Guillaume Girault

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

BackgroundSingle 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.ResultsA 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.ConclusionsThis 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.

A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for Bacillus anthracis

Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and represent informative DNA markers extensively used to analyze phylogenetic relationships between strains. Medium to high throughput, open methodologies able to test many SNPs in a minimum time are therefore in great need. By using the versatile Luminex® xTAG technology, we developed an efficient multiplexed SNP genotyping assay to score 13 phylogenetically informative SNPs within the genome of Bacillus anthracis. The Multiplex Oligonucleotide Ligation-PCR procedure (MOL-PCR) described by Deshpande et al., 2010 has been modified and adapted for simultaneous interrogation of 13 biallelic canonical SNPs in a 13-plex assay. Changes made to the originally published method include the design of allele-specific dual-priming-oligonucleotides (DPOs) as competing detection probes (MOLigo probes) and use of asymmetric PCR reaction for signal amplification and labeling of ligation products carrying SNP targets. These innovations significantly reduce cross-reactivity observed when initial MOLigo probes were used and enhance hybridization efficiency onto the microsphere array, respectively. When evaluated on 73 representative samples, the 13-plex assay yielded unambiguous SNP calls and lineage affiliation. Assay limit of detection was determined to be 2ng of genomic DNA. The reproducibility, robustness and easy-of-use of the present method were validated by a small-scale proficiency testing performed between four European laboratories. While cost-effective compared to other singleplex methods, the present MOL-PCR method offers a high degree of flexibility and scalability. It can easily accommodate newly identified SNPs to increase resolving power to the canSNP typing of B. anthracis.

Whole Genome-Sequencing and Phylogenetic Analysis of a Historical Collection of Bacillus anthracis Strains from Danish Cattle

Bacillus anthracis, the causative agent of anthrax, is known as one of the most genetically monomorphic species. Canonical single-nucleotide polymorphism (SNP) typing and whole-genome sequencing were used to investigate the molecular diversity of eleven B. anthracis strains isolated from cattle in Denmark between 1935 and 1988. Danish strains were assigned into five canSNP groups or lineages, i.e. A.Br.001/002 (n = 4), A.Br.Ames (n = 2), A.Br.008/011 (n = 2), A.Br.005/006 (n = 2) and A.Br.Aust94 (n = 1). The match with the A.Br.Ames lineage is of particular interest as the occurrence of such lineage in Europe is demonstrated for the first time, filling an historical gap within the phylogeography of the lineage. Comparative genome analyses of these strains with 41 isolates from other parts of the world revealed that the two Danish A.Br.008/011 strains were related to the heroin-associated strains responsible for outbreaks of injection anthrax in drug users in Europe. Eight novel diagnostic SNPs that specifically discriminate the different sub-groups of Danish strains were identified and developed into PCR-based genotyping assays.

Draft Genomes of Three Strains Representative of the Bacillus anthracis Diversity Found in France

ABSTRACT We report here the draft genomes of three Bacillus anthracis strains isolated in France: 08-8_20 (A.Br.001/002), 99-100 (A.Br.011/009), and 00-82 (B.Br CNEVA). The total lengths of assemblies are 5,440,708 bp, 5,446,472 bp, and 5,436,014 bp for 08-8_20, 99-100, and 00-82, respectively.

The characterization of Brucella strains isolated from cattle in Algeria reveals the existence of a B. abortus lineage distinct from European and Sub-Saharan Africa strains

Brucellosis is a zoonosis caused by bacteria of the genus Brucella that causes important economic losses and human suffering worldwide. Brucellosis control requires an understanding of the Brucella species circulating in livestock and humans and, although prevalent in African countries of the Mediterranean basin, data for this area are mostly restricted to isolates obtained from humans and small ruminants. Here, we report the characterization of twenty-four Brucella strains isolated from Algerian cattle. Bruce-ladder multiplex PCR and conventional biotyping showed that Algerian cattle are infected mostly by B. abortus biovar 3, and to less extent by B. abortus biovar 1 and B. melitensis biovar 3. Extended AMOS-ERY PCR showed that all Algerian B. abortus biovar 3 strains were of the subgroup 3b. Although by multi locus variable number of tandem repeats analysis (MLVA) most isolates were closer to the European counterparts, five strains displayed characteristics distinct from the European isolates and those of countries across the Sahara, including three repetitions of marker Bruce55. These five strains, plus an earlier isolate from an Algerian human patient, may represent a lineage close to clades previously described in Africa. These data provide the basis for additional molecular epidemiology studies in northern Africa and indicate that further bacteriological and molecular investigations are necessary for a complete understanding of the epidemiology of cattle brucellosis in countries north and south of the Sahara.

Comparative Genomics and in vitro Infection of Field Clonal Isolates of Brucella melitensis Biovar 3 Did Not Identify Signature of Host Adaptation

Brucella spp. are responsible for brucellosis, a widespread zoonosis causing reproductive disorders in animals. Species-classification within this monophyletic genus is based on bacteriological and biochemical phenotyping. Traditionally, Brucella species are reported to have a preferential, but not exclusive mammalian host. However, this concept can be challenged since many Brucella species infect a wide range of animal species. Adaptation to a specific host can be a driver of pathogen variation. It is generally thought that Brucella species have highly stable and conserved genomes, however the degree of genomic variation during natural infection has not been documented. Here, we investigated potential genetic diversity and virulence of Brucella melitensis biovar 3 field isolates obtained from a single outbreak but from different host species (human, bovine, small ruminants). A unique MLVA-16 pattern suggested all isolates were clonal. Comparative genomic analyses showed an almost non-existent genetic diversity among isolates (only one SNP; no architectural rearrangements) and did not highlight any signature specific to host adaptation. Similarly, the strains showed identical capacities to enter and replicate in an in vitro model of macrophage infection. In our study, the absence of genomic variability and similar virulence underline that B. melitensis biovar 3 is a broad-host-range pathogen without the need to adapt to different hosts.

S1 Fig. Evolutionary analyses of 52 strains of B. anthracis based on 29906 chromosomal SNPs.

S1 Fig. Evolutionary analyses of 52 strains of B. anthracis based on 29906 chromosomal SNPs. Phylogenetic relationships were inferred using the Maximum Likelihood method (A), the Maximum Parsimony method (B), the Neighbor-Joining method (C) and the UPGMA method (D). Bootstrap values (100 iterations) higher than 70% are shown next to the branches. All evolutionary analyses were conducted in MEGA6 [28]. The maximum likelihood tree with the highest log likelihood (0.000) is shown in A. Tree #1 out of 2 most parsimonious trees (length = 7469, consistency index = 0.815, retention index = 0.947) is shown in B. The optimal neighbour-joining tree with the sum of branch length = 0.251 is shown in C. The optimal UPGMA tree with the sum of branch length = 0.239 is shown in D. The later tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.

Fig. 1 Position of the eleven Danish strains within the B. anthracis phylogenetic tree based on whole-genome SNP analysis.

Figure 1. Position of the eleven Danish strains from cattle within the B. anthracis phylogenetic tree based on whole-genome SNP analysis. Minimum spanning tree based on 6596 chromosomal SNPs (A), 222 pXO1 SNPs (B) and 166 pXO2 SNPs (C). The 13 different canSNP groups are color-coded: C.Br.A1055 in white, B.Br.CNEVA in yellow, B.Br.001/002 and B.Br.Kruger in orange, A.Br.011/009 in light blue, A.Br.008/011 in blue, A.Br.WNA in dark blue, A.Br.005/006 in pink, A.Br.003/004 in red, A.Br.001/002 in green, A.Br.WNA in dark green, A.Br.Aust94 in brown and A.Br.Vollum in purple. The position of the 11 Danish isolates (in bold and underligned), the African IEMVT89 and 40 available whole genome-sequenced strains is marked. The length of each branch is proportional (logarithmic scale) to the number of SNPs identified between strains. Indicated in red are the position and name of some new or published SNPs specific to various canSNP groups: A05 (A.Br.005/006 group); A.Br.008 (A.Br.008/009 group); A08/D and A08/D1 (A.Br.008/011); A.Br.011 (A.Br.011/009); A.Br.009 (A.Br.WNA); A.Br.002 and A01 (A.Br.001/002 and A.Br.Ames); A02, A02/A, A02/B, A02/B1 (A.Br.001/002 subgroup A02); A.Br.001 and A01/A-DK (A.Br.Ames); A.Br.013, A.Br.015a, A.Br.15b, A.Br.026 (A.Br.Aust94). Based on a parsimony approach, the trees sizes are, respectively, 6730 (A), 227 (B) and 168 SNPs (C), i.e. containing approximately 1.9 (A), 2.2 (B) or 1.2 (C) % of homoplasia.The minimum spanning tree was drawn in BioNumerics version 6.6 (Applied Maths) using default settings.