Identification and Discrimination of Burkholderia pseudomallei , B. mallei , and B. thailandensis by Real-Time PCR Targeting Type III Secretion System Genes

Abstract

Burkholderia pseudomallei and B. mallei are two highly pathogenic bacteria, responsible for melioidosis and glanders, respectively. The two are closely related and can also be mistaken for B. thailandensis, a nonpathogenic species. To improve their differential identification, we describe a hydrolysis probe-based real-time PCR method using the uneven distribution of type III secretion system genes among these three species.

Using real-time PCR to specifically detect Burkholderia mallei

“…An ELISA has been developed using irradiation-killed B. mallei whole cells; however, cross-reactivity with irradiation-killed B. pseudomallei was observed, suggesting that this The development of PCR-based methodologies for the identification and differentiation of B. mallei from B. pseudomallei and B. thailandensis has been troublesome due to the level of genetic similarity between these Burkholderia species (Brett et al, 1998;Godoy et al, 2003;Holden et al, 2004;Nierman et al, 2004). Various chromosomal markers have been used to detect B. pseudomallei, and include the 16S and 23S rRNA, 16S-23S intergenic region, the filamentforming flagellin fliC gene, heat-shock protein Hsp70, and a putative type three secretion operon (Antonov et al, 2005;Bauernfeind et al, 1998;Gee et al, 2003;Hagen et al, 2002;Lee et al, 2005;Sprague et al, 2002;Thibault et al, 2004;Tomaso et al, 2004Tomaso et al, , 2005Tyler et al, 1995;U'Ren et al, 2005). Several of the assays that have been described are targeted toward single-nucleotide differences in the 23S rDNA region, the 16S region, and a putative antibiotic resistance gene.…”

“…For B. pseudomallei, the 16S and 23S rRNA, 16S-23S intergenic region, fliC, heat-shock protein 70 (Hsp70), and a type three secretion (TTS) system have been targeted for the development of molecular-based assays (Antonov et al, 2005;Bauernfeind et al, 1998;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, 2004Tomaso et al, , 2005Tyler et al, 1995;U'Ren et al, 2005). One study required the use of two separate assays and a negative PCR result to discriminate B. mallei from B. pseudomallei.…”

PCR-based identification of Burkholderia pseudomallei

“…Where only conventional PCR is available, the conventional PCR method is still fast enough to replace the confirmatory role proposed for gas liquid chromatography of bacterial fatty acid methyl ester derivatives 7 . In the absence of other confirmatory methods; whether phenotypic or 15,17,18,19,20 . While a study comparing the performance of these assays has yet to be performed it is likely that one or more will find a place in an expanded laboratory discovery pathway, possibly being incorporated into a multiplex real time PCR method.…”

“…Where ambiguities or discrepancies could not be resolved, the PCR product was re-sequenced until a clear read was obtained. The sequences were then aligned using ClustalX v1.83 15 and presented in graphic format using Bioedit v7.0.0 (Isis Pharmaceuticals Inc.) 3 . Lastly, phylogenetic analysis of phaC gene alignments was conducted using Phylodraw v0.82 (Pusan National University, South Korea).…”

Pathogenicity and biotechnological applications of the genus Burkholderia

“…pseudomallei and B. mallei are two highly pathogenic bacteria, responsible for melioidosis and glanders, respectively [3,7]. Melioidosis, which affects humans, is endemic in Southern Asia and northern Australia, and its etiological agent, B. pseudomallei, is an environmental saprophyte that is commonly found in wet soils and stagnant waters throughout the endemic regions [3].…”