Development of rRNA-Based PCR Assays for Identification of Burkholderia cepacia Complex Isolates Recovered from Cystic Fibrosis Patients

Abstract

PCR assays targeting rRNA genes were developed to identify species (genomovars) within the Burkholderia cepacia complex. Each assay was tested with 177 bacterial isolates that also underwent taxonomic analysis by whole-cell protein profile. These isolates were from clinical and environmental sources and included 107 B. cepacia complex strains, 23 Burkholderia gladiolistrains, 20 Ralstonia pickettii strains, 10Pseudomonas aeruginosa strains, 8 Stenotrophomonas maltophilia strains, and 9 isolates belonging to nine other species. The sensitivity and specificity of the 16S rRNA-based assay for Burkholderia multivorans (genomovar II) were 100 and 99%, respectively; for Burkholderia vietnamiensis(genomovar V), sensitivity and specificity were 87 and 92%, respectively. An assay based on 16S and 23S rRNA gene analysis ofB. cepacia ATCC 25416 (genomovar I) was useful in identifying genomovars I, III, and IV as a group (sensitivity, 100%, and specificity, 99%). Another assay, designed to be specific at the genus level, identified all but one of the Burkholderia andRalstonia isolates tested (sensitivity, 99%, and specificity, 96%). The combined use of these assays offers a significant improvement over previously published PCR assays forB. cepacia.

Arbitrarily primed PCR and sequencing of 16S rDNA for epidemiological typing and species identification of Burkholderia cepacia isolates from Swedish patients with cystic fibrosis reveal genetic heterogeneityNote

“…As expected, the two B. multivorans isolates were highly similar to the B. vietnamiensis strains, appearing in the vicinity of the reference strains of these species. These were all identical by analysis of the V3 region; the single-base species-level differences were detected elsewhere as described by LiPuma et al (23). Two 16S rDNA clusters were clearly distinguishable: one for B. cepacia, the other for B. vietnamiensis and B. multivorans.…”

“…Primers designed for ribosomal gene sequences give reliable results for identification to genus and often to species level of most bacteria due to conserved and variable sequences in the ribosomal 16S and 23S RNA genes. Recently introduced rDNA-based PCR methods using multiple primer pairs for identification of Burk-holderia allow discrimination of not only different species but also subspecies (22)(23)(24)(25).…”

Assessment of the specificity of Burkholderia and Pseudomonas qPCR assays for detection of these genera in soil using 454 pyrosequencing

“…The two soils are amended with two different fertilizers; sludge and household compost. The last years' rapid development of fully sequenced bacteria and changing phylogenetic trees has called for a revision of the previously used Burkholderia and Pseudomonas primers, because they were designed using a limited number of sequences, which makes these genusspecific primers unspecific or too specific not covering the entire genera of Pseudomonas and Burkholderia (Widmer et al, 1998;Johnsen et al, 1999;LiPuma et al, 1999;Khan & Yadav, 2004;Lloyd-Jones et al, 2005). Furthermore, some of the published primers for Burkholderia and Pseudomonas are based on the use of one specific primer and one general primer, which increases the possibility of false positives.…”

Evaluation of restriction fragment length polymorphism analysis of 16S rDNA as a tool for genomovar characterisation within the Burkholderia cepacia complex

“…Accurate and simple identi¢cation of B. cepacia complex and subsequent genomovar determination is not straightforward in the routine clinical microbiological laboratory [2,13,17]. Genomovar-speci¢c PCR tests based on the 16S and 23S rRNA genes [18,19] or the RecA gene sequences [14] have recently been developed. The main disadvantage of such an approach with this complex comprising at least nine species are the need to perform multiple PCR tests and the presence of false negative results [20].…”