Phylogeography and Genetic Diversity of Francisella tularensis subsp. holarctica in France (1947-2018)

In France, tularemia is caused by Francisella tularensis subsp. holarctica and is a sporadic disease affecting mainly wildlife animals and humans. F. tularensis species presents low genetic diversity that remains poorly described in France, as only a few genomes of isolates from the country are available so far. The objective of this study was to characterize the genetic diversity of F. tularensis in France and describe the phylogenetic distribution of isolates through whole-genome sequencing and molecular typing. Whole genomes of 350 strains of human or animal origin, collected from 1947 to 2018 in France and neighboring countries, were sequenced. A preliminary classification using the established canonical single nucleotide polymorphism (canSNP) nomenclature was performed. All isolates from France (except four) belonged to clade B.44, previously described in Western Europe. To increase the resolution power, a whole-genome SNP analysis was carried out. We were able to accurately reconstruct the population structure according to the global phylogenetic framework, and highlight numerous novel subclades. Whole-genome SNP analysis identified 87 new canSNPs specific to these subclades, among which 82 belonged to clade B.44. Identifying genomic features that are specific to sublineages is highly relevant in epidemiology and public health. We highlighted a large number of clusters among a single clade (B.44), which shows for the first time some genetic diversity among F. tularensis isolates from France, and the star phylogeny observed in clade B.44-subclades revealed that F. tularensis biodiversity in the country is relatively recent and resulted from clonal expansion of a single population. No association between clades and hosts or clinical forms of the disease was detected, but spatiotemporal clusters were identified for the first time in France. This is consistent with the hypothesis of persistence of F. tularensis strains found in Western Europe in the environment, associated with slow replication rates. Moreover, the presence of identical genotypes across long periods of time, and across long distances, supports this hypothesis but also suggests long-distance dispersal of the bacterium.This work was supported by the French National Research Agency (ANR) and the Direction Générale de l’Armement (DGA) (No. ANR-15-ASTR-0021-01). MK is a Ph.D. student co-supported by Université Paris-Est and DGA grants

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

Phenotypic and Molecular Characterization of Brucella microti-Like Bacteria From a Domestic Marsh Frog (Pelophylax ridibundus)

Several Brucella isolates have been described in wild-caught and “exotic” amphibians from various continents and identified as B. inopinata-like strains. On the basis of epidemiological investigations conducted in June 2017 in France in a farm producing domestic frogs (Pelophylax ridibundus) for human consumption of frog's legs, potentially pathogenic bacteria were isolated from adults showing lesions (joint and subcutaneous abscesses). The bacteria were initially misidentified as Ochrobactrum anthropi using a commercial identification system, prior to being identified as Brucella spp. by MALDI-TOF assay. Classical phenotypic identification confirmed the Brucella genus, but did not make it possible to conclude unequivocally on species determination. Conventional and innovative bacteriological and molecular methods concluded that the investigated strain was very close to B. microti species, and not B. inopinata-like strains, as expected. The methods included growth kinetic, antimicrobial susceptibility testing, RT-PCR, Bruce-Ladder, Suis-Ladder, RFLP-PCR, AMOS-ERY, MLVA-16, the ectoine system, 16S rRNA and recA sequence analyses, the LPS pattern, in silico MLST-21, comparative whole-genome analyses (including average nucleotide identity ANI and whole-genome SNP analysis) and HRM-PCR assays. Minor polyphasic discrepancies, especially phage lysis and A-dominant agglutination patterns, as well as, small molecular divergences suggest the investigated strain should be considered a B. microti-like strain, raising concerns about its environmental persistence and unknown animal pathogenic and zoonotic potential as for other B. microti strains described to date

Structural and Barrier Properties of Compatibilized PE/PA6 Multinanolayer Films

The barrier performance and structural lightening of organic materials are increasingly desired and constitute a major challenge for manufacturers, particularly for transport and packaging. A promising technique which tends to emerge in recent years is that of multinanolayer coextrusion. The advantage is that it can produce multilayers made of thousands of very thin layers, leading to new properties due to crystalline morphology changes induced by confinement. This paper is focusing on the study of multinanolayered films with alternated polyethylene (PE), compatibilizer (PEgMA) and polyamide 6 (PA6) layers and made by a forced assembly coextrusion process equipped with layer multiplying elements (LME). PE/PA6 multilayer films consisting of 5 to 2049 layers (respectively 0 to 9 LME) were successfully obtained with well-organized multilayered structure. The evolution of the morphology and the microstructure of these two semi-crystalline polymers, when the thickness of each polymer layer decreases from micro-scale to nano-scale, was correlated to the water and gas transport properties of the PE/PA multilayers. The expected improvement of barrier properties was limited due to the on-edge orientation of crystals in very thin PE and PA6 layers. Despite this change of crystalline morphology, a slight improvement of the gas barrier properties was shown by comparing experimental results with permeabilities predicted on the basis of a serial model developed by considering a PE/PA6 interphase. This interphase observed by TEM images and the on-edge crystal orientation in multilayers were evidenced from mechanical properties showing an increase of the stiffness and the strength

Convergence of a Lagrange--Galerkin method for a fluid-rigid body system in ALE formulation

International audienceIn this paper, we propose a numerical scheme to compute the motion of a two-dimensional rigid body in a viscous fluid, modeled by the Navier-Stokes equations. Our method combines a finite element approximations and the use of the method of characteristics to solve an Arbitrary Lagrangian Eulerian formulation of the problem. We derive error estimates for this scheme which imply its convergence

Développement d'outils de typage moléculaire de haute résolution pour la détection et la différenciation de Bacillus anthracis

Bacillus anthracis is a pathogenic bacterium with a worldwide repartition. It is the causative agent of a zoonosis named Anthrax. Belonging to the Bacillus genus, B. anthracis has the ability to sporulate: this particularity allows the bacterium to stay as quiescent spores into the soils and to resist against different kind of stresses (UV, heat treatment…). Mammals are principally infected and human or animal outbreaks are reported annually in the world. Several regions are endemic while some others, like France, report more sporadic cases. Even if Anthrax incidence is in constant decrease all over the world, B. anthracis is still a pathogen of interest for many countries because of its potential use as a biological weapon. The study of this bacterium has a two-tier purpose: first, it is the cause of a relative mortality in livestock and wildlife, and second it is potentially used as a weapon of mass destruction. This bacterial species is considered highly monomorphic and all strains are extremely closed genetically. In order to precisely identify strains during outbreaks or bioterrorism attempt and track the source of infection, several diagnosis and typing methods are available. However, not all of these methods have the discrimination power, the robustness or the ease of use that is required in the laboratory. During this work, I have studied the diversity of European isolates of B. anthracis. A whole genome sequencing approach for approximately 250 strains has been done with a great diversity into strains (France, Europe). A comparative bioinformatic analysis allowed genomes reconstruction and polymorphisms identification among European isolates (Single Nucleotide Polymorphism = SNP). These markers leaded to establish a precise phylogeny among 292 B. anthracis isolates at a world level. Several hypotheses concerning the origins and the evolution of this pathogen have been proposed. A new sublineage has been potentially discovered. A panel of sixty SNPs has been identified and confirmed to genotype the major groups and lineages phylogenetically related in Europe. Two molecular typing approaches based on PCR amplification have been developed. Using the identified SNP, they can be used to discriminate strains between each others. The first one is a quick and low cost approach (PCR HRM) whereas the second can be used to multiplex a lot of analyses (Luminex). My work allowed a significative increase into B. anthracis knowledge. The typing tools developed will allow traceability and quick identification of the strains involved in an Anthrax outbreak or in a suspected bioterrorist attempt in France.Bacillus anthracis est une bactérie pathogène de répartition mondiale qui est responsable d’une zoonose, le charbon bactéridien. Appartenant au genre Bacillus, elle possède la capacité de sporuler : cette particularité lui permet de rester en dormance dans les sols et de résister à de nombreux stress (UV, chaleur…). Infectant principalement les mammifères, cette bactérie est responsable de foyers animaux et humains recensés annuellement à travers le monde. Certaines régions sont endémiques, d’autres, comme la France, ne connaissent que quelques cas sporadiques par an. Malgré l’incidence décroissante de la maladie à travers le monde, B. anthracis reste néanmoins d’un intérêt majeur pour de nombreux pays étant donné son usage potentiel en tant qu’arme biologique. L’étude de cette bactérie a une finalité duale, d’une part pour la mortalité qu’elle cause au sein des cheptels bovins et de la faune sauvage, et d’autre part, pour son potentiel en tant qu’arme de destruction massive. Cette espèce bactérienne est considérée comme très clonale : toutes les souches sont extrêmement proches d’un point de vue génétique. Afin de pouvoir identifier précisément les souches et remonter à la source d’infection, diverses méthodes de diagnostic et de typage moléculaire sont disponibles. Cependant, toutes ne possèdent pas le pouvoir de résolution souhaité, la robustesse ou la simplicité d’utilisation. Mon travail a consisté à étudier la diversité des souches de B. anthracis disponibles en Europe. Pour cela, un séquençage du génome complet a été réalisé sur un panel d’environ 250 souches (France, Europe) afin de disposer d’une très grande diversité. Une analyse bioinformatique comparative a permis de reconstruire les génomes des isolats européens et l’identification de polymorphismes de séquence spécifiques à ces souches (ici, les Single Nucleotide Polymorphims = SNP). Ces marqueurs ont conduit à établir avec précision la phylogénie de 292 souches de B. anthracis au niveau mondial. Plusieurs hypothèses concernant les origines et l’évolution de ce pathogène ont été proposées. Une proposition d’une nouvelle sous-lignée a également été faite. Enfin, un panel de soixante nouveaux marqueurs de type SNP a été identifié et validé pour génotyper les groupes et lignées phylogénétiquement apparentés présents en Europe. Deux méthodes de biologie moléculaire basées sur l’amplification PCR de ces marqueurs SNP ont été mises au point. La première permet une analyse rapide à faible coût (PCR HRM) alors que la seconde permet un multiplexage des tests (Luminex). Mon travail a permis un accroissement significatif de la connaissance de ce pathogène qu’est B. anthracis. Les outils de typage mis en place permettront à la France de disposer d’un outil de diagnostic performant, assurant la traçabilité et l’identification rapide des souches impliquées lors de foyers de fièvre charbonneuse ou lors de cas importés

Development of high resolution molecular typing tools for detection and differentiation of Bacillus anthracis

Bacillus anthracis est une bactérie pathogène de répartition mondiale qui est responsable d’une zoonose, le charbon bactéridien. Appartenant au genre Bacillus, elle possède la capacité de sporuler : cette particularité lui permet de rester en dormance dans les sols et de résister à de nombreux stress (UV, chaleur…). Infectant principalement les mammifères, cette bactérie est responsable de foyers animaux et humains recensés annuellement à travers le monde. Certaines régions sont endémiques, d’autres, comme la France, ne connaissent que quelques cas sporadiques par an. Malgré l’incidence décroissante de la maladie à travers le monde, B. anthracis reste néanmoins d’un intérêt majeur pour de nombreux pays étant donné son usage potentiel en tant qu’arme biologique. L’étude de cette bactérie a une finalité duale, d’une part pour la mortalité qu’elle cause au sein des cheptels bovins et de la faune sauvage, et d’autre part, pour son potentiel en tant qu’arme de destruction massive. Cette espèce bactérienne est considérée comme très clonale : toutes les souches sont extrêmement proches d’un point de vue génétique. Afin de pouvoir identifier précisément les souches et remonter à la source d’infection, diverses méthodes de diagnostic et de typage moléculaire sont disponibles. Cependant, toutes ne possèdent pas le pouvoir de résolution souhaité, la robustesse ou la simplicité d’utilisation. Mon travail a consisté à étudier la diversité des souches de B. anthracis disponibles en Europe. Pour cela, un séquençage du génome complet a été réalisé sur un panel d’environ 250 souches (France, Europe) afin de disposer d’une très grande diversité. Une analyse bioinformatique comparative a permis de reconstruire les génomes des isolats européens et l’identification de polymorphismes de séquence spécifiques à ces souches (ici, les Single Nucleotide Polymorphims = SNP). Ces marqueurs ont conduit à établir avec précision la phylogénie de 292 souches de B. anthracis au niveau mondial. Plusieurs hypothèses concernant les origines et l’évolution de ce pathogène ont été proposées. Une proposition d’une nouvelle sous-lignée a également été faite. Enfin, un panel de soixante nouveaux marqueurs de type SNP a été identifié et validé pour génotyper les groupes et lignées phylogénétiquement apparentés présents en Europe. Deux méthodes de biologie moléculaire basées sur l’amplification PCR de ces marqueurs SNP ont été mises au point. La première permet une analyse rapide à faible coût (PCR HRM) alors que la seconde permet un multiplexage des tests (Luminex). Mon travail a permis un accroissement significatif de la connaissance de ce pathogène qu’est B. anthracis. Les outils de typage mis en place permettront à la France de disposer d’un outil de diagnostic performant, assurant la traçabilité et l’identification rapide des souches impliquées lors de foyers de fièvre charbonneuse ou lors de cas importés.Bacillus anthracis is a pathogenic bacterium with a worldwide repartition. It is the causative agent of a zoonosis named Anthrax. Belonging to the Bacillus genus, B. anthracis has the ability to sporulate: this particularity allows the bacterium to stay as quiescent spores into the soils and to resist against different kind of stresses (UV, heat treatment…). Mammals are principally infected and human or animal outbreaks are reported annually in the world. Several regions are endemic while some others, like France, report more sporadic cases. Even if Anthrax incidence is in constant decrease all over the world, B. anthracis is still a pathogen of interest for many countries because of its potential use as a biological weapon. The study of this bacterium has a two-tier purpose: first, it is the cause of a relative mortality in livestock and wildlife, and second it is potentially used as a weapon of mass destruction. This bacterial species is considered highly monomorphic and all strains are extremely closed genetically. In order to precisely identify strains during outbreaks or bioterrorism attempt and track the source of infection, several diagnosis and typing methods are available. However, not all of these methods have the discrimination power, the robustness or the ease of use that is required in the laboratory. During this work, I have studied the diversity of European isolates of B. anthracis. A whole genome sequencing approach for approximately 250 strains has been done with a great diversity into strains (France, Europe). A comparative bioinformatic analysis allowed genomes reconstruction and polymorphisms identification among European isolates (Single Nucleotide Polymorphism = SNP). These markers leaded to establish a precise phylogeny among 292 B. anthracis isolates at a world level. Several hypotheses concerning the origins and the evolution of this pathogen have been proposed. A new sublineage has been potentially discovered. A panel of sixty SNPs has been identified and confirmed to genotype the major groups and lineages phylogenetically related in Europe. Two molecular typing approaches based on PCR amplification have been developed. Using the identified SNP, they can be used to discriminate strains between each others. The first one is a quick and low cost approach (PCR HRM) whereas the second can be used to multiplex a lot of analyses (Luminex). My work allowed a significative increase into B. anthracis knowledge. The typing tools developed will allow traceability and quick identification of the strains involved in an Anthrax outbreak or in a suspected bioterrorist attempt in France

Les modes de vie dans les écoquartiers. Phase 2 : les éco-quartiers à fort portage et à visibilité institutionnels (ZAC de Bonne à Grenoble et La Courrouze à Rennes)

Le présent rapport constitue le tome 2 de la démarche pluriannuelle menée pour le bureau AD4. Après avoir appréhendé modes de vie, représentations sociales et schémas de valeurs dans des cas d’initiatives habitantes et/ou d’orientation sociale (la Résidence du Parc à Graulhet dans le Tarn, les Courtils à Hédé-Bazouges en Ille-et-Vilaine, l’Eco-quartier du Four à Pain à Faux-la-Montagne dans la Creuse), la présente recherche est consacrée à des éco-quartiers à fort portage et à forte visibilité institutionnelle : ZAC de Bonne à Grenoble et la Courrouze à Rennes. Dans les deux cas, ces explorations ont été conduites sur une base pluridisciplinaire (sociologie, psychologie environnementale, géographie sociale et sciences politiques), à partir, pour le premier temps, d’entretiens qualitatifs allant de 40 minutes à 2 heures 30 chez les habitants (42) ou avec les acteurs (15), et de groupe de discussion (en cumulé 5 heures), et dans le second, de 40 entretiens allant de 1 à 2 heures, chez l’habitant

La fabrique métropolitaine au défi de ses périphéries : vers une infrapolitique de l’habiter ?

International audienc

Les éco-quartiers : vers une infrapolitique par les modes de vie et leurs communs

International audienc