What matters in chronic <i>Burkholderia cenocepacia</i> infection in cystic fibrosis: Insights from comparative genomics

<div><p><i>Burkholderia cenocepacia</i> causes severe pulmonary infections in cystic fibrosis (CF) patients. Since the bacterium is virtually untreatable by antibiotics, chronic infections persist for years and might develop into fatal septic pneumonia (cepacia syndrome, CS). To devise new strategies to combat chronic <i>B</i>. <i>cenocepacia</i> infections, it is essential to obtain comprehensive knowledge about their pathogenesis. We conducted a comparative genomic analysis of 32 Czech isolates of epidemic clone <i>B</i>. <i>cenocepacia</i> ST32 isolated from various stages of chronic infection in 8 CF patients. High numbers of large-scale deletions were found to occur during chronic infection, affecting preferentially genomic islands and nonessential replicons. Recombination between insertion sequences (IS) was inferred as the mechanism behind deletion formation; the most numerous IS group was specific for the ST32 clone and has undergone transposition burst since its divergence. Genes functionally related to transition metal metabolism were identified as hotspots for deletions and IS insertions. This functional category was also represented among genes where nonsynonymous point mutations and indels occurred parallelly among patients. Another category exhibiting parallel mutations was oxidative stress protection; mutations in catalase KatG resulted in impaired detoxification of hydrogen peroxide. Deep sequencing revealed substantial polymorphism in genes of both categories within the sputum <i>B</i>. <i>cenocepacia</i> ST32 populations, indicating extensive adaptive evolution. Neither oxidative stress response nor transition metal metabolism genes were previously reported to undergo parallel evolution during chronic CF infection. Mutations in <i>katG</i> and copper metabolism genes were overrepresented in patients where chronic infection developed into CS. Among professional phagocytes, macrophages use both hydrogen peroxide and copper for their bactericidal activity; our results thus tentatively point to macrophages as suspects in pathogenesis towards the fatal CS.</p></div

Localization of mutations in proteins under parallel evolution during chronic infection.

<p>Protein chains under parallel evolution are denoted in light blue, their functional domains are denoted in dark blue (DHp in CusS, βi4 in RpoB). Cofactors are denoted as forest green spheres, catalytic and active site amino acids are denoted as lime green spheres. Amino acids homologous to residues affected by mutations during chronic Bcc infection (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006762#ppat.1006762.s013" target="_blank">S10 Table</a>) are denoted as spheres and colored as explained in the legend (for ST32 WGS data, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006762#ppat.1006762.g003" target="_blank">Fig 3</a>; for ST32 DPS data, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006762#ppat.1006762.t001" target="_blank">Table 1</a>; for IIIA WGS data, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006762#ppat.1006762.s012" target="_blank">S9 Table</a>). Visualizations were carried out in Chimera [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006762#ppat.1006762.ref083" target="_blank">83</a>].</p

3D-plot of parallel mutations among 8 patients with chronic ST32 infections resulting in CS.

<p>All genes where nonsynonymous substitutions occurred in at least 1 isolate from at least 3 patients are depicted. Reported mutations have arisen during within-patient evolution, <i>i</i>.<i>e</i>. are missing in ancestral ST32 genotype which initially colonized the patient (inferred from WGS phylogeny, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006762#ppat.1006762.g001" target="_blank">Fig 1B</a>).</p

Sensitivity of ST32 isolates to hydrogen peroxide.

<p>Minimum inhibitory concentrations (MIC) towards H₂O₂ were recorded after aerobic growth in Luria broth at 37°C for 24 h. MIC values were constant during 3 biological replicates.</p

Parallel mutations in <i>B</i>. <i>cenocepacia</i> ST32 sputum populations from 12 chronically infected, non-CS patients.

<p>Parallel mutations in <i>B</i>. <i>cenocepacia</i> ST32 sputum populations from 12 chronically infected, non-CS patients.</p

Overview of sequenced <i>B</i>. <i>cenocepacia</i> ST32 isolates.

<p>(A) Sample collection chart. Bacteria were isolated at indicated time points and named by patient IDs (1 to 8) and chronology of isolation A to D (i.e., isolates 1A to 1D, 2A to 2D etc.). Isolates 2C and 2D, 5C and 5D, 7C and 7D, and 8C and 8D overlapped due to their concurrent isolation at the time of CS (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006762#ppat.1006762.s004" target="_blank">S1 Table</a> for details). (B) Whole-genome phylogeny. Consensus genomic sequences obtained by mapping sequencing reads onto the complete reference genome were used for tree inference. The Maximum-Likelihood tree was constructed from 2,754 variant nucleotide positions using the CSIPhylogeny pipeline [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006762#ppat.1006762.ref074" target="_blank">74</a>]. (C) SNP accumulation during chronic infection. SNPs informative of within-patient evolution (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006762#sec012" target="_blank">Materials and Methods</a>) were plotted against time elapsed from the first Bcc culture positivity to the point of bacterial isolation.</p

Insertion sequences (IS), genomic islands (GI) and large-scale deletions (LSD) in ST32 isolates.

<p>The inner circles denote particular isolates´ genomes and reference genome, ordered as indicated. IS insertions and LSDs over 10 kb in size are colored as explained in the legend. The outer circles denote GC skew, GC content and GIs as explained in the legend. The four replicons are not plotted to scale; their relative sizes are denoted in the middle. Visualizations were carried out in BRIG [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006762#ppat.1006762.ref082" target="_blank">82</a>]. For source data, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006762#ppat.1006762.s006" target="_blank">S3</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006762#ppat.1006762.s009" target="_blank">S6</a> Tables.</p