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Vibrio parahaemolyticus is a marine bacterium coming from estuarine environments, where the migratory birds can easily be colonized by V. parahaemolyticus. Migratory birds may be important reservoirs of V. parahaemolyticus by growth and re-entry into the environment. To further explore the spreading mechanism of V. parahaemolyticus among marine life, human beings, and migratory birds, we aimed to investigate the characteristics of the genetic diversity, antimicrobial resistance, virulence genes, and a potentially informative gene marker of V. parahaemolyticus isolated from migratory birds in China. This study recovered 124 (14.55%) V. parahaemolyticus isolates from 852 fecal and environmental (water) samples. All of the 124 strains were classified into 85 known sequence types (STs), of which ST-2738 was most frequently identified. Analysis of the population structure using whole-genome variation of the 124 isolates illustrated that they grouped into 27 different clonal groups (CGs) belonging to the previously defined geographical populations VppX and VppAsia. Even though these genomes have high diversity, an extra copy of tRNA-Gly was presented in all migratory bird-carried V. parahaemolyticus isolates, which could be used as a potentially informative marker of the V. parahaemolyticus strains derived from birds. Antibiotic sensitivity experiments revealed that 47 (37.10%) isolates were resistant to ampicillin. Five isolates harbored the plasmid-mediated quinolone resistance (PMQR) gene qnrD , which has not previously been identified in this species. The investigation of antibiotic resistance provides the basic knowledge to further evaluate the risk of enrichment and reintroduction of pathogenic V. parahaemolyticus strains in migratory birds. IMPORTANCE The presence of V. parahaemolyticus in migratory birds' fecal samples implies that the human pathogenic V. parahaemolyticus strains may also potentially infect birds and thus pose a risk for zoonotic infection and food safety associated with re-entry into the environment. Our study firstly highlights the extra copy of tRNA as a potentially informative marker for identifying the bird-carried V. parahaemolyticus strains. Also, we firstly identify the plasmid-mediated quinolone resistance (PMQR) gene qnrD in V. parahaemolyticus. To further evaluate the risk of enrichment and reintroduction of pathogenic strains carried by migratory birds, we suggest conducting estuarine environmental surveillance to monitor the antibiotic resistance and virulence factors of bird-carried V. parahaemolyticus isolates., Competing Interests: The authors declare no conflict of interest.

Controlling foodborne diseases requires robust outbreak detection and a comprehensive understanding of outbreak dynamics. Here, by integrating large-scale phylogenomic analysis of 3,642 isolates and epidemiological data, we performed 'data-driven' outbreak detection and described the long-term outbreak dynamics of the leading seafood-associated pathogen, Vibrio parahaemolyticus, in Shenzhen, China, over a 17-year period. Contradictory to the widely accepted notion that sporadic patients and independent point-source outbreaks dominated foodborne infections, we found that 71% of isolates from patients grouped into within-1-month clusters that differed by ≤6 single nucleotide polymorphisms, indicating putative outbreaks. Furthermore, we showed that despite the long time spans between clusters, 70% of them were genomically closely related and were inferred to arise from a small number of common sources, which provides evidence that hidden persistent reservoirs generated most of the outbreaks rather than independent point-sources. Phylogeographical analysis further revealed the geographical heterogeneity of outbreaks and identified a coastal district as the potential hotspot of outbreaks and as the hub and major source of cross-district spread events. Our findings provide a comprehensive picture of the long-term spatiotemporal dynamics of foodborne outbreaks and present a different perspective on the major source of foodborne infections, which will inform the design of future disease control strategies., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Investigating fitness interactions in natural populations remains a considerable challenge. We take advantage of the unique population structure of Vibrio parahaemolyticus , a bacterial pathogen of humans and shrimp, to perform a genome-wide screen for coadapted genetic elements. We identified 90 interaction groups (IGs) involving 1,560 coding genes. 82 IGs are between accessory genes, many of which have functions related to carbohydrate transport and metabolism. Only 8 involve both core and accessory genomes. The largest includes 1,540 SNPs in 82 genes and 338 accessory genome elements, many involved in lateral flagella and cell wall biogenesis. The interactions have a complex hierarchical structure encoding at least four distinct ecological strategies. One strategy involves a divergent profile in multiple genome regions, while the others involve fewer genes and are more plastic. Our results imply that most genetic alliances are ephemeral but that increasingly complex strategies can evolve and eventually cause speciation., Competing Interests: YC, CY, HQ, HW, RY, DF No competing interests declared, (© 2020, Cui et al.)

Vibrio parahaemolyticus is the leading bacterial cause of seafood-associated gastroenteritis worldwide. Moreover, infections and outbreaks caused by V. parahaemolyticus has kept increasing over the last two decades. In this study, we investigated the genetic diversity, virulence factors and farm-to-table spread pattern of V. parahaemolyticus by analyzing 383 genomes of food-associated isolates. These strains were isolated from diverse sample types from six provinces of China in 2014, being classified into three tiers of the farm-to-table spread process: food production, circulation and consumption. The genetic diversity of V. parahaemolyticus in different classifications, including geographical location, sample type, source and spread tier, was similar, as the median number of pairwise SNPs within each classification was between 33,013 and 33,659. Specifically, there was no clear boundaries in genetic diversity of the isolates from inland vs. coastal provinces, as well as of those from freshwater vs. seawater products. Moreover, the virulence genes and genomic islands were only found in a small number of isolates, indicating a low disease risk of the food-associated isolates in this study. By further exploring 28 recently emerged clonal groups, we identified seven farm-to-table spread events, showing a common pattern of single-source radial spread accompanied with occasional gene gain/loss events. Generally speaking, our work highlighted the colonization of V. parahaemolyticus in inland provinces and freshwater environment, and provided a snapshot of the farm-to-table spread pattern of V. parahaemolyticus food-associated isolates. Our results showed the feasibility of tracking the farm-to-table spread of foodborne pathogen, which would help construct the whole genome sequencing-based molecular tracking network in the future., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Humans have profoundly affected the ocean environment but little is known about anthropogenic effects on the distribution of microbes. Vibrio parahaemolyticus is found in warm coastal waters and causes gastroenteritis in humans and economically significant disease in shrimps. Based on data from 1103 genomes of environmental and clinical isolates, we show that V. parahaemolyticus is divided into four diverse populations, VppUS1, VppUS2, VppX and VppAsia. The first two are largely restricted to the US and Northern Europe, while the others are found worldwide, with VppAsia making up the great majority of isolates in the seas around Asia. Patterns of diversity within and between the populations are consistent with them having arisen by progressive divergence via genetic drift during geographical isolation. However, we find that there is substantial overlap in their current distribution. These observations can be reconciled without requiring genetic barriers to exchange between populations if long-range dispersal has increased dramatically in the recent past. We found that VppAsia isolates from the US have an average of 1.01% more shared ancestry with VppUS1 and VppUS2 isolates than VppAsia isolates from Asia itself. Based on time calibrated trees of divergence within epidemic lineages, we estimate that recombination affects about 0.017% of the genome per year, implying that the genetic mixture has taken place within the last few decades. These results suggest that human activity, such as shipping, aquatic products trade and increased human migration between continents, are responsible for the change of distribution pattern of this species.

Vibrio parahaemolyticus, the leading cause of seafood-associated gastroenteritis worldwide, requires the two type-III secretion systems (T3SS1 and T3SS2) and a thermostable direct hemolysin (encoded by tdh1 and tdh2) for full virulence. The tdh genes and the T3SS2 gene cluster constitute an 80 kb pathogenicity island known as Vp-PAI located on the chromosome II. Expression of T3SS1 and Vp-PAI is regulated in a quorum sensing (QS)-dependent manner but its detailed mechanisms remain unknown. Herein, we show that three factors (QS regulators AphA and OpaR and an AraC-type transcriptional regulator QsvR) form a complex regulatory network to control the expression of T3SS1 and Vp-PAI genes. At low cell density (LCD), whereas Vp-PAI expression is repressed, T3SS1 genes are induced by AphA, which directly binds (an operator region of) the exsBAD-vscBCD operon. At high cell density (HCD), the bacterium turns off T3SS1 expression by replacing AphA with OpaR, triggering the induction of Vp-PAI. Furthermore, QsvR binds to the regulatory regions of all the tested T3SS1 and Vp-PAI genes to activate their transcription at HCD. Taken together, our data highlight how multiple QS regulators contribute to the pathogenicity of V. parahaemolyticus by precisely controlling the expression of major virulence determinants during different stages of growth., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Vibrio parahaemolyticus , the leading causative agent of seafood-associated gastroenteritis, harbors two major virulence gene loci T3SS1 and Vp-PAI (T3SS2 and tdh2 ). ToxR is a virulence regulator of vibrios. Cell density-dependent transcriptional pattern of toxR and its regulatory actions on T3SS1 and Vp-PAI have been previously reported, but the detailed regulatory mechanisms are still obscure. In the present work, we showed that the highest transcription level of toxR occurs at an OD 600 = 0.2-0.4, which may be due to the subtle repression of ToxR and the quorum-sensing (QS) master regulator AphA. We also showed that ToxR is involved in regulating the mouse lethality, enterotoxicity, cytotoxicity, and hemolytic activity of V. parahaemolyticus . ToxR binds to the multiple promoter-proximal DNA regions within the T3SS1 locus to repress their transcription. In addition, ToxR occupies the multiple promoter-proximal DNA regions of Vp-PAI locus to activate their transcription. Thus, ToxR regulates the multiple virulence phenotypes via directly acting on the T3SS1 and Vp-PAI genes. Data presented here provide a deeper understanding of the regulatory patterns of ToxR in V. parahaemolyticus .

Bacteroides fragilis is an anaerobic, Gram-negative, commensal bacterium of the human gut. It plays an important role in promoting the maturation of the immune system, as well as suppressing abnormal inflammation. Many recent studies have focused on the relationship between B. fragilis and human immunity, and indicate that B. fragilis has many useful probiotic effects. As inhibition of intestinal pathogens is an important characteristic of probiotic strains, this study examined whether B. fragilis could inhibit pathogenic bacteria. Results showed that Vibrio parahaemolyticus was inhibited by B. fragilis in vitro , and that B. fragilis could protect both RAW 264.7 and LoVo cells from damage caused by V. parahaemolyticus . Using in vivo imaging, we constructed a light-emitting V. parahaemolyticus strain and showed that B. fragilis might shorten the colonization time and reduce the number of lux -expressing bacteria in a mouse model. These results provide useful information for developing B. fragilis into a probiotic product, and also indicate that this commensal bacterium might aid in the clinical treatment of gastroenteritis caused by V. parahaemolyticus .

We investigated global patterns of variation in 157 whole-genome sequences of Vibrio parahaemolyticus, a free-living and seafood associated marine bacterium. Pandemic clones, responsible for recent outbreaks of gastroenteritis in humans, have spread globally. However, there are oceanic gene pools, one located in the oceans surrounding Asia and another in the Mexican Gulf. Frequent recombination means that most isolates have acquired the genetic profile of their current location. We investigated the genetic structure in the Asian gene pool by calculating the effective population size in two different ways. Under standard neutral models, the two estimates should give similar answers but we found a 27-fold difference. We propose that this discrepancy is caused by the subdivision of the species into a hundred or more ecotypes which are maintained stably in the population. To investigate the genetic factors involved, we used 51 unrelated isolates to conduct a genome-wide scan for epistatically interacting loci. We found a single example of strong epistasis between distant genome regions. A majority of strains had a type VI secretion system associated with bacterial killing. The remaining strains had genes associated with biofilm formation and regulated by cyclic dimeric GMP signaling. All strains had one or other of the two systems and none of isolate had complete complements of both systems, although several strains had remnants. Further "top down" analysis of patterns of linkage disequilibrium within frequently recombining species will allow a detailed understanding of how selection acts to structure the pattern of variation within natural bacterial populations., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Objective: The DNA-binding domain of ToxR protein of Vibrio parahaemolyticus was expressed using the Escherichia coli BL21lambdaDE3 protein expression system, and its DNA-binding activity was characterized., Methods: The fragment of DNA-binding domain at N-terminal of ToxR (ToxR-N) was amplified by PCR from V. parahaemolyticus strain RIMD2210633, and then cloned into the BamHI and Hind III sites of the vector pET28a. The recombinant plasmid pET28a was transformed into BL21lambdaDE3. Over-expression of His-ToxR-N in the LB medium was induced by adding 1 mmol/L IPTG (isopropyl-b-D-thiogalactoside). The over-expressed protein was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham), and then the His-tag was removed by using restricted thrombin. The electrophoretic mobility shift assay was used to analyze the DNA-binding activity of ToxR-N to the promoter-proximal DNA regions of calR and VP1687, respectively. The promoter-proximal regions of calR and VP1687 were separately cloned into the pHRP309 vector containing a promoterless lacZ gene. Then, each of the two recombinant LacZ reporter plasmids was transformed into the wide-type strain (WT) and the toxR null mutant strain (DeltatoxR), respectively, to measure the promoter activity (the beta-Galactosidase activity) of the target genes in WT and DeltatoxR by using the beta-Galactosidase Enzyme Assay System., Results: The purified ToxR-N protein had the ability to bind to the upstream DNA regions of calR but not VP1687. The LacZ fusion results showed that the transcription of calR and VP1687 was positively and negatively regulated by ToxR in V. parahaemolyticus, respectively., Conclusion: The recombinant ToxR-N protein could be used for studying the transcriptional regulation mechanism in V. parahaemolyticus. ToxR fulfills a mechanism of negative regulation of T3SS1 genes by activating the expression of calR through protein-proximal promoter DNA association.

Objective: To study the regulation mechanism of biofilm formation c-di-GMP synthesisby AphA in Vibrio parahaemolyticus, by using phenotypic and molecular biochemical experiments., Methods: Colony morphology and crystal violet staining assays were used to analyze the phenotypic changes between the aphA null mutant (delta aphA) and the wide-type (WT) parent strain. The intracellular levels of c-di-GMP in the delta aphA and WT strains were determined by a chromatography-coupled tandem mass spectrometry (HPLC-MS/MS) method. Total RNAs were extracted from delta aphA and WT. Quantitative RT-PCR was applied to calculate the transcriptional variation of scrABC and scrG between delta aphA and WT. The promoter-proximal regions of scrABC and scrG were cloned into the pHRP309 vector containing a promoterless lacZ gene, respectively. Then, each of the two recombinant LacZ reporter plasmids was transformed into delta aphA and WT, respectively, to measure the promoter activity (the beta-Galactosidase activity) of the target genes in AaphA and WT by using the beta-Galactosidase Enzyme Assay System. The over-expressed His-AphA was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham). Then, the electrophoretic mobility shift assay (EMSA) was applied to analyze the DNA-binding activity of His-AphA to scrABC and scrG promoter regions in vitro. [Results] The phenotypic experiments disclosed that AphA was an activator of c-di-GMP synthesis and biofilm formation in Vibrio parahaemolyticus. The quantitative RT-PCR and LacZ fusion results showed that the transcription of scrABC and scrG was under negative control of AphA. However, the purified His-AphA could not bind to the upstream DNA regions of scrABC and scrG, as determined by EMSA., Conclusion: The fact that AphA represses the transcription of scrABC and scrG will at least partially account for the positive regulation of e-di-GMP synthesis and biofilm formation by AphA in Vibrio parahaemolyticus.

Objective: To establish a method for gene complementation in Vibrio parahaemolyticus using the plasmid pBAD33., Methods: The entire coding region of opaR or aphA was amplified by PCR and cloned into pBAD33. The recombinant plasmid was transformed into δopaR and δaphA (the opaR or aphA null mutant strain, respectively) separately to construct the complemented mutant strain C-δaphA and C-δopaR, respectively. RT-PCR was used to verify the transcription of opaR and aphA in the corresponding complemented mutant strains. Primer extension experiments were performed to determine the relative mRNA levels of mfpA (a gene previously characterized to be negatively regulated by AphA and positively by OpaR) in the wild-type strain, δopaR, δaphA, C-δaphA, and C-δopaR., Results: opaR and aphA were transcribed in the corresponding complemented mutant strains, and their mRNA levels were comparable to those detected in the wild-type strains., Conclusion: A method has been established for gene complementation in Vibrio parahaemolyticus using the plasmid pBAD33.

Vibrio parahaemolyticus AphA and OpaR are the two master regulators of quorum sensing (QS) that are abundantly produced and operate at low cell density (LCD) and high cell density (HCD), respectively, with an outcome of reciprocally gradient production of these two proteins with transition between LCD and HCD. The cpsQ-mfpABC gene cluster is transcribed as two operons cpsQ-mfpABC and mfpABC in V. parahaemolyticus. MfpABC is a putative membrane fusion transporter that contributes to biofilm development. CpsQ is a c-di-GMP-binding regulator that activates the expression of capsular polysaccharide genes and mfpABC and, thus, induces biofilm development. As shown in this study, OpaR and AphA bind to the promoter region of mfpABC to enhance and repress its transcription, respectively. In contrast, the positive and negative regulation of cpsQ-mfpABC by AphA and OpaR, respectively, achieves probably through acting of AphA or OpaR on additional unknown regulator(s) of cpsQ-mfpABC. The transcriptional levels of cpsQ-mfpABC and mfpABC enhance gradually with transition from LCD to HCD due to the above reciprocal regulatory action of OpaR and AphA. Data presented here present a novel paradigm of combined action of the two master QS regulators in controlling expression of the QS regulon members., (© 2013 Published by Elsevier B.V.)

Background: Vibrio parahaemolyticus AphA and OpaR are the two master quorum sensing (QS) regulators that are abundantly expressed at low cell density (LCD) and high cell density (HCD), respectively, with a feature of reciprocally gradient production of them with transition between LCD and HCD. The type VI secretion system 2 (T6SS2) gene cluster can be assigned into three putative operons, namely VPA1027-1024, VPA1043-1028, and VPA1044-1046. T6SS2 contributes to adhesion of V. parahaemolyticus to host cells., Methodology/principal Findings: OpaR box-like sequences were found within the upstream promoter regions of all the above three operons, while none of AphA box-like elements could be identified for them. The subsequent primer extension, LacZ fusion, electrophoretic mobility shift, and DNase I footprinting assays disclosed that OpaR bound to the promoter regions of these three operons to stimulate their transcription, while AphA negatively regulated their transcription most likely through acting on OpaR. This regulation led to a gradient increase of T6SS2 transcription with transition from LCD to HCD., Conclusions/significance: V. parahaemolyticus OpaR and AphA positively and negatively regulate T6SS2 expression, respectively, leading to a gradient elevation of T6SS2 expression with transition from LCD to HCD. T6SS2 genes are thus assigned as the QS regulon members in V. parahaemolyticus.

AphA is a small PadR-family DNA-binding regulator in vibrios. AphA has been shown to be involved in transcriptional auto-repression, intestinal colonization and lethality in mice, biofilm formation, and quorum sensing in Vibrio cholerae. The AphA protein of Vibrio parahaemolyticus has 85% identity to that of V. cholerae with the same number of amino acids. In this work, the aphA null mutant was constructed from a wild-type pandemic strain of V. parahaemolyticus for characterization of the phenotypic changes. AphA is required for biofilm formation in V. parahaemolyticus, and a decreased production of biofilm exopolysaccharide matrix in the aphA mutant relative to the wild-type parent strain accounts for its reduced biofilm formation. AphA is also necessary for the optimal swimming and swarming motility of V. parahaemolyticus. In addition, AphA is essential for lethality in mice and cytotoxic activity, but the aphA deletion did not have effect on enterotoxicity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: AphA is the master quorum-sensing (QS) regulator operating at low cell density in vibrios. Molecular regulation of target genes by AphA has been characterized in Vibrio harveyi and V. cholerae, but it is still poorly understood in V. parahaemolyticus., Methodology/principal Findings: The AphA proteins are extremely conserved in V. parahaemolyticus, Vibrio sp. Ex25, Vibrio sp. EJY3, V. harveyi, V. vulnificus, V. splendidus, V. anguillarum, V. cholerae, and V. furnissii. The above nine AphA orthologs appear to recognize conserved cis-acting DNA signals which can be represented by two consensus constructs, a 20 bp box sequence and a position frequency matrix. V. parahaemolyticus AphA represses the transcription of ahpA, qrr4, and opaR through direct AphA-target promoter DNA association, while it inhibits the qrr2-3 transcription in an indirect manner. Translation and transcription starts, core promoter elements for sigma factor recognition, Shine-Dalgarno sequences for ribosome recognition, and AphA-binding sites (containing corresponding AphA box-like sequences) were determined for the three direct AphA targets ahpA, qrr4, and opaR in V. parahaemolyticus., Conclusions/significance: AphA-mediated repression of ahpA, qrr2-4, and opaR was characterized in V. parahaemolyticus by using multiple biochemical and molecular experiments. The computational promoter analysis indicated the conserved mechanism of transcriptional regulation of QS regulator-encoding genes ahpA, qrr4, and opaR in vibrios.

Background: Vibrio parahaemolyticus is a leading cause of infectious diarrhea and enterogastritis via the fecal-oral route. V. harveyi is a pathogen of fishes and invertebrates, and has been used as a model for quorum sensing (QS) studies. LuxR is the master QS regulator (MQSR) of V. harveyi, and LuxR-dependent expression of its own gene, qrr2-4 and aphA have been established in V. harveyi. Molecular regulation of target genes by the V. parahaemolyticus MQSR OpaR is still poorly understood., Methodology/principal Findings: The bioinformatics analysis indicated that V. parahaemolyticus OpaR, V. harveyi LuxR, V. vulnificu SmcR, and V. alginolyticus ValR were extremely conserved, and that these four MQSRs appeared to recognize the same conserved cis-acting signals, which was represented by the consensus constructs manifesting as a position frequency matrix and as a 20 bp box, within their target promoters. The MQSR box-like sequences were found within the upstream DNA regions of opaR, qrr2-4 and aphA in V. parahaemolyticus, and the direct transcriptional regulation of these target genes by OpaR were further confirmed by multiple biochemical experiments including primer extension assay, gel mobility shift assay, and DNase I footprinting analysis. Translation and transcription starts, core promoter elements for sigma factor recognition, Shine-Dalgarno sequences for ribosome recognition, and OpaR-binding sites were determined for the five target genes of OpaR, which gave a structural map of the OpaR-dependent promoters. Further computational promoter analysis indicated the above regulatory circuits were shared by several other closely related Vibrios but with slight exceptions., Conclusions/significance: This study gave a comprehensive computational and characterization of the direct transcriptional regulation of five target genes, opaR, qrr2-4 and ahpA, by OpaR in V. parahaemolyticus. These characterized regulatory circuits were conserved in V. harveyi and V. parahaemolyticus.

The type VI secretion system (T6SS) is bacterial protein injection machinery with roles in virulence, symbiosis, interbacterial interaction, antipathogenesis, and environmental stress responses. There are two T6SS loci, T6SS1 and T6SS2, in the two chromosomes of Vibrio parahaemolyticus, respectively. This work disclosed that the master quorum sensing (QS) regulator OpaR repressed the transcription of hcp1 encoding the structural component Hcp1 of T6SS1 in V. parahaemolyticus, indicating that QS had a negative regulatory action on T6SS1. A single σ(54)-dependent promoter was transcribed for hcp1 in V. parahaemolyticus, and its activity was repressed by the OpaR regulator. Since the OpaR protein could not bind to the upstream region of hcp1, OpaR would repress the transcription of hcp1 in an indirect manner.

A total of 18 variably-presented gene clusters (LVPCs) and nine previously characterized variable-number tandem repeats (VNTRs), and all known virulence markers were screened for their frequency and/or copy number in 251 global strains of Vibrio parahaemolyticus using PCR and gel or capillary electrophoresis. A two-step genotyping approach combining the use of LVPCs and VNTRs was established accordingly. The frequency profiles of LVPCs and virulence markers were primarily used to group the strains into six distinct complexes with different potential pathogenicity natures. The strains from each of these complexes were further analyzed with VNTRs to give a much more detailed discrimination of the strains. A genetic fingerprint-like database of a large collection of strains established with this two-stage approach would be very useful for identification, genotyping, origin tracing, and risk estimation of V. parahaemolyticus., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A collection of 174 global isolates of Vibrio parahaemolyticus were analyzed by multilocus sequence typing (MLST) on the basis of ten conserved genes. The results showed a high level of nucleotide and allelic diversity with the evidence of purifying selection and of frequent recombination. Recombination played a much greater role than mutation in generating genetic heterogeneity. The 174 strains could be assigned into 89 different sequence types, which could be further separated into six clonal complexes (CCs; CC1 to CC6) plus 71 singletons. The three major CCs, namely CC1 to CC3, corresponded to the groups of pre-1996 clinical old-O3:K6 strains (trh(+), T3SS2β(+), tdh(-), T3SS2α(-), and GS-PCR(-)), post-1996 pandemic strains (trh(-), T3SS2β(-), tdh(+), T3SS2α(+), and GS-PCR(+)) and non-clinical isolates (trh(-), T3SS2β(-), tdh(-), T3SS2α(-), and GS-PCR(-)), respectively. The MLST data enable the construction of a phylogenetic structure from the allelic profiles rather the nucleotide sequences, so as to reduce the affect of frequent recombination. The six CCs arose on a background of mutation and recombination, and according to the previously reported data, this bacterium could be evolved fast due to lateral acquisition of foreign genes especially including those encoding virulence determinants. V. parahaemolyticus had a typical epidemic population structure that is driven by mutation, recombination and lateral gene transfer., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Objective: We developed and evaluated a whole-genome DNA microarray of Vibrio parahaemolyticus., Methods: Based on the genomic sequences of V. parahaemolyticus, we chose a total number of 4770 genes, amplified them by PCR with specific primers, purified the PCR products and printed them onto glass slides. We performed two sets of hybridizations by the method of two-fluorescence comparative hybridization to evaluate the microarray quality, followed by PCR method to validate parts of microarray results., Results: Microarray hybridization results were completely consistent with theory expectations and PCR verification results., Conclusion: We successfully developed a batch of good quality whole-genome DNA microarrays of V. parahaemolyticus, built up a method of microarray-based comparative genomic hybridization of V. parahaemolyticus and a set of microarray data analysis standard method.

Vibrio parahaemolyticus is the leading cause of seafood-associated bacterial gastroenteritis and is a moderately halophilic, salt-requiring bacterium. Global gene expression profiles of V. parahaemolyticus grown under 2% and 0.66% NaCl were compared to define the low-salt stimulon. The ectABC-lysC operon for synthesis of the compatible solute ectoine, as well as three compatible solute transport systems, namely ProU (glycine betaine), OpuD1 (glycine betaine) and Pot2 (spermidine), was up-regulated under 2% NaCl relative to 0.66% NaCl. The 2% NaCl condition favored the inducible expression of OmpW, OmpN and OmpA2, while repressed the expression of OmpA1, OmpU and VP1008. These results indicated that, to master the hyperosmotic stress of saline environments, V. parahaemolyticus might not only accumulate osmoprotectants through uptake or endogenous synthesis of compatible solutes, but also remodel its profiles of outer membrane protein to restore its cell membrane. The above differentially regulated genes will provide novel candidates for the further investigation of the molecular mechanisms of osmoadaptation in V. parahaemolyticus., (Copyright 2009 Elsevier B.V. All rights reserved.)

A whole-genome DNA microarray was constructed to dissect expression profiles of Vibrio parahaemolyticus in response to a sudden temperature downshift from 37 to 10 degrees C. The mRNA level of each gene at each of three time points (20, 40 and 60 min after temperature downshift) was compared with that just before cold treatment. Clustering analysis of time-course data revealed nine gene clusters with different time-dependent expression patterns. Downregulation of metabolism-related genes was obviously dominant over upregulation at all time points. The distinct negative regulation of metabolism-related genes would account for a generally reduced cellular protein pool resulting from the sudden temperature downshift. In contrast, cold shock had a 'neutral and balanced' regulatory action on nonmetabolic cellular pathways, which likely brought about the remodelling of cell envelope structures and transport/binding functions. We identified a 171-bp 5'-untranslated region in the cspA transcript. The cspA gene encoded cold shock protein A (CspA), and CspA was shown to be the major cold shock protein in V. parahaemolyticus. Evident regulatory motifs were conserved within the cspA promoter regions of Escherichia coli and V. parahaemolyticus. These two bacteria likely use the same mechanism to regulate the cold-inducible expression of cspA.

Background: Outbreak of V. parahaemolyticus infections occurred since 1996 was linked to a proposed clonal complex, the pandemic group. The whole genome sequence provides an unprecedented opportunity for dissecting genome plasticity and phylogeny of the populations of V. parahaemolyticus. In the present work, a whole-genome cDNA microarray was constructed to compare the genomic contents of a collection of 174 strains of V. parahaemolyticus., Results: Genes that present variably in the genome accounted for about 22% of the whole gene pool on the genome. The phylogenetic analysis of microarray data generated a minimum spanning tree that depicted the phylogenetic structure of the 174 strains. Strains were assigned into five complexes (C1 to C5), and those in each complex were related genetically and phylogenetically. C3 and C4 represented highly virulent clinical clones. C2 and C3 constituted two different clonal complexes 'old-O3:K6 clone' and 'pandemic clone', respectively. C3 included all the 39 pandemic strains tested (trh-, tdh+ and GS-PCR+), while C2 contained 12 pre-1996 'old' O3:K6 strains (trh+, tdh- and GS-PCR-) tested herein. The pandemic clone (post-1996 'new' O3:K6 and its derivates O4:K68, O1:K25, O1:KUT and O6:K18) might be emerged from the old-O3:K6 clone, which was promoted by acquisition of toxRS/new sequence and genomic islands. A phylogenetic intermediate O3:K6 clade (trh-, tdh- and GS-PCR+) was identified between the pandemic and old-O3:K6 clones., Conclusion: A comprehensive overview of genomic contents in a large collection of global isolates from the microarray-based comparative genomic hybridization data enabled us to construct a phylogenetic structure of V. parahaemolyticus and an evolutionary history of the pandemic group (clone) of this pathogen.