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Journal of Clinical Microbiology, December 2001, p. 4247-4255, Vol. 39, No. 12
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.12.4247-4255.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

PCR-Based Detection and Identification of Burkholderia cepacia Complex Pathogens in Sputum from Cystic Fibrosis Patients

Andrew McDowell,1,* Eshwar Mahenthiralingam,2 John E. Moore,1 Kerstin E. A. Dunbar,1 A. Kevin Webb,3 Mary E. Dodd,3 S. Lorraine Martin,4 B. Cherie Millar,1 Christopher J. Scott,4 Mary Crowe,1 and J. Stuart Elborn5

Molecular Epidemiology Research Unit, Northern Ireland Public Health Laboratory, Department of Bacteriology,1 and Northern Ireland Regional Adult Cystic Fibrosis Center,5 Belfast City Hospital, Belfast, Northern Ireland, United Kingdom BT9 7AB; Cardiff School of Biosciences, Cardiff University, Cardiff, Wales, United Kingdom CF1 3US2; Bradbury Adult Cystic Fibrosis Center, Wythenshawe Hospital, Manchester, England, United Kingdom M23 9LT3; and Biomolecular Sciences Group, School of Pharmacy, The Queen's University of Belfast, Belfast, Northern Ireland, United Kingdom BT9 7BL4

Received 22 June 2001/Returned for modification 5 August 2001/Accepted 8 September 2001

PCR amplification of the recA gene followed by restriction fragment length polymorphism (RFLP) analysis was investigated for the rapid detection and identification of Burkholderia cepacia complex genomovars directly from sputum. Successful amplification of the B. cepacia complex recA gene from cystic fibrosis (CF) patient sputum samples containing B. cepacia genomovar I, Burkholderia multivorans, B. cepacia genomovar III, Burkholderia stabilis, and Burkholderia vietnamiensis was demonstrated. In addition, the genomovar identifications determined directly from sputum were the same as those obtained after selective culturing. Sensitivity experiments revealed that recA-based PCR could reliably detect B. cepacia complex organisms to concentrations of 106 CFU g of sputum-1. To fully assess the diagnostic value of the method, sputum samples from 100 CF patients were screened for B. cepacia complex infection by selective culturing and recA-based PCR. Selective culturing identified 19 samples with presumptive B. cepacia complex infection, which was corroborated by phenotypic analyses. Of the culture-positive sputum samples, 17 were also detected directly by recA-based PCR, while 2 samples were negative. The isolates cultured from both recA-negative sputum samples were subsequently identified as Burkholderia gladioli. RFLP analysis of the recA amplicons revealed 2 patients (12%) infected with B. multivorans, 11 patients (65%) infected with B. cepacia genomovar III-A, and 4 patients (23%) infected with B. cepacia genomovar III-B. These results demonstrate the potential of recA-based PCR-RFLP analysis for the rapid detection and identification of B. cepacia complex genomovars directly from sputum. Where the sensitivity of the assay proves a limitation, sputum samples can be analyzed by selective culturing followed by recA-based analysis of the isolate.

* Corresponding author. Present address: Biomolecular Sciences Group, School of Pharmacy, The Queen's University of Belfast, Medical Biology Center, 97 Lisburn Rd., Belfast, Northern Ireland, United Kingdom BT9 7BL. Phone: 44 (0)2890 272047. Fax: 44 (0)2890 247794. E-mail: a.mcdowell{at}qub.ac.uk.

Journal of Clinical Microbiology, December 2001, p. 4247-4255, Vol. 39, No. 12
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.12.4247-4255.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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