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Classification of Ralstonia pickettii-like isolates from the environment and clinical samples as Ralstonia insidiosa sp. nov.

¹ Department of Pediatrics and Communicable Diseases, University of Michigan, 1150 W. Med. Ctr. Dr., MSRB III, Rm 8323, Ann Arbor, MI 48109-0646, USA
² Laboratorium voor Microbiologie, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium

Correspondence
Tom Coenye
Tom.Coenye{at}UGent.be

	ABSTRACT

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Thirteen Ralstonia pickettii-like isolates from the environment (water, soil and activated sludge) and human clinical samples (including respiratory secretions of cystic fibrosis patients) were investigated in a polyphasic taxonomic study that employed 16S rDNA sequence analysis, DNA–DNA hybridization, determination of DNA base composition, whole-cell protein analysis, biochemical characterization and PCR-based assays. All isolates were classified as a novel Ralstonia species, for which the name Ralstonia insidiosa sp. nov. is proposed. The type strain, LMG 21421^T (=CCUG 46789^T), was isolated from the sputum of a patient with acute lymphoblastic leukaemia. R. insidiosa can be differentiated from other species of the genus Ralstonia and phenotypically similar species (including the Burkholderia cepacia complex and Achromobacter xylosoxidans) by a variety of biochemical tests, whole-cell protein analysis and several PCR-based assays. Some outstanding issues in the taxonomy of the genus Ralstonia are also discussed.

Published online ahead of print on 29 November 2002 as DOI 10.1099/ijs.0.02555-0.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Ralstonia insidiosa LMG 21421^T is AF488779.

Figures showing normalized whole-cell protein profiles and PCR analysis with primer pair Rp-F1/R38R1 of Ralstonia strains are available as supplementary material in IJSEM Online.

	INTRODUCTION

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The genus Ralstonia was proposed in 1995 to accommodate the generically misplaced species Burkholderia pickettii, Burkholderia solanacearum and Alcaligenes eutrophus (Yabuuchi et al., 1995). Since its proposal, a number of additional Ralstonia species have been described: Ralstonia basilensis (Steinle et al., 1998), Ralstonia gilardii (Coenye et al., 1999), Ralstonia paucula (Vandamme et al., 1999), Ralstonia oxalatica (Sahin et al., 2000), Ralstonia mannitolilytica (De Baere et al., 2001), Ralstonia campinensis (Goris et al., 2001), Ralstonia metallidurans (Goris et al., 2001) and Ralstonia taiwanensis (Chen et al., 2001). Thus, at the time of writing, the genus contains 11 species with validly published names.

Species of the genus Ralstonia occupy diverse ecological niches. Ralstonia solanacearum is an important phytopathogen that has an unusually broad host range and causes bacterial wilt on a variety of economically important crops (Hayward, 1991). Ralstonia eutropha, R. oxalatica, R. basilensis, R. campinensis and R. metallidurans are environmental organisms; strains that belong to several of these species have considerable importance as possible agents for the bioremediation of soil and water contaminated with heavy metals or chlorinated organic compounds (Steinle et al., 1998; Goris et al., 2001). Ralstonia pickettii has been isolated from various clinical sources (Riley & Weaver, 1975; McNeil et al., 1985) and has been found to be responsible for pseudobacteraemia (Verschraegen et al., 1985). Strains of R. mannitolilytica (formerly known as ‘Pseudomonas thomasii’ and R. pickettii biovar 3) have also been isolated from various clinical sources, including nosocomial recurrent meningitis (Philips et al., 1972; De Baere et al., 2001; Vaneechoutte et al., 2001). R. gilardii, R. paucula and R. taiwanensis have been isolated both from human clinical samples and from the environment (Coenye et al., 1999; Vandamme et al., 1999; Chen et al., 2001; Wauters et al., 2001). Recent studies have demonstrated that R. pickettii, R. mannitolilytica, R. gilardii and R. taiwanensis can also be recovered from the respiratory tract of cystic fibrosis (CF) patients (Chen et al., 2001; Coenye et al., 2002a, b).

Here we report on the polyphasic taxonomic analysis of 13 strains that were initially identified as R. pickettii, Ralstonia sp. or Burkholderia cepacia complex. The strains were isolated from various environmental samples (river and pond water, soil and activated sludge) and from clinical samples, including the respiratory secretions of CF patients. The results of our study allow us to classify these 13 isolates as members of a novel Ralstonia species, for which we propose the name Ralstonia insidiosa sp. nov.

	METHODS

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Bacterial strains and growth conditions.
The R. insidiosa strains used in this study, several of which have been deposited in the culture collections of the BCCM/LMG (Laboratorium voor Microbiologie Gent, Belgium) and CCUG (University of Göteborg, Department of Clinical Bacteriology, Göteborg, Sweden), are listed in Table 1. Type and reference strains of all other Ralstonia species have been described previously (Coenye et al., 1999; Vandamme et al., 1999; Chen et al., 2001; De Baere et al., 2001; Goris et al., 2001). Strains were grown aerobically on Mueller–Hinton broth (Becton Dickinson) supplemented with 1·8 % (w/v) agar and incubated for 24 h at 32 °C, unless otherwise indicated.

16S rDNA sequencing.
Preparation of DNA, PCR amplification of the 16S rRNA gene and 16S rDNA sequencing were performed as described previously (Coenye et al., 2002a). A phylogenetic tree based on the neighbour-joining method (Saitou & Nei, 1987) was constructed by using Kodon software (Applied Maths).

Determination of DNA base composition and DNA–DNA hybridization.
DNA preparation, determination of DNA base composition by HPLC (Coenye et al., 2001) and DNA–DNA hybridizations with photobiotin-labelled probes in microplate wells (Ezaki et al., 1989) were performed as described previously. The hybridization temperature was 50 °C.

PCR assays.
PCR assays were performed in 25 µl reaction mixtures that contained 2 µl DNA solution, 1 U Taq polymerase (Gibco-BRL), 250 mM (each) dNTP (Gibco-BRL), 1·5 mM MgCl₂, 1x PCR buffer (Gibco-BRL) and 20 pmol (each) oligonucleotide primer. Specific primers for R. pickettii (Rp-F1/Rp-R1) and R. mannitolilytica (Rm-F1/Rm-R1) were developed in a previous study (Coenye et al., 2002b). Amplification was carried out using a PTC-100 programmable thermal cycler (MJ Research). After initial denaturation was performed for 2 min at 94 °C, 30 amplification cycles were completed, each consisting of 1 min at 94 °C, 1 min at the appropriate annealing temperature and 1 min 30 s at 72 °C. A final extension step of 10 min at 72 °C was applied. Negative PCR controls, containing all reaction mixture components except template DNA, were included for every experiment. The annealing temperatures used were 55 °C (for the identification of R. insidiosa and R. pickettii) or 57 °C (for the identification of R. mannitolilytica).

Evaluation of PCR assays.
For evaluation of the PCR assay, 108 isolates were tested. These included 13 R. insidiosa strains (listed in Table 1), 71 strains that represented all other Ralstonia species and 24 type or reference strains that represented phylogenetically related species, including all members of the B. cepacia complex (B. cepacia genomovars I, III and VI, Burkholderia multivorans, Burkholderia stabilis, Burkholderia vietnamiensis, Burkholderia ambifaria, Burkholderia anthina and Burkholderia pyrrocinia), Burkholderia gladioli, Pandoraea apista, Pandoraea norimbergensis, Pandoraea pnomenusa, Pandoraea sputorum, Pandoraea pulmonicola, Achromobacter xylosoxidans, Achromobacter denitrificans, Alcaligenes faecalis, Bordetella avium, Bordetella hinzii, Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis.

Phenotypic characterization.
For conventional biochemical testing, bacteria were grown on Mueller–Hinton agar plates for 24–48 h and then inoculated into the test reagent. Oxidase and catalase tests were performed with 1 % tetramethyl p-phenylenediamine dihydrochloride and 3 % hydrogen peroxide, respectively. Lysine decarboxylase, ONPG and oxidation–fermentation sugars (sucrose and lactose) were obtained from Remel. These test reactions were incubated at 37 °C and examined daily for 7 days except for ONPG, which was examined at 24 and 48 h. The RapID NF Plus (Remel) and API 20 NE (bioMérieux) commercial identification systems were used, according to the manufacturers' instructions. Growth on B. cepacia selective agar (BCSA) that contained 600 U polymyxin B ml^-1, 10 µg gentamicin ml^-1 and 2·5 µg vancomycin ml^-1 was determined at 32 °C.

	RESULTS AND DISCUSSION

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Taxonomic structure and phylogenetic position of R. insidiosa
The reproducibility of SDS-PAGE of whole-cell proteins was checked by preparing protein extracts in duplicate. The correlation level between patterns obtained with different extracts of the same strain was >93 % (data not shown). By visual comparison of the protein patterns, all R. insidiosa isolates were virtually indistinguishable. However, after numerical analysis of the patterns, the R. insidiosa isolates clustered in two subgroups (see supplementary figure available in IJSEM Online). This is due to slight but reproducible distortions and variations in the protein pattern (mainly in the region between 40 and 60 kDa), which can have a profound effect on numerical analysis, as discussed previously (Vandamme et al., 1997, 1999; Goris et al., 2001). Reference strains of other Ralstonia species were characterized by distinct protein patterns and occupied separate positions in the dendrogram (see supplementary figure available in IJSEM Online).

The 16S rRNA gene of R. insidiosa LMG 21421^T (GenBank no. AF488779) has high similarity to the 16S rRNA genes of reference strains of R. pickettii (97·8 %) and R. mannitolilytica (97·5 %) (Fig. 1). Similarity to the 16S rRNA genes of other Ralstonia species (including R. solanacearum) is <97·0 % (Fig. 1). This clearly indicates that this organism belongs to the genus Ralstonia. The DNA G+C content of R. insidiosa strains LMG 21421^T and LMG 18319 was 64·3 and 63·9 %, respectively. This DNA base ratio of 63·9–64·3 % is within the range of the genus Ralstonia [63–70 % (Yabuuchi et al., 1995; Goris et al., 2001)].

View larger version (39K): [in this window] [in a new window]   Fig. 1. Phylogenetic tree based on 16S rDNA sequence data, showing the position of R. insidiosa within the genus Ralstonia. GenBank accession numbers are shown in parentheses. Bar, 10 % sequence dissimilarity.

Identification of R. insidiosa
For the development of species-specific PCR primers, 16S rDNA sequences of all Ralstonia species and representatives of related genera were retrieved from GenBank and aligned using MegAlign software (DNAStar). Based on this alignment, reverse primer R38R1 (5'-CACACCTAATATTAGTAAGTGCG-3') was developed. Primer R38R1 was used in combination with the previously developed forward primer Rp-F1 (5'-ATGATCTAGCTTGCTAGATTGAT-3') (Coenye et al., 2002b) and allowed the specific amplification of a 403 bp 16S rDNA fragment from all R. insidiosa strains, without cross-reactivity with any other Ralstonia, Burkholderia, Pandoraea, Achromobacter or Bordetella species tested (data not shown). None of the R. insidiosa strains reacted with primer pair Rm-F1/Rm-R1 (developed for the identification of R. mannitolilytica), but all R. insidiosa strains cross-reacted with primer pair Rp-F1/Rp-B1 (developed for the identification of R. pickettii) (data not shown). Therefore, the combined use of these three PCR assays will allow the sensitive and specific identification of R. insidiosa, R. pickettii and R. mannitolilytica.

All R. insidiosa strains grew at 28, 32 and 37 °C and on BCSA, showed oxidase, catalase, lipase, phosphatase, proline aminopeptidase, pyrrollidonyl aminopeptidase and -L-glutamyl aminopeptidase activities, and assimilated glucose, gluconate, caprate, adipate, malate and citrate. All R. insidiosa strains reduced nitrate. Activities of lysine decarboxylase, arginine dihydrolase, -glucosidase, -glucosidase, tryptophan aminopeptidase and N-benzylarginine aminopeptidase were not observed. None of the R. insidiosa strains assimilated arabinose, mannose, mannitol or maltose. Indole production or the production of acid from glucose, sucrose or lactose was not observed. None of the isolates showed -galactosidase activity when tested with the API 20 NE or RapID NF Plus systems, but tube ONPG tests gave weakly positive results after 48 h incubation. Assimilation of N-acetylglucosamine and phenylacetate and the presence of urease activity were strain-dependent characteristics.

With the API 20 NE system, R. insidiosa strains were identified as A. xylosoxidans (‘good identification’, profile 1040477) or R. pickettii (‘good identification’, profile 5240477 or ‘low discrimination’, profile 1240575). With the RapID NF Plus system, all R. insidiosa strains were identified as Shewanella putrefaciens (‘adequate identification’, profile 430616 or ‘questionable identification’, profile 434616). Phenotypically, R. insidiosa can be differentiated from its closest neighbours R. pickettii and R. mannitolilytica by lack of assimilation of arabinose, mannitol and N-acetylglucosamine. Differentiation of R. indisiosa from R. solanacearum is possible by the lack of assimilation of phenylacetate and caprate in the latter. Characteristics that are useful for differentiation of R. insidiosa from other Ralstonia species are shown in Table 2. The absence of lysine decarboxylase activity allows differentiation from most members of the B. cepacia complex (except B. multivorans and B. cepacia genomovar VI); differentiation from the two latter species is possible (Coenye et al., 2001) due to the lack of acid production from lactose in R. insidiosa. Several R. insidiosa strains were identified as A. xylosoxidans by using the API 20 NE system, but unlike A. xylosoxidans (von Graevenitz, 1995), R. insidiosa does not produce acid from glucose. All R. insidiosa strains were identified as S. putrefaciens with the RapID NF Plus system. However, R. insidiosa can easily be differentiated from S. putrefaciens by the lack of citrate and malate assimilation in the latter species (Venkateswaran et al., 1999).

It has previously been reported that the phytopathogenic organisms [Pseudomonas] syzygii (causal agent of Sumatra disease of cloves) and the ‘blood disease bacterium’ (BDB, causal agent of blood disease of bananas) are closely related to R. solanacearum (Taghavi et al., 1996; Anzai et al., 2000); this is confirmed in our study (Fig. 1). However, previous studies using DNA–DNA hybridization, 16S rDNA sequencing and AFLP (amplified fragment length polymorphism) fingerprinting have indicated that R. solanacearum is taxonomically diverse (Palleroni & Doudoroff, 1971; Hayward, 1991; Taghavi et al., 1996; Poussier et al., 2000) and a thorough reassessment of the relationships between the phylogenetic subdivisions within R. solanacearum, and between R. solanacearum, [P.] syzygii and the BDB, seems necessary before any taxonomic changes are made.

The species Cupriavidus necator was proposed by Makkar & Casida (1987). This organism (represented by a single isolate, ATCC 43291^T) is highly resistant to copper and is a non-obligate predator of bacteria in soil. Based on 16S rDNA sequence analysis, this organism clearly belongs to the genus Ralstonia, with R. eutropha as its closest relative (99·3 % sequence identity; Fig. 1). However, its DNA G+C content was reported to be 57±1 mol%, which is significantly below the range reported for the genus Ralstonia; additional taxonomic data will be required to accurately determine the status of this organism.

A survey of GenBank revealed the presence of several 16S rDNA sequences derived from as-yet-unclassified Ralstonia species. Most of these have been collected from soil or water polluted with various organic compounds or heavy metals. Alignment of these 16S rDNA sequences with those of representatives of all Ralstonia species revealed that several of these isolates probably belong to existing Ralstonia species, while others were more distantly related and probably represent novel taxa (data not shown). Polyphasic taxonomic studies including DNA–DNA hybridization will ultimately be required to determine the status of these isolates and to determine whether strains with potential bioremediation applications are distinct from pathogens of humans and plants.

Description of Ralstonia insidiosa sp. nov.
Ralstonia insidiosa (in.si.di.o'sa. L. fem. adj. insidiosa deceitful, dangerous, referring to the fact that these seemingly harmless environmental organisms can be isolated from, and possibly cause infection in, humans).

Cells are Gram-negative, non-sporulating, aerobic, non-fermentative, motile rods. Growth occurs at 28, 32 and 37 °C and on BCSA. Catalase, oxidase, lipase, phosphatase, proline aminopeptidase, pyrrolidonyl aminopeptidase and -L-glutamyl aminopeptidase activities are present. No lysine decarboxylase, arginine dihydrolase, gelatinase, -glucosidase, -glucosidase, tryptophan aminopeptidase or N-benzylarginine aminopeptidase activity is detected. No indole production or production of acid from glucose, sucrose or lactose occurs. Assimilates glucose, gluconate, caprate, adipate, malate and citrate but not arabinose, mannose, mannitol or maltose. Reduces nitrate. Characteristics that differentiate R. insidiosa from other Ralstonia species are summarized in Table 2. The DNA base composition is 63·9–64·3 mol% G+C.

The type strain, LMG 21421^T (=CCUG 46789^T), was isolated from the sputum of a patient with acute lymphoblastic leukaemia in the USA in 2001. In addition to the characteristics described for the species, the type strain shows no urease activity, does not assimilate N-acetylglucosamine and assimilates phenylacetate.

	ACKNOWLEDGEMENTS

 
This work was supported by a grant from the Cystic Fibrosis Foundation (United States) to J. J. L. T. C. is supported by the Caroll Haas Research Fund in Cystic Fibrosis. J. G. was supported by a ‘Concerted Research Action’ project grant from the Flemish Government. P. V. is indebted to the Fund for Scientific Research – Flanders for financial support. We thank Liesbeth Lebbe for excellent technical assistance.

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