ABSTRACT
Abstract The virulence genes in invasive aspergillosis (IA) have not been analyzed adequately. The present study was designed to evaluate the expression of gpaB and sidA genes, which are important virulence genes in Aspergillus spp. from bronchoalveolar lavage (BAL) samples. Direct examination and culture on Czapek Agar and Sabouraud Dextrose Agar media were performed for 600 BAL specimens isolated from patients with possible aspergillosis. A Galactomannan ELISA assay was also carried out. The expression levels of the gpaB and sidA genes in isolates were analyzed using quantitative real-time PCR (qRT-PCR). We identified 2 species, including Aspergillus flavus (A. flavus) and Aspergillus fumigatus (A. fumigatus) in 25 positive samples for invasive aspergillosis as validated using GM-ELISA. A. flavus is the main pathogen threatening transplant recipients and cancer patients worldwide. In this study, A. flavus had low levels of the gpaB gene expression compared to A. fumigatus (p = 0.006). The highest sidA expression was detected in transplant recipients (p = 0.05). There was no significant correlation between sidA expression and underlying disease (p = 0.15). The sidA and gpaB gene expression patterns may provide evidence that these virulence genes play important roles in the pathogenicity of Aspergillus isolates; however, there are several regulatory genes responsible for the unexpressed sidA and gpaB genes in the isolates.
Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Aged, 80 and over , Aspergillosis/microbiology , Aspergillus flavus/metabolism , Aspergillus flavus/pathogenicity , Aspergillus fumigatus/metabolism , Aspergillus fumigatus/pathogenicity , Bacterial Proteins/metabolism , Aspergillus flavus/isolation & purification , Aspergillus flavus/genetics , Aspergillus fumigatus/isolation & purification , Aspergillus fumigatus/genetics , Bacterial Proteins/genetics , VirulenceABSTRACT
Aflatoxin contamination of peanut, due to infection by
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 AnalysisABSTRACT
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 PurificationABSTRACT
Bacillus species constitute a diverse group of bacteria widely distributed in soil and the aquatic environment. In this study, Bacillus strains isolated from the coastal environment of Cochin, India were identified by detailed conventional biochemical methods, fatty acid methyl ester (FAME) analysis and partial 16S rDNA sequencing. Analysis of the data revealed that Bacillus pumilus was the most predominant species in the region under study followed by B. cereus and B. sphaericus. The B. pumilus isolates were further characterized by arbitrarily primed PCR (AP-PCR), antibiotic sensitivity profiling and PCR screening for known toxin genes associated with Bacillus spp. All B. pumilus isolates were biochemically identical, exhibited high protease and lipase activity and uniformly sensitive to antibiotics tested in this study. One strain of B. pumilus harboured cereulide synthetase gene cesB of B. cereus which was indistinguishable from rest of the isolates biochemically and by AP-PCR. This study reports, for the first time, the presence of the emetic toxin gene cesB in B. pumilus.
As espécies de Bacillus constituem um grupo diversificado de bactérias amplamente distribuídas no solo e no ambiente aquático. Neste estudo, cepas de Bacillus isoladas do ambiente costeiro de Cochin, Índia, foram identificadas através de métodos bioquímicos convencionais, análise de ésteres metílicos de ácidos graxos (FAME) e sequenciamento de 16S rDNA. A análise dos dados revelou que Bacillus pumilus foi a espécie predominante na região estudada, seguido de B. cereus e B. sphaericus. Os isolados de B. pumilus foram caracterizados através da reação em cadeia da polimerase com primers arbitrários (AP-PCR), perfil de sensibilidade a antibióticos e triagem por PCR de genes de toxinas associadas com Bacillus spp. Todos os isolados de B. pumilus foram bioquimicamente idênticos, apresentaram elevada atividade de protease e lipase e foram uniformemente sensíveis aos antibióticos estudados. Um dos isolados de B. pumilus apresentou o gene cesB de B. cereus, que não foinão distinguível dos demais isolados por testes bioquímicos nem por AP-PCR. Este é o primeiro relato da presença do gene cesB da toxina eméticaem B. pumilus.
Subject(s)
Aspergillus flavus/genetics , Bacillus/isolation & purification , In Vitro Techniques , Lipase/genetics , Peptide Hydrolases/genetics , Pimenta/genetics , Polymerase Chain Reaction , Base Sequence , Fatty Acids/analysis , Aquatic Environment , Methods , Soil , MethodsABSTRACT
Aspergillus flavus is a very important toxigenic fungus that produces aflatoxins, a group of extremely toxic substances to man and animals. Toxigenic fungi can grow in feed crops, such as maize, peanuts, and soybeans, being thus of high concern for public health. There are toxigenic and non-toxigenic A. flavus variants, but the necessary conditions for expressing the toxigenic potential are not fully understood. Therefore, we have studied total-DNA polymorphism from toxigenic and non toxigenic A. flavus strains isolated from maize crops and soil at two geographic locations, 300 km apart, in the Southeast region of Brazil. Total DNA from each A. flavus isolate was extracted and subjected to polymerase chain reaction amplification with five randomic primers through the RAPD (random amplified polymorphic DNA) technique. Phenetic and cladistic analyses of the data, based on bootstrap analyses, led us to conclude that RAPD was not suitable to discriminate toxigenic from non toxigenic strains. But the present results support the use of RAPD for strain characterization, especially for preliminary evaluation over extensive collections.