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

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

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

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

INTRODUCTIONUntil recently, vancomycin-resistant enterococcus (VRE) infection or colonisation was a rare occurrence in Singapore. The first major VRE outbreak involving a 1500-bed tertiary care institution in March 2005 presented major challenges in infection control and came at high costs. This study evaluates the predictors of VRE carriage based on patients' clinical and demographic profiles.

MATERIALS AND METHODSStudy patients were selected from the hospital inpatient census population during the VRE outbreak (aged 16 years or more). Clinical information from 84 cases and 377 controls were analysed.

RESULTSSignificant predictors of VRE carriage included: age>65 years Odds ratio (OR), 1.98; 95% CI (confidence interval), 1.14 to 3.43); female gender (OR, 2.15; 95% CI, 1.27 to 3.65); history of diabetes mellitus (OR, 1.94; 95% CI, 1.14 to 3.30), and staying in a crowded communal ward (OR, 2.75; 95% CI, 1.60 to 4.74). Each additional day of recent hospital stay also posed increased risk (OR, 1.03; 95% CI, 1.01 to 1.04).

CONCLUSIONElderly diabetic females with prolonged hospitalisation in crowded communal wards formed the profile that significantly predicted VRE carriage in this major hospital-wide outbreak of VRE in Singapore. It is imperative that active VRE surveillance and appropriate infection control measures be maintained in these wards to prevent future VRE outbreaks.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Case-Control Studies ; Cross Infection ; drug therapy ; epidemiology ; microbiology ; Disease Outbreaks ; Enterococcus ; drug effects ; Enterococcus faecalis ; isolation & purification ; Enterococcus faecium ; isolation & purification ; Female ; Humans ; Infection Control ; Male ; Medical Audit ; Middle Aged ; Risk Factors ; Singapore ; epidemiology ; Streptococcal Infections ; drug therapy ; epidemiology ; Vancomycin ; pharmacology ; therapeutic use ; Vancomycin Resistance

OBJECTIVETo investigate the changes in fecal flora and its correlation with the occurrence and progression of inflammatory bowel disease (IBD).

METHODSWe collected fresh fecal specimens from 167 IBD patients (including 113 with ulcerative colitis and 54 with Crohn's disease) and 54 healthy volunteers. The fecal flora was analyzed by gradient dilution method and the data of inflammatory markers including WBC, PLT, CRP and ESR were collected to assess the association between the fecal flora and the inflammatory markers.

RESULTSThe species Enterrococcus (6.60∓0.23, P<0.01), Saccharomyces (2.22∓0.27, P<0.05), Bacteriodes (5.57∓0.28, P<0.001), Bifidobacterium (5.08∓0.30, P<0.01), Peptococcus (6.22∓0.25, P<0.001), Lactobacillus (6.00∓0.26, P<0.001), and Clostridium (3.57∓0.30, P<0.05) all increased significantly, while Eubacterium (1.56∓0.24, P<0.01) reduced markedly in patients with ulcerative colitis compared with those in the control subjects. Enterrococcus (6.93∓0.28, P<0.01), Saccharomyces (2.73∓0.37, P<0.01), Bacteriodes (4.32∓0.52, P<0.05), Bifidobacterium (4.88∓0.42, P<0.05), Peptococcus (6.19∓0.32, P<0.01) and Lactobacillus (4.73∓0.47, P<0.001) all increased significantly and Eubacterium (1.01∓0.29, P<0.01) and Clostridium (0.87∓0.31, P<0.01) decreased in patients with Crohn's disease. The positivity rates of bacterial culture were consistent with the results of quantitative analysis of the fecal flora. The changes in fecal flora did not show a significant correlation with these inflammatory markers.

CONCLUSIONIBD patients have fecal flora imbalance compared with the healthy controls, and this imbalance may contribute to the occurrence and progression of IBD. The decline of Eubacterium contributes to the occurrence and development of IBD.

Adult ; Bacteria ; isolation & purification ; Bacteroides ; isolation & purification ; Bifidobacterium ; isolation & purification ; Biomarkers ; analysis ; Clostridium ; isolation & purification ; Colitis, Ulcerative ; microbiology ; Crohn Disease ; microbiology ; Enterococcus ; isolation & purification ; Eubacterium ; isolation & purification ; Feces ; microbiology ; Female ; Humans ; Inflammatory Bowel Diseases ; etiology ; microbiology ; Lactobacillus ; isolation & purification ; Male ; Peptococcus ; isolation & purification ; Saccharomyces ; isolation & purification

Primary aortoenteric fistula (PAEF) is a rare but catastrophic cause of massive gastrointestinal bleeding. Diagnosis of PAEF is difficult to make and is frequently delayed without strong clinical suspicion. Timely surgical intervention is essential for patient's survival. We report on a case of an 86-year-old woman with no history of abdominal surgery, who presented with abdominal pain. Initially, computed tomography scan showed an intra-abdominal abscess, located anterior to the aortic bifurcation. However, she was discharged without treatment because of spontaneous improvement on a follow-up computed tomography scan, which showed a newly developed right common iliac artery aneurysm. One week later, she was readmitted due to recurrent abdominal pain. On the second day of admission, sudden onset of gastrointestinal bleeding occurred for the first time. After several endoscopic examinations, an aortoenteric fistula bleeding site was found in the sigmoid colon, and aortography showed progression of a right common iliac artery aneurysm. We finally concluded that intra-abdominal abscess induced an infected aortic aneurysm and enteric fistula to the sigmoid colon. This case demonstrated an extremely rare type of PAEF to the sigmoid colon caused by an infected abdominal aortic aneurysm, which has rarely been reported.
Abdominal Abscess/*diagnosis/microbiology ; Aged, 80 and over ; Aorta, Abdominal/radiography ; Aortic Aneurysm, Abdominal/*diagnosis/etiology ; Bacteroides/isolation & purification ; Bacteroides fragilis/isolation & purification ; Colon, Sigmoid/radiography ; Colonoscopy ; Enterococcus/isolation & purification ; Female ; Fistula/*diagnosis ; Humans ; Tomography, X-Ray Computed

OBJECTIVETo study the prevalence of Parvimonas micra (Pm) and the associations between Pm and pulp dominant pathogens in order to reflect the colonization of Pm in the infected root canals with chronic periradicular periodontitis.

METHODSA total of 120 teeth diagnosed as chronic periradicular periodontitis from 104 patients were included into the study. The teeth were allocated into untreated (primary infectious) and root-canal- treated (secondary infectious) groups with 60 in either group. Samples were collected from the root canals using sterile files and paper points, and subsequent extraction of bacterial DNA was undertaken. The Pm 16S rDNA level was evaluated using 16S rDNA PCR. The prevalence of Pm in chronic periradicular periodontitis was determined accordingly. Then, the associations of Pm and Enterococcus faecalis (Ef), Porphyromonas endodontalis (Pe) as well as Porphyromonas gingivalis (Pg) were analysed.

RESULTSPm was detected in 40% (24/60) of the samples from the primary infectious group, 5% (3/60) from the secondary infectious group. The prevalences of Pm from the two groups were different significantly (χ² = 21.06, P < 0.05). Significant correlations (untreated group OR = 5.98, root-canal-treated group OR = 33.50) between Pm and Pe were identified in both groups, while the correlations between Pm and Pg as well as Ef were not of significance, respectively.

CONCLUSIONSA significantly higher relevance ratio of Pm was estimated in the primary infectious group than the secondary infectious one. Pm and Pe were correlated significantly in the infected root canals, suggesting a symbiotic relation between these two bacteria.

Chronic Periodontitis ; DNA, Bacterial ; Dental Pulp Cavity ; microbiology ; Enterococcus faecalis ; isolation & purification ; Humans ; Periapical Periodontitis ; microbiology ; Polymerase Chain Reaction ; Porphyromonas endodontalis ; isolation & purification ; Porphyromonas gingivalis ; isolation & purification ; Root Canal Therapy

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

BACKGROUND: Patients infected with vancomycin-resistant enterococci (VRE) are kept in isolation to prevent the spread of VRE in medical facilities. However, decision-making regarding isolation can be challenging at the time of re-admission of previously VRE-colonized or infected patients who have not been examined for VRE infections for a long time. This study focused on providing guidelines for isolating VRE patients based on the analysis of risk factors for prolonged carriage and reacquisition of VRE. METHODS: A retrospective review was performed on medical records of patients who were diagnosed with VRE infections at a university hospital in 2009. Durations of colonization and negative conversion of VRE were estimated by Kaplan-Meier methods. Prolonged duration of VRE infections and risk factors for reacquisition were analyzed using Cox's proportional hazard model. RESULTS: Among 220 VRE-colonized patients, 132 were cleared, and 30 reacquired after negative conversion of VRE. The median duration of colonization was 33.1 weeks, and the median clearance period was 19.4 weeks. Patients who were admitted via the emergency department and treated with glycopeptides tended to have prolonged duration of VRE colonization. Prolonged hospitalization and metronidazole therapy increased the risk of reacquisition more rapidly. CONCLUSION: Treatment with glycopeptides, metronidazole antibiotic therapy, history of admission via the emergency department, and prolonged hospitalization can affect to prolonged carriage and reacquisition of VRE. Consider carefully the release of isolation of VRE patients with these risk factors.
Colon ; Emergency Service, Hospital ; Enterococcus ; Glycopeptides ; Hospitalization ; Humans ; Medical Records ; Metronidazole ; Patient Isolation ; Proportional Hazards Models ; Retrospective Studies ; Risk Factors* ; Vancomycin Resistance