To establish a simple, sensitive and effective technique for the identification of six common dermatophytes, polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) targeting Topoisomerase II gene were used. The DNA of 6 common dermatophytes was amplified by primer dPsD1 and then primers dPsD2. The products generated by dPsD2 were digested with restriction enzyme Hinc II and Hinf I separately. A DNA fragment of about 3390 bp was amplified by using primer dPsD1 from the genomic DNA of each dermatophyte species. The product of dPsD2 was 2380 bp and the restriction profiles of Hinc II and Hinf I were between 58-1670 bp. By using PCR-RFLP, all of the 6 dermatophytoses were diagnosed to species level and no obvious difference identification between Hinc II and Hinf I. It is concluded that the PCR-RFLP identification of dermatophytes by Hinc II or Hinf I is efficient and rapid in clinical practice.
Arthrodermataceae/*classification ; Arthrodermataceae/genetics ; Arthrodermataceae/*isolation & purification ; DNA Topoisomerases, Type II/genetics ; DNA, Fungal/analysis ; DNA, Fungal/genetics ; Dermatomycoses/*microbiology ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length

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

BACKGROUND: We evaluated the efficacy of multilocus sequence typing (MLST) for assessing the genetic relationship among Candida albicans isolates from patients with candidemia in a hospital setting. METHODS: A total of 45 C. albicans isolates from 21 patients with candidemia were analyzed. The MLST results were compared with results obtained by Southern blot hybridization (C1 fingerprinting) and pulsed-field gel electrophoresis (PFGE). PFGE analysis included karyotyping and restriction endonuclease analysis of genomic DNAs using BssHII (REAG-B) and SfiI (REAG-S). RESULTS: The 45 isolates yielded 20 unique diploid sequence types (DSTs) by MLST, as well as 12 karyotypes, 15 REAG-B patterns, 13 REAG-S patterns, and 14 C1 fingerprinting types. Microevolution among intra-individual isolates was detected in 6, 5, 3, 5, and 7 sets of isolates by MLST (1 or 2 allelic differences), REAG-B, REAG-S, C1 fingerprinting, and a combination of all methods, respectively. Among 20 DSTs, 17 were unique, and 3 were found in more than 1 patient. The results of 2 DSTs obtained from 9 patient isolates were in agreement with REAG and C1 fingerprinting patterns. However, the remaining DST, which was shared by 2 patient isolates, showed 2 different PFGE and C1 fingerprinting patterns. In addition, 3 sets of isolates from different patients, which differed in only 1 or 2 alleles by MLST, also exhibited different PFGE or C1 fingerprinting patterns. CONCLUSIONS: MLST is highly discriminating among C. albicans isolates, but it may have some limitations in typing isolates from different patients, which may necessitate additional analysis using other techniques.
Alleles ; Blotting, Southern ; Candida albicans/*classification/genetics/isolation & purification ; Candidemia/*microbiology ; DNA, Fungal/*analysis ; Electrophoresis, Gel, Pulsed-Field ; Genotype ; Humans ; Karyotyping ; Multilocus Sequence Typing/*methods

OBJECTIVE: The aim of this study was to compare the sensitivity of conventional culture and PCR method. METHOD: DNA samples were collected from the sinus of 20 patients with chronic sinusitis and 18 patients diagnosed as fungal sinusitis clinically. Fungal specific PCR analysis and standard culture were performed on every sample. chi2 analysis was used to determine the statistical differences between groups. RESULT: Fungal DNA were detected in 78% of patients with fungal sinusitis and 5% of patients with chronic sinusitis in PCR analysis, while the positive rate were 44% and 0% in standard cultures respectively. CONCLUSION PCR is more rapid and sensitive than nasal swab culture in detecting the fungi in sinus mucosa. Our study suggests that it is an efficient method in diagnosis of fungal sinusitis.
Adult ; Chronic Disease ; DNA, Fungal ; analysis ; Fungi ; genetics ; isolation & purification ; Humans ; Middle Aged ; Paranasal Sinuses ; microbiology ; Polymerase Chain Reaction ; methods ; Sinusitis ; microbiology ; 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

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

AIMTo identify heterogeneity of Candida albicans (C. albicans) isolated from the population with cancer in China by using identification medium, subculture molecular typing, and antifungal susceptibility test.

METHODOLOGYOral cheek mucosal specimens from 52 cancer patients receiving chemotherapy were cultured on CHROMagar Candida plates for Candida identification. All the C. albicans colonies on the plates were subcultured and reconfirmed by API20C, then submitted to the antifungal drug susceptibility test with fluconazole and molecular typing using randomly amplified polymorphic DNA-PCR (RAPD) with primers RSD6 and RSD12.

RESULTS54% (28/52) patients were oral yeast carriage in which C. albicans predominated. More than 7 C. albicans colonies were isolated from each of 12 patients (Group A), while less than 5 colonies were isolated from each of 16 patients (Group B). RSD6 and RSD12 were successful in eliciting 17 (A1-A17) and 2 (B1-B2) genotypes, respectively from among the 205 isolates. The two primers were combined to generate 21 genotypes. The C. albicans isolates obtained from the same patient and episode showed a diversity for fluconazole revealed by MIC50 and MIC90.

CONCLUSIONThe heterogeneity of the C. albicans colonies isolated from the same patients can be detected. C. albicans with varied fluconazole susceptibility and genotypic characteristics may coexist in the same oral Candida population.

Adult ; Aged ; Antifungal Agents ; pharmacology ; Candida albicans ; classification ; genetics ; isolation & purification ; Candida glabrata ; classification ; isolation & purification ; Candidiasis, Oral ; microbiology ; China ; DNA, Fungal ; analysis ; Drug Resistance, Fungal ; genetics ; Female ; Fluconazole ; pharmacology ; Genetic Heterogeneity ; Genotype ; Hematologic Neoplasms ; drug therapy ; Humans ; Male ; Microbial Sensitivity Tests ; Middle Aged ; Mouth Mucosa ; microbiology ; Mycology ; methods ; Neoplasms ; drug therapy ; Young Adult

ATP sulfurylase (ATPS,EC 2.7.7.4) reversibly catalyzes the reaction between ATP and sulfate to produce APS and pyrophosphate (PPi), and has been used in pyrosequencing. The gene coding ATP sulfurylase was amplified from the genomic DNA of Saccharomyces cerevisias (CICC 1202), and cloned into prokaryotic expression plasmid pET28a( + ) to provide a recombinant expression plasmid pET28a( + )-ATPS. Upon IPTG induction, ATP sulfurylase was produced by E. coli BL21 (DE3) harboring the recombinant expression plasmid pET28a( + )-ATPS. The relative molecular weight of recombinant ATP sulfurylase with His tag was about 60 kD. The recombinant ATP sulfurylase with electrophoretic pure grade was obtained only by two purification steps: His * Bind Resin affinity chromatography and ultrafiltration. The specific activity of the purified recombinant ATP sulfurylase was as high as 5.1 x 10(4) u/mg. The successful application of the enzyme in pyrosequencing was also demostrated.
Escherichia coli ; genetics ; metabolism ; Fungal Proteins ; genetics ; metabolism ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Saccharomyces cerevisiae ; enzymology ; genetics ; Sequence Analysis, DNA ; methods ; Sulfate Adenylyltransferase ; biosynthesis ; genetics

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

OBJECTIVETo screen out fungus strains with acetylcholinesterase inhibitory activity from Huperzia serrata.

METHODEndophytic fungi fermentation products from 59 H. serrata strains were stained with acetylcholinesterase hydrolyzed alpha-naphthaleneacetic ethyl ester and fast blue B salt, and screened for acetylcholinesterase inhibitory activity with thin-layer chromatography-bioautography. Target strains were classified and identified through the sequence analysis on 18s rDNA and 5.8s rDNA combined with morphological characteristics.

RESULTFungus strain LQ2F01 from H. serrata showed positive color reaction in the screening for acetylcholinesterase inhibitory activity. The sequence analysis on 18s rDNA and 5.8s rDNA combined with morphological characteristics showed the strain LQ2F01 belonged to Acremonium.

CONCLUSIONEndophytic Fungi LQ2F01 from H. serrata shows identical acetylcholinesterase inhibitory activity with the host plant, which is of great significance to the development of natural medicines and the studies on the relationship between the endophytic gungi and the host plant.

Acetylcholinesterase ; metabolism ; Acremonium ; genetics ; metabolism ; Cholinesterase Inhibitors ; isolation & purification ; metabolism ; Chromatography, Thin Layer ; DNA, Fungal ; chemistry ; genetics ; DNA, Ribosomal ; chemistry ; genetics ; Diazonium Compounds ; metabolism ; Fungi ; classification ; genetics ; metabolism ; Huperzia ; microbiology ; Hydrolysis ; Naphthaleneacetic Acids ; metabolism ; Phylogeny ; RNA, Ribosomal, 18S ; classification ; genetics ; RNA, Ribosomal, 5.8S ; classification ; genetics ; Sequence Analysis, DNA