Transoceanic spreading of pathogenic strains of Vibrio parahaemolyticus with distinctive genetic signatures in the recA gene

Abstract

Vibrio parahaemolyticus is an important human pathogen whose transmission is associated with the consumption of contaminated seafood. Consistent multilocus sequence typing for V. parahaemolyticus has shown difficulties in the amplification of the recA gene by PCR associated with a lack of amplification or a larger PCR product than expected. In one strain (090-96, Peru, 1996), the produced PCR product was determined to be composed of two recA fragments derived from different Vibrio species. To better understand this phenomenon, we sequenced the whole genome of this strain. The hybrid recA gene was found to be the result of a fragmentation of the original lineage-specific recA gene resulting from a DNA insertion of approximately 30 kb in length. This insert had a G+C content of 38.8%, lower than that of the average G+C content of V. parahaemolyticus (45.2%), and contained 19 ORFs, including a complete recA gene. This new acquired recA gene deviated 24% in sequence from the original recA and was distantly related to recA genes from bacteria of the Vibrionaceae family. The reconstruction of the original recA gene (recA3) identified the precursor as belonging to ST189, a sequence type reported previously only in Asian countries. The identification of this singular genetic feature in strains from Asia reveals new evidence for genetic connectivity between V. parahaemolyticus populations at both sides of the Pacific Ocean that, in addition to the previously described pandemic clone, supports the existence of a recurrent transoceanic spreading of pathogenic V. parahaemolyticus with the corresponding potential risk of pandemic expansion.

Fig 1. V. parahaemolyticus “snapshot” of CC345 obtained using eBURST v3.

Other CCs were defined using stringent criteria (6/7 shared alleles) (Figure B in S1 File). Among those groups or CCs, ST345 was identified as the predicted clonal ancestor of CC345. STs that are SLV of each other are shown connected by lines (black). DLV STs are shown as connected light gray lines. CC—Clonal Complex. R- Recombination event and the number of SNPs between ST345 (founder) and the other STs are between brackets. All the differences were located at the recA allele, except for ST812 that were located at the gyrB allele.

Fig 2. Schematic representation of the recA gene region between four V. parahaemolyticus genomes.

Comparison includes RIMD2210633 (prototypic “pandemic” strain O3:K6), BB22OP (closest relative ST88), strains 090–96 (Peruvian, fragmented recA gene), and S134 (Chinese strain containing a similar insert at recA gene). Predicted genes are depicted by horizontal arrows and named according the legend. Red bars represent inverted regions flanking the insertion. The percentage DNA sequence identity is indicated in grey bars and represents the BLASTn matches between sequences.

Fig 3. Sequence types (STs) identified at both side of the Pacific Ocean and route for the movement of waters associated with El Niño event (red arrow).

STs in red indicate those genetic variants detected both in Asia and Peru (ST189a), including the ST189 for the strain 090–96 with the recA insertion.

Fig 4. NeighborNet graph generated using the BIGSdb Genome Comparator tool implemented within the PubMLST website [34,35] with 2005 variable loci identified after using as a reference (ref) RIMD2210633 chromosome I (3079 genes) of available V. parahaemolyticus genomes at Genbank (Table A in S1 File for the list of the strains) and strain 090–96 (Peru 1996), showing that this strain is closed related to BB22OP (ST88) and the two other ST189 strains and far related to other strains containing fragmented recA genes with similar insertion (S130 and S134, both ST527).

This clearly suggests acquisition of this region by HGT. A similar graph was obtained with chromosome II of the same strain as reference (data not shown). Distances between taxa are calculated as the number of loci with different allele sequences.