OBJECTIVETo determine the antibiotics resistance, aminoglycoside-modifying enzymes and homology of high-level gentamycin resistant enterococcus in clinical specimens.

METHODSThe high-level gentamicin resistant (HLGR) isolates were screened by the agar method and the resistance of 14 antimicrobial agents was determined by K-B method. The aminoglycoside-modifying enzyme genes were detected by polymerase chain reaction (PCR). Pulsed-field gel electrophoresis (PFGE) was used to analyze the homology of HLGR isolates.

RESULTSThe ratio of HLGR was 64.2% (68/106). Among the HLGR,there were no isolates resistant to linezolid, vancomycin and tecoplanin, and Enterococcus faecium was more resistant to beta-lactam antibiotics and quinolone than Enterococcus faecalis. The positive rate of aac(6')-Ie-aph(2')-Ia was 92.6% and 3 isolates had the resistance gene mostly similar to aph(2')-Id. And among 51 HLGR isolates from the hospitalized patients, PFGE grouped 17 E. faecalis isolates into 4 clusters (A-D), and 33 E. faecium isolates into 8 clusters (A-H) with A cluster as predominant.

CONCLUSIONHLGR has become the important antibiotic resistance bacteria which results in nosocomial infection; and aac(6')-Ie-aph(2')-Ia is the main aminoglycoside-modifying enzyme gene which causes HLGR.

Drug Resistance, Bacterial ; genetics ; Electrophoresis, Gel, Pulsed-Field ; Enterococcus ; drug effects ; genetics ; Enterococcus faecalis ; drug effects ; genetics ; Enterococcus faecium ; drug effects ; genetics ; Gentamicins ; pharmacology ; Humans ; Kanamycin Kinase ; genetics ; Microbial Sensitivity Tests

An increase in vancomycin-resistant enterococcal (VRE) bacteremia in hemato-oncological patients (n=19) in our institution from 2000 through 2001 led us to analyze the molecular epidemiologic patterns and clinical features unique to our cases. The pulsed field gel electrophoresis of the isolates revealed that the bacteremia was not originated from a single clone but rather showed endemic pattern of diverse clones with small clusters. A different DNA pattern of blood and stool isolates from one patient suggested exogenous rather than endogenous route of infection. Enterococcus faecium carrying vanA gene was the causative pathogen in all cases. Patients with VRE bacteremia showed similar clinical courses compared with those with vancomycin-susceptible enterococcal (VSE) bacteremia. Vancomycin resistance did not seem to be a poor prognostic factor because of similar mortality (5/8, 62.5%) noted in VSE bacteremia. Initial disease severity and neutropenic status may be major determinants of prognosis in patients with VRE bacteraemia.
Adolescent ; Adult ; Bacteremia/*drug therapy/microbiology ; Bacterial Proteins/genetics ; Carbon-Oxygen Ligases/genetics ; Electrophoresis, Gel, Pulsed-Field ; Enterococcus/*drug effects/genetics ; Female ; Humans ; Male ; Middle Aged ; *Vancomycin Resistance

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

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

BACKGROUNDVancomycin-resistant Enterococci (VRE) cause serious infections that are difficult to treat. We carried out this study to determine the mutant prevention concentration (MPC) of linezolid when combined with minocycline against VRE strains, to determine the mechanism of drug resistance in vitro, and to provide a theoretical basis for the rational use of drugs against VRE.

METHODSThe minimum inhibitory concentrations (MICs) of linezolid and minocycline against 30 Enterococci (E.) isolates (including 20 VRE strains) were determined by the broth microdilution method. Drug interactions were assessed by the checkerboard microdilution tests and confirmed by time-kill studies. Two vancomycin-susceptible strains N27 and N40 (linezolid MIC, 2 g/ml; minocycline MIC, 4 µg/ml) and control strains E. faecalis ATCC 29212 and ATCC 51299 were also tested. The MPCs of linezolid and minocycline (alone and combined) were determined using the agar dilution method. Strains showing stable resistance were analyzed by polymerase chain reaction (PCR) amplification of domain V of the 23S rRNA gene.

RESULTSCheckerboard titration studies revealed synergistic effects of combination therapy in 26.7% of 30 E. isolates. Antagonism was not observed. The G2576U mutation was detected in stable linezolid-resistant strains of ATCC 29212, N40, and N27 before and after resistance screening, and MIC values increased with the number of G2576U mutations. The MPC of linezolid against E. decreased dramatically when combined with minocycline, and vice versa.

CONCLUSIONLinezolid or minocycline alone produce resistant strains; however, their joint use may reduce the MPC of each agent against VRE, thereby decreasing resistant mutants and bacterial infections.

Acetamides ; pharmacology ; Anti-Bacterial Agents ; pharmacology ; Anti-Infective Agents ; pharmacology ; Drug Therapy, Combination ; Enterococcus ; drug effects ; genetics ; Linezolid ; Microbial Sensitivity Tests ; Minocycline ; pharmacology ; Mutation ; Oxazolidinones ; pharmacology ; Vancomycin Resistance

BACKGROUND: Active screening for vancomycin-resistant enterococci (VRE) using rectal specimens is recommended to limit the spread of antimicrobial resistance within certain high-risk populations. We evaluated the diagnostic performance of Vancomycin Resistance 3 Multiplexed Tandem PCR assay (AusDiagnostics, Australia), a rapid multiplex real-time PCR assay that detects vanA and/or vanB. METHODS: Two-hundred-and-eleven rectal swabs from Hematology and Oncology unit were submitted for VRE surveillance via direct detection of vanA and/or vanB by culture and by using Vancomycin Resistance 3 Multiplexed Tandem PCR assay. Enterococci were identified to the species level by using standard biochemical tests and BD Phoenix Automated Microbiology System (BD Diagnostic Systems, USA). Vancomycin susceptibility of enterococci was determined using Etest (BioMerieux, France). RESULTS: Compared to the culture method, Vancomycin Resistance 3 Multiplexed Tandem PCR assay had a sensitivity of 84.0%, specificity of 98.8%, positive predictive value (PPV) of 91.3%, and negative predictive value (NPV) of 97.6%. The assay failed to detect 18 (8.5%) specimens because of the presence of PCR inhibitors; of the remaining 193 specimens, 25 (12.9%) were positive, 23 for vanA, and 2 for vanB. Although both sensitivity and specificity for vanA VRE was 100% compared to the culture method, all vanB-positive specimens tested negative by VRE culture. CONCLUSIONS: Vancomycin Resistance 3 Multiplexed Tandem PCR assay is a rapid and laborsaving option for VRE surveillance for direct use on rectal swabs. However, the high rate of PCR failure owing to the inhibitors in the specimens and the low specificity for vanB should be considered when interpreting the results.
Bacterial Proteins/genetics ; Carbon-Oxygen Ligases/genetics ; DNA, Bacterial/*analysis ; Enterococcus/*drug effects/*genetics/growth & development/metabolism ; Humans ; *Multiplex Polymerase Chain Reaction ; Reagent Kits, Diagnostic ; Rectum/*microbiology ; Sensitivity and Specificity ; Vancomycin/*pharmacology ; Vancomycin Resistance/*genetics

BACKGROUNDThe incidence of vancomycin-resistant enterococci (VRE) appeared to be increasing in China, but very few nosocomial outbreaks have been reported. Our hospital had experienced an outbreak of VRE since March 2008 to March 2009. The objective of this study was to analyze the molecular features of the isolates and the control measures used to eradicate a VRE outbreak in a tertiary institution in China.

METHODSWe characterized VRE isolates from 21 infected and 11 colonized inpatients from a single hospital by pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), the analysis of Tn1546-like elements and virulence genes detection. Infection control measures, including more environmental disinfection, screening for VRE colonization, contact precautions, education and strict antibiotic restriction, were implemented to control the outbreak.

RESULTSDuring the outbreak, a total of 32 VRE strains were obtained. There were 21 strains found in Emergency Intensive Care Unit (EICU), 9 isolates from Geriatric Ward, and two from other units. All the isolates harbored the vanA gene, however, four of them exhibited the VanB phenotype. Meanwhile, MLST analysis revealed that all isolates belonged to clonal complex (CC) 17. With the infection-control measures, the epidemic was constrained in two units (EICU and Geriatric Ward). After March 2009, no further case infected with VRE was detected in the following one-year period.

CONCLUSIONThe outbreak was controlled by continuous implementation of the infection control programme, and more rigorous infection control policy is needed.

China ; Electrophoresis, Gel, Pulsed-Field ; Enterococcus faecium ; drug effects ; genetics ; pathogenicity ; Gram-Positive Bacterial Infections ; microbiology ; transmission ; Hospitals ; Humans ; Microbial Sensitivity Tests ; Multilocus Sequence Typing ; Polymerase Chain Reaction ; Vancomycin Resistance ; genetics ; physiology

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

BACKGROUNDNosocomial infection caused by methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) could lead to increased morbidity and mortality. In 2006, VRE nosocomial spread became a reality in our hospital since the first VRE nosocomial infection in 2003. Little is known about the prevalence of coexistence with VRE and MRSA in the patients. The primary objective of the study was to identify the molecular characteristics of epidemic MRSA clones in our hospital and the prevalence of the coexistence with MRSA and VRE in same patients during the 2-year period, 2006 - 2007.

METHODSThe clinical features, laboratory test results, and therapeutic outcomes of 129 cases who isolated MRSA collected from January 2006 to December 2007 were retrospectively analyzed. Polymerase chain reaction (PCR) was used to determine mecA-femB type and staphylococcal cassette chromosome mec (SCCmec) type. All the participants were screened for clinical and microbiological data to identify the coexistence of VRE strains with MRSA.

RESULTSOne hundred and twenty-nine MRSA isolates were included in the study: 71 (55%) from the intensive care unit, 35 (27.2%) from the surgical wards and 23 (17.8%) from the medical wards. The most frequent source of isolation of MRSA was sputum (76.7%). From seven patients we isolated MRSA and VRE (E. faecium) simultaneously during their inpatient stay. One hundred and twenty-seven (127/129, 98.4%) MRSA isolates harboured SCCmec type III, only 2 MRSA strains contained SCCmec type II. All of the 129 MRSA isolates remained sensitive to vancomycin, teicoplanin and linezolid. Higher sensitivity rates were noted for chloramphenicol 99.2% (128/129). Only 20.2% (26/129) of the MRSA isolates were sensitive to rifampin. All isolates presented resistance to multiple antimicrobial agents with high minimum inhibitory concentrations (MICs), including: beta-lactams (penicillin, oxacillin, cefoxitin, and cefazolin), tetracycline, erythromycin, gentamicin, and quinolones (ciprofloxacin, levofloxacin, and moxifloxacin).

CONCLUSIONSThe predominant MRSA clone at Beijing Chaoyang Hospital from 2006 to 2007 had the type III SCCmec element. All of the MRSA isolates were multiresistant to antimicrobial agents. Emergence of coexistence of MRSA and VRE in the same patient was not rare. Physicians should pay more attention to infections resulting from MRSA and VRE. Aggressive infection control measures should be taken to prevent the transmission of the multidrug resistance organism.

Adult ; Aged ; Aged, 80 and over ; Anti-Bacterial Agents ; pharmacology ; China ; Chromosomes, Bacterial ; genetics ; Enterococcus ; drug effects ; genetics ; physiology ; Gram-Positive Bacterial Infections ; epidemiology ; Hospitals ; statistics & numerical data ; Humans ; Male ; Methicillin-Resistant Staphylococcus aureus ; drug effects ; genetics ; physiology ; Microbial Sensitivity Tests ; Middle Aged ; Polymerase Chain Reaction ; Prevalence ; Staphylococcal Infections ; epidemiology ; Vancomycin Resistance

No abstract available.
Aged ; Anti-Bacterial Agents/pharmacology/therapeutic use ; Bacteremia/blood/*diagnosis/microbiology ; C-Reactive Protein/analysis ; Cholecystitis/blood/cerebrospinal fluid/microbiology ; Electrophoresis, Gel, Pulsed-Field ; Enterococcus faecium/drug effects/isolation & purification/metabolism ; Humans ; Male ; Microbial Sensitivity Tests ; Microscopy, Electron, Scanning ; Ochrobactrum/drug effects/isolation & purification/*metabolism ; RNA, Ribosomal, 16S/analysis/genetics/metabolism ; Sequence Analysis, DNA ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization