ABSTRACT
Restriction fragment length polymorphism (RFLP) analysis of a PCR-amplified fragment of the 16S rRNA gene was performed on reference strains belonging to 21 different enterococcal species and on 75 Enterococcus isolates recovered from poultry meat, pasteurised milk and fresh cheese. PCR amplification generated a 275 bp fragment, which was digested with three restriction endonucleases (DdeI, HaeIII, HinfI). The strains were divided into five groups (groups A-E) on the basis of their restriction patterns. Five biochemical tests (arabinose, arginine, manitol, methyl-beta-D-glucopyranoside and raffinose) were then performed in addition to RFLP analysis to narrow the identification of enterococcal strains to the species level. PCR-RFLP, in conjunction with the selected biochemical tests, allowed the precise identification of the 21 species of Enterococcus included in the present study. This proposed method is relatively simple and rapid and can be useful as an adjunct tool for accurate identification of Enterococcus.
Subject(s)
DNA, Bacterial/genetics , Enterococcus/genetics , Polymorphism, Restriction Fragment Length/genetics , RNA, Ribosomal, 16S/genetics , Animals , Base Sequence , DNA Restriction Enzymes , Enterococcus/classification , Enterococcus/isolation & purification , Food Microbiology , Polymerase Chain ReactionABSTRACT
The present study was designed to characterize beta-lactamase genes and evaluate polymerase chain reaction (PCR) typing for multidrug-resistant Pseudomonas aeruginosa pulsed-field gel electrophoresis (PFGE) genotype A isolates from Rio de Janeiro, Brazil, collected between April 1999 and March 2000 and one additional isolate collected in June 2002. As reported previously, all of the genotype A isolates produced non-characterized metallo-beta-lactamase. These isolates (22) were screened for the bla(SPM) gene by PCR and dot-blotting. Isolates were typed by PCR fingerprinting with primers RAPD-1, 272, 208, 1290, ERIC-1 and ERIC-2. The bla(SPM) gene was detected in 18 (82%) of the 22 isolates. PCR fingerprinting gave results that correlated with PFGE, except with primer 1290. In Rio de Janeiro and other Brazilian states, nearly all SPM-producing P. aeruginosa isolates belong to a single PFGE type accounting for a large proportion of drug-resistant P. aeruginosa hospital infections. RAPD PCR fingerprinting may be a useful technique to screen for an epidemic multidrug-resistant strain in Brazil.