Evolution of Burkholderia pseudomallei in recurrent melioidosis

Abstract

Burkholderia pseudomallei, the etiologic agent of human melioidosis, is capable of causing severe acute infection with overwhelming septicemia leading to death. A high rate of recurrent disease occurs in adult patients, most often due to recrudescence of the initial infecting strain. Pathogen persistence and evolution during such relapsing infections are not well understood. Bacterial cells present in the primary inoculum and in late infections may differ greatly, as has been observed in chronic disease, or they may be genetically similar. To test these alternative models, we conducted whole-genome comparisons of clonal primary and relapse B. pseudomallei isolates recovered six months to six years apart from four adult Thai patients. We found differences within each of the four pairs, and some, including a 330 Kb deletion, affected substantial portions of the genome. Many of the changes were associated with increased antibiotic resistance. We also found evidence of positive selection for deleterious mutations in a TetR family transcriptional regulator from a set of 107 additional B. pseudomallei strains. As part of the study, we sequenced to base-pair accuracy the genome of B. pseudomallei strain 1026b, the model used for genetic studies of B. pseudomallei pathogenesis and antibiotic resistance. Our findings provide new insights into pathogen evolution during long-term infections and have important implications for the development of intervention strategies to combat recurrent melioidosis.

Figure 1. Schematic circular diagrams of the large and small chromosomes of the 1026b genome.

Rings from outside to inside: scale with tick marks positioned every 200 Kb, annotated forward CDS and reverse CDS colored-coded by COG functional categories (Table S1), pseudogenes (olive), genomic islands (red), IS elements (blue), rRNA and tRNA (green), GC% plot and (G – C)/(G + C) deviation plot.

Figure 2. Fosmid tilings from 1258a and 1258b spanning the location of the 330 Kb deletion.

Tilings of 1258a (top) and 1258b (bottom) fosmids against the 1026b reference genome as shown. Sequence coordinates of 1026b chromosome 2 appear along the top horizontal line below which three black lines indicate 1x, 2x and 3x fosmid coverage. Two additional horizontal black lines visually separate coverage lines from clone positions, which are represented by rectangles. Four colors within each rectangle illustrate features of the clone position as described in Hayden et al. . Blue dots at the lower left and right edges of rectangles symbolize the x and y fosmid paired-end sequences aligned to the 1026b sequence. In 1258a, five clones (yellow first quarter) spanned the deletion breakpoint and flanked a large coverage gap in the tiling. The x (at 2.0 Mb) and y (at 1.7 Mb) anchored positions of a single clone are circled. In contrast, clones from 1258b tiled throughout this region indicating that there is no deletion in this isolate.

Figure 3. Depth of coverage of 1710a Sanger sequence reads and fosmids across chromosome 2.

The number of 1710a sequence reads (red) and fosmids (blue) was counted in 500 bp and 10 Kb sliding windows, respectively, along chromosome 2 of the complete 1710b reference sequence. The spike in coverage of fosmids and sequence reads between 200 and 400 Kb is due to the tandem repeat of 17 genes. The smaller spike of sequence reads between 1800 and 2000 Kb is the result of a repeat of the Rhs element Vgr protein gene BURPS1710b_A0207.