Bacillus anthracis evolutionary history: taking advantage of polytomies and human history to propose dating points

International audienceThe document deposited here is a copy of the slides presented in support of my talk during the "Biology of Anthrax" meeting held in Bari, Italy (September 3 rd-6 th 2019). The talk was a synthesis and global analysis of our recent investigations taking advantage of the existence of rare remarkable features in the phylogeny of Bacillus anthracis, polytomies, to propose tentative dating points.Le document déposé ici est une copie des diapositives que j'ai utilisées lors du congrès "Biology of Anthrax" (Bari, Italie, 3 au 6 septembre 2019). Bacillus anthracis est une espèce à évolution strictement clonale, ce qui permet de calculer des arbres phylogénétiques très robustes à partir des centaines de génomes complets disponibles. Mais du fait de la capacité de B. anthracis à sporuler, nous ne savons pas actuellement déduire de datation à partir de l'analyse des divergences génétiques. La présentation était une synthèse et une analyse globale de nos investigations récentes qui tirent parti de l'existence de radiations en étoile dans la structure de population de B. anthracis pour proposer des dates. Ces "polytomies" qui sont très rares coïncideraient avec des évènement historiques majeurs, tels que les conquêtes Mongoles au 13eme siècle. Ainsi l'analyse de la répartition géographique des souches constituant les différentes branches de ces polytomies permettrait d'en dater certaines. Les trois pages d'introduction aux diapositives fournissent en principe les éléments de contexte. Trois polytomies ont été étudiées. La présentation comporte deux hypothèses inédites et une prédiction. La première hypothèse est que l'expédition au Québec en 1665 du Régiment de Carignan-Salières a conduit à introduire le lignage de B. anthracis qui est actuellement largement prédominant en Amérique du Nord. La seconde est que les douze premiers chevaux parmi les environ 80 qui seront expédiés de France au Canada entre 1665 et 1671 et constitueront l'origine du "Cheval Canadien" ont été sélectionnés parmi les chevaux du régiment. Le régiment était un régiment d'infanterie, mais à cette époque, un régiment d'infanterie comptait quelques chevaux par compagnie. Le régiment aurait transporté le pathogène à son insu depuis son lieu de garnison (Marsal en Lorraine, région dans laquelle on trouve aujourd'hui les souches de B. anthracis les plus proches de souches d'Amérique du Nord et d'Afrique de l'Ouest) qu'il quitte en Janvier 1665 jusqu'au Québec atteint à l'été et automne 1665. Ce n'est pas la nourriture qui aura pu être porteuse de la contamination puisque le régiment achetait ses provisions à chaque étape de son déplacement en France. Le plus plausible serait alors une contamination d'équipements, voire même une contamination des chevaux qui auraient pu être porteurs sains. Cependant cette dernière option est actuellement encore loin d'être scientifiquement établie

Identification of polymorphic tandem repeats by direct comparison of genome sequence from different bacterial strains : a web-based resource

BACKGROUND: Polymorphic tandem repeat typing is a new generic technology which has been proved to be very efficient for bacterial pathogens such as B. anthracis, M. tuberculosis, P. aeruginosa, L. pneumophila, Y. pestis. The previously developed tandem repeats database takes advantage of the release of genome sequence data for a growing number of bacteria to facilitate the identification of tandem repeats. The development of an assay then requires the evaluation of tandem repeat polymorphism on well-selected sets of isolates. In the case of major human pathogens, such as S. aureus, more than one strain is being sequenced, so that tandem repeats most likely to be polymorphic can now be selected in silico based on genome sequence comparison. RESULTS: In addition to the previously described general Tandem Repeats Database, we have developed a tool to automatically identify tandem repeats of a different length in the genome sequence of two (or more) closely related bacterial strains. Genome comparisons are pre-computed. The results of the comparisons are parsed in a database, which can be conveniently queried over the internet according to criteria of practical value, including repeat unit length, predicted size difference, etc. Comparisons are available for 16 bacterial species, and the orthopox viruses, including the variola virus and three of its close neighbors. CONCLUSIONS: We are presenting an internet-based resource to help develop and perform tandem repeats based bacterial strain typing. The tools accessible at now comprise four parts. The Tandem Repeats Database enables the identification of tandem repeats across entire genomes. The Strain Comparison Page identifies tandem repeats differing between different genome sequences from the same species. The "Blast in the Tandem Repeats Database" facilitates the search for a known tandem repeat and the prediction of amplification product sizes. The "Bacterial Genotyping Page" is a service for strain identification at the subspecies level

The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats

<p>Abstract</p> <p>Background</p> <p>In Archeae and Bacteria, the repeated elements called CRISPRs for "clustered regularly interspaced short palindromic repeats" are believed to participate in the defence against viruses. Short sequences called spacers are stored in-between repeated elements. In the current model, motifs comprising spacers and repeats may target an invading DNA and lead to its degradation through a proposed mechanism similar to RNA interference. Analysis of intra-species polymorphism shows that new motifs (one spacer and one repeated element) are added in a polarised fashion. Although their principal characteristics have been described, a lot remains to be discovered on the way CRISPRs are created and evolve. As new genome sequences become available it appears necessary to develop automated scanning tools to make available CRISPRs related information and to facilitate additional investigations.</p> <p>Description</p> <p>We have produced a program, CRISPRFinder, which identifies CRISPRs and extracts the repeated and unique sequences. Using this software, a database is constructed which is automatically updated monthly from newly released genome sequences. Additional tools were created to allow the alignment of flanking sequences in search for similarities between different loci and to build dictionaries of unique sequences. To date, almost six hundred CRISPRs have been identified in 475 published genomes. Two Archeae out of thirty-seven and about half of Bacteria do not possess a CRISPR. Fine analysis of repeated sequences strongly supports the current view that new motifs are added at one end of the CRISPR adjacent to the putative promoter.</p> <p>Conclusion</p> <p>It is hoped that availability of a public database, regularly updated and which can be queried on the web will help in further dissecting and understanding CRISPR structure and flanking sequences evolution. Subsequent analyses of the intra-species CRISPR polymorphism will be facilitated by CRISPRFinder and the dictionary creator. CRISPRdb is accessible at <url>http://crispr.u-psud.fr/crispr</url></p

CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats

Clustered regularly interspaced short palindromic repeats (CRISPRs) constitute a particular family of tandem repeats found in a wide range of prokaryotic genomes (half of eubacteria and almost all archaea). They consist of a succession of highly conserved regions (DR) varying in size from 23 to 47 bp, separated by similarly sized unique sequences (spacer) of usually viral origin. A CRISPR cluster is flanked on one side by an AT-rich sequence called the leader and assumed to be a transcriptional promoter. Recent studies suggest that this structure represents a putative RNA-interference-based immune system. Here we describe CRISPRFinder, a web service offering tools to (i) detect CRISPRs including the shortest ones (one or two motifs); (ii) define DRs and extract spacers; (iii) get the flanking sequences to determine the leader; (iv) blast spacers against Genbank database and (v) check if the DR is found elsewhere in prokaryotic sequenced genomes. CRISPRFinder is freely accessible at http://crispr.u-psud.fr/Server/CRISPRfinder.php

Pseudolysogeny and sequential mutations build multiresistance to virulent bacteriophages in Pseudomonas aeruginosa

International audienceCoevolution between bacteriophages and their prey is the result of mutualistic interactions. Here we show that pseudolysogeny is a frequent outcome of infection by virulent phages of Pseudomonas aeruginosa, and that selection of resistant bacterial mutants is favored by continuous production of phages. We investigated the frequency and characteristics of P. aeruginosa strain PAO1 variants resisting infection by different combinations of virulent phages belonging to four genera. The frequency of resistant bacteria was 10-5 for single phage infection and 10-6 for infections with combinations of two or four phages. The genome of 27 variants was sequenced and the comparison with the genome of the parental PAO1 strain allowed the identification of point mutations or small indels. Four additional variants were characterized by a candidate gene approach. In total, 27 independent mutations were observed affecting 14 genes and a regulatory region. The mutations affected genes involved in biosynthesis of type IV pilus, alginate, LPS and O-antigen. Half of the variants possessed changes in homopolymer tracts responsible for frameshift mutations, and these phase variation mutants were shown to be unstable. Eleven double mutants were detected. The presence of free phage DNA was observed in association with exclusion of superinfection in half of the variants, and in three of them no chromosomal mutation could be found. Upon further growth of these pseudolysogens, some variants with new chromosomal mutations were recovered presumably due to continuous evolutionary pressure

High resolution, on-line identification of strains from the Mycobacterium tuberculosis complex based on tandem repeat typing

BACKGROUND: Currently available reference methods for the molecular epidemiology of the Mycobacterium tuberculosis complex either lack sensitivity or are still too tedious and slow for routine application. Recently, tandem repeat typing has emerged as a potential alternative. This report contributes to the development of tandem repeat typing for M. tuberculosis by summarising the existing data, developing additional markers, and setting up a freely accessible, fast, and easy to use, internet-based service for strain identification. RESULTS: A collection of 21 VNTRs incorporating 13 previously described loci and 8 newly evaluated markers was used to genotype 90 strains from the M. tuberculosis complex (M. tuberculosis (64 strains), M. bovis (9 strains including 4 BCG representatives), M. africanum (17 strains)). Eighty-four different genotypes are defined. Clustering analysis shows that the M. africanum strains fall into three main groups, one of which is closer to the M. tuberculosis strains, and an other one is closer to the M. bovis strains. The resulting data has been made freely accessible over the internet to allow direct strain identification queries. CONCLUSIONS: Tandem-repeat typing is a PCR-based assay which may prove to be a powerful complement to the existing epidemiological tools for the M. tuberculosis complex. The number of markers to type depends on the identification precision which is required, so that identification can be achieved quickly at low cost in terms of consumables, technical expertise and equipment

Evaluation and selection of tandem repeat loci for Streptococcus pneumoniae MLVA strain typing

BACKGROUND: Precise identification of bacterial pathogens at the strain level is essential for epidemiological purposes. In Streptococcus pneumoniae, the existence of 90 different serotypes makes the typing particularly difficult and requires the use of highly informative tools. Available methods are relatively expensive and cannot be used for large-scale or routine typing of any new isolate. We explore here the potential of MLVA (Multiple Loci VNTR Analysis; VNTR, Variable Number of Tandem Repeats), a method of growing importance in the field of molecular epidemiology, for genotyping of Streptococcus pneumoniae. RESULTS: Available genome sequences were searched for polymorphic tandem repeats. The loci identified were typed across a collection of 56 diverse isolates and including a group of serotype 1 isolates from Africa. Eventually a set of 16 VNTRs was proposed for MLVA-typing of S. pneumoniae. These robust markers were sufficient to discriminate 49 genotypes and to aggregate strains on the basis of the serotype and geographical origin, although some exceptions were found. Such exceptions may reflect serotype switching or horizontal transfer of genetic material. CONCLUSION: We describe a simple PCR-based MLVA genotyping scheme for S. pneumoniae which may prove to be a powerful complement to existing tools for epidemiological studies. Using this technique we uncovered a clonal population of strains, responsible for infections in Burkina Faso. We believe that the proposed MLVA typing scheme can become a standard for epidemiological studies of S. pneumoniae

Genetic diversity of Francisella tularensis subsp. holarctica in Kazakhstan

Tularemia is a highly dangerous zoonotic infection due to the bacteria Francisella tularensis. Low genetic diversity promoted the use of polymorphic tandem repeats (MLVA) as first-line assay for genetic description. Whole genome sequencing (WGS) is becoming increasingly accessible, opening the perspective of a time when WGS might become the universal genotyping assay. The main goal of this study was to describe F. tularensis strains circulating in Kazakhstan based on WGS data and develop a MLVA assay compatible with in vitro and in silico analysis. In vitro MLVA genotyping and WGS were performed for the vaccine strain and for 38 strains isolated in Kazakhstan from natural water bodies, ticks, rodents, carnivores, and from one migratory bird, an Isabellina wheatear captured in a rodent burrow. The two genotyping approaches were congruent and allowed to attribute all strains to two F. tularensis holarctica lineages, B.4 and B.12. The seven tandem repeats polymorphic in the investigated strain collection could be typed in a single multiplex PCR assay. Identical MLVA genotypes were produced by in vitro and in silico analysis, demonstrating full compatibility between the two approaches. The strains from Kazakhstan were compared to all publicly available WGS data of worldwide origin by whole genome SNP (wgSNP) analysis. Genotypes differing at a single SNP position were collected within a time interval of more than fifty years, from locations separated from each other by more than one thousand kilometers, supporting a role for migratory birds in the worldwide spread of the bacteria.Peer reviewe

Fieldable genotyping of Bacillus anthracis and Yersinia pestis based on 25-loci Multi Locus VNTR Analysis

<p>Abstract</p> <p>Background</p> <p>Anthrax and plague are diseases caused by <it>Bacillus anthracis </it>and <it>Yersinia pestis </it>respectively. These bacteria are etiological agents for worldwide zoonotic diseases and are considered among the most feared potential bioterror agents. Strain differentiation is difficult for these microorganisms because of their high intraspecies genome homogeneity. Moreover, fast strain identification and comparison with known genotypes may be crucial for naturally occurring outbreaks versus bioterrorist events discrimination.</p> <p>Results</p> <p>Thirty-nine <it>B. anthracis </it>and ten <it>Y. pestis </it>strains, representative of the species genetic diversity, were genotyped by Agilent 2100 Bioanalyzer using previously described Multiple Locus VNTR Analysis assays (MLVA). Results were compared to previous data obtained by standard genotyping system (capillary electrophoresis on automatic sequencer) and, when necessary, direct amplicon sequencing. A reference comparison table containing actual fragment sizes, sequencer sizes and Agilent sizes was produced.</p> <p>Conclusion</p> <p>In this report an automated DNA electrophoresis apparatus which provides a cheaper alternative compared to capillary electrophoresis approaches was applied for genotyping of <it>B. anthracis </it>and <it>Y. pesti</it>s. This equipment, uses pre-cast gels and provides easy transportation, low maintenance and overall general logistic requirements and costs, is easy to set up and provides rapid analysis. This platform is a candidate for on-site MLVA genotyping of biothreat agents as well as other bacterial pathogens. It is an alternative to the more expensive and demanding capillary electrophoresis methods, and to the less expensive but more time-consuming classical gel electrophoresis approach.</p

Molecular characterization of Coxiella burnetii isolates by infrequent restriction site-PCR and MLVA typing

BACKGROUND: Coxiella burnetii, the causative agent of Q fever, has a wide host range. Few epidemiological tools are available, and they are often expensive or not easily standardized across laboratories. In this work, C. burnetii isolates from livestock and ticks were typed using infrequent restriction site-PCR (IRS-PCR) and multiple loci variable number of tandem repeats (VNTR) analysis (MLVA). RESULTS: By applying IRS-PCR, 14 C. burnetii isolates could be divided into six groups containing up to five different isolates. Clustering as deduced from MLVA typing with 17 markers provided an increased resolution with an excellent agreement to IRS-PCR, and with the plasmid type of each strain. MLVA was then applied to 28 additional C. burnetii isolates of different origin and 36 different genotypes were identified among the 42 isolates investigated. The clustering obtained is in agreement with published Multiple Locus Sequence Typing (MLST) data. Two panels of markers are proposed, panel 1 which can be confidently typed on agarose gel at a lower cost and in any laboratory setting (10 minisatellite markers with a repeat unit larger than 9 bp), and panel 2 which comprises 7 microsatellites and provides a higher discriminatory power. CONCLUSION: Our analyses demonstrate that MLVA is a powerful and promising molecular typing tool with a high resolution and of low costs. The consistency of the results with independent methods suggests that MLVA can be applied for epidemiological studies. The resulting data can be queried on a dedicated MLVA genotyping Web service