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Background   Burkholderia cenocepacia  is the most prominent species of the  B. cepacia  complex (Bcc), a group of nine closely related and difficult to identify bacteria that cause serious infections in patients with cystic fibrosis. Despite its clinical relevance, identification of  B. cenocepacia  as a single species is unavailable, as it splits by a widely used  recA  gene-based PCR identification method into discrete phylogenetic subgroups IIIA, IIIB, IIIC and IIID. With the aim of identifying gene targets suitable for unified detection of  B. cenocepacia  strains, we examined sequence polymorphisms in the  repA  and  parB  genes. These essential genes are involved in the replication and partitioning of bacterial replicons, hence we also had the opportunity for the first time to investigate the evolution of the multireplicon (three chromosome) structure of Bcc genomes.    Results  Alignment of the  repA  and  parB  genes from publicly available Bcc genome sequences enabled the design of primers for their amplification and sequence analysis. Multilocus sequencing typing, a highly discriminatory method for Bcc species and strain discrimination, was used to select strains of unique sequence types (STs) that spanned the known Bcc genetic diversity. Sequence datasets of  repA  (83 isolates, 67 STs) and  parB  (120 isolates, 95 STs) genes from the second chromosome were aligned and examined phylogenetically to identify polymorphisms suitable for identification of  B. cenocepacia . In contrast to  parB , the Bcc  repA  sequences demonstrated distinct clustering of  B. cenocepacia  from other species, which enabled the design a species-specific multiplex PCR. The novel single-reaction  B. cenocepacia  detection method was tested on a panel of 142 different Bcc strains (142 STs) and distinguished  recA  groups IIIA, IIIB and IIID, from all other Bcc members with 100% sensitivity and 93% specificity.    Conclusion  The  repA -based multiplex PCR is a useful aid to the rapid identification of the most clinically relevant  B. cenocepacia recA  subgroups IIIA, IIIB and IIID. Phylogenetic analysis of  repA  and  parB  genes demonstrated that acquisition of the second and third replicons of Bcc genomes occurred prior to their differentiation into discrete species and that the sharing of replicons across species had not occurred.

Diversity of the parB and repA genes of the Burkholderia cepacia complex and their utility for rapid identification of Burkholderia cenocepacia