BACKGROUND: We developed and evaluated the utility of a multiplex real-time PCR assay that uses melting curve analysis and allows simultaneous identification of vancomycin-resistant genotypes and clinically relevant enterococci. METHODS: The specificity of the assay was tested using 4 reference strains of vancomycin-resistant enterococci (VRE) and 2 reference strains of vancomycin-susceptible enterococci. Ninety-three clinical isolates of enterococci with different glycopeptide-resistant phenotypes were genotyped and identified using a multiplex real-time PCR assay and melting curve analysis. RESULTS: Representative melting curves were obtained for Enterococcus faecium, Enterococcus faecalis, vanA-containing E. faecium, vanB-containing E. faecalis, Enterococcus gallinarum, and Enterococcus casseliflavus. Phenotypic and genotypic analysis of the isolates revealed same results for 82 enterococcal isolates, while in 4 isolates, the glycopeptide-resistant phenotypes were inconsistent with the glycopeptide-resistant genotypes and in the 4 other isolates, species could not be accurately identified. Three isolates with mixed strains, which were detected by the PCR assay, could not be correctly identified using phenotypic methods. CONCLUSIONS: VRE genotyping and identification of clinically relevant enterococci were rapidly and correctly performed using multiplex real-time PCR assay and melting curve analysis.
Bacterial Proteins/genetics ; Carbon-Oxygen Ligases/genetics ; DNA, Bacterial/genetics ; Enterococcus/genetics/*isolation &purification ; Enterococcus faecalis/genetics/isolation &purification ; Enterococcus faecium/genetics/isolation &purification ; Genotype ; Nucleic Acid Denaturation ; Peptide Synthases/genetics ; Phenotype ; *Polymerase Chain Reaction ; Vancomycin Resistance/*genetics

OBJECTIVETo investigate the presence of pathogenicity island (PAI)-associated genes in the enterococcal isolates.

METHODSUsing PCR and hybridization methods, PAI-associated genes were detected in 155 enteococcal strains isolated from clinical patients and healthy individuals.

RESULTSAmong the 155 enterococcal isolates, 137 (88.39%) carried at least one of PAI-associated genes, namely hyd (positivity rate of 81.94%), psaA (78.06%), nuc (57.42%), esp (53.55%), cylB (52.90%), and gls24-like (38.06%) genes. Expect for esp gene, the other 5 genes showed higher positivity rates in the E. faecalis strains than in the E. faecium strains, and this difference was statistically significant for the genes nuc, cylB, and gls24-like. The positivity rates and the number of these genes in the E. faecalis from clinical isolates were both significantly higher than those in the strains isolated from healthy individuals.

CONCLUSIONThe data show a wide distribution of the PAI-associated genes among the enterococcal strains, and E. faecalis strains are more likely than E. faecium strains to be positive for the 6 genes, which are present at significant higher rates in the clinically isolated samples than in that from healthy individuals.

Bacterial Proteins ; chemistry ; genetics ; Enterococcus ; genetics ; isolation & purification ; pathogenicity ; Enterococcus faecalis ; genetics ; isolation & purification ; pathogenicity ; Genomic Islands ; genetics ; Gram-Positive Bacterial Infections ; microbiology ; Humans ; Membrane Proteins ; genetics ; Virulence ; genetics

BACKGROUND: Accurate and early detection of vancomycin-resistant enterococci (VRE) is critical for controlling nosocomial infection. In this study, we evaluated the usefulness of a selective chromogenic agar medium and of multiplex PCR for detection of VRE, and both these techniques were compared with the conventional culture method for VRE detection. METHODS: We performed the following 3 methods for detecting VRE infection in stool specimens: the routine culture method, culturing in selective chromogenic agar medium (chromID VRE, bioMerieux, France), and multiplex PCR using the Seeplex(R) VRE ACE Detection kit (Seegene Inc., Korea) with additional PCR for vanC genes. RESULTS: We isolated 109 VRE strains from 100 stool specimens by the routine culture method. In chromID VRE, all the isolates showed purple colonies, including Enterococcus gallinarum and E. raffinosus, which were later identified using the Vitek card. All VRE isolates were identified by the multiplex PCR method; 100 were vanA-positive E. faecium, 8 were vanA- and vanC-1-positive E. gallinarum, and 1 was vanA-positive E. raffinosus. CONCLUSIONS: For VRE surveillance, culturing the isolates in chromID VRE after broth enrichment appears to be an accurate, rapid, and easy method for routine screening test. Multiplex PCR is relatively expensive and needs skilled techniques for detecting VRE, but it can be an auxiliary tool for rapid detection of genotype during a VRE outbreak.
Agar/chemistry ; Chromogenic Compounds/*chemistry ; Enterococcus/drug effects/genetics/*isolation & purification ; Enterococcus faecium/genetics/isolation & purification ; Feces/microbiology ; Genotype ; Humans ; Phenotype ; Polymerase Chain Reaction/*methods ; Reagent Kits, Diagnostic ; *Vancomycin Resistance

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

Screening of microorganisms producing flocculating substances was carried out. A strain secreting a large amount of bioflocculant was isolated from wastewater samples collected from the Little Moon River in Beijing. Based on the morphological properties and 16S rDNA sequence analysis, the isolate (designated W31) was classified as Vagococcus sp. A bioflocculant (named MBFW31) produced by W31 was extracted from the culture broth by ethanol precipitation and purified by gel chromatography. MBFW31 was heat-stable and had strong flocculating activity in a wide range of pH with relatively low dosage requirement. MBFW31 was identified as a polysaccharide with molecular weight over 2 x 10(6). It contained neutral sugar and uronic acid as its major and minor components, respectively. Infrared spectra showed the presence of hydroxyl, carboxyl and methoxyl group in its molecules. The present results suggested that MBFW31 had potential application in wastewater treatment.
Carbohydrates ; analysis ; chemistry ; Enterococcus ; genetics ; isolation & purification ; metabolism ; Flocculation ; Species Specificity ; Waste Disposal, Fluid ; methods ; Water Microbiology ; Water Pollutants ; isolation & purification

OBJECTIVETo investigate the homology and resistant mechanism of vancomycin-resistant Enterococci (VRE) isolates.

METHODSA total of 9 VRE isolates were collected from 2006 to 2007 at PUMC hospital. The susceptibility of these isolates to 10 different antibiotics including vancomycin was tested by E-test. These strains were processed by brain heart infusion agar screening in the presence of vancomycin (6 microg/ml), and were analyzed for genotypic characteristics using the multiplex PCR. The homology of the isolates was determined by pulsed-field gel electrophoresis (PFGE).

RESULTSAll the 9 VRE isolates were identified as Enterococci faecium. The visual analysis of PFGE patterns revealed 6 different PFGE types. The vanA gene was confirmed by PCR and sequencing in 9 VRE isolates, which were consistent between phenotype and genotype for glycopeptides resistance.

CONCLUSIONSOnly vanA genotype was detected in PUMC hospital. Clonal dissemination, horizontal gene transfer, and the selective pressure of antimicrobial agents may contribute to the increase of VRE.

Bacterial Proteins ; genetics ; Bacterial Typing Techniques ; Drug Resistance, Multiple, Bacterial ; Enterococcus faecium ; classification ; drug effects ; genetics ; isolation & purification ; Gram-Positive Bacterial Infections ; microbiology ; Humans ; Vancomycin Resistance

OBJECTIVETo identify a new peptide deformylase (PDF) gene (Genebank Accession AY238515) from Enterococcus faecium and to establish a new screening model targeted on PDF.

METHODSA new PDF gene was identified by BLAST analysis and PCR and was subsequently over-expressed in the prokaryotic expression host E. coli B121(DE3). Over-expressed protein was purified for enzymatic assay by metal affinity chromatography and a new screening model was established for novel antibiotics.

RESULTA new PDF gene of Enterococcus faecium was identified successfully. Ten positive samples were picked up from 8000 compound library and the microbial fermentation broth samples.

CONCLUSIONA new PDF of gene Enterococcus faecium was first identified and the model had a high efficacy. Positive samples screened may be antibacterial agents of broad spectrum.

Amidohydrolases ; antagonists & inhibitors ; genetics ; isolation & purification ; Amino Acid Sequence ; Anti-Bacterial Agents ; therapeutic use ; Cloning, Molecular ; Drug Evaluation, Preclinical ; Enterococcus faecium ; enzymology ; genetics ; Fluorescamine ; Fluorescence ; Hydrogen-Ion Concentration ; Indicators and Reagents ; Kinetics ; Models, Molecular ; Molecular Sequence Data ; Temperature

BACKGROUND: The aim of this study was to evaluate a newly developed PCR-based reverse blot hybridization assay (PCR-REBA), REBA Sepsis-ID (M&D, Wonju, Korea), to rapidly detect the presence of bacteremia and antimicrobial resistance gene in blood culture samples. METHODS: One thousand four hundred consecutive blood culture samples from patients with a delta neutrophil index greater than 2.7% were selected from March to July in 2013. Three hundred positive and 1,100 negative for bacterial growth in blood culture bottles samples were tested by conventional and real-time PCR-REBA, respectively. RESULTS: The overall agreement between the conventional identification test and the REBA Sepsis-ID test was 95.3% (286/300). Agreement for gram-positive bacteria, gram-negative bacteria, fungi, and polymicrobials was 94.5% (190/201), 97.3% (71/73), 100% (14/14), and 91.7% (11/12), respectively. The detection rate of the mecA gene from methicillin-resistant Staphylococcus isolates was 97.8% (90/92). The vanA gene was detected in one blood culture sample from which vancomycin-resistant Enterococcus was isolated. When the cycle threshold for real-time PCR was defined as 30.0, 2.4% (26/1,100) of negative blood culture samples tested positive by real-time PCR. CONCLUSIONS: The REBA Sepsis-ID test is capable of simultaneously and quickly detecting both causative agents and antimicrobial resistance genes, such as mecA and van, in blood culture positive samples.
Bacteremia/microbiology ; Bacterial Proteins/*genetics ; Bacteriological Techniques/*methods ; Carbon-Oxygen Ligases/*genetics ; Drug Resistance, Bacterial/genetics ; Enterococcus/*genetics/isolation & purification ; Humans ; Methicillin-Resistant Staphylococcus aureus/*genetics/isolation & purification ; *Nucleic Acid Hybridization ; RNA, Ribosomal, 16S/analysis ; Reagent Kits, Diagnostic ; *Real-Time Polymerase Chain Reaction

The crude extracts of the fermentation broth from a marine sediment-derived actinomycete strain, Saccharothrix sp. 10-10, showed significant antibacterial activities against drug-resistant pathogens. A genome-mining PCR-based experiment targeting the genes encoding key enzymes involved in the biosynthesis of secondary metabolites indicated that the strain 10-10 showed the potential to produce tetracenomycin-like compounds. Further chemical investigation of the cultures of this strain led to the identification of two antibiotics, including a tetracenomycin (Tcm) analogs, Tcm X (1), and a tomaymycin derivative, oxotomaymycin (2). Their structures were identified by spectroscopic data analysis, including UV, 1D-NMR, 2D-NMR and MS spectra. Tcm X (1) showed moderate antibacterial activities against a number of drug-resistant pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) pathogens, with the MIC values in the range of 32-64 microg x mL(-1). In addition, 1 also displayed significant cytotoxic activities against human cancer cell lines, including HL60 (leukemia), HepG2 (liver), and MCF-7 (breast) with the IC 50 values of 5.1, 9.7 and 18.0 micromol x L(-1), respectively. Guided by the PCR-based gene sequence analysis, Tcm X (1) and oxotomaymycin (2) were identified from the genus of Saccharothrix and their 13C NMR data were correctly assigned on the basis of 2D NMR spectroscopic data analysis for the first time.
Actinomycetales ; chemistry ; genetics ; Anti-Bacterial Agents ; chemistry ; isolation & purification ; pharmacology ; Antineoplastic Agents ; chemistry ; isolation & purification ; pharmacology ; Benzodiazepinones ; chemistry ; isolation & purification ; pharmacology ; Cell Line, Tumor ; Data Mining ; methods ; Drug Resistance, Bacterial ; Enterococcus faecalis ; drug effects ; Fermentation ; Genomics ; Humans ; Inhibitory Concentration 50 ; Marine Biology ; Methicillin-Resistant Staphylococcus aureus ; drug effects ; Microbial Sensitivity Tests ; Molecular Structure ; Naphthacenes ; chemistry ; isolation & purification ; pharmacology ; Phylogeny ; Staphylococcus epidermidis ; drug effects

In July 2010, we identified an outbreak of vancomycin-resistant enterococci (VRE) in our 26-bed neonatal intensive care unit. We performed an epidemiological investigation after clinical cultures of 2 neonates were positive for VRE. Identification, susceptibility testing, and molecular characterization were performed. Cultures of 3 surveillance stool samples of inpatients and 5 environmental samples were positive for VRE. All isolates were identified as Enterococcus faecium containing the vanA gene. Two distinct clones were identified by performing pulsed-field gel electrophoresis. The 2 clones exhibited different pulsotypes, but they represented identical Tn1546 types. Two sequence types, ST18 and ST192, were identified among all of the isolates with multilocus sequence typing. Our investigation determined that the outbreak in the neonatal intensive care unit was caused by 2 genetically different clones. The outbreak may have occurred through clonal spread and horizontal transfer of the van gene.
Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/genetics ; Bacterial Typing Techniques ; Carbon-Oxygen Ligases/genetics ; DNA, Bacterial/analysis ; *Disease Outbreaks ; Electrophoresis, Gel, Pulsed-Field ; Enterococcus faecium/drug effects/*genetics/isolation & purification ; Feces/microbiology ; Genotype ; Gram-Positive Bacterial Infections/diagnosis/epidemiology/*microbiology ; Humans ; Infant, Newborn ; Intensive Care Units, Neonatal ; Male ; Multilocus Sequence Typing ; Vancomycin/pharmacology ; *Vancomycin Resistance