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Journal of Clinical Microbiology, September 2000, p. 3165-3173, Vol. 38, No. 9
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

DNA-Based Diagnostic Approaches for Identification of Burkholderia cepacia Complex, Burkholderia vietnamiensis, Burkholderia multivorans, Burkholderia stabilis, and Burkholderia cepacia Genomovars I and III

Eshwar Mahenthiralingam,1,* Jocelyn Bischof,1,dagger Sean K. Byrne,2 Christopher Radomski,3 Julian E. Davies,3,4 Yossef Av-Gay,5 and Peter Vandamme6

Department of Pediatrics, University of British Columbia and British Columbia's Children's Hospital, British Columbia's Research Institute for Children's and Women's Health,1 Molecular Diagnostics Laboratory, British Columbia's Centre for Disease Control, and Department of Pathology, University of British Columbia,2 Terragen Diversity Incorporated,3 Department of Microbiology, University of British Columbia,4 and Department of Medicine, University of British Columbia,5 Vancouver, British Columbia, Canada, and Laboratory for Microbiology, Faculty of Science, University of Ghent, Ghent, Belgium6

Received 17 February 2000/Returned for modification 28 April 2000/Accepted 12 June 2000

Bacteria of the Burkholderia cepacia complex consist of five discrete genomic species, including genomovars I and III and three new species: Burkholderia multivorans (formerly genomovar II), Burkholderia stabilis (formerly genomovar IV), and Burkholderia vietnamiensis (formerly genomovar V). Strains of all five genomovars are capable of causing opportunistic human infection, and microbiological identification of these closely related species is difficult. The 16S rRNA gene (16S rDNA) and recA gene of these bacteria were examined in order to develop rapid tests for genomovar identification. Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S rDNA revealed sequence polymorphisms capable of identifying B. multivorans and B. vietnamiensis but insufficient to discriminate strains of B. cepacia genomovars I and III and B. stabilis. RFLP analysis of PCR-amplified recA demonstrated sufficient nucleotide sequence variation to enable separation of strains of all five B. cepacia complex genomovars. Complete recA nucleotide sequences were obtained for 20 strains representative of the diversity of the B. cepacia complex. Construction of a recA phylogenetic tree identified six distinct clusters (recA groups): B. multivorans, B. vietnamiensis, B. stabilis, genomovar I, and the subdivision of genomovar III isolates into two recA groups, III-A and III-B. Alignment of recA sequences enabled the design of PCR primers for the specific detection of each of the six latter recA groups. The recA gene was found on the largest chromosome within the genome of B. cepacia complex strains and, in contrast to the findings of a previous study, only a single copy of the gene was present. In conclusion, analysis of the recA gene of the B. cepacia complex provides a rapid and robust nucleotide sequence-based approach to identify and classify this taxonomically complex group of opportunistic pathogens.

* Corresponding author. Present address: Cardiff School of Biosciences, Main Building, Cardiff University, P.O. Box 915, Cardiff CF10 3TL, United Kingdom. Phone: 44 (029) 20875875. Fax: 44 (029) 20874305. E-mail: MahenthiralingamE{at}cardiff.ac.uk.

dagger Present address: Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada.

Journal of Clinical Microbiology, September 2000, p. 3165-3173, Vol. 38, No. 9
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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