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

Cystic fibrosis (CF) predisposes patients to bacterial colonization and inf ection of the lower airways. Several species belonging to the genus Burkhol deria are potential CF-related pathogens, but microbiological identificatio n 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 a vailable diagnostic assays is required. ri total of 114 geographically dive rse bacterial isolates, previously identified in reference laboratories as Burkholderia cepacia (n = 51), B. gladioli ill = Iii, Ralstonia pickettii ( n = 6), B. multivorans (n = 2), Stenotrophomonas maltophilia (n = 3), and P seudomonas aeruginosa (n = 11), were collected from environmental, clinical , and reference sources, In addition, 27 clinical isolates putatively. iden tified as Burkholderia spp, were recovered from the sputum of Dutch CF pati ents. till isolates were used to evaluate the accuracy of tn B selective gr owth media, four systems far biochemical identification (API 20NE, Vitek GN I, Vitek NFC, and MicroScan), and three different PCR-based assays. The PCR assays amplify different parts of the ribosomal DNA operation, either alon e or in combination with cleavage by various restriction enzymes (PCR-restr iction fragment length polymorphism [RFLP] analysis). The best system for t he biochemical identification of B, cepacia appeared to be the API ZONE tes t. None of the biochemical assays successfully grouped the B. gladioli stra ins. The PCR-RFLP method appeared to be the optimal method for accurate nuc leic acid-mediated identification of the different Burkholderia spp, With t his method, B. gladioli was also reliably classified in a separate group. F or the laboratory diagnosis of B. cepacia, we recommend parallel cultures o n blood agar medium and selective agar plates. Further identification of co lonies with a Burkholderia phenotype should be performed with the API ZONE test, For final confirmation of species identities, PCR amplification of th e small-subunit rRNA gene followed by RFLP analysis with various enzymes is recommended.