OBJECTIVE: A strain of Aspergillus niger (A. niger), capable of releasing bound phenolic acids from wheat bran, was isolated. This strain was identified by gene sequence identification. The antioxidant and anti-inflammatory capacity of ferulic acid released from wheat bran by this A. niger strain (FA-WB) were evaluated. METHODS: Molecular identification techniques based on PCR analysis of specific genomic sequences were conducted; antioxidant ability was examined using oxygen radical absorbance capacity (ORAC), cellular antioxidant activity (CAA) assays, and erythrocyte hemolysis assays. RAW264.7 cells were used as a model to detect anti-inflammatory activity. RESULTS: The filamentous fungal isolate was identified to be A. niger. ORAC and CAA assay showed that FA-WB had better antioxidant activity than that of the ferulic acid standard. The erythrocyte hemolysis assay results suggested that FA-WB could attenuate AAPH-induced oxidative stress through inhibition of reactive oxy gen species (ROS) generation. FA-WB could significantly restore the AAPH-induced increase in intracellular antioxidant enzyme activities to normal levels as well as inhibit the intracellular malondialdehyde formation. TNF-a, IL-6, and NO levels indicated that FA-WB can inhibit the inflammation induced by lipopolysaccharide (LPS). CONCLUSION Ferulic acid released from wheat bran by a new strain of A. niger had good anti-inflammatory activity and better antioxidant ability than standard ferulic acid.
Animals ; Anti-Inflammatory Agents ; metabolism ; pharmacology ; Antioxidants ; metabolism ; pharmacology ; Aspergillus niger ; genetics ; isolation & purification ; metabolism ; Coumaric Acids ; metabolism ; pharmacology ; DNA, Fungal ; analysis ; Dietary Fiber ; microbiology ; Erythrocytes ; drug effects ; metabolism ; Fermentation ; Hep G2 Cells ; Humans ; Interleukin-6 ; metabolism ; Lipopolysaccharides ; pharmacology ; Mice ; RAW 264.7 Cells ; Sheep ; Tumor Necrosis Factor-alpha ; metabolism

BACKGROUND: Next-generation sequencing (NGS) can detect many more microorganisms of a microbiome than traditional methods. This study aimed to analyze the vaginal microbiomes of Korean women by using NGS that included bacteria and other microorganisms. The NGS results were compared with the results of other assays, and NGS was evaluated for its feasibility for predicting vaginitis. METHODS: In total, 89 vaginal swab specimens were collected. Microscopic examinations of Gram staining and microbiological cultures were conducted on 67 specimens. NGS was performed with GS junior system on all of the vaginal specimens for the 16S rRNA, internal transcribed spacer (ITS), and Tvk genes to detect bacteria, fungi, and Trichomonas vaginalis. In addition, DNA probe assays of the Candida spp., Gardnerella vaginalis, and Trichomonas vaginalis were performed. Various predictors of diversity that were obtained from the NGS data were analyzed to predict vaginitis. RESULTS: ITS sequences were obtained in most of the specimens (56.2%). The compositions of the intermediate and vaginitis Nugent score groups were similar to each other but differed from the composition of the normal score group. The fraction of the Lactobacillus spp. showed the highest area under the curve value (0.8559) in ROC curve analysis. The NGS and DNA probe assay results showed good agreement (range, 86.2-89.7%). CONCLUSIONS: Fungi as well as bacteria should be considered for the investigation of vaginal microbiome. The intermediate and vaginitis Nugent score groups were indistinguishable in NGS. NGS is a promising diagnostic tool of the vaginal microbiome and vaginitis, although some problems need to be resolved.
Area Under Curve ; Bacteria/*genetics/isolation & purification ; Bacterial Proteins/genetics ; Candida/*genetics/isolation & purification ; Female ; Fungal Proteins/genetics ; Gardnerella vaginalis/genetics/isolation & purification ; High-Throughput Nucleotide Sequencing ; Humans ; *Microbiota ; RNA, Ribosomal, 16S/chemistry/genetics/metabolism ; ROC Curve ; Sequence Analysis, DNA ; Trichomonas vaginalis/genetics/isolation & purification ; Vagina/*microbiology ; Vaginitis/*diagnosis/microbiology

BACKGROUND: We investigated the species distribution and amphotericin B (AMB) susceptibility of Korean clinical Aspergillus isolates by using two Etests and the CLSI broth microdilution method. METHODS: A total of 136 Aspergillus isolates obtained from 11 university hospitals were identified by sequencing the internal transcribed spacer (ITS) and beta-tubulin genomic regions. Minimal inhibitory concentrations (MICs) of AMB were determined in Etests using Mueller-Hinton agar (Etest-MH) and RPMI agar (Etest-RPG), and categorical agreement with the CLSI method was assessed by using epidemiological cutoff values. RESULTS: ITS sequencing identified the following six Aspergillus species complexes: Aspergillus fumigatus (42.6% of the isolates), A. niger (23.5%), A. flavus (17.6%), A. terreus (11.0%), A. versicolor (4.4%), and A. ustus (0.7%). Cryptic species identifiable by beta-tubulin sequencing accounted for 25.7% (35/136) of the isolates. Of all 136 isolates, 36 (26.5%) had AMB MICs of > or =2 microg/mL by the CLSI method. The categorical agreement of Etest-RPG with the CLSI method was 98% for the A. fumigatus, A. niger, and A. versicolor complexes, 87% for the A. terreus complex, and 37.5% for the A. flavus complex. That of Etest-MH was < or =75% for the A. niger, A. flavus, A. terreus, and A. versicolor complexes but was higher for the A. fumigatus complex (98.3%). CONCLUSIONS: Aspergillus species other than A. fumigatus constitute about 60% of clinical Aspergillus isolates, and reduced AMB susceptibility is common among clinical isolates of Aspergillus in Korea. Molecular identification and AMB susceptibility testing by Etest-RPG may be useful for characterizing Aspergillus isolates of clinical relevance.
Amphotericin B/*pharmacology ; Antifungal Agents/*pharmacology ; Aspergillus/*drug effects/isolation & purification ; DNA, Fungal/chemistry/genetics/metabolism ; Hospitals ; Humans ; Microbial Sensitivity Tests ; Mycoses/diagnosis/microbiology ; Republic of Korea ; Sequence Analysis, DNA ; Tubulin/genetics

Microsporidia are eukaryotic organisms that cause zoonosis and are major opportunistic pathogens in HIV-positive patients. However, there is increasing evidence that these organisms can also cause gastrointestinal and ocular infections in immunocompetent individuals. In Korea, there have been no reports on human infections with microsporidia to date. In the present study, we used real-time PCR and nucleotide sequencing to detect Encephalitozoon intestinalis infection in seven of 139 human diarrheal stool specimens (5%) and Encephalitozoon hellem in three of 34 farm soil samples (8.8%). Genotype analysis of the E. hellem isolates based on the internal transcribed spacer 1 and polar tube protein genes showed that all isolates were genotype 1B. To our knowledge, this is the first report on human E. intestinalis infection in Korea and the first report revealing farm soil samples as a source of E. hellem infection. Because microsporidia are an important public health issue, further large-scale epidemiological studies are warranted.
AIDS-Related Opportunistic Infections/parasitology ; Adolescent ; Adult ; Aged ; Agriculture ; Base Sequence ; Child ; Child, Preschool ; DNA, Intergenic/genetics ; DNA, Protozoan/genetics ; Encephalitozoon/*genetics/*isolation & purification ; Encephalitozoonosis/*epidemiology ; Feces/*parasitology ; Female ; Fungal Proteins/genetics ; Humans ; Infant ; Infant, Newborn ; Male ; Middle Aged ; Molecular Typing ; Real-Time Polymerase Chain Reaction ; Republic of Korea/epidemiology ; Sequence Alignment ; Sequence Analysis, DNA ; Soil/*parasitology ; Young Adult

In the present study, terminal-restriction fragment length polymorphism (T-RFLP) technique was applied to assess the diversity and tissue distribution of the fungal endophyte communities of Alpinia officinarum collected from Longtang town in Xuwen county, Guangdong province, China, at which the pharmacological effect of the medicine plant is traditional considered to be the significantly higher than that in any other growth areas in China. A total of 28 distinct Terminal-Restriction Fragment (T-RFs) were detected with HhaI Mono-digestion targeted amplified fungal nuclear ribosomal internal transcribed spacer region sequences (rDNA ITS) from the root, rhizome, stem, and leaf internal tissues of A. officinarum plant, indicating that at least 28 distinct fungal species were able to colonize the internal tissue of the host plant. The rDNA ITS-T-RFLP profiles obtained from different tissues of the host plant were obvious distinct. And the numbers of total T-RFs, and the dominant T-RFs detected from various tissues were significantly different. Based on the obtained T-RFLP profiles, Shannon's diversity index and the Shannon's evenness index were calculated, which were significantly different among tissues (P < 0.05). Furthermore, two types of active chemicals, total volatile oils by water vapor distillation method and galangin by methanol extraction-HPLC method, were examined in the each tissue of the tested plant. Both of tested components were detected in all of the four tissues of the medicine plant with varying contents. And the highest was in rhizome tissue. Correlation analysis revealed there were significant negative correlations between both of the tested active components contents and calculated Shannon's diversity index, as well as the Shannon's evenness index of the fungal endophyte communities of the host plant (P = 0, Pearson correlation coefficient ≤ -0.962), and significant positive correlations between both of the tested active components contents and 325 bp dominant T-RF linkage to Pestalotiopsis (P = 0, Pearson correlation coefficient ≥ 0.975). In conclusion, A. officinarum is colonized by diverse fungal endophytes communities. The diversity of the fungal endophytes was found in the A. officinarum varied with differences of the tissue types of the host plants and was closely correlated with the accumulation of main active components, total volatile oils and galangin contents in the host plant tissue.
Alpinia ; chemistry ; microbiology ; Biodiversity ; China ; DNA, Fungal ; genetics ; DNA, Ribosomal ; genetics ; Drugs, Chinese Herbal ; analysis ; Endophytes ; classification ; genetics ; growth & development ; isolation & purification ; Fungi ; classification ; genetics ; growth & development ; isolation & purification ; Phylogeny ; Plants, Medicinal ; chemistry ; microbiology ; Polymorphism, Restriction Fragment Length

OBJECTIVETo promote development and utilization of Ophiocordyceps gracilis in xinjiang and provide basic data for researching and sustainable developing medicine fungus related to O. gracilis.

METHODA white strain SFYT002 isolated from the sclerotium of O. gracilis in Xinjiang was researched by morphological observation, ITS and 18SrDNA sequencing. The ITS and 18SrDNA sequences of the strain were determined, BLAST was compared with the other sequences of Tolypocladium in GenBank. The phylogenetic trees of ITS and 18SrDNA sequences were analyzed in Tolypocladium. In addition, the filter paper method was used to study the antibacterial effects.

RESULTThe main morphological characters of this strain were white cotton-like colonies, phialide with inflated base, drastically sharping with partially bending tips, small and transparent budding spores with being always assemble to spearhead and globular, subglobular or ellipse conidiospores. The phylogenetic trees of ITS and 18SrDNA sequences were constructed and analyzed in Tolypocladium. It was resulted that Tolypocladium was confirmed to be monophyletic, and the strain SFYT002 was the same as the systematic position of others of T. inflatum. Meanwhile, the antibacterial test was performed against the 4 common pathogenic bacteria. It was showed that both fermentation and its extracts of different polar from this strain possessed good anti-bacteria capacities.

CONCLUSIONThe strain SFYT02 was identified as T. inflatum, and inhibited effectively growth of bacteria.

Anti-Bacterial Agents ; isolation & purification ; pharmacology ; China ; DNA, Fungal ; genetics ; DNA, Intergenic ; genetics ; Hypocreales ; genetics ; isolation & purification ; physiology ; Medicine, Chinese Traditional ; methods ; Mycelium ; Phylogeny

Child ; Child, Preschool ; Clinical Laboratory Techniques ; DNA, Fungal ; genetics ; Evidence-Based Medicine ; Fungi ; genetics ; isolation & purification ; Humans ; Infant ; Mycological Typing Techniques ; Mycoses ; diagnosis ; microbiology ; Polymerase Chain Reaction ; Serologic Tests ; Specimen Handling

BACKGROUND: The identification of molds in clinical laboratories is largely on the basis of phenotypic criteria, the classification of which can be subjective. Recently, molecular methods have been introduced for identification of pathogenic molds in clinical settings. Here, we employed comparative sequence analysis to identify molds. METHODS: A total of 47 clinical mold isolates were used in this study, including Aspergillus and Trichophyton. All isolates were identified by phenotypic properties, such as growth rate, colony morphology, and reproductive structures. PCR and direct sequencing, targeting the internal transcribed spacer (ITS) region, the D1/D2 region of the 28S subunit, and the beta-tubulin gene, were performed using primers described previously. Comparative sequence analysis by using the GenBank database was performed with the basic local alignment search tool (BLAST) algorithm. RESULTS: For Aspergillus, 56% and 67% of the isolates were identified to the species level by using ITS and beta-tubulin analysis, respectively. Only D1/D2 analysis was useful for Trichophyton identification, with 100% of isolates being identified to the species level. Performances of ITS and D1/D2 analyses were comparable for species-level identification of molds other than Aspergillus and Trichophyton. In contrast, the efficacy of beta-tubulin analysis was limited to genus identification because of the paucity of database information for this gene. CONCLUSIONS: The molecular methods employed in this study were valuable for mold identification, although the different loci used had variable usefulness, according to mold genus. Thus, a tailored approach is recommended when selecting amplification targets for molecular identification of molds.
Aspergillus/genetics/isolation & purification ; DNA, Fungal/analysis/isolation & purification ; Databases, Genetic ; Fungi/genetics/*isolation & purification ; Humans ; Polymerase Chain Reaction ; RNA, Ribosomal, 28S/*genetics ; Sequence Analysis, DNA ; Trichophyton/genetics/isolation & purification ; Tubulin/*genetics

OBJECTIVETo evaluate the feasibility of PCR/reverse line blot hybridization (RLB) assay in the detection and identification of clinical pathogens in fungal sinusitis (FS).

METHODSTwenty-six formalin-fixed and paraffin-embedded tissues and 8 fresh tissues of FS were collected from Beijing Tongren Hospital, Capital Medical University from May 2009 to February 2010. Pathological examination, fungal culture and ITS2 region sequencing were carried out. The DNA of all samples was extracted by standard procedure and fungal universal primers ITS3 and ITS4 were used for PCR amplification of all tissues. Then the amplified products were used for RLB with five fungal species-specific probes. The results of PCR/RLB were compared with ITS region sequencing, fungal culture and pathological examination.

RESULTSFor the biopsy tissues, fungal cultures were positive in 14 cases (41.2%); pathologic examination demonstrated fungal hyphae in all cases; ITS2 region sequencing was successful in 16 cases (47.1%); PCR/RLB showed A. flavus in 14 cases, A. fumigatus in 10 cases, A. niger in four cases, A. nidulans in one case, A. flavus and A. fumigatus in three cases, and A. fumigatus and A. niger in two cases.

CONCLUSIONSThe PCR/RLB assay is suitable for rapid and accurate detection and identification of the pathogenic fungus of FS. Compared with the conventional fungal culture and microscopy, pathologic examination and DNA sequencing, the PCR/RLB has the advantages of more economy, time saving, and higher sensitivity, specificity and throughput.

Aspergillus ; classification ; genetics ; isolation & purification ; Aspergillus flavus ; genetics ; isolation & purification ; Aspergillus fumigatus ; genetics ; isolation & purification ; Aspergillus niger ; genetics ; isolation & purification ; DNA Primers ; DNA, Fungal ; genetics ; Humans ; Mycoses ; diagnosis ; microbiology ; Nucleic Acid Hybridization ; methods ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Sinusitis ; diagnosis ; microbiology

OBJECTIVETo identify the endophytic fungal strain PR35 separated from Paeonia delavayi and study chemical constituents of its secondary metabolites.

METHODThe fungal strain PR35 was identified by morphological observation and ITS rDNA sequence analysis. Various chromatographic methods were adopted to separate and purify its secondary metabolites, and their structures were identified by physiochemical properties and spectral data

RESULTThe fungal strain PR35 was identified as Trichoderma longibrachiatum. Five compounds were separated from fermentation products of fungal strain PR35 and identified as 1-(2,6-dihydroxyphenyl)-3-hydroxybutan-1-one (1), 1-(2,6-dihydroxypheny) propan-1-one (2), 1-(2,4,6-trihydroxyphenyl) butan-1-one (3), 4-methoxy-1-naphthol (4), and cerevisterol (5). Among them, compounds 1-3 showed notable antifungal activities against Botrytis cinerea, Fusarium avenaceum and Hormodendrum compactum.

CONCLUSIONThe endophytic fungus T. longibrachiatum was separated from the plant P. delavayi for the first time. Five compounds were first separated from endophytic fungus of P. delavayi. Among them, compound 4 was separated from microbial fermentation products for the first time.

DNA, Fungal ; genetics ; DNA, Intergenic ; genetics ; Endophytes ; classification ; genetics ; isolation & purification ; metabolism ; Paeonia ; microbiology ; Phylogeny ; Trichoderma ; classification ; genetics ; isolation & purification ; metabolism