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

Abstract

Discrimination of Burkholderia (B.) pseudomallei and B. mallei from environmental B. thailandensis is challenging. We describe a discrimination method based on sequence comparison of the ribosomal protein S21 (rpsU) gene. The rpsU gene was sequenced in ten B. pseudomallei, six B. mallei, one B. thailandensis reference strains, six isolates of B. pseudomallei, and 37 of B. thailandensis. Further rpsU sequences of six B. pseudomallei, three B. mallei, and one B. thailandensis were identified via NCBI GenBank. Three to four variable base-positions were identified within a 120-base-pair fragment, allowing discrimination of the B. pseudomallei/mallei-cluster from B. thailandensis, whose sequences clustered identically. All B. mallei and three B. pseudomallei sequences were identical, while 17/22 B. pseudomallei strains differed in one nucleotide (78A>C). Sequences of the rpsU fragment of ‘out-stander’ reference strains of B. cepacia, B. gladioli, B. plantarii, and B. vietnamensis clustered differently. Sequence comparison of the described rpsU gene fragment can be used as a supplementary diagnostic procedure for the discrimination of B. mallei/pseudomallei from B. thailandensis as well as from other species of the genus Burkholderia, keeping in mind that it does not allow for a differentiation between B. mallei and B. pseudomallei.

rpsU-based discrimination within the genusBurkholderia

“…Recently, sequence analysis of a 120-base-pair fragment of rpsU coding for a ribosomal protein S21 homolog with a length of 70 amino acids upstream of the B. pseudomallei fl iC [25] was described as a method for discriminating B. mallei and B. pseudomallei from apathogenic B. thailandensis within the B. pseudomallei complex [26]. The protein belongs to the eubacterial macromolecular synthesis (MMS) operon playing a role in the initiation of protein, DNA, and RNA synthesis.…”

“…The protein belongs to the eubacterial macromolecular synthesis (MMS) operon playing a role in the initiation of protein, DNA, and RNA synthesis. The species-specifi c variations within the Burkholderia genus seem to be low [26,27]. So far, the power of rpsU sequencing to discriminate agents of the B. pseudomallei complex from other Burkholderia spp.…”

Comparison of mast Burkholderia cepacia, ashdown + gentamicin, and Burkholderia pseudomallei selective agar for the selective growth of Burkholderia spp.

“…Focusing on combined approaches for the detection of B. pseudomallei and B. mallei , the causative agents of melioidosis and glanders, respectively [1, 2], Ashdown agar was described to be most sensitive for the isolation of B. pseudomallei [25]. The finding of that study [25] that growth of B. mallei generally fails on this medium, however, could not be confirmed by the here presented data using Ashdown + G agar, although the agar did not allow the growth of all B. mallei strains investigated.…”

“…mallei , the causative agent of glanders, and Burkholderia pseudomallei , the causative agent of melioidosis [1, 2], species with relevance for cystic fibrosis patients, e.g., the Burkholderia cepacia complex, and environmental species [3, 4]. …”

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

“…Several methods have been developed for detection of various combinations of these species, which include serologic tests (Samosornsuk et al, 1999;Steinmetz et al, 1999;Anuntagool et al, 2000;Chenthamarakshan et al, 2001;Cheng et al, 2006), commercial biochemical tests (Inglis et al, 1998;Lowe et al, 2002;Glass and Popovic, 2005;Amornchai et al, 2007), GC-FAME (Inglis et al, 2003), GLC-FAME (Inglis et al, 2005), microscopic methods (Walsh et al, 1994;Wuthiekanun et al, 2005;Hagen et al, 2011), MALDI-TOF (Inglis et al, 2012), PCR subtractive hybridization technique , PCR-RFLP (Tanpiboonsak et al, 2004) and gene sequencing (Woo et al, 2002;Gee et al, 2003;Frickmann et al, 2012). Serologic tests may be unreliable in endemic areas due to seroconversion (White, 2003) for those previously exposed to the organism.…”