Whole-genome comparison between reference sequences and oyster Vibrio vulnificus C-genotype strains

Whole-genome sequences of Vibrio vulnificus clinical genotype (C-genotype) from the CICESE Culture Collection, isolated from oysters, were compared with reference sequences of CMCP6 and YJ016 V. vulnificus C-genotype strains of clinical origin. The RAST web server estimated the whole genome to be ~4.8 Mb in CICESE strain 316 and ~4.7 Mb in CICESE strain 325. No plasmids were detected in the CICESE strains. Based on a phylogenetic tree that was constructed with the whole-genome results, we observed high similarity between the reference sequences and oyster C-genotype isolates and a sharp contrast with environmental genotype (E-genotype) reference sequences, indicating that the differences between the C- and E-genotypes do not necessarily correspond to their isolation origin. The CICESE strains share 3488 genes (63.2%) with the YJ016 strain and 3500 genes (63.9%) with the CMCP6 strain. A total of 237 pathogenicity associated genes were selected from reference clinical strains, where—92 genes were from CMCP6, 126 genes from YJ016, and 19 from MO6-24/O; the presence or absence of these genes was recorded for the CICESE strains. Of the 92 genes that were selected for CMCP6, 67 were present in both CICESE strains, as were as 86 of the 126 YJ016 genes and 13 of the 19 MO6-24/O genes. The detection of elements that are related to virulence in CICESE strains—such as the RTX gene cluster, vvhA and vvpE, the type IV pili cluster, the XII genomic island, and the viuB genes, suggests that environmental isolates with the C-genotype, have significant potential for infection.


Genome sequencing
A single colony of the CICESE strains was used to inoculate Zobell´s marine broth and was grown overnight at 35˚C. Genomic DNA was extracted from the cultures with the Wizard Genomics TM DNA Purification Kit (Promega, Madison, WI, USA), according to the manufacturer's instructions. The genomic DNA was sequenced (paired-end) on a Miseq TM platform (Illumina Inc., USA).
The contigs that were obtained from the CICESE strains were submitted to the Rapid Annotation Using Subsystem Technology (RAST) [34] and PathoSystems Resource Integration Center (PATRIC) web servers [35], to determine the annotation of the genes.
Because we focused on determining the presence or absence of C-genotype and pathogenesis-related genes, as reported by Chen et al. [26], Gulig et al. [36], and Morrison et al. [23], each gene from the CMCP6, YJ016, and MO6-24/O, was evaluated by alignment with the annotated contigs that were generated for the CICESE strains using the RAST and PATRIC web servers.

Genomic comparison
A genotype phylogenetic tree was constructed with whole-genome data from 2 C-genotype CICESE strains and data from Morrison et al.
The number of common genes between the CICESE genomes and the YJ016 and CMCP6 reference sequences was evaluated using PATRIC web server annotations, and then compared with the Venny web application (http://bioinfogp.cnb.csic.es/tools/venny/index.html).

Results
Sequencing of CICESE-316 and CICESE-325 resulted in 2,117,568 and 3,238,599 (2x~140 bp) paired-end reads, with N50 = 14,356 and 20,259 respectively. The depth of sequencing coverage was equivalent to 50.52x and 73.35x, with a GC content of 46.9% and 46.8%; the sequences were assembled in 847 and 677 contigs for CICESE-316 and CICESE-325 (Table 1). Using the RAST web server, the estimated size of the entire genome for these strains was~4.76 and~4.71 Mb. A total of 4,234 and 4,217 coding sequences (CDS) were detected for CICESE-316 and CICESE-325, respectively as well as 13 and 15 RNAs. No plasmids were detected in the CICESE strains ( Table 1).
The phylogenetic tree in Fig 1 shows the association between the CICESE strains and Cgenotype reference genomes (CMCP6, YJ016, and MO6-24/O) and a clear separation from those with the E-genotype (E64MW, JY1305 and JY1701).
The CDS that we obtained were classified into 26 categories (RAST web server). Categories that were related to virulence, disease, and defense were enriched in 88 and 83 CDS for CICESE-316 and CICESE-325, respectively, whereas resistance to antibiotics and toxic compounds were represented in 70 and 66 CDS.  (Table 2).
Sequences were assembled for the 2 chromosomes for the CICESE strains and CMCP6 and compared with the reference genome of YJ016. Compararison show gaps in different zones in both chromosomes (Fig 3). The most notorious gaps for CICESE strains and CMCP6 were found between 200 to around 2250 kpb in the chromosome I. Chromosome II showed several differences among CICESE strains and CMCP6 with YJ016, most notably at 920 kbp. A region with low GC content was also detected in chromosome I, located between 1750 and 1950 kpb (Fig 3) corresponding to a super-integron (SI). This region has high homology with a genomic SI region on chromosome I of YJ016 (VV1745 to VV1941).
Three secretions systems were also detected in both CICESE strains: T1SS, T2SS, and T6SS. The genomic island XII, located on the small chromosome, was also detected in both CICESE strains, with >49.8% GC content and differences in nucleotides of 1.8% or less (477 SNP in CICESE-316 and 586 SNP in CICESE-325) with respect to nucleotides reported for YJ016 in genomic island XII. The viuB gene was detected in both strains, but the rsbRST operon was only present in CICESE-316.

Discussion
The assembly and annotation of the CICESE strains (Table 1) were similar to those for the V. vulnificus reference genomes. The estimated sizes for the entire CICESE-316 and CICESE-325    Fig 1 shows that select C-genotype reference genomes (CMCP6, YJ016, MO6-24/O) grouped with the C-genotype CICESE strains and differed sharply from the E-genotype reference genomes (JY1305, E64MW, JY1701). These wholegenome results confirm that the C-genotype cluster of V. vulnificus strains does not necessarily correspond to their clinical or environmental isolation. Guerrero et al. [8], have reported close homology between environmental isolates of C-genotype strains by PFGE, MLST, and rtxA1 , YJ016 has 777 and CMCP6 has 332 genes that are exclusive to these reference strains, similar to the number of not-shared genes (Fig 2A and 2B) for CICESE-316 and CICESE-325 (between 290 to 337 genes). These findings indicate that the quantity of not-shared genes recorded in the CICESE and reference strains is normally reported in comparative genomic analyses of C-genotype strains.
Most of the pathogen-related genes associated to pathogenic strains with C-genotype that were identified by Chen et al.
Most groups of genes that are related to pathogenicity clusters [26] were detected in the CICESE strains. In the alignment with the YJ016 chromosomes (Fig 3), the CICESE and CMCP6 sequences had nearly the same differences in both chromosomes with respect to YJ016. According to Thiaville et al. [25], both reference strains show high virulence for skin and liver infections and have similar lethality; thus, the differences between CMCP6 and CICESE strains, with YJ016, might not be essential for virulence.
The RTX gene cluster; the vvhA and vvpE genes; the secretion systems T1SS, T2SS, and T6SS; the viuB gene; the type IV pili cluster; and the genomic island XII-all of which were detected in the CICESE strains-have been identified as important virulence factors.
The MARTXvv toxin has been described as the main virulence factor of V. vulnificus; this toxin is involved in apoptosis and necrosis and is essential in the early stages of infection and its dissemination to the bloodstream [42][43][44]. Partial or total deletion of rtxA1, decrease its cytotoxicity and ability to infect and results in a 2600-fold increase in its LD 50 in an animal model [22,42,43,45].
The rtxB, rtxD, rtxE, and tolC genes encode for structural proteins in the type I secretion system (T1SS), which mediates the release of the MARTX toxin to the surrounding environment [46,47]. The deletion of rtxE affects the secretion of MARTX, and mutant strains have lower cytotoxic activity in cell lines [48].

vvhA and vvpE
The vvhA and vvpE genes, which have been reported in all V. vulnificus strains, were detected in the CICESE strains. vvhA (VV2_0404, CMCP6), an extracellular hemolysin, and vvpE (VV2_0032, CMCP6), a metalloprotease, have been implicated in necrosis, vascular permeability, apoptosis, pore formation, and tissue damage [49]. The secretion of vvhA and vvpE into the environment, is mediated by the type II (T2SS) secretion system [50], which was detected in both CICESE strains.

Type IV pili cluster
The CICESE strains contained the type IV pilus gene cluster (pilA, pilB, pilC, and pilD; Fig 3). pilA variated significantly within VV2778, but few variations were found in VV2779, VV2780, and VV2781 compared with the YJ016 strain. pilA has been implicated in the adherence to host cells, biofilm formation, and virulence [51]. Chattopadhyay et al. [52], have suggested that the variability in pilA in V. vulnificus is associated with several functions, allowing it to adapt to various hosts. Therefore, these differences could be associated with the isolation of CICESE strains from oyster, compared with the clinical origin of reference strains.

Genomic island XII
The genomic island XII, located on the small chromosome (VVA1613 to VVA1636) [53], was present in both CICESE strains, with few differences compared with YJ016 (Fig 3). This 33-kb region, which has an aberrant GC content of 50%, correlates with high-virulence C-genotype strains and confers a selective advantage in the environment or human host [53]. Morrison et al. [23], reported that this region is present in C-genotypes but not in E-genotypes. This region harbors 2 chondroitinase genes, an ABC transport system, the arylsulfatase A gene cluster, and hypothetical proteins [53]. The arylsulfatase (aslA) gene has been implicated in the invasion of the blood-brain barrier in E. coli [54], and chondroitinase has been described as a virulent factor in certain fungi, such as Paracoccidioides brasiliensis [55].

Additional pathogenic-associated genes
Strains with the C-genotype are more resistant to stressful conditions than E-genotype strains [56]. The ability to survive under stressful conditions has been associated with the presence of the RsbRST stress module genes (rsbR, rsbS, rsbT, and rsbU) and the siderophore-encoding (viuB) gene [23,24]. Williams et al. [57] evaluated strains that contained the rsbRST operon and found it to be specific to C-genotypes, wherein 75% of C-genotypes and no E-genotypes harbored the entire operon. Bogard and Oliver [24], have reported that in the C-genotype strains that they studied, they detected the viuB gene, whereas few E-genotype strains were positive for this gene. The presence of both elements-viuB and the rsbRST operon-in CICESE-316 (Fig 3), indicates a greater ability to survive under stressful conditions. The T1SS, T2SS, T4SS, and T6SS secretion systems, has been identified in V. vulnificus [27]. These systems are involved in the secretion of proteins, such as toxins, and the transport of DNA [58]. In addition to T1SS and T2SS, most of the genes associated with T6SS (VVA0970 to VVA0996, for YJ016), were also detected in CICESE strains (Fig 3).The genes associated to T4SS were not detected in the CICESE strains.
The region with low GC content between 1750 to 1950 Mb on chromosome I corresponds to the SI in the CICESE strains (Fig 3). This region is analogous to the genes that have been reported for YJ016 (VV1745 to VV1941) but differs from those of CMCP6 (VV1_2401 to VV1_2501), likely because the genes that are encoded within the SI are primarily strain-specific [36].
The CICESE strains that were isolated from oyster samples, showed high genomic similarity to reference C-genotype V. vulnificus strains. The detection of elements that are related to virulence-such as the rxt gene cluster, vvhA and vvpE, the type IV pili cluster, the genomic island XII, viuB, and the genes in Table 2-in the CICESE strains, suggests that environmental isolates with C-genotype, have a high potential for virulence and infection; this hypothesis should be tested in a future study on virulence.