Genome-Wide SNP-genotyping array to study the evolution of the human pathogen Vibrio vulnificus Biotype 3

Vibrio vulnificus is an aquatic bacterium and an important human pathogen. Strains Of V. vulnificus are classified into three different biotypes. The newly emerged biotype 3 has been found to be clonal and restricted to Israel. In the family Vibrionaceae , horizontal gene transfer is the main mechanism responsible for the emergence of new pathogen groups. To better understand the evolution of the bacterium, and in particular to trace the evolution of biotype 3, we performed genome-wide SNP genotyping of 254 clinical and environmental V. vulnificus isolates with worldwide distribution recovered over a 30-year period, representing all phylogeny groups. A custom single-nucleotide polymorphism (SNP) array implemented on the Illumina GoldenGate platform was developed based on 570 SNPs randomly distributed throughout the genome. In general, the genotyping results divided the V. vulnificus species into three main phylogenetic lineages and an additional subgroup, clade B, consisting of environmental and clinical isolates from Israel. Data analysis suggested that 69% of biotype 3 SNPs are similar to SNPs from clade B, indicating that biotype 3 and clade B have a common ancestor. The rest of the biotype 3 SNPs were scattered along the biotype 3 genome, probably representing multiple chromosomal segments that may have been horizontally inserted into the clade B recipient core genome from other phylogroups or bacterial species sharing the same ecological niche. Results emphasize the continuous evolution of V. vulnificus and support the emergence of new pathogenic groups within this species as a recurrent phenomenon. Our findings contribute to a broader understanding of the evolution of this human pathogen

Phylogenetic relationships among 30 <i>V. vulnificus</i> strains inferred from GoldenGate genotyping results compared to sequencing.

<p>Results were based on Illuminus-P genotype calls obtained by the GoldenGate assay at 31 SNPs compared to sequencing analysis of the same 12 intergenic loci analyzed as sequence types. These loci served for array quality control. A nonparametric analysis of allelic variation was used to calculate the Nei coefficient of association and to generate the corresponding matrix with SAS 8.02, followed by minimum-evolution (ME) cluster analysis using MEGA5.</p

PCA of genotyping data of Israeli biotype_1 strains processed in relation to synthetic biotype_3 haplotype.

<p>Principal component analysis (PCA) was carried out on datasets containing 530 SNPs. The scatter diagram of a cluster analysis performed with PCA is shown; the strains (dots) are classified into two or three groups with the following color code: LI – light blue, azure, LIII – black, LI-clade-B – dark blue. Together, components PC1 and PC2 explain 50% of the variance (38 and 12%, respectively); 95% confidence ellipses around each group are indicated.</p

Phylogenetic relationships among 254 <i>V. vulnificus</i> isolates based on variation data at 570 SNP loci.

<p>(A) Parametric analysis: the evolutionary history was inferred using the neighbor-joining method and the evolutionary distances were computed using the Kimura two-parameter method with bootstrap analysis of 1,000 replicates. (B) Nonparametric analysis: genetic relationships among strains were inferred using the Jaccard similarity coefficient, and the distance matrix was generated with PAST software (version 1.94b). The phylogenetic tree was constructed by using the minimum evolution (ME) cluster analysis.</p

The group origin map of the biotype 3 haplotype in comparison to the synthetic haplotype.

<p>Data are arranged in three rows representing the 530 SNPs along the <i>V. vulnificus</i> chromosomes (genome). (1) The group origin map based on PCA results (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114576#pone-0114576-g004" target="_blank">Fig. 4</a>). Blue – SNPs originated in LI-clade-B, light blue – SNPs originated in LI, black – SNPs originated in LIII; SNPs that were not found in any of the 59 tested biotype 1 strains were not colored. (2) The synthetic haplotype of biotype 3. The different alleles are color coded as follows: allele 1 – orange, allele 2 – green, and “no product” allele – yellow.</p

Variation within <i>V. vulnificus</i> groups with worldwide distribution isolated between 1979 and 2009 (254 isolates).

<p>Variation within <i>V. vulnificus</i> groups with worldwide distribution isolated between 1979 and 2009 (254 isolates).</p

Heat map of genotyping data at 570 SNPs of 127 strains isolated in Israel.

<p><i>V. vulnificus</i> strains (vertical axis) are arranged according to their phylogenetic relationships and marked by biogroups (68 biotype 3 isolates and 59 representative biotype 1 isolates). Allelic variations of SNPs (horizontal axis) are ordered according to their position along the two bacterial chromosomes (according to the YJ106 genome). The last 40 SNPs (on the right) were selected as unique to biotype 3 based on its draft genome (VVyb1(BT3)). Different colors represent the different alleles: allele 1 – orange, allele 2 – green, and “no product” allele – yellow.</p