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Journal of Clinical Microbiology, July 1999, p. 2158-2164, Vol. 37, No. 7
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Identification of Burkholderia spp. in the Clinical Microbiology Laboratory: Comparison of Conventional and Molecular Methods

Cindy van Pelt,1,* Cees M. Verduin,1 Wil H. F. Goessens,1 Margreet C. Vos,1 Burkhard Tümmler,2 Christine Segonds,3 Frans Reubsaet,4 Henri Verbrugh,1 and Alex van Belkum1

Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center Rotterdam EMCR, 3015 GD Rotterdam,1 and National Institute of Public Health and the Environment RIVM, 3720 BA Bilthoven,4 The Netherlands; Klinische Forschergruppe Molekulare Pathologie der Mukoviszidose, Zentrum Biochemie und Zentrum Kinderheilkunde, Medizinische Hochschule Hannover, D-30623 Hannover, Germany2; and Observatoire Cepacia, Laboratoire de Bacteriologie-Virologie-Hygiene, Hôpital Rangueil, 31403 Toulouse Cédex 4, France3

Received 30 November 1998/Returned for modification 4 February 1999/Accepted 26 March 1999

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.

* Corresponding author. Mailing address: Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center Rotterdam EMCR, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands. Phone: 31-10-4633668. Fax: 31-10-4633875. E-mail: vanpelt{at}bacl.azr.nl.

Journal of Clinical Microbiology, July 1999, p. 2158-2164, Vol. 37, No. 7
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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