Bacillus anthracis in France : phylogenetic relationships at local, regional and national levels and its involvement in forensic and epidemiologic investigation. – FRANthracis

B. anthracis is an evolutionarily young pathogen that shows an extremely high genetic homology. Due to this lack of diversity, only modern molecular characterization techniques with high discrimination power such as genome-wide SNPs analyses or whole-genome sequencing are effective to differentiate strains.
Next-generation sequencing technologies are powerful tools for the unbiased typing of any pathogen. By interrogating nearly every base of a genome, individualizing genetic markers can be reliably discovered, thus facilitating high-resolution strain tracking. The dramatic improvement of Next-Generation-Sequencing technologies allows now its use at a reasonable cost in systematic characterization of B. anthracis. Large-scale comparative genome sequencing of hundreds of strains from around Europe offers a powerful approach for identifying novel SNPs. New DNA signatures that discriminate further within sub-lineages or specifically define a particular strain will be searched for in silico and developed into new genotyping tools. Recent technologies, such as High Resolution Melting (HRM) or suspension array (Luminex platform) will be used. PCR-HRM assays are cost-effective and can be rapidly designed around SNPs to determine the extent to which each marker varies in the B. anthracis population.

In order to construct a simplified SNP-typing method to facilitate the identification, sub-typing and tracking of French strains, next-generation sequencing technology was applied for parallel microbial whole-genome characterization. 123 French strains from different geographic areas have been sequenced and comparative analysis carried out. By interrogating nearly every base of these genomes, rare SNP signatures have been discovered that precisely discriminate the three sub-lineages present in France (B.Br.CNEVA, A.Br.011/009 and A.Br.001 / 002) or specifically define particular strains.
561 diagnostic SNPs defining the major B.Br CNEVA sub-lineage were identified. A geographic clustering into 3 to 4 groups (Alps, Pyrenees, Massif Central and the Saône-et-Loire sub-cluster) was observed. Five SNPs, representative of the different French sub-groups affiliated to B.Br CNEVA, were selected and validated. Concerning the A.Br.011/009 subgroup, a less apparent geographic clustering was observed based on 841 SNP markers. The minor A.Br.001/002 subgroup will require a comparative analysis of foreign strains to highlight unique “French” DNA signatures. The sequencing of 80 strains isolated in Europe, resulting from collaborations established with nine European laboratories or institutes, is in progress for this purpose.
Key diagnostic SNPs identified throughout this work will be used to develop precise, fast and inexpensive genotyping tools useful for applications in microbiological forensics and epidemiological investigations of anthrax outbreaks. More fundamentally, the mass production of genomic data will contribute to a better understanding of the biodiversity found within B. anthracis strains in Europe. In this respect, the project will go far beyond what was originally planned.

As a consequence of this project, France will possess the tools and required know-how to react rapidly when a strain identity needs to be established. The work undertaken will also contribute to a better understanding of the B. anthracis population present in Europe and enhance our ability to trace an outbreak strain back to its possible origin.
The current work establishes a high-resolution picture of the genetic diversity of B. anthracis in France and Europe (thanks to a new-built network of foreign partners). It provides valuable datasets for future epidemiological or forensic studies. Facing a suspect case of anthrax, extensive typing data set about autochthonous but also worldwide isolates are indeed required for comparison so that accurate hypotheses can be made about isolate origins. Without in-depth understanding of the anthrax “background”, we stand in danger of drawing false conclusions and making wrongful incriminations.

Genotyping of French Bacillus anthracis strains based on 30-loci Multi Locus VNTR analysis : epidemiology, marker evaluation, and update of the internet genotype database (manuscript submited for publication)

Bacillus anthracis, a spore-forming pathogenic bacterium found throughout the world, is the causative agent of anthrax. In France, a few sporadic cases are recorded each year in areas where outbreaks had been reported previously, but large outbreaks could occur.

Bacillus anthracis is one of the most feared microorganisms due to its high rate of infectivity and lethality, to its global dispersal pattern and to the extreme stability of its spores. It is considered as the main threat in what concerns bioterrorism. Different strategies exist to counter this threat. This project aims at improving forensics and bio-traceability of this potential biowarfare agent, thus discouraging its intentional use.

B. anthracis is considered as an evolutionarily young species, recently emerged from the B. cereus group. It presents very low molecular diversity among strains. Due to this genetic homogeneity, only a combination of genotyping methods with high discrimination power (multiple-locus variable-number tandem repeat analysis (MLVA), single nucleotide repeat (SNR), genome-wide single nucleotide polymorphism (SNP) analyses or even whole-genome sequencing) has proved to be successful in differentiating strains.

The recent and dramatic improvement of whole genome sequencing technology should allow its more frequent use for systematic characterization of B. anthracis. Our project aims at improving knowledge of the existing diversity in B. anthracis isolates in France in order to develop a highly precise and robust subtyping system and know-how for the primary investigation of strain traceability. High-resolution whole-genome sequencing and comparative genomics will be applied to 25 to 50 French strains from different geographic areas to identify their key genetic features. The clonal nature of B. anthracis makes SNPs, which are scattered throughout the whole genome, ideal signatures for subtyping this pathogen. Only rare diagnostic markers can provide the level of discrimination required for microbial forensics and for precise strain identification. As a consequence of this project, France will possess the required know-how to react rapidly when a strain identity needs to be established.

High-Resolution Melting (HRM) will be used to validate these potentially diagnostic SNPs across a hundred of diverse strains. DNA signatures that discriminate lineages and strain groups, as well as specifically define a particular strain, will be selected. HRM capability to detect and distinguish SNRs will also be evaluated. Screening of a few SNR markers could add resolving power to the genotyping tool under development and might be part of the HRM assays..

Discriminative SNPs will be developed into a molecular typing assay and used to provide further data on the whole ANSES collection. Precise identification of B. anthracis isolates enables epidemiological analyses of natural anthrax cases or forensic investigations of criminal acts as part of the Biotox-Piratox network. Studies will be undertaken (1) to describe the historical, geographic and genetic diversity of the three lineages established in France (A.Br.008/009, A.Br.001/002 and B.Br.CNEVA), (2) to establish the phylogenetic relationships among B. anthracis isolates inside and outside France and (3) to investigate major epizootic recently occurred, such as those in Doubs in 2008 (involving strains affiliated with the A.Br.001/002 sub-group) and in Alps in 2009 (strains allocated into the B.Br.CNEVA sub-lineage). An extensive genotype data set will be created.