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1.
J. appl. oral sci ; 28: e20190578, 2020. tab, graf
Article in English | LILACS, BBO | ID: biblio-1101256

ABSTRACT

Abstract Objective This study sought to analyze the gene expression of Candida albicans in sound root surface and root caries lesions, exploring its role in root caries pathogenesis. Methodology The differential gene expression of C. albicans and the specific genes related to cariogenic traits were studied in association with samples of biofilm collected from exposed sound root surface (SRS, n=10) and from biofilm and carious dentin of active root carious lesions (RC, n=9). The total microbial RNA was extracted, and the cDNA libraries were prepared and sequenced on the Illumina Hi-Seq2500. Unique reads were mapped to 163 oral microbial reference genomes including two chromosomes of C. albicans SC5314 (14,217 genes). The putative presence of C. albicans was estimated (sum of reads/total number of genes≥1) in each sample. Count data were normalized (using the DESeq method package) to analyze differential gene expression (using the DESeq2R package) applying the Benjamini-Hochberg correction (FDR<0.05). Results Two genes (CaO19.610, FDR=0.009; CaO19.2506, FDR=0.018) were up-regulated on SRS, and their functions are related to biofilm formation. Seven genes ( UTP20 , FDR=0.018; ITR1 , FDR=0.036; DHN6 , FDR=0.046; CaO19.7197 , FDR=0.046; CaO19.7838 , FDR=0.046; STT4 , FDR=0.046; GUT1 , FDR=0.046) were up-regulated on RC and their functions are related to metabolic activity, sugar transport, stress tolerance, invasion and pH regulation. The use of alternative carbon sources, including lactate, and the ability to form hypha may be a unique trait of C. albicans influencing biofilm virulence. Conclusions C. albicans is metabolically active in SRS and RC biofilm, with different roles in health and disease.


Subject(s)
Humans , Tooth Root/microbiology , Candida albicans/genetics , DNA, Fungal/genetics , Root Caries/microbiology , Biofilms/growth & development , Candida albicans/isolation & purification , Candida albicans/growth & development , Gene Expression , Gene Expression Regulation, Fungal , Up-Regulation , Sequence Analysis, RNA , Transcriptome , Morphogenesis
2.
Mem. Inst. Oswaldo Cruz ; 115: e200208, 2020. tab, graf
Article in English | SES-SP, LILACS, SES-SP | ID: biblio-1135227

ABSTRACT

Paracoccidioides spp. isolation from environmental samples is rare and hardly reproducible. Molecular techniques have facilitated the fungal detection. However, it can be still difficult. Some strategies to enhance the capacity of DNA detection have been adopted, including the analysis of soil samples belonging to the habitat of animals from which Paracoccidioides spp. have already been isolated, notably armadillo burrows. To date, the detection of Paracoccidioides spp. has not yet been reported from outbreak hotspots. Clusters and outbreaks of acute paracoccidioidomycosis (PCM), usually a more severe clinical form, have currently occurred in urban areas being associated to climate changes, deforestation, and great constructions. These occurrences potentially signalise the fungus' environmental niche, a riddle not yet solved. The authors performed an environmental investigation in a deeply disturbed area, after a highway construction in Rio de Janeiro, Brazil, where a recent outbreak of acute PCM occurred. Specific DNA sequences of Paracoccidioides brasiliensis were detected in shallow soil samples around the highway, reinforcing the association between the road construction and this PCM outbreak.


Subject(s)
Animals , Paracoccidioides/isolation & purification , Paracoccidioidomycosis/microbiology , Armadillos , DNA, Fungal/genetics , Paracoccidioides/growth & development , Paracoccidioides/genetics , Soil Microbiology , Brazil , Base Sequence , Sequence Analysis, DNA , Ecosystem
3.
Rev. Soc. Bras. Med. Trop ; 52: e20180473, 2019. tab
Article in English | LILACS | ID: biblio-990445

ABSTRACT

Abstract INTRODUCTION: Candidiasis is the most frequent opportunistic mycosis in humans and can cause mortality, particularly in immunodeficient patients. One major concern is the increasing number of infections caused by drug-resistant Candidas trains, as these cannot be efficiently treated with standard therapeutics. The most common mechanism of fluconazole resistance in Candida is mutation of ERG11, a gene involved in the biosynthesis of ergosterol, a compound essential for cell integrity and membrane function. METHODS: Based on this knowledge, we investigated polymorphisms in the ERG11 gene of 3 Candida species isolated from immunocompromised and immunocompetent patients. In addition, we correlated the genetic data with the fluconazole susceptibility profile of the Candida isolates. RESULTS: A total of 80 Candida albicans, 8 Candida tropicalis and 6 Candida glabrata isolates were obtained from the saliva of diabetic, kidney transplant and immunocompetent patients. Isolates were considered susceptible to fluconazole if the minimum inhibitory concentration was lower than 8 μg/mL. The amino acid mutations F105L, D116E, K119N, S137L, and K128T were observed in C. albicans isolates, and T224C and G263A were found in C. tropicalis isolates. CONCLUSIONS: Despite the high number of polymorphisms observed, the mutations occurred in regions that are not predicted to interfere with ergosterol synthesis, and therefore are not related to fluconazole resistance.


Subject(s)
Humans , Male , Female , Adult , Aged , Polymorphism, Genetic/drug effects , Candida/drug effects , Candida/genetics , Fluconazole/pharmacology , Kidney Transplantation , Diabetes Mellitus/microbiology , Antifungal Agents/pharmacology , Reference Values , Saliva/microbiology , Candida/isolation & purification , DNA, Fungal/genetics , Microbial Sensitivity Tests , Polymerase Chain Reaction , Drug Resistance, Fungal/genetics , Immunocompetence , Middle Aged , Mutation/drug effects
4.
Mem. Inst. Oswaldo Cruz ; 113(10): e180340, 2018. tab, graf
Article in English | LILACS | ID: biblio-1040583

ABSTRACT

Histoplasmosis is a systemic mycosis infection caused by Histoplasma capsulatum, a heterothallic ascomycete. The sexual reproduction of this fungus is regulated by the mating type (MAT1) locus that contains MAT1-1 and MAT1-2 idiomorphs, which were identified by uniplex polymerase chain reaction (PCR). This study aimed to optimise single-step multiplex PCR for the accurate detection of the distinct mating types of H. capsulatum. Among the 26 isolates tested, 20 had MAT1-1 genotype, while six showed MAT1-2 genotype, in agreement with the uniplex PCR results. These results suggest that multiplex PCR is a fast and specific tool for screening H. capsulatum mating types.


Subject(s)
DNA, Fungal/genetics , DNA Primers/genetics , Histoplasma/genetics , Reproducibility of Results , Sequence Analysis, DNA , Multiplex Polymerase Chain Reaction , Genotype , Histoplasma/classification
5.
Mem. Inst. Oswaldo Cruz ; 112(6): 403-410, June 2017. tab, graf
Article in English | LILACS | ID: biblio-841808

ABSTRACT

BACKGROUND Enterocytozoon bieneusi are the most common microsporidia associated with different clinical manifestations such as diarrhoea, respiratory tract inflammation and acalculous cholecystitis, especially in immunocompromised patients. Infection usually occurs by ingestion of food and water contaminated with spores, but can also result from direct contact with spores through broken skin, eye lesions, and sexual transmission, depending on the microsporidian species. Although there are reports of E. bieneusi found in humans and animals in Brazil, there are no published studies of environmental samples examined by molecular methods. OBJECTIVES The purpose of this study was to verify the presence of E. bieneusi in raw sewage and treated effluent from a combined system by molecular methods. METHODS Raw sewage and treated effluent samples collected from a combined system were analysed for the presence of E. bieneusi using the internal transcriber spacer (ITS) region of E. bieneusi by nested polymerase chain reaction. FINDINGS The analysis revealed E. bieneusi presence and a novel genotype (EbRB) in one raw sewage sample and one treated effluent. MAIN CONCLUSIONS The presence of E. bieneusi in final effluent indicates that the combined system may not remove microsporidian spores. This study is the first report of E. bieneusi in environmental samples in Brazil.


Subject(s)
DNA, Fungal/genetics , Polymerase Chain Reaction , Enterocytozoon/isolation & purification , Enterocytozoon/genetics , Phylogeny , Brazil , Sequence Analysis , Genotype
6.
Article in English | LILACS | ID: biblio-842772

ABSTRACT

ABSTRACT The aim of this study was to assess a collection of yeasts to verify the presence of Candida dubliniensis among strains isolated from the oral mucosa of AIDS pediatric patients which were initially characterized as Candida albicans by the traditional phenotypic method, as well as to evaluate the main phenotypic methods used in the discrimination between the two species and confirm the identification through genotypic techniques, i.e., DNA sequencing. Twenty-nine samples of C. albicans isolated from this population and kept in a fungi collection were evaluated and re-characterized. In order to differentiate the two species, phenotypic tests (Thermotolerance tests, Chromogenic medium, Staib agar, Tobacco agar, Hypertonic medium) were performed and genotypic techniques using DNA sequencing were employed for confirmation of isolated species. Susceptibility and specificity were calculated for each test. No phenotypic test alone was sufficient to provide definitive identification of C. dubliniensis or C. albicans, as opposed to results of molecular tests. After amplification and sequencing of specific regions of the 29 studied strains, 93.1% of the isolates were identified as C. albicans and 6.9% as C. dubliniensis. The Staib agar assay showed a higher susceptibility (96.3%) in comparison with other phenotypic techniques. Therefore, genotypic methods are indispensable for the conclusive identification and differentiation between these species.


Subject(s)
Humans , Child , AIDS-Related Opportunistic Infections/microbiology , Candida/genetics , Candidiasis, Oral/microbiology , DNA, Fungal/genetics , Candida albicans/genetics , Candida albicans/isolation & purification , Candida/classification , Candida/isolation & purification , Genotype , Mouth Mucosa/microbiology , Mycological Typing Techniques , Phenotype , Polymerase Chain Reaction
7.
Braz. j. microbiol ; 47(1): 18-24, Jan.-Mar. 2016. tab, graf
Article in English | LILACS | ID: lil-775112

ABSTRACT

Abstract Phenol and phenolic compounds are environmental pollutants present in industrial wastewaters such as coal tar, oil refineries and petrochemical plants. Phenol removal from industrial effluents is extremely important for the protection of environment. Usually, phenol degradation is carried out by physicochemical methods that are costly and produce hazardous metabolites. Recently, phenol biodegradation has been considered. Yeasts are the most important phenol biodegraders. In this study, the phenol-degrading yeast from environmental samples (soil and wastewater) was isolated from the coking plant of Zarand, Kerman. Then total heterotrophic yeasts were counted. The soil samples had higher rates of yeast degrader, in comparison to wastewater samples. After three passages, four yeasts (K1, K2, K7 and K11) that had the highest growth rate were selected for further study. Also, these yeasts were able to remove phenol measured by Gibbs reagent. The effect of four different concentrations of phenol (50, 125, 200 and 275) mg L−1 was measured and three degradation patterns in these yeasts were observed. The hydrophobicity and emulsification activity were measured in all eleven yeasts. Finally, strong yeasts in phenol degrading yeasts were identified by molecular method using amplification of 18S rRNA gene region. The sequencing results showed that these isolated yeasts belonged to Candida tropicalis strain K1, Pichia guilliermondii strain K2, Meyerozyma guilliermondii strain K7 and C. tropicalis strain K11.


Subject(s)
Industrial Waste , Phenol/metabolism , Waste Water/microbiology , Water Pollutants, Chemical/metabolism , Yeasts/classification , Yeasts/metabolism , Biotransformation , Cluster Analysis , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Iran , Molecular Sequence Data , Phylogeny , /genetics , Sequence Analysis, DNA , Soil Microbiology , Yeasts/genetics , Yeasts/isolation & purification
8.
Braz. j. microbiol ; 47(1): 172-176, Jan.-Mar. 2016. tab
Article in English | LILACS | ID: lil-775126

ABSTRACT

Abstract Candida species, especially C. albicans, are commensals on human mucosal surfaces, but are increasingly becoming one of the important invasive pathogens as seen by a rise in its prevalence in immunocompromised patients and in antibiotic consumption. Thus, an accurate identification of Candida species in patients with pulmonary symptoms can provide important information for effective treatment. A total of 75 clinical isolates of Candida species were obtained from the bronchoalveolar lavage fluid of both immunocompromised and immunocompetent patients with pulmonary symptoms. Candida cultures were identified based on nuclear ribosomal Internal Transcribed Spacer (ITS1-ITS2 rDNA) sequence analysis by polymerase chain reaction–restriction fragment length polymorphisms (PCR-RFLP). Molecular identification indicated that the isolates belonged predominantly to C. albicans (52%), followed by C. tropicalis (24%), C. glabrata (14.7%), C. krusei (5.3%), C. parapsilosis (1.3%), C. kefyr (1.3%) and C. guilliermondii (1.3%). Given the increasing complexity of disease profiles and their management regimens in diverse patients, rapid and accurate identification of Candida species can lead to timely and appropriate antifungal therapy.


Subject(s)
Humans , Bronchoalveolar Lavage Fluid/microbiology , Candida/isolation & purification , Candidiasis/diagnosis , Lung Diseases, Fungal/diagnosis , Candida/classification , Candida/genetics , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Ribosomal Spacer/chemistry , DNA, Ribosomal Spacer/genetics , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Sequence Analysis, DNA , Time Factors
9.
Braz. j. microbiol ; 46(3): 903-910, July-Sept. 2015. tab, ilus
Article in English | LILACS | ID: lil-755814

ABSTRACT

Nineteen fungi and seven yeast strains were isolated from sugarcane bagasse piles from an alcohol plant located at Brazilian Cerrado and identified up to species level on the basis of the gene sequencing of 5.8S-ITS and 26S ribosomal DNA regions. Four species were identified: Kluyveromyces marxianus, Aspergillus niger, Aspergillus sydowii and Aspergillus fumigatus, and the isolates were screened for the production of key enzymes in the saccharification of lignocellulosic material. Among them, three strains were selected as good producers of hemicellulolitic enzymes: A. niger (SBCM3), A. sydowii (SBCM7) and A. fumigatus (SBC4). The best β-xylosidase producer was A. niger SBCM3 strain. This crude enzyme presented optimal activity at pH 3.5 and 55 °C (141 U/g). For β-glucosidase and xylanase the best producer was A. fumigatus SBC4 strain, whose enzymes presented maximum activity at 60 °C and pH 3.5 (54 U/g) and 4.0 (573 U/g), respectively. All these crude enzymes presented stability around pH 3.0–8.0 and up to 60 °C, which can be very useful in industrial processes that work at high temperatures and low pHs. These enzymes also exhibited moderate tolerance to ethanol and the sugars glucose and xylose. These similar characteristics among these fungal crude enzymes suggest that they can be used synergistically in cocktails in future studies of biomass conversion with potential application in several biotechnological sectors.

.


Subject(s)
Aspergillus fumigatus/enzymology , Aspergillus niger/enzymology , Cellulose/metabolism , /metabolism , Kluyveromyces/enzymology , Saccharum/microbiology , Xylosidases/metabolism , beta-Glucosidase/metabolism , Aspergillus fumigatus/isolation & purification , Aspergillus fumigatus/metabolism , Aspergillus niger/isolation & purification , Aspergillus niger/metabolism , Base Sequence , Biomass , Brazil , DNA, Fungal/genetics , DNA, Intergenic/genetics , Fermentation , Kluyveromyces/isolation & purification , Kluyveromyces/metabolism , Lignin/metabolism , Molecular Typing , Mycological Typing Techniques , RNA, Ribosomal/genetics , Sequence Analysis, DNA
10.
Braz. j. microbiol ; 46(3): 673-682, July-Sept. 2015. tab, ilus
Article in English | LILACS | ID: lil-755832

ABSTRACT

Aflatoxin contamination of peanut, due to infection by Aspergillus flavus, is a major problem of rain-fed agriculture in India. In the present study, molecular characterisation of 187 Aspergillus flavus isolates, which were sampled from the peanut fields of Gujarat state in India, was performed using AFLP markers. On a pooled cluster analysis, the markers could successfully discriminate among the ‘A’, ‘B’ and ‘G’ group A. flavus isolates. PCoA analysis also showed equivalent results to the cluster analysis. Most of the isolates from one district could be clustered together, which indicated genetic similarity among the isolates. Further, a lot of genetic variability was observed within a district and within a group. The results of AMOVA test revealed that the variance within a population (84%) was more than that between two populations (16%). The isolates, when tested by indirect competitive ELISA, showed about 68.5% of them to be atoxigenic. Composite analysis between the aflatoxin production and AFLP data was found to be ineffective in separating the isolate types by aflatoxigenicity. Certain unique fragments, with respect to individual isolates, were also identified that may be used for development of SCAR marker to aid in rapid and precise identification of isolates.

.


Subject(s)
Aspergillus flavus , Aflatoxins/metabolism , Arachis/microbiology , Agriculture , Amplified Fragment Length Polymorphism Analysis , Aspergillus flavus/classification , Aspergillus flavus/genetics , Aspergillus flavus/isolation & purification , DNA, Fungal/genetics , Enzyme-Linked Immunosorbent Assay , Genes, Fungal , Genetic Variation/genetics , India , Molecular Typing , Mycological Typing Techniques , Principal Component Analysis
11.
Braz. j. infect. dis ; 19(4): 376-383, July-Aug. 2015. tab, ilus
Article in English | LILACS | ID: lil-759281

ABSTRACT

Paracoccidioidomycosis is a systemic and endemic mycosis, restricted to tropical and subtropical areas of Latin America. The infection is caused by the thermal dimorphic fungus Paracoccidioides brasiliensisand Paracoccidioides lutzii. The diagnosis of paracoccidioidomycosis is usually performed by microscopic examination, culture and immunodiagnostic tests to respiratory specimens, body fluids and/or biopsies; however these methods require laboratory personnel with experience and several days to produce a result. In the present study, we have validated and evaluated a nested PCR assay targeting the gene encoding the Paracoccidioides gp43membrane protein in 191 clinical samples: 115 samples from patients with proven infections other than paracoccidioidomycosis, 51 samples as negative controls, and 25 samples from patients diagnosed with paracoccidioidomycosis. Additionally, the specificity of the nested PCR assay was also evaluated using purified DNA isolated from cultures of different microorganisms (n= 35) previously identified by culture and/or sequencing. The results showed that in our hands, this nested PCR assay for gp43 protein showed specificity and sensitivity rates of 100%. The optimized nested PCR conditions in our laboratory allowed detection down to 1 fg of P. brasiliensisDNA.


Subject(s)
Humans , DNA, Fungal/genetics , Fungal Proteins/genetics , Paracoccidioides/genetics , Paracoccidioidomycosis/diagnosis , Colombia , Polymerase Chain Reaction , Paracoccidioides/isolation & purification , Sensitivity and Specificity
12.
Braz. j. microbiol ; 46(2): 359-366, Apr-Jun/2015. tab, graf
Article in English | LILACS | ID: lil-749714

ABSTRACT

Endophytes are being considered for use in biological control, and the enzymes they secrete might facilitate their initial colonization of internal plant tissues and direct interactions with microbial pathogens. Microbial proteases are also biotechnologically important products employed in bioremediation processes, cosmetics, and the pharmaceutical, photographic and food industries. In the present study, we evaluated antagonism and competitive interactions between 98 fungal endophytes and Alternaria alternata, Colletotrichum sp., Phyllosticta citricarpa and Moniliophthora perniciosa. We also examined the proteolytic activities of endophytes grown in liquid medium and conducted cup plate assays. The results showed that certain strains in the assemblage of P. hispidum endophytes are important sources of antifungal properties, primarily Lasiodiplodia theobromae JF766989, which reduced phytopathogen growth by approximately 54 to 65%. We detected 28 endophytes producing enzymatic halos of up to 16.40 mm in diameter. The results obtained in the present study highlight the proteolytic activity of the endophytes Phoma herbarum JF766995 and Schizophyllum commune JF766994, which presented the highest enzymatic halo diameters under at least one culture condition tested. The increased activities of certain isolates in the presence of rice or soy flour as a substrate (with halos up to 17.67 mm in diameter) suggests that these endophytes have the potential to produce enzymes using agricultural wastes.


Subject(s)
Antifungal Agents/metabolism , Endophytes/isolation & purification , Endophytes/metabolism , Fungi/isolation & purification , Fungi/metabolism , Peptide Hydrolases/metabolism , Piper/microbiology , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Ribosomal Spacer/chemistry , DNA, Ribosomal Spacer/genetics , Fungi/classification , Fungi/drug effects , Microbiological Techniques , Molecular Sequence Data , Sequence Analysis, DNA
13.
Braz. j. microbiol ; 46(2): 397-406, Apr-Jun/2015. tab, graf
Article in English | LILACS | ID: lil-749738

ABSTRACT

Penicillum janthinellum SDX7 was isolated from aged petroleum hydrocarbon-affected soil at the site of Anand, Gujarat, India, and was tested for different pH, temperature, agitation and concentrations for optimal growth of the isolate that was capable of degrading upto 95%, 63% and 58% of 1%, 3% and 5% kerosene, respectively, after a period of 16 days, at optimal growth conditions of pH 6.0, 30 °C and 180 rpm agitation. The GC/MS chromatograms revealed that then-alkane fractions are easily degraded; however, the rate might be lower for branched alkanes, n-alkylaromatics, cyclic alkanes and polynuclear aromatics. The test doses caused a concentration-dependent depletion of carbohydrates of P. janthinellum SDX7 by 3% to 80%, proteins by 4% to 81% and amino acids by 8% to 95% upto 16 days of treatment. The optimal concentration of 3% kerosene resulted in the least reduction of the metabolites of P. janthinellum such as carbohydrates, proteins and amino acids with optimal growth compared to 5% and 1% (v/v) kerosene doses on the 12th and 16th day of exposure. Phenols were found to be mounted by 43% to 66% at lower and higher concentrations during the experimental period. Fungal isolate P. janthinellum SDX7 was also tested for growth on various xenobiotic compounds.


Subject(s)
Kerosene , Penicillium/growth & development , Penicillium/metabolism , Soil Microbiology , Soil Pollutants/metabolism , Xenobiotics/metabolism , Base Composition , Biotransformation , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Gas Chromatography-Mass Spectrometry , Genes, rRNA , Hydrogen-Ion Concentration , India , Molecular Sequence Data , Penicillium/genetics , Penicillium/isolation & purification , RNA, Fungal/genetics , /genetics , Sequence Analysis, DNA , Temperature
14.
Braz. j. microbiol ; 46(1): 59-65, 05/2015. tab, graf
Article in English | LILACS | ID: lil-748250

ABSTRACT

Thermophilic and thermotolerant micro-organisms strains have served as the natural source of industrially relevant and thermostable enzymes. Although some strains of the Trametes genus are thermotolerant, few Trametes strains were studied at the temperature above 30 °C until now. In this paper, the laccase activity and the mycelial growth rate for Trametes trogii LK13 are superior at 37 °C. Thermostability and organic cosolvent tolerance assays of the laccase produced at 37 °C indicated that the enzyme possessed fair thermostability with 50% of its initial activity at 80 °C for 5 min, and could remain 50% enzyme activity treated with organic cosolvent at the concentration range of 25%–50% (v/v). Furthermore, the test on production of laccase and lignocellulolytic enzymes showed the crude enzymes possessed high laccase level (1000 U g−1) along with low cellulose (2 U g−1) and xylanase (140 U g−1) activity. Thus, T. trogii LK13 is a potential strain to be applied in many biotechnological processes.


Subject(s)
Laccase/metabolism , Trametes/enzymology , Trametes/growth & development , Cluster Analysis , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Ribosomal Spacer/chemistry , DNA, Ribosomal Spacer/genetics , Enzyme Stability , Laccase/chemistry , Microscopy , Molecular Sequence Data , Phylogeny , Sequence Analysis, DNA , Solvents , Temperature , Trametes/cytology , Trametes/radiation effects
15.
Braz. j. microbiol ; 46(1): 285-292, 05/2015. tab, graf
Article in English | LILACS | ID: lil-748256

ABSTRACT

Aspergillus flavus was isolated from soil and exhibited laccase activity under both constitutive and copper induced conditions. Spiking the medium with 1 mM copper sulfate resulted in an increase in the activity which reached 51.84 U/mL, a distinctive protein band was detected at 60 kDa. The extracellular enzyme was purified 81 fold using gel filtration chromatography and resulted in two different laccase fractions L1 and L2, the latter had a higher enzymatic activity which reached 79.57 U/mL and specific activity of 64.17 U/μg protein. The analysis of the spectrum of the L2 fraction showed a shoulder at 330 nm which is characteristic for T2/T3 copper centers; both copper and zinc were detected suggesting that this is an unconventional white laccase. Primers of laccase gene were designed and synthesized to recover specific gene from A. flavus. Sequence analysis indicated putative laccase (Genbank ID: JF683612) at the amino acid level suggesting a close identity to laccases from other genera containing the copper binding site. Decolorization of textile waste water under different conditions showed possible application in bioremediation within a short period of time. The effect of copper on A. flavus was concentration dependent.


Subject(s)
Aspergillus flavus/drug effects , Aspergillus flavus/enzymology , Copper/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Laccase/biosynthesis , Transcriptional Activation/drug effects , Aspergillus flavus/genetics , Aspergillus flavus/isolation & purification , Chromatography, Gel , Culture Media/chemistry , DNA, Fungal/genetics , Electrophoresis, Polyacrylamide Gel , Industrial Waste , Laccase/chemistry , Laccase/isolation & purification , Molecular Sequence Data , Molecular Weight , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Soil Microbiology , Spectrum Analysis , Water Purification
16.
Braz. j. microbiol ; 45(4): 1211-1220, Oct.-Dec. 2014. graf, mapas, tab
Article in English | LILACS | ID: lil-741270

ABSTRACT

A systematized survey was conducted to find soil-borne microbes that degrade cellulose in soils from unique ecosystems, such as the Superpáramo, Páramo, and the High Andean Forest in the Nevados National Natural Park (NNNP), Colombia. These high mountain ecosystems represent extreme environments, such as high levels of solar radiation, low atmospheric pressure, and extreme daily changes in temperature. Cellulolytic activity of the microorganisms was evaluated using qualitative tests, such as growth in selective media followed by staining with congo red and iodine, and quantitative tests to determine the activity of endoglucanase, β-glucosidase, exoglucanase, and total cellulase. Microorganisms were identified using molecular markers, such as the 16S rRNA gene for bacteria and the internal transcribed spacer region (ITS) of ribosomal DNA for fungi. Multivariate statistical analysis (MVA) was used to select microorganisms with high cellulolytic capacity. A total of 108 microorganisms were isolated from the soils and, in general, the enzymatic activities of fungi were higher than those of bacteria. Our results also found that none of the organisms studied were able to degrade all the components of the cellulose and it is therefore suggested that a combination of bacteria and/or fungi with various enzymatic activities be used to obtain high total cellulolytic activity. This study gives an overview of the potential microorganism that could be used for cellulose degradation in various biotechnological applications and for sustainable agricultural waste treatment.


Subject(s)
Bacteria/isolation & purification , Bacteria/metabolism , Cellulose/metabolism , Fungi/isolation & purification , Fungi/metabolism , Soil Microbiology , Bacteria/classification , Bacteria/genetics , Colombia , Cellulase/analysis , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Helminth/chemistry , DNA, Helminth/genetics , DNA, Ribosomal Spacer/chemistry , DNA, Ribosomal Spacer/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Fungi/classification , Fungi/genetics , Hydrolysis , /genetics , Sequence Analysis, DNA
17.
Braz. j. microbiol ; 45(4): 1293-1302, Oct.-Dec. 2014. ilus, graf, tab
Article in English | LILACS | ID: lil-741279

ABSTRACT

Xylanase (EC 3. 2. 1. 8), hydrolyzes xylo-oligosaccharides into D-xylose and required for complete hydrolysis of native cellulose and biomass conversion. It has broad range of applications in the pulp and paper, pharmaceutical and Agri-food industries. Fifty fungal species were isolated from the fouled soil around an oil refinery and screened for the production of xylanase enzyme by enrichment culture techniques. The isolated fungal strain was identified as Hypocrea lixii SS1 based on the results of biochemical tests and 18s rRNA sequencing. The phylogenetic tree was constructed using the MEGA 5 software. Further, Hypocrea lixii SS1 was tested for the ability to utilize the sunflower oil sludge (waste from the oil industry) as the sole carbon source for xylanase production. The growth characteristics of Hypocrea lixii SS1 were also studied and maximum growth was found on the 7th day of incubation. The fungus showed a remarkable xylanase production of 38.9 U/mL. Xylanase was purified using a combination of 0-50% NH4SO2 precipitation, DEAE-sepharose and Sephacryl S-200 chromatography. Single peak obtained in RP-HPLC confirms the purity of xylanase. Further the enzyme produced was affirmed as xylanase with its molecular weight (29 kDa) using SDS-PAGE.


Subject(s)
Soil Microbiology , Trichoderma/classification , Trichoderma/isolation & purification , Xylosidases/analysis , Chromatography, Liquid , Cluster Analysis , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Molecular Weight , Mycological Typing Techniques , Phylogeny , /genetics , Sequence Analysis, DNA , Trichoderma/enzymology , Trichoderma/growth & development , Xylosidases/chemistry , Xylosidases/isolation & purification
18.
Rev. biol. trop ; 62(3): 1197-1208, jul.-sep. 2014. ilus, tab
Article in Spanish | LILACS | ID: lil-753682

ABSTRACT

Ganoderma includes species of great economic and ecological importance, but taxonomists judge the current nomenclatural situation as chaotic and poorly studied in the neotropics. From this perspective, phylogenetic analyses inferred from ribosomal DNA sequences have aided the clarification of the genus status. In this study, 14 specimens of Ganoderma and two of Tomophagus collected in Brazil were used for DNA extraction, amplification and sequencing of the ITS and LSU regions (rDNA). The phylogenetic delimitation of six neotropical taxa (G. chalceum, G. multiplicatum, G. orbiforme, G. parvulum, G. aff. oerstedtii and Tomophagus colossus) was determined based on these Brazilian specimens and found to be distinct from the laccate Ganoderma from Asia, Europe, North America and from some specimens from Argentina. Phylogenetic reconstructions confirmed that the laccate Ganoderma is distinct from Tomophagus, although they belong to the same group. The use of taxonomic synonyms Ganoderma subamboinense for G. multiplicatum, G. boninense for G. orbiforme and G. chalceum for G. cupreum was not confirmed. However, Ganoderma parvulum was confirmed as the correct name for specimens called G. stipitatum. Furthermore, the name G. lucidum should be used only for European species. The use of valid published names is proposed according to the specimen geographical distribution, their morphological characteristics and rDNA analysis. Rev. Biol. Trop. 62 (3): 1197-1208. Epub 2014 September 01.


Ganoderma incluye especies de gran importancia económica y ecológica, sin embargo, su nomenclatura actual es caótica y poco estudiada en el neotrópico. En este estudio se utilizaron 14 muestras de Ganoderma y dos de Tomophagus recolectados en Brasil para la extracción de ADN, amplificación y secuenciación de las regiones ITS y LSU. La delimitación filogenética de seis táxones neotropicales fue discutida con base en especímenes brasileños y secuencias del GenBank. Estas especies mostraron ser distintas de los Ganoderma lacados de Asia, Europa, América del Norte y de algunos ejemplares de Argentina. Las reconstrucciones filogenéticas confirman que los Ganoderma lacados son distintos de Tomophagus, aunque pertenecen al mismo grupo. No se confirman los sinónimos de G. subamboinense a G. multiplicatum, de G. boninense a G. orbiforme y G. chalceum a G. cupreum. G. parvulum se confirma como el nombre correcto para G. stipitatum. G. lucidum sólo se debe utilizar para especies europeas. Por lo tanto, se propone el uso de nombres publicados válidamente de acuerdo con la distribución geográfica de las muestras, características morfológicas y análisis de ADNr.


Subject(s)
DNA, Fungal/genetics , DNA, Ribosomal Spacer/genetics , Ganoderma/cytology , Ganoderma/genetics , Phylogeny , Polymerase Chain Reaction , Sequence Analysis, DNA
19.
Braz. j. microbiol ; 45(3): 977-983, July-Sept. 2014. ilus, tab
Article in English | LILACS | ID: lil-727029

ABSTRACT

Extraneous DNA interferes with PCR studies of endophytic fungi. A procedure was developed with which to evaluate the removal of extraneous DNA. Wheat (Triticum aestivum) leaves were sprayed with Saccharomyces cerevisiae and then subjected to physical and chemical surface treatments. The fungal ITS1 products were amplified from whole tissue DNA extractions. ANOVA was performed on the DNA bands representing S. cerevisiae on the agarose gel. Band profile comparisons using permutational multivariate ANOVA (PERMANOVA) and non-metric multidimensional scaling (NMDS) were performed on DGGE gel data, and band numbers were compared between treatments. Leaf surfaces were viewed under variable pressure scanning electron microscopy (VPSEM). Yeast band analysis of the agarose gel showed that there was no significant difference in the mean band DNA quantity after physical and chemical treatments, but they both differed significantly (p < 0.05) from the untreated control. PERMANOVA revealed a significant difference between all treatments (p < 0.05). The mean similarity matrix showed that the physical treatment results were more reproducible than those from the chemical treatment results. The NMDS showed that the physical treatment was the most consistent. VPSEM indicated that the physical treatment was the most effective treatment to remove surface microbes and debris. The use of molecular and microscopy methods for the post-treatment detection of yeast inoculated onto wheat leaf surfaces demonstrated the effectiveness of the surface treatment employed, and this can assist researchers in optimizing their surface sterilization techniques in DNA-based fungal endophyte studies.


Subject(s)
Endophytes/isolation & purification , Microbiological Techniques/methods , Sterilization/methods , Triticum/microbiology , Denaturing Gradient Gel Electrophoresis , DNA, Fungal/genetics , DNA, Fungal/isolation & purification , DNA, Plant/chemistry , DNA, Plant/genetics , DNA, Ribosomal Spacer/genetics , DNA, Ribosomal Spacer/isolation & purification , Microscopy, Electron, Scanning , Polymerase Chain Reaction , Plant Leaves/microbiology , Plant Leaves/ultrastructure , Surface Properties , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/isolation & purification , Saccharomyces cerevisiae/ultrastructure , Triticum/ultrastructure
20.
Mem. Inst. Oswaldo Cruz ; 109(4): 436-441, 03/07/2014. tab, graf
Article in English | LILACS | ID: lil-716301

ABSTRACT

Over the last decades, Candida spp have been responsible for an increasing number of infections, especially in patients requiring intensive care. Knowledge of local epidemiology and analysis of the spread of these pathogens is important in understanding and controlling their transmission. The aim of this study was to evaluate the genetic diversity of 31 Candida albicans and 17 Candida glabrata isolates recovered from intensive care unit patients from the tertiary hospital in Krakow between 2011-2012. The strains were typed by random amplified polymorphic DNA (RAPD) polymerase chain reaction using five primers (CD16AS, HP1247, ERIC-2, OPE-3 and OPE-18). The results of the present investigation revealed a high degree of genetic diversity among the isolates. No clonal relationship was found among the C. albicans strains, whereas two C. glabrata isolates were identical. The source of Candida infection appeared to be mostly endogenous; however, the presence of two clonal C. glabrata strains suggested the possibility of cross-transmission of these pathogens. Our study confirmed the high discriminatory power of the RAPD technique in the molecular typing of Candida clinical isolates. This method may be applied to the evaluation of transmission routes of pathogenic fungi on a local level.


Subject(s)
Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Young Adult , Candida albicans/genetics , Candida glabrata/genetics , Candidiasis/epidemiology , Antifungal Agents/pharmacology , Candida albicans/drug effects , Candida glabrata/drug effects , Candidiasis/microbiology , DNA Primers/genetics , DNA, Fungal/genetics , Intensive Care Units , Microbial Sensitivity Tests , Molecular Typing , Poland/epidemiology , Random Amplified Polymorphic DNA Technique
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