In the course of this study a number of bacteria previously identified phenotypically as R. pickettii were subsequently identified as R. insidiosa using species-specific PCR. These bacteria are hard to distinguish from each other phenotypically . R. insidiosa, the closest related bacteria to R. pickettii , has been isolated from the respiratory tracts of cystic fibrosis patients , river and pond water, soil, activated sludge  and has also been detected in water distribution systems  and laboratory purified water systems . It has also been the causative agent of two cases of serious hospital infection in two immunocompromised individuals .
Each of the four DNA-based fingerprinting and sequencing methods were suitable for distinguishing and grouping the isolates, although the sensitivity of the methods varied. Of the three phenotypic methods examined, the API 20NE system was more discriminatory than the Remel RapID NF Plus system or the Vitek NFC. However, the Remel RapID NF Plus system and the Vitek NFC did prove more useful for the accurate identification of R. pickettii isolates, as previously reported . The API 20NE gave thirty-five different biotypes for fifty-nine isolates (Table 3, Figure 1), which grouped together isolates from different environments. These results broadly agree with those of Dimech et al who found homogeneity in physiological parameters .
Genotypic studies carried out by both Dimech et al. and Chetoui et al. hinted that R. pickettii also had genotypic homogeneity [25, 26]. This was investigated in this study using the methods described above. Our data based on the sequence of 16S-23S spacer regions of nineteen isolates indicated that Ralstonia pickettii is a homogenous species with little difference between isolates from different environmental niches. Clearly using these methods we can however determine differences between R. pickettii and R. insidiosa.
The fliC gene has been used for bacterial strain differentiation in multiple studies such as for Ralstonia solanacearum  and Burkholderia cepacia complex . Four different types of flagellin gene have been found in R. pickettii isolates analysed in this study (Groups 1, 2, 3 and 4). This is similar to data from P. aeruginosa where two different types of fliC gene have been found  and from the B. cepacia complex where again two different types of fliC gene have also been found [55, 56]. The fliC gene appears however not to be useful for distinguishing between R. pickettii and R. insidiosa based on our findings. The division of the groups did not correlate to clinical or environmental association or to their location of isolation. The reasons for the variation between the 16S-23S spacer region and the fliC gene could be potentially due to the structure of the fliC gene. This is demonstrated by Burkholderia flagellin sequences, which exhibit high levels of homology in the conserved terminal regions but differ considerably in the central region . Variation is a common feature of flagellin proteins, which are believed to fold into a hairpin-like conformation, with the terminal domains being responsible for defining the basic filament structure lying on the inner surface and the central, variable region being surface exposed .
In a previous epidemiological study involving sixteen isolates of R. pickettii, eight different RAPD profiles were observed for isolates coming from blood culture, distilled water and an aqueous chlorhexidine solution . In another study, involving fourteen isolates of R. pickettii from various biological samples the same RAPD pattern was found in all instances , while Pasticci et al., carried out a study involving fifteen isolates of R. pickettii that gave three patterns . The results of our study with a larger number of isolates indicated that there is some diversity in the studied populations but that this is limited and isolates from different environments grouped together.
The results obtained with BOX-PCR showed nineteen different profiles among the fifty-nine isolates examined again demonstrating limited diversity (Figure 3b). To the best of our knowledge this is the first reported study of the diversity of R. pickettii and R. insidiosa carried out with BOX-PCR. A similar study carried by Coenye et al., on ninety-seven B. cepacia Genomovar III isolates found 20 different patterns with a DI value of 0.821 .
The molecular fingerprinting methods used here yielded rapid and reproducible fingerprints for clinical and environmental isolates of R. pickettii and R. insidiosa. Presently, little is known regarding the source of R. pickettii isolates occurring in hospital environments. Investigations by other authors have reported no evidence of patient-to-patient transmission, and they suggest that multiple independent acquisitions from environmental sources could be an important mode of transmission of R. pickettii . The most common sites of contamination were blood-sampling tubes, dialysis machines, nebulizers and other items frequently in contact with water .