelium growth substrates. In conclusion, application of FT-IR spectroscopy provided a fast, reliable, and cost-efficient solution for the classification of pure cultures from closely related mushroom species.     

具有细胞毒活性红树林真菌094811的鉴定

为鉴定合成细胞毒活性及多羟基甾醇脂肪酸酯新结构代谢产物的红树林真菌094811,采用形态观察、BIOLOG生理特征鉴定及核糖体18S rDNA测序对菌株094811进行了鉴定。菌株094811与模式菌株黑曲霉As3.40、泡盛曲霉As3.44形态特征极为相似,难以区分。扫描电镜下分别观察且比较菌株094811、模式菌株黑曲霉As3.40、泡盛曲霉As3.44分生孢子的形态,判断菌株094811为泡盛曲霉;根据BIOLOG微生物鉴定系统给出的相似菌种名称及综合形态和生理生化特征,推断菌株094811为泡盛曲霉;通过18S rDNA部分序列测序、GenBank比对,及系统演化分析进一步确认菌株094811为泡盛曲霉。菌株094811的形态、生理特征及其核糖体18S rDNA的序列特征给出了一致的结论。首次鉴定发现1株具有合成细胞毒活性及新的多羟基甾醇脂肪酸酯结构化合物的能力的泡盛曲霉。     

Comparative study of Aspergillus mycotoxin production on enriched media and construction material

Isolates of Aspergillus flavus and Aspergillus fumigatus from indoor air were compared with a known mycotoxin producer for their capacity to produce mycotoxins on a variety of enrichment media and with growth on indoor substrates such as ceiling tile and wall board. In enrichment media, four of seven isolates of A. flavus produced at least one aflatoxin and both isolates of A. fumigatus produced mycotoxins. The spectrum of mycotoxins and their concentrations varied with the strain and medium. When the mycotoxin-positive strains were grown to a dense concentration on indoor construction and finishing materials such as ceiling tile and wall boards, mycotoxins were not detected in extracts of the materials. Colonization of indoor surfaces by mycotoxin-producing strains of A. flavus and A. fumigatus may not necessarily expose inhabitants to mycotoxins or result in production of mycotoxins. Received 09 February 1999/ Accepted in revised form 28 June 1999