Molecular identification and typing of Burkholderia pseudomallei and Burkholderia mallei: when is enough enough?

Abstract

Burkholderia mallei and B. pseudomallei are highly pathogenic microorganisms for both humans and animals. Moreover, they are regarded as potential agents of bioterrorism. Thus, rapid and unequivocal detection and identification of these dangerous pathogens is critical. In the present study, we describe the use of an optimized protocol for the early diagnosis of experimental glanders and melioidosis and for the rapid differentiation and typing of Burkholderia strains. This experience with PCR-based identification methods indicates that single PCR targets (23S and 16S rRNA genes, 16S-23S intergenic region, fliC and type III secretion gene cluster) should be used with caution for identification of B. mallei and B. pseudomallei, and need to be used alongside molecular methods such as gene sequencing. Several molecular typing procedures have been used to identify genetically related B. pseudomallei and B. mallei isolates, including ribotyping, pulsed-field gel electrophoresis and multilocus sequence typing. However, these methods are time consuming and technically challenging for many laboratories. RAPD, variable amplicon typing scheme, Rep-PCR, BOX-PCR and multiple-locus variable-number tandem repeat analysis have been recommended by us for the rapid differentiation of B. mallei and B. pseudomallei strains.

Workshop on Treatment of and Postexposure Prophylaxis forBurkholderia pseudomalleiandB. malleiInfection, 2010

“…Any positive culture is considered diagnostic for melioidosis because B. pseudomallei is not considered to be a member of the colonizing microbiota. PCR to detect B. pseudomallei and B. mallei in clinical samples has been described, but is less sensitive than culture ( 14 , 15 ). …”

Development of a real-time loop-mediated isothermal amplification assay for detection ofBurkholderia mallei

“…In year 2006, A PCR assay and a 5ʹ nuclease real‐time PCR test targeting flagellar biosynthesis protein‐insertion sequence ( fliP‐ IS 407 A) were reported for specific and direct identification of B. mallei (Scholz et al., ; Tomaso et al., ). Other molecular methods for identification and differentiation of B. mallei from B. pseudomallei include pulse‐field gel electrophoresis, 16S rRNA sequencing, MLVA, PCR‐restriction fragment length polymorphism, randomly amplified polymorphic DNA (RAPD), MLST, BurkDiff and one gene pyrosequencing (Antonov et al., , ; Bowers et al., ; Chantratita et al., ; Gilling, Luna, & Pflugradt, ; Harvey & Minter, ; Scholz et al., ; Tanpiboonsak, Paemanee, Bunyarataphan, & Tungpradabkul, ). The described molecular methods are expensive, time‐consuming and labour intensive and require technical expertise, making them non‐viable for many laboratories with resource‐poor settings.…”

PCR-based Methodologies Used to Detect and Differentiate theBurkholderia pseudomalleicomplex:B. pseudomallei,B. mallei, andB. thailandensis

“…One follow-up study evaluated various procedures for their ability to detect B. pseudomallei and B. mallei in purified DNA, environmental and inoculated clinical samples (Antonov et al, 2008). All of the assays evaluated had 100% accuracy on purified DNA and inoculated clinical samples, with a detection limit of 10-10 2 genomic equivalents.…”