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.

FIG. 1

RFLP analysis of the 1-kb recA gene amplified from strains of B. cepacia genomovar I (GI), B. multivorans (Bm), B. cepacia genomovar III (GIII), B. stabilis (Bs), and B. vietnamiensis (Bv). (A) HaeIII RFLP analysis. The alphabetical RFLP types, along with their genomovar status, are shown above the lanes. Lanes (left to right): Ma, molecular size markers (100-bp ladder); D, LMG1222; E, CEP509; F, C5393; C, C1576; G, LMG12614; G, C5424; H, C1394; J, PC184; I, CEP511; J, C7322; J, LMG14294; A, LMG16230; and B, C2822. (B) MnlI RFLP analysis. The alphabetical RFLP types, along with their genomovar status, are shown above the lanes. Lanes are as described for panel A, except for C1576, which was replaced with LMG14273.

FIG. 2

PCR amplification of the 1-kb B. cepacia complex recA gene from the sputum of CF patients infected with genomovar I (GI), B. multivorans (Bm), genomovar III (GIII), B. stabilis (Bs), and B. vietnamiensis (Bv). The genomovar status of each sample is shown above each lane. Molecular size markers (100-bp ladder) were run in lane Ma.

FIG. 3

Comparison of recA-based PCR-RFLP identification results (with HaeIII) produced directly from CF sputum and after selective culturing. (A) Patient infected with B. multivorans. The RFLP type produced directly from analysis of the patient's sputum is shown in lane S. The RFLP types produced upon analysis of 15 colonies isolated by selective culturing are shown in lanes 1 to 15. Molecular size markers (100-bp ladder) were run in lane Ma. (B) Patient infected with genomovar III (recA group III-A). The RFLP type obtained directly from analysis of the patient's sputum is shown in lane S. The RFLP types produced upon analysis of 15 colonies isolated by selective culturing are shown in lanes 1 to 15. Molecular size markers (100-bp ladder) were run in lane Ma.

FIG. 4

Experimental approach for recA-based detection and identification of B. cepacia complex genomovars in CF sputum.