The Identification and Differentiation between Burkholderia mallei and Burkholderia pseudomallei Using One Gene Pyrosequencing

Abstract

The etiologic agents for melioidosis and glanders, Burkholderia mallei and Burkholderia pseudomallei respectively, are genetically similar making identification and differentiation from other Burkholderia species and each other challenging. We used pyrosequencing to determine the presence or absence of an insertion sequence IS407A within the flagellin P (fliP) gene and to exploit the difference in orientation of this gene in the two species. Oligonucleotide primers were designed to selectively target the IS407A-fliP interface in B. mallei and the fliP gene specifically at the insertion point in B. pseudomallei. We then examined DNA from ten B. mallei, ten B. pseudomallei, 14 B. cepacia, eight other Burkholderia spp., and 17 other bacteria. Resultant pyrograms encompassed the target sequence that contained either the fliP gene with the IS407A interruption or the fully intact fliP gene with 100% sensitivity and 100% specificity. These pyrosequencing assays based upon a single gene enable investigators to reliably identify the two species. The information obtained by these assays provides more knowledge of the genomic reduction that created the new species B. mallei from B. pseudomallei and may point to new targets that can be exploited in the future.

Development of a rapid and sensitive recombinase polymerase amplification‐lateral flow assay for detection ofBurkholderia mallei

“…Molecular methods viz. randomly amplified polymorphic DNA (RAPD), MLST, BurkDiff, pulse‐field gel electrophoresis, multiple locus variable number of tandem repeats, MLVA and one gene pyrosequencing have also been developed for identification and differentiation of B. mallei and B. pseudomallei (Antonov et al., ; Bowers et al., ; Chantratita et al., ; Gilling, Luna, & Pflugradt, ; Scholz et al., ). Polymerase chain reaction and other molecular assays are laboratory based, time consuming, labour intensive and require complicated instrumentation and technical expertise.…”

“…The fliP-IS407A genomic region has earlier been used for the development of PCR and real-time PCR assays for direct and specific detection of B. mallei Tomaso et al, 2006). Polymerase chain reaction-based assays reported prior to the year 2006 provided indirect proof of B. mallei detection based on the absence of B. pseudomallei-specific DNA sequences in B. mallei (Lee, Wang, & Yap, 2005;Thibault, Valade, & Vidal, 2004 pseudomallei (Antonov et al, 2007;Bowers et al, 2010;Chantratita et al, 2006;Gilling, Luna, & Pflugradt, 2014;Scholz et al, 2014 (Mirzai, Safi, Mossavari, Afshar, & Bolourchian, 2016;Pal et al, 2018). One LAMP assay has limitation regarding specificity (Mirzai et al, 2016) and the other has a detection limit of 1 pg genomic DNA of B. mallei (Pal et al, 2018).…”

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.…”

Patogenesis dan virulensi Burkholderia pseudomallei penyebab melioidosis dan Burkholderia cepacia sebagai patogen oportunis

“…Namun berdasarkan literatur, diketahui bahwa mutasi frame-shift pada yang dikode oleh gen motB milik B. mallei dapat digunakan sebagai primer duplex PCR. (15,16)…”