RESUMO
BACKGROUND: Vancomycin-resistant enterococci (VRE) are a serious problem all over the world. The present study was conducted to investigate antimicrobial resistance patterns, genotypes, clonal relationship, and virulence fac- tors of VRE species isolated from rectal swab samples of hospitalized patients, patient's relatives, and medical staff at Istanbul University Cerrahpasa Medical School hospital. METHODS: The VRE isolates were typed with an automated VITEK system and their antibiotic sensibilities were analysed by disc diffusion and Etest® method. The molecular characterization and clonal relationships were per- formed using a PCR method and virulence genes by sequence typing. RESULTS: A total of 100 (10.3%) of the 971 patients were colonized with VRE. None of the investigated 25 patient's relatives and 45 medical staff carried VRE. All VRE strains were identified as E. faecium. They were vanA genotype and originated from a single clone. VRE strains exhibited multi-drug resistance. High-level gentamicin-resistance was 93%. However, lower resistance rates were found for linezolid (40%) and quinopristin-dalfopristin (11%). The enterococcal surface protein gene esp was found positive in 87 of 100 isolates, and four strains were positive for the cylB (cytolysin) gene. CONCLUSIONS: The identification of VRE strains to the species level and detection of virulence genes will assist in infection control practices.
Assuntos
Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Bactérias Gram-Positivas/microbiologia , Hospitalização , Hospitais Universitários , Reto/microbiologia , Resistência a Vancomicina/genética , Enterococos Resistentes à Vancomicina/genética , Enterococos Resistentes à Vancomicina/isolamento & purificação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/uso terapêutico , Sequência de Bases , Criança , Pré-Escolar , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Feminino , Genótipo , Infecções por Bactérias Gram-Positivas/diagnóstico , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/epidemiologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Dados de Sequência Molecular , Fenótipo , Reação em Cadeia da Polimerase , Ribotipagem , Turquia/epidemiologia , Enterococos Resistentes à Vancomicina/classificação , Enterococos Resistentes à Vancomicina/efeitos dos fármacos , Enterococos Resistentes à Vancomicina/patogenicidade , Virulência/genética , Fatores de Virulência/genética , Adulto JovemRESUMO
In an attempt to obtain facile methods to purify the heterotetrameric ADP-glucose pyrophosphorylase (AGPase), polyhistidine tags were attached to either the large (LS) or small (SS) subunits of this oligomeric enzyme. The addition of polyhistidine tag to the N-terminus of the LS or SS and co-expression with its unmodified counterpart subunit resulted in substantial induction of enzyme activity. In contrast, attachment of a polyhistidine-containing peptide through the use of a commercially available pET vector or addition of polyhistidine tags to the C-terminal ends of either subunit resulted in poor expression and/or production of enzyme activity. Preliminary experiment showed that these polyhistidine N-terminal-tagged enzymes interacted with Ni-NTA-agarose, indicating that immobilized metal affinity chromatography (IMAC) would be useful for efficient purification of the heterotetrameric AGPases. When ion-exchange chromatography step was employed prior to the IMAC, the polyhistidine-tagged AGPases were purified to near homogeneity. Comparison of kinetic parameters between AGPases with and without the polyhistidine tags revealed that attachment of the polyhistidine did not alter the allosteric and catalytic properties of the enzymes. These results indicate that polyhistidine tags will be useful for the rapid purification of preparative amounts of AGPases for biochemical and physical studies.