Accurate identification of members of the Burkholderia cepacia complex in cystic fibrosis sputum

The Burkholderia cepacia complex (BCC) is a group of closely related species which includes opportunistic pathogens causing chronic respiratory infections in immunocompromised patients, or individuals affected by cystic fibrosis (CF). Other Burkholderia species causing infection in the CF population are Burkholderia gladioli and Burkholderia pseudomallei. Traditional phenotypic analyses have been demonstrated to be inadequate for reliable identifications of isolates of BCC and B.gladioli. A pan-genomic analysis approach was used to design species-specific probes for Burkholderia cenocepacia, B.cepacia, Burkholderia multivorans, Burkholderia vietnamiensis, Burkholderia ambifaria, Burkholderia dolosa, Burkholderia pyrrocinia and B.gladioli. Multiplex real-time PCR assay was developed and tested using sputum specimens collected from CF patients spiked with Burkholderia species. The assay exhibited 100% sensitivity for all eight target species and detected 10(2) to 10(3)CFUml(-1) when applied to spiked sputum. Our PCR assay resulted highly specific for each of the Burkholderia species tested, allowing discrimination among Burkholderia and non-Burkholderia pathogens. Analysis carried out on 200 sputa positive for the presence of Burkholderia revealed that PCR assay and recA sequencing were fully comparable for identification of Burkholderia at the level of species.