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

Abstract

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 10 ⁶ 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.