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Front. Microbiol. | doi: 10.3389/fmicb.2019.01904

Corrigendum: Phylogeny of Vibrio vulnificus from the analysis of the core-genome: implications for intra-species taxonomy

  • 1University of Valencia, Spain
  • 2University of Bath, United Kingdom
  • 3Biotechvana SL, Spain
  • 4Centre for Environment, Fisheries and Aquaculture Science (CEFAS), United Kingdom
  • 5University of North Carolina at Charlotte, United States
  • 6Technion Israel Institute of Technology, Israel
  • 7University of Florida, United States

Vibrio vulnificus is an emerging zoonotic pathogen that inhabits brackish water ecosystems from temperate and tropical areas and whose geographical distribution has recently extended to Northern countries due to global warming (Baker-Austin et al., 2013, 2017). The pathogen survives in water, either associated with the mucous surfaces of algae and aquatic animals or as a free-living bacterium that can be concentrated by filtering organisms such as oysters (Oliver, 2015).
V. vulnificus was defined as a bacterial species in 1976 (Farmer 3rd, 1979) and was later split into three biotypes (Bt) on the basis of differences in genotypic and phenotypic traits as well as in host range (Bisharat et al., 1999; Tison et al., 1982). The three Bts are opportunistic human pathogens but Bt2 is also pathogenic for aquatic animals. The zoonotic strains belong to the same serovar (Ser) and are classified as Bt2-SerE (Biosca et al., 1997).
The various diseases caused by this species are known as vibriosis. Human vibriosis presents two main forms depending on the pathogen´s route of entry into the body, ingestion or contact (Oliver, 2015). In the first case, the pathogen is ingested with raw seafood, colonizes the intestine and causes gastroenteritis and/or primary septicemia. In the second case, the pathogen crosses the skin barrier during an injury or directly colonizes a preexisting wound causing local but severe necrosis and/or secondary septicemia. The main factor that predisposes to death by sepsis is a high level of serum iron as a consequence of multiple pathologies (e.g. hemochromatosis, diabetes, cirrhosis and viral hepatitis) (Oliver, 2015).
Epidemiological data on human vibriosis from the Centers for Disease Control and Prevention (CDC) estimates that around 80,000 people are infected by Vibrio spp. each year in the USA and that, of these, V. vulnificus is responsible for most of the fatal cases (>50% for septicemia cases (Jones and Oliver, 2009)). Thus, V. vulnificus is responsible for over 95% of seafood-related deaths (Jones and Oliver, 2009), the highest fatality rate of any known food-borne pathogen (Rippey, 1994). In addition, increasing incidents of infections are occurring globally, with cases reported in Europe and Asia (Baker-Austin et al., 2013; Hlady and Klontz, 1996; Lee et al., 2013b). Crucially, infections currently appear to be increasing in both the USA and in Europe (Baker-Austin et al., 2013; Newton et al., 2012).

Regarding fish vibriosis, eels seem to be the most susceptible hosts, especially under farming conditions (Amaro et al., 2015). The pathogen colonizes the eel gills, enters the blood and causes death by septicemia even in healthy individuals (Marco-Noales et al., 2001). Eel vibriosis occurs in farms as epizootics or outbreaks of high mortality that can lead to the closure of the farm if the disease is not controlled promptly. Epizootiological data suggest that outbreaks of eel vibriosis have been registered in all the countries where eels are cultured in brackish waters, including fish farms located in Northern-European countries (Haenen et al., 2013).

The genetic basis for human virulence is only partially known although most studies suggest that all the strains of the species may have the ability to infect humans regardless of their origin, clinical or environmental (Gulig et al., 2005). In contrast, the ability to infect fish is dependent on a virulence plasmid (pVvBt2) that is only present in Bt2 strains (Lee et al., 2008; Roig and Amaro, 2009). The plasmid encodes resistance to the innate immunity of eels, and probably other teleost (Lee et al., 2008, 2013a; Pajuelo et al., 2015). Interestingly, pVvbt2 can be transmitted among bacteria aided by a second, conjugative plasmid (pVv) which is widespread in the species (Lee et al., 2008).

The aim of this study is to describe the phylogenetic groups within the species and compare them with the current intraspecific groups from the characterization the core genome of species (CGS), of pVvbt2 (CGP) and of human virulence-related genes (CGV). Our results highlight the importance of aquaculture industry in the recent evolution and epidemic spread of the species and support the intra-specific classification in lineages instead of Bts as well as the inclusion of a pathovar grouping all fish pathogenic isolates for which we propose the name “piscis”.

Bacterial isolates, culture conditions, DNA extraction and sequencing
The genomes used in this study and the main features of the corresponding strains are detailed in Table 1. The strains whose genome was sequenced are marked in Table 1. These strains were routinely grown in Tryptone Soy Broth or agar plus 5 g/l NaCl (TSB-1 or TSA-1, Pronadisa, Spain) at 28ºC for 24 h. The strains were maintained both as lyophilized stocks and as frozen stocks at -80º C in marine broth (Difco) plus 20% (v/v) glycerol.

DNA was extracted using GenElute™ Bacterial Genomic DNA (Sigma, Spain) from bacteria grown with shaking at 28ºC for 12 h. Samples with a DNA concentration of 10-15 ng/µl were used for sequencing with Illumina Genome Analyzer technology GAII (Illumina MiSeq) flow cell in the Genome Analysis Centre in Norwich (UK) and the SCSIE of the University of Valencia (Spain). To this end, unique index-tagged libraries for each sample (up 96 strains) were created using TruSeq DNA Sample Preparation for subsequent cluster generation (Illumina cBot) and up to 12 separate libraries were sequenced in each of eight channels in Illumina Genome Analyser GAII cells with 100-base paired-end reads. The index-tag sequence information was used for downstream processing to assign reads to the individual samples (Harris et al., 2010).

Genome sequence assembly
The genomes of the Bt1 strain YJ016 (NC_005139 and NC_005140), the Bt2 strain CECT 4999 (NZ_CP014636.1 andNZ_CP014637.1), the Bt3 strain vvby1 (NZ_CM001799.1 and NZ_CM001800.1) and the plasmids pR99 (AM293858) and p4602-2 (AM293860) were used as references. After sequencing, reads from each genome (average of 31x106 reads [3,000 Mbp] per genome) were mapped onto the reference plasmid/genome and draft plasmids/genomes were generated using Geneious 6.1.6 (Biomatters) software.

Multiple sequence alignments were obtained using the progressive algorithm of the Mauve software with default options (Darling et al., 2004). Locally Collinear Blocks (LCBs) with a size larger than 1 kb and present in all the genomes were used to define the core genomes. The selected LCBs were concatenated to be used in subsequent analyses to build a core genome multiple alignment.

Core genome
The genomes of the Bt1 strain YJ016 and of pR99 were selected as references to define the CGS and the CGP, respectively. The YJ016 genome contains 5,097 genes (3,387 in chromosome I and 1,710 in chromosome II) and pR99 contains 71 genes. In addition, we selected 3 Vibrio species (V. parahaemolyticus, V. anguillarum and V. cholerae) that had at least one fully sequenced and annotated genome to define the core genome of the genus (CGG). Table S5 summarizes the characteristics of the closed Vibrio genomes selected for the study.

The search and identification of each gene was performed using local BLAST searches (Wang et al., 2003). Three independent searches, one per chromosome and plasmid, were performed and a database was generated for each genome. The resulting sequences were mapped onto reference genes using Geneious 6.1.6 (Biomatters) software. As the sequences used for the generation of the databases were not closed genomes (draft genomes/contigs), only the genes present in all the genomes, with a minimum length of 80% of the total length of the homologous gene in the reference genome and with a DNA identity higher than 70%, were included in the CGS, the CGP or the CGG for further analysis.

In order to refine the preliminary alignment for the entire core, individual alignments for each gene were performed using the program MAFFT (Katoh and Standley, 2013). The elimination of unaligned ends and genes that did not match the above requirements was performed using an in-house Python script. Once all the genes were aligned, a concatenated sequence was generated for the CGS, the CGP and the CGG.

Functional analysis of the CGS
Functional annotation was performed using Blast2GO (Conesa et al., 2005) and InterProScan (Quevillon et al., 2005). Gene descriptions were obtained by blasting the predicted protein sequences against the b2g_sep13 public database. The COG terms for phylogenetic classification of the proteins and clusters of orthologous groups of proteins for each gene were determined using the COGnitor software and COG database, (Tatusov, 2000).

Single nucleotide polymorphisms (SNPs) identification.
SNPs were identified in the genomes as described previously (Harris et al., 2010) with appropriate SNP cutoffs to minimize the number of false-positive/negative calls. SNPs were filtered to remove those at sites with an SNP quality score lower than 40 and/or with read coverage below 25X in this region (the average coverages by strain are listed in Table 1). SNPs at sites with heterogeneous mappings were also filtered out if the SNP was present in fewer than 85% of reads for that position.

Virulence genes: PCoA analysis
We identified human virulence-related genes in the CGS from previously published data (Alice et al., 2008; Bogard and Oliver, 2007; Brown and Gulig, 2009; Chakrabarti et al., 1999; Chen et al., 2004; Chen and Chung, 2011; Gulig et al., 2005; Horstman et al., 2004; Kim et al., 2011; Lee et al., 2010; Liu et al., 2009; Oh et al., 2008). Then, we identified the SNPs and performed a principal coordinate analysis (PCoA) (Orloci, 1975) for the calculated genetic distance via the covariance matrix (with data standardization using GenAlEx 6.5(Peakall and Smouse, 2012)) to explore and visualize similarities in the virulence-related genes on the basis of pairwise individual genetic distances (Smouse and Peakall, 1999).

Phylogenetic reconstruction and congruence analysis
All the phylogenetic trees for CGG, CGS, CGP and VCGS were reconstructed using the maximum-likelihood (ML) method with PhyML software (Guindon et al., 2009). The best evolutionary model for each dataset was determined with jModelTest (Posada, 2008) and considering the Akaike information criterion (AIC) (Akaike, 1974). The selected models were the generalized time-reversible model (GTR) with Gamma (+G) distribution and invariant positions (+I) (Tavaré, 1986) for MLSA analysis, GTR+G for SNP analysis, Tamura-Nei (Tn93) (Tamura, 1992) with Gamma distribution for the core genome and core genome genes analyses, and Kimura-2-parameter (K2) model for V. cholerae SNP analysis (Kimura, 1980). The pVvBt2 phylogeny was evaluated using the Hasegawa-Kishino-Yano model (Hasegawa et al., 1985). Support for the nodes derived in these reconstructions was evaluated by bootstrapping using 1,000 replicates (Felsenstein, 1985).

The congruence among the different phylogenetic reconstructions was evaluated using Shimodaira-Hasegawa (SH) (Shimodaira and Hasegawa, 1999) and expected-likelihood weight (ELW) tests as implemented in TreePuzzle version 5.2 (Schmidt et al., 2002; Strimmer and Rambaut, 2002).


Core genome.
We obtained almost complete genome sequences of 38 V. vulnificus strains (Table 1). The CGS was inferred from these genomes and additional genomes taken from the database (80 in total). The analysis included strains of the three Bts, isolated from a variety of hosts and habitats all over the world (Table 1). The main characteristics of the analyzed genomes, and the CGS as well as the number of SNPs identified per chromosome are detailed in Tables 2 and 3, respectively. The average gene identities in the CGS were 91 % and 89% for chromosome I and II, respectively, which supports previous observations pertaining to the greater variability of chromosome II in Vibrio spp (Kirkup et al., 2010).

The genes of CGS and the associated metabolic pathways are shown in Tables S1 and S2, respectively. The genes present in all the strains that did not match the criteria used to define the CGS (spanning more than 80% of the length and showing more than 70% identity with respect to the homologous gene in the reference YJ016 genome) are shown in Table S3. CGS includes practically all the genes for glycolysis, TCA cycle and pentose phosphate pathway, aerobic and anaerobic respiration, nitrate respiration, as well as for biosynthesis of metabolic intermediates, cofactors, nucleotides, amino acids and cell building blocks (Table S1 and Table S2). Interestingly, all the common genes for butanoate metabolism, glycerolipid metabolism, peptidoglycan biosynthesis and pyrimidine metabolism were located on chromosome I while those for biosynthesis of siderophores, fatty acid biosynthesis/elongation, glycerophospolipid metabolism, glyoxilate/dicarboxylate metabolism, nicotinate/nicotinamide metabolism, porphyrin metabolism, and valine/leucine/isoleucine metabolism were located on chromosome II (Table S2).

The CGS also includes genes involved in survival in different environments (water, animal intestine, chitinous surfaces), such as genes for resistance to different stressors (cold, heat, toxic oxygen forms, antimicrobials, tellurite….), genes for chitin degradation (various chitinases) and genes for surface colonization (pilus MSHA [mannose sensitive hemagglutination], flagellum biogenesis,...). Some of these genes are located in only one of the two chromosomes (e.g., genes for flagellum and MSHA pilus in chromosome I and for Flp/Tad pilus in chromosome II) (Table S1).

Figure 1 presents the distribution of gene ontology terms. Most of the genes in the CGS encode proteins associated to cell membranes that are related with regulation of transcription, transport or metabolic/oxido-reduction process and present catalytic/hydrolase, transferase or transcription-factor activity (Figure 1). Again, many functions associated to CGS genes in chromosome I were not found in chromosome II such as kinase activity, metal ion transport and phospho-relay response regulator activity etc. (Figure 1). In addition, no CGS gene related to proteolysis, phospho-relay signal transduction, carbohydrate transport, phosphorylation, ATP catabolic process, chemotaxis, biosynthesis, DNA recombination and repair, and phosphoenolpyruvate-dependent sugar phosphotransferase system were identified in chromosome II (Figure 1).

To determine the CGP, the plasmid was reconstructed from the sequenced genomes of the Bt2 strains indicated in Table 1. We found six variants of the plasmid, two previously described (types II and IV) (Lee et al., 2008; Roig and Amaro, 2009) and four new ones (Table 4). The list of genes in the CGP is shown in Table S4. The CGP includes three virulence genes, two host-specific, vep07 and ftbp (fish transferrin binding protein), involved in resistance to and growth in eel serum, respectively (Lee et al., 2008; Pajuelo et al., 2015), and one host-nonspecific, rtxA1. This is a mosaic gene related with resistance to phagocytosis by murine and eel phagocytes (Lee et al., 2013a; Satchell, 2015) that presents at least seven different forms in V. vulnificus (Roig et al., 2011; Satchell, 2015).

Phylogenomic analysis
To root the V. vulnificus phylogenetic tree within the genus, we first reconstructed a phylogenetic tree from the closed genomes of 15 strains belonging to seven Vibrio species (V. parahaemolyticus, V. anguillarum, V. algynolyticus, V. campbellii, V. furnissii, V. cholerae and V. splendidus-clade) (main genome characteristics shown in Table S5) together with 27 selected V. vulnificus genomes (Table 1). Maximum-likelihood (ML) trees for chromosome I, II and I+II were reconstructed based on the common genes of these Vibrio spp. (Figure S1). The ML trees showed V. vulnificus as a very compact and independent group. Next, we inferred the phylogeny of the species from the 80 V. vulnificus genomes indicated in Table 1 by using ML reconstruction obtained from the SNPs of coding regions.

The phylogenetic trees for V. vulnificus are shown in Figure 2. The rooted V. vulnificus phylogenetic reconstructions for chromosomes I and II were compared by congruence tests (Table 5). The results clearly indicate that chromosomes I and II were not congruent with one another, which strongly suggests that both chromosomes have suffered inter and/or intra chromosomal rearrangement since the emergence of V. vulnificus ancestor. In fact, several strains changed their relationships in the tree for each chromosome (Figure 2). Among them, we highlight the well-known human clinical isolates C7184, YJ016 and CMCP6 (Figure 2). Despite the global incongruence between the two chromosomes phylogenies, all the phylogenetic trees divided the species into five well-defined, highly supported lineages, two of them (lineages IV and V) including only a few strains. No strain changed of lineage between the two trees (Figure 2).
The average pairwise similarity for the strains of all the lineages was 82.5%. Lineage I (LI) (average pairwise similarity 93.1%) includes clinical (50% of isolates) and environmental (50% of isolates) Bt1 isolates from the USA, South Korea, Taiwan, Israel and Spain. The clinical LI isolates were recovered from human infections in the USA, South Korea and Taiwan, with 80% of these derived from blood (related to primary septicemia, when the etiology is known), 10% from stools (related to gastroenteritis) and 10% from wound samples. The environmental LI isolates were recovered from oysters (40%), water (30%), and fish (30%) in Spain, Israel, Taiwan, South Korea and the USA. Interestingly, the Israeli strain yv158, although environmental, belongs to a previously described highly clonal group (clade A), which includes both environmental and clinical isolates (wound samples), all of vcg C-type (clinical type according to the polymorphism in virulence correlated gene (Rosche et al., 2005)), supporting the potential virulence of this specific strain (Broza et al., 2012).

Lineage II includes all the Bt3 strains, which form a highly homogeneous group (average pairwise similarity 97.8%). These Bt3 strains were isolated in Israel from outbreaks of human vibriosis associated with the handling of farmed-tilapia (year of isolation, 1996-2003) and from aquaculture fishponds of tilapia (2002-2004). In consequence, all of them are associated to the aquaculture industry.

LIII (average pairwise similarity 87.4%) includes Bt1 and Bt2 strains from environmental sources (39.6% of the isolates; 21% from water, 21% from fish, 53.5% from seafood and 4.5% from sediment), diseased humans (29.2% of the isolates; 57% from human blood [none of them with known etiology] and 43% from wounds) and diseased fish (31.2%) origins. All the zoonotic strains (Bt2-SerE), regardless their source (environment, diseased animal or diseased human), country (France, Australia, Denmark and Spain) or year of isolation (from 1980 to 2004), cluster in a highly homogeneous group (average pairwise similarity 97.7%). The non-typable Bt2 strain (960426-1 4C) formed a subgroup together with the SerE clonal-complex while SerI and A strains formed another subgroup both within LIII (Figure 2).

Interestingly, all human clinical cases of known aetiology caused by LII and III isolates in Europe and Israel were related to farmed-fish handling regardless the Bt of the isolate.

LIV (similarity 98.7%) is formed by two Spanish isolates from the Ebro-Delta (a nature park by the Mediterranean Sea) area, one from seawater and the other from a human clinical (leg wound) case, both of Bt1. Finally, LV is formed by a unique isolate of Bt1 from Israel and clinical origin that is considered as representative of a highly virulent clone designated as Clade B (Efimov et al., 2015).

The phylogenetic tree for pVvBt2 based on the CGP shows relationships among isolates that did not match the previous phylogenetic relationships (Figures 2 and 3). Further, the congruence analyses revealed that phylogenetic trees from the CGP and CGS were not congruent to each other, suggesting a different evolutionary history for the plasmid and the chromosomes (Table 4)

Virulence-related genes
We searched for human virulence-related genes described in the literature for V. vulnificus, and we found that 75% of them were present in the CGS. This finding allowed us to define the human virulence-related core genome (VCGS) (Table S6). The VCGS includes genes for the flagellum, capsule, LPS- and cell-wall biosynthesis, motility, hemolysin and proteases (such as VvhA and a collagenase), resistance to human serum (trkA (Chen et al., 2004)), heme uptake, biosynthesis and uptake of vulnibactin, several genes for transcriptional regulators such as Fur, and finally genes for MARTX (Multifunctional Autoprocessive Repeat in Toxin) transport and modification systems, genes that are duplicated in pVvbt2 (Alice et al., 2008; Bogard and Oliver, 2007; Brown and Gulig, 2009; Chakrabarti et al., 1999; Chen and Chung, 2011; Gulig et al., 2005; Horstman et al., 2004; Kim et al., 2011; Lee et al., 2008; Liu et al., 2009; Oh et al., 2008; Oliver, 2015; Satchell, 2015; Yu-Chung et al., 2004).

The core genome has been demonstrated to be an optimum data set for determining the phylogeny of a bacterial species since it primarily includes the essential genes of a species and excludes the non-essential ones, such as those present in MGE (mobile genetic element: genomic islands, prophages and plasmids) (Juhas et al., 2009; Segerman, 2012; Wolf et al., 2013). In the current study, we have used this approach to analyze the phylogeny of V. vulnificus and compare the phylogenetic groups with the current biotypes of the species.

Our phylogenomic analysis suggests that V. vulnificus has diverged in five well-defined and separate lineages that do not correspond with the current Bts. LI is currently formed by the most dangerous strains from a public health perspective. All of them correspond to Bt1 and were mostly isolated from human blood in North America and Asia, presumably from primary septicemia cases after ingestion of raw seafood. LII and LIII comprise strains of the three Bts, mostly isolated from fish-farming related environments, including humans infected through handling of farm-fish in Europe and Israel (Bisharat et al., 1999; Haenen et al., 2013).

LII includes all Bt3 strains regardless of their origin (human infections related to fish-farms or environmental), which constitute a clonal group. Bt3 emerged in Israel in 1990 in farms of tilapia and is the only that has produced outbreaks of human infections, all of them through severe wound infections or secondary septicemia cases (Bisharat et al., 1999). By using different genomic approaches, Efimov et al. (2013) and Koton et al. (2014) have hypothesized that Bt3 emerged in the nutrient-enriched environment represented by the aquaculture industry from a Bt1 ancestor that acquired a rather small number of genes from different donors, probably belonging to the genus Shewanella. The proposed ancestor was v252, a representative strain from a highly virulent clade designated as clade B (Efimov et al., 2015). Our analysis does not support this hypothesis. Instead, clade B shares the closest common ancestor with LIII and not with LII.

LIII includes Bt1 and Bt2 strains. Sanjuán et al. (2011) proposed that Bt2 is a polyphyletic group subdivided in Ser-related subgroups, one of which is a clonal complex (Bt2-SerE). Our phylogenomic study confirms that Bt2 is polyphyletic and that the SerE-subgroup is highly homogeneous (identity value of 97.7%). Bt2 was defined in 1982 based on the differential properties of the first fish-pathogenic strains, all of which belonged to SerE and were isolated from eel-farms in Japan (Muroga et al., 1976a, 1976b; Tison et al., 1982). Later, Bt2-SerE isolates were recovered from human infections of unknown etiology from the USA and Europe as well as from different epizootic events of high mortality affecting different farmed-eels in Europe. Bt2-SerA and I emerged simultaneously in Spanish and Danish farms after the industry initiated the change from brackish- to fresh-waters in order to control the severity of vibriosis outbreaks due to Bt2-SerE as well as the probability of human infections (Fouz and Amaro, 2003). These new serovars are adapted to infect through and to survive in fresh-waters (Fouz et al., 2006).

All the analyzed Bt2 strains contained the virulence plasmid pVvbt2, SerE strains present the two variants previously described (Lee et al., 2008) and a new one. SerA and I strains showed three new variants. It was previously hypothesized that pVvbt2 could have been acquired several times in the fish-farming industry by Bt1 strains (Sanjuán et al., 2011). To test this hypothesis, we compared the chromosomal phylogenetic trees reconstructed for Bt2 strains from the CGS with those from the CGP and found that they were not congruent. This result strongly supports the hypothesis of Sanjuán et al. (2011) and suggests that pVvbt2 has been acquired independently by different clones within LIII. One of these plasmid-carrier clones successfully amplified in eel-farms and spread to other places and countries, probably in carrier fishes, giving rise to the worldwide expanded, current clonal complex. This clonal complex is supposed to be zoonotic because all Bt2-SerE isolates examined to date are virulent for both fish and mice, including the environmental isolates (Sanjuán et al., 2004).

Comparisons of the core genome between clinical and environmental strains of the closely related species V. cholerae reveal that this species is divided into two linages, with most of the epidemic strains appearing closely related, regardless of their geographical origins (Eppinger et al., 2014). The only clonal V. vulnificus group with a worldwide distribution is that formed by Bt2-SerE strains, a group that combines the ability to infect fish with that of infecting humans and of surviving in the environment without nutrients for years in a viable but non-culturable state (Marco-Noales et al., 1999). Moreover, in V. cholerae the ability to cause cholera epidemics lies on mobile genetic elements, such as phages and pathogenicity islands that carry the genes encoding the cholera toxin, TCP pilus, etc. (Das et al., 2016; Ramamurthy and Bhattacharya, 2011). In contrast, our phylogenomic analysis, as well as those based on MLSA (Cohen et al., 2007; Sanjuán et al., 2011) and microarray hybridization (Raz et al., 2014), show that environmental and clinical strains of V. vulnificus are distributed throughout the phylogenetic lineages, regardless the Bt, country of origin, or year of isolation. This result is compatible with the hypothesis that all V. vulnificus isolates, unlike V. cholerae, have the ability to infect humans. To confirm this, we investigated which virulence-related genes were present in the CGS and found that most of them belong to the core genome.
Summarizing, all the phylogenetic reconstructions from the core genome of the species, the fish-virulence plasmid and the human-virulence genes strongly suggest that V. vulnificus emerged from an ancestor potentially virulent for humans that diverged in five lineages that do not correspond with the current Bts. Our results also highlight the importance of the aquaculture industry in the recent evolution and epidemic spread of the species and, finally, support the intra-specific classification in lineages instead of in Bts as well as the inclusion of a pathovar grouping all fish pathogenic isolates for which we propose the name “piscis”.

Keywords: Vibrio vulnificus, Filogenomics, evolution, Pathovar, core genome, Virulence plasmid

Received: 17 Jul 2019; Accepted: 02 Aug 2019.

Copyright: © 2019 Roig, Gonzalez-Candelas, Sanjuan, Fouz, Feil, Llorens, Baker-Austin, Oliver, Danin-Poleg, Gibas, Kashi, Gulig, Morrison and Amaro. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Prof. Carmen Amaro, University of Valencia, Valencia, 46010, Valencian Community, Spain,