An objective approach for Burkholderia pseudomallei strain selection as challenge material for medical countermeasures efficacy testing

Burkholderia pseudomallei is the causative agent of melioidosis, a rare disease of biodefense concern with high mortality and extreme difficulty in treatment. No human vaccines are available that protect against B. pseudomallei infection, and with the current limitations of antibiotic treatment, the development of new preventative and therapeutic interventions is crucial. Although clinical trials could be used to test the efficacy of new medical countermeasures (MCMs), the high mortality rates associated with melioidosis raises significant ethical issues concerning treating individuals with new compounds with unknown efficacies. The US Food and Drug Administration (FDA) has formulated a set of guidelines for the licensure of new MCMs to treat diseases in which it would be unethical to test the efficacy of these drugs in humans. The FDA “Animal Rule” 21 CFR 314 calls for consistent, well-characterized B. pseudomallei strains to be used as challenge material in animal models. In order to facilitate the efficacy testing of new MCMs for melioidosis using animal models, we intend to develop a well-characterized panel of strains for use. This panel will comprise of strains that were isolated from human cases, have a low passage history, are virulent in animal models, and are well-characterized phenotypically and genotypically. We have reviewed published and unpublished data on various B. pseudomallei strains to establish an objective method for selecting the strains to be included in the panel of B. pseudomallei strains with attention to five categories: animal infection models, genetic characterization, clinical and passage history, and availability of the strain to the research community. We identified 109 strains with data in at least one of the five categories, scored each strain based on the gathered data and identified six strains as candidate for a B. pseudomallei strain panel

Анімізм (з Матэрыялаў да «Тлумачальнага слоўніка славянскай міфалогіі»)

<div><p><i>Burkholderia pseudomallei</i> is the causative agent of melioidosis and a potential bioterrorism agent. In the development of medical countermeasures against <i>B</i>. <i>pseudomallei</i> infection, the US Food and Drug Administration (FDA) animal Rule recommends using well-characterized strains in animal challenge studies. In this study, whole genome sequence data were generated for 6 <i>B</i>. <i>pseudomallei</i> isolates previously identified as candidates for animal challenge studies; an additional 5 isolates were sequenced that were associated with human inhalational melioidosis. A core genome single nucleotide polymorphism (SNP) phylogeny inferred from a concatenated SNP alignment from the 11 isolates sequenced in this study and a diverse global collection of isolates demonstrated the diversity of the proposed Animal Rule isolates. To understand the genomic composition of each isolate, a large-scale blast score ratio (LS-BSR) analysis was performed on the entire pan-genome; this demonstrated the variable composition of genes across the panel and also helped to identify genes unique to individual isolates. In addition, a set of ~550 genes associated with pathogenesis in <i>B</i>. <i>pseudomallei</i> were screened against the 11 sequenced genomes with LS-BSR. Differential gene distribution for 54 virulence-associated genes was observed between genomes and three of these genes were correlated with differential virulence observed in animal challenge studies using BALB/c mice. Differentially conserved genes and SNPs associated with disease severity were identified and could be the basis for future studies investigating the pathogenesis of <i>B</i>. <i>pseudomallei</i>. Overall, the genetic characterization of the 11 proposed Animal Rule isolates provides context for future studies involving <i>B</i>. <i>pseudomallei</i> pathogenesis, differential virulence, and efficacy to therapeutics.</p></div

Correlations of LS-BSR values with observed differential virulence in BALB/c mice.

<p>*High, intermediate and low virulence determined by intranasal challenge at ~10 colony forming units.</p><p>Correlations of LS-BSR values with observed differential virulence in BALB/c mice.</p

Annotation for unique genes identified in genomes sequenced in the current study.

<p>Annotation for unique genes identified in genomes sequenced in the current study.</p

Nucleotide variant information for re-sequencing projects conducted in current study.

<p>Nucleotide variant information for re-sequencing projects conducted in current study.</p

Details of isolates sequenced in current study.

<p>*genome has been sequenced previously</p><p>Details of isolates sequenced in current study.</p

A heatmap of blast score ratio (BSR) values [44] calculated from a known set of virulence factors characterized in <i>B</i>. <i>pseudomallei</i> (S3 Table) with the large-scale blast score ratio (LS-BSR) pipeline [43].

<p>A maximum likelihood phylogeny was inferred on a concatenation of single nucleotide polymorphisms (SNPs) and was correlated to the heatmap.</p

A maximum likelihood phylogeny inferred from a concatenation of ~63,000 core-genome single nucleotide polymorphisms (SNPs) identified in the eleven genomes sequenced in this study, shown in red, and a reference set of genomes (S2 Table).

<p>The tree was inferred with RAxML v8 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121052#pone.0121052.ref031" target="_blank">31</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121052#pone.0121052.ref032" target="_blank">32</a>] using the ASC_GTRGAMMA model and 100 bootstrap replicates. Filled circles are placed at nodes where the bootstrap support values are >90%.</p

Plots of single nucleotide polymorphism (SNP) density and homoplasy density (HD), across the two chromosomes of the reference isolate, K96243 [30].

<p>The outer ring represents the number of informative SNPs across 1-kb genomic intervals. The inner ring indicates the number of homoplasious SNPs, as determined by a retention index (RI) value <0.5 calculated by Paup [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121052#pone.0121052.ref035" target="_blank">35</a>], divided by the total number of informative SNPs over the same 1-kb genomic interval. HD and SD values were visualized with Circos [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121052#pone.0121052.ref036" target="_blank">36</a>].</p