O-antigen type A has been described as a disaccharide glucose-talose repeat in B. pseudomallei, B. mallei, and B. thailandensis and these structures differ only by side group modification. B. pseudomallei modifies the talose residue with a 2-O methyl/4-O acetyl group or with a 2-O acetyl/4-O hydroxyl group [15, 16]. In B. mallei, regardless of whether the 2-O position is methylated or acetylated, the 4-O position remains in its native hydroxyl state . B. thailandensis has been reported to have the same modification patterns as B. pseudomallei[12, 14, 22], but a recent study by Ngugi, et al., suggests that B. thailandensis E264 features a different pattern. Utilizing gas chromatography/mass spectrometry (GC/MS) to examine methylation patterns, they concluded this strain does not methylate the 2-O position. Brett, et al., generated mutants of oacA, the 4-O acetyltransferase gene, which also had the unexpected result of a lack of methylation at the 2-O position. This suggests that the methyl group may be lost during GC/MS or the E264 strain utilized by Ngugi, et al., may have undergone mutation in oacA, losing its methylase capabilities.
In our current study, 21 out of 23 B. mallei strains expressed intact type A O-antigens while the remaining two (ATCC10399 and NCTC120) were rough. Two previous studies showed that B. mallei ATCC10399 had a full ladder pattern by silver staining and immunoblotting [13, 20]. Our genomic analysis has shown that wbiG gene which is known to be involved in the biosynthesis of the type A O-antigen, was disrupted in this strain by IS407A. This supported our finding that ATCC10399 produced rough LPS. B. mallei NCTC120 was also known as a rough LPS type due to the disruption of its wbiE, the glycosyltransferase gene, by IS407A[13, 20]. DNA sequencing of this strain in our current study revealed the absence of this insertion element, however, a 22 base pair artifact remains in the 3′ end of this gene (GenBank: JN581992), suggesting, IS407A remains active in this strain. We believe that the artifact sequence of the IS407A is disruptive enough to yield the same phenotype as the full insertion.
Eleven strains of B. ubonensis, all Australian environmental isolates, were found to express type B. This O-antigen type is present in approximately 14% of all B. pseudomallei isolates of which the vast majority are Australian . We report here the first discovery of B. pseudomallei type B O-antigen in a near-neighbor species. Previously, B. ubonenesis was known in Australia from only two strains, only one of which has been sequenced and contains an unknown O-antigen biosynthesis gene cluster (NZ_ABBE01000374) . Environmental sampling in northern Australia yielded 44 total B. ubonensis strains, which was the species most commonly isolated. Conversely, only two B. thailandensis strains were isolated, the same number as Levy, et al., found . While no study has examined the abundance of B. ubonensis in Southeast Asia, it is possible that these two species occupy a similar environmental niche where B. ubonensis is able to outcompete B. thailandensis in Australia. In support this, B. ubonensis isolated from Papua New Guinea exhibited antibiosis against B. pseudomallei. These Australian isolates may produce a similar compound against B. thailandensis.
B. thailandensis-like species, a new member of the Pseudomallei group, expresses type B2 and a novel ladder pattern seropositive for type B, thus far unknown in any other species or strain. Curiously, B. thailandensis 82172 expresses type B2, as well, marking the first description of another O-antigen type in this species. This strain belongs to a distinct phylogenetic cluster along with four other geographically diverse B. thailandensis strains, only one of which was isolated in Asia. This cluster has been suggested as the beginning of a possible speciation event and the discovery of type B2 LPS lends further credence to this idea .
Burkholderia sp. MSMB175 is another Australian environmental isolate which clusters with the Pseudomallei group on the basis of recA and 16S sequence and may represent a new species (data not shown). The presence of type B2 O-antigen (Table 1) supports the possibility that this strain belongs to the Pseudomallei group.
A 1993 study of northeastern Thai children by Kanaphun, et al., revealed that 80% are seropositive for antibodies against B. pseudomallei by the age of four. Accordingly, over 25% of environmental Burkholderia isolates in Thailand are B. thailandensis. This suggests that some of these children have instead been exposed to this species and not B. pseudomallei, especially given the noted inaccuracies and high background of indirect hemagglutination assays . Little work has examined the seropositive rates in Australia, but two studies in Northern Queensland returned rates of 2.5-5.7% [30, 31]. The high clinical relevance of B. pseudomallei expressing type B or B2 O-antigen, along with the new apparent abundance of these types in Australian near-neighbors, suggest similar exposures may result in false positive diagnoses, as is likely the case in Thailand.
These near-neighbor species are avirulent, B. mallei excepted, and as such are not limited to the biosafety regulations that B. pseudomallei is as a biosafety level 3 (BSL-3) organism. Few laboratories worldwide are properly equipped to handle BSL-3 work and so the finding of B. pseudomallei type LPS in these non-pathogenic Burkholderia species will allow many additional laboratories the opportunity to work towards vaccine development for melioidosis.