Identification of Burkholderia spp. in the clinical microbiology laboratory: comparison of conventional and molecular methods

Abstract

Cystic fibrosis (CF) predisposes patients to bacterial colonization and infection of the lower airways. Several species belonging to the genus Burkholderia are potential CF-related pathogens, but microbiological identification may be complicated. This situation is not in the least due to the poorly defined taxonomic status of these bacteria, and further validation of the available diagnostic assays is required. A total of 114 geographically diverse bacterial isolates, previously identified in reference laboratories as Burkholderia cepacia (n = 51), B. gladioli (n = 14), Ralstonia pickettii (n = 6), B. multivorans (n = 2), Stenotrophomonas maltophilia (n = 3), and Pseudomonas aeruginosa (n = 11), were collected from environmental, clinical, and reference sources. In addition, 27 clinical isolates putatively identified as Burkholderia spp. were recovered from the sputum of Dutch CF patients. All isolates were used to evaluate the accuracy of two selective growth media, four systems for biochemical identification (API 20NE, Vitek GNI, Vitek NFC, and MicroScan), and three different PCR-based assays. The PCR assays amplify different parts of the ribosomal DNA operon, either alone or in combination with cleavage by various restriction enzymes (PCR-restriction fragment length polymorphism [RFLP] analysis). The best system for the biochemical identification of B. cepacia appeared to be the API 20NE test. None of the biochemical assays successfully grouped the B. gladioli strains. The PCR-RFLP method appeared to be the optimal method for accurate nucleic acid-mediated identification of the different Burkholderia spp. With this method, B. gladioli was also reliably classified in a separate group. For the laboratory diagnosis of B. cepacia, we recommend parallel cultures on blood agar medium and selective agar plates. Further identification of colonies with a Burkholderia phenotype should be performed with the API 20NE test. For final confirmation of species identities, PCR amplification of the small-subunit rRNA gene followed by RFLP analysis with various enzymes is recommended.

FIG. 1

PCR-RFLP analysis of Burkholderia spp. and other gram-negative bacilli. The four panels display the results obtained by restriction of small-subunit rRNA amplicons with the restriction endonucleases indicated below the panels. The lanes show the RFLP types found in each enzyme assay. DNA templates were derived from the following strains (from left to right) (RFLP type): B. cepacia Dutch patient isolate (AAAA), B. cepacia ATCC 25416 (AAAB), B. cepacia H134-6 (ABBB), B. gladioli ATCC 10248 (BBBC), B. gladioli RIVM 95-665 (BBEC), R. pickettii CCUG3314 (DDDE), P. aeruginosa ATCC 27853 (EEFH), and S. maltophilia RIVM 96-330 (CCCD). Lane MW, molecular weight (MW) standard.

FIG. 2

Prediction of different restriction sites in the ribosomal genes for Burkholderia spp. Numbering identifies nucleotides in the consensus small-subunit rRNA gene sequences available through GenBank. Restriction sites are highlighted by boxes, and the nature of the restriction enzyme is indicated by the appropriate abbreviation.