Developpment of a new multilocus Variable-Number Tandem Repeat typing scheme for the global surveillance of #Xanthomonas citri pv. Citri# : Poster 19

International audienceMultiLocus Variable number of tandem repeats Analysis (MLVA) has been extensively used for addressing epidemiological and evolutionary questions 0 11 bacteria pathogenic to humans (Ciammaruconi el al., 2008). This typing scheme was shown especially useful for the so-called monomorphic pathogens, which can be efficiently genotyped with a high throughput by MLVA. In contrast, it has been scarcely used for bacterial plant pathogens despite the tremendous impact of some of them. We developed an optimized MLVA scheme targeting long tandem repeats (2: 10 bp) for assessing the global epidemiology of the c itrus pathogen Xanthomonas citri pv. citri (Xcc). Xcc is a quarantine organism in several countries and a significant threat for the citrus industry worldwide. We screened the genome of Xcc strain IAI'AR 306 and of phylogenetically related xanthomonads for tandem repeals. The resolution at 31 polymorphie loci (MLVA3 1) was assessed using 129 strains of Xcc representative of the currently known genetic and pathological diversity of the pathogen worldwide. Based on Discriminant Analys is of Principal Components (DAPC), seven mostly pathotypespecifie genetic clusters were defined. DAPC c1uster 1 had a much wider geographical distribution than others. This single DAPC c1uster has been primarily implicated in the major geographical extension of Xcc during the XXth century. Because of the suboptimal inter-laboratory reproducibility of AFLP and rep-PCR, MLVA-31 represents an opportunity for international genotyping data sharing. (Texte intégral

A MLVA genotyping scheme for global surveillance of the citrus pathogen Xanthomonas citri pv. citri suggests a worldwide geographical expansion of a single genetic lineage

International audienceMultiLocus Variable number of tandem repeat Analysis (MLVA) has been extensively used to examine epidemiological and evolutionary issues on monomorphic human pathogenic bacteria, but not on bacterial plant pathogens of agricultural importance albeit such tools would improve our understanding of their epidemiology, as well as of the history of epidemics on a global scale. Xanthomonas citri pv. citri is a quarantine organism in several countries and a major threat for the citrus industry worldwide. We screened the genomes of Xanthomonas citri pv. citri strain IAPAR 306 and of phylogenetically related xanthomonads for tandem repeats. From these in silico data, an optimized MLVA scheme was developed to assess the global diversity of this monomorphic bacterium. Thirty-one minisatellite loci (MLVA-31) were selected to assess the genetic structure of 129 strains representative of the worldwide pathological and genetic diversity of X. citri pv. citri. Based on Discriminant Analysis of Principal Components (DAPC), four pathotype-specific clusters were defined. DAPC cluster 1 comprised strains that were implicated in the major geographical expansion of X. citri pv. citri during the 20th century. A subset of 12 loci (MLVA-12) resolved 89% of the total diversity and matched the genetic structure revealed by MLVA-31. MLVA-12 is proposed for routine epidemiological identification of X. citri pv. citri, whereas MLVA-31 is proposed for phylogenetic and population genetics studies. MLVA-31 represents an opportunity for international X. citri pv. citri genotyping and data sharing. The MLVA-31 data generated in this study was deposited in the Xanthomonas citri genotyping database (http://www.biopred.net/MLVA/)

Congruence analysis of 5 sets of VNTR markers: MLVA7, MLVA8, MLVA15, MLVA25 and MLVA31.

<p>a congruence analysis in the “France” dataset (this study); b congruence analysis in the “Namibia” dataset <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095131#pone.0095131-Beyer1" target="_blank">[16]</a>.</p

Genotyping of French <i>Bacillus anthracis</i> Strains Based on 31-Loci Multi Locus VNTR Analysis: Epidemiology, Marker Evaluation, and Update of the Internet Genotype Database

<div><p>Background</p><p><i>Bacillus anthracis</i> is known to have low genetic variability. In spite of this lack of diversity, multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) and single nucleotide polymorphisms (SNPs) including the canonical SNPs assay (canSNP) have proved to be highly effective to differentiate strains. Five different MLVA schemes based on a collection of 31 VNTR loci (MLVA8, MLVA15, MLVA20, MLVA25 and MLVA31) with increased resolving power have been described.</p><p>Results</p><p>MLVA31 was applied to characterize the French National Reference Laboratory collection. The total collection of 130 strains is resolved in 35 genotypes. The 119 veterinary and environmental strains collection in France were resolved into 26 genotypes belonging to three canSNP lineages and four MLVA clonal complexes (CCs) with particular geographical clustering. A subset of seven loci (MLVA7) is proposed to constitute a first line assay. The loci are compatible with moderate resolution equipment such as agarose gel electrophoresis and show a good congruence value with MLVA31. The associated MLVA and SNP data was imported together with published genotyping data by taking advantage of major enhancements to the MLVAbank software and web site.</p><p>Conclusions</p><p>The present report provides a wide coverage of the genetic diversity of naturally occurring <i>B. anthracis</i> strains in France as can be revealed by MLVA. The data obtained suggests that once such coverage is achieved, it becomes possible to devise optimized first-line MLVA assays comprising a sufficiently low number of loci to be typed either in one multiplex PCR or on agarose gels. Such a selection of seven loci is proposed here, and future similar investigations in additional countries will indicate to which extend the same selection can be used worldwide as a common minimum set. It is hoped that this approach will contribute to an efficient and low-cost routine surveillance of important pathogens for biosecurity such as <i>B. anthracis</i>.</p></div

Electrophoresis gel of the MLVA7 panel on four strains.

<p>Well 1: Sterne strain, wells 2 to 4: French bovine strains. Migration on 3% standard agarose gel at 110 V during 4 hours. A 100 bp ladder was used running from 100 bp up to 1000 bp, the 500 bp and 1000 bp bands are more intense.</p

Comparison of the diversity index (DI) of individual loci in the “France” versus “Namibia” datasets.

<p>Comparison of the diversity index (DI) of individual loci in the “France” versus “Namibia” datasets.</p

(a) Minimum spanning tree of MLVA31 data from 119 animal and environmental <i>B. anthracis</i> strains.

<p>Each circle represents a unique genotype. The diameter of each circle corresponds to the number of isolates with the same genotype. Genotypes connected by a shaded background differ by a maximum of 3 of the 31 VNTR markers and could be considered as a “clonal complex”. Thick connecting lines represent one locus differences; regular connecting lines represent two loci differences. The length of each branch is proportional to the number of differences. Each epidemiological situation is represented by a specific color as defined in part b.(<b>b</b>). Localization of the 119 animal and environmental <i>B. anthracis</i> strains.</p

Geographical repartition of the strains for each clonal complex (CC).

<p>^aCC1 strains. ^bCC2 (black circle), and CC3 (grey circle) strains. ^cCC4 strains.</p

Discriminatory power and linkage disequilibrium analysis results for 5 sets of VNTR markers: the newly proposed MLVA7 and MLVA8, MLVA15, MLVA25, MLVA31.

<p>*Values as calculated from MLVA31 data published by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095131#pone.0095131-Beyer1" target="_blank">[16]</a>.</p

List of 32 published <i>B. anthracis</i> VNTR markers.

a<p>Keim et al. 2000 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095131#pone.0095131-Keim2" target="_blank">[6]</a>;</p>b<p>Le Flèche et al. 2001 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095131#pone.0095131-LeFlche1" target="_blank">[10]</a>;</p>c<p>Keim et al. 2004 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095131#pone.0095131-Keim3" target="_blank">[15]</a>;</p>d<p>Lista et al. 2006 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095131#pone.0095131-Lista1" target="_blank">[11]</a>;</p>e<p>Leski et al. 2009 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095131#pone.0095131-Leski1" target="_blank">[43]</a>.</p><p>*Locus not currently used due to some large amplicon sizes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095131#pone.0095131-LeFlche1" target="_blank">[10]</a>. The numerical allele coding convention is as published by Keim et al., <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095131#pone.0095131-Keim2" target="_blank">[6]</a>, Lista et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095131#pone.0095131-Lista1" target="_blank">[11]</a> and Antwerpen et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095131#pone.0095131-Antwerpen1" target="_blank">[26]</a>, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095131#pone.0095131.s001" target="_blank">Data S1</a> for more details on the coding conventions and comparison with alternative coding conventions.</p