DNA hybridization and contour-clamped homogeneous electric field electrophoresis for identification of enterococci to the species level

Abstract

In this study, 113 Enterococcus faecium, 37 Enterococcus faecalis, 24 Enterococcus gallinarum, 15 Enterococcus raffinosus, and 13 Enterococcus casseliflavus clinical isolates and American Type Culture Collection (ATCC) strains were evaluated by contour-clamped homogeneous electric field electrophoresis. Thirty-one of the E. faecium, 22 of the E. faecalis, 24 of the E. gallinarum, 15 of the E. raffinosus, and 13 of the E. casseliflavus isolates were also evaluated by DNA-DNA hybridization. Genomic DNAs from type strains E. faecalis ATCC 19433, E. faecium ATCC 19434, E. gallinarum ATCC 49573, E. raffinosus ATCC 49427, and E. casseliflavus ATCC 25788 were labeled with biotin for use as probes. E. faecalis differed from all other species in always having a largest fragment of >400 kb. E. gallinarum was different from all other species in having all SmaI fragments of <200 kb. Biotin-labeled probes showed a high degree of hybridization with genomic DNA from the same species and a low degree of hybridization when hybridized to genomic DNA from different species for all isolates tested except for four isolates identified as E. faecium by conventional biochemical methods. The DNA from these four isolates hybridized strongly to DNA from E. gallinarum ATCC 49573 and weakly to E. faecium ATCC 19434 DNA and had all SmaI fragments of <200 kb in size. These data suggest that these isolates are nonmotile E. gallinarum. DNA from each ATCC type strain hybridized strongly with itself and had only a low degree of hybridization with DNA from other ATCC type strains tested. These results suggest that contour-clamped homogeneous electric field electrophoresis patterns and DNA-DNA hybridization with biotin-labeled probes may be of use for species differentiation of some enterococci.

Identification of Enterococcus faecalis strains by DNA hybridization and pulsed-field gel electrophoresis

“…We read with interest the recently published article by Donabedian et al reporting the use of contour-clamped homogeneous electric field (CHEF) electrophoresis for species differentiation of enterococci (2). At that time, we agreed with the authors' statement that ''Enterococcus faecalis differed from all other species in always having a largest fragment of Ͼ400 kb,'' as these findings were similar to our experience.…”

“…1. The largest fragment of SmaI-digested genomic DNA from these isolates was approximately 309 kb, which is almost 100 kb smaller than the largest fragment for E. faecalis published by Donabedian et al (2).…”

“…Unfortunately, a recent report describes vancomycin-resistant strains of E. faecium with atypical biochemical characteristics, which makes the identification of these strains more difficult (8). Some authors have used other tools for characterization of enterococci (whole-cell protein profiles, pulsed-field electrophoresis gel, and gene probes) (2,8); however, these techniques usually have been performed with typical strains. Our results widen the definition of atypical vancomycin-resistant enterococci, in this case, E. faecalis isolates which were ''atypical'' on the basis of the SmaI-digestion patterns, and suggest that the use of DNA banding patterns for identification purposes must be undertaken with caution.…”

A novel gentamicin resistance gene in Enterococcus

“…SF9117 is a strain of E. gallinarum isolated from an abdominal wound infection in a horse at the University of Pennsylvania. It was identified by using conventional biochemical and motility criteria (12). Enterococcus faecalis FA2-2 (8) was used as the plasmid-free recipient strain in mating experiments.…”

“…For detection of DNA-DNA homology, probes were biotin labeled with a nick translation kit (BRL Life Technologies, Gaithersburg, Md. ), and DNA was transferred to nitrocellulose by the method of Southern and was exposed to probes for hybridization (12). Nested deletions of cloned DNA were made by using the Erase-a-Base System from Promega (Madison, Wis.).…”