In order to develop a rapid and reliable method for B. cereus genotyping, factors influencing PFGE results, including preparation of bacterial cells embedded in agarose, lysis of embedded cells, enzymatic digestion of intact genomic DNA, and electrophoresis parameters allowing for reproducible and meaningful DNA fragment separation, were controlled. Optimal cellular growth (Luria-Bertani agar plates for 12-18 h) and lysis conditions (4 h incubation with 500 µg/mL lysozyme) produced sharp bands on the gel. Restriction enzyme NotI was chosen as the most suitable. Twenty-two isolates were analyzed by NotI digestion, using three electrophoretic parameters (EPs). The EP-a was optimal for distinguishing between isolates. The optimized protocol could be completed within 40 h which is a significant improvement over the previous methods.
Bacillus cereus ; genetics ; isolation & purification ; Bacteriological Techniques ; DNA, Bacterial ; chemistry ; genetics ; Electrophoresis, Gel, Pulsed-Field ; methods

Naringin has been reported to possess a wild range of biological activities. However, the route and metabolites of naringin produced by intestinal bacteria are not well understood. In this paper, different bacteria were isolated from human feces and their abilities to convert naringin to different metabolites were studied. Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) with automated data analysis software (MetaboLynx) was applied to fast analysis of naringin metabolites. Using MSE and mass defect filter techniques, three metabolites were detected and tentatively identified. The results indicated that acetylation, hydrolyzation and hydrolyzation with hydrogenation were the major metabolic pathways of naringin in vitro. Then, we studied the gene sequence of the 16S rRNA of the bacteria by extraction of genomic DNA of the strain, PCR amplification and clone of the 16S rRNA. The consequence proved that Enterococcus sp.30, Bacillus sp.46, Escherichia sp.54 and Escherichia sp.63 have the peculiar metabolism characteristic of naringin.
Bacillus ; genetics ; isolation & purification ; metabolism ; Chromatography, High Pressure Liquid ; Enterococcus ; genetics ; isolation & purification ; metabolism ; Escherichia ; genetics ; isolation & purification ; metabolism ; Feces ; microbiology ; Female ; Flavanones ; metabolism ; Humans ; Metabolic Networks and Pathways ; Phylogeny ; RNA, Ribosomal, 16S ; genetics ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Bacillus (B.) anthracis, the etiological agent of anthrax, is one of the most genetically monomorphic bacteria species in the world. Due to the very limited genetic diversity of this species, classification of isolates of this bacterium requires methods with high discriminatory power. Single nucleotide repeat (SNR) analysis is a type of variable-number tandem repeat assay that evaluates regions with very high mutation rates. To subtype a collection of 21 isolates that were obtained during a B. anthracis outbreak in Korea, we analyzed four SNR marker loci using nucleotide sequencing analysis. These isolates were obtained from soil samples and the Korean Center for Disease Control and Prevention. The SNR analysis was able to detect 13 subgenotypes, which allowed a detailed evaluation of the Korean isolates. Our study demonstrated that the SNR analysis was able to discriminate between strains with the same multiple-locus variable-number tandem repeat analysis genotypes. In summary, we obtained SNR results for four SNR marker loci of newly acquired strains from Korea. Our findings will be helpful for creating marker systems and help identify markers that could be used for future forensic studies.
Bacillus anthracis/*classification/*genetics/isolation & purification ; *Genetic Variation ; *Minisatellite Repeats ; Polymerase Chain Reaction/veterinary ; Republic of Korea ; Sequence Analysis, DNA/*methods/veterinary ; *Soil Microbiology

BACKGROUND: The aims of this study were to compare several DNA extraction methods and 16S rDNA primers and to evaluate the clinical utility of broad-range PCR in continuous ambulatory peritoneal dialysis (CAPD) culture fluids. METHODS: Six type strains were used as model organisms in dilutions from 10(8) to 100 colony-forming units (CFU)/mL for the evaluation of 5 DNA extraction methods and 5 PCR primer pairs. Broad-range PCR was applied to 100 CAPD culture fluids, and the results were compared with conventional culture results. RESULTS: There were some differences between the various DNA extraction methods and primer sets with regard to the detection limits. The InstaGene Matrix (Bio-Rad Laboratories, USA) and Exgene Clinic SV kits (GeneAll Biotechnology Co. Ltd, Korea) seem to have higher sensitivities than the others. The results of broad-range PCR were concordant with the results from culture in 97% of all cases (97/100). Two culture-positive cases that were broad-range PCR-negative were identified as Candida albicans, and 1 PCR-positive but culture-negative sample was identified as Bacillus circulans by sequencing. Two samples among 54 broad-range PCR-positive products could not be sequenced. CONCLUSIONS: There were differences in the analytical sensitivity of various DNA extraction methods and primers for broad-range PCR. The broad-range PCR assay can be used to detect bacterial pathogens in CAPD culture fluid as a supplement to culture methods.
Bacillus/genetics/isolation & purification ; Bacteria/genetics/*isolation & purification ; Candida albicans/genetics/isolation & purification ; DNA Primers/genetics ; DNA, Bacterial/*analysis/isolation & purification ; *Genetic Techniques/standards ; Humans ; Peritoneal Dialysis, Continuous Ambulatory ; Peritonitis/*microbiology ; Polymerase Chain Reaction ; Reagent Kits, Diagnostic ; Sequence Analysis, DNA

OBJECTIVETo isolate and characterize endophytic fungi from seven Dendrobium species, and detect their antimicrobial activities.

METHODFungal endophytes were isolated by strictly sterile sample preparation and fungal identification methods were based on their ITS ribosomal DNA (ITS rDNA gene) sequences. The agar well diffusion method was then employed to evaluate the antimicrobial activity against six pathogenic organisms and the phylogenetic tree of active isolates was constructed by the MEGA.

RESULTNinety-eight endophytic fungi obtained from seven Dendrobium spp., and among them twenty-four isolates, representing 11 genera and 14 species, displayed anti-microbial activities. The phylogenetic assay based on ITS-rDNA showed that 24 active isolates were sorted to 7 taxonomic orders: Hypocreales, Sordariales, Capnodiales, Eurotiales, Botryosphaeriales, Xylariales and Mucorales. The results of antimicrobial activity assay revealed that 1.02%, 10.2%, 18.4%, 1.02%, 1.02% and 10.2% of fermentation broths of 98 isolates displayed significant antimicrobial activities against E. coli, B. subtilis, S. aureus, C. albicans, C. neoformans and A. fumigatus, respectively. Four strains DL-R-3, DL-S-6, DG-R-10 and DN-S-1 displayed strong and broad antimicrobial spectrum.

CONCLUSIONEndophytic fungi associated with Dendrobium species have fungal diversity, and possess diverse antimicrobial activity.

Anti-Infective Agents ; metabolism ; pharmacology ; Aspergillus fumigatus ; drug effects ; Bacillus subtilis ; drug effects ; Base Sequence ; Biodiversity ; Candida albicans ; drug effects ; China ; Cryptococcus neoformans ; drug effects ; DNA, Fungal ; chemistry ; isolation & purification ; DNA, Ribosomal Spacer ; chemistry ; genetics ; Dendrobium ; microbiology ; physiology ; Endophytes ; classification ; genetics ; isolation & purification ; physiology ; Escherichia coli ; drug effects ; Fungi ; classification ; genetics ; isolation & purification ; physiology ; Microbial Sensitivity Tests ; Molecular Sequence Data ; Phylogeny ; Plant Roots ; microbiology ; physiology ; Plant Stems ; microbiology ; physiology ; Sequence Alignment ; Sequence Analysis, DNA ; Staphylococcus aureus ; drug effects

OBJECTIVETo isolate novel actinomycetes and to evaluate their antibacterial activity.

METHODSThree soil samples were collected from Vengodu (village) in Kanchipuram district, Tamil Nadu, India. Actinomycetes were isolated using serial dilution and plating method on actinomycetes isolation agar.

RESULTSTotally 35 isolates were obtained on the basis of colony characteristics on actinomycetes isolation agar. All the isolates were screened for antibacterial activity by cross streak method. Medium and optimization of day were done for the potent strains using Nathan's agar well diffusion method. Isolation of bioactive compounds from significant active isolates was done by using different media. The most active isolate VAS 10 was identified as Actinobacterium Loyola PBT VAS 10 (accession No. JF501398) using 16s rRNA sequence method. The hexane, ethyl acetate, dichloromethane and butanol extracts of VAS 10 were tested against bacteria. The maximum antibacterial activity was observed in dichloromethane and ethyl acetate; maximum zones of inhibition were observed against Enterococcus durans. The rRNA secondary structure and the restriction sites of Actinobacterium Loyola VAS 10 were predicted using Genebee and NEBCutter online tools respectively.

CONCLUSIONSThe present study showed that among the isolated actinomycetes, Actinobacterium Loyola PBT VAS 10 (accession No. JF501398) showed good antibacterial activity against the tested bacteria.

Actinobacteria ; chemistry ; isolation & purification ; physiology ; Animals ; Anti-Bacterial Agents ; pharmacology ; Antibiosis ; physiology ; Bacillus subtilis ; drug effects ; Enterobacter aerogenes ; drug effects ; Escherichia coli ; drug effects ; India ; Microbial Sensitivity Tests ; Phylogeny ; RNA, Ribosomal, 16S ; genetics ; Soil Microbiology ; Species Specificity ; Vibrio parahaemolyticus ; drug effects

We screened a strain NK13 for a certain extent asymmetric hydrolysis the rac-ketoprofen Chloroethyl ester to (S)-Ketoprofen. As identified, NK13 was Bacillus megaterium. Digested NK13 genomic DNA with Sau3AI partially and recovered the fragment from 2 kb to 6 kb, cleaved the plasmid of pUC18 with BamH I, ligated the 2-6 kb fragment of NK13 genomic DNA into pUC18 plasmid, and then transformed an Escherichia coli strain DH5alpha. We created the gene library of NK13 and obtained a positive clone, pUC-NK1 in the library from the tributyrin flat. The result of sequencing showed that there was a whole open read frame (ORF) of 633 bp lipase gene in the plasmid of pUC-NK1. To compare with the genes of GenBank, this lipase gene was reported firstly (GenBank Accession No. EU381317). The lipase gene was amplified by PCR, using pUC-NK1 plasmid as template, and subcloned into the high expression vector pET21b(+) under the control of T7 promoter. The recombinant plasmid, pET-NKest1, was then transformed into an Escherichia coli strain BL21 (DE3) for the production of recombinant lipase protein. After 3 hours of induction by isopropyl-beta-D-thiogalactoside (IPTG), lipase was expressed. SDS-PAGE analysis showed that the relative molecular mass of the lipase protein was about 20 kDa. The result of high performance liquid chromatography (HPLC) showed that the conversion rate of the recombinant strain was fifty times than the wild strain NK13's. The (S)-Ketoprofen enantiomeric excess of the recombinant strain was 75.28%, which indicated that the lipase could hydrolyze (S)-Ketoprofen Chloroethyl ester firstly. If we research the conditions of the hydrolysis rac-ketoprofen Chloroethyl ester of this lipase further, maybe it could offer a foundation to product (S)-Ketoprofen industrially.
Amino Acid Sequence ; Bacillus megaterium ; genetics ; isolation & purification ; metabolism ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Ketoprofen ; analogs & derivatives ; chemistry ; isolation & purification ; Lipase ; biosynthesis ; genetics ; Molecular Sequence Data ; Open Reading Frames ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Stereoisomerism

OBJECTIVETo develop a fast, high-throughput screening method with suspension array technique for simultaneous detection of biothreat bacteria.

METHODS16 S rDNA universal primers for Bacillus anthracis, Francisella tularensis, Yersinia pestis, Brucella spp.and Burkholderia pseudomallei were selected to amplify corresponding regions and the genus-specific or species-specific probes were designed. After amplification of chromosomal DNA by 16 S rDNA primers 341A and 519B, the PCR products were detected by suspension array technique. The sensitivity, specificity, reproducibility and detection power were also analyzed.

RESULTSAfter PCR amplification by 16 S rDNA primers and specific probe hybridization, the target microorganisms could be identified at genus level, cross reaction was recognized in the same genus. The detection sensitivity of the assay was 1.5 pg/microl (Burkholderia pseudomallei), 20 pg/microl (Brucella spp.), 7 pg/microl (Bacillus anthracis), 0.1 pg/microl (Francisella tularensis), and 1.1 pg/microl (Yersinia pestis), respectively. The coefficient of variation for 15 test of different probes was ranged from 5.18% to 17.88%, it showed good reproducibility. The assay could correctly identify Bacillus anthracis and Yersinia pestis strains in simulated white powder samples.

CONCLUSIONThe suspension array technique could be served as an opening screening method for biothreat bacteria rapid detection.

Bacillus anthracis ; isolation & purification ; Bioterrorism ; prevention & control ; DNA Primers ; DNA, Bacterial ; analysis ; Francisella tularensis ; isolation & purification ; Oligonucleotide Array Sequence Analysis ; methods ; Polymerase Chain Reaction ; methods ; RNA, Ribosomal, 16S ; genetics ; Yersinia pestis ; isolation & purification

Laser scanning confocal microscope (LSCM) is currently the only equipment to observe fluorescence. However, this technique has disadvantages such as high cost and long test process. In this study, we developed a new system of laser-induced fluorescence (LIF) for microfluidic chip applied to detecting the expression of green fluorescent protein (GFP) in Bacillus subtilis. This novel system was comprised of laser device, optics unit, microfluidic chip, photomultiplier and computer treatment unit. The tests indicated that microfluidic chip could detect the expression of GFP as sensitively as LSCM in Bacillus subtilis. Moreover, this LIF detection system could instead of PCR to identify the positive clone in this special case. Nevertheless, the LIF system only was suitable to detect the fluorescent strength of GFP, and could not meet the request of some cases for example protein location. Therefore, this system will be applied in environmental detection with microbe, drug discovery and other cases.
Bacillus subtilis ; isolation & purification ; metabolism ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Microfluidic Analytical Techniques ; methods

OBJECTIVETo characterize the heterotrophic nitrifying bacteria.

METHODSThe bacteria were isolated from membrane bioreactor for treating synthetic wastewater using the method newly introduced in this study. Fluorescence in situ hybridization (FISH) was used to validate the nonexistence of autotrophic ammonia oxidizers and nitrite oxidizers. Batch tests were carried out to investigate the capability of heterotrophic nitrification by the pure culture. Phylogenetic analysis of the pure culture was performed.

RESULTSA heterotrophic nitrifier, named Bacillus sp. LY, was newly isolated from the membrane bioreactor system in which the efficiency of TN removal was up to 80%. After 24-day, incubation, the removal efficiency of COD by Bacillus sp. LY was 71.7%. The ammonium nitrogen removal rate after assimilation nearly ceased by Bacillus sp. LY was 74.7%. The phylogenetic tree of Bacillus sp. LY and the neighbouring nitrifiers were given.

CONCLUSIONSThe batch test results indicate that Bacillus sp. LY can utilize the organic carbon as the source of assimilation when it grows on glucose and ammonium chloride medium accompanying the formation of oxidized-nitrogen. It also can denitrify nitrate while nitrifying. Bacillus sp. LY may become a new bacterial resource for heterotrophic nitrification and play a bioremediation role in nutrient removal.

Bacillus ; classification ; genetics ; isolation & purification ; metabolism ; Base Sequence ; DNA Primers ; DNA, Ribosomal ; genetics ; Environmental Restoration and Remediation ; methods ; Nitrates ; metabolism ; Phylogeny ; RNA, Ribosomal, 16S ; genetics