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

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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^, 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 andthree new species: Burkholderia multivorans (formerly genomovarII), Burkholderia stabilis (formerly genomovar IV), and Burkholderiavietnamiensis (formerly genomovar V). Strains of all five genomovarsare capable of causing opportunistic human infection, and microbiologicalidentification of these closely related species is difficult.The 16S rRNA gene (16S rDNA) and recA gene of these bacteria wereexamined in order to develop rapid tests for genomovar identification.Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified16S rDNA revealed sequence polymorphisms capable of identifyingB. multivorans and B. vietnamiensis but insufficient to discriminatestrains of B. cepacia genomovars I and III and B. stabilis. RFLPanalysis of PCR-amplified recA demonstrated sufficient nucleotidesequence variation to enable separation of strains of all fiveB. cepacia complex genomovars. Complete recA nucleotide sequenceswere obtained for 20 strains representative of the diversity ofthe B. cepacia complex. Construction of a recA phylogenetic treeidentified six distinct clusters (recA groups): B. multivorans,B. vietnamiensis, B. stabilis, genomovar I, and the subdivisionof genomovar III isolates into two recA groups, III-A and III-B.Alignment of recA sequences enabled the design of PCR primersfor the specific detection of each of the six latter recA groups.The recA gene was found on the largest chromosome within the genomeof B. cepacia complex strains and, in contrast to the findingsof a previous study, only a single copy of the gene was present.In conclusion, analysis of the recA gene of the B. cepacia complexprovides a rapid and robust nucleotide sequence-based approachto identify and classify this taxonomically complex group of opportunisticpathogens.

^* Corresponding author. Present address: Cardiff School of Biosciences, Main Building, Cardiff University, P.O. Box 915, CardiffCF10 3TL, United Kingdom. Phone: 44 (029) 20875875. Fax: 44 (029)20874305. E-mail: MahenthiralingamE{at}cardiff.ac.uk.

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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