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

Abstract

Burkholderia cepacia, which is an important pathogen in cystic fibrosis (CF) owing to the potential severity of the infections and the high transmissibility of some clones, has been recently shown to be a complex of five genomic groups, i.e., genomovars I, II (B. multivorans), III, and IV and B. vietnamiensis. B. gladioli is also involved, though rarely, in CF. Since standard laboratory procedures fail to provide an accurate identification of these organisms, we assessed the ability of restriction fragment length polymorphism (RFLP) analysis of amplified 16S ribosomal DNA (rDNA), with the combination of the patterns obtained with six endonucleases, to differentiate Burkholderia species. This method was applied to 16 type and reference strains of the genus Burkholderia and to 51 presumed B. cepacia clinical isolates, each representative of one clone previously determined by PCR ribotyping. The 12 Burkholderia type strains tested were differentiated, including B. cepacia, B. multivorans, B. vietnamiensis, and B. gladioli, but neither the genomovar I and III reference strains nor the genomovar IV reference strain and B. pyrrociniaT were distinguishable. CF clinical isolates were mainly distributed in RFLP group 2 (which includes B. multivoransT) and RFLP group 1 (which includes B. cepacia genomovar I and III reference strains, as well as nosocomial clinical isolates). Two of the five highly transmissible clones in French CF centers belonged to RFLP group 2, and three belonged to RFLP group 1. The remaining isolates either clustered with other Burkholderia species (B. cepacia genomovar IV or B. pyrrocinia, B. vietnamiensis, and B. gladioli) or harbored unique combinations of patterns. Thus, if further validated by hybridization studies, PCR-RFLP of 16S rDNA could be an interesting identification tool and contribute to a better evaluation of the respective clinical risks associated with each Burkholderia species or genomovar in patients with CF.

FIG. 1

DdeI restriction patterns (type and reference strains). Lanes: 1 to 5 (cepacia complex strains), B. cepacia genomovar I ATCC 25416^T, B. cepacia genomovar II (B. multivorans) LMG 13010^T, B. cepacia genomovar III LMG 12615, B. cepacia genomovar IV LMG 14294, and B. vietnamiensis TVV75^T, respectively; 6 to 14 (other Burkholderia species), B. gladioli pv. gladioli CFBP 2427^T, B. gladioli pv. alliicola CFBP 2422^T, B. cocovenenans LMG 11626^T, B. pyrrocinia LMG 14191^T, B. caryophilli LMG 2155^T, B. andropogonis LMG 2129^T, B. plantarii LMG 9035^T, B. glumae CFBP 2430^T, and B. glathei LMG 14190^T, respectively; 15 and 16 (other genera), R. pickettii ATCC 27511^T and Stenotrophomonas maltophilia ATCC 13637^T, respectively; M, molecular weight marker VIII (Boehringer Mannheim).