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

doi:10.1128/JCM.39.12.4247-4255.2001

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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 forthe rapid detection and identification of Burkholderia cepaciacomplex genomovars directly from sputum. Successful amplificationof the B. cepacia complex recA gene from cystic fibrosis (CF)patient sputum samples containing B. cepacia genomovar I, Burkholderiamultivorans, B. cepacia genomovar III, Burkholderia stabilis,and Burkholderia vietnamiensis was demonstrated. In addition,the genomovar identifications determined directly from sputumwere the same as those obtained after selective culturing. Sensitivityexperiments revealed that recA-based PCR could reliably detectB. cepacia complex organisms to concentrations of 10⁶ CFU g of sputum¹. To fully assess the diagnostic value of the method, sputum samplesfrom 100 CF patients were screened for B. cepacia complex infectionby selective culturing and recA-based PCR. Selective culturingidentified 19 samples with presumptive B. cepacia complex infection,which was corroborated by phenotypic analyses. Of the culture-positivesputum samples, 17 were also detected directly by recA-based PCR,while 2 samples were negative. The isolates cultured from bothrecA-negative sputum samples were subsequently identified as Burkholderiagladioli. RFLP analysis of the recA amplicons revealed 2 patients(12%) infected with B. multivorans, 11 patients (65%) infectedwith B. cepacia genomovar III-A, and 4 patients (23%) infectedwith B. cepacia genomovar III-B. These results demonstrate thepotential of recA-based PCR-RFLP analysis for the rapid detectionand identification of B. cepacia complex genomovars directly fromsputum. Where the sensitivity of the assay proves a limitation,sputum samples can be analyzed by selective culturing followedby recA-based analysis of theisolate.

^* 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)2890247794. E-mail: a.mcdowell{at}qub.ac.uk.

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