Assuntos
Transtorno Bipolar/imunologia , Corticosteroides/imunologia , Corticosteroides/metabolismo , Transtorno Bipolar/diagnóstico , Transtorno Bipolar/metabolismo , Citocinas/sangue , Humanos , Monócitos/metabolismo , Sistema Hipófise-Suprarrenal/imunologia , Sistema Hipófise-Suprarrenal/metabolismo , Receptores de Interleucina-2/sangue , Linfócitos T/citologia , Linfócitos T/metabolismo , Tireoidite Autoimune/imunologia , Tireoidite Autoimune/metabolismoRESUMO
Species-specific PCR assays with primers targeted to D-alanine:D-alanine ligase (ddl) encoding genes were developed for the identification of Enterococcus durans and E. hirae. The specificity of the primers was validated in a multiplex PCR on well characterised E. durans (n=30) and E. hirae (n=16) strains, all of which were identified correctly. This PCR procedure offers a reliable and rapid alternative to conventional phenotypic methods for speciation of these enterococci of growing clinical importance.
Assuntos
Enterococcus/classificação , Enterococcus/genética , Peptídeo Sintases/genética , Reação em Cadeia da Polimerase/métodos , Técnicas de Tipagem Bacteriana , Primers do DNA , Enterococcus/enzimologia , Humanos , Especificidade da Espécie , Fatores de TempoRESUMO
In the present study, 124 enterococcal strains, isolated from traditional Italian cow, goat and buffalo cheeses, were characterized using phenotypic features and randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR). The RAPD-PCR profiles obtained with four primers and five different amplification conditions were compared by numerical analysis and allowed an inter- and intraspecific differentiation of the isolates. Whole cell protein analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used as a reference method for species identification. The strains were identified as Enterococcus faecalis (82 strains), E. faecium (27 strains), E. durans (nine strains), E. gallinarum (four strains) and E. hirae (two strains). Species recognition by means of RAPD-PCR was in agreement with the SDS-PAGE results except for eight strains of E. faecium that clustered in separated groups. On the other hand, phenotypic identification based on carbohydrate fermentation profiles, using the rapid ID 32 STREP galleries, gave different results from SDS-PAGE in 12.1% of the cases. The majority of the strains had weak acidifying and proteolytic activities in milk. One E. faecium strain showed vanA (vancomycin resistance) genotype while four strains showed a beta-haemolytic reaction on human blood. Several strains showed antagonistic activity towards indicator strains of Listeria innocua, Clostridium tyrobutyricam and Propionibacterium freudenreichii subsp. shermanii.
Assuntos
Queijo/microbiologia , Enterococcus/classificação , Enterococcus/genética , Animais , Búfalos , Bovinos , Eletroforese em Gel de Poliacrilamida/veterinária , Enterococcus/efeitos dos fármacos , Genótipo , Cabras , Itália , Fenótipo , Filogenia , Técnica de Amplificação ao Acaso de DNA Polimórfico/métodos , Técnica de Amplificação ao Acaso de DNA Polimórfico/veterinária , Especificidade da Espécie , Resistência a VancomicinaRESUMO
A rapid and reliable polymerase chain reaction (Specific and Random Amplification (SARA)-PCR) has been developed to identify enterococcal species and to detect the vanA gene in one single reaction. This technique was based on the use of the primer set previously designed to amplify a part of the vanA gene (Dutka-Malen et al., J. Clin. Microbiol., 33 (1995) 24-27). In the chosen low stringency conditions complex patterns were obtained, with a sharp band of approximately 700 bp in cases where the vanA gene was present. Discrimination at the species and strain level was achieved by applying the SARA-PCR assay to a collection of 55 enterococcal isolates and type strains. Simultaneously the vanA gene was detected in all strains showing high resistance to vancomycin.
