Comparison of genomic DNAs of different enterococcal isolates using restriction endonucleases with infrequent recognition sites

A cloned 1.8-kb probe containing the 3' end of 16S ribosomal RNA and the 5' end of 23S ribosomal RNA from Enterococcus hirae was used to analyze various endonuclease digests of enterococci. In the ATCC strains tested we observed a remarkable conservation of the ApaI sites in the rrn operons, and a partial conservation of EcoRI sites. Using a number of other endonuclease digestions with the ApaI rrn probe, we estimate the number of rrn operons in enterococci to be between five and six.

Section: Methodsmentioning

confidence: 99%

Ribosomal RNA gene (rrn) organization in enterococci

Sechi

1994

“…Agarose plugs were prepared [8] and digested overnight with 40-80 units of SfiI or NotI (Boehringer) according to the manufacturer's recommendations. The software-assisted ZIFE electrophoresis apparatus (Auto Base, Q-Life System Inc.) was used with ROM cards that optimize DNA fragment separations in the 100-700 kb (card no.…”

Section: Zero Integrated Field Gel Electrophoresis (Zife)mentioning

confidence: 99%

Conjugal transfer of the vancomycin resistance determinant vanB between enterococci involves the movement of large genetic elements from chromosome to chromosome

Quintiliani¹

1994

Resistance to various levels of vancomycin and susceptibility to teicoplanin in enterococci (VanB phenotype) is mediated by the vanB gene cluster. VanB-type resistance was transferred by intra- and inter-specific conjugation between different strains of Enterococcus. Analysis of SfiI-digested genomic DNA by zero integrated-field electrophoresis followed by Southern hybridization revealed vanB-containing chromosomal insertions of approximately 90-250 kb in the transconjugants. Thus, transfer of VanB-type resistance is associated with the movement of large genetic elements from chromosome to chromosome.

“…Several approaches for identi¢cation below the species level were developed, among others pulsed ¢eld gel electrophoresis (PFGE) of DNA macrorestriction fragments [2]. The ¢rst study involving enterococci demonstrated the usefulness of the procedure for typing strains of Enterococcus faecalis [3]. Also, for Enterococcus faecium it was demonstrated that PFGE provided an e¤cient procedure for mapping genomic polymorphism in an epidemiologically concordant fashion [4].…”

Section: Introductionmentioning

confidence: 99%

Random amplification of polymorphic DNA versus pulsed field gel electrophoresis of SmaI DNA macrorestriction fragments for typing strains of vancomycin-resistant enterococci

Braak

Power

Anthony

et al. 2000

Genetic typing of vancomycin-resistant enterococci (VRE) can be performed using a variety of methods, but comparative analyses of the quality of these methods are still relatively scarce. We here compare random amplification of polymorphic DNA (RAPD) analysis with pulsed field gel electrophoresis (PFGE) of DNA macrorestriction fragments as examples of two of the recent and well-accepted molecular typing methods. For the latter method, empirical guidelines for the interpretation of the DNA fingerprints have been proposed in the international literature. Based on our experimental analyses, we define similar criteria for RAPD fingerprinting. A collection of 100 strains of VRE, comprising Enterococcus faecium, Enterococcus faecalis, Enterococcus avium, Enterococcus gallinarum and Enterococcus casseliflavus, was assembled. Fifty isolates were Dutch, another 50 were isolated in the UK. Strains were selected on the basis of previously determined putative identity, close relatedness or uniqueness. The strains were analysed using well-standardised RAPD and PFGE protocols. Resulting fingerprints were interpreted with computerised methods involving band positioning and we show that typing of VRE by PFGE and RAPD generates highly congruent DNA fingerprint clustering. When the proposed international criteria for interpretation of PFGE fingerprints were applied in our case, 86% PFGE homology as discriminating value between close relatedness and uniqueness, a 75% homology cut-off for the comparison of the RAPD-generated DNA fingerprints revealed essentially identical strain clusters. As a spin-off it is revealed that strains from the different species can be efficiently discriminated, that strains from the UK and The Netherlands form separate clusters and that strains from veterinary origin can be identified separately as well.