Multilocus sequence typing and evolutionary relationships among the causative agents of melioidosis and glanders, Burkholderia pseudomallei and Burkholderia mallei

Abstract

A collection of 147 isolates of Burkholderia pseudomallei, B. mallei, and B. thailandensis was characterized by multilocus sequence typing (MLST). The 128 isolates of B. pseudomallei, the causative agent of melioidosis, were obtained from diverse geographic locations, from humans and animals with disease, and from the environment and were resolved into 71 sequence types. The utility of the MLST scheme for epidemiological investigations was established by analyzing isolates from captive marine mammals and birds and from humans in Hong Kong with melioidosis. MLST gave a level of resolution similar to that given by pulsed-field gel electrophoresis and identified the same three clones causing disease in animals, each of which was also associated with disease in humans. The average divergence between the alleles of B. thailandensis and B. pseudomallei was 3.2%, and there was no sharing of alleles between these species. Trees constructed from differences in the allelic profiles of the isolates and from the concatenated sequences of the seven loci showed that the B. pseudomallei isolates formed a cluster of closely related lineages that were fully resolved from the cluster of B. thailandensis isolates, confirming their separate species status. However, isolates of B. mallei, the causative agent of glanders, recovered from three continents over a 30-year period had identical allelic profiles, and the B. mallei isolates clustered within the B. pseudomallei group of isolates. Alleles at six of the seven loci in B. mallei were also present within B. pseudomallei isolates, and B. mallei is a clone of B. pseudomallei that, on population genetics grounds, should not be given separate species status.

FIG. 1.

Variable sites within the alleles at the seven MLST loci. The sequences of all of the alleles at each locus that are represented among the 147 Burkholderia isolates are shown. Only the variable sites are shown, and these are numbered in vertical format. For allele 1 at each locus, the nucleotide present at each variable site is shown. For other alleles, only those sites where the nucleotides differ from those in allele 1 are shown; sites that have the same nucleotide as that in allele 1 are shown by a dot. The alleles in normal font are from B. pseudomallei, those in boldface are from B. thailandensis, and the final allele at each locus (in italics) is from the Oklahoma isolates.

FIG. 2.

Relationships among Burkholderia isolates. A UPGMA tree was constructed from the matrix of pairwise differences in the allelic profiles of the 147 Burkholderia isolates. The nodes from which all B. pseudomallei and B. thailandensis isolates descend are marked. The five B. mallei isolates (ST40) have identical allelic profiles and cluster among the B. pseudomallei isolates. Two isolates that were assigned to the species B. pseudomallei but which in this study were found to be closely allied with B. thailandensis (shown as B. thailandensis?) and three isolates from Oklahoma that originally were tentatively assigned to the species B. pseudomallei had divergent allelic profiles and differed from all B. pseudomallei and B. thailandensis isolates at all seven loci. The STs that include at least four isolates and the strain used to obtain the genome sequence (K96243) are shown.

FIG. 3.

Tree constructed from the concatenated sequences of the seven MLST loci. The concatenated sequences from each of the 81 STs represented among the 147 Burkholderia isolates were used to construct a minimum-evolution tree. The percent recoveries of the major nodes in 1,000 bootstrap replicates are shown. The positions of the B. mallei clone (ST40) within the B. pseudomallei STs and of the isolate used for genome sequencing (ST10) are shown. The bar indicates differences at 0.5% of nucleotide sites. The position of B. cepacia genomovar III on the tree is shown by a dotted line, since a homolog of narK was not identified in the B. cepacia genome sequence (www.sanger.ac.uk/Projects/B_cepacia/), and its relationship to the other Burkholderia isolates was obtained on a tree (data not shown) constructed by using the concatenated sequences of only six of the seven MLST loci.

FIG. 4.

Correlation between molecular typing results obtained by MLST and PFGE. A set of 17 isolates of B. pseudomallei from captive animals with melioidosis and their environments and from cases of human disease in Hong Kong were analyzed by both PFGE (A) and MLST (B). The STs are shown on the dendrogram produced from the MLST data.