Differentiation of Burkholderia species by PCR-restriction fragment lengthpolymorphism analysis of the 16S rRNA gene and application to cystic fibrosis isolates

Burkholderia cepacia, which is an important pathogen in cystic fibrosis (CF ) owing to the potential severity of the infections and the high transmissi bility of some clones, has been recently shown to be a complex of five geno mic groups, i.e,, genomovars I, II. (B. multivorans), III, and IV and B. vi etnamiensis. B. gladioli is also involved, though rarely, in CF, Since stan dard laboratory procedures fail to provide an accurate identification of th ese organisms, we assessed the ability of restriction fragment length polym orphism (RFLP) analysis of amplified 16S ribosomal DNA (rDNA), with the com bination of the patterns obtained with six endonucleases, to differentiate Burkholderia species. This method was applied to 16 type and reference stra ins of the genus Burkholderia and to 51 presumed B. cepacia clinical isolat es, each representative of one clone previously determined by PCR ribotypin g. The 12 Burkholderia type strains tested were differentiated, including B . cepacia, B. multivorans, B. vietnamiensis, and B. gladioli, but neither t he genomovar I and III reference strains nor the genomovar TV reference str ain and B. pyrrocinia(T) were distinguishable. CF clinical isolates were ma inly distributed in RFLP group 2 (which includes B. multivorans(T)) and RFL P group 1 (which includes B. cepacia genomovar I and III reference strains, as well as nosocomial clinical isolates). Two of the five highly transmiss ible clones in French CF centers belonged to RFLP group 2, and three belong ed to RFLP group 1. The remaining isolates either clustered with other Burk holderia species (B. cepacia genomovar TV or B. pyrrocinia, B. vietnamiensi s, and B. gladioli) or harbored unique combinations of patterns. Thus, if f urther validated by hybridization studies, PCR-RFLP of 16S rDNA could be an interesting identification tool and contribute to a better evaluation of t he respective clinical risks associated with each Burkholderia species or g enomovar in patients with CF.