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Environmental isolates from central and north-eastern Thailand were collected and tentatively identified as B. pseudomallei strains, based on their growth on selective media, biochemical profiling and latex agglutination assays (Smith et al., 1995, Wuthiekanun et al., 1995a). However, Brett and colleagues, whilst attempting to develop a transposon based mutagenesis system for B. pseudomallei observed a number of phenotypic and genotypic dissimilarities between clinical isolates and some of the environmental isolates, specifically the differences associated with the decreased virulence for Syrian golden hamsters in comparison to the true B. pseudomallei strains (Brett et al., 1997, DeShazer et al., 1997). These environmental isolates had a more than 105 fold decrease in virulence relative to B. pseudomallei in this animal model of acute melioidosis (Brett et al., 1997). Wuthiekanun and co-workers demonstrated differences in the biochemical characteristics of B. pseudomallei isolates and true isolates, suggesting that these environmental isolates may represent a new species of B. pseudomallei (Brett et al., 1998). Brett and colleagues cloned and sequenced more than 95% of the 16S rDNA from B. pseudomallei 1026b, which is a virulent clinical isolate similar to K96243 and the newly isolated B. pseudomallei-like E264, to assess the relatedness of these organisms to each other, finding 15 nucleotide dissimilarities in all 1488 base pairs of the 16S rRNA upon alignment of the two 16S rRNA alleles, thus confirming the presence of a new Burkholderia species to which they proposed the name B. thailandensis.

B. thailandensis has colonies which are smooth and glossy with pink pigmentation, in comparison to the rough, wrinkled and dark purple pigmentation found in B. pseudomallei colonies and is capable of assimilating L-arabinose (Brett et al., 1997). It is sensitive to tetracycline, ceftayidime and trimethoprim, but resistant to aminoglycosidases. B. thailandensis and B. pseudomallei strains are structurally and immunologically similar to one another and the differences in virulence were further investigated to identify the genetic factors responsible for the enhanced virulence of B. pseudomallei (Brett et al., 1998).

The genomes of B. pseudomallei and B. thailandensis both comprise two highly syntenic chromosomes with comparable numbers of coding regions, protein family distributions and horizontally acquired genomic islands. They share an extensive repertoire of genes involved in core metabolism, accessory pathways, structure-based superfamilies and bacterial virulence factors, however virulence related genes appear to have undergone accelerated change, to adapt to the challenge of infecting and surviving in human or animal hosts in B. pseudomallei. The B. thailandensis chromosomes are 3.8Mb and 2.9Mb (Yu et al., 2006).

The B. thailandensis genome contains at least 15 regions exhibiting either atypical GC content or stretches of bacteriophage related genes and phage-like integrases, which collectively encompass 4-5% of the entire genome. These genomic islands are not found in B. pseudomallei and B. mallei, consistent with their acquisition by B. thailandensis subsequent to the B. pseudomallei-B. thailandensis divergence. The genomic islands of B. thailandensis seem to occur in the same relative genomic location as those in B. pseudomallei, suggesting that these locations may represent genomic hot spots or landmarks for the acquisition of horizontally acquired sequences. Thus the acquisition and loss of large-scale genomic material seems to represent a major driving force in bacterial evolution and often plays a critical role in the development of novel microbial phenotypes.

Although B. thailandensis and B. pseudomallei occupy similar ecological niches, biochemical analysis has identified several phenotypic differences between the species including the ability of B. thailandensis but not B. pseudomallei to assimilate the carbon sources arabinose and xylose (Moore et al., 2004, Smith et al., 1997). B. thailandensis was shown to contain an eight-gene arabinose assimilation operon on chromosome 2 that is absent in B. pseudomallei, where this region has been replaced by protein clusters. Introduction of this operon into B. pseudomallei resulted in the downregulation of a number of type III secretion genes and the strain displayed reduced virulence in Syrian hamsters (Brett et al., 1998, Reckseidler et al., 2001, Wuthiekanun et al., 1996, Wiersinga and van der Poll, 2009). Similarly B. thailandensis contains a 64kb region on chromosome 1 encoding several genes involved in xylose metabolism, which is absent and replaced in B. pseudomallei. This suggests an evolutionary model where the horizontal acquisition by B. pseudomallei may have also resulted in the deletion of the xylose gene cluster, demonstrating how horizontal transfer events can often result in simultaneous gene acquisition and loss (Whitfield and Roberts, 1999).

Variations in surface component proteins have been shown to contribute to virulence in several pathogenic species, such as is seen with fimbriae, short pilus structures allowing bacteria to adhere to environmental surfaces and host cells (Kespichayawattana et al., 2004). B. pseudomallei contains twice as many fimbrial gene clusters as B. thailandensis and has been shown to be more efficient than B. thailandensis in adhering to and invading host cells. Furthermore it has been shown that B. pseudomallei contains a large gene cluster involved in the synthesis and export of capsular polysaccharides, a major determinant of virulence and this cluster is absent in B. thailandensis. The specific location of the B. pseudomallei capsular gene cluster within its genome is likely to be non-random, as it replaces a pre-existing 10-gene cluster in B. thailandensis already dedicated toward the metabolism and processing of polysaccharide structures (Figure 1.9). The original cluster in B. thailandensis contains several genes involved in polysaccharide assembly, raising the possibility that a key event in the pathogenic evolution of B. pseudomallei was to replace a pre-existing or ancestral polysaccharide coat with an alternative pathogenic variant capable of resisting challenges by the immune system of infected hosts (Reckseidler et al., 2001).

Strong conservation between the B. pseudomallei and B. thailandensis proteomes were found in the core metabolic pathways such as amino acid metabolism, cofactor and carrier synthesis, nucleotide and protein biosynthesis, consistent with the ability of B. pseudomallei and B. thailandensis to occupy similar environmental niches (Smith et al., 1997). Unexpectedly, the B. pseudomallei and B. thailandensis proteomes also appear to share significant similarities in their virulence components and 71% of potential virulence genes found in B. pseudomallei are also present in B. thailandensis at an average similarity of greater than 80%, including two type III secretion systems (T3SS2 and T3SS3), antibiotic resistance genes, type IV pili-generating proteins, hemolysin-related genes ad several adhesion factors and proteases (Holden et al., 2004). However, even though both B. pseudomallei and B. thailandensis share two T3SS, required for the full virulence of B. pseudomallei in a hamster model of infection (Warawa and Woods, 2002), it has recently been shown that arabinose exposure may downregulate T3SS expression and activity (Moore et al., 2004). The absence of an arabinose assimilation operon in B. pseudomallei might thus have contributed to the increased virulence of this species.

Figure 1.9 – B. pseudomallei and B. thailandensis polysaccharide biosynthesis clusters.

The schematic was adapted from the genomic comparison study by Yu assessing the genome of B. pseudomallei K96243 and

B. thailandensis E264 (Yu et al., 2006). The diagram shows the capsular polysaccharide biosynthesis cluster of B. pseudomallei K96243 (top – pink) with an ancestral polysaccharide cluster of B. thailandensis E264 (bottom – red).

Genomic comparisons between pathogens and non-pathogenic relatives have played an important role in identifying the mechanisms responsible for acquisition of virulence in the natural environment. Yu and colleagues discuss that gene mutation, gene deletion and gene acquisition on the part of B. pseudomallei are likely to represent the major evolutionary drivers of virulence and that other proposed mechanisms of pathogen evolution including chromosomal rearrangement and bacteriophage-mediated recombination may thus play a less relevant role in the pathogenic evolution (Yu et al., 2006).

B. pseudomallei can only be experimentally manipulated under biosafety level 3 (BSL 3) conditions, but B. thailandensis is non-pathogenic for humans and animals although it displays several phenotypic characteristics similar to B. pseudomallei. As suggested by Nierman and colleagues research on the 16S rRNA phylogeny studies, B. thailandensis is closely related to B. pseudomallei, whilst the phylogenetic similarity between B. pseudomallei and B. mallei suggested it to be a derivative or clone of B. pseudomallei (Nierman et al., 2004). B. thailandensis on the other hand, was thought to have diverged from B. pseudomallei and B. mallei approximately 47 Million years ago, suggesting that although B. thailandensis is avirulent, it is likely to be highly evolutionary related to virulent B. pseudomallei and thus a good candidate for comparative genomic analysis (Yu et al., 2006).