BACKGROUND
vibrio parahaemolyticus is a halophilic bacterium which has long been recognized  <cit>  as a human pathogen that causes gastroenteritis and, occasionally, wound infections and sepsis in immunocompromised patients.
it is the leading etiologic agent for bacterial foodborne disease in japan and other parts of asia, and it is the most common bacterial cause of seafood-associated disease in the united states.
prior to  <dig>  there was no specific serotype of v. parahaemolyticus that was associated with disease outbreaks, and the bacterium had never been reported to cause a pandemic.
however, during that year, a major outbreak occurred in india, > 50% of the v. parahaemolyticus strains isolated from patients were serotype o3:k <dig>  <cit> .
also, the outbreak rapidly spread to other countries in asia, south america, north america, africa and europe, resulting in a pandemic affecting tens of thousands of people  <cit> .
during its global dissemination, >  <dig> serovariants  is recognized  <cit>  as the most important virulence factor of v. parahaemolyticus, and a tdh-related hemolysin  is believed to account for the virulence of strains that do not produce tdh.
prior whole-genome sequencing  <cit>  of a serotype o3:k <dig>  pandemic isolate designated rimd <dig> identified two type iii secretion systems .
t3ssi is present in all v. parahaemolyticus isolates examined and is required for the bacterium's cytolytic activity  <cit> ; whereas, t3ssii is required for enterotoxicity and is located in the tdh-containing pathogenicity island  <cit> .
outbreaks of diarrheal disease caused by v. parahaemolyticus may pose a significant health threat.
thus far, the most affected country  has been chile, where >  <dig>  cases were reported during  <dig>  this observation suggests that, under appropriate conditions, v. parahaemolyticus may cause large-scale outbreaks comparable to those elicited by v. cholerae.
at the present time, the reasons for the pandemic strains' rapid increase in virulence/prevalence have not been rigorously determined.
in addition, the mechanism for rapid serotype conversion warrant further study.
furthermore, it is not clear whether the virulence mechanisms of tdh+ and trh+ strains are similar.
therefore, in order to address some of these questions, we performed rigorous genomic analyses of two pre-pandemic and four pandemic isolates of v. parahaemolyticus.
RESULTS
comparative genomics of v. parahaemolyticus
prior to this study, an o3:k <dig> pandemic isolate  was sequenced to completion  <cit>  and an o3:k <dig> non-pandemic isolate  was sequenced to draft status  <cit> .
in this study, we sequenced four additional isolates of v. parahaemolyticus to at least 8-fold draft coverage, for a total of six clinical isolates; two non-pandemic and four pandemic .
the two non-pandemic strains, aq <dig> and aq <dig>  were isolated in  <dig> and  <dig>  respectively, and both originated from southeast asia.
throughout the remainder of this study, we will refer to these two non-pandemic isolates as "pre-pandemic" because they were isolated prior to the documented start of the pandemic.
three of the pandemic isolates were from southeast asia, including strain rimd <dig> in  <dig>  strain an <dig> in  <dig>  and strain k <dig> in  <dig>  while the fourth pandemic isolate  was isolated from peru in  <dig>  therefore, the isolates represented two geographic areas where major outbreaks occurred.
in addition, they also have different serotypes and toxin profiles.
all of the pandemic strains were  and the pre-pandemic strains were either  or , thus representing two potentially different virulence mechanisms.
to improve our understanding of the pandemic clone's evolution during their global dissemination, the genome of a peruvian isolate   <cit>  was sequenced and compared to the genomes of asian isolates collected at different time points during the pandemic.
in the later stage of the pandemic, there were fewer cases of infection in south asia; thus, v. parahaemolyticus isolated during this time seems to be less virulent .
therefore, an isolate  collected in  <dig> from india was included and considered a "less virulent" late stage pandemic isolate in this study.
also, the genome of a never-before-sequenced serotype o4:k <dig> pandemic isolate  was characterized in order to advance our understanding of the mechanism for its serotype conversion.
†the contig n <dig> is the length of the smallest contig in the set that contains the fewest  contigs whose combined length represents at least 50% of the assembly  <cit> .
§the contig n <dig> was calculated from the celera assembly, not the contigs submitted to genbank.
‡pre-pandemic years.
n.a.
not applicable.
the pan genome of the six v. parahaemolyticus strains we examined had  <dig>  chromosomal coding genes, and each individual genome  had an average of  <dig>  coding genes .
three thousand twenty eight genes, ca.
71% of the coding genes were present in all the strains .
however, that number may be lower than the actual number because the genomes, except for the genome of rimd <dig>  were not sequenced to completion.
therefore, some of the open-reading frames  that bordered contigs may have failed to meet the cut-offs and, subsequently, were treated as not present.
the four newly sequenced genomes displayed a high degree of synteny with rimd <dig> .
there was very little rearrangement of the genome of the pre-pandemic strain aq <dig> and essentially no rearrangement in the pandemic strains.
because the gaps are not closed for five of the genomes, our report of synteny represents our best estimation.
super integron
v. parahaemolyticus harbors a super integron  on chromosome i. the si is about  <dig> kb long and contains ca.
<dig> orfs, which is much smaller than the sis in v. cholerae  and in v. vulnificus .
most of the orfs in the si regions encode hypothetical proteins.
the si integrases were identical in the six v. parahaemolyticus strains examined, but the cassettes in the si regions of the pre-pandemic strains varied greatly from those of the pandemic strains.
for example, only  <dig> and  <dig> of the  <dig> orfs in the pandemic strains' si regions were present in those of pre-pandemic strains aq <dig> and aq <dig>  respectively.
however, the cassettes in the si regions of the four pandemic strains were nearly identical; i.e., they contained only a few point mutations.
the only exception was isolate k <dig>  which had an additional six hypothetical proteins inserted between the integrase and the rest of the cassettes.
these observations indicate that the integrase is active in v. parahaemolyticus and contributes to species evolution.
however, the fact that its si region is smaller than those of other pathogenic vibrio species, and the presence of highly conserved cassettes in the pandemic strains, suggests that the genomes of v. parahaemolyticus may be more stable than those of other pathogenic vibrios.
pathogenicity islands, prophages, and integrated elements
only the pandemic strains examined in this study contained the pathogenicity islands previously described  <cit>  for v. parahaemolyticus .
in addition, we detected various prophages and integrated elements using phage_finder  <cit> .
prophage f <dig>  which has been widely used as a genetic marker for the pandemic clone  <cit> , was present in chromosome i  of all the pandemic strains we examined, but it was absent from the pre-pandemic strains .
however, a prophage similar to f <dig> was present in pre-pandemic strain aq <dig>  in the same location occupied by f <dig> in the pandemic strains .
in addition to f <dig>  another prophage was identified adjacent to f <dig>  in all of the pandemic strains and in pre-pandemic strain aq <dig>  but it was absent from strain aq <dig>  also, a second copy of that prophage was present in chromosome ii of the pandemic strains and in strain aq <dig> .
in addition, a prophage region unique to the serotype o4:k <dig>  pandemic strain an <dig>  was identified by phage_finder .
in the first column, if genes are absent from rimd, the gene numbers in one of the other genomes are indicated in the parenthesis.
* these two phages are very similar.
v variable contents in the super integron
each of the strains we studied had one or two integrated elements targeting the tmrna gene .
for example, peru <dig>  an <dig>  and k <dig> had two different, integrated elements inserted in tandem into the 3' end of their tmrna genes.
the element closest to the tmrna gene contained two genes that may influence virulence: a putative cyclic diguanylate phosphodiesterase eal domain protein and an arac superfamily putative fimbrial transcriptional activator.
the second element was distinguished by the presence of a ribonuclease h-encoding gene.
the first element was present in strains aq <dig> and aq <dig>  but not in strain rimd <dig>  however, strains aq <dig> and aq <dig> lacked the ribonuclease h-encoding element present in strain rimd <dig>
characterization of the pathogenicity islands
pre-pandemic strain aq <dig> is tdh-, trh+ and urease-positive, and its genome sequence revealed a pathogenicity island  containing  <dig> orfs .
another pathogenicity island  was previously identified in chromosome ii of pandemic strain rimd <dig> and includes loci vpa1310- <dig> .
tdhpai contains a type iii secretion system  and two copies of tdh; whereas, trhpai contains trh, an integrase, transposases, a urease gene cluster, a peptide/nickel transportation system, and a t3ss that is different from the one in tdhpai .
the t3ss in aq4037's trhpai is similar to t3ssiiβ in v. parahaemolyticus th <dig>  which is related to the t3ss in non-o <dig>  non-o <dig> strains of v. cholerae  <cit> .
interestingly, trhpai was found in chromosome ii of strain th <dig>  but it was located in chromosome i of strain aq <dig>  this discrepancy in chromosomal location may be providing a clue to the pathogenicity island's mobility.
close examination of the tdhpai region in the six genomes revealed major differences between the pre-pandemic and pandemic strains .
the four epidemic strains' tdhpais were very similar to one another; however, the entire pathogenicity island was absent from the pre-pandemic, tdh- strain aq <dig>  instead, that strain contained a pre-pandemic-specific region of  <dig> orfs important for the uptake and metabolism of carbon sources and other nutrients.
in addition, although pre-pandemic strain aq <dig> contained both tdhpai and the pre-pandemic nutrient uptake region, it had an inverted tdhs gene .
thus, the pre-pandemic and pandemic strains exhibited major differences between the region upstream of the pathogenicity island and in tdh's orientation.
whether those variations affect the expression of the pathogenicity island's genes, which contributes to differences between the pre-pandemic and pandemic strains' virulence, remains to be determined.
the pathogenicity island's organization suggests that an ancestral strain possessing the o3:k <dig> serotype may have recruited a tdhpai next to vpa <dig>  which yielded a transient strain that subsequently lost the pre-pandemic island and gave rise to the pandemic strains.
another possibility is that the tdh+, pre-pandemic strain and the pandemic strains independently recruited the pathogenicity islands into the same location.
the tdh and trh genes are related but vary substantially  <cit> , and many variants of tdh and two trh genes have been described.
they have been found in various vibrio species and their phylogenetic relationships are not in accordance with the relationship of the host species  <cit> .
although most of them are in the chromosomes, some of them are present in plasmids, a finding that is consistent with their proposed mobility.
also, the presence of trh and tdh on both chromosomes of v. parahaemolyticus supports the idea that they may have been acquired by lateral gene transfer and may have integrated into the bacterium's genome during independent events.
also, since v. parahaemolyticus strains that have both tdh and trh have been described  <cit> , it might be worthwhile to sequence those strains' genomes in order to understand the evolutionary history of tdh and trh in their hosts.
tdh and trh are the only confirmed virulence factors of v. parahaemolyticus; however, their precise roles are not well understood.
genomic sequencing revealed that the genes encoding those toxins are in close proximity to a t3ssii system, which suggests they may have the same origin as that transport system.
thus, it is tempting to speculate that tdh  and t3ssii have coordinating activities related to the virulence of v. parahaemolyticus.
some translocon proteins and effectors have been identified for t3ssii  <cit> ; however, the putative relationship between tdh, trh, and t3ssii needs further investigation.
another t3ss  identified in the v. parahaemolyticus genome was demonstrated  <cit>  to be required for cytolytic activity.
however, t3ssi was conserved in all of the genomes we examined.
variability within the o3:k <dig> genetic locus
since  <dig>  serotype o3:k <dig> has predominated among clinical isolates of v. parahaemolyticus, thus that serotype has been associated with the bacterium's increased virulence.
however, strains of o3:k <dig> serotype had been isolated more than a decade before the pandemic initiated.
it is not clear if there are variations within the o3:k <dig> genetic determinants between non-pandemic and pandemic strains, and thus causing subtle structural difference of the o and k antigens that could not be detected by the serotyping techniques.
therefore, we examined the o- and k-antigen-encoding gene clusters in the pre-pandemic and pandemic o3:k <dig> strains for any variations that may explain the observed increase in virulence.
the o- and k-antigen-encoding gene clusters are juxtaposed in v. parahaemolyticus o3:k <dig>  <cit> .
in strain rimd <dig>  they are located at loci vp0190- <dig> in chromosome i   <cit> .
however, that region was conserved in all o3:k <dig> strains , which suggests that the serotype of v. parahaemolyticus may not be directly related to the pandemic strain's increased virulence.
serotype conversion from o3:k <dig> to o4:k68
in addition to serotype o3:k <dig>  >  <dig> other serotypes of v. parahaemolyticus were detected among pandemic strains  <cit> .
the mechanism for this serotype conversion remains unknown.
since the o- and k-antigen loci are tightly linked on the chromosome  <cit> , a recombination event involving this region would enable rapid conversion of serotypes.
before comparing the o- and k-antigen region between o3:k <dig> and o4:k <dig> serotypes, we first wanted to determine the variability of this region within the o4:k <dig> serotype as we did above for the o3:k <dig> serotype.
we compared the o- and k- region of strain an <dig> , an5034_1842- <dig>  to the o- and k- region of another o4:k <dig> strain designated niid242- <dig>  <cit> .
both clusters were nearly identical , as observed above for the o3:k <dig> loci.
when comparing the o- and k-antigen regions between strain an <dig>  and strain rimd <dig> , this region varied substantially, except for the first seven genes, suggesting recombination as a method for serotype conversion.
to help identify the scope of the recombination event, we analyzed the distribution of single-nucleotide polymorphisms  in the genome of strain an <dig> compared to strain rimd <dig>  compared to the other pandemic isolates, strain an <dig> had  <dig>  snps ,  <dig>   of which clustered in a  <dig> kb region , corresponding to between 2-kb upstream and 88-kb downstream of the o- and k-antigen-encoding gene clusters .
this observation suggests that a recombination event involving a much larger region than the o-antigen- and k-antigen loci occurred and gave rise to the new o4:k <dig> serotype during the pandemic.
origin of the south american outbreaks
our results indicated that the pandemic strains are closely related to one another.
strain rimd <dig> differed from strain k <dig> and the peruvian strain  by  <dig> and  <dig> snps, respectively .
peru <dig> was isolated at approximately the same time the first outbreak  was reported in india.
also, its gene content is almost identical to that of a pandemic asian strain  isolated at the same time, which supports the idea that the pandemic strain spread from asia to south america very soon after it emerged.
considering the proximity, it is likely that the  <dig> outbreak in chile was caused by strains descended from the peruvian isolates.
we speculate that the strains from south american outbreaks are closely related to the strains from indian outbreaks.
this hypothesis will require genomic sequencing of additional strains from south america, specifically those from chile, to confirm.
fewer cases of disease caused by v. parahaemolyticus were reported from asia during the later stages of the pandemic than during its early stages ; thus, we suspect that the strains isolated during the later stages were not as virulent as those obtained during its early stages.
therefore, we sequenced and analyzed the genome of strain k <dig>  which was isolated from india almost a decade after the pandemic started, to look for gene deletions and variations.
major gene deletions were not identified in strain k5030; instead, several insertions were detected .
in addition to the cassettes added to the strain's si, k <dig> had an insertion of  <dig> orfs  containing a dna phosphorothioation  system, which incorporates sulphur into the dna backbone,  <cit> .
recent evidence supports the lateral transfer of dnd genes among bacterial genomes  <cit> .
besides the dnd genes, there were also two transposases and one integrase in the insertion.
however, it is not clear whether the addition of those orfs reduced the strain's virulence by altering the structure and function of virulence factors.
comparative genomic analyses of the three major pathogenic vibrio species
in order to characterize important genetic differences between v. parahaemolyticus, v. cholerae and v. vulnificus, we compared their core genomes.
the v. cholerae group consisted of four completely sequenced genomes of toxigenic, serotype o <dig> strains: n <dig>  m66- <dig>  mj- <dig> and o <dig>  <cit> .
the v. vulnificus group contained two completely sequenced genomes of strains cmcp <dig> and yj <dig>  <cit> , and the v. parahaemolyticus group consisted of strain rimd <dig> and the two pre-pandemic isolates .
the orthologs from each group were extracted by comparing all members of the group, after which the core genes from each group were compared to each other.
forty-nine to 59% of the core genes in each species were common to all three species .
however, 14-24% of the core genes in each group were only conserved in its own group and were in different cog  categories .
these are likely to be the genes that define the bacteria at the species level.
furthermore, each species had specific genes and transporters required for various metabolic pathways, which indicates that they have different requirements for transporting various ions, nutrients, and other metabolites across their outer membranes.
in addition, each species had unique two-component regulatory systems and chemotaxis genes, which indicate that they have specific signal pathways that respond to various environmental stimuli.
the three species also differ in their outer membrane structure and virulence genes .
furthermore, v. vulnificus has a unique set of genes for flp pilus synthesis, and toxigenic strains of v. cholerae have genes for toxin co-regulated pilus synthesis, a well-studied virulence factor  <cit>  .
v. parahaemolyticus possesses genes encoding two unique flagella, in addition to the genes required for the biosynthesis of flagella possessed by all three species.
v. cholerae also has two different t3ss-containing islands  <cit> .
t3ss has been reported to be closely related to t3ssii in v. parahaemolyticus and to be present in non-o <dig>  non-o <dig> strains of v. cholerae  <cit> , but we did not detect it in the toxigenic strains of v. cholerae.
however, the possession of t3ss by v. parahaemolyticus and non-o <dig>  non-o <dig> strains of v. cholerae suggests that this transport system is required for colonization of their unknown environmental hosts and reservoirs.
evolution of v. parahaemolyticus
in order to advance our understanding of the relationships between the v. parahaemolyticus strains we characterized, a set of  <dig> single-copy genes present in all six strains , taken from the analysis of three pathogenic vibrios, was compiled, and a nucleotide maximum-likelihood tree was inferred for the concatenated set of  <dig> genes .
our expectation that a resolved phylogeny supported by the majority of genes would be found and interpreted as an estimate of the strains' phylogenies was not met, a result similar to that of boyd et al  <dig>  <cit>  who also found that all of the pandemic strains were intermixed with pre-pandemic strains, but used just three genes in their analysis.
inspection of the aligned genomes with the snps marked in each one  revealed that the snps were often clustered, possibly indicative of recombination events.
recombination events involving multiple snps would distort the branch lengths and provide a poor estimate of the phylogeny.
in retrospect, this result might have been expected because we have shown that recombination events can involve a large number of variable nucleotides in numerous genes and each one of those events will distort the distance measure used for phylogenetic trees that assume all mutations are independently acquired.
in the sequenced strains, the single recombination event that converted the serotype and replaced the neighboring  <dig> kb contained  <dig> times the total number of variable nucleotides observed in the rest of the genome .
thus, if only one of the  <dig> genes from this region were included with the rest of the genes in the genome to calculate a tree, that one gene, on average, would contribute 11% of the total variation.
in order to calculate accurate trees, each recombination event and independent mutation event must be given equal weight.
CONCLUSIONS
this study helps to improve our understanding of how v. parahaemolyticus evolved during a pandemic.
the results of our multiple genome analyses are consistent with the idea that pandemic strains of v. parahaemolyticus evolved from pre-pandemic strains by numerous deletions and acquisitions of genetic material.
pandemic strains differ from pre-pandemic strains mostly in mobile genetic elements and the structure of the pathogenicity islands.
serotype conversion to o4:k <dig> was likely due to a recombination event involving a region much larger than the o-antigen- and k-antigen-encoding gene clusters.
in addition, this study revealed that  and  strains not only have different toxin genes, but also differ in the structures and locations of their pathogenicity islands.
lateral gene transfer seems to be the major force shaping the virulence of v. parahaemolyticus, as evidenced by the diversity in the locations and nucleotide sequences of the virulence factor-encoding genes.
in addition, previous studies had shown that insertion sequences in v. parahaemolyticus could change the genome structure and result in the loss of a major virulence factor  <cit> .
however, the pandemic strains we studied are almost monomorphic  and the outbreaks in asia and south america are closely related.
during the pandemic's later development, pandemic strains with the o3:k <dig> serotype  were no longer prevalent in india, where the pandemic originated .
instead, massive outbreaks were reported in chile.
this observation suggests that the virulent clone that spread to south america during the pandemic's early stage has persisted in that area and continues to cause outbreaks.
there was no loss of known virulence genes in the later stage pandemic  isolate from south asia; therefore, its virulence status needs further evaluation.
genetic changes in the etiologic agent may not be the only factor leading to v. parahaemolyticus-mediated pandemics .
for example, an outbreak of foodborne diarrheal disease caused by v. parahaemolyticus was reported on an alaskan cruise ship during  <dig>  and the source of the infection was v. parahaemolyticus-contaminated oysters harvested following warm weather in alaska, where v. parahaemolyticus had not been previously isolated  <cit> .
in addition, although there are major genetic differences between pre-pandemic and pandemic strains of v. parahaemolyticus, and they may, to some extent, contribute to the pandemic strains' increased virulence, a pathogenicity island  is also present in the pre-pandemic strain aq <dig>  thus, it is likely that the recent v. parahaemolyticus-mediated pandemic resulted from the convergence of genetic changes in the etiologic agent and the presence of optimal conditions for survival and growth in its natural reservoirs.
materials and methods
strain isolation and verification
the v. parahaemolyticus strains were isolated on tcbs  agar medium followed by their presumptive identification with a multitest medium  <cit> .
the strains' identities were confirmed by a species-specific toxr assay  <cit> , a commercially available v. parahaemolyticus antiserum kit  was employed for serological typing, and tdh and trh were identified by pcr  <cit> .
strains were cultured overnight in luria-bertani broth, and dna was obtained by lysing the bacteria with proteinase k followed by dna extraction and purification with a qiagen maxi kit .
genome sequencing
the genomes of v. parahaemolyticus strains an <dig>  aq <dig>  k <dig>  and peru <dig> were sequenced by the sanger whole-genome random shotgun method  <cit> .
briefly, one small insert plasmid library  and one medium insert plasmid library  were constructed by random nebulization and cloning of genomic dna.
during the initial random-sequencing phase, 8-fold sequence coverage was achieved with the small- and medium-size libraries sequenced to yield 5-fold and 3-fold coverage, respectively.
the sequences were assembled using the celera assembler  <cit> , and ordered scaffolds were generated by using nucmer  <cit>  to align the contigs to the genome of v. parahaemolyticus rimd <dig>  followed by hierarchical scaffolding with bambus  <cit> .
the contig n <dig> was determined as described  <cit> .
an initial set of open-reading frames  was identified using glimmer  <cit> , and orfs shorter than  <dig> bp  were eliminated.
a region containing the likely origin of replication was identified, and base pair  <dig> was designated adjacent to the dnaa gene located in that region  <cit> .
the orfs were searched against a nonredundant protein database, and the orf predictions and gene family identifications were done as previously described  <cit> .
two sets of hidden markov models  were used to determine orfs membership in families and super families.
these included  <dig> hmms from pfam v <dig>  and  <dig> hmms from the tigr ortholog resource.
a transmembrane hidden markov model   <cit>  was used to identify membrane-spanning domains in proteins, and putative functional role categories were assigned internally as previously described  <cit> .
the nucleotide sequences and the corresponding automated annotations for the genomes of v. parahaemolyticus strains an <dig>  aq <dig>  k <dig>  and peru <dig> were submitted to ncbi, with accession numbers , respectively.
comparative genomics
the database and cut-offs mentioned above were used, as previously described  <cit> , to  produce an ortholog match table,  construct a venn diagram, and  bin the relationships within the venn diagram.
synteny plots using promer  <cit>  were computed as previously described  <cit> .
snps were identified by mapping chromosomal contigs to the complete reference genome of rimd <dig> using nucmer  <cit>  with default setting and displayed using the show-snps tool with the -c  and -i  options.
show-snps is part of the mummer  <dig> distribution http://mummer.sourceforge.net/.
dna maximum likelihood trees were created  for each of the  <dig> entries in the above mentioned ortholog table that had orthologs for v. vulnificus cmcp <dig> and the six v. parahaemolyticus strains.
in order to ensure proper alignment of the coding regions, the trees were based on dna alignments back-aligned from the proteins' alignments.
competing interests
the authors declare that they have no competing interests.
authors' contributions
ocs, rh, gbn, aig, mn, and def conceived and organized the project; gbn, aig, and mn provided strains; ocs and yc isolated dna; def organized the sequencing studies; def, jhb, and yc analyzed the genomic data; and yc, def, jhb, and ocs prepared the manuscript.
all authors read and approved the final manuscript.
supplementary material
additional file 1
vibrio parahaemolyticus ortholog match table.
blast-based ortholog match table of v. parahaemolyticus strains
click here for file
additional file 2
vibrio parahaemolyticus single nucleotide polymorphisms.
single nucleotide polymorphisms of v. parahaemolyticus genomes relative to reference strain rimd2210633
click here for file
additional file 3
relationships of v. parahaemolyticus strains.
dna maximum likelihood tree based on  <dig> orthologs of the six v. parahaemolyticus strains.
v. vulnificus strain cmcp <dig> was used as an outlier.
click here for file
acknowledgements
we are grateful to the j. craig venter institute's sequencing, bioinformatics and it departments for supporting the infrastructure required to determine the genomes' sequences and annotation.
we especially thank a. scott durkin for contributing to the genomes' annotation.
we also thank arnold s. kreger and stephanie zafonte for reading and editing the manuscript.
this project was supported by the  national institute of allergy and infectious diseases , under contract number n01-ai- <dig>  and  university of maryland clinical research unit of the food and waterborne diseases integrated research network , under contract number n01-ai- <dig>

 vibrio parahaemolyticus is a halophilic bacterium which has long been recognized as a human pathogen that causes gastroenteritis and, occasionally, wound infections and sepsis in immunocompromised patients. it is the leading etiologic agent for bacterial foodborne disease in japan and other parts of asia, and it is the most common bacterial cause of seafood-associated disease in the united states. prior to there was no specific serotype of v. parahaemolyticus that was associated with disease outbreaks, and the bacterium had never been reported to cause a pandemic. however, during that year, a major outbreak occurred in india, > 50% of the v. parahaemolyticus strains isolated from patients were serotype o3:k. also, the outbreak rapidly spread to other countries in asia, south america, north america, africa and europe, resulting in a pandemic affecting tens of thousands of people. during its global dissemination, > serovariants is recognized as the most important virulence factor of v. parahaemolyticus, and a tdh-related hemolysin is believed to account for the virulence of strains that do not produce tdh. prior whole-genome sequencing of a serotype o3:k pandemic isolate designated rimd identified two type iii secretion systems. t3ssi is present in all v. parahaemolyticus isolates examined and is required for the bacterium's cytolytic activity; whereas, t3ssii is required for enterotoxicity and is located in the tdh-containing pathogenicity island. outbreaks of diarrheal disease caused by v. parahaemolyticus may pose a significant health threat. thus far, the most affected country has been chile, where > cases were reported during this observation suggests that, under appropriate conditions, v. parahaemolyticus may cause large-scale outbreaks comparable to those elicited by v. cholerae. at the present time, the reasons for the pandemic strains' rapid increase in virulence/prevalence have not been rigorously determined. in addition, the mechanism for rapid serotype conversion warrant further study. furthermore, it is not clear whether the virulence mechanisms of tdh+ and trh+ strains are similar. therefore, in order to address some of these questions, we performed rigorous genomic analyses of two pre-pandemic and four pandemic isolates of v. parahaemolyticus.  comparative genomics of v. parahaemolyticus prior to this study, an o3:k pandemic isolate was sequenced to completion and an o3:k non-pandemic isolate was sequenced to draft status. in this study, we sequenced four additional isolates of v. parahaemolyticus to at least 8-fold draft coverage, for a total of six clinical isolates; two non-pandemic and four pandemic. the two non-pandemic strains, aq and aq were isolated in and respectively, and both originated from southeast asia. throughout the remainder of this study, we will refer to these two non-pandemic isolates as "pre-pandemic" because they were isolated prior to the documented start of the pandemic. three of the pandemic isolates were from southeast asia, including strain rimd in strain an in and strain k in while the fourth pandemic isolate was isolated from peru in therefore, the isolates represented two geographic areas where major outbreaks occurred. in addition, they also have different serotypes and toxin profiles. all of the pandemic strains were and the pre-pandemic strains were either or, thus representing two potentially different virulence mechanisms. to improve our understanding of the pandemic clone's evolution during their global dissemination, the genome of a peruvian isolate was sequenced and compared to the genomes of asian isolates collected at different time points during the pandemic. in the later stage of the pandemic, there were fewer cases of infection in south asia; thus, v. parahaemolyticus isolated during this time seems to be less virulent. therefore, an isolate collected in from india was included and considered a "less virulent" late stage pandemic isolate in this study. also, the genome of a never-before-sequenced serotype o4:k pandemic isolate was characterized in order to advance our understanding of the mechanism for its serotype conversion. †the contig n is the length of the smallest contig in the set that contains the fewest contigs whose combined length represents at least 50% of the assembly. §the contig n was calculated from the celera assembly, not the contigs submitted to genbank. ‡pre-pandemic years. n.a. not applicable. the pan genome of the six v. parahaemolyticus strains we examined had chromosomal coding genes, and each individual genome had an average of coding genes. three thousand twenty eight genes, ca. 71% of the coding genes were present in all the strains. however, that number may be lower than the actual number because the genomes, except for the genome of rimd were not sequenced to completion. therefore, some of the open-reading frames that bordered contigs may have failed to meet the cut-offs and, subsequently, were treated as not present. the four newly sequenced genomes displayed a high degree of synteny with rimd. there was very little rearrangement of the genome of the pre-pandemic strain aq and essentially no rearrangement in the pandemic strains. because the gaps are not closed for five of the genomes, our report of synteny represents our best estimation. super integron v. parahaemolyticus harbors a super integron on chromosome i. the si is about kb long and contains ca. orfs, which is much smaller than the sis in v. cholerae and in v. vulnificus. most of the orfs in the si regions encode hypothetical proteins. the si integrases were identical in the six v. parahaemolyticus strains examined, but the cassettes in the si regions of the pre-pandemic strains varied greatly from those of the pandemic strains. for example, only and of the orfs in the pandemic strains' si regions were present in those of pre-pandemic strains aq and aq respectively. however, the cassettes in the si regions of the four pandemic strains were nearly identical; i.e., they contained only a few point mutations. the only exception was isolate k which had an additional six hypothetical proteins inserted between the integrase and the rest of the cassettes. these observations indicate that the integrase is active in v. parahaemolyticus and contributes to species evolution. however, the fact that its si region is smaller than those of other pathogenic vibrio species, and the presence of highly conserved cassettes in the pandemic strains, suggests that the genomes of v. parahaemolyticus may be more stable than those of other pathogenic vibrios. pathogenicity islands, prophages, and integrated elements only the pandemic strains examined in this study contained the pathogenicity islands previously described for v. parahaemolyticus. in addition, we detected various prophages and integrated elements using phage_finder. prophage f which has been widely used as a genetic marker for the pandemic clone, was present in chromosome i of all the pandemic strains we examined, but it was absent from the pre-pandemic strains. however, a prophage similar to f was present in pre-pandemic strain aq in the same location occupied by f in the pandemic strains. in addition to f another prophage was identified adjacent to f in all of the pandemic strains and in pre-pandemic strain aq but it was absent from strain aq also, a second copy of that prophage was present in chromosome ii of the pandemic strains and in strain aq. in addition, a prophage region unique to the serotype o4:k pandemic strain an was identified by phage_finder. in the first column, if genes are absent from rimd, the gene numbers in one of the other genomes are indicated in the parenthesis. * these two phages are very similar. v variable contents in the super integron each of the strains we studied had one or two integrated elements targeting the tmrna gene. for example, peru an and k had two different, integrated elements inserted in tandem into the 3' end of their tmrna genes. the element closest to the tmrna gene contained two genes that may influence virulence: a putative cyclic diguanylate phosphodiesterase eal domain protein and an arac superfamily putative fimbrial transcriptional activator. the second element was distinguished by the presence of a ribonuclease h-encoding gene. the first element was present in strains aq and aq but not in strain rimd however, strains aq and aq lacked the ribonuclease h-encoding element present in strain rimd characterization of the pathogenicity islands pre-pandemic strain aq is tdh-, trh+ and urease-positive, and its genome sequence revealed a pathogenicity island containing orfs. another pathogenicity island was previously identified in chromosome ii of pandemic strain rimd and includes loci vpa1310-. tdhpai contains a type iii secretion system and two copies of tdh; whereas, trhpai contains trh, an integrase, transposases, a urease gene cluster, a peptide/nickel transportation system, and a t3ss that is different from the one in tdhpai. the t3ss in aq4037's trhpai is similar to t3ssiiβ in v. parahaemolyticus th which is related to the t3ss in non-o non-o strains of v. cholerae. interestingly, trhpai was found in chromosome ii of strain th but it was located in chromosome i of strain aq this discrepancy in chromosomal location may be providing a clue to the pathogenicity island's mobility. close examination of the tdhpai region in the six genomes revealed major differences between the pre-pandemic and pandemic strains. the four epidemic strains' tdhpais were very similar to one another; however, the entire pathogenicity island was absent from the pre-pandemic, tdh- strain aq instead, that strain contained a pre-pandemic-specific region of orfs important for the uptake and metabolism of carbon sources and other nutrients. in addition, although pre-pandemic strain aq contained both tdhpai and the pre-pandemic nutrient uptake region, it had an inverted tdhs gene. thus, the pre-pandemic and pandemic strains exhibited major differences between the region upstream of the pathogenicity island and in tdh's orientation. whether those variations affect the expression of the pathogenicity island's genes, which contributes to differences between the pre-pandemic and pandemic strains' virulence, remains to be determined. the pathogenicity island's organization suggests that an ancestral strain possessing the o3:k serotype may have recruited a tdhpai next to vpa which yielded a transient strain that subsequently lost the pre-pandemic island and gave rise to the pandemic strains. another possibility is that the tdh+, pre-pandemic strain and the pandemic strains independently recruited the pathogenicity islands into the same location. the tdh and trh genes are related but vary substantially, and many variants of tdh and two trh genes have been described. they have been found in various vibrio species and their phylogenetic relationships are not in accordance with the relationship of the host species. although most of them are in the chromosomes, some of them are present in plasmids, a finding that is consistent with their proposed mobility. also, the presence of trh and tdh on both chromosomes of v. parahaemolyticus supports the idea that they may have been acquired by lateral gene transfer and may have integrated into the bacterium's genome during independent events. also, since v. parahaemolyticus strains that have both tdh and trh have been described, it might be worthwhile to sequence those strains' genomes in order to understand the evolutionary history of tdh and trh in their hosts. tdh and trh are the only confirmed virulence factors of v. parahaemolyticus; however, their precise roles are not well understood. genomic sequencing revealed that the genes encoding those toxins are in close proximity to a t3ssii system, which suggests they may have the same origin as that transport system. thus, it is tempting to speculate that tdh and t3ssii have coordinating activities related to the virulence of v. parahaemolyticus. some translocon proteins and effectors have been identified for t3ssii; however, the putative relationship between tdh, trh, and t3ssii needs further investigation. another t3ss identified in the v. parahaemolyticus genome was demonstrated to be required for cytolytic activity. however, t3ssi was conserved in all of the genomes we examined. variability within the o3:k genetic locus since serotype o3:k has predominated among clinical isolates of v. parahaemolyticus, thus that serotype has been associated with the bacterium's increased virulence. however, strains of o3:k serotype had been isolated more than a decade before the pandemic initiated. it is not clear if there are variations within the o3:k genetic determinants between non-pandemic and pandemic strains, and thus causing subtle structural difference of the o and k antigens that could not be detected by the serotyping techniques. therefore, we examined the o- and k-antigen-encoding gene clusters in the pre-pandemic and pandemic o3:k strains for any variations that may explain the observed increase in virulence. the o- and k-antigen-encoding gene clusters are juxtaposed in v. parahaemolyticus o3:k. in strain rimd they are located at loci vp0190- in chromosome i. however, that region was conserved in all o3:k strains, which suggests that the serotype of v. parahaemolyticus may not be directly related to the pandemic strain's increased virulence. serotype conversion from o3:k to o4:k68 in addition to serotype o3:k > other serotypes of v. parahaemolyticus were detected among pandemic strains. the mechanism for this serotype conversion remains unknown. since the o- and k-antigen loci are tightly linked on the chromosome, a recombination event involving this region would enable rapid conversion of serotypes. before comparing the o- and k-antigen region between o3:k and o4:k serotypes, we first wanted to determine the variability of this region within the o4:k serotype as we did above for the o3:k serotype. we compared the o- and k- region of strain an, an5034_1842- to the o- and k- region of another o4:k strain designated niid242-. both clusters were nearly identical, as observed above for the o3:k loci. when comparing the o- and k-antigen regions between strain an and strain rimd, this region varied substantially, except for the first seven genes, suggesting recombination as a method for serotype conversion. to help identify the scope of the recombination event, we analyzed the distribution of single-nucleotide polymorphisms in the genome of strain an compared to strain rimd compared to the other pandemic isolates, strain an had snps, of which clustered in a kb region, corresponding to between 2-kb upstream and 88-kb downstream of the o- and k-antigen-encoding gene clusters. this observation suggests that a recombination event involving a much larger region than the o-antigen- and k-antigen loci occurred and gave rise to the new o4:k serotype during the pandemic. origin of the south american outbreaks our results indicated that the pandemic strains are closely related to one another. strain rimd differed from strain k and the peruvian strain by and snps, respectively. peru was isolated at approximately the same time the first outbreak was reported in india. also, its gene content is almost identical to that of a pandemic asian strain isolated at the same time, which supports the idea that the pandemic strain spread from asia to south america very soon after it emerged. considering the proximity, it is likely that the outbreak in chile was caused by strains descended from the peruvian isolates. we speculate that the strains from south american outbreaks are closely related to the strains from indian outbreaks. this hypothesis will require genomic sequencing of additional strains from south america, specifically those from chile, to confirm. fewer cases of disease caused by v. parahaemolyticus were reported from asia during the later stages of the pandemic than during its early stages; thus, we suspect that the strains isolated during the later stages were not as virulent as those obtained during its early stages. therefore, we sequenced and analyzed the genome of strain k which was isolated from india almost a decade after the pandemic started, to look for gene deletions and variations. major gene deletions were not identified in strain k5030; instead, several insertions were detected. in addition to the cassettes added to the strain's si, k had an insertion of orfs containing a dna phosphorothioation system, which incorporates sulphur into the dna backbone,. recent evidence supports the lateral transfer of dnd genes among bacterial genomes. besides the dnd genes, there were also two transposases and one integrase in the insertion. however, it is not clear whether the addition of those orfs reduced the strain's virulence by altering the structure and function of virulence factors. comparative genomic analyses of the three major pathogenic vibrio species in order to characterize important genetic differences between v. parahaemolyticus, v. cholerae and v. vulnificus, we compared their core genomes. the v. cholerae group consisted of four completely sequenced genomes of toxigenic, serotype o strains: n m66- mj- and o. the v. vulnificus group contained two completely sequenced genomes of strains cmcp and yj, and the v. parahaemolyticus group consisted of strain rimd and the two pre-pandemic isolates. the orthologs from each group were extracted by comparing all members of the group, after which the core genes from each group were compared to each other. forty-nine to 59% of the core genes in each species were common to all three species. however, 14-24% of the core genes in each group were only conserved in its own group and were in different cog categories. these are likely to be the genes that define the bacteria at the species level. furthermore, each species had specific genes and transporters required for various metabolic pathways, which indicates that they have different requirements for transporting various ions, nutrients, and other metabolites across their outer membranes. in addition, each species had unique two-component regulatory systems and chemotaxis genes, which indicate that they have specific signal pathways that respond to various environmental stimuli. the three species also differ in their outer membrane structure and virulence genes. furthermore, v. vulnificus has a unique set of genes for flp pilus synthesis, and toxigenic strains of v. cholerae have genes for toxin co-regulated pilus synthesis, a well-studied virulence factor. v. parahaemolyticus possesses genes encoding two unique flagella, in addition to the genes required for the biosynthesis of flagella possessed by all three species. v. cholerae also has two different t3ss-containing islands. t3ss has been reported to be closely related to t3ssii in v. parahaemolyticus and to be present in non-o non-o strains of v. cholerae, but we did not detect it in the toxigenic strains of v. cholerae. however, the possession of t3ss by v. parahaemolyticus and non-o non-o strains of v. cholerae suggests that this transport system is required for colonization of their unknown environmental hosts and reservoirs. evolution of v. parahaemolyticus in order to advance our understanding of the relationships between the v. parahaemolyticus strains we characterized, a set of single-copy genes present in all six strains, taken from the analysis of three pathogenic vibrios, was compiled, and a nucleotide maximum-likelihood tree was inferred for the concatenated set of genes. our expectation that a resolved phylogeny supported by the majority of genes would be found and interpreted as an estimate of the strains' phylogenies was not met, a result similar to that of boyd et al who also found that all of the pandemic strains were intermixed with pre-pandemic strains, but used just three genes in their analysis. inspection of the aligned genomes with the snps marked in each one revealed that the snps were often clustered, possibly indicative of recombination events. recombination events involving multiple snps would distort the branch lengths and provide a poor estimate of the phylogeny. in retrospect, this result might have been expected because we have shown that recombination events can involve a large number of variable nucleotides in numerous genes and each one of those events will distort the distance measure used for phylogenetic trees that assume all mutations are independently acquired. in the sequenced strains, the single recombination event that converted the serotype and replaced the neighboring kb contained times the total number of variable nucleotides observed in the rest of the genome. thus, if only one of the genes from this region were included with the rest of the genes in the genome to calculate a tree, that one gene, on average, would contribute 11% of the total variation. in order to calculate accurate trees, each recombination event and independent mutation event must be given equal weight.  this study helps to improve our understanding of how v. parahaemolyticus evolved during a pandemic. the results of our multiple genome analyses are consistent with the idea that pandemic strains of v. parahaemolyticus evolved from pre-pandemic strains by numerous deletions and acquisitions of genetic material. pandemic strains differ from pre-pandemic strains mostly in mobile genetic elements and the structure of the pathogenicity islands. serotype conversion to o4:k was likely due to a recombination event involving a region much larger than the o-antigen- and k-antigen-encoding gene clusters. in addition, this study revealed that and strains not only have different toxin genes, but also differ in the structures and locations of their pathogenicity islands. lateral gene transfer seems to be the major force shaping the virulence of v. parahaemolyticus, as evidenced by the diversity in the locations and nucleotide sequences of the virulence factor-encoding genes. in addition, previous studies had shown that insertion sequences in v. parahaemolyticus could change the genome structure and result in the loss of a major virulence factor. however, the pandemic strains we studied are almost monomorphic and the outbreaks in asia and south america are closely related. during the pandemic's later development, pandemic strains with the o3:k serotype were no longer prevalent in india, where the pandemic originated. instead, massive outbreaks were reported in chile. this observation suggests that the virulent clone that spread to south america during the pandemic's early stage has persisted in that area and continues to cause outbreaks. there was no loss of known virulence genes in the later stage pandemic isolate from south asia; therefore, its virulence status needs further evaluation. genetic changes in the etiologic agent may not be the only factor leading to v. parahaemolyticus-mediated pandemics. for example, an outbreak of foodborne diarrheal disease caused by v. parahaemolyticus was reported on an alaskan cruise ship during and the source of the infection was v. parahaemolyticus-contaminated oysters harvested following warm weather in alaska, where v. parahaemolyticus had not been previously isolated. in addition, although there are major genetic differences between pre-pandemic and pandemic strains of v. parahaemolyticus, and they may, to some extent, contribute to the pandemic strains' increased virulence, a pathogenicity island is also present in the pre-pandemic strain aq thus, it is likely that the recent v. parahaemolyticus-mediated pandemic resulted from the convergence of genetic changes in the etiologic agent and the presence of optimal conditions for survival and growth in its natural reservoirs. materials and methods strain isolation and verification the v. parahaemolyticus strains were isolated on tcbs agar medium followed by their presumptive identification with a multitest medium. the strains' identities were confirmed by a species-specific toxr assay, a commercially available v. parahaemolyticus antiserum kit was employed for serological typing, and tdh and trh were identified by pcr. strains were cultured overnight in luria-bertani broth, and dna was obtained by lysing the bacteria with proteinase k followed by dna extraction and purification with a qiagen maxi kit. genome sequencing the genomes of v. parahaemolyticus strains an aq k and peru were sequenced by the sanger whole-genome random shotgun method. briefly, one small insert plasmid library and one medium insert plasmid library were constructed by random nebulization and cloning of genomic dna. during the initial random-sequencing phase, 8-fold sequence coverage was achieved with the small- and medium-size libraries sequenced to yield 5-fold and 3-fold coverage, respectively. the sequences were assembled using the celera assembler, and ordered scaffolds were generated by using nucmer to align the contigs to the genome of v. parahaemolyticus rimd followed by hierarchical scaffolding with bambus. the contig n was determined as described. an initial set of open-reading frames was identified using glimmer, and orfs shorter than bp were eliminated. a region containing the likely origin of replication was identified, and base pair was designated adjacent to the dnaa gene located in that region. the orfs were searched against a nonredundant protein database, and the orf predictions and gene family identifications were done as previously described. two sets of hidden markov models were used to determine orfs membership in families and super families. these included hmms from pfam v and hmms from the tigr ortholog resource. a transmembrane hidden markov model was used to identify membrane-spanning domains in proteins, and putative functional role categories were assigned internally as previously described. the nucleotide sequences and the corresponding automated annotations for the genomes of v. parahaemolyticus strains an aq k and peru were submitted to ncbi, with accession numbers, respectively. comparative genomics the database and cut-offs mentioned above were used, as previously described, to produce an ortholog match table, construct a venn diagram, and bin the relationships within the venn diagram. synteny plots using promer were computed as previously described. snps were identified by mapping chromosomal contigs to the complete reference genome of rimd using nucmer with default setting and displayed using the show-snps tool with the -c and -i options. show-snps is part of the mummer distribution http://mummer.sourceforge.net/. dna maximum likelihood trees were created for each of the entries in the above mentioned ortholog table that had orthologs for v. vulnificus cmcp and the six v. parahaemolyticus strains. in order to ensure proper alignment of the coding regions, the trees were based on dna alignments back-aligned from the proteins' alignments. competing interests the authors declare that they have no competing interests. authors' contributions ocs, rh, gbn, aig, mn, and def conceived and organized the project; gbn, aig, and mn provided strains; ocs and yc isolated dna; def organized the sequencing studies; def, jhb, and yc analyzed the genomic data; and yc, def, jhb, and ocs prepared the manuscript. all authors read and approved the final manuscript. supplementary material additional file 1 vibrio parahaemolyticus ortholog match table. blast-based ortholog match table of v. parahaemolyticus strains click here for file additional file 2 vibrio parahaemolyticus single nucleotide polymorphisms. single nucleotide polymorphisms of v. parahaemolyticus genomes relative to reference strain rimd2210633 click here for file additional file 3 relationships of v. parahaemolyticus strains. dna maximum likelihood tree based on orthologs of the six v. parahaemolyticus strains. v. vulnificus strain cmcp was used as an outlier. click here for file acknowledgements we are grateful to the j. craig venter institute's sequencing, bioinformatics and it departments for supporting the infrastructure required to determine the genomes' sequences and annotation. we especially thank a. scott durkin for contributing to the genomes' annotation. we also thank arnold s. kreger and stephanie zafonte for reading and editing the manuscript. this project was supported by the national institute of allergy and infectious diseases, under contract number n01-ai- and university of maryland clinical research unit of the food and waterborne diseases integrated research network, under contract number n01-ai- 
we sequenced four additional genomes of v. parahaemolyticus in this study that were isolated from different geographical regions and time points. comparative genomic analyses of six strains of v. parahaemolyticus isolated from asia and peru were performed in order to advance knowledge concerning the evolution of v. parahaemolyticus; specifically, the genetic changes contributing to serotype conversion and virulence. the "core" genes of v. parahaemolyticus were also compared to those of v. cholerae and v. vulnificus, in order to delineate differences between these three pathogenic species. our data support the idea that the pandemic strains are closely related and that recent south american outbreaks of foodborne disease caused by v. parahaemolyticus are closely linked to outbreaks in india. major differences between pathogenicity islands and mobile elements are also likely driving the evolution of v. parahaemolyticus. in addition, the results of single nucleotide polymorphism analysis showed that 94% of the snps between o3:k and o4:k pandemic isolates were within a kb region surrounding the o- and k-antigen-encoding gene clusters. serotype conversion from o3:k to o4:k was likely due to a recombination event involving a region much larger than the o-antigen- and k-antigen-encoding gene clusters. in addition, our analyses categorized genes that may be useful in differentiating pathogenic vibrios at the species level. the sixth pandemic strain sequenced in this study was serotype o4:k genomic analyses revealed that the trh+ and tdh+ strains had different types of pathogenicity islands and mobile elements as well as major structural differences between the tdh pathogenicity islands of the pre-pandemic and pandemic strains. beginning in a more virulent strain having serotype o3:k caused major outbreaks in india and other parts of the world, resulting in the emergence of a pandemic. two pre-pandemic strains and three pandemic strains, isolated from different geographical regions, were serotype o3:k and either toxin profiles or. two genomes, one of this virulent strain and one pre-pandemic strain have been sequenced. approximately one-half of each species' core genes were conserved in all three species, and 14-24% of the core genes were species-specific and in different functional categories. vibrio parahaemolyticus is a common cause of foodborne disease.
 vibrio parahaemolyticus is a common cause of foodborne disease. beginning in a more virulent strain having serotype o3:k caused major outbreaks in india and other parts of the world, resulting in the emergence of a pandemic. other serovariants of this strain emerged during its dissemination and together with the original o3:k were termed strains of the pandemic clone. two genomes, one of this virulent strain and one pre-pandemic strain have been sequenced. we sequenced four additional genomes of v. parahaemolyticus in this study that were isolated from different geographical regions and time points. comparative genomic analyses of six strains of v. parahaemolyticus isolated from asia and peru were performed in order to advance knowledge concerning the evolution of v. parahaemolyticus; specifically, the genetic changes contributing to serotype conversion and virulence. two pre-pandemic strains and three pandemic strains, isolated from different geographical regions, were serotype o3:k and either toxin profiles or. the sixth pandemic strain sequenced in this study was serotype o4:k  genomic analyses revealed that the trh+ and tdh+ strains had different types of pathogenicity islands and mobile elements as well as major structural differences between the tdh pathogenicity islands of the pre-pandemic and pandemic strains. in addition, the results of single nucleotide polymorphism analysis showed that 94% of the snps between o3:k and o4:k pandemic isolates were within a kb region surrounding the o- and k-antigen-encoding gene clusters. the "core" genes of v. parahaemolyticus were also compared to those of v. cholerae and v. vulnificus, in order to delineate differences between these three pathogenic species. approximately one-half of each species' core genes were conserved in all three species, and 14-24% of the core genes were species-specific and in different functional categories.  our data support the idea that the pandemic strains are closely related and that recent south american outbreaks of foodborne disease caused by v. parahaemolyticus are closely linked to outbreaks in india. serotype conversion from o3:k to o4:k was likely due to a recombination event involving a region much larger than the o-antigen- and k-antigen-encoding gene clusters. major differences between pathogenicity islands and mobile elements are also likely driving the evolution of v. parahaemolyticus. in addition, our analyses categorized genes that may be useful in differentiating pathogenic vibrios at the species level. 