Discrimination of Burkholderia gladioli from other Burkholderia species detectable in cystic fibrosis patients by PCR

Abstract

A procedure for molecular identification of Burkholderia gladioli is described. Specific 16S and 23S rRNA gene signature sequences were defined as primers for PCR. The method allows rapid and specific discrimination of B. gladioli from related species (B. cepacia, B. multivorans, B. vietnamiensis, B. mallei, B. pseudomallei, Ralstonia pickettii, and R. eutropha) and should contribute to the clarification of its role as a human pathogen, e.g., in cystic fibrosis.

FIG. 1

Alignment of the 16S rDNA within the variable helix 18 region and the variable helix 37 and 38 region. The underlined bases are the target signatures for B. gladioli- and B. cepacia-specific PCR primers. Nucleotide numbering is in accordance with Escherichia coli rDNA numbering (4). Bg.DSM, B. gladioli DSM 4285; Bg.X67038, B. gladioli ATCC 10248^T (13); Bg.S55001, B. gladioli EY3258 (27); Bg.FC329, Bg.H129, Bg.H176, and Bg.CEP025, B. gladioli cystic fibrosis isolates (this study); Bc.type, B. cepacia LMG 1222^T (this study); Bc.M22518, B. cepacia LMG 1222^T (8); Bc.X87275, B. cepacia DSM 50181 (15); Bc.L28675, B. cepacia G4 (16); Bc.IN 122, B. cepacia cystic fibrosis isolate (this study). Wobble base: Y = C or T.

FIG. 2

Alignment of the 23S rDNA within the helix 9 and 10 regions and within the variable helix 45 region. The underlined bases are the target signature for B. gladioli- and B. cepacia-specific PCR primers. Nucleotide numbering is in accordance with E. coli rDNA numbering (5). Bg.DSM, B. gladioli DSM 4285; Bg.FC329, Bg.H129, Bg.H176, and Bg.CEP025, B. gladioli cystic fibrosis isolates (this study); Bc.type, B. cepacia LMG 1222^T; Bc.X16368, B. cepacia DSM 50181 (11); Bc.IN 122 and Bc.C5424, B. cepacia cystic fibrosis isolates (this study). The asterisks indicate that there was no nucleotide at this position, in contrast to E. coli.

FIG. 3

Primer combinations and PCR products of two different sizes. Lanes: SM, DNA molecular size markers; 1, CMG-16-1 plus G-16-2 and CMG-23-1 plus CM-23-2 (PCR products obtained with B. gladioli [468 bp] and B. cepacia [388 bp]); 2, CMG-16-1 plus G-16-2 (B. gladioli product [468 bp]); 3, CMG-23-1 plus G-23-2 (B. gladioli product [388 bp]); 4, CMG-16-1 plus G-16-2 (no PCR product obtained with B. cepacia); 5, CMG-23-1 plus G-23-2 (no PCR product obtained with B. cepacia); 6, CMG-16-1 plus CM-16-2 (B. cepacia product [468 bp]); 7, CMG-23-1 plus CM-2-2 (B. cepacia product [388 bp]); 8, CMG-16-1 plus CM-16-2 (no PCR product obtained with B. gladioli); 9, CMG-23-1 plus CM-23-2 (no PCR product obtained with B. gladioli); 10, CMG-16-1 plus CM-16-2 and CMG-23-1 plus G-23-2 (PCR products obtained with B. cepacia [468 bp] and B. gladioli [388 bp]). The first letters(s) of the species from which PCR products were obtained is underlined.