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1 Laboratory for Pharmaceutical Microbiology, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium
2 Laboratory for Microbiology, Ghent University, Ledeganckstraat 35, B-9000 Ghent, Belgium
3 Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, MI 48109-0646, USA
4 Cardiff School of Biosciences, PO Box 915, Cardiff University, Cardiff CF10 3TL, UK
Correspondence
Peter Vandamme
Peter.Vandammme{at}UGent.be
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the recA genes of strains LMG 21820 and LMG 21443 are AY324807 and AY324808.
Details of the 18 isolates of B. dolosa sp. nov. included in this study are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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, b; Vandamme et al., 1997, 2002, 2003). In the present study, we describe biochemical tests, a 16S rDNA-based specific PCR test and several PCR-RFLP approaches for differentiating B. cepacia genomovar VI from other B. cepacia-complex bacteria, and propose to classify this bacterium formally as Burkholderia dolosa sp. nov. Details of 18 B. dolosa isolates collected in the course of a long-term study on the biodiversity of B. cepacia-complex bacteria are available as supplementary material in IJSEM Online. The isolates were obtained from specimens from cystic fibrosis patients from the USA, Canada, France and the UK and from environmental samples. Isolates were grown aerobically on trypticase soy agar at 30 °C.
DNA was prepared as described by Pitcher et al. (1989). Amplification of the recA gene and subsequent digestion of the amplicon with HaeIII and MnlI were performed as described by Mahenthiralingam et al. (2000). Three HaeIII restriction patterns could be distinguished. Most isolates (including all isolates described by Coenye et al., 2001a) were characterized by a previously described RFLP type designated Q (Vandamme et al., 2002). Two other RFLP types, one represented only by isolate LMG 21443 and the other represented by isolates LMG 21820 and R-17373, were observed (data not shown). MnlI restriction patterns of type Q isolates and of isolates LMG 21820 and R-17373 were identical, but distinct from that of isolate LMG 21443 (data not shown).
DNA–DNA hybridization experiments were performed with photobiotin-labelled probes in microplate wells as described by Ezaki et al. (1989), using an HTS7000 Bio Assay reader (Perkin Elmer) for the fluorescence measurements. The hybridization temperature was 50 °C. DNA–DNA binding values between isolates representing the three HaeIII recA RFLP types (LMG 18941, LMG 21443 and LMG 21820) were above 87 %. Combined with the previously reported high values (>97 %) obtained between type Q isolates LMG 18941, LMG 18942 and LMG 18943T (Coenye et al., 2001a), these high DNA–DNA hybridization values indicate that B. dolosa forms a homogeneous genospecies.
Analysis of the recA gene sequences of isolates LMG 21820 and LMG 21443 was performed as described by Mahenthiralingam et al. (2000). The sequences were aligned and compared with recA gene sequences from B. dolosa and other B. cepacia-complex isolates analysed in previous studies (Coenye et al., 2001b; LiPuma et al., 2001; Mahenthiralingam et al., 2000; Vandamme et al., 2000; Vermis et al., 2002a). The recA sequence of Burkholderia sp. LB400 was obtained from the LB400 genome sequencing project (http://genome.jgi-psf.org/draft_microbes/burfu/burfu.home.html) and was used as the outgroup. A phylogenetic tree based on the neighbour-joining method (Saitou & Nei, 1987) was constructed by using the KODON (Applied Maths) software package. All B. dolosa isolates formed a single recA lineage (Fig. 1).
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. Subsequent digestion of the amplicon using the restriction enzymes AluI, CfoI and DdeI was performed as described by Mahenthiralingam et al. (2000). The B. dolosa isolates generated two restriction-profile combinations that were not observed for other bacteria. Thirteen cystic fibrosis isolates, all from the USA, revealed a CfoI restriction profile distinct from that of isolates R-8868, R-15304, LMG 21443, LMG 21820 and R-17373. These two sets of restriction profiles correspond to the AluI–CfoI–DdeI restriction patterns ACC and ADC, respectively (Vermis et al., 2002b).
On the basis of previously published 16S rDNA sequences (Coenye et al., 2001a), a B. dolosa-specific PCR assay was developed. The assay was performed in a final volume of 25 µl composed of 50 mM KCl, 20 mM Tris/HCl, 1·5 mM MgCl2, 1 U Taq polymerase (Invitrogen), 0·4 µM each primer (G6F1, 5'-GATTAATACCGCATACGATCCAC-3'; G6B2, 5'-GGATTTCACATCGGTCTTAGGG-3'), 200 µM each dNTP and 2 µl lysed whole bacteria as template. Amplification was carried out using a PTC-100 programmable thermal cycler (BIOzym). After initial denaturation for 3 min at 95 °C, 30 amplification cycles were completed, each consisting of 1 min at 94 °C, 1 min at 62 °C and 2 min at 72 °C, followed by a final extension for 6 min at 72 °C. A total of 160 isolates were tested: all 18 B. dolosa isolates from the present study and an additional 20 B. dolosa isolates from US patients with cystic fibrosis, nine B. cepacia (genomovar I) isolates, 10 B. multivorans isolates, 18 B. cenocepacia isolates, seven B. stabilis isolates, 10 B. vietnamiensis isolates, nine B. ambifaria isolates, 19 B. anthina isolates, nine B. pyrrocinia isolates, four Burkholderia gladioli isolates, 13 Pandorea isolates, five Ralstonia isolates, three Achromobacter isolates and six Pseudomonas isolates. All B. dolosa isolates, except one, generated an amplicon of 448 bp. There were no false-positive reactions, resulting in sensitivity and specificity of 97 and 100 %, respectively.
A B. dolosa-specific recA primer was also designed. Published recA genes from all current genomovars were aligned and the B. dolosa-diagnostic primer G6N (positions 136–120 in the recA gene of LMG 18943T; 5'-CGAGCGAGCCGGTCGAT-3') was designed. The primer contained three bases specific to B. dolosa recA and absent at those positions in all other B. cepacia-complex recA genes: the 3' base (T; A in coding sequence) and the two further mismatched bases shown underlined in the primer sequence given above. In combination with recA primer BCR1 as the forward primer (5'-TGACCGCCGAGAAGAGCAA-3'; Mahenthiralingam et al., 2000) and primer G6N as the reverse primer, a PCR under standard conditions (Mahenthiralingam et al., 2000) with an annealing temperature of 67 °C produced a 135 bp amplicon from B. dolosa strains R-2879, LMG 18942, LMG 18943T, R-6140 and R-8869. The B. dolosa recA PCR failed to produce any amplification products when tested against controls representing genomovars I–VII (data not shown). The B. dolosa recA diagnostic PCR was not tested against B. anthina or B. pyrrocinia strains. However, analysis of recA sequence from these species demonstrated that the mismatches described above were present, and indicated that the diagnostic PCR would not amplify recA from these species.
The carbon-source-assimilation patterns and growth ability on B. cepacia-selective media [BCSA (Henry et al., 1997), PCAT (Burbage & Sasser, 1982)] and B. cepacia-selective agar (Mast Diagnostics) of 142 B. cepacia-complex isolates, representing all genomovars, were evaluated in a separate study (Vermis et al., 2003). All B. dolosa isolates could be grown on BCSA and MAST selective agar, but not on PCAT agar; in contrast, more than 85 % of the remaining B. cepacia-complex isolates did. B. dolosa isolates did not assimilate azelaic acid or tryptamine, the two carbon sources present in PCAT. In addition, none of the B. dolosa isolates could utilize salicin, but they were all able to grow on L-arabinose. In comparison, all other B. cepacia-complex genomovars were able to utilize azelaic acid. Growth on tryptamine, salicin and L-arabinose was strain dependent for B. vietnamiensis (growth was observed for 1 of 15 isolates), B. multivorans (5 of 19 isolates) and B. stabilis (4 of 14 isolates).
Description of Burkholderia dolosa sp. nov.
Burkholderia dolosa (do.lo'sa. L. fem. adj. dolosa deceitful, unwilling, referring to the absence of growth on a B. cepacia-selective medium).
Gram-negative, small (1·5–2·5 µm long), motile, rod-shaped cells. Biochemical characteristics are listed by Coenye et al. (2001a). In addition, isolates fail to grow on the B. cepacia-selective medium PCAT and are unable to utilize azelaic acid, tryptamine or salicin. Major fatty acid components are 16 : 0 (about 26 %), 17 : 0 cyclo (about 16 %), 19 : 0 cyclo 
8c (about 15 %), 18 : 1 (about 10 %), summed feature 3 (comprising 14 : 0 3-OH, 16 : 1 iso, 12 : 0 alde, an unidentified fatty acid with equivalent chain length of 10·928 or a combination of these fatty acids) (about 9 %) and 16 : 0 3-OH (about 6 %). The G+C content of the DNA is 66·9–67·7 mol%. Compared with other B. cepacia-complex bacteria, strains generate unique 16S rDNA RFLP and recA RFLP patterns and can be identified using a specific 16S rDNA-based PCR assay. Isolates have been obtained from the environment and from sputum of patients with cystic fibrosis.
The type strain is LMG 18943T (=CCUG 47727T). The type strain and other B. dolosa reference strains are available from the BCCM/LMG and CCUG culture collections.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Coenye, T., LiPuma, J. J., Henry, D., Hoste, B., Vandemeulebroecke, K., Gillis, M., Speert, D. P. & Vandamme, P. (2001a). Burkholderia cepacia genomovar VI, a new member of the Burkholderia cepacia complex isolated from cystic fibrosis patients. Int J Syst Evol Microbiol 51, 271–279.[Abstract]
Coenye, T., Mahenthiralingam, E., Henry, D., LiPuma, J. J., Laevens, S., Gillis, M., Speert, D. P. & Vandamme, P. (2001b). Burkholderia ambifaria sp. nov., a novel member of the Burkholderia cepacia complex including biocontrol and cystic fibrosis-related isolates. Int J Syst Evol Microbiol 51, 1481–1490.[Abstract]
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
Henry, D., Campbell, M., LiPuma, J. & Speert, D. (1997). Identification of Burkholderia cepacia isolates from patients with cystic fibrosis and use of a simple new selective medium. J Clin Microbiol 35, 614–619.
LiPuma, J. J., Spliker, T., Gill, L. H., Campbell, P. W., III, Liu, L. & Mahenthiralingam, E. (2001). Disproportionate distribution of Burkholderia cepacia complex species and transmissibility markers in cystic fibrosis. Am J Respir Crit Care Med 164, 92–96.
Mahenthiralingam, E., Bischof, J., Byrne, S. K., Radomski, C., Davies, J. E., Av-Gay, Y. & Vandamme, P. (2000). DNA-based diagnostic approaches for identification of Burkholderia cepacia complex, Burkholderia vietnamiensis, Burkholderia multivorans, Burkholderia stabilis, and Burkholderia cepacia genomovars I and III. J Clin Microbiol 38, 3165–3173.
Pitcher, D. G., Saunders, N. A. & Owen, R. J. (1989). Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 8, 151–156.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Ségonds, C., Heulin, T., Marty, N. & Chabanon, G. (1999). Differentiation of Burkholderia species by PCR-restriction fragment length polymorphism analysis of the 16S rRNA gene and application to cystic fibrosis isolates. J Clin Microbiol 37, 2201–2208.
Vandamme, P., Holmes, B., Vancanneyt, M. & 8 other authors (1997). Occurrence of multiple genomovars of Burkholderia cepacia in cystic fibrosis patients and proposal of Burkholderia multivorans sp. nov. Int J Syst Bacteriol 47, 1188–1200.
Vandamme, P., Mahenthiralingam, E., Holmes, B., Coenye, T., Hoste, B., De Vos, P., Henry, D. & Speert, D. P. (2000). Identification and population structure of Burkholderia stabilis sp. nov. (formerly Burkholderia cepacia genomovar IV). J Clin Microbiol 38, 1042–1047.
Vandamme, P., Henry, D., Coenye, T., Nzula, S., Vancanneyt, M., LiPuma, J. J., Speert, D. P., Govan, J. R. W. & Mahenthiralingam, E. (2002). Burkholderia anthina sp. nov. and Burkholderia pyrrocinia, two additional Burkholderia cepacia complex bacteria, may confound results of new molecular diagnostic tools. FEMS Immunol Med Microbiol 33, 143–149.[CrossRef][Medline]
Vandamme, P., Holmes, B., Coenye, T., Goris, J., Mahenthiralingam, E., LiPuma, J. J. & Govan, J. R. W. (2003). Burkholderia cenocepacia sp. nov. – a new twist to an old story. Res Microbiol 154, 91–96.[Medline]
Vermis, K., Coenye, T., Mahenthiralingam, E., Nelis, H. J. & Vandamme, P. (2002a). Evaluation of species-specific recA-based PCR tests for genomovar level identification within the Burkholderia cepacia complex. J Med Microbiol 51, 937–940.
Vermis, K., Vandekerckhove, C., Nelis, H. J. & Vandamme, P. A. R. (2002b). Evaluation of restriction fragment length polymorphism analysis of 16S rDNA as a tool for genomovar characterisation within the Burkholderia cepacia complex. FEMS Microbiol Lett 214, 1–5.[CrossRef][Medline]
Vermis, K., Vandamme, P. & Nelis, H. J. (2003). Burkholderia cepacia complex genomovars: utilization of carbon sources, susceptibility to antimicrobial agents and growth on selective media. J Appl Microbiol 95, 1191–1199.[CrossRef][Medline]
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