Genotypic and phenotypic diversity of Ralstonia pickettii and Ralstonia insidiosa isolates from clinical and environmental sources including High-purity Water. Diversity in Ralstonia pickettii

Abstract

Background: Ralstonia pickettii is a nosocomial infectious agent and a significant industrial contaminant. It has been found in many different environments including clinical situations, soil and industrial High Purity Water. This study compares the phenotypic and genotypic diversity of a selection of strains of Ralstonia collected from a variety of sources.

Results: Ralstonia isolates (fifty-nine) from clinical, industrial and environmental origins were compared genotypically using i) Species-specific-PCR, ii) PCR and sequencing of the 16S-23S rRNA Interspatial region (ISR) iii) the fliC gene genes, iv) RAPD and BOX-PCR and v) phenotypically using biochemical testing. The species specific-PCR identified fifteen out of fifty-nine designated R. pickettii isolates as actually being the closely related species R. insidiosa. PCR-ribotyping of the 16S-23S rRNA ISR indicated few major differences between the isolates. Analysis of all isolates demonstrated different banding patterns for both the RAPD and BOX primers however these were found not to vary significantly.

Conclusions: R. pickettii species isolated from wide geographic and environmental sources appear to be reasonably homogenous based on genotypic and phenotypic characteristics. R. insidiosa can at present only be distinguished from R. pickettii using species specific PCR. R. pickettii and R. insidiosa isolates do not differ significantly phenotypically or genotypically based on environmental or geographical origin.

Figure 1

Cluster analysis of API 20NE results. B: Biotype 1 to 35- numbers assigned to API 20NE profile, isolates belonging to each biotype can be seen in Table 1. Scale is a measure of the phenotypic relatedness of isolates.

Figure 2

Phylogenetic trees. A) Phylogenetic tree of R. pickettii and R. insidiosa 16S-23S ISR of nineteen sequenced isolates and sequence data available on the Genbank database. The tree was rooted with the ISR of Ralstonia solanacearum (Genbank Accession No AJ277280), Cupriavidus necator (AJ783978) and Burkholderia cepacia (L28154). B) Phylogenetic tree of R. pickettii and R. insidiosa fliC genes of nineteen sequenced isolates and sequence data available on the Genbank database. The tree was rooted with the fliC of Burkholderia cepacia (L28154). Cluster analysis was based upon the neighbour-joining method. Numbers at branch-points are percentages of 1000 bootstrap resamplings that support the topology of the tree.

Figure 3

RAPD analysis with primer OPA03U and BOX analysis. A) RAPD analysis with primer OPA03U B) BOX analysis. Dendrogram of fifty-nine isolates of R. pickettii and R. insidiosa by the Pearson correlation using the UPGMA linkage method.