High-throughput sequencing of Bacillus anthracis in France: investigating genome diversity and population structure using whole-genome SNP discovery

BMC Genomics

Sylviane Derzelle¹  Gilles Vergnaud²  Yann Blouin²  Guillaume Girault¹ 

[1] University Paris-Est, Anses, Animal Health Laboratory, Bacterial Zoonoses Unit, Maisons-Alfort 94706, France;University Paris-Sud, Institut de Génétique et Microbiologie, UMR 8621, Orsay 91405, France

关键词: HRM;    Comparative genomics;    Molecular typing;    Single nucleotide polymorphism;    Whole genome sequencing;    Bacillus anthracis;   

Others  :  1217468
DOI  :  10.1186/1471-2164-15-288

 received in 2013-11-21, accepted in 2014-04-09,  发布年份 2014

【 摘 要 】

Background

Single nucleotide polymorphisms (SNPs) are ideal signatures for subtyping monomorphic pathogens such as Bacillus anthracis. Here we report the use of next-generation sequencing technology to investigate the historical, geographic and genetic diversity of Bacillus anthracis in France. 122 strains isolated over a 60-years period throughout the country were whole-genome sequenced and comparative analyses were carried out with a focus on SNPs discovery to discriminate regional sub-groups of strains.

Results

A total of 1581 chromosomal SNPs precisely establish the phylogenetic relationships existing between the French strains. Phylogeography patterns within the three canSNP sub-lineages present in France (i.e. B.Br.CNEVA, A.Br.011/009 and A.Br.001/002) were observed. One of the more remarkable findings was the identification of a variety of genotypes within the A.Br.011/009 sub-group that are persisting in the different regions of France. The 560 SNPs defining the A.Br.011/009- affiliated French strains split the Trans-Eurasian sub-group into six distinct branches without any intermediate nodes. Distinct sub-branches, with some geographic clustering, were resolved. The 345 SNPs defining the major B.Br CNEVA sub-lineage clustered three main phylogeographic clades, the Alps, the Pyrenees, and the Massif Central, with a small Saône-et-Loire sub-cluster nested within the latter group. The French strains affiliated to the minor A.Br.001/002 group were characterized by 226 SNPs. All recent isolates collected from the Doubs department were closely related. Identification of SNPs from whole-genome sequences facilitates high-resolution strain tracking and provides the level of discrimination required for outbreak investigations. Eight diagnostic SNPs, representative of the main French-specific phylogeographic clusters, were therefore selected and developed into high-resolution melting SNP discriminative assays.

Conclusions

This work has established one of the most accurate phylogenetic reconstruction of B. anthracis population structure in a country. An extensive next-generation sequencing (NGS) dataset of 122 French strains have been created that allowed the identification of novel diagnostic SNPs useful to rapidly determine the geographic origin of any strain found in France.

【 授权许可】

   
2014 Girault et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

【 参考文献 】

• [1]Hugh-Jones M, Blackburn J: The ecology of Bacillus anthracis. Mol Aspects Med 2009, 30:356-367.
• [2]Pearson T, Busch JD, Ravel J, Read TD, Rhoton SD, U'Ren JM, Simonson TS, Kachur SM, Leadem RR, Cardon ML, Van Ert MN, Huynh LY, Fraser CM, Keim P: Phylogenetic discovery bias in Bacillus anthracis using single-nucleotide polymorphisms from whole-genome sequencing. Proc Natl Acad Sci U S A 2004, 101:13536-13541.
• [3]Van Ert MN, Easterday WR, Huynh LY, Okinaka RT, Hugh-Jones ME, Ravel J, Zanecki SR, Pearson T, Simonson TS, U'Ren JM, Kachur SM, Leadem-Dougherty RR, Rhoton SD, Guenevier Z, Farlow J, Coker PR, Smith KL, Wang B, Kenefic LJ, Fraser-Liggett CM, Wagner DM, Keim P: Global genetic population structure of Bacillus anthracis. PLoS One 2007, 2:e461.
• [4]Marston CK, Allen CA, Beaudry J, Price EP, Wolken SR, Pearson T, Keim P, Hoffmaster AR: Molecular epidemiology of anthrax cases associated with recreational use of animal hides and yarn in the United States. PLoS One 2011, 6:e28274.
• [5]Price EP, Seymour ML, Sarovich DS, Latham J, Wolken SR, Mason J, Vincent G, Drees KP, Beckstrom-Sternberg SM, Phillippy AM, Koren S, Okinaka RT, Chung WK, Schupp JM, Wagner DM, Vipond R, Foster JT, Bergman NH, Burans J, Pearson T, Brooks T, Keim P: Molecular epidemiologic investigation of an anthrax outbreak among heroin users, Europe. Emerg Infect Dis 2012, 18:1307-1313.
• [6]Van Ert MN, Easterday WR, Simonson TS, U'Ren JM, Pearson T, Kenefic LJ, Busch JD, Huynh LY, Dukerich M, Trim CB, Beaudry J, Welty-Bernard A, Read T, Fraser CM, Ravel J, Keim P: Strain-specific single-nucleotide polymorphism assays for the Bacillus anthracis Ames strain. J Clin Microbiol 2007, 45:47-53.
• [7]Kenefic LJ, Pearson T, Okinaka RT, Schupp JM, Wagner DM, Ravel J, Hoffmaster AR, Trim CP, Chung WK, Beaudry JA, Foster JT, Mead JI, Keim P: Pre-Columbian origins for North American anthrax. PLoS One 2009, 4:e4813.
• [8]Birdsell DN, Pearson T, Price EP, Hornstra HM, Nera RD, Stone N, Gruendike J, Kaufman EL, Pettus AH, Hurbon AN, Buchhagen JL, Harms NJ, Chanturia G, Gyuranecz M, Wagner DM, Keim PS: Melt analysis of mismatch amplification mutation assays (Melt-MAMA): a functional study of a cost-effective SNP genotyping assay in bacterial models. PLoS One 2012, 7:e32866.
• [9]Vaissaire J, Mock M, Le Doujet C, Levy M: Le charbon bactéridien. Epidémiologie de la maladie en France. Méd Mal Infect 2001, 31:257-271.
• [10]Madani N, Mendy C, Moutou F, Garin-Bastuji B: La fièvre charbonneuse en France. Episodes de l'été 2009 et foyers enregistrés sur la dernière décennie (1999–2009). Bulletin épidémiologique 2010, 38:17-19.
• [11]Derzelle S, Laroche S, Le Fleche P, Hauck Y, Thierry S, Vergnaud G, Madani N: Characterization of genetic diversity of Bacillus anthracis in France by using high-resolution melting assays and multilocus variable-number tandem-repeat analysis. J Clin Microbiol 2011, 49:4286-4292.
• [12]NCBI http://www.ncbi.nlm.nih.gov/ webcite
• [13]Pearson T, Okinaka RT, Foster JT, Keim P: Phylogenetic understanding of clonal populations in an era of whole genome sequencing. Infect Genet Evol 2009, 9:1010-1019.
• [14]Smith KL, DeVos V, Bryden H, Price LB, Hugh-Jones ME, Keim P: Bacillus anthracis diversity in Kruger National Park. J Clin Microbiol 2000, 38:3780-3784.
• [15]Simonson TS, Okinaka RT, Wang B, Easterday WR, Huynh L, U'Ren JM, Dukerich M, Zanecki SR, Kenefic LJ, Beaudry J, Schupp JM, Pearson T, Wagner DM, Hoffmaster A, Ravel J, Keim P: Bacillus anthracis in China and its relationship to worldwide lineages. BMC Microbiol 2009, 9:71. BioMed Central Full Text
• [16]Calavas D, Sala C, Vaissaire J, Condé J, Thien-Aubert H, Hessemann M, Woronoff-Rhen N: Retour d'expérience sur un épisode de fièvre charbonneuse chez les bovins dans le Doubs au cours de l'été 2008. Bulletin épidémiologique 2009, 32:1-7.
• [17]IMAGIF http://www.imagif.cnrs.fr webcite
• [18]Kolsto AB, Tourasse NJ, Okstad OA: What sets Bacillus anthracis apart from other Bacillus species? Annu Rev Microbiol 2009, 63:451-476.
• [19]Primer 3+ software http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/ webcite