CDC group IV c-2: a new Ralstonia species close to Ralstonia eutropha

Abstract

CDC group IV c-2, an environmental gram-negative bacillus recently proposed for inclusion in the genus Ralstonia, has been isolated in several human infections. Biochemical characterization and 16S ribosomal DNA (rDNA) sequencing with phylogenetic analysis were used to characterize eight clinical isolates and four type strains. Other typing tools, such as pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) analysis, were also used. PFGE typing of clinical isolates was unsuccessful because the DNA was degraded, and RAPD analysis was poorly discriminatory. In contrast, the type strains were clearly distinguished with both PFGE and RAPD analysis. All of the 16S rDNA sequences were identical. Comparison of the 16S rDNA sequences to the GenBank sequences showed that they were consistent with CDC group IV c-2 belonging to the genus Ralstonia. The closest matches were obtained with Ralstonia eutropha. However, four differences in 32 biochemical tests separated R. eutropha from CDC group IV c-2, which suggests that CDC group IV c-2 is a new species of the genus Ralstonia.

FIG. 1

XbaI PFGE patterns of four C IV-2 type strains and eight C IV-2 clinical isolates. Phage lambda DNA was used as a size ladder. Lanes 1 through 4, type strains G6817, G3900, G608, and F; lane 5, Armand-Trousseau Hospital isolate; lane 6, Antoine-Béclère Hospital isolate; lane 7, Paul Brousse Hospital isolate; lane 8, Saint Vincent-de-Paul Hospital isolate; lane 9, Saint-Antoine Hospital isolate. The four type strains showed four different patterns. In contrast, no pattern was obtained for the clinical isolates, probably because of DNase produced by these strains.

FIG. 2

RAPD patterns with primer AP3 (I) and the universal primer M13 (II). Lanes 1 through 4, type strains G6817, G3900, G608, and F; lanes 5 and 6, Armand-Trousseau Hospital isolates; lane 7, Antoine-Béclère Hospital isolate; lane 8, Paul Brousse Hospital isolate; lane 9, Saint Vincent-de-Paul Hospital isolate; lanes 10 through 12, Saint-Antoine Hospital isolates. The four type strains showed four different patterns. Seven of the eight clinical isolates had identical banding patterns; only one isolate (lane 10) showed a slightly different pattern. Lanes M, molecular size markers; sizes are in base pairs.

FIG. 3

Unrooted tree reconstruction based on 979 bases of the 16S rDNA sequences of 24 bacteria. FRA01, sequence of clinical isolates and type strains. JHH, sequence obtained by Osterhout et al. (17). Trees shown were obtained by neighbor joining. Parsimony, maximum-likelihood, and quartet-puzzling methods yielded very similar topologies. Support for clades was evaluated by bootstrap resampling (10,000 times). Radial representation (a) accounts for the distance between strains, expressed as the expected number of substitutions per site. Relevant cluster support values are indicated on the cladogram (b). Sequences obtained from C IV-2 isolates cluster strongly with sequences obtained from R. eutropha strains.