A possible pitfall in the identification of Burkholderia mallei using molecular identification systems based on the sequence of the flagellin fliC gene

Abstract

Amotile Burkholderia mallei and motile Burkholderia pseudomallei display a high similarity with regard to phenotype and clinical syndromes, glanders and melioidosis. The aim of this study was to establish a fast and reliable molecular method for identification and differentiation. Despite amotility, the gene of the filament forming flagellin (fliC) could be completely sequenced in two B. mallei strains. Only one mutation was identified discriminating between B. mallei and B. pseudomallei. A polymerase chain reaction-restriction fragment length polymorphism assay was designed making use of the absence of an AvaII recognition site in B. mallei. All seven B. mallei, 12 out of 15 B. pseudomallei and 36 closely related apathogenic Burkholderia thailandensis strains were identified correctly. However, in three B. pseudomallei strains a point mutation at gene position 798 (G to C) disrupted the AvaII site. Therefore, molecular systems based on the fliC sequence can be used for a reliable proof of strains of the three species but not for the differentiation of B. mallei and B. pseudomallei isolates.

Discrimination ofBurkholderia mallei/pseudomalleifromBurkholderia thailandensisby sequence comparison of a fragment of the ribosomal protein S21 (rpsU) gene

“…Thirteen B. pseudomallei reference strains and all six isolates from Thailand showed a 78A>C shift. The strain 844 and the isolates UE 16 and UE 19, which have the same fliC sequence as B. mallei strains as previously demonstrated [11], were identical regarding the rpsU sequence with the other investigated B. pseudomallei isolates with the 78A>C shift. Three to four mutations in the nucleotide sequence of the rpsU gene of B. thailandensis allow for a differentiation of this non-pathogenic saprophyte from the dangerous agents B. mallei and B. pseudomallei as well as from other pathogenic species as B. cepacia and B. gladioli.…”

“…However, DNA-DNA-hybridization studies suggested that they are merely pathovars of one single species [9,10]. Sequence analysis of the gene of the filament-forming flagellin, fliC, corroborated this theory [11].…”

“…The rpsU gene sequences of the following strains were obtained from NCBI GenBank (accession number in brackets) (www.ncbi.nlm.nih.gov/genbank/): B. pseudomallei 1026b (U73848), MSHR 346 (CP001408), 1106a (CP000572), 1710b (CP000124), K96243 (BX571965), 668 (CP000570), B. mallei NCTC 10247 (CP000548), NCTC 10229 (CP000546), SAVP1 (CP000526), B. thailandensis E264 (CP000086) or from previous studies of the authors [11,28] [38] were performed using the open source software ClustalW2 version 2.1 and ClustalX2 version 2.1 (www.clustal.org) [39]. The phylogenetic tree based on the ClustalX2 analysis was printed using the open source NJplot software (http://pbil.univ-lyon1.fr/software/njplot.html).…”

Development of a polymerase chain reaction assay for the specific identification of Burkholderia mallei and differentiation from Burkholderia pseudomallei and other closely related Burkholderiaceae

“…There are several reports in the literature that describe PCR-based assays for identifying B. pseudomallei; however, none of the assays are currently being used for melioidosis diagnosis (Bauernfeind et al, 1998;Gee et al, 2003;Hagen et al, 2002;Holden et al, 2004;Lee et al, 2005;Sprague et al, 2002;Tanpiboonsak et al, 2004;Thibault et al, 2004;Tomaso et al, 2004).…”

“…There have been several reports in the literature that have targeted the B. pseudomallei 16S and 23S rRNA, 16S-23S intergenic region, flagellin C (fliC), heat shock protein 70, and a type 3 secretion (TTS) system for the molecular identification of B. pseudomallei and possible differentiation from B. mallei (Antonov et al, 2004;Gee et al, 2003;Hagen et al, 2002;Lee et al, 2005;Sprague et al, 2002;Tanpiboonsak et al, 2004;Thibault et al, 2004;Tomaso et al, 2005;Tomaso et al, 2004;Tyler et al, 1995). Despite the numerous misleading titles in the literature (i.e., titles that imply B. mallei-specific), there are no B. mallei-specific molecular assays for discriminating this obligate mammalian pathogen from B. pseudomallei.…”