Species-specific PCR as a tool for the identification of Burkholderia gladioli

Abstract

Burkholderia gladioli colonizes the respiratory tracts of patients with cystic fibrosis and chronic granulomatous disease. However, due to the high degree of phenotypic similarity between this species and closely related species in the Burkholderia cepacia complex, accurate identification is difficult. Incorrect identification of these species may have serious repercussions for the management of patients with cystic fibrosis. To develop an accurate procedure for the identification of B. gladioli, a molecular method to discriminate between this species and other species commonly isolated from the sputa of patients with cystic fibrosis was investigated. The 23S ribosomal DNA was cloned from several clinical isolates of B. gladioli, and the nucleotide sequence was determined. Computer-assisted sequence comparisons indicated four regions of the 23S rRNA specific for this species; these regions were used to design three primer pairs for species-specific PCR. Two of the primer pairs showed 100% sensitivity and specificity for B. gladioli when tested against a panel of 47 isolates comprising 19 B. gladioli isolates and 28 isolates of 16 other bacterial species. One of the primer pairs was further assessed for species specificity by using a panel of 102 isolates obtained from the Burkholderia cepacia Research Laboratory and Repository. The species-specific PCR was positive for 70 of 74 isolates of B. gladioli and was negative for all other bacterial species examined. Overall, this primer pair displayed a sensitivity and specificity of 96% (89 of 93) and 100%, respectively. These data demonstrate the potential of species-specific PCR for the identification of B. gladioli.