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1. Zhu  BC, Lo  JY, Li  YT, Li  SC, Jaynes  JM, Gildemeister  OS, Laine  RA, Ou  CY,     ( 1992 )

Thermostable, salt tolerant, wide pH range novel chitobiase from Vibrio parahemolyticus: isolation, characterization, molecular cloning, and expression.

Journal of biochemistry 112 (1)
PMID : 1429506  :   DOI  :   10.1093/oxfordjournals.jbchem.a123857    
Abstract >>
A chitobiase gene from Vibrio parahemolyticus was cloned into plasmid pUC18 in Escherichia coli strain DH5 alpha. The plasmid construct, pC120, contained a 6.4 kb Vibrio DNA insert. The recombinant gene expressed chitobiase [EC 3.2.1.30] activity similar to that found in the native Vibrio. The enzyme was purified by ion exchange, hydroxylapatite and gel permeation chromatographies, and exhibited an apparent molecular weight of 80 kDa on SDS-polyacrylamide gel electrophoresis. Chitobiose and 6 more substrates, including beta-N-acetyl galactosamine glycosides, were hydrolyzed by the recombinant chitobiase, indicating its putative classification as an hexosaminidase [EC 3.2.1.52]. The enzyme was resistant to denaturation by 2 M NaCl, thermostable at 45 degrees C and active over a very unusual (for glycosyl hydrolases) pH range, from 4 to 10. The purified cloned chitobiase gave 4 closely focussed bands on an isoelectric focusing gel, at pH 4 to 6.5. The N-terminal 43 amino acid sequence shows no homology with other proteins in commercial databanks or in the literature, and from its N-terminal sequence, appears to be a novel protein, unrelated in sequence to chitobiases from other Vibrios reported and unrelated to hexosaminidases from other organisms.
KeywordMeSH Terms
2. Fukunaga  N, Imagawa  S, Sahara  T, Ishii  A, Suzuki  M,     ( 1992 )

Purification and characterization of monomeric isocitrate dehydrogenase with NADP(+)-specificity from Vibrio parahaemolyticus Y-4.

Journal of biochemistry 112 (6)
PMID : 1295895  :   DOI  :   10.1093/oxfordjournals.jbchem.a123988    
Abstract >>
NADP(+)-dependent isocitrate dehydrogenase [IDH: EC 1.1.1.42] was purified to electrophoretic homogeneity from Vibrio parahaemolyticus Y-4, and shown to be a monomeric protein of molecular weight 80,000 with a pI of 5.0. The amino acid composition and partial sequence at the N-terminus resembled those reported for other bacterial monomeric IDHs. Immunotitration with antisera to the monomeric and dimeric enzymes (antisera to IDH-II and -I of Vibrio ABE-1) showed an immunochemical distinction between the monomeric and dimeric IDHs, but there is similarity within the IDHs of each group. The circular dichroism spectra of the native and heat-denatured enzyme are also similar to those of monomeric IDH (IDH-II of Vibrio ABE-1). These monomeric IDHs are proteins comprising 17-22% helix and 25-35% beta-pleated sheet in the native state.
KeywordMeSH Terms
3. Güvener  ZT, McCarter  LL,     ( 2003 )

Multiple regulators control capsular polysaccharide production in Vibrio parahaemolyticus.

Journal of bacteriology 185 (18)
PMID : 12949095  :   DOI  :   10.1128/jb.185.18.5431-5441.2003     PMC  :   PMC193756    
Abstract >>
Vibrio parahaemolyticus, a biofouling marine bacterium and human pathogen, undergoes phase variation displaying translucent (TR) and opaque (OP) colony morphologies. Prior studies demonstrated that OP colonies produce more capsular polysaccharide (CPS) than TR colonies and that opacity is controlled by the Vibrio harveyi LuxR-type transcriptional activator OpaR. CPS has also been shown to be regulated by the scrABC signaling pathway, which involves a GGDEF-EAL motif-containing sensory protein. The present study identifies cps genes and examines their regulation. Transposon insertions in the cps locus, which contains 11 genes, abolished opacity. Such mutants failed to produce CPS and were defective in pellicle formation in microtiter wells and in a biofilm attachment assay. Reporter fusions to cpsA, the first gene in the locus, showed approximately 10-fold-enhanced transcription in the OP (opaR+) strain compared to a TR (deltaopaR) strain. Two additional transcriptional regulators were discovered. One potential activator, CpsR, participates in the scrABC GGDEF-EAL-signaling pathway; CpsR was required for the increased cps expression observed in scrA deltaopaR strains. CpsR, which contains a conserved module found in members of the AAA+ superfamily of ATP-interacting proteins, is homologous to Vibrio cholerae VpsR; however, unlike VpsR, CpsR was not essential for cps expression. CpsS, the second newly identified regulator, contains a CsgD-type DNA-binding domain and appears to act as a repressor. Mutants with cpsS defects have greatly elevated cps transcription; their high level of cpsA expression was CpsR dependent in TR strains and primarily OpaR dependent in OP strains. Thus, a network of positive and negative regulators modulates CPS production in V. parahaemolyticus.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Transcription Factors
4. Stewart  BJ, McCarter  LL,     ( 2003 )

Lateral flagellar gene system of Vibrio parahaemolyticus.

Journal of bacteriology 185 (15)
PMID : 12867460  :   DOI  :   10.1128/jb.185.15.4508-4518.2003     PMC  :   PMC165745    
Abstract >>
Vibrio parahaemolyticus possesses dual flagellar systems adapted for movement under different circumstances. A single polar flagellum propels the bacterium in liquid (i.e., swimming) with a motor that is powered by the sodium motive force. Multiple proton-driven lateral flagella enable translocation over surfaces (i.e., swarming). The polar flagellum is produced continuously, while production of lateral flagella is induced when the organism is grown on surfaces. This work describes the isolation of mutants with insertions in the structural and regulatory laf genes. A Tn5-based lux transcriptional reporter transposon was constructed and used for mutagenesis and subsequent transcriptional analysis of the laf regulon. Twenty-nine independent insertions were distributed within 16 laf genes. DNA sequence analysis identified 38 laf genes in two loci. Among the mutants isolated, 11 contained surface-induced lux fusions. A hierarchy of laf gene expression was established following characterization of the laf::lux transcriptional fusion strains and by mutational and primer extension analyses of the laf regulon. The laf system is like many enteric systems in that it is a proton-driven, peritrichous flagellar system; however, laf regulation was different from the Salmonella-Escherichia coli paradigm. There is no apparent flhDC counterpart that encodes master regulators known to control flagellar biosynthesis and swarming in many enteric bacteria. A potential sigma(54)-dependent regulator, LafK, was demonstrated to control expression of early genes, and a lateral-specific sigma(28) factor controls late flagellar gene expression. Another notable feature was the discovery of a gene encoding a MotY-like product, which previously had been associated only with the architecture of sodium-type polar flagellar motors.
KeywordMeSH Terms
DNA-Binding Proteins
Gene Expression Regulation, Bacterial
5. Makino  K, Oshima  K, Kurokawa  K, Yokoyama  K, Uda  T, Tagomori  K, Iijima  Y, Najima  M, Nakano  M, Yamashita  A, Kubota  Y, Kimura  S, Yasunaga  T, Honda  T, Shinagawa  H, Hattori  M, Iida  T,     ( 2003 )

Genome sequence of Vibrio parahaemolyticus: a pathogenic mechanism distinct from that of V cholerae.

Lancet (London, England) 361 (9359)
PMID : 12620739  :   DOI  :   10.1016/S0140-6736(03)12659-1    
Abstract >>
Vibrio parahaemolyticus, a gram-negative marine bacterium, is a worldwide cause of food-borne gastroenteritis. V parahaemolyticus strains of a few specific serotypes, probably derived from a common clonal ancestor, have lately caused a pandemic of gastroenteritis. The organism is phylogenetically close to V cholerae, the causative agent of cholera. The whole genome sequence of a clinical V parahaemolyticus strain RIMD2210633 was established by shotgun sequencing. The coding sequences were identified by use of Gambler and Glimmer programs. Comparative analysis with the V cholerae genome was undertaken with MUMmer. The genome consisted of two circular chromosomes of 3288558 bp and 1877212 bp; it contained 4832 genes. Comparison of the V parahaemolyticus genome with that of V cholerae showed many rearrangements within and between the two chromosomes. Genes for the type III secretion system (TTSS) were identified in the genome of V parahaemolyticus; V cholerae does not have these genes. The TTSS is a central virulence factor of diarrhoea-causing bacteria such as shigella, salmonella, and enteropathogenic Escherichia coli, which cause gastroenteritis by invading or intimately interacting with intestinal epithelial cells. Our results suggest that V parahaemolyticus and V cholerae use distinct mechanisms to establish infection. This finding explains clinical features of V parahaemolyticus infections, which commonly include inflammatory diarrhoea and in some cases systemic manifestations such as septicaemia, distinct from those of V cholerae infections, which are generally associated with non-inflammatory diarrhoea.
KeywordMeSH Terms
6. Kwok  AY, Wilson  JT, Coulthart  M, Ng  LK, Mutharia  L, Chow  AW,     ( 2002 )

Phylogenetic study and identification of human pathogenic Vibrio species based on partial hsp60 gene sequences.

Canadian journal of microbiology 48 (10)
PMID : 12489780  :   DOI  :   10.1139/w02-089    
Abstract >>
The use of hsp60 gene sequences for phylogenetic study and identification of pathogenic marine vibrios was investigated. A 600-bp partial hsp60 gene was amplified by PCR and sequenced from 29 strains representing 15 Vibrio species within the family Vibrionaceae. Sequence comparison of the amplified partial hsp60 gene revealed 71-82% sequence identity among different Vibrio species and 96-100% sequence identity among epidemiologically distinct strains with the same species designation. This degree of discrimination allows unambiguous differentiation of all Vibrio species included in the current study from each other, as well as from Aeromonas hydrophila and Plesiomonas shigelloides, which are often misidentified as Vibrio species by conventional biochemical methods. Based on the hsp60 gene sequences, two previously unidentified shrimp isolates were found to be more closely related to Vibrio alginolyticus (93-94% sequence identity) than to Vibrio parahaemolyticus (89% sequence identity), whereas 16S rRNA gene analysis was unable to differentiate among these closely related species (95-97% sequence identity). Our results indicate that the hsp60 gene may be a useful alternative target for phylogenetic analysis and species identification of marine Vibrios to complement more conventional identification systems.
KeywordMeSH Terms
7. Boles  BR, McCarter  LL,     ( 2002 )

Vibrio parahaemolyticus scrABC, a novel operon affecting swarming and capsular polysaccharide regulation.

Journal of bacteriology 184 (21)
PMID : 12374828  :   DOI  :   10.1128/jb.184.21.5946-5954.2002     PMC  :   PMC135390    
Abstract >>
Swarming is an adaptation of many bacteria to growth on surfaces. A search for genes controlling swarmer cell differentiation of Vibrio parahaemolyticus identified a novel three-gene operon that potentially encodes a pyridoxal-phosphate-dependent enzyme, an extracellular solute-binding protein, and a membrane-bound GGDEF- and EAL-motif sensory protein. The functions of these motifs, which are named after conserved amino acid sequences, are unknown, although the domains are found singly and in combination in a variety of bacterial signaling proteins. Studies with translational fusions supported the predicted localization of the gene products. When the operon was overexpressed, swarmer cell gene transcription was induced in liquid culture. Mutants with defects in any of the three genes exhibited decreased swarming and lateral flagellar (laf) gene expression. Complementation studies confirmed an operon organization and suggested that all three genes participated in laf regulation. The lesions that decreased swarming increased capsular polysaccharide (CPS) production, and overexpression of the operon inhibited transcription of the CPS gene cpsA. Thus, the scrABC locus appears to inversely regulate two gene systems that are pertinent to colonization of surface swarming and CPS.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
8. Chen  J, Morita  Y, Huda  MN, Kuroda  T, Mizushima  T, Tsuchiya  T,     ( 2002 )

VmrA, a member of a novel class of Na(+)-coupled multidrug efflux pumps from Vibrio parahaemolyticus.

Journal of bacteriology 184 (2)
PMID : 11751837  :   DOI  :   10.1128/jb.184.2.572-576.2002     PMC  :   PMC139572    
Abstract >>
Gene vmrA, cloned from Vibrio parahaemolyticus, made Escherichia coli resistant to 4',6-diamino-2-phenylindol, tetraphenylphosphonium chloride, acriflavine, and ethidium bromide. VmrA belongs to the DinF branch of MATE family efflux transporters. VmrA catalyzed acriflavine efflux and showed Na(+)/drug transporter activity because the addition of tetraphenylphosphonium to Na(+)-loaded cells caused Na(+) efflux.
KeywordMeSH Terms
9. Yeung  PS, Hayes  MC, DePaola  A, Kaysner  CA, Kornstein  L, Boor  KJ,     ( 2002 )

Comparative phenotypic, molecular, and virulence characterization of Vibrio parahaemolyticus O3:K6 isolates.

Applied and environmental microbiology 68 (6)
PMID : 12039748  :   DOI  :   10.1128/aem.68.6.2901-2909.2002     PMC  :   PMC123939    
Abstract >>
Historically, Vibrio parahaemolyticus infections have been characterized by sporadic cases caused by multiple, diverse serotypes. However, since 1996, V. parahaemolyticus serotype O3:K6 strains have been associated with several large-scale outbreaks of illness, suggesting the emergence of a "new" group of organisms with enhanced virulence. We have applied three different molecular subtyping techniques to identify an appropriate method for differentiating O3:K6 isolates from other serotypes. Pulsed-field gel electrophoresis (PFGE) following NotI digestion differentiated seven closely related subtypes among O3:K6 and related strains, which were distinct from PFGE patterns for non-O3:K6 isolates. Ribotyping and tdh sequencing were less discriminatory than PFGE, but further confirmed close genetic relationships among recent O3:K6 isolates. In vitro adherence and cytotoxicity studies with human epithelial cells showed that O3:K6 isolates exhibited statistically higher levels of adherence and cytotoxicity to host cells than non-O3:K6 isolates. Epithelial cell cytotoxicity patterns were determined with a lactate dehydrogenase release assay. At 3 h postinfection, high relative cytotoxicities (>50% maximum lactate dehydrogenase activity) were found among a greater proportion of recently isolated O3:K6 and closely related strains (75%) than among the non-O3:K6 isolates (23%). A statistically significant relationship between adherence and cytotoxicity suggests that the pathogenic potential of some isolates may be associated with increased adherence to epithelial cells. Our findings suggest that enhanced adherence and cytotoxicity may contribute to the apparent unique pathogenic potential of V. parahaemolyticus O3:K6 strains.
KeywordMeSH Terms
10. Hervio-Heath  D, Colwell  RR, Derrien  A, Robert-Pillot  A, Fournier  JM, Pommepuy  M,     ( 2002 )

Occurrence of pathogenic vibrios in coastal areas of France.

Journal of applied microbiology 92 (6)
PMID : 12010553  :  
Abstract >>
This study was carried out to investigate the occurrence of potentially pathogenic species of Vibrio in French marine and estuarine environments. Samples of coastal waters and mussels collected between July and September 1999 were analysed by culture, using selective media including thiosulphate-citrate-bile salts-sucrose and modified cellobiose-polymixin B-colistin agar. Presumptive Vibrio colonies were isolated and identified using selected biochemical tests. Specific primers based on flanking sequences of the cytolysin, vvhA gene, pR72H DNA fragment and 16S-23S rRNA intergenic spacer region (ISR) were used in a polymerase chain reaction (PCR) to confirm the identification of Vibrio vulnificus, V. parahaemolyticus and V. cholerae, respectively. In this study, V. alginolyticus (99 of 189) was the predominant species, followed by V. parahaemolyticus (41 of 189), V. vulnificus (20 of 189) and non-O1/non-O139 V. cholerae (three of 189). All 20 V. vulnificus isolates showed PCR amplification of the vvhA gene, 16 of which had been isolated from estuarine water. The PCR amplification of the pR72H DNA fragment in 41 V. parahaemolyticus isolates generated two unique amplicons of 387 and 320 bp. The latter, present in 24.4% of these isolates, had not previously been found in V. parahaemolyticus strains examined to date. Amplification of the trh gene in two of the isolates suggested these to be virulent strains. Three strains identified as V. cholerae by amplification of the 16S-23S rRNA ISR were confirmed to be non-cholera (non-O1/non-O139) strains. The results of this study demonstrated the presence of pathogenic Vibrio species in French coastal waters. Furthermore, the PCR approach proved useful for the rapid and reliable confirmation of species identification. These findings indicate the potential sanitary risk associated with the presence of pathogenic Vibrio spp. in cultivated mussels and in the aquatic environment. The PCR can be used to detect pathogenic vibrios directly in environmental samples.
KeywordMeSH Terms
11. Kim  SK, Yang  JY, Cha  J,     ( 2002 )

Cloning and sequence analysis of a novel metalloprotease gene from Vibrio parahaemolyticus 04.

Gene 283 (1��2��)
PMID : 11867235  :   DOI  :   10.1016/s0378-1119(01)00882-4    
Abstract >>
The metalloprotease gene (vppC) from Vibrio parahaemolyticus 04 has been cloned and sequenced. The vppC gene contains an open reading frame of 2442 nucleotides encoding a polypeptide of 814 amino acids with a calculated molecular mass of 89,833 Da. The predicted amino acid sequence of VppC containing a zinc metalloprotease HEXXH consensus motif displays extensive homology to the collagenase from Vibrio alginolyticus. The activity of the recombinant protease produced in Escherichia coli was examined by gelatin zymography and proteolytic activity assays. The substrate specificity study showed that the type I collagen and synthetic collagenase substrate carbobenzoxy-glycyl-L-prolyl-glycyl-glycyl-L-prolyl-L-alanine were the best substrates, indicating that the cloned metalloprotease is indeed a collagenase. Multiple alignment analysis of the amino acid sequences and the enzymatic properties such as molecular mass and substrate specificity revealed three distinct classes of Vibrio metalloproteases. The identification of a new metalloprotease gene expands the role of Vibrio metalloproteases as a virulence factor for host infection.
KeywordMeSH Terms
12. Maeda  T, Furushita  M, Hamamura  K, Shiba  T,     ( 2001 )

Structures of ribonuclease P RNAs of Vibrio core species.

FEMS microbiology letters 198 (2)
PMID : 11430405  :   DOI  :   10.1111/j.1574-6968.2001.tb10633.x    
Abstract >>
The structures of an RNA component of ribonuclease P (RNase P RNA) were examined for Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio carchariae, Vibrio natriegens, Vibrio campbellii, Vibrio proteolyticus, Vibrio pelagius and Vibrio harveyi to clearly determine their genetic differences. The RNase P RNAs ranged from 382 to 454 nucleotides (nt) in size, and were remarkably different from each other in the structure of two helices, P3 and P12. The P3 helices were comprised of tandem repeats of a palindromic sequence (24 nt), resulting in the longitudinal repetition of a stem structure. The number of repetitions ranged from four in V. harveyi, to one in both V. alginolyticus and V. proteolyticus. The genes for the RNase P RNAs of all species were located between two open reading frames, the amino acid sequences of which were similar to the hypothetical proteins located at 70.92 and 1.94 min in the Escherichia coli chromosome.
KeywordMeSH Terms
Escherichia coli Proteins
Phylogeny
13. Rowe-Magnus  DA, Guerout  AM, Ploncard  P, Dychinco  B, Davies  J, Mazel  D,     ( 2001 )

The evolutionary history of chromosomal super-integrons provides an ancestry for multiresistant integrons.

Proceedings of the National Academy of Sciences of the United States of America 98 (2)
PMID : 11209061  :   DOI  :   10.1073/pnas.98.2.652     PMC  :   PMC14643    
Abstract >>
Integrons are genetic elements that acquire and exchange exogenous DNA, known as gene cassettes, by a site-specific recombination mechanism. Characterized gene cassettes consist of a target recombination sequence (attC site) usually associated with a single open reading frame coding for an antibiotic resistance determinant. The affiliation of multiresistant integrons (MRIs), which contain various combinations of antibiotic resistance gene cassettes, with transferable elements underlies the rapid evolution of multidrug resistance among diverse Gram-negative bacteria. Yet the origin of MRIs remains unknown. Recently, a chromosomal super-integron (SI) harboring hundreds of cassettes was identified in the Vibrio cholerae genome. Here, we demonstrate that the activity of its associated integrase is identical to that of the MRI integrase, IntI1. We have also identified equivalent integron superstructures in nine distinct genera throughout the gamma-proteobacterial radiation. Phylogenetic analysis revealed that the evolutionary history of the system paralleled that of the radiation, indicating that integrons are ancient structures. The attC sites of the 63 antibiotic-resistance gene cassettes identified thus far in MRIs are highly variable. Strikingly, one-fifth of these were virtually identical to the highly related yet species-specific attC sites of the SIs described here. Furthermore, antimicrobial resistance homologues were identified among the thousands of genes entrapped by these SIs. Because the gene cassettes of SIs are substrates for MRIs, these data identify SIs as the source of contemporary MRIs and their cassettes. However, our demonstration of the metabolic functions, beyond antibiotic resistance and virulence, of three distinct SI gene cassettes indicates that integrons function as a general gene-capture system for bacterial innovation.
KeywordMeSH Terms
Evolution, Molecular
Genome, Bacterial
14. Stine  OC, Sozhamannan  S, Gou  Q, Zheng  S, Morris  JG, Johnson  JA,     ( 2000 )

Phylogeny of Vibrio cholerae based on recA sequence.

Infection and immunity 68 (12)
PMID : 11083852  :   DOI  :   10.1128/iai.68.12.7180-7185.2000     PMC  :   PMC97837    
Abstract >>
We sequenced a 705-bp fragment of the recA gene from 113 Vibrio cholerae strains and closely related species. One hundred eighty-seven nucleotides were phylogenetically informative, 55 were phylogenetically uninformative, and 463 were invariant. Not unexpectedly, Vibrio parahaemolyticus and Vibrio vulnificus strains formed out-groups; we also identified isolates which resembled V. cholerae biochemically but which did not cluster with V. cholerae. In many instances, V. cholerae serogroup designations did not correlate with phylogeny, as reflected by recA sequence divergence. This observation is consistent with the idea that there is horizontal transfer of O-antigen biosynthesis genes among V. cholerae strains.
KeywordMeSH Terms
15. Yamaichi  Y, Park  KS, Yokoyama  K, Sugahara  T, Iida  T, Nasu  H,     ( 2000 )

A filamentous phage associated with recent pandemic Vibrio parahaemolyticus O3:K6 strains.

Journal of clinical microbiology 38 (6)
PMID : 10834969  :   PMC  :   PMC86752    
Abstract >>
A specific serotype, O3:K6, of Vibrio parahaemolyticus has recently been causing epidemics of gastroenteritis in Southeast Asia, Japan, and North America. To examine whether the new O3:K6 strains possess characteristics that may exacerbate outbreaks, we compared V. parahaemolyticus O3:K6 strains with non-O3:K6 strains using strains isolated from individuals with traveler's diarrhea at Kansai Airport Quarantine Station, Osaka, Japan. All 24 O3:K6 strains possessed a common plasmid, pO3K6 (DNA size, 8,782 bp, with 10 open reading frames [ORFs]). The gene organization of pO3K6 was similar to that of Vf33, a filamentous phage previously described in V. parahaemolyticus. We isolated a phage (phage f237) from the culture supernatant of V. parahaemolyticus O3:K6 strain KXV237, which formed a turbid plaque on an indicator strain. The genome of f237 was single-stranded DNA, and the double-stranded DNA obtained by treatment of the genome with DNA polymerase was identical to that of pO3K6 when analyzed by agarose gel electrophoresis after HindIII digestion. Furthermore, the N-terminal amino acid sequence of the f237 major coat protein was found in ORF4 of pO3K6. Our results showed that pO3K6 is a replicative form of f237. Among the ORFs found in the f237 genome, the sequence of ORF8 had no significant homology to those of any proteins in databases. ORF8 was located on a region corresponding to the distinctive region of Vf33, and its G+C content was apparently lower than that of the remaining DNA sequence of f237. By colony hybridization, ORF8 was detected only in O3:K6 strains isolated since 1996 and was not found in O3:K6 strains isolated before 1996 and clinical V. parahaemolyticus strains other than those of serotype O3:K6. Thus, this study shows that f237 is exclusively associated with recent V. parahaemolyticus O3:K6 strains. The ORF8 gene can be a useful genetic marker for the identification of the recently widespread O3:K6 strains of V. parahaemolyticus.
KeywordMeSH Terms
Diarrhea
Disease Outbreaks
Travel
16. Eskandari  S, Lam  JT, Whitelegge  JP, le Coutre  J, Kim  O, Turk  E,     ( 2000 )

Molecular characterization of Vibrio parahaemolyticus vSGLT: a model for sodium-coupled sugar cotransporters.

The Journal of biological chemistry 275 (33)
PMID : 10835424  :   DOI  :   10.1074/jbc.M003127200    
Abstract >>
The Na(+)/galactose cotransporter (vSGLT) of Vibrio parahaemolyticus, tagged with C-terminal hexahistidine, has been purified to apparent homogeneity by Ni(2+) affinity chromatography and gel filtration. Resequencing the vSGLT gene identified an important correction: the N terminus constitutes an additional 13 functionally essential residues. The mass of His-tagged vSGLT expressed under its native promoter, as determined by electrospray ionization-mass spectrometry (ESI-MS), verifies these 13 residues in wild-type vSGLT. A fusion protein of vSGLT and green fluorescent protein, comprising a mass of over 90 kDa, was also successfully analyzed by ESI-MS. Reconstitution of purified vSGLT yields proteoliposomes active in Na(+)-dependent galactose uptake, with sugar preferences (galactose > glucose > fucose) reflecting those of wild-type vSGLT in vivo. Substrates are transported with apparent 1:1 stoichiometry and apparent K(m) values of 129 mm (Na(+)) and 158 microm (galactose). Freeze-fracture electron microscopy of functional proteoliposomes shows intramembrane particles of a size consistent with vSGLT existing as a monomer. We conclude that vSGLT is a suitable model for the study of sugar cotransporter mechanisms and structure, with potential applicability to the larger SGLT family of important sodium:solute cotransporters. It is further demonstrated that ESI-MS is a powerful tool for the study of proteomics of membrane transporters.
KeywordMeSH Terms
17. Park  KS, Iida  T, Yamaichi  Y, Oyagi  T, Yamamoto  K, Honda  T,     ( 2000 )

Genetic characterization of DNA region containing the trh and ure genes of Vibrio parahaemolyticus.

Infection and immunity 68 (10)
PMID : 10992480  :   DOI  :   10.1128/iai.68.10.5742-5748.2000     PMC  :   PMC101532    
Abstract >>
We have demonstrated that possession of the gene for thermostable direct hemolysin-related hemolysin (trh) coincides with the presence of the urease gene among clinical Vibrio parahaemolyticus strains and that the location of the two genes are in close proximity on the chromosome. Here, we cloned and sequenced the 15,754-bp DNA region containing the trh gene and the gene cluster for urease production from the chromosome of clinical V. parahaemolyticus (TH3996). We found 16 open reading frames (ORFs) and a lower G+C content (41%) compared with the total genome of this bacterium (46 to 47%). The ure cluster consisted of eight genes, namely, ureDABCEFG and ureR. ureR was located 5.2 kb upstream of the other seven genes in the opposite direction. The genetic organization and sequences of the ure genes resembled those found in Proteus mirabilis. Between ureR and the other ure genes, there were five ORFs, which are homologous with the nickel transport operon (nik) of Escherichia coli. We disrupted each of the ureR, ureC, and nikD genes in TH3996 by homologous recombination and analyzed the phenotype of the mutants. In the presence of urea these mutant strains had dramatically less urease activity than the strain they were derived from. Disruption of ureR, nikD, or ureC, however, had no effect on TRH production. The DNA region containing the trh, nik, and ure genes was found in only trh-positive strains and not in Kanagawa phenomenon-positive and environmental V. parahaemolyticus strains. At the end of the region, an insertion sequence-like element existed. These results suggest that the DNA region was introduced into V. parahaemolyticus in the past through a mechanism mediated by insertion sequences. This is the first reported case that the genes for an ATP-binding cassette-type nickel transport system, which may play a role in nickel transport through bacterial cytoplasmic membrane, are located adjacent to the ure cluster on the genome of an organism.
KeywordMeSH Terms
Bacterial Proteins
Genes, Bacterial
18. Whitelegge  JP, Gross  A, Turk  E, le Coutre  J,     ( 2000 )

Proteomics on full-length membrane proteins using mass spectrometry.

Biochemistry 39 (15)
PMID : 10757971  :   DOI  :   10.1021/bi000150m    
Abstract >>
A general technique has been developed that allows rapid mass spectrometric analysis of full-length membrane proteins [Whitelegge, J. P., le Coutre, J., et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 10695-10698]. Using in-line HPLC electrospray ionization mass spectrometry (LC-MS), different native and recombinant bacterial membrane proteins of up to 61 kDa are characterized. Mass spectrometric data of four entirely different membrane proteins from three bacterial organisms, two transporters, a channel, and a porin protein are presented. In addition to determination of the molecular mass with an accuracy of +/-0.01%, the technique monitors alkylation or oxidation of single Cys residues and errors in deduced amino acid sequences. Finally, using in-line LC-MS, unknown proteins can be identified from solubilized Escherichia coli membranes without prior purification.
KeywordMeSH Terms
Escherichia coli Proteins
Proteome
Symporters
19. Boles  BR,     ( 2000 )

Insertional inactivation of genes encoding components of the sodium-type flagellar motor and switch of Vibrio parahaemolyticus.

Journal of bacteriology 182 (4)
PMID : 10648530  :   DOI  :   10.1128/jb.182.4.1035-1045.2000     PMC  :   PMC94380    
Abstract >>
Vibrio parahaemolyticus possesses two types of flagella, polar and lateral, powered by distinct energy sources, which are derived from the sodium and proton motive forces, respectively. Although proton-powered flagella in Escherichia coli and Salmonella enterica serovar Typhimurium have been extensively studied, the mechanism of torque generation is still not understood. Molecular knowledge of the structure of the sodium-driven motor is only now being developed. In this work, we identify the switch components, FliG, FliM, and FliN, of the sodium-type motor. This brings the total number of genes identified as pertinent to polar motor function to seven. Both FliM and FliN possess charged domains not found in proton-type homologs; however, they can interact with the proton-type motor of E. coli to a limited extent. Residues known to be critical for torque generation in the proton-type motor are conserved in the sodium-type motor, suggesting a common mechanism for energy transfer at the rotor-stator interface regardless of the driving force powering rotation. Mutants representing a complete panel of insertionally inactivated switch and motor genes were constructed. All of these mutants were defective in sodium-driven swimming motility. Alkaline phosphatase could be fused to the C termini of MotB and MotY without abolishing motility, whereas deletion of the unusual, highly charged C-terminal domain of FliM disrupted motor function. All of the mutants retained proton-driven, lateral motility over surfaces. Thus, although central chemotaxis genes are shared by the polar and lateral systems, genes encoding the switch components, as well as the motor genes, are distinct for each motility system.
KeywordMeSH Terms
20. Shyu  YC,     ( 1999 )

Cloning and characterization of manganese superoxide dismutase gene from Vibrio parahaemolyticus and application to preliminary identification of Vibrio strains.

IUBMB life 48 (3)
PMID : 10690650  :   DOI  :   10.1080/713803521    
Abstract >>
The sodA gene coding for manganese superoxide dismutase (Mn-SOD) from the marine microorganism Vibrio parahaemolyticus was cloned, sequenced, and overexpressed in Escherichia coli by use of the pET20b (+) expression vector. The full-length gene consisted of a 588-bp open reading frame and encoded a polypeptide of 196 amino acid residues, with a calculated molecular mass of 21,713 Da. The recombinant enzyme was efficiently purified from crude E. coli cell lysate by metal ion affinity chromatography. The recombinant VPMn-SOD resisted thermo-denaturation up to 60 degrees C and was insensitive to such inhibitors as EDTA, NaN3 and diethyldithiocarbamic acid. The specificity of V. parahaemolyticus Mn-SOD gene probe was analyzed by cross-species polymerase chain reaction to provide information for Vibrio strain identification.
KeywordMeSH Terms
Genes, Bacterial
21. Okuda  J, Nishino  T,     ( 1999 )

Sequence analysis of the gyrA and parC homologues of a wild-type strain of Vibrio parahaemolyticus and its fluoroquinolone-resistant mutants.

Antimicrobial agents and chemotherapy 43 (5)
PMID : 10223929  :   PMC  :   PMC89126    
Abstract >>
Vibrio parahaemolyticus causes seafood-borne gastroenteritis in humans. It is particularly important in Japan, where raw seafood is frequently consumed. Fluoroquinolone is one of the current drugs of choice for treating patients infected by V. parahaemolyticus because resistant strains are rarely found. To study a possible fluoroquinolone resistance mechanism in this organism, nucleotide sequences that are homologous to known gyrA and parC genes have been cloned from V. parahaemolyticus AQ3815 and sequenced by amplification with degenerate primers of the quinolone resistance-determining region (QRDR), followed by cassette ligation-mediated PCR. Open reading frames encoding polypeptides of 878 and 761 amino acid residues were detected in the gyrA and parC homologues, respectively. The V. parahaemolyticus GyrA and ParC sequences were most closely related to Erwinia carotovora GyrA (76% identity) and Escherichia coli ParC (69% identity) sequences, respectively. Ciprofloxacin-resistant mutants of AQ3815 were obtained on an agar medium by multistep selection with increasing levels of the quinolone. One point mutation only in the gyrA QRDR was detected among mutants with low- to intermediate-level resistance, while point mutations in both the gyrA and parC QRDRs were detected only in strains with high-level resistance. These results strongly suggest that, as in other gram-negative bacteria, GyrA and ParC are the primary and secondary targets, respectively, of ciprofloxacin in V. parahaemolyticus.
KeywordMeSH Terms
22. Laine  RA,     ( 1999 )

Sequence of the V. parahaemolyticus gene for cytoplasmic N, N'-diacetylchitobiase and homology with related enzymes.

Journal of biochemistry 125 (6)
PMID : 10348911  :   DOI  :   10.1093/oxfordjournals.jbchem.a022390    
Abstract >>
The nucleotide sequence of the gene encoding the cytoplasmic N, N'-diacetylchitobiase [EC 3.2.1.14] from Vibrio parahaemolyticus (ATCC #27969) has been determined. The deduced peptide sequence of this unusual beta-hexosaminidase surprisingly shows minimum evolutionary relationship to two other reported N, N'-diacetylchitobiases from vibrios, except in highly conserved regions which are also homologous to lysosomal beta-hexosaminidases from eukaryotes including humans. In contrast, the two other beta-hexosaminidases from vibrios with reported sequences are much more closely related to each other. This novel 85 kDa cytoplasmic glycosyl hydrolase with restricted specificity participates in the high level utilization of chitin-derived 2-deoxy-2-acetamido-D-glucose (GlcNAc) by vibrios as one of two parallel pathways for the metabolism of N,N'-diacetylchitobiose [Bassler, B.L., Yu, C., Lee, Y.C., and Roseman, S. (1991) J. Biol. Chem. 266, 24276-24286]. These pathways use chitin-binding proteins for the adherence of the bacterial chitinase to the substrate, and an extracellular chitinase and a periplasmic chitodextrinase to produce N,N'-diacetylchitobiose. The V. parahaemolyticus cytoplasmic N,N'-diacetyl-chitobiase reported herein appears to be a unique protein, lacking a signal sequence, and genetically distant from other known chitinoclastic beta-N,N'-diacetyl-hexosaminidases. This is consistent with its limited substrate specificity to small GlcNAc terminated oligosaccharides and its cytoplasmic rather than periplasmic localization.
KeywordMeSH Terms
Genes, Bacterial
23. Jaques  S, Kim  YK,     ( 1999 )

Mutations conferring resistance to phenamil and amiloride, inhibitors of sodium-driven motility of Vibrio parahaemolyticus.

Proceedings of the National Academy of Sciences of the United States of America 96 (10)
PMID : 10318954  :   DOI  :   10.1073/pnas.96.10.5740     PMC  :   PMC21930    
Abstract >>
The bacterial flagellum is powered by a rotary motor capable of turning the helical flagellar propeller at very high speeds. Energy to drive rotation is derived from the transmembrane electrochemical potential of specific ions. Ions passing through a channel component are thought to generate the force to power rotation. Two kinds of motors, dependent on different coupling ions, have been described: proton-driven and sodium-driven motors. There are four known genes encoding components of the sodium-powered polar flagellar motor in Vibrio parahaemolyticus. Two, which are characterized here, are homologous to genes encoding constituents of the proton-type motor (motA and motB), and two encode components unique to the sodium-type motor (motX and motY). The sodium-channel-blocking drugs phenamil and amiloride inhibit rotation of the polar flagellum and therefore can be used to probe the architecture of the motor. Mutants were isolated that could swim in the presence of phenamil or amiloride. The majority of the mutations conferring phenamil-resistant motility alter nucleotides in the motA or motB genes. The resultant amino acid changes localize to the cytoplasmic face of the torque generator and permit identification of potential sodium-interaction sites. Mutations that confer motility in the presence of amiloride do not alter any known component of the sodium-type flagellar motor. Thus, evidence supports the existence of more than one class of sodium-interaction site at which inhibitors can interfere with sodium-driven motility.
KeywordMeSH Terms
24. Henderson  DP, Payne  SM, Rashidi  CE, Fuson  KL, Rashidi  JR, Deanda  MT, Mouton  SL, Occhino  DA,     ( 1999 )

Comparison of the heme iron utilization systems of pathogenic Vibrios.

Journal of bacteriology 181 (11)
PMID : 10348876  :   PMC  :   PMC93831    
Abstract >>
Vibrio alginolyticus, Vibrio fluvialis, and Vibrio parahaemolyticus utilized heme and hemoglobin as iron sources and contained chromosomal DNA similar to several Vibrio cholerae heme iron utilization genes. A V. parahaemolyticus gene that performed the function of V. cholerae hutA was isolated. A portion of the tonB1 locus of V. parahaemolyticus was sequenced and found to encode proteins similar in amino acid sequence to V. cholerae HutW, TonB1, and ExbB1. A recombinant plasmid containing the V. cholerae tonB1 and exbB1D1 genes complemented a V. alginolyticus heme utilization mutant. These data suggest that the heme iron utilization systems of the pathogenic vibrios tested, particularly V. parahaemolyticus and V. alginolyticus, are similar at the DNA level, the functional level, and, in the case of V. parahaemolyticus, the amino acid sequence or protein level to that of V. cholerae.
KeywordMeSH Terms
25. Kadokura  K, Sakamoto  Y, Saito  K, Ikegami  T, Hirano  T, Hakamata  W, Oku  T, Nishio  T,     ( 2007 )

Production of a recombinant chitin oligosaccharide deacetylase from Vibrio parahaemolyticus in the culture medium of Escherichia coli cells.

Biotechnology letters 29 (8)
PMID : 17479220  :   DOI  :   10.1007/s10529-007-9386-6    
Abstract >>
An open reading frame (ORF) encoding chitin oligosaccharide deacetylase (Pa-COD) gene and its signal sequence was cloned from the Vibrio parahaemolyticus KN1699 genome and its sequence was analyzed. The ORF encoded a 427 amino acid protein, including the 22 amino acid signal sequence. The deduced amino acid sequence was highly similar to several bacterial chitin oligosaccharide deacetylases in carbohydrate esterase family 4. An expression plasmid containing the gene was constructed and inserted into Escherichia coli cells and the recombinant enzyme was secreted into the culture medium with the aid of the signal peptide. The concentration of the recombinant enzyme in the E. coli culture medium was 150 times larger than that of wild-type enzyme produced in the culture medium by V. parahaemolyticus KN1699. The recombinant enzyme was purified to homogeneity from culture supernatant in an overall yield of 16%. Substrate specificities of the wild-type and the recombinant enzymes were comparable.
KeywordMeSH Terms
26. Criminger  JD, Hazen  TH, Sobecky  PA, Lovell  CR,     ( 2007 )

Nitrogen fixation by Vibrio parahaemolyticus and its implications for a new ecological niche.

Applied and environmental microbiology 73 (18)
PMID : 17675440  :   DOI  :   10.1128/AEM.00981-07     PMC  :   PMC2074916    
Abstract >>
A Vibrio parahaemolyticus strain isolated from the rhizosphere of the ecosystem dominant estuarine grass, Spartina alterniflora, was characterized and shown to carry nifH, the gene encoding the nitrogenase iron protein, and to fix N(2). Nitrogen fixation may contribute substantially to the adaptability, niche breadth, and ecological significance of V. parahaemolyticus.
KeywordMeSH Terms
Ecosystem
27. Mao  Z, Yu  L, You  Z, Wei  Y, Liu  Y,     ( 2007 )

Cloning, expression and immunogenicty analysis of five outer membrane proteins of Vibrio parahaemolyticus zj2003.

Fish & shellfish immunology 23 (3)
PMID : 17451968  :   DOI  :   10.1016/j.fsi.2007.01.004    
Abstract >>
Genes of five outer membrane proteins of Vibrio parahaemolyticus zj2003, including OmpW, OmpV, OmpK, OmpU and TolC, were cloned and expressed as N-terminal His(6)-tagged proteins in Escherichia coli. The recombinant fusion proteins were purified with nickel chelate affinity chromatography. To analyze the immunogenicity of these proteins, large yellow croaker (Pseudosciaena crocea) were immunized by intraperitoneal injection. Antibody response was assessed by method of enzyme-linked immunosorbent assay. Titres to all five recombinant proteins increased during 4 to 8 weeks post immunization, within the range of log 2 values of 5.75 to 10.8. Recorded relative survival percent (RPS) of the vaccinated groups varied from 80% to 90%, while 10 fish in control group all died. Western blot tests were undertaken with the serum of survival fish after experimental infection. Except for recombinant TolC, the other four recombinant proteins were recognized by the serum. It is indicated that four outer membrane proteins of V. parahaemolyticus zj2003, including OmpW, OmpV, OmpU and OmpK, are immunogenic during in vivo infection, which would be of some significance in developing efficient vaccine in aquaculture. This is the first report of successful vaccination against V. parahaemolyticus with purified recombinant outer membrane proteins.
KeywordMeSH Terms
28. Yamanaka  H, Tadokoro  S, Miyano  M, Takahashi  E, Kobayashi  H, Okamoto  K,     ( N/A )

Studies on the region involved in the transport activity of Escherichia coli TolC by chimeric protein analysis.

Microbial pathogenesis 42 (5��6��)
PMID : 17350794  :   DOI  :   10.1016/j.micpath.2007.01.006    
Abstract >>
Gram-negative bacteria possess the outer membrane protein TolC which acts as an exit duct across the outer membrane. However, the region involved in the transport activity of TolC has remained unclear. We analyzed this region by creating chimeric TolCs. First, we expressed the genes for TolCs of Vibrio parahaemolyticus (vp-tolC) and Salmonella typhimurium (sal-tolC) in Escherichia coli. The levels of sequence identity in the mature region of VP-TolC/EC-TolC and Sal-TolC/EC-TolC with maximum matching are 43% and 90%, respectively. We found that the transport activity of VP-TolC was weak compared with that of TolC of E. coli (EC-TolC) although the transport activity of Sal-TolC was similar to that of EC-TolC. A comparison of the sequence of the three tolCs showed that the sequence around the periplasmic region covering Asn-188 to Lys-214 of EC-TolC is lowly identical to that of VP-TolC although the region of EC-TolC is almost identical to that of Sal-TolC. We think, therefore, that the region covering Asn-188 to Lys-214 of EC-TolC may have an important role to express its transport activity in E. coli. To examine the possibility, we divided the region of EC-TolC into three and exchanged the gene for each portion with that of vp-tolC. These mutant ec-tolCs were expressed in E. coli and the activity of each chimeric TolC was measured. The results showed that the portion covering Val-198 to Lys-214 of EC-TolC is deeply involved in the transport activity.
KeywordMeSH Terms
29. Hou  XL, Cao  QY, Pan  JC, Chen  Z,     ( 2006 )

[Classification and identification of vibrio cholerae and vibrio parahaemolyticus isolates based on gyrB gene phylogenetic analysis].

Wei sheng wu xue bao = Acta microbiologica Sinica 46 (6)
PMID : 17302148  :  
Abstract >>
In order to validate the usefulness of gyrB genotype for the classification and identification of Vibrio cholerae and Vibrio parahaemolyticus isolates, the phylogenetic analysis of 13 V. cholerae, 8 V. parahaemolyticus, 2 Aeromonas hydrophila and 1 Plesiomonas shigelloides strains was carried out using the partial coding sequence of gyrB, a gene that encodes the B subunit of DNA gyrase (topoisomerase type II) in bacteria. These strains were separately clustered at species level and typed by the DNA sequences of reference strains from GenBank. CtxA positive V. cholerae strains including 8 clincical isolates of 0139 and 2 clinical isolates of 01 formed one cluster. Four V. parahaemolyticus strains of 1 isolate from 2002 Zhejiang outbreak patient (tdh positive), 2 clinical isoltates from 2004 and 1 strain from Japan were grouped with an environmental isolate (trh positive) from 2001. GyrB genotype is applicable to species identification of V. cholerae, V. parahaemolyticus, A. hydrophila and P. shigelloides isolates. The ctxA positive 0139 and 01 group of V. cholerae are closely related, as reflected by gyrB sequence divergence. Furthermore, the toxigenic V. parahaemolyticus strain isolated from environments may be the potential pathogen to the local prevalent and sporadic cases.
KeywordMeSH Terms
30. Nhung  PH, Shah  MM, Ohkusu  K, Noda  M, Hata  H, Sun  XS, Iihara  H, Goto  K, Masaki  T, Miyasaka  J, Ezaki  T,     ( 2007 )

The dnaJ gene as a novel phylogenetic marker for identification of Vibrio species.

Systematic and applied microbiology 30 (4)
PMID : 17207598  :   DOI  :   10.1016/j.syapm.2006.11.004    
Abstract >>
The utility of the dnaJ gene for identifying Vibrio species was investigated by analyzing dnaJ sequences of 57 type strains and 22 clinical strains and comparing sequence homologies with those of the 16S rDNA gene and other housekeeping genes (recA, rpoA, hsp60). Among the 57 Vibrio species, the mean sequence similarity of the dnaJ gene (77.9%) was significantly less than that of the 16S rDNA gene (97.2%), indicating a high discriminatory power of the dnaJ gene. Most Vibrio species were, therefore, differentiated well by dnaJ sequence analysis. Compared to other housekeeping genes, the dnaJ gene showed better resolution than recA or rpoA for differentiating Vibrio coralliilyticus from Vibrio neptunius and Vibrio harveyi from Vibrio rotiferianus. Among the clinical strains, all 22 human pathogenic strains, including an atypical strain, were correctly identified by the dnaJ sequence. Our findings suggest that analysis of the dnaJ gene sequence can be used as a new tool for the identification of Vibrio species.
KeywordMeSH Terms
Genes, Bacterial
HSP40 Heat-Shock Proteins
31. González-Escalona  N, Blackstone  GM, DePaola  A,     ( 2006 )

Characterization of a Vibrio alginolyticus strain, isolated from Alaskan oysters, carrying a hemolysin gene similar to the thermostable direct hemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus.

Applied and environmental microbiology 72 (12)
PMID : 17056701  :   DOI  :   10.1128/AEM.01548-06     PMC  :   PMC1694234    
Abstract >>
A Vibrio strain isolated from Alaskan oysters and classified by its biochemical characteristics as Vibrio alginolyticus possessed a thermostable direct hemolysin-related hemolysin (trh) gene previously reported only in Vibrio parahaemolyticus. This trh-like gene was cloned and sequenced and was 98% identical to the trh2 gene of V. parahaemolyticus. This gene seems to be functional since it was transcriptionally active in early-stationary-phase growing cells. To our knowledge, this is the first report of V. alginolyticus possessing a trh gene.
KeywordMeSH Terms
32. Tarr  CL, Patel  JS, Puhr  ND, Sowers  EG, Bopp  CA, Strockbine  NA,     ( 2007 )

Identification of Vibrio isolates by a multiplex PCR assay and rpoB sequence determination.

Journal of clinical microbiology 45 (1)
PMID : 17093013  :   DOI  :   10.1128/JCM.01544-06     PMC  :   PMC1828960    
Abstract >>
Vibrio, a diverse genus of aquatic bacteria, currently includes 72 species, 12 of which occur in human clinical samples. Of these 12, three species--Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus-account for the majority of Vibrio infections in humans. Rapid and accurate identification of Vibrio species has been problematic because phenotypic characteristics are variable within species and biochemical identification requires 2 or more days to complete. To facilitate the identification of human-pathogenic species, we developed a multiplex PCR that uses species-specific primers to amplify gene regions in four species (V. cholerae, V. parahaemolyticus, V. vulnificus, and V. mimicus). The assay was tested on a sample of 309 Vibrio isolates representing 26 named species (including 12 human pathogens) that had been characterized by biochemical methods. A total of 190 isolates that had been identified as one of the four target species all yielded results consistent with the previous classification. The assay identified an additional four V. parahaemolyticus isolates among the other 119 isolates. Sequence analysis based on rpoB was used to validate the multiplex results for these four isolates, and all clustered with other V. parahaemolyticus sequences. The rpoB sequences for 12 of 15 previously unidentified isolates clustered with other Vibrio species in a phylogenetic analysis, and three isolates appeared to represent unnamed Vibrio species. The PCR assay provides a simple, rapid, and reliable tool for identification of the major Vibrio pathogens in clinical samples, and rpoB sequencing provides an additional identification tool for other species in the genus Vibrio.
KeywordMeSH Terms
Sequence Analysis, DNA
33. Tracz  DM, Backhouse  PG, Olson  AB, McCrea  JK, Walsh  JA, Ng  LK, Gilmour  MW,     ( 2007 )

Rapid detection of Vibrio species using liquid microsphere arrays and real-time PCR targeting the ftsZ locus.

Journal of medical microbiology 56 (Pt 1)
PMID : 17172518  :   DOI  :   10.1099/jmm.0.46759-0    
Abstract >>
The development of rapid and sensitive molecular techniques for the detection of Vibrio species would be useful for the surveillance of sporadic infections and management of major outbreaks. Comparative sequence analysis of the ftsZ gene in the predominant Vibrio species that cause human disease revealed distinct alleles for each examined species, including Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus. Light Upon eXtension (LUX) real-time PCR assays were developed to target these species-specific polymorphisms, and were successful in rapidly differentiating the major pathogenic Vibrio species. Luminex liquid microsphere array technology was used to develop a comprehensive assay capable of simultaneously detecting V. cholerae, V. parahaemolyticus and V. vulnificus. These assays permitted the identification of a presumptive V. parahaemolyticus isolate as Vibrio alginolyticus, which was verified using additional molecular characterization.
KeywordMeSH Terms
34. Wang  HZ, Wong  MM, O'Toole  D, Mak  MM, Wu  RS, Kong  RY,     ( 2006 )

Identification of a DNA methyltransferase gene carried on a pathogenicity island-like element (VPAI) in Vibrio parahaemolyticus and its prevalence among clinical and environmental isolates.

Applied and environmental microbiology 72 (6)
PMID : 16751568  :   DOI  :   10.1128/AEM.02095-05     PMC  :   PMC1489626    
Abstract >>
In this study we identified a putative virulence-associated DNA methyltransferase (MTase) gene carried on a novel 22.79-kb pathogenicity island-like element (VPAI) in V. parahaemolyticus. The V. parahaemolyticus MTase gene was shown by PCR to be prevalent (>98%) in pandemic thermostable direct hemolysin gene-positive isolates, which suggests that VPAI may confer unique virulence traits to pandemic strains of V. parahaemolyticus.
KeywordMeSH Terms
35. Parvathi  A, Kumar  HS, Bhanumathi  A, Ishibashi  M, Nishibuchi  M, Karunasagar  I, Karunasagar  I,     ( 2006 )

Molecular characterization of thermostable direct haemolysin-related haemolysin (TRH)-positive Vibrio parahaemolyticus from oysters in Mangalore, India.

Environmental microbiology 8 (6)
PMID : 16689720  :   DOI  :   10.1111/j.1462-2920.2006.00990.x    
Abstract >>
Pathogenic Vibrio parahaemolyticus strains producing either or both of a thermostable direct haemolysin (TDH) and a TDH-related haemolysin (TRH) encoded by tdh and trh genes, respectively, are isolated at a low rate from the environment. However, recently we observed that a considerable percentage of APW (alkaline peptone water) enrichment broths of oysters collected off Mangalore India, were trh(+), rather than tdh(+) by PCR. In order to further investigate the prevalence and genetic diversity of trh bearing V. parahaemolyticus in our coast, we attempted to isolate and characterize trh(+)V. parahaemolyticus from oysters. A total of 27 trh(+) strains were isolated during the period between March 2002 and February 2004, of which nine were also tdh(+). All the trh(+) isolates were positive for urease phenotype. The isolates belonged to diverse phenotypes. In order to explore the possible presence of heterogeneity in the trh gene region among trh(+)V. parahaemolyticus, a 1.5 kb region around trh gene was PCR amplified and restriction digested using selected restriction enzymes. The whole genome comparison of strains was performed by randomly amplified polymorphic DNA PCR (RAPD PCR). The PCR-RFLP results revealed fairly well conserved nature of the trh gene region studied in different serotypes. Though 11 strains were positive by PCR for a genomic fragment that has been reported to be amplified in pandemic strains, all strains were negative by group-specific PCR (GS-PCR), orf8 PCR and showed a different RAPD pattern compared with pandemic strains. The results suggest that genetically diverse V. parahaemolyticus carrying virulence genes are associated with the aquatic environment in this region.
KeywordMeSH Terms
36. Nakasone  N, Iwanaga  M,     ( 1991 )

Purification and characterization of pili isolated from Vibrio parahaemolyticus Na2.

Infection and immunity 59 (2)
PMID : 1670933  :   PMC  :   PMC257821    
Abstract >>
Pili from Vibrio parahaemolyticus Na2 isolated from a patient with diarrhea were purified and characterized. The organisms were hemagglutinative, but the purified pili were not. Na2 pili were physicochemically and immunologically quite different from the previously described V. parahaemolyticus Ha7 pili. Nevertheless, there was a high degree of homology between their N-terminal amino acid sequences.
KeywordMeSH Terms
37. Nakaguchi  Y, Nishibuchi  M,     ( 2005 )

The promoter region rather than its downstream inverted repeat sequence is responsible for low-level transcription of the thermostable direct hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus.

Journal of bacteriology 187 (5)
PMID : 15716457  :   DOI  :   10.1128/JB.187.5.1849-1855.2005     PMC  :   PMC1063991    
Abstract >>
We determined the transcriptional start site of the thermostable direct hemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus by using a PCR-based method and identified the promoter. Mutagenic analysis indicated that the promoter-bearing region rather than its downstream inverted repeat sequence was responsible for the low-revel of trh transcription.
KeywordMeSH Terms
38. Thompson  FL, Gevers  D, Thompson  CC, Dawyndt  P, Naser  S, Hoste  B, Munn  CB, Swings  J,     ( 2005 )

Phylogeny and molecular identification of vibrios on the basis of multilocus sequence analysis.

Applied and environmental microbiology 71 (9)
PMID : 16151093  :   DOI  :   10.1128/AEM.71.9.5107-5115.2005     PMC  :   PMC1214639    
Abstract >>
We analyzed the usefulness of rpoA, recA, and pyrH gene sequences for the identification of vibrios. We sequenced fragments of these loci from a collection of 208 representative strains, including 192 well-documented Vibrionaceae strains and 16 presumptive Vibrio isolates associated with coral bleaching. In order to determine the intraspecies variation among the three loci, we included several representative strains per species. The phylogenetic trees constructed with the different genetic loci were roughly in agreement with former polyphasic taxonomic studies, including the 16S rRNA-based phylogeny of vibrios. The families Vibrionaceae, Photobacteriaceae, Enterovibrionaceae, and Salinivibrionaceae were all differentiated on the basis of each genetic locus. Each species clearly formed separated clusters with at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively. The genus Vibrio was heterogeneous and polyphyletic, with Vibrio fischeri, V. logei, and V. wodanis grouping closer to the Photobacterium genus. V. halioticoli-, V. harveyi-, V. splendidus-, and V. tubiashii-related species formed groups within the genus Vibrio. Overall, the three genetic loci were more discriminatory among species than were 16S rRNA sequences. In some cases, e.g., within the V. splendidus and V. tubiashii group, rpoA gene sequences were slightly less discriminatory than recA and pyrH sequences. In these cases, the combination of several loci will yield the most robust identification. We can conclude that strains of the same species will have at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively.
KeywordMeSH Terms
Bacterial Typing Techniques
Phylogeny
Sequence Analysis, DNA
39. Wong  HC, Chen  CH, Chung  YJ, Liu  SH, Wang  TK, Lee  CL, Chiou  CS, Nishibuchi  M, Lee  BK,     ( 2005 )

Characterization of new O3:K6 strains and phylogenetically related strains of Vibrio parahaemolyticus isolated in Taiwan and other countries.

Journal of applied microbiology 98 (3)
PMID : 15715859  :   DOI  :   10.1111/j.1365-2672.2004.02478.x    
Abstract >>
We analysed the genetic divergence in the pandemic new O3:K6 and phylogenetically related (new O3:K6-like) strains and compare these two groups in terms of virulence and other biological traits. A total of 160 new O3:K6, new O3:K6-like and other strains of Vibrio parahaemolyticus isolated in Taiwan and other countries were collected and their clonal relationships analysed using SfiI-pulsed-field gel electrophoresis. All of the new O3:K6 and new O3:K6-like strains were grouped in cluster I with five new patterns identified. A O6:K18 strain was identified as a new member of the new O3:K6-like strains in addition to O4:K68, O1:KUT and O1:K25 strains. All of the lipopolysaccharide preparations of the selected strains exhibited closely spaced quadruplet banding patterns with similar mobility. The two groups of strains exhibited 100% sequence identity in the internal sequences of the toxR and laf genes, and also displayed similar virulence properties as determined with a suckling mouse model. The new O3:K6 and new O3:K6-like strains were highly similar in virulence and in several other phenotypical and genotypical traits. This work demonstrated the spread and divergence of the pandemic and related clone of V. parahaemolyticus with similar virulence.
KeywordMeSH Terms
Food Microbiology
Genes, Bacterial
40. Park  KS, Arita  M, Iida  T, Honda  T,     ( 2005 )

vpaH, a gene encoding a novel histone-like nucleoid structure-like protein that was possibly horizontally acquired, regulates the biogenesis of lateral flagella in trh-positive Vibrio parahaemolyticus TH3996.

Infection and immunity 73 (9)
PMID : 16113292  :   DOI  :   10.1128/IAI.73.9.5754-5761.2005     PMC  :   PMC1231141    
Abstract >>
A histone-like nucleoid structure (H-NS) is a major component of the bacterial nucleoid and plays a crucial role in the global gene regulation of enteric bacteria. Here, we cloned and characterized the gene for the H-NS-like protein VpaH in Vibrio parahaemolyticus. vpaH encodes a protein of 134 amino acids that shows approximately 55%, 54%, and 41% identities with VicH in Vibrio cholerae, H-NS in V. parahaemolyticus, and H-NS in Escherichia coli, respectively. The vpaH gene was found in only trh-positive V. parahaemolyticus strains and not in Kanagawa-positive or in trh-negative environmental strains. Moreover, the G+C content of the vpaH gene was 38.6%, which is lower than the average G+C content of the whole genome of this bacterium (45.4%). These data suggest that vpaH was transmitted to trh-possessing V. parahaemolyticus strains by lateral transfer. The vpaH gene was located about 2.6 kb downstream of the trh gene, in the convergent direction of the trh transcription. An in-frame deletion mutant of vpaH lacked motility on semisolid motility assay plates. Western blot analysis and electron microscopy observations revealed that the mutant was deficient in lateral flagella biogenesis, whereas there was no defect in the expression of polar flagella. Additionally, the vpaH mutant showed a decreased adherence to HeLa cells and a decrease in biofilm formation compared with the wild-type strain. Introduction of the vpaH gene in the vpaH-negative strain increased the expression of lateral flagella compared with the wild-type strain. In conclusion, our findings suggest that VpaH affects lateral flagellum biogenesis in trh-positive V. parahaemolyticus strain TH3996.
KeywordMeSH Terms
Gene Transfer, Horizontal
41. Xie  ZY, Hu  CQ, Chen  C, Zhang  LP, Ren  CH,     ( 2005 )

Investigation of seven Vibrio virulence genes among Vibrio alginolyticus and Vibrio parahaemolyticus strains from the coastal mariculture systems in Guangdong, China.

Letters in applied microbiology 41 (2)
PMID : 16033522  :   DOI  :   10.1111/j.1472-765X.2005.01688.x    
Abstract >>
To investigate the distribution of the virulence of two Vibrio species among different strains obtained from the mariculture systems on the coast of Guangdong in China and the correlation between the virulence strains and the virulence genes among Vibrio alginolyticus. Besides three strains, 72 V. alginolyticus strains and seven Vibrio parahaemolyticus strains were examined by PCR or semi-nested PCR for the virulence genes (tlh, trh, tdh, toxR, toxRS, ctxA, VPI). Additionally, the virulence of 18 V. alginolyticus strains was tested. Virulence genes homologous to those in the V. parahaemolyticus and Vibrio cholerae are widely distributed among V. alginolyticus and V. parahaemolyticus in the coastal mariculture systems in Guangdong, China. Some of the V. alginolyticus strains are pathogenic to aquatic animals, and might have derived their virulence genes from V. parahaemolyticus or V. cholerae, representing a possible reservoir of these genes. However, there is no correlation between presence and absence of the virulence genes used to investigate V. alginolyticus and its virulent strains. In this report, we also show that tlh is distributed among V. alginolyticus.
KeywordMeSH Terms
Genes, Bacterial
42. Trosky  JE, Mukherjee  S, Burdette  DL, Roberts  M, McCarter  L, Siegel  RM, Orth  K,     ( 2004 )

Inhibition of MAPK signaling pathways by VopA from Vibrio parahaemolyticus.

The Journal of biological chemistry 279 (50)
PMID : 15459200  :   DOI  :   10.1074/jbc.M407001200    
Abstract >>
During infection, bacterial pathogens utilize a type III secretion system to inject effectors into the cytoplasm of a target cell where they disrupt the defense system of the host cell. Vibrio parahaemolyticus, a causative agent of gastroenteritis endemic in Southeast Asia, has a type III secretion system that encodes a novel member of the YopJ-like protein effector family, VopA (Vibrio outer protein A). Our studies revealed that Vibrio VopA encodes an evolutionarily conserved activity that is extremely potent and requires an intact catalytic site to abrogate signaling pathways in a manner distinct from that of other YopJ-like effectors. We observed that VopA efficiently inhibits the MAPK signaling pathways but not the NFkappaB pathway in mammalian cells. When expressed in yeast, VopA induces a growth arrest phenotype and also blocks yeast MAPK signaling pathways. Our observations provide insight into the immense diversity of targets utilized by YopJ-like effectors to manipulate eukaryotic signaling machineries that are important for the response and survival of the host cell during infection and/or symbiosis.
KeywordMeSH Terms
MAP Kinase Signaling System
43. Thompson  CC, Thompson  FL, Vandemeulebroecke  K, Hoste  B, Dawyndt  P, Swings  J,     ( 2004 )

Use of recA as an alternative phylogenetic marker in the family Vibrionaceae.

International journal of systematic and evolutionary microbiology 54 (Pt 3)
PMID : 15143042  :   DOI  :   10.1099/ijs.0.02963-0    
Abstract >>
This study analysed the usefulness of recA gene sequences as an alternative phylogenetic and/or identification marker for vibrios. The recA sequences suggest that the genus Vibrio is polyphyletic. The high heterogeneity observed within vibrios was congruent with former polyphasic taxonomic studies on this group. Photobacterium species clustered together and apparently nested within vibrios, while Grimontia hollisae was apart from other vibrios. Within the vibrios, Vibrio cholerae and Vibrio mimicus clustered apart from the other genus members. Vibrio harveyi- and Vibrio splendidus-related species formed compact separated groups. On the other hand, species related to Vibrio tubiashii appeared scattered in the phylogenetic tree. The pairs Vibrio coralliilyticus and Vibrio neptunius, Vibrio nereis and Vibrio xuii and V. tubiashii and Vibrio brasiliensis clustered completely apart from each other. There was a correlation of 0.58 between recA and 16S rDNA pairwise similarities. Strains of the same species have at least 94 % recA sequence similarity. recA gene sequences are much more discriminatory than 16S rDNA. For 16S rDNA similarity values above 98 % there was a wide range of recA similarities, from 83 to 99 %.
KeywordMeSH Terms
Genes, Bacterial
44. Kishishita  M, Matsuoka  N, Kumagai  K, Yamasaki  S, Takeda  Y, Nishibuchi  M,     ( 1992 )

Sequence variation in the thermostable direct hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus.

Applied and environmental microbiology 58 (8)
PMID : 1514791  :   PMC  :   PMC195802    
Abstract >>
Our previous molecular epidemiologic study with gene probes (H. Shirai, H. Ito, T. Hirayama, Y. Nakamoto, N. Nakabayashi, K. Kumagai, Y. Takeda, and M. Nishibuchi, Infect. Immun. 58:3568-3573, 1990) demonstrated that the gene (trh) encoding a thermostable direct hemolysin-related hemolysin was strongly associated with clinical strains of Vibrio parahaemolyticus. Strain-to-strain variation in the intensities of the hybridization signals observed in the above study also suggested that the trh genes in different strains may have significantly divergent nucleotide sequences. To assess the public health significance of the rare environmental strains which exhibited very weak hybridization signals with the trh gene-specific DNA probe, the trh-like sequence was cloned from one of the environmental strains and the nucleotide sequence was determined in this study. A hemolysin gene (trh2) which was 84% homologous to the trh gene (newly named trh1) and 54.8 to 68.8% homologous to the genes (tdh) encoding thermostable direct hemolysins was detected in the cloned sequence. The trh2 gene product showed a profile of hemolytic activities against various animal erythrocytes different from that of the trh1 gene product. The trh2 gene product was antigenically related (partially identical) to the trh1 and tdh gene products. DNA colony blot and Southern blot hybridization analyses with trh1- and trh2-specific DNA probes showed that the trh1 probe-positive strains exhibiting hybridization signals with varying intensities could be clustered into trh1 and trh2 subgroups. In addition, hybridization analysis with oligonucleotide probes demonstrated significant strain-to-strain variation in the trh1 and trh2 gene sequences.(ABSTRACT TRUNCATED AT 250 WORDS)
KeywordMeSH Terms
45. Chowdhury  NR, Stine  OC, Morris  JG, Nair  GB,     ( 2004 )

Assessment of evolution of pandemic Vibrio parahaemolyticus by multilocus sequence typing.

Journal of clinical microbiology 42 (3)
PMID : 15004094  :   DOI  :   10.1128/jcm.42.3.1280-1282.2004     PMC  :   PMC356825    
Abstract >>
The genetic relatedness of 81 isolates of Vibrio parahaemolyticus was assessed by multilocus sequence typing. The strain with serotype O3:K6 emerged as a pandemic pathogen in 1996, with subsequent expansion to include strains having serotypes O1:KUT, O4:K68, and O1:K25. Sequence data from gyrB, recA, dnaE, and gnd revealed that 16 distinct serogroups isolated prior to the pandemic were highly variable and only isolates of serogroup O3:K6 shared two alleles with the pandemic strains. The pandemic strains regardless of serotype were clonal, with 51 of 54 isolates having the identical allelic profile (AP). Serotype alone did not adequately define a pandemic strain: among O1:KUT strains tested, seven strains with the identical pandemic AP carried previously described pandemic markers, while five nonpandemic strains had five distinct APs. Our sequence data provide strong molecular support for the clonal origin of pandemic V. parahaemolyticus O3:K6 and suggest that strains within such a clonal group may acquire previously identified serotypes.
KeywordMeSH Terms
46. Nishiguchi  MK, Nair  VS,     ( 2003 )

Evolution of symbiosis in the Vibrionaceae: a combined approach using molecules and physiology.

International journal of systematic and evolutionary microbiology 53 (Pt 6)
PMID : 14657139  :   DOI  :   10.1099/ijs.0.02792-0    
Abstract >>
The family Vibrionaceae is considered to be one of the most diverse and well-studied groups of bacteria. Here, evolution is assessed within the Vibrionaceae to determine whether multiple origins of eukaryotic associations have occurred within this diverse group of bacteria. Analyses were based on a large molecular dataset, along with a matrix that consisted of 100 biochemical and restriction digest characters. By using direct optimization methods to analyse both datasets individually and in combination, a total-evidence cladogram has been produced, which supports the hypothesis that several important symbionts (both mutualistic and pathogenic) within the Vibrionaceae are not monophyletic. This leads us to consider that symbiosis (and subsequently, associations with Eukarya) has evolved multiple times within the Vibrionaceae lineage.
KeywordMeSH Terms
Biological Evolution
Phylogeny
47. Liu  R, Chen  J, Li  K, Zhang  X,     ( 2011 )

Identification and evaluation as a DNA vaccine candidate of a virulence-associated serine protease from a pathogenic Vibrio parahaemolyticus isolate.

Fish & shellfish immunology 30 (6)
PMID : 21536140  :   DOI  :   10.1016/j.fsi.2011.04.005    
Abstract >>
A putative serine protease gene was cloned from the genomic DNA of Vibrio parahaemolyticus FYZ8621.4. The gene consisted of 1779 base pairs and encoded a 592 amino acid protein. The gene was expressed in Escherichia coli. The expressed protease was purified by Ni-NTA His-Bind Resin column and showed a 63 kDa band on SDS-PAGE. The protease exhibited proteolytic activity on gelatin agar plate and showed maximal proteolytic activity at pH 8.0 and 37 �XC. It hydrolyzed N-�\-benzoyl-L-tyrosine p-nitroanilide (BAPNA), but did not N-benzoyl-L-arginine ethylester (BAEE), N-benzoyl-L-tyrosine ethylester (BTEE) and N-acetyl-L-tyrosine ethylester (ATEE). Mutants at conserved residues Asp(51) (Asp(51)-Asn), His(89) (His(89)-Asp) and Ser(318) (Ser(318)-Leu, Ser(318)-Pro) lost proteolytic activities completely. The protein was confirmed to belong to serine protease. The purified serine protease was toxic to zebrafish with a LD(50) of 15.4 �gg/fish. A DNA vaccine was constructed by inserting the mutated serine protease (Ser(318)-Pro) gene into pEGFP-N1 plasmid. The pEGFP-N1/m-vps was transfected in HeLa cells. The serine protease was confirmed to be expressed by fluorescence microscopy observation and Western blotting analysis. The pEGFP-N1/m-vps was further observed to express in muscle of the injected turbot (Scophthalmus maximus) by Western blotting seven days after immunization. Efficient protection against lethal V. parahaemolyticus challenge was observed on the vaccinated turbot with pEGFP-N1/m-vps, with the highest relative percent survival (RPS) of 96.11%. Significant specific antibody responses were also observed in the turbot vaccinated with the DNA vaccine. The results indicated that the serine protease might be a potential virulence factor and could be used as an efficient vaccine candidate for the disease control caused by V. parahaemolyticus.
KeywordMeSH Terms
48. Izumiya  H, Matsumoto  K, Yahiro  S, Lee  J, Morita  M, Yamamoto  S, Arakawa  E, Ohnishi  M,     ( 2011 )

Multiplex PCR assay for identification of three major pathogenic Vibrio spp., Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus.

Molecular and cellular probes 25 (4)
PMID : 21530641  :   DOI  :   10.1016/j.mcp.2011.04.004    
Abstract >>
A multiplex PCR assay was developed based on atpA-sequence diversification for molecular identification of 3 major pathogenic Vibrio species: Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus. It specifically identified them from among 133 strains of various Vibrio species and other genera, and was applicable for testing seawater, suggesting its usefulness.
KeywordMeSH Terms
49. Kadhim  HM, Miah  A, Munn  CB, Gilpin  ML,     ( 2012 )

Development of a polymerase chain reaction (PCR) test for the detection of virulent forms of Vibrio parahaemolyticus.

European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology 31 (4)
PMID : 21725864  :   DOI  :   10.1007/s10096-011-1324-9    
Abstract >>
Vibrio parahaemolyticus is a marine bacterium and some strains cause gastroenteritis in humans. Clinical isolates are thought to possess virulence factors that are absent from the majority of environmental isolates. Use of randomly amplified polymorphic DNA (RAPD)-PCR produced a unique 600 bp amplicon (band Y) in the majority of clinical isolates and rarely in environmental isolates tested. The DNA from band Y was cloned and sequenced and found to code for an outer membrane protein (OMP). Two polymerase chain reaction (PCR) primers were designed to specifically amplify a 200 bp unique sequence from presumptive virulent strains (PCR-OMP). The virulence of 23 clinical and 32 environmental isolates was assessed in cytotoxicity tests by treatment of Caco-2 cells with extracellular products (ECPs). All but two of the clinical isolates (91%) were positive for the 200 bp PCR-OMP and their ECPs produced a significantly higher (p < 0.05) lactate dehydrogenase (LDH) release (mean 72.88%) than the ECPs of environmental isolates (mean 15.3%) with the exception of one environmental isolate that produced the 200 bp amplicon. A positive 200 bp PCR-OMP is strongly correlated with virulence, as determined by the cytotoxicity assay, and identified virulent forms better than current PCR tests for tdh, trh or T3SS2.
KeywordMeSH Terms
50. Oberbeckmann  S, Wichels  A, Maier  T, Kostrzewa  M, Raffelberg  S, Gerdts  G,     ( 2011 )

A polyphasic approach for the differentiation of environmental Vibrio isolates from temperate waters.

FEMS microbiology ecology 75 (1)
PMID : 21118277  :   DOI  :   10.1111/j.1574-6941.2010.00998.x    
Abstract >>
Climate change and marine traffic lead to changing species communities in the oceans. Due to increasing seawater temperatures, pathogenic Vibrio species could become significant even in temperate waters. We classified mesophilic Vibrio isolates from the German Bight (North Sea) using a polyphasic approach with special emphasis on Vibrio parahaemolyticus. Matrix-assisted laser desorption/ionization time-of-flight MS was used as a primary screen to classify isolates, 16S rRNA gene and rpoB gene sequencing to identify species. Potential V. parahaemolyticus isolates were screened for regulatory or virulence-related genes (toxR, tlh, tdh, trh). To investigate genomic diversity, we applied repetitive-sequence-based PCRs. Results were evaluated and methods compared using multivariate statistical analysis. Most isolates were classified as V. parahaemolyticus or Vibrio alginolyticus. Reliable differentiation between both species was achieved by rpoB sequencing and toxR detection. Among the fingerprinting methods, ERIC-PCR showed the highest discriminatory power, displaying three separated clusters. These clusters represent the species V. parahaemolyticus, V. alginolyticus and one group in between. The frequent detection of V. parahaemolyticus in the German Bight reveals the urgency for further monitoring. In this context, a polyphasic approach, such as defined in this study, is needed to differentiate populations of V. parahaemolyticus and V. alginolyticus.
KeywordMeSH Terms
Water Microbiology
51. Ansede-Bermejo  J, Gavilan  RG, Triñanes  J, Espejo  RT, Martinez-Urtaza  J,     ( 2010 )

Origins and colonization history of pandemic Vibrio parahaemolyticus in South America.

Molecular ecology 19 (18)
PMID : 20735744  :   DOI  :   10.1111/j.1365-294X.2010.04782.x    
Abstract >>
The dynamics of dissemination of the environmental human pathogen Vibrio parahaemolyticus are uncertain. The O3:K6 clone was restricted to Asia until its detection along the Peruvian coasts and in northern Chile in 1997 in phase with the arrival of El Ni?o waters. A subsequent emergence of O3:K6 strains was detected in austral Chile in 2004. The origin of these 1997 and 2004 population radiations has not yet been conclusively determined. Multiple loci VNTR analysis using seven polymorphic loci was carried out with a number of representative strains from Asia, Peru and Chile to determine their genetic characteristics and population structure. Asian and Chilean subpopulations were the most genetically distant groups with an intermediate subpopulation in Peru. Population structure inferred from a minimum-spanning tree and Bayesian analysis divided the populations into two genetically distinct groups, consistent with the epidemic dynamics of the O3:K6 clone in South America. One group comprised strains from the original Asiatic population and strains arriving in Peru and Chile in 1997. The second group included the remaining Peruvian Strains and Chilean strains obtained from Puerto Montt in 2004. The analysis of the arrival of the O3:K6 clone at the Pacific coasts of South America has provided novel insights linking the origin of the invasion in 1997 to Asian populations and describing the successful establishment of the O3:K6 populations, first in Peru and subsequently in the South of Chile owing to a possible radiation of Peruvian populations.
KeywordMeSH Terms
Genetic Variation
Minisatellite Repeats
52. Haldar  S, Chatterjee  S, Sugimoto  N, Das  S, Chowdhury  N, Hinenoya  A, Asakura  M, Yamasaki  S,     ( 2011 )

Identification of Vibrio campbellii isolated from diseased farm-shrimps from south India and establishment of its pathogenic potential in an Artemia model.

Microbiology (Reading, England) 157 (Pt 1)
PMID : 20847009  :   DOI  :   10.1099/mic.0.041475-0    
Abstract >>
Shrimp diseases are frequently reported to be caused by closely related vibrios, and in many cases they are tentatively but inaccurately identified as Vibrio harveyi and related vibrios. In the present study, 28 biochemically identified V. harveyi-related strains isolated from diseased shrimps were randomly selected for further characterization by molecular tools. Twenty-six strains were identified as Vibrio campbellii and two as V. harveyi by sequence analysis of 16S rRNA and uridylate kinase genes. Haemolysin-gene-based species-specific multiplex PCR also confirmed these results. Experimental challenge studies using Artemia as a model showed that eight isolates were highly pathogenic, three were moderately pathogenic and the remaining 17 were non-pathogenic. Ribotyping with BglI clearly distinguished V. campbellii from V. harveyi, but it failed to separate pathogenic and non-pathogenic clusters. Artemia nauplii challenged with a fluorescently labelled highly pathogenic strain (IPEY54) showed patches in the digestive tract. However, no patches were observed for a non-pathogenic strain (IPEY41). Direct bacterial counts also supported colonization potential for the highly pathogenic strain. To our knowledge, this is the first report on the isolation and accurate identification of large numbers of V. campbellii associated with shrimp disease in aquacultural farms. V. campbellii has long been considered to be non-pathogenic and classified with V. harveyi-related bacteria. However, we show that this species may be an emerging aquaculture pathogen. This study will help to formulate suitable strategies to combat this newly identified pathogen.
KeywordMeSH Terms
53. Caburlotto  G, Gennari  M, Ghidini  V, Tafi  M, Lleo  MM,     ( 2010 )

Serological and molecular characterization of Vibrio parahaemolyticus marine strains carrying pandemic genetic markers.

The ISME journal 4 (8)
PMID : 20393570  :   DOI  :   10.1038/ismej.2010.34    
Abstract >>
In 2005, pandemic Vibrio parahaemolyticus was reported to have been introduced in Europe: O3:K6 strains were isolated from clinical cases in France and Spain, and were found to be associated with consumption of contaminated seafood. On the contrary, pandemic strains were not isolated from seafood or from the environment itself. Analysis of two V. parahaemolyticus strains isolated in May 2007 from Northern Italy seawater and plankton samples revealed the presence of the virulence gene tdh and the pandemic-specific markers orf8 and toxRS/new sequence (group-specific PCR). The two strains showed serotypes not included in the 'pandemic group', but their molecular typing proved that they represent a single clone showing a genetic profile very similar to that of pandemic O3:K6 reference isolates. Moreover, the two marine strains carried three virulence-related genes associated with clinical strains and, to date, hardly ever or never detected in environmental strains. The presence, in strains isolated from the marine environment, of genetic pandemic markers and virulence genes normally associated with clinical isolates proves that marine strains might constitute a public health concern.
KeywordMeSH Terms
Disease Outbreaks
54. Honda  T, Abad-Lapuebla  MA, Ni  YX, Yamamoto  K, Miwatani  T,     ( 1991 )

Characterization of a new thermostable direct haemolysin produced by a Kanagawa-phenomenon-negative clinical isolate of Vibrio parahaemolyticus.

Journal of general microbiology 137 (2)
PMID : 2016584  :   DOI  :   10.1099/00221287-137-2-253    
Abstract >>
The production of two haemolysins, thermostable direct haemolysin (Vp-TDH) and a Vp-TDH-related haemolysin (Vp-TRH), by clinical isolates of Vibrio parahaemolyticus has previously been reported. Here we describe a third type of haemolysin (named Vp-TDH/I), which is produced by a clinical isolate (strain TH012) that is Kanagawa phenomenon negative. Vp-TDH/I was purified by a series of column chromatographies on DEAE-Sephadex A25, hydroxyapatite, Sepharose 4B and Mono Q. By physicochemical, biological and immunological analyses, Vp-TDH/I was demonstrated to be similar, but not identical, to Vp-TDH and Vp-TRH. The gene encoding Vp-TDH/I was cloned and the deduced amino acid sequence of Vp-TDH/I confirmed that Vp-TDH/I has a sequence different from those of previously known Vp-TDH and Vp-TRH. Not only purified Vp-TDH/I but also live cells of the Vp-TDH/I-producing strain induced fluid accumulation in ligated rabbit intestine. We conclude that this clinical isolate produces a new type of Vp-TDH-related haemolysin, which may be involved in the pathogenesis of this organism.
KeywordMeSH Terms
55. Chimetto  LA, Cleenwerck  I, Alves  N, Silva  BS, Brocchi  M, Willems  A, De Vos  P, Thompson  FL,     ( 2011 )

Vibrio communis sp. nov., isolated from the marine animals Mussismilia hispida, Phyllogorgia dilatata, Palythoa caribaeorum, Palythoa variabilis and Litopenaeus vannamei.

International journal of systematic and evolutionary microbiology 61 (Pt 2)
PMID : 20305064  :   DOI  :   10.1099/ijs.0.019729-0    
Abstract >>
Eight Vibrio isolates originating from the marine corals Mussismilia hispida and Phyllogorgia dilatata and the zoanthids Palythoa caribaeorum and Palythoa variabilis in Brazil and the Pacific white shrimp (Litopenaeus vannamei) in Ecuador were studied by means of a polyphasic approach. The novel isolates formed a tight monophyletic group in the genus Vibrio and were closely related to species of the Vibrio harveyi group, to which they showed more than 99 % 16S rRNA gene sequence similarity. Analysis based on concatenated sequences of the following seven genes, 16S rRNA, gyrB, recA, rpoA, topA, pyrH and mreB (5633 bp in length), showed clear separation between the isolates and species of the V. harveyi group. Amplified fragment length polymorphism (AFLP) analysis, performed previously, revealed that a representative isolate of this group, LMG 20370, was clearly separate from known Vibrio species (it belonged to the so-called AFLP cluster A31). DNA-DNA hybridization (DDH) experiments with representative isolates and type strains of the V. harveyi species group revealed high DDH between the novel isolates (more than 74 %) and less than 70 % DDH towards type strains of related Vibrio species, proving the novel species status of the isolates. Phenotypically, the novel species belongs to the arginine dihydrolase (A)-negative, lysine decarboxylase (L)-positive and ornithine decarboxylase (O)-positive (A-/L+/O+) cluster reported previously. Most species of the V. harveyi group (i.e. Vibrio rotiferianus, V. harveyi, V. parahaemolyticus and V. alginolyticus) also belong to this A-/L+/O+ cluster. However, several phenotypic features can be used for the identification of the novel species. In contrast to its closest phylogenetic neighbours, the novel species exhibits esterase (C4) and N-acetyl-�]-glucosaminidase activities, but it does not produce acetoin, does not use citrate, �\-ketoglutaric acid or propionic acid and does not ferment melibiose. The novel species can also be differentiated on the basis of the presence of the fatty acids C(17 : 0,) C(17 : 1)�s8c, iso-C(17 : 0) and iso-C(13 : 0) and the absence of the fatty acid C(18 : 0). The name Vibrio communis sp. nov. is proposed for this taxon. Strain R-40496(T) (=LMG 25430(T) =CAIM 1816(T)) is the type strain.
KeywordMeSH Terms
Phylogeny
56. Espiñeira  M, Atanassova  M, Vieites  JM, Santaclara  FJ,     ( 2010 )

Validation of a method for the detection of five species, serogroups, biotypes and virulence factors of Vibrio by multiplex PCR in fish and seafood.

Food microbiology 27 (1)
PMID : 19913702  :   DOI  :   10.1016/j.fm.2009.09.004    
Abstract >>
In this work a sequential multiplex PCR system was designed and validated for the detection of most frequent foodborne pathogen Vibrio species in fish and seafood (Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginoliticus and Vibrio mimicus). The method proposed functions in a hierarchical way, being composed of an end-point multiplex PCR to detect the presence of DNA belonging to the studied species, followed by multiplex PCR and fragment analysis allowing the viability assessment of the detected strains. The final multiplex PCR step of the method may be applied if identification of the serogroup, biotype and/or virulence factor level is necessary. Forty samples of commercial fish and seafood products were used at the method validation stage. Sixty three marine organism samples obtained from various estuarine areas of Spain including shrimps, crabs, bivalve mollusks and fishes were screened for presence of Vibrio species and 2 mussel samples were found positive for V. parahaemolyticus. On the whole, the proposed method is robust and readily adaptable in routine molecular diagnostic laboratories, allowing monitoring and simultaneous detection of all these bacterial pathogens in seafood samples, reducing the expenses and time consumed by other analytical methods.
KeywordMeSH Terms
57. Roque  A, Lopez-Joven  C, Lacuesta  B, Elandaloussi  L, Wagley  S, Furones  MD, Ruiz-Zarzuela  I, de Blas  I, Rangdale  R, Gomez-Gil  B,     ( 2009 )

Detection and identification of tdh- and trh-positive Vibrio parahaemolyticus strains from four species of cultured bivalve molluscs on the Spanish Mediterranean Coast.

Applied and environmental microbiology 75 (23)
PMID : 19801467  :   DOI  :   10.1128/AEM.00772-09     PMC  :   PMC2786417    
Abstract >>
Presented here is the first report describing the detection of potentially diarrheal Vibrio parahaemolyticus strains isolated from cultured bivalves on the Mediterranean coast, providing data on the presence of both tdh- and trh-positive isolates. Potentially diarrheal V. parahaemolyticus strains were isolated from four species of bivalves collected from both bays of the Ebro delta, Spain.
KeywordMeSH Terms
58. Hazen  TH, Martinez  RJ, Chen  Y, Lafon  PC, Garrett  NM, Parsons  MB, Bopp  CA, Sullards  MC, Sobecky  PA,     ( 2009 )

Rapid identification of Vibrio parahaemolyticus by whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Applied and environmental microbiology 75 (21)
PMID : 19749061  :   DOI  :   10.1128/AEM.01171-09     PMC  :   PMC2772414    
Abstract >>
Vibrio parahaemolyticus is a pathogenic marine bacterium that is the main causative agent of bacterial seafood-borne gastroenteritis in the United States. An increase in the frequency of V. parahaemolyticus-related infections during the last decade has been attributed to the emergence of an O3:K6 pandemic clone in 1995. The diversity of the O3:K6 pandemic clone and its serovariants has been examined using multiple molecular techniques including multilocus sequence analysis, pulsed-field gel electrophoresis, and group-specific PCR analysis. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has become a powerful tool for rapidly distinguishing between related bacterial species. In the current study, we demonstrate the development of a whole-cell MALDI-TOF MS method for the distinction of V. parahaemolyticus from other Vibrio spp. We identified 30 peaks that were present only in the spectra of the V. parahaemolyticus strains examined in this study that may be developed as MALDI-TOF MS biomarkers for identification of V. parahaemolyticus. We detected variation in the MALDI-TOF spectra of V. parahaemolyticus strains isolated from different geographical locations and at different times. The MALDI-TOF MS spectra of the V. parahaemolyticus strains examined were distinct from those of the other Vibrio species examined including the closely related V. alginolyticus, V. harveyi, and V. campbellii. The results of this study demonstrate the first use of whole-cell MALDI-TOF MS analysis for the rapid identification of V. parahaemolyticus.
KeywordMeSH Terms
59. Ki  JS, Zhang  R, Zhang  W, Huang  YL, Qian  PY,     ( 2009 )

Analysis of RNA polymerase beta subunit (rpoB) gene sequences for the discriminative power of marine Vibrio species.

Microbial ecology 58 (4)
PMID : 19418092  :   DOI  :   10.1007/s00248-009-9519-7    
Abstract >>
In the present study, we sequenced the RNA polymerase beta subunit (rpoB) gene of marine Vibrio species and assessed its discriminative power in identifying vibrios. Both the rpoB and 16S rRNA sequences of 29 phenotypically different Vibrio strains isolated from coastal waters were determined. Molecular and phylogenetic comparisons of the sequences of these two genes classified the 29 strains into 11 different species. The resolution of the Vibrio spp. on the rpoB phylogenetic tree was approximately three times greater than that on the 16S rRNA phylogenetic tree. Moreover, by comparing the rpoB sequences of 98 marine gamma-Proteobacteria, including 38 marine Vibrio species, Vibrio-specific primers were developed to amplify a 730-bp fragment of the rpoB gene. Using these primers, we successfully detected Vibrio signals in environmental samples and determined their relative abundances via comparisons with known standards. This rpoB-targeting polymerase chain reaction assay can be used efficiently to monitor relative Vibrio abundance in marine waters.
KeywordMeSH Terms
Phylogeny
60. Harth  E, Matsuda  L, Hernández  C, Rioseco  ML, Romero  J, González-Escalona  N, Martínez-Urtaza  J, Espejo  RT,     ( 2009 )

Epidemiology of Vibrio parahaemolyticus outbreaks, southern Chile.

Emerging infectious diseases 15 (2)
PMID : 19193258  :   DOI  :   10.3201/eid1502.071269     PMC  :   PMC2657608    
Abstract >>
Disease outbreaks caused by Vibrio parahaemolyticus in Puerto Montt, Chile, began in 2004 and reached a peak in 2005 at 3,600 clinical cases. Until 2006, every analyzed case was caused by the serovar O3:K6 pandemic strain. In the summer of 2007, only 475 cases were reported; 73% corresponded to the pandemic strain. This decrease was associated with a change in serotype of many pandemic isolates to O3:K59 and the emergence of new clinical strains. One of these strains, associated with 11% of the cases, was genotypically different from the pandemic strain but contained genes that were identical to those found on its pathogenicity island. These findings suggest that pathogenicity-related genes were laterally transferred from the pandemic strain to one of the different V. parahaemolyticus groups comprising the diverse and shifting bacterial population in shellfish in this region.
KeywordMeSH Terms
Disease Outbreaks
61. Chimetto  LA, Brocchi  M, Gondo  M, Thompson  CC, Gomez-Gil  B, Thompson  FL,     ( 2009 )

Genomic diversity of vibrios associated with the Brazilian coral Mussismilia hispida and its sympatric zoanthids (Palythoa caribaeorum, Palythoa variabilis and Zoanthus solanderi).

Journal of applied microbiology 106 (6)
PMID : 19291243  :   DOI  :   10.1111/j.1365-2672.2009.04149.x    
Abstract >>
A taxonomic survey of the vibrios associated with the Brazilian endemic coral Mussismilia hispida and the sympatric zoanthids (i.e. Palythoa caribaeorum, Palythoa variabilis and Zoanthus solanderi). Mucus of 54 cnidarian specimens collected in three different places at S?o Sebasti?o in two consecutive years (i.e. 2005 and 2006) was used for taxonomic characterization of the cnidarian microbiota. Ninety-eight of the 151 vibrio isolates fell within the vibrio core group according to partial 16S rDNA sequences. We performed the sequencing of recA and pyrH genes of all vibrio isolates. The most abundant taxa belonged to the vibrio core group (Vibrio harveyi, Vibrio rotiferianus, Vibrio campbellii and Vibrio alginolyticus), Vibrio mediterranei (=Vibrio shillonii) and Vibrio chagasii. With the exception of V. chagasii which was found only in the mucus of M. hispida, the other species appeared in different hosts with no evidence for the presence of host-specific clones or species. Using rep-PCR analysis, we observed a high genomic heterogeneity within the vibrios. Each vibrio isolate generated a different rep-PCR fingerprint pattern. There was a complete agreement between the grouping based on rep-PCR and concatenated sequences of pyrH, recA and 16S rDNA, but the pyrH gene has the highest discriminatory power for vibrio species identification. The vibrio core group is dominant in the mucus of these cnidarians. There is a tremendous diversity of vibrio lineages within the coral mucus. pyrH gene sequences permit a clear-cut identification of vibrios. The taxonomic resolution provided by pyrH (but not recA) appears to be enough for identifying species of vibrios and for disclosing putative new taxa. The vibrio core group appears to be dominant in the mucus of the Brazilian cnidarians. The overrepresentation of these vibrios may reflect as yet unknown ecological functions in the coral holobiont.
KeywordMeSH Terms
62. Okada  N, Iida  T, Park  KS, Goto  N, Yasunaga  T, Hiyoshi  H, Matsuda  S, Kodama  T, Honda  T,     ( 2009 )

Identification and characterization of a novel type III secretion system in trh-positive Vibrio parahaemolyticus strain TH3996 reveal genetic lineage and diversity of pathogenic machinery beyond the species level.

Infection and immunity 77 (2)
PMID : 19075025  :   DOI  :   10.1128/IAI.01184-08     PMC  :   PMC2632016    
Abstract >>
Vibrio parahaemolyticus is a bacterial pathogen causative of food-borne gastroenteritis. Whole-genome sequencing of V. parahaemolyticus strain RIMD2210633, which exhibits Kanagawa phenomenon (KP), revealed the presence of two sets of the genes for the type III secretion system (T3SS) on chromosomes 1 and 2, T3SS1 and T3SS2, respectively. Although T3SS2 of the RIMD2210633 strain is thought to be involved in human pathogenicity, i.e., enterotoxicity, the genes for T3SS2 have not been found in trh-positive (KP-negative) V. parahaemolyticus strains, which are also pathogenic for humans. In the study described here, the DNA region of approximately 100 kb that surrounds the trh gene of a trh-positive V. parahaemolyticus strain, TH3996, was sequenced and its genetic organization determined. This revealed the presence of the genes for a novel T3SS in this region. Animal experiments using the deletion mutant strains of a gene (vscC2) for the novel T3SS apparatus indicated that the T3SS is essential for the enterotoxicity of the TH3996 strain. PCR analysis showed that all the trh-positive V. parahaemolyticus strains tested possess the novel T3SS-related genes. Phylogenetic analysis demonstrated that although the novel T3SS is closely related to T3SS2 of KP-positive V. parahaemolyticus, it belongs to a distinctly different lineage. Furthermore, the two types of T3SS2 lineage are also found among pathogenic Vibrio cholerae non-O1/non-O139 strains. Our findings demonstrate that these two distinct types are distributed not only within a species but also beyond the species level and provide a new insight into the pathogenicity and evolution of Vibrio species.
KeywordMeSH Terms
Genetic Variation
63. Ansaruzzaman  M, Chowdhury  A, Bhuiyan  NA, Sultana  M, Safa  A, Lucas  M, von Seidlein  L, Barreto  A, Chaignat  CL, Sack  DA, Clemens  JD, Nair  GB, Choi  SY, Jeon  YS, Lee  JH, Lee  HR, Chun  J, Kim  DW,     ( 2008 )

Characteristics of a pandemic clone of O3 : K6 and O4 : K68 Vibrio parahaemolyticus isolated in Beira, Mozambique.

Journal of medical microbiology 57 (Pt 12)
PMID : 19018020  :   DOI  :   10.1099/jmm.0.2008/004275-0    
Abstract >>
The genetic characteristics of Vibrio parahaemolyticus strains isolated in 2004 and 2005 in Mozambique were assessed in this study to determine whether the pandemic clone of V. parahaemolyticus O3 : K6 and O4 : K68 serotypes has spread to Mozambique. Fifty-eight V. parahaemolyticus strains isolated from hospitalized diarrhoea patients in Beira, Mozambique, were serotyped for O : K antigens and genotyped for toxR, tdh and trh genes. A group-specific PCR, a PCR that detects the presence of ORF8 of the filamentous phage f237, arbitrarily primed PCR, PFGE and multilocus sequence typing were performed to determine the pandemic status of the strains and their ancestry. All strains of serovars O3 : K6 (n=38) and O4 : K68 (n=4) were identified as a pandemic clonal group by these analyses. These strains are closely related to the pandemic reference strains of O3 : K6 and O4 : K68, which emerged in Asia in 1996 and were later found globally. The pandemic serotypes O3 : K6 and O4 : K68 including reference strains grouped into a single cluster indicating emergence from a common ancestor. The O3 : K58 (n=8), O4 : K13 (n=6), O3 : KUT (n=1) and O8 : K41 (n=1) strains showed unique characteristics different from the pandemic clone.
KeywordMeSH Terms
Disease Outbreaks
Vibrio parahaemolyticus
64. Kamruzzaman  M, Bhoopong  P, Vuddhakul  V, Nishibuchi  M,     ( 2008 )

Detection of a functional insertion sequence responsible for deletion of the thermostable direct hemolysin gene (tdh) in Vibrio parahaemolyticus.

Gene 421 (1��2��)
PMID : 18598741  :   DOI  :   10.1016/j.gene.2008.06.009    
Abstract >>
The thermostable direct hemolysin coded by the tdh gene is a marker of virulent strains of Vibrio parahaemolyticus. The tdh genes are flanked by insertion sequences collectively named as ISVs or their remnants; but the ISVs so far examined have accumulated mutations in the transposase genes and underwent structural arrangements and their transposition activity could not be expected; the tdh gene was thus considered to have been acquired by V. parahaemolyticus through horizontal transfer in the past during evolution. We recently isolated from the same patient tdh+ strains and a tdh(-) strain (PCR examination) that were otherwise indistinguishable. The purpose of this study was to examine the hypothesis that the tdh(-) strain was derived from the tdh+ strain by a deletion of the tdh gene mediated by a functional ISV. Southern blot hybridization showed tdh+ sequences in the tdh(-) strain (PSU-1466). Nucleotide sequence analysis of the tdh and its flanking sequences revealed the tdh gene was split into two parts and they were located 3182-bp apart in PSU-1466. The two tdh sequences were flanked by one of the ISVs, named as ISVpa3, in PSU-1466. This genetic structure could be explained by an ISVpa3-mediated partial tdh deletion from a tdh+ strain followed by transposition of the duplicated ISVpa3 and the deleted tdh sequence into a neighboring location. The ISVpa3 of PSU-1466 coded for a full-length transposase and a DDE motif. We were able to demonstrate transposition activity of the ISVpa3 cloned from PSU-1466 using the replicon fusion assay with the conjugal transfer of a cointegrate from Escherichia coli to V. parahaemolyticus. Our data support ISVpa3-mediated partial tdh deletion resulted in the emergence of the tdh(-) strain.
KeywordMeSH Terms
DNA Transposable Elements
Gene Deletion
65. Okura  M, Osawa  R, Tokunaga  A, Morita  M, Arakawa  E, Watanabe  H,     ( 2008 )

Genetic analyses of the putative O and K antigen gene clusters of pandemic Vibrio parahaemolyticus.

Microbiology and immunology 52 (5)
PMID : 18557895  :   DOI  :   10.1111/j.1348-0421.2008.00027.x    
Abstract >>
Pandemic V. parahaemolyticus strains have rapidly changed their serotypes, but its determinants, especially K antigen, and the genes involved in serotype have been an open question. The purpose of this study was to gain insights into these points. Although V. parahaemolyticus is known to be lacking O-side chain on its lipopolysaccharide, and O antigens are thought to be represented by core OS, the genome sequence of V. parahaemolyticus O3:K6 strain RIMD2210633 suggests that this bacterium potentially synthesizes O-side chain. To explore possible relatedness between this O-side chain biosynthesis gene cluster, which is similar in the serotypes of Vibrio cholerae, and of V. parahaemolyticus, we amplified both core OS and O-side chain gene clusters of the strains belonging to various serotypes of V. parahaemolyticus by long PCR and performed PCR RFLP analyses. The results of our RFLP analyses suggest that the core OS biosynthesis gene cluster is related to the O antigens of pandemic V. parahaemolyticus and that the putative O-side chain gene cluster is related to K antigens of pandemic V. parahaemolyticus. We then determined the sequence of these regions of a pandemic O4:K68 strain, and compared it with the corresponding sequence of RIMD2210633. In addition, PCR analysis showed the putative O4 and K68 antigen gene clusters are unique to the strains belonging to the O4 and K68 serotype respectively. The data implies that the pandemic O4:K68 V. parahaemolyticus strain emerged from the pandemic O3:K6 strain by replacement of the putative O and K antigen gene clusters.
KeywordMeSH Terms
Multigene Family
66. Boyd  EF, Cohen  AL, Naughton  LM, Ussery  DW, Binnewies  TT, Stine  OC, Parent  MA,     ( 2008 )

Molecular analysis of the emergence of pandemic Vibrio parahaemolyticus.

BMC microbiology 8 (N/A)
PMID : 18590559  :   DOI  :   10.1186/1471-2180-8-110     PMC  :   PMC2491623    
Abstract >>
Vibrio parahaemolyticus is abundant in the aquatic environment particularly in warmer waters and is the leading cause of seafood borne gastroenteritis worldwide. Prior to 1995, numerous V. parahaemolyticus serogroups were associated with disease, however, in that year an O3:K6 serogroup emerged in Southeast Asia causing large outbreaks and rapid hospitalizations. This new highly virulent strain is now globally disseminated. We performed a four-way BLAST analysis on the genome sequence of V. parahaemolyticus RIMD2210633, an O3:K6 isolate from Japan recovered in 1996, versus the genomes of four published Vibrio species and constructed genome BLAST atlases. We identified 24 regions, gaps in the genome atlas, of greater than 10 kb that were unique to RIMD2210633. These 24 regions included an integron, f237 phage, 2 type III secretion systems (T3SS), a type VI secretion system (T6SS) and 7 Vibrio parahaemolyticus genomic islands (VPaI-1 to VPaI-7). Comparative genomic analysis of our fifth genome, V. parahaemolyticus AQ3810, an O3:K6 isolate recovered in 1983, identified four regions unique to each V. parahaemolyticus strain. Interestingly, AQ3810 did not encode 8 of the 24 regions unique to RMID, including a T6SS, which suggests an additional virulence mechanism in RIMD2210633. The distribution of only the VPaI regions was highly variable among a collection of 42 isolates and phylogenetic analysis of these isolates show that these regions are confined to a pathogenic clade. Our data show that there is considerable genomic flux in this species and that the new highly virulent clone arose from an O3:K6 isolate that acquired at least seven novel regions, which included both a T3SS and a T6SS.
KeywordMeSH Terms
Disease Outbreaks
Genome, Bacterial
67. Vongxay  K, Pan  Z, Zhang  X, Wang  S, Cheng  S, Mei  L, Xu  C, Fang  W,     ( 2008 )

Occurrence of pandemic clones of Vibrio parahaemolyticus isolates from seafood and clinical samples in a Chinese coastal province.

Foodborne pathogens and disease 5 (2)
PMID : 18370608  :   DOI  :   10.1089/fpd.2007.0045    
Abstract >>
Fifty-four isolates of Vibrio parahaemolyticus were examined for hemolytic and urease-producing phenotypes as well as presence of virulence markers by polymerase chain reaction (PCR). All clinical isolates (11/11, 100%) and one out of 42 isolates from seafood (2.4%) possessed the tdh gene and showed hemolysis. This tdh-positive seafood isolate as well as four clinical isolates belonged to the new pandemic clone O3:K6 according to serotyping and sequencing of the toxRS locus. The new O3:K6 clone, O1:KUT, and O4:K68 shared the same characteristic variations from the old O3:K6 clone at six base positions from 576 to 1244 of the toxRS locus. Seven clinical isolates (63.6%) were positive on both toxRS- and orf8-based PCRs. Our results indicate that the new pandemic O3:K6 clonal group of V. parahaemolyticus isolates from clinical and seafood sources are present in the southeastern coastal Chinese province Zhejiang and the pandemic clones of V. parahaemolyticus should be targeted for control of seafood-related transmission to humans.
KeywordMeSH Terms
68. Urbanczyk  H, Ast  JC, Kaeding  AJ, Oliver  JD, Dunlap  PV,     ( 2008 )

Phylogenetic analysis of the incidence of lux gene horizontal transfer in Vibrionaceae.

Journal of bacteriology 190 (10)
PMID : 18359809  :   DOI  :   10.1128/JB.00101-08     PMC  :   PMC2394989    
Abstract >>
Horizontal gene transfer (HGT) is thought to occur frequently in bacteria in nature and to play an important role in bacterial evolution, contributing to the formation of new species. To gain insight into the frequency of HGT in Vibrionaceae and its possible impact on speciation, we assessed the incidence of interspecies transfer of the lux genes (luxCDABEG), which encode proteins involved in luminescence, a distinctive phenotype. Three hundred three luminous strains, most of which were recently isolated from nature and which represent 11 Aliivibrio, Photobacterium, and Vibrio species, were screened for incongruence of phylogenies based on a representative housekeeping gene (gyrB or pyrH) and a representative lux gene (luxA). Strains exhibiting incongruence were then subjected to detailed phylogenetic analysis of horizontal transfer by using multiple housekeeping genes (gyrB, recA, and pyrH) and multiple lux genes (luxCDABEG). In nearly all cases, housekeeping gene and lux gene phylogenies were congruent, and there was no instance in which the lux genes of one luminous species had replaced the lux genes of another luminous species. Therefore, the lux genes are predominantly vertically inherited in Vibrionaceae. The few exceptions to this pattern of congruence were as follows: (i) the lux genes of the only known luminous strain of Vibrio vulnificus, VVL1 (ATCC 43382), were evolutionarily closely related to the lux genes of Vibrio harveyi; (ii) the lux genes of two luminous strains of Vibrio chagasii, 21N-12 and SB-52, were closely related to those of V. harveyi and Vibrio splendidus, respectively; (iii) the lux genes of a luminous strain of Photobacterium damselae, BT-6, were closely related to the lux genes of the lux-rib(2) operon of Photobacterium leiognathi; and (iv) a strain of the luminous bacterium Photobacterium mandapamensis was found to be merodiploid for the lux genes, and the second set of lux genes was closely related to the lux genes of the lux-rib(2) operon of P. leiognathi. In none of these cases of apparent HGT, however, did acquisition of the lux genes correlate with phylogenetic divergence of the recipient strain from other members of its species. The results indicate that horizontal transfer of the lux genes in nature is rare and that horizontal acquisition of the lux genes apparently has not contributed to speciation in recipient taxa.
KeywordMeSH Terms
Gene Transfer, Horizontal
69. González-Escalona  N, Martinez-Urtaza  J, Romero  J, Espejo  RT, Jaykus  LA, DePaola  A,     ( 2008 )

Determination of molecular phylogenetics of Vibrio parahaemolyticus strains by multilocus sequence typing.

Journal of bacteriology 190 (8)
PMID : 18281404  :   DOI  :   10.1128/JB.01808-07     PMC  :   PMC2293261    
Abstract >>
Vibrio parahaemolyticus is an important human pathogen whose transmission is associated with the consumption of contaminated seafood. There is a growing public health concern due to the emergence of a pandemic strain causing severe outbreaks worldwide. Many questions remain unanswered regarding the evolution and population structure of V. parahaemolyticus. In this work, we describe a multilocus sequence typing (MLST) scheme for V. parahaemolyticus based on the internal fragment sequences of seven housekeeping genes. This MLST scheme was applied to 100 V. parahaemolyticus strains isolated from geographically diverse clinical (n = 37) and environmental (n = 63) sources. The sequences obtained from this work were deposited and are available in a public database (http://pubmlst.org/vparahaemolyticus). Sixty-two unique sequence types were identified, and most (50) were represented by a single isolate, suggesting a high level of genetic diversity. Three major clonal complexes were identified by eBURST analysis. Separate clonal complexes were observed for V. parahaemolyticus isolates originating from the Pacific and Gulf coasts of the United States, while a third clonal complex consisted of strains belonging to the pandemic clonal complex with worldwide distribution. The data reported in this study indicate that V. parahaemolyticus is genetically diverse with a semiclonal population structure and an epidemic structure similar to that of Vibrio cholerae. Genetic diversity in V. parahaemolyticus appears to be driven primarily by frequent recombination rather than mutation, with recombination ratios estimated at 2.5:1 and 8.8:1 by allele and site, respectively. Application of this MLST scheme to more V. parahaemolyticus strains and by different laboratories will facilitate production of a global picture of the epidemiology and evolution of this pathogen.
KeywordMeSH Terms
70. Luan  X, Chen  J, Zhang  XH, Li  Y, Hu  G,     ( 2007 )

Expression and characterization of a metalloprotease from a Vibrio parahaemolyticus isolate.

Canadian journal of microbiology 53 (10)
PMID : 18026209  :   DOI  :   10.1139/W07-085    
Abstract >>
The extracellular zinc metalloprotease from Vibrio parahaemolyticus (VPM) is a putative virulence factor for host infection. It is synthesized from the vpm gene of V. parahaemolyticus as a polypeptide of 814 amino acids with an estimated molecular mass of 89,833 Da, containing a zinc metalloprotease HEXXH consensus motif. To investigate the enzymatic properties of V. parahaemolyticus metalloprotease, the mature vpm gene was overexpressed in Escherichia coli, and the recombinant protein (rVPM) was purified by a His-binding metal affinity column (>95% purity). The activity of the recombinant protease produced in E. coli was examined by gelatin activity staining and proteolytic activity assays using gelatin and azocasein as substrates. rVPM showed maximum activity at about 37 degrees C and pH 8. The cytotoxicity against flounder gill cells and fish pathogenicity indicated a potential role in pathogenesis.
KeywordMeSH Terms
Metalloproteases
71. Kamruzzaman  M, Nishibuchi  M,     ( 2008 )

Detection and characterization of a functional insertion sequence, ISVpa2, in Vibrio parahaemolyticus.

Gene 409 (1��2��)
PMID : 18164873  :   DOI  :   10.1016/j.gene.2007.11.012    
Abstract >>
PCR analysis of the pandemic strain of Vibrio parahaemolyticus, KX-V237 (total genome sequenced) showed a subculture where the size of the amplicons had increased. The purpose of this study was to analyze the mechanism of this change. We found a 1,243-bp DNA sequence inserted in one of the pandemic marker genes in this strain. The inserted DNA sequence possessed the genetic structures shared by insertion sequences (ISs) of the IS3 family. This IS had 26-bp imperfect terminal inverted repeats (IRs) and two partially overlapping reading frames, orfA and orfB. OrfA codes for a helix-turn-helix, OrfA and OrfAB produced by translational frameshifting code for leucine zipper motifs, and OrfB codes for a DDE motif. orfA and orfB were homologous to those in the IS3 family. This IS was named ISVpa2. Southern blot analysis showed the copy number of ISVpa2 in our stock culture and its subculture of KX-V237 was three and four, respectively; whereas it was only one in the reported genome sequence. Analysis of the flanking sequences for seven ISVpa2 copies showed ISVpa2 is capable of inserting at multiple sites and ISVpa2 causes genetic rearrangements including insertional inactivation of the target gene and adjacent deletion. ISVpa2 created 3-base duplications upon insertion. PCR, hybridization, and nucleotide sequence analyses showed ISVpa2 homologs were detected in all of the 62 other strains of V. parahaemolyticus examined; and in some strains of Vibrio vulnificus (98% identity), Vibrio penaeicida (86% identity), and Vibrio splendidus (87% identity); but was not in 25 other species in the genus Vibrio. The data demonstrate that ISVpa2 is a transpositionally active IS discovered for the first time in V. parahaemolyticus and suggest that ISVpa2 may be transferred among the species of the genus Vibrio.
KeywordMeSH Terms
DNA Transposable Elements
72. Sugiyama  T, Iida  T, Izutsu  K, Park  KS, Honda  T,     ( 2008 )

Precise region and the character of the pathogenicity island in clinical Vibrio parahaemolyticus strains.

Journal of bacteriology 190 (5)
PMID : 18156272  :   DOI  :   10.1128/JB.01293-07     PMC  :   PMC2258670    
Abstract >>
In this study, we determined the borders of the pathogenicity island in V. parahaemolyticus RIMD2210633 (Vp-PAI). Vp-PAI has features in common with Tn7 and other related elements at both terminal ends. Our findings indicate that the mobile element with a transposase which contains the DDE motif may have been involved in Vp-PAI formation.
KeywordMeSH Terms
73. Tanaka  Y, Kimura  B, Takahashi  H, Watanabe  T, Obata  H, Kai  A, Morozumi  S, Fujii  T,     ( 2008 )

Lysine decarboxylase of Vibrio parahaemolyticus: kinetics of transcription and role in acid resistance.

Journal of applied microbiology 104 (5)
PMID : 18031521  :   DOI  :   10.1111/j.1365-2672.2007.03652.x    
Abstract >>
The aim of this study was to investigate the detailed mechanisms of acid resistance in Vibrio parahaemolyticus. All 11 strains of V. parahaemolyticus survived lethal acidic conditions following acid adaptation, and accumulation of cadaverine was detected. The addition of lysine improved survival, suggesting that lysine decarboxylase plays a role in the adaptive acid tolerance response. Two open reading frames (ORF) in V. parahaemolyticus, which are separated by a noncoding region, were found to be highly homologous to bacterial lysine decarboxylase (cadA) and lysine/cadaverine antiporter (cadB) genes. Transcriptional analyses of this operon revealed acid induction and enhanced induction by external lysine. The relative expression ratio of each transcript was found to follow the trend of cadA mRNA > cadB mRNA > cadBA bi-cistronic mRNA. A mutated strain, with a disrupted cadA gene, showed attenuated acid survival. We identified the lysine decarboxylase gene operon of V. parahaemolyticus. Expression of this operon was induced under acidic conditions. The cadA-mutated strain constructed in this study showed weaker tolerance to acidic conditions than the wild-type strain. Vibrio parahaemolyticus utilizes the lysine decarboxylation pathway for survival in acidic conditions.
KeywordMeSH Terms
Food Microbiology
Transcription, Genetic
74. Thompson  CC, Thompson  FL, Vicente  AC, Swings  J,     ( 2007 )

Phylogenetic analysis of vibrios and related species by means of atpA gene sequences.

International journal of systematic and evolutionary microbiology 57 (Pt 11)
PMID : 17978204  :   DOI  :   10.1099/ijs.0.65223-0    
Abstract >>
We investigated the use of atpA gene sequences as alternative phylogenetic and identification markers for vibrios. A fragment of 1322 bp (corresponding to approximately 88% of the coding region) was analysed in 151 strains of vibrios. The relationships observed were in agreement with the phylogeny inferred from 16S rRNA gene sequence analysis. For instance, the Vibrio cholerae, Vibrio halioticoli, Vibrio harveyi and Vibrio splendidus species groups appeared in the atpA gene phylogenetic analyses, suggesting that these groups may be considered as separate genera within the current Vibrio genus. Overall, atpA gene sequences appeared to be more discriminatory for species differentiation than 16S rRNA gene sequences. 16S rRNA gene sequence similarities above 97% corresponded to atpA gene sequences similarities above 80%. The intraspecies variation in the atpA gene sequence was about 99% sequence similarity. The results showed clearly that atpA gene sequences are a suitable alternative for the identification and phylogenetic study of vibrios.
KeywordMeSH Terms
Bacterial Typing Techniques
Phylogeny
Sequence Analysis, DNA
75. Kadokura  K, Sakamoto  Y, Saito  K, Ikegami  T, Hirano  T, Hakamata  W, Oku  T, Nishio  T,     ( 2007 )

Production and secretion of a recombinant Vibrio parahaemolyticus chitinase by Escherichia coli and its purification from the culture medium.

Bioscience, biotechnology, and biochemistry 71 (11)
PMID : 17986788  :   DOI  :   10.1271/bbb.70389    
Abstract >>
An open reading frame encoding the chitinase gene and its signal sequence was cloned from the Vibrio parahaemolyticus KN1699 genome. An expression plasmid containing the gene was introduced into Escherichia coli cells, and recombinant chitinase (Pa-rChi) was produced and secreted into the culture medium with the aid of the signal peptide. Pa-rChi was purified and its substrate specificity was determined.
KeywordMeSH Terms
76. Hunt  DE, Gevers  D, Vahora  NM, Polz  MF,     ( 2008 )

Conservation of the chitin utilization pathway in the Vibrionaceae.

Applied and environmental microbiology 74 (1)
PMID : 17933912  :   DOI  :   10.1128/AEM.01412-07     PMC  :   PMC2223224    
Abstract >>
Vibrionaceae are regarded as important marine chitin degraders, and attachment to chitin regulates important biological functions; yet, the degree of chitin pathway conservation in Vibrionaceae is unknown. Here, a core chitin degradation pathway is proposed based on comparison of 19 Vibrio and Photobacterium genomes with a detailed metabolic map assembled for V. cholerae from published biochemical, genomic, and transcriptomic results. Further, to assess whether chitin degradation is a conserved property of Vibrionaceae, a set of 54 strains from 32 taxa were tested for the ability to grow on various forms of chitin. All strains grew on N-acetylglucosamine (GlcNAc), the monomer of chitin. The majority of isolates grew on alpha (crab shell) and beta (squid pen) chitin and contained chitinase A (chiA) genes. chiA sequencing and phylogenetic analysis suggest that this gene is a good indicator of chitin metabolism but appears subject to horizontal gene transfer and duplication. Overall, chitin metabolism appears to be a core function of Vibrionaceae, but individual pathway components exhibit dynamic evolutionary histories.
KeywordMeSH Terms
77. Mao  Z, Yu  L, You  Z, Wei  Y, Liu  Y,     ( 2007 )

Expression and immunogenicity analysis of two iron-regulated outer membrane proteins of Vibrio parahaemolyticus.

Acta biochimica et biophysica Sinica 39 (10)
PMID : 17928925  :   DOI  :   10.1111/j.1745-7270.2007.00339.x    
Abstract >>
Genes of two iron-regulated outer membrane proteins of Vibrio parahaemolyticus zj2003, a pathogenic strain isolated from large yellow croaker (Pseudosciaena crocea), psuA and pvuA, were cloned and expressed as N-terminal His(6)-tagged proteins in Escherichia coli BL(21)(DE(3)). The recombinant fusion proteins were purified with nickel chelate affinity chromatography. To analyze the immunogenicity of the proteins, groups of large yellow croaker were immunized with the purified recombinant psuA, pvuA or both, by intraperitoneal injection. Antibody response was assessed by enzyme-linked immunosorbent assay. Titers to the recombinant proteins increased from log(2) 3.25 to log(2) 9.80, 4-8 weeks following immunization. The relative percent survival of the groups vaccinated with psuA, pvuA, or a combination of the two, reached 50%, 62.5% and 75%, respectively. Western blot analysis was carried out with the serum from unvaccinated survival fish after infection. Both recombinant proteins were detected, indicating that these two proteins of V. parahaemolyticus zj2003 were immunogenic and could produce synergistic effects during in vivo infection, and they might be considered as important components for developing an aquaculture vaccine against this pathogen.
KeywordMeSH Terms
78. Coutard  F, Lozach  S, Pommepuy  M, Hervio-Heath  D,     ( 2007 )

Real-time reverse transcription-PCR for transcriptional expression analysis of virulence and housekeeping genes in viable but nonculturable Vibrio parahaemolyticus after recovery of culturability.

Applied and environmental microbiology 73 (16)
PMID : 17557845  :   DOI  :   10.1128/AEM.02776-06     PMC  :   PMC1950994    
Abstract >>
A real-time reverse transcription-PCR method was developed to determine whether the recovery of culturability of viable but nonculturable (VBNC) Vibrio parahaemolyticus induced the expression of virulence genes coding for the thermostable direct hemolysin and for type III secretion system 2 (TTSS2). The culturability of clinical strain Vp5 of V. parahaemolyticus in artificial seawater at 4 degrees C was monitored, and the VBNC state was obtained. One day after entry into the VBNC state, temperature upshifts to 20 and 37 degrees C allowed the recovery of culturability. Standard curves for the relative quantification of expression of the housekeeping genes rpoS, pvsA, fur, and pvuA; the tdh2 gene; and the TTSS2 genes (VPA1354, VPA1346, and VPA1342) were established. The levels of expression of the pvsA and pvuA genes were stable and were used to normalize the levels of expression of the other genes. No transcriptional induction of the selected virulence genes under the temperature conditions used to recover the culturability of the VBNC bacteria was observed. The study results demonstrate that the recovery of culturability of VBNC cells of pathogenic V. parahaemolyticus is restricted to regrowth, without correlation with the induction of virulence gene expression. Disease induction would depend mainly on host-pathogen interactions that allow the expression of the virulence genes. This is the first time that the use of mRNA to detect viable cells was evaluated by computing the half-lives of multiple mRNA species under conditions inducing the VBNC state.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
79. Li  R, Ye  L, Zheng  Z, Chan  EWC, Chen  S,     ( 2017 )

Genetic Characterization of Broad-Host-Range IncQ Plasmids Harboring blaVEB-18 in Vibrio Species.

Antimicrobial agents and chemotherapy 61 (7)
PMID : 28507104  :   DOI  :   10.1128/AAC.00708-17     PMC  :   PMC5487652    
Abstract >>
N/A
KeywordMeSH Terms
IncQ plasmids
Vibrio spp.
blaVEB-18
IncQ plasmids
Vibrio spp.
blaVEB-18
80. Shinoda  S, Matsuoka  H, Tsuchie  T, Miyoshi  S, Yamamoto  S, Taniguchi  H, Mizuguchi  Y,     ( 1991 )

Purification and characterization of a lecithin-dependent haemolysin from Escherichia coli transformed by a Vibrio parahaemolyticus gene.

Journal of general microbiology 137 (12)
PMID : 1791426  :   DOI  :   10.1099/00221287-137-12-2705    
Abstract >>
Lecithin-dependent haemolysin (LDH) of Vibrio parahaemolyticus was purified from Escherichia coli C600 transformed with a plasmid (pHL591) ligated with a 1.5 kb DNA fragment of V. parahaemolyticus. The final preparation comprised two LDH proteins with different molecular masses which were immunologically cross-reactive and had the same enzymic activity. The LDH was a phospholipase hydrolysing both fatty acid esters of phospholipid, i.e. it hydrolysed phosphatidylcholine (PC) to lysophosphatidylcholine (LPC) and then LPC to glycerophosphorylcholine (GPC). From this point of view, LDH should be classified as a phospholipase B. Phospholipase B, however, does not usually show haemolytic activity, because the intermediate (LPC), which is the actual haemolytic agent, is immediately hydrolysed to the final product (GPC). On the other hand, LPC formed by LDH action was comparatively stable, because the rates of the two reactions catalysed by LDH, PC to LPC and LPC to GPC, are almost the same. This is the reason that LDH shows haemolytic activity. Therefore, LDH of V. parahaemolyticus is an atypical phospholipase to be designated as phospholipase A2/lysophospholipase.
KeywordMeSH Terms
Transformation, Bacterial
81. Theethakaew  C, Nakamura  S, Motooka  D, Matsuda  S, Kodama  T, Chonsin  K, Suthienkul  O, Iida  T,     ( 2017 )

Plasmid dynamics in Vibrio parahaemolyticus strains related to shrimp Acute Hepatopancreatic Necrosis Syndrome (AHPNS).

Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 51 (N/A)
PMID : 28404482  :   DOI  :   10.1016/j.meegid.2017.04.007    
Abstract >>
Vibrio parahaemolyticus is a causative agent of acute hapatopancreatic necrosis syndrome (AHPNS) which causes early mortality in white shrimp. Emergence of AHPNS has caused tremendous economic loss for aquaculture industry particularly in Asia since 2010. Previous studies reported that strains causing AHPNS harbor a 69-kb plasmid with possession of virulence genes, pirA and pirB. However, genetic variation of the 69-kb plasmid among AHPNS related strains has not been investigated. This study aimed to analyze genetic composition and diversity of the 69-kb plasmid in strains isolated from shrimps affected by AHPNS. Plasmids recovered from V. parahaemolyticus strain VPE61 which represented typical AHPNS pathogenicity, strain VP2HP which did not represent AHPNS pathogenicity but was isolated from AHPNS affected shrimp and other AHPNS V. parahaemolyticus isolates in Genbank were investigated. Protein coding genes of the 69-kb plasmid from the strain VPE61 were identical to that of AHPNS strain from Vietnam except the inverted complement 3.4-kb transposon covering pirA and pirB. The strain VP2HP possessed remarkable large 183-kb plasmid which shared similar protein coding genes to those of the 69-kb plasmid from strain VPE61. However, the 3.4-kb transposon covering pirA and pirB was absent from the 183-kb plasmid in strain VP2HP. A number of protein coding genes from the 183-kb plasmid were also detected in other AHPNS strains. In summary, this study identified a novel 183-kb plasmid that is related to AHPNS causing strains. Homologous recombination of the 69-kb AHPNS plasmid and other naturally occurring plasmids together with loss and gain of AHPNS virulence genes in V. parahaemolyticus were observed. The outcome of this research enables understanding of plasmid dynamics that possibly affect variable degrees of AHPNS pathogenicity.
KeywordMeSH Terms
Acute hepatopancreatic necrosis syndrome
Genetic evolution
Plasmid
V. parahaemolyticus
82. Li  R, Ye  L, Zheng  Z, Chan  EW, Chen  S,     ( 2016 )

Genetic Characterization of a blaVEB-2-Carrying Plasmid in Vibrio parahaemolyticus.

Antimicrobial agents and chemotherapy 60 (11)
PMID : 27645248  :   DOI  :   10.1128/AAC.01749-16     PMC  :   PMC5075119    
Abstract >>
This report describes the first detection of a blaVEB-2 gene in a Vibrio parahaemolyticus strain isolated from a shrimp sample. The blaVEB-2 gene was carried on a novel Inc-type plasmid that was likely to have originated from aquatic organisms, as indicated by a comparison with other known genetic elements in the GenBank database. However, the plasmid contains resistance elements usually harbored by members of the family Enterobacteriaceae, suggesting that gene transfer events occurred and contributed to the formation of this multidrug resistance-encoding plasmid.
KeywordMeSH Terms
83. Nilsson  WB, Turner  JW,     ( 2016 )

The thermostable direct hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus: Sequence variation and implications for detection and function.

Journal of microbiological methods 126 (N/A)
PMID : 27094247  :   DOI  :   10.1016/j.mimet.2016.04.007    
Abstract >>
Vibrio parahaemolyticus is a leading cause of bacterial food-related illness associated with the consumption of undercooked seafood. Only a small subset of strains is pathogenic. Most clinical strains encode for the thermostable direct hemolysin (TDH) and/or the TDH-related hemolysin (TRH). In this work, we amplify and sequence the trh gene from over 80 trh+strains of this bacterium and identify thirteen genetically distinct alleles, most of which have not been deposited in GenBank previously. Sequence data was used to design new primers for more reliable detection of trh by endpoint PCR. We also designed a new quantitative PCR assay to target a more conserved gene that is genetically-linked to trh. This gene, ureR, encodes the transcriptional regulator for the urease gene cluster immediately upstream of trh. We propose that this ureR assay can be a useful screening tool as a surrogate for direct detection of trh that circumvents challenges associated with trh sequence variation.
KeywordMeSH Terms
Detection
PCR
Sequence variation
V. parahaemolyticus
trh
Genetic Variation
Real-Time Polymerase Chain Reaction
84. Li  R, Wong  MH, Zhou  Y, Chan  EW, Chen  S,     ( 2015 )

Complete nucleotide sequence of a conjugative plasmid carrying bla(PER-1).

Antimicrobial agents and chemotherapy 59 (6)
PMID : 25779581  :   DOI  :   10.1128/AAC.00518-15     PMC  :   PMC4432139    
Abstract >>
The nucleotide sequence of a self-transmissible plasmid pVPH1 harboring bla(PER-1) from Vibrio parahaemolyticus was determined. pVPH1 was 183,730 bp in size and shared a backbone similar to pAQU1 and pAQU2, differing mainly in an ?40-kb multidrug resistance (MDR) region. A complex class 1 integron was identified together with ISCR1 and bla(PER-1) (ISCR1-bla(PER-1)-gst-abct-qacE�G1-sul1), which was shown to form a circular intermediate playing an important role in the dissemination of bla(PER-1).
KeywordMeSH Terms
85. Leoni  F, Talevi  G, Masini  L, Ottaviani  D, Rocchegiani  E,     ( 2016 )

Trh (tdh-/trh+) gene analysis of clinical, environmental and food isolates of Vibrio parahaemolyticus as a tool for investigating pathogenicity.

International journal of food microbiology 225 (N/A)
PMID : 26990408  :   DOI  :   10.1016/j.ijfoodmicro.2016.02.016    
Abstract >>
Sequencing analysis of the trh gene encoding the TDH-related haemolysin of tdh-/trh+ Vibrio parahaemolyticus isolated in Italy between 2002 and 2011 from clinical, environmental, and food samples revealed the presence of the trh2 variant in all isolates. The trh2 of the clinical isolate was 100% identical to other clinical tdh-/trh2 V. parahaemolyticus from Europe. Nucleotide and amino acid differences in the trh2 sequences of clinical isolates from Italy and other countries allowed a differentiation of the clinical strains from the majority of environmental or food strains isolated in Italy. Aspartic acid and isoleucine at positions 113 and 115, encoded by nucleotide triplets GAT and ATT at positions 337-339 and 343-345 of the complete trh gene sequence, were present in clinical strains from Europe (Italy, Norway and Germany), Asia and the United States. Only 35.5% of the tdh-/trh2 V. parahaemolyticus of environmental or food origin from Italy shared the same triplets/amino acid detected in clinical isolates, while 64.5% of isolates from the marine environment were different from those of clinical origins, demonstrating that differences occur amongst the trh2 sequences of strains from the environment and these polymorphisms may differentiate potentially pathogenic from less or non-pathogenic cultures found in the environment and seafood. In addition the distribution of T3SS2 genes was investigated in this group of tdh-/trh+ V. parahaemolyticus from different sources and in three clinical tdh+/trh- V. parahaemolyticus isolates. All tdh-/trh+ V. parahaemolyticus of environmental or food source, independent of year of isolation or geographical origin, amplified all the screened T3SS2�] genes and tested negative to PCR assays for all five T3SS2�\ genes, as the tdh-/trh+ clinical V. parahaemolyticus isolate. The vopC genes, encoding for one of the effector proteins of T3SS2, were partially sequenced and compared to clinical tdh-/trh+ and tdh+/trh+ V. parahaemolyticus isolates from other countries. Analysis of T3SS2�] vopC sequences revealed variation in tdh-/trh2 isolates from Italy, which were separated from a group of vopC sequences derived from trh2 V. parahaemolyticus from the USA.
KeywordMeSH Terms
Gastrointestinal infection
Haemolysin
Pathogenicity-related genes
T3SS2
Trh gene sequence
V. parahaemolyticus
Environmental Microbiology
Food Microbiology
86. Caburlotto  G, Suffredini  E, Toson  M, Fasolato  L, Antonetti  P, Zambon  M, Manfrin  A,     ( 2016 )

Occurrence and molecular characterisation of Vibrio parahaemolyticus in crustaceans commercialised in Venice area, Italy.

International journal of food microbiology 220 (N/A)
PMID : 26773255  :   DOI  :   10.1016/j.ijfoodmicro.2015.12.007    
Abstract >>
Infections due to the pathogenic human vibrios, Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus, are mainly associated with consumption of raw or partially cooked bivalve molluscs. At present, little is known about the presence of Vibrio species in crustaceans and the risk of vibriosis associated with the consumption of these products. The aim of the present study was to evaluate the prevalence and concentration of the main pathogenic Vibrio spp. in samples of crustaceans (n=143) commonly eaten in Italy, taking into account the effects of different variables such as crustacean species, storage conditions and geographic origin. Subsequently, the potential pathogenicity of V. parahaemolyticus strains isolated from crustaceans (n=88) was investigated, considering the classic virulence factors (tdh and trh genes) and four genes coding for relevant proteins of the type III secretion systems 2 (T3SS2�\ and T3SS2�]). In this study, the presence of V. cholerae and V. vulnificus was never detected, whereas 40 samples (28%) were positive for V. parahaemolyticus with an overall prevalence of 41% in refrigerated products and 8% in frozen products. The highest prevalence and average contamination levels were detected in Crangon crangon (prevalence 58% and median value 3400 MPN/g) and in products from the northern Adriatic Sea (35%), with the samples from the northern Venetian Lagoon reaching a median value of 1375 MPN/g. While genetic analysis confirmed absence of the tdh gene, three of the isolates contained the trh gene and, simultaneously, the T3SS2�] genes. Moreover three possibly clonal tdh-negative/trh-negative isolates carried the T3SS2�\ apparatus. The detection of both T3SS2�\ and T3SS2�] apparatuses in V. parahaemolyticus strains isolated from crustaceans emphasised the importance of considering new genetic markers associated with virulence besides the classical factors. Moreover this study represents the first report dealing with Vibrio spp. in crustaceans in Italy, and it may provide useful information for the development of sanitary surveillance plans to prevent the risk of vibriosis in seafood consumers.
KeywordMeSH Terms
Enumeration
Most probable number (MPN)
Prevalence
T3SS2
tdh
trh
Food Microbiology
87. Whistler  CA, Hall  JA, Xu  F, Ilyas  S, Siwakoti  P, Cooper  VS, Jones  SH,     ( 2015 )

Use of Whole-Genome Phylogeny and Comparisons for Development of a Multiplex PCR Assay To Identify Sequence Type 36 Vibrio parahaemolyticus.

Journal of clinical microbiology 53 (6)
PMID : 25832299  :   DOI  :   10.1128/JCM.00034-15     PMC  :   PMC4432066    
Abstract >>
Vibrio parahaemolyticus sequence type 36 (ST36) strains that are native to the Pacific Ocean have recently caused multistate outbreaks of gastroenteritis linked to shellfish harvested from the Atlantic Ocean. Whole-genome comparisons of 295 genomes of V. parahaemolyticus, including several traced to northeastern U.S. sources, were used to identify diagnostic loci, one putatively encoding an endonuclease (prp), and two others potentially conferring O-antigenic properties (cps and flp). The combination of all three loci was present in only one clade of closely related strains of ST36, ST59, and one additional unknown sequence type. However, each locus was also identified outside this clade, with prp and flp occurring in only two nonclade isolates and cps in four. Based on the distribution of these loci in sequenced genomes, prp identified clade strains with >99% accuracy, but the addition of one more locus increased accuracy to 100%. Oligonucleotide primers targeting prp and cps were combined in a multiplex PCR method that defines species using the tlh locus and determines the presence of both the tdh and trh hemolysin-encoding genes, which are also present in ST36. Application of the method in vitro to a collection of 94 clinical isolates collected over a 4-year period in three northeastern U.S. states and 87 environmental isolates revealed that the prp and cps amplicons were detected only in clinical isolates identified as belonging to the ST36 clade and in no environmental isolates from the region. The assay should improve detection and surveillance, thereby reducing infections.
KeywordMeSH Terms
88. Xu  F, Ilyas  S, Hall  JA, Jones  SH, Cooper  VS, Whistler  CA,     ( 2015 )

Genetic characterization of clinical and environmental Vibrio parahaemolyticus from the Northeast USA reveals emerging resident and non-indigenous pathogen lineages.

Frontiers in microbiology 6 (N/A)
PMID : 25904905  :   DOI  :   10.3389/fmicb.2015.00272     PMC  :   PMC4387542    
Abstract >>
Gastric infections caused by the environmentally transmitted pathogen, Vibrio parahaemolyticus, have increased over the last two decades, including in many parts of the United States (US). However, until recently, infections linked to shellfish from the cool northeastern US waters were rare. Cases have risen in the Northeast, consistent with changes in local V. parahaemolyticus populations toward greater abundance or a shift in constituent pathogens. We examined 94 clinical isolates from a period of increasing disease in the region and compared them to 200 environmental counterparts to identify resident and non-indigenous lineages and to gain insight into the emergence of pathogenic types. Genotyping and multi-locus sequence analysis (MLSA) of clinical isolates collected from 2010 to 2013 in Massachusetts, New Hampshire, and Maine revealed their polyphyletic nature. Although 80% of the clinical isolates harbored the trh hemolysin either alone or with tdh, and were urease positive, 14% harbored neither hemolysin exposing a limitation for these traits in pathogen detection. Resident sequence type (ST) 631 strains caused seven infections, and show a relatively recent history of recombination with other clinical and environmental lineages present in the region. ST34 and ST674 strains were each linked to a single infection and these strain types were also identified from the environment as isolates harboring hemolysin genes. Forty-two ST36 isolates were identified from the clinical collection, consistent with reports that this strain type caused a rise in regional infections starting in 2012. Whole-genome phylogenies that included three ST36 outbreak isolates traced to at least two local sources demonstrated that the US Atlantic coastal population of this strain type was indeed derived from the Pacific population. This study lays the foundation for understanding dynamics within natural populations associated with emergence and invasion of pathogenic strain types in the region.
KeywordMeSH Terms
MLSA
Vibriosis
disease ecology
emergent pathogen
hemolysin
pathogen evolution
population structure
89. Hazen  TH, Lafon  PC, Garrett  NM, Lowe  TM, Silberger  DJ, Rowe  LA, Frace  M, Parsons  MB, Bopp  CA, Rasko  DA, Sobecky  PA,     ( 2015 )

Insights into the environmental reservoir of pathogenic Vibrio parahaemolyticus using comparative genomics.

Frontiers in microbiology 6 (N/A)
PMID : 25852665  :   DOI  :   10.3389/fmicb.2015.00204     PMC  :   PMC4371758    
Abstract >>
Vibrio parahaemolyticus is an aquatic halophilic bacterium that occupies estuarine and coastal marine environments, and is a leading cause of seafood-borne food poisoning cases. To investigate the environmental reservoir and potential gene flow that occurs among V. parahaemolyticus isolates, the virulence-associated gene content and genome diversity of a collection of 133 V. parahaemolyticus isolates were analyzed. Phylogenetic analysis of housekeeping genes, and pulsed-field gel electrophoresis, demonstrated that there is genetic similarity among V. parahaemolyticus clinical and environmental isolates. Whole-genome sequencing and comparative analysis of six representative V. parahaemolyticus isolates was used to identify genes that are unique to the clinical and environmental isolates examined. Comparative genomics demonstrated an O3:K6 environmental isolate, AF91, which was cultured from sediment collected in Florida in 2006, has significant genomic similarity to the post-1995 O3:K6 isolates. However, AF91 lacks the majority of the virulence-associated genes and genomic islands associated with these highly virulent post-1995 O3:K6 genomes. These findings demonstrate that although they do not contain most of the known virulence-associated regions, some V. parahaemolyticus environmental isolates exhibit significant genetic similarity to clinical isolates. This highlights the dynamic nature of the V. parahaemolyticus genome allowing them to transition between aquatic and host-pathogen states.
KeywordMeSH Terms
O3:K6
Vibrio parahaemolyticus
environment
genomics
phylogenomics
90. Chiou  J, Li  R, Chen  S,     ( 2015 )

CARB-17 family of �]-lactamases mediates intrinsic resistance to penicillins in Vibrio parahaemolyticus.

Antimicrobial agents and chemotherapy 59 (6)
PMID : 25801555  :   DOI  :   10.1128/AAC.00047-15     PMC  :   PMC4432138    
Abstract >>
Vibrio parahaemolyticus is commonly resistant to ampicillin, yet the mechanisms underlying this phenomenon are not clear. In this study, a novel class A carbenicillin-hydrolyzing �]-lactamase (CARB) family of �]-lactamases, bla(CARB-17), was identified and found to be responsible for the intrinsic penicillin resistance in V. parahaemolyticus. Importantly, bla(CARB-17)-like genes were present in all 293 V. parahaemolyticus genome sequences available in GenBank and detectable in all 91 V. parahaemolyticus food isolates, further confirming the intrinsic nature of this gene.
KeywordMeSH Terms
91. Hirano  T, Sugiyama  K, Sakaki  Y, Hakamata  W, Park  SY, Nishio  T,     ( 2015 )

Structure-based analysis of domain function of chitin oligosaccharide deacetylase from Vibrio parahaemolyticus.

FEBS letters 589 (1)
PMID : 25479092  :   DOI  :   10.1016/j.febslet.2014.11.039    
Abstract >>
The X-ray crystal structure of chitin oligosaccharide deacetylase from Vibrio parahaemolyticus (Vp-COD) was determined at an 1.35 ? resolution. The amino acid sequence and structure of Vp-COD show that the enzyme comprises one polysaccharide deacetylase domain (PDD) and two carbohydrate-binding domains (CBDs). On the basis of a chitin-binding assay with Vp-COD and its CBDs-deleted mutant, it was confirmed that CBDs can adhere to chitin. The catalytic activity of the CBDs-deleted mutant was only mildly depressed compared with that of Vp-COD, indicating that CBDs are unlikely to affect the configuration of the active center residues in active site of PDD.
KeywordMeSH Terms
Binding assay
Chitin oligosaccharide deacetylase
Domain function
Enzyme mutation
Kinetics
Protein structure
92. Rykovskaia  OA, Smolikova  LM, Monakhova  EV, Chemisova  OS, Podoĭnitsyna  OA, Golenishcheva  EN, Sanamiants  EM, Sagakiants  MM, Dalikova  RR,     ( N/A )

[Collection of Vibrio parahaemolyticus species members: phenotypic and genotypic characteristics].

Zhurnal mikrobiologii, epidemiologii, i immunobiologii N/A (6)
PMID : 25816506  :  
Abstract >>
Formation of Vibrio parahaemolyticus collection according to modern methodical opportunities and understanding of causative agent biology. Traditional biochemical tests and PCR-testing of species-specific genes were used to confirm species membership. Catalase, DNAse, proteolytic and tweenase activity was determined by common methods. Virulence was evaluated by a complex method: hemolytic activity was determined in Kanagawa test (KT), urease--in Christensen medium, PRC-testing of tdh and-trh genes. Serotyping was carried out with a commercial O/K-sera kit. PCR-genotyping was carried out by marker genes of 7 pathogenicity islands (VPaI-1-7). Species membership was confirmed for the studied strains. Serologic typing allowed to detect members of 18 serologic groups among the collection strains. All the collection cultures were divided into 4 groups based on KT-Ure-tdh-trh features recombination. A number of genetic variants were detected, strains belonging to a pandemic group and O3:K6 serogroup were determined. A collection of V. parahaemolyticus cultures was formed and characterized by a large set of pheno- and genotypic features. A database was developed including information on strain origins, pheno- and genetic features, with genetic variants given, for ease of use of the collection.
KeywordMeSH Terms
Genes, Bacterial
Genotype
Phenotype
93. Esteves  K, Mosser  T, Aujoulat  F, Hervio-Heath  D, Monfort  P, Jumas-Bilak  E,     ( 2015 )

Highly diverse recombining populations of Vibrio cholerae and Vibrio parahaemolyticus in French Mediterranean coastal lagoons.

Frontiers in microbiology 6 (N/A)
PMID : 26236294  :   DOI  :   10.3389/fmicb.2015.00708     PMC  :   PMC4503927    
Abstract >>
Vibrio parahaemolyticus and Vibrio cholerae are ubiquitous to estuarine and marine environments. These two species found in Mediterranean coastal systems can induce infections in humans. Environmental isolates of V. cholerae (n = 109) and V. parahaemolyticus (n = 89) sampled at different dates, stations and water salinities were investigated for virulence genes and by a multilocus sequence-based analysis (MLSA). V. cholerae isolates were all ctxA negative and only one isolate of V. parahaemolyticus displayed trh2 gene. Most Sequence Types (ST) corresponded to unique ST isolated at one date or one station. Frequent recombination events were detected among different pathogenic species, V. parahaemolyticus, V. cholerae, Vibrio mimicus, and Vibrio metoecus. Recombination had a major impact on the diversification of lineages. The genetic diversity assessed by the number of ST/strain was higher in low salinity condition for V. parahaemolyticus and V. cholerae whereas the frequency of recombination events in V. cholerae was lower in low salinity condition. Mediterranean coastal lagoon systems housed V. cholerae and V. parahaemolyticus with genetic diversities equivalent to the worldwide diversity described so far. The presence of STs found in human infections as well as the frequency of recombination events in environmental vibrios populations could predict a potential epidemiological risk.
KeywordMeSH Terms
French coastal lagoons
Vibrio
human pathogens
multi-locus sequence analysis
phylogeny
recombination
virulence factor
French coastal lagoons
Vibrio
human pathogens
multi-locus sequence analysis
phylogeny
recombination
virulence factor
94. Lee  CT, Chen  IT, Yang  YT, Ko  TP, Huang  YT, Huang  JY, Huang  MF, Lin  SJ, Chen  CY, Lin  SS, Lin  SS, Lightner  DV, Wang  HC, Wang  AH, Wang  HC, Hor  LI, Lo  CF,     ( 2015 )

The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin.

Proceedings of the National Academy of Sciences of the United States of America 112 (34)
PMID : 26261348  :   DOI  :   10.1073/pnas.1503129112     PMC  :   PMC4553777    
Abstract >>
Acute hepatopancreatic necrosis disease (AHPND) is a severe, newly emergent penaeid shrimp disease caused by Vibrio parahaemolyticus that has already led to tremendous losses in the cultured shrimp industry. Until now, its disease-causing mechanism has remained unclear. Here we show that an AHPND-causing strain of V. parahaemolyticus contains a 70-kbp plasmid (pVA1) with a postsegregational killing system, and that the ability to cause disease is abolished by the natural absence or experimental deletion of the plasmid-encoded homologs of the Photorhabdus insect-related (Pir) toxins PirA and PirB. We determined the crystal structure of the V. parahaemolyticus PirA and PirB (PirA(vp) and PirB(vp)) proteins and found that the overall structural topology of PirA(vp)/PirB(vp) is very similar to that of the Bacillus Cry insecticidal toxin-like proteins, despite the low sequence identity (<10%). This structural similarity suggests that the putative PirAB(vp) heterodimer might emulate the functional domains of the Cry protein, and in particular its pore-forming activity. The gene organization of pVA1 further suggested that pirAB(vp) may be lost or acquired by horizontal gene transfer via transposition or homologous recombination.
KeywordMeSH Terms
AHPND
Pir toxin
Vibrio parahaemolyticus
shrimp
virulence plasmid
95. Han  JE, Tang  KF, Tran  LH, Lightner  DV,     ( 2015 )

Photorhabdus insect-related (Pir) toxin-like genes in a plasmid of Vibrio parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease (AHPND) of shrimp.

Diseases of aquatic organisms 113 (1)
PMID : 25667334  :   DOI  :   10.3354/dao02830     PMC  :   PMC4785170    
Abstract >>
The 69 kb plasmid pVPA3-1 was identified in Vibrio parahaemolyticus strain 13?028/A3 that can cause acute hepatopancreatic necrosis disease (AHPND). This disease is responsible for mass mortalities in farmed penaeid shrimp and is referred to as early mortality syndrome (EMS). The plasmid has a GC content of 45.9% with a copy number of 37 per bacterial cell as determined by comparative quantitative PCR analyses. It consists of 92 open reading frames that encode mobilization proteins, replication enzymes, transposases, virulence-associated proteins, and proteins similar to Photorhabdus insect-related (Pir) toxins. In V. parahaemolyticus, these Pir toxin-like proteins are encoded by 2 genes (pirA- and pirB-like) located within a 3.5 kb fragment flanked with inverted repeats of a transposase-coding sequence (1 kb). The GC content of these 2 genes is only 38.2%, substantially lower than that of the rest of the plasmid, which suggests that these genes were recently acquired. Based on a proteomic analysis, the pirA-like (336 bp) and pirB-like (1317 bp) genes encode for 13 and 50 kDa proteins, respectively. In laboratory cultures of V. parahaemolyticus 13-028/A3, both proteins were secreted into the culture medium. We developed a duplex PCR diagnostic method, with a detection limit of 10(5) CFU ml(-1) and targeting pirA- and pirB-like genes in this strain of V. parahaemolyticus. This PCR protocol can reliably detect AHPND-causing strains of V. parahaemolyticus and does not cross react with non-pathogenic strains or with other species of Vibrio isolated from shrimp ponds.
KeywordMeSH Terms
96. Machado  H, Gram  L,     ( 2015 )

The fur gene as a new phylogenetic marker for Vibrionaceae species identification.

Applied and environmental microbiology 81 (8)
PMID : 25662978  :   DOI  :   10.1128/AEM.00058-15     PMC  :   PMC4375339    
Abstract >>
Microbial taxonomy is essential in all areas of microbial science. The 16S rRNA gene sequence is one of the main phylogenetic species markers; however, it does not provide discrimination in the family Vibrionaceae, where other molecular techniques allow better interspecies resolution. Although multilocus sequence analysis (MLSA) has been used successfully in the identification of Vibrio species, the technique has several limitations. They include the fact that several locus amplifications and sequencing have to be performed, which still sometimes lead to doubtful identifications. Using an in silico approach based on genomes from 103 Vibrionaceae strains, we demonstrate here the high resolution of the fur gene in the identification of Vibrionaceae species and its usefulness as a phylogenetic marker. The fur gene showed within-species similarity higher than 95%, and the relationships inferred from its use were in agreement with those observed for 16S rRNA analysis and MLSA. Furthermore, we developed a fur PCR sequencing-based method that allowed identification of Vibrio species. The discovery of the phylogenetic power of the fur gene and the development of a PCR method that can be used in amplification and sequencing of the gene are of general interest whether for use alone or together with the previously suggested loci in an MLSA.
KeywordMeSH Terms
Phylogeny
97. Erler  R, Wichels  A, Heinemeyer  EA, Hauk  G, Hippelein  M, Reyes  NT, Gerdts  G,     ( 2015 )

VibrioBase: A MALDI-TOF MS database for fast identification of Vibrio spp. that are potentially pathogenic in humans.

Systematic and applied microbiology 38 (1)
PMID : 25466918  :   DOI  :   10.1016/j.syapm.2014.10.009    
Abstract >>
Mesophilic marine bacteria of the family Vibrionaceae, specifically V. cholerae, V. parahaemolyticus and V. vulnificus, are considered to cause severe illness in humans. Due to climate-change-driven temperature increases, higher Vibrio abundances and infections are predicted for Northern Europe, which in turn necessitates environmental surveillance programs to evaluate this risk. We propose that whole-cell matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling is a promising tool for the fast and reliable species classification of environmental isolates. Because the reference database does not contain sufficient Vibrio spectra we generated the VibrioBase database in this study. Mass spectrometric data were generated from 997 largely environmental strains and filed in this new database. MALDI-TOF MS clusters were assigned based on the species classification obtained by analysis of partial rpoB (RNA polymerase beta-subunit) sequences. The affiliation of strains to species-specific clusters was consistent in 97% of all cases using both approaches, and the extended VibrioBase generated more specific species identifications with higher matching scores compared to the commercially available database. Therefore, we have made the VibrioBase database freely accessible, which paves the way for detailed risk assessment studies of potentially pathogenic Vibrio spp. from marine environments.
KeywordMeSH Terms
Database
MALDI-TOF MS
Marine
Pathogenic
Vibrio
Whole-cell fingerprinting
Database
MALDI-TOF MS
Marine
Pathogenic
Vibrio
Whole-cell fingerprinting
Database
MALDI-TOF MS
Marine
Pathogenic
Vibrio
Whole-cell fingerprinting
Database
MALDI-TOF MS
Marine
Pathogenic
Vibrio
Whole-cell fingerprinting
Database
MALDI-TOF MS
Marine
Pathogenic
Vibrio
Whole-cell fingerprinting
Database
MALDI-TOF MS
Marine
Pathogenic
Vibrio
Whole-cell fingerprinting
Database
MALDI-TOF MS
Marine
Pathogenic
Vibrio
Whole-cell fingerprinting
Database
MALDI-TOF MS
Marine
Pathogenic
Vibrio
Whole-cell fingerprinting
Database
MALDI-TOF MS
Marine
Pathogenic
Vibrio
Whole-cell fingerprinting
Database
MALDI-TOF MS
Marine
Pathogenic
Vibrio
Whole-cell fingerprinting
Database
MALDI-TOF MS
Marine
Pathogenic
Vibrio
Whole-cell fingerprinting
Database
MALDI-TOF MS
Marine
Pathogenic
Vibrio
Whole-cell fingerprinting
Database
MALDI-TOF MS
Marine
Pathogenic
Vibrio
Whole-cell fingerprinting
Database
MALDI-TOF MS
Marine
Pathogenic
Vibrio
Whole-cell fingerprinting
Bacterial Typing Techniques
Databases, Chemical
98. Rahman  MS, Martino  ME, Cardazzo  B, Facco  P, Bordin  P, Mioni  R, Novelli  E, Fasolato  L,     ( 2014 )

Vibrio trends in the ecology of the Venice lagoon.

Applied and environmental microbiology 80 (8)
PMID : 24487545  :   DOI  :   10.1128/AEM.04133-13     PMC  :   PMC3993166    
Abstract >>
Vibrio is a very diverse genus that is responsible for different human and animal diseases. The accurate identification of Vibrio at the species level is important to assess the risks related to public health and diseases caused by aquatic organisms. The ecology of Vibrio spp., together with their genetic background, represents an important key for species discrimination and evolution. Thus, analyses of population structure and ecology association are necessary for reliable characterization of bacteria and to investigate whether bacterial species are going through adaptation processes. In this study, a population of Vibrionaceae was isolated from shellfish of the Venice lagoon and analyzed in depth to study its structure and distribution in the environment. A multilocus sequence analysis (MLSA) was developed on the basis of four housekeeping genes. Both molecular and biochemical approaches were used for species characterization, and the results were compared to assess the consistency of the two methods. In addition, strain ecology and the association between genetic information and environment were investigated through statistical models. The phylogenetic and population analyses achieved good species clustering, while biochemical identification was demonstrated to be imprecise. In addition, this study provided a fine-scale overview of the distribution of Vibrio spp. in the Venice lagoon, and the results highlighted a preferential association of the species toward specific ecological variables. These findings support the use of MLSA for taxonomic studies and demonstrate the need to consider environmental information to obtain broader and more accurate bacterial characterization.
KeywordMeSH Terms
Ecosystem
Seawater
99. Jiang  W, Han  X, Wang  Q, Li  X, Yi  L, Liu  Y, Ding  C,     ( 2014 )

Vibrio parahaemolyticus enolase is an adhesion-related factor that binds plasminogen and functions as a protective antigen.

Applied microbiology and biotechnology 98 (11)
PMID : 24430205  :   DOI  :   10.1007/s00253-013-5471-z    
Abstract >>
Vibrio parahaemolyticus, an emerging food and waterborne pathogen, is a leading cause of seafood poisoning worldwide. Surface proteins can directly participate in microbial virulence by facilitating pathogen dissemination via interactions with host factors. Screening and identification of protective antigens is important for developing therapies against V. parahaemolyticus infections. Here, we systematically characterized a novel immunogenic enolase of V. parahaemolyticus. The enolase gene of V. parahaemolyticus ATCC33847 was cloned, sequenced, and expressed in Escherichia coli BL21. Enzymatic assays revealed that the purified recombinant V. parahaemolyticus enolase protein catalyzes the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate. Western blot analysis showed that V. parahaemolyticus enolase was detectable in the extracellular, outer membrane (OM) and cytoplasmic protein fractions using antibodies against the recombinant enolase. Surface expression of enolase was further confirmed by immunogold staining and mass spectrometry (liquid chromatography-tandem mass spectrometry) analysis of OM protein profiles. Notably, V. parahaemolyticus enolase was identified as a human plasminogen-binding protein with the enzyme-linked immunosorbent assay. The values obtained for adherence and inhibition suggest a role of surface-exposed enolase in epithelial adherence of V. parahaemolyticus. We further showed that enolase confers efficient immunity against challenge with a lethal dose of V. parahaemolyticus in a mouse model. To our knowledge, this is the first study to demonstrate the plasminogen-binding activity of enolase that is an adhesion-related factor of V. parahaemolyticus. Our findings collectively imply that enolase plays important roles in pathogenicity, supporting its utility as a novel vaccine candidate against V. parahaemolyticus infection.
KeywordMeSH Terms
100. Kumar  BK, Deekshit  VK, Rai  P, Shekar  M, Karunasagar  I, Karunasagar  I,     ( 2014 )

Presence of T3SS2�] genes in trh? Vibrio parahaemolyticus isolated from seafood harvested along Mangalore coast, India.

Letters in applied microbiology 58 (5)
PMID : 24372411  :   DOI  :   10.1111/lam.12210    
Abstract >>
Vibrio parahaemolyticus is a seafood-borne pathogen autochthonous to the marine and estuarine ecosystem, responsible for gastroenteritis when contaminated raw seafood is consumed. The pathogenicity has been associated with thermostable direct haemolysin (TDH) and TDH-related haemolysin (TRH). Of late, the presence of T3SS2�\ and T3SS2�] gene clusters has been well documented in clinical isolates of Vibrio parahaemolyticus and known to play an essential role in pathogenesis. However, reports on the presence of T3SS�] genes in V. parahaemolyticus isolated from the seafood and/or environmental samples are scanty. In this study, we have identified and analysed the distribution of the T3SS2�] genes in V. parahaemolyticus isolated from seafood harvested along southwest coast of India. Results showed that T3SS2�] genes are solely associated with trh? and tdh? /trh? strains of V. parahaemolyticus. Reverse transcriptase PCR (RT-PCR) showed that the T3SS2�] genes identified in trh? V. parahaemolyticus were transcriptionally active. To our knowledge, this study appears to be the first description on the presence of T3SS2�]-positive V. parahaemolyticus isolated from seafood in India. The study of T3SS2 along with other virulence factors will help in better understanding of the risk of seafood-borne illness due to V. parahaemolyticus. T3SSs (�\ or �]) are the important virulence factors of Vibrio parahaemolyticus that contribute to their pathogenicity in humans. This study demonstrated the presence of T3SS2�] genes in V. parahaemolyticus isolated from the seafood harvested along Mangalore coast. RT-PCR showed that the T3SS2�] genes identified in seafood isolates of V. parahaemolyticus were found to be functional. To the best of our knowledge, this is the first description of T3SS2�] genes in trh? V. parahaemolyticus isolated from seafood in India. The presence of T3SS2 along with other virulence factors such as TDH and/or TRH highlights a potential health risk for seafood consumers.
KeywordMeSH Terms
TDH-related haemolysin
V. parahaemolyticus
seafood
thermostable direct haemolysin
type III secretion system
Food Contamination
101. Liu  M, Wong  MH, Chen  S,     ( 2013 )

Mechanisms of fluoroquinolone resistance in Vibrio parahaemolyticus.

International journal of antimicrobial agents 42 (2)
PMID : 23751355  :   DOI  :   10.1016/j.ijantimicag.2013.04.024    
Abstract >>
N/A
KeywordMeSH Terms
102. Li  L, Lin  SL, Deng  L, Liu  ZG,     ( 2013 )

Potential use of chitosan nanoparticles for oral delivery of DNA vaccine in black seabream Acanthopagrus schlegelii Bleeker to protect from Vibrio parahaemolyticus.

Journal of fish diseases 36 (12)
PMID : 24093149  :   DOI  :   10.1111/jfd.12032    
Abstract >>
To develop an effective and easy-to-administer vaccine against vibriosis of fish, the chitosan nanoparticles-loaded DNA vaccine against Vibrio parahaemolyticus was studied. A DNA vaccine was constructed using the outer membrane protein K (ompK) gene of V. parahaemolyticus strain (OS4) and pEGFP-N2 , a eukaryotic expression vector, and the construct was named pEGFP-N2 -OMPK (pDNA). The pDNA was encapsulated in chitosan particles (chitosan/pDNA). The effective diameter, mean diameter and polydispersity of the particles were 284.4 nm, 218.9 nm and 0.160, respectively. Scanning electron microscopy showed that the particles are dispersed as individual nanoparticles with spherical shape of around 200 nm and are homogeneously distributed. Encapsulation efficiency and loading percentage of nanoparticles were 91.5% and 2.08%, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) showed that RNA-containing information of the ompK gene existed in mid-intestine, liver, kidney and muscle 3 weeks after oral administration in black seabream Acanthopagrus schlegelii Bleeker. Expression of the reporter gene, green fluorescent protein (GFP), was observed in the above-mentioned tissues by fluorescence microscopy. Expression of the ompK gene within 3 weeks evoked an immune response. Black seabream was protected from V. parahaemolyticus (OS4), with 72.3% relative percentage survival (RPS) 3 weeks post-vaccination with chitosan/pDNA. The direct agglutination test indicated that oral administration with chitosan/pDNA induced an antibody immune response in fish against V. parahaemolyticus (OS4). Data obtained, here and in other related studies, suggest that chitosan nanoparticles are promising carriers for an oral pDNA vaccine.
KeywordMeSH Terms
DNA vaccine
Vibrio parahaemolyticus
chitosan
oral vaccination
outer membrane protein K
Sea Bream
103. Klein  SL, Gutierrez West  CK, Mejia  DM, Lovell  CR,     ( 2014 )

Genes similar to the Vibrio parahaemolyticus virulence-related genes tdh, tlh, and vscC2 occur in other vibrionaceae species isolated from a pristine estuary.

Applied and environmental microbiology 80 (2)
PMID : 24212573  :   DOI  :   10.1128/AEM.02895-13     PMC  :   PMC3911104    
Abstract >>
Detection of the human pathogen Vibrio parahaemolyticus often relies on molecular biological analysis of species-specific virulence factor genes. These genes have been employed in determinations of V. parahaemolyticus population numbers and the prevalence of pathogenic V. parahaemolyticus strains. Strains of the Vibrionaceae species Photobacterium damselae, Vibrio diabolicus, Vibrio harveyi, and Vibrio natriegens, as well as strains similar to Vibrio tubiashii, were isolated from a pristine salt marsh estuary. These strains were examined for the V. parahaemolyticus hemolysin genes tdh, trh, and tlh and for the V. parahaemolyticus type III secretion system 2�\ gene vscC2 using established PCR primers and protocols. Virulence-related genes occurred at high frequencies in non-V. parahaemolyticus Vibrionaceae species. V. diabolicus was of particular interest, as several strains were recovered, and the large majority (>83%) contained virulence-related genes. It is clear that detection of these genes does not ensure correct identification of virulent V. parahaemolyticus. Further, the occurrence of V. parahaemolyticus-like virulence factors in other vibrios potentially complicates tracking of outbreaks of V. parahaemolyticus infections.
KeywordMeSH Terms
Estuaries
104. Zhang  H, You  C, Ren  J, Xu  D, Han  M, Liao  W,     ( 2014 )

A simple one-step PCR walking method and its application of bacterial rRNA for sequencing identification.

Current microbiology 68 (2)
PMID : 24126601  :   DOI  :   10.1007/s00284-013-0462-y    
Abstract >>
There are many PCR walking methods applied currently, and they all have examples of successful application in organisms which are more complex than bacteria. However, to a certain extent, it will be more convenient for researchers if the complicated operation and poor specificity for bacteria can be improved. Here, we introduced an improved one-step PCR walking method of bacteria. Using a specific primer of the known sequence together with a universal semi-random primer, the unknown sequence adjacent to a known sequence can be obtained easily by just one ordinary round PCR. The products can be gel-purified and directly sequenced. Specific primers were designed according to the gene sequence of bacterial rRNA, and the variable and adjacent gene sequences were obtained by this method. The sequence analysis of the product showed that it can improve the resolution of bacterial identification to the species level.
KeywordMeSH Terms
RNA, Bacterial
RNA, Ribosomal
105. Liu  M, Wong  MH, Chen  S,     ( 2013 )

Molecular characterisation of a multidrug resistance conjugative plasmid from Vibrio parahaemolyticus.

International journal of antimicrobial agents 42 (6)
PMID : 24139885  :   DOI  :   10.1016/j.ijantimicag.2013.08.014    
Abstract >>
Vibrio parahaemolyticus is a major causative agent of gastroenteritis and is the leading cause of food-borne illness in Hong Kong. Recent studies of resistance to extended-spectrum �]-lactams and fluoroquinolones in V. parahaemolyticus have caused huge concern. This work reports the characterisation of a multidrug resistance conjugative plasmid in V. parahaemolyticus isolated from shrimp samples from Hong Kong. The plasmid is ca. 200 kb and carries multidrug resistance genes, including a novel plasmid-mediated quinolone resistance gene qnrVC6 surrounded by several known and novel insertion sequence (IS) elements, an extended-spectrum �]-lactamase gene bla(PER-1) mediated by ISCR1, and a ca. 3-kb four-gene cassette (aacA3, catB2, dfrA1 and aadA1) class 1 integron. Transmission of this multidrug resistance conjugative plasmid among Vibrio spp. would compromise the effectiveness of Vibrio infection control and pose a huge threat to public health.
KeywordMeSH Terms
Multidrug resistance
Novel integron
Vibrio parahaemolyticus
bla(PER-1)
qnrVC6
Conjugation, Genetic
Drug Resistance, Multiple, Bacterial
Plasmids
106. Gavilan  RG, Zamudio  ML, Martinez-Urtaza  J,     ( 2013 )

Molecular epidemiology and genetic variation of pathogenic Vibrio parahaemolyticus in Peru.

PLoS neglected tropical diseases 7 (5)
PMID : 23696906  :   DOI  :   10.1371/journal.pntd.0002210     PMC  :   PMC3656152    
Abstract >>
Vibrio parahaemolyticus is a foodborne pathogen that has become a public health concern at the global scale. The epidemiological significance of V. parahaemolyticus infections in Latin America received little attention until the winter of 1997 when cases related to the pandemic clone were detected in the region, changing the epidemic dynamics of this pathogen in Peru. With the aim to assess the impact of the arrival of the pandemic clone on local populations of pathogenic V. parahaemolyticus in Peru, we investigated the population genetics and genomic variation in a complete collection of non-pandemic strains recovered from clinical sources in Peru during the pre- and post-emergence periods of the pandemic clone. A total of 56 clinical strains isolated in Peru during the period 1994 to 2007, 13 strains from Chile and 20 strains from Asia were characterized by Multilocus Sequence Typing (MLST) and checked for the presence of Variable Genomic Regions (VGRs). The emergence of O3:K6 cases in Peru implied a drastic disruption of the seasonal dynamics of infections and a shift in the serotype dominance of pathogenic V. parahaemolyticus. After the arrival of the pandemic clone, a great diversity of serovars not previously reported was detected in the country, which supports the introduction of additional populations cohabitating with the pandemic group. Moreover, the presence of genomic regions characteristic of the pandemic clone in other non-pandemic strains may represent early evidence of genetic transfer from the introduced population to the local communities. Finally, the results of this study stress the importance of population admixture, horizontal genetic transfer and homologous recombination as major events shaping the structure and diversity of pathogenic V. parahaemolyticus.
KeywordMeSH Terms
Genetic Variation
107. Gode-Potratz  CJ, McCarter  LL,     ( 2011 )

Quorum sensing and silencing in Vibrio parahaemolyticus.

Journal of bacteriology 193 (16)
PMID : 21705592  :   DOI  :   10.1128/JB.00432-11     PMC  :   PMC3147687    
Abstract >>
The quorum regulatory cascade is poorly characterized in Vibrio parahaemolyticus, in part because swarming and virulence factors--the hallmarks of the organism--are repressed by this scheme of gene control, and quorum sensing seems to be silenced in many isolates. In these studies, we examine a swarming-proficient, virulent strain and identify an altered-function allele of the quorum regulator luxO that is demonstrated to produce a constitutively active mimic of LuxO?P. We find that LuxO* affects the expression of three small regulatory RNAs (Qrrs) and the activity of a translational fusion in opaR, the output regulator. Tests for epistasis showed that luxO* is dominant over luxO and that opaR is dominant over luxO. Thus, information flow through the central elements of the V. parahaemolyticus quorum pathway is proven for the first time. Quorum-sensing output was explored using microarray profiling: the OpaR regulon encompasses ?5.2% of the genome. OpaR represses the surface-sensing and type III secretion system 1 (T3SS1) regulons. One novel discovery is that OpaR strongly and oppositely regulates two type VI secretion systems (T6SS). New functional consequences of OpaR control were demonstrated: OpaR increases the cellular cyclic di-GMP (c-di-GMP) level, positively controls chitin-induced DNA competency, and profoundly blocks cytotoxicity toward host cells. In expanding the previously known quorum effects beyond the induction of the capsule and the repression of swarming to elucidate the global scope of genes in the OpaR regulon, this study yields many clues to distinguishing traits of this Vibrio species; it underscores the profoundly divergent survival strategies of the quorum On/Off phase variants.
KeywordMeSH Terms
Gene Silencing
108. Cariani  A, Piano  A, Consolandi  C, Severgnini  M, Castiglioni  B, Caredda  G, Candela  M, Serratore  P, De Bellis  G, Tinti  F,     ( 2012 )

Detection and characterization of pathogenic vibrios in shellfish by a Ligation Detection Reaction-Universal Array approach.

International journal of food microbiology 153 (3)
PMID : 22177227  :   DOI  :   10.1016/j.ijfoodmicro.2011.11.010    
Abstract >>
Vibrios are a group of major foodborne pathogens widely distributed in marine environment. Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus are the pathogenic species of Vibrio that pose the greatest threat to human health. However, other vibrios, e.g. Vibrio alginolyticus, Vibrio mimicus and Grimontia hollisae, apparently less relevant in the group of foodborne pathogens, have been sporadically found in outbreaks. For seafood safety and economic purposes, a rapid and powerful method for the specific identification of harmful Vibrio strains is needed. We developed a PCR-Ligase Detection Reaction-Universal Array (PCR-LDR-UA) assay for the simultaneous identification of pathogenic vibrios and detection of virulence coding genes. The entire procedure was validated on a total of 31 reference strains and isolates from clinical and environmental samples, as well as on bivalve tissue homogenates infected with different strains of target Vibrio species. Twenty-three shellfish samples directed to human consumption were successfully screened, thus demonstrating that the developed microarray-based platform could be a reliable and sensitive detection tool for the identification of harmful Vibrio strains in seafood.
KeywordMeSH Terms
109. Figge  MJ, Robertson  LA, Ast  JC, Dunlap  PV,     ( 2011 )

Historical microbiology: revival and phylogenetic analysis of the luminous bacterial cultures of M. W. Beijerinck.

FEMS microbiology ecology 78 (3)
PMID : 22066815  :   DOI  :   10.1111/j.1574-6941.2011.01177.x    
Abstract >>
Luminous bacteria isolated by Martinus W. Beijerinck were sealed in glass ampoules in 1924 and 1925 and stored under the names Photobacterium phosphoreum and 'Photobacterium splendidum'. To determine if the stored cultures were viable and to assess their evolutionary relationship with currently recognized bacteria, portions of the ampoule contents were inoculated into culture medium. Growth and luminescence were evident after 13 days of incubation, indicating the presence of viable cells after more than 80 years of storage. The Beijerinck strains are apparently the oldest bacterial cultures to be revived from storage. Multi-locus sequence analysis, based on the 16S rRNA, gapA, gyrB, pyrH, recA, luxA, and luxB genes, revealed that the Beijerinck strains are distant from the type strains of P. phosphoreum, ATCC 11040(T), and Vibrio splendidus, ATCC 33125(T), and instead form an evolutionarily distinct clade of Vibrio. Newly isolated strains from coastal seawater in Norway, France, Uruguay, Mexico, and Japan grouped with the Beijerinck strains, indicating a global distribution for this new clade, designated as the beijerinckii clade. Strains of the beijerinckii clade exhibited little sequence variation for the seven genes and approximately 6300 nucleotides examined despite the geographic distances and the more than 80 years separating their isolation. Gram-negative bacteria therefore can survive for many decades in liquid storage, and in nature, they do not necessarily diverge rapidly over time.
KeywordMeSH Terms
Luminescence
Phylogeny
110. Collin  B, Rehnstam-Holm  AS,     ( 2011 )

Occurrence and potential pathogenesis of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus on the South Coast of Sweden.

FEMS microbiology ecology 78 (2)
PMID : 21692819  :   DOI  :   10.1111/j.1574-6941.2011.01157.x    
Abstract >>
During the summer of 2006, several wound infections - of which three were fatal - caused by Vibrio cholerae were reported from patients who had been exposed to water from the Baltic Sea. Before these reports, we initiated a sampling project investigating the occurrence of potential human pathogenic V. cholerae, Vibrio vulnificus and Vibrio parahaemolyticus in The Sound between Sweden and Denmark. The Blue mussel (Mytilus edulis) was used as an indicator to follow the occurrence of vibrios over time. Molecular analyses showed high frequencies of the most potent human pathogenic Vibrio spp.; 53% of mussel samples were positive for V. cholerae (although none were positive for the cholera toxin gene), 63% for V. vulnificus and 79% for V. parahaemolyticus (of which 47% were tdh(+) and/or trh(+)). Viable vibrios were also isolated from the mussel meat and screened for virulence by PCR. The mortality of eukaryotic cells when exposed to bacteria was tested in vivo, with results showing that the Vibrio strains, independent of species and origin, were harmful to the cells. Despite severe infections and several deaths, no report on potential human pathogenic vibrios in this area had been published before this study.
KeywordMeSH Terms
Water Microbiology
111. Chen  W, Xie  Y, Xu  J, Wang  Q, Gu  M, Yang  J, Zhou  M, Wang  D, Shi  C, Shi  X,     ( 2012 )

Molecular typing of Vibrio parahaemolyticus isolates from the middle-east coastline of China.

International journal of food microbiology 153 (3)
PMID : 22225982  :   DOI  :   10.1016/j.ijfoodmicro.2011.12.001    
Abstract >>
The occurrence of outbreaks of Vibrio parahaemolyticus gastroenteritis in China highlights the need for strain characterization and subtyping of this pathogenic species. A total of 56 epidemiologically-unrelated strains of V. parahaemolyticus were isolated from clinical samples, seafood and various environmental sites in the middle-east coastline of China from 2006 to 2008. The isolates were characterized using four molecular typing methods, including ribotyping, enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), pulsed-field gel electrophoresis (PFGE), and sequence analysis of the gyrB gene. Genetic profiles of cluster analysis from these molecular typing tests clearly showed that there were differences in potential pathogenicity among isolates from seafood and its environments. Genetic characterization of two isolates (F13 and QS2) that originated from seafood demonstrated that they were potentially pathogenic. Discriminatory indices of four typing methods for the 56 V. parahaemolyticus isolates were differentiated by Simpson's Index of Diversity. The discriminatory index of ERIC-PCR typing was maximal (D=0.942), while that of sequence analysis of the gyrB gene was minimal (D=0.702). The discriminatory ability was greatly enhanced (D=0.966) when ERIC-PCR was coupled with sequence analysis of the gyrB gene. These results suggest that ERIC-PCR combined with sequence analysis of gyrB gene may be a reliable, rapid typing strategy for V. parahaemolyticus strains.
KeywordMeSH Terms
Environmental Monitoring
112. Hoffmann  M, Monday  SR, Fischer  M, Brown  EW,     ( 2012 )

Genetic and phylogenetic evidence for misidentification of Vibrio species within the Harveyi clade.

Letters in applied microbiology 54 (2)
PMID : 22118600  :   DOI  :   10.1111/j.1472-765X.2011.03183.x    
Abstract >>
This report describes the use of a six-gene multi-locus sequence analysis (MLSA) to correctly identify Vibrio strains of the Harveyi clade. Vibrio isolates were characterized using a six housekeeping gene MLSA. The study provided evidence supporting: (i) a substantial number of reference strains maintained within commercial culture collections are misidentified taxonomically at the species level; (ii) two V. alginolyticus subclades retain species-level divergence; and (iii) V. communis and V. owensii likely are the same species. A significant number (n = 10) of Harveyi clade Vibrio strains have been inaccurately identified, including evidence that V. communis and V. owensii strains, two recently discovered species assigned to the Harveyi clade, comprise a single species. As Harveyi clade vibrios have an enormous impact on human and aquatic animal health, it is of paramount importance to identify members of the Harveyi clade correctly.
KeywordMeSH Terms
Phylogeny
113. Oberbeckmann  S, Wichels  A, Wiltshire  KH, Gerdts  G,     ( 2011 )

Occurrence of Vibrio parahaemolyticus and Vibrio alginolyticus in the German Bight over a seasonal cycle.

Antonie van Leeuwenhoek 100 (2)
PMID : 21598011  :   DOI  :   10.1007/s10482-011-9586-x    
Abstract >>
Bacteria of the genus Vibrio are an important component of marine ecosystems worldwide. The genus harbors several human pathogens, for instance the species Vibrio parahaemolyticus, a main cause for foodborne gastroenteritis in Asia and the USA. Pathogenic V. parahaemolyticus strains emerged also in Europe, but little is known about the abundance, pathogenicity and ecology of V. parahaemolyticus especially in Northern European waters. This study focuses on V. parahaemolyticus and its close relative Vibrio alginolyticus in the North Sea (Helgoland Roads, Germany). Free-living, plankton-attached and shellfish-associated Vibrio spp. were quantified between May 2008 and January 2010. CFUs up to 4.3 �� 10(3) N l(-1) and MPNs up to 240 N g(-1) were determined. Phylogenetic classification based on rpoB gene sequencing revealed V. alginolyticus as the dominant Vibrio species at Helgoland Roads, followed by V. parahaemolyticus. We investigated the intraspecific diversity of V. parahaemolyticus and V. alginolyticus using ERIC-PCR. The fingerprinting disclosed three distinct groups at Helgoland Roads, representing V. parahaemolyticus, V. alginolyticus and one group in between. The species V. parahaemolyticus occurred mainly in summer months. None of the strains carried the virulence-associated genes tdh or trh. We further analyzed the influence of nutrients, secchi depth, temperature, salinity, chlorophyll a and phytoplankton on the abundance of Vibrio spp. and the population structure of V. parahaemolyticus. Spearman Rank analysis revealed that particularly temperature correlated significantly with Vibrio spp. numbers. Based on multivariate statistical analyses we report that the V. parahaemolyticus population was structured by a complex combination of environmental parameters. To further investigate these influences is the key to understanding the dynamics of Vibrio spp. in temperate European waters, where this microbial group and especially the pathogenic species, are likely to gain in importance.
KeywordMeSH Terms
114.     ( 1996 )

Vibrio parahaemolyticus FlaJ, a homologue of FliS, is required for production of a flagellin.

Molecular microbiology 20 (1)
PMID : 8861212  :   DOI  :   10.1111/j.1365-2958.1996.tb02496.x    
Abstract >>
The flaA locus of Vibrio parahaemolyticus encodes one of the four polar flagellin genes, the flagellum-capping protein HAP2, and three additional flagellar genes. Sequence analysis downstream of the gene encoding HAP2 revealed the region to be similar to the fliD (HAP2) locus of Pseudomonas aeruginosa. The deduced protein sequences for the newly identified genes suggest that one protein belongs to the family of transcriptional regulatory proteins known to interact with sigma (54), one may be a rod component of the flagellum, and one resembles the FliS protein. fliS is an essential flagellar gene in many bacteria; however its function is not clear. The V. parahaemolyticus polar flaC flagellin gene was poorly expressed in Escherichia coli Production of FlaC was stimulated by provision of the flaA locus in trans. Dissection of this locus revealed that the fliS-like gene, flaJ, was required for increased expression of flaC. Stimulation by FlaJ occurred in E.coli mutants defective in either the master flagellar-controlling operon or the gene encoding the flagellar sigma (28). Therefore the effect of FlaJ was not mediated through flagellar proteins. Nor was it mediated through sigma (54) for enhanced FlaC production was observed in mutants with defects in the gene encoding sigma (54).
KeywordMeSH Terms
DNA-Binding Proteins
115.     ( 1996 )

Sequence of a Na+/glucose symporter gene and its flanking regions of Vibrio parahaemolyticus.

Biochimica et biophysica acta 1281 (1)
PMID : 8652595  :   DOI  :   10.1016/0005-2736(96)00025-9    
Abstract >>
The nucleotide sequence of an approximately 6 kbp segment of chromosomal DNA of Vibrio parahaemolyticus was determined. The nucleotide sequence revealed four open reading frames (ORFs) in this region. Hydropathy profiles of the deduced amino acid sequence of the ORFs indicate that ORF1 encodes a hydrophobic polypeptide with typical characteristics of a membrane transport protein. All other ORFs encode hydrophilic polypeptides. ORF1 showed significant amino acid sequence similarity to proteins of the SGLT (Na+/glucose symporter) family, and the amino acid sequence of ORF4 showed very high similarity to several bacterial transcriptional repressor proteins (GalR-LacI family). We observed elevated glucose transport activity in cells harboring a plasmid carrying the DNA region corresponding to ORF1, and the glucose transport was greatly stimulated by Na+. Thus, we believe that ORF1 encodes a Na+/glucose symporter.
KeywordMeSH Terms
116.     ( 1995 )

Cloning and sequence analysis of Vibrio parahaemolyticus ompK gene encoding a 26-kDa outer membrane protein, OmpK, that serves as receptor for a broad-host-range vibriophage, KVP40.

FEMS microbiology letters 134 (2��3��)
PMID : 8586275  :   DOI  :   10.1111/j.1574-6968.1995.tb07945.x    
Abstract >>
The ompK gene of Vibrio parahaemolyticus 1010 (RIMD 2210001) encoding an outer membrane protein (OMP), OmpK, which serves as the receptor for a broad-host-range vibriophage, KVP40, was cloned and sequenced. The gene consisted of 789 nucleotides encoding 263 amino acids. Since the first 20 amino acids most likely constitute the signal peptide, mature OmpK would consist of 243 amino acids with a calculated molecular mass of 27458 Da. Sequence comparisons indicate that OmpK is unique among Vibrio OMPs so far sequenced, but may be distantly related to Tsx of enteric bacteria and is homologous to an Aeromonas hydrophila OMP, protein IV.
KeywordMeSH Terms
Genes, Bacterial
117.     ( 1994 )

Demonstration and characterization of simultaneous production of a thermostable direct hemolysin (TDH/I) and a TDH-related hemolysin (TRHx) by a clinically isolated Vibrio parahaemolyticus strain, TH3766.

Infection and immunity 62 (1)
PMID : 8262624  :   PMC  :   PMC186082    
Abstract >>
Simultaneous production of a thermostable direct hemolysin (TDH)-like toxin (TDHx) and a TDH-related hemolysin (TRH)-like toxin (TRHx) by a clinical isolate (strain TH3766) of Kanagawa phenomenon-positive Vibrio parahaemolyticus was demonstrated and characterized. The two hemolysins were differentially purified by column chromatography on hydroxyapatite and immunoaffinity columns. The molecular weight of the two hemolysins were estimated to be 23,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE). The purified TDHx was indistinguishable from the previously reported TDH/I (from strain TH012) but was different from the authentic TDH of a Kanagawa phenomenon-positive strain (T4750) physicochemically. The mobility of TRHx in nondenaturing PAGE differed from all the known TDHs and TRHs. The genes (tdhX and trhX) coding for TDHx and TRHx were cloned and sequenced. Homologies of nucleotide sequences of the coding regions between tdhX and tdhA (a gene for the authentic TDH) and between trhX and trh (a gene for the authentic TRH) were 98.1 and 99.1%, respectively, and homology between tdhX and trhX was 68.1%. At the amino acid level, TdhX was completely identical to TDH/I, although two base differences were found in the nucleotide sequences between tdhX and tdh/I. Two amino acid differences were observed between TrhX and Trh. Thus, these findings suggest that the TH3766 strain produces two types of hemolysins simultaneously. This is the first evidence that a strain of V. parahaemolyticus produces two types of toxins of the TDH-TRH family at the same time.
KeywordMeSH Terms
118.     ( 1993 )

Identification of genes encoding components of the swarmer cell flagellar motor and propeller and a sigma factor controlling differentiation of Vibrio parahaemolyticus.

Journal of bacteriology 175 (11)
PMID : 8501040  :   DOI  :   10.1128/jb.175.11.3361-3371.1993     PMC  :   PMC204733    
Abstract >>
Vibrio parahaemolyticus possesses two distinct motility systems, the polar system used for swimming in liquid environments and the lateral system used for swarming over surfaces. Growth on surfaces induces swarmer cell differentiation and expression of the lateral motility system. Mutants, created by transposon mutagenesis of a clone expressing lateral flagellin and gene disruption in V. parahaemolyticus, were unable to swarm and failed to make lateral flagellin; therefore, unlike the case for the polar system, there is one gene (lafA) encoding lateral flagellin. In addition to lafA, other genes required for swarming but not for swimming were identified by gene replacement mutagenesis. The nucleotide sequence of the clone determined open reading frames (ORFs) and deduced amino acid sequences showed similarities to flagellar components of other bacteria: flagellin, hook-associated protein (HAP2), motor components, and flagellar sigma factor (sigma 28). Many sigma 28 factors have been shown to recognize cognate promoters; however, expression of lafA in Escherichia coli required LafS, and E. coli sigma 28 did not substitute. Also, there were no sequences preceding genes encoding flagellin or HAP2 resembling the sigma 28 consensus promoter. The product of the sigma-like gene seems to be a unique member of the sigma 28 cluster. It appears the result of requiring expression for immunodetection of flagellin clones was that the sigma locus was fortuitously cloned, since the sigma and lafA loci were not contiguous in the chromosome. This work initiates identification and placement of genes in a scheme of control for swarmer cell differentiation; three levels have been identified in the transcriptional hierarchy.
KeywordMeSH Terms
119.     ( 1994 )

MotY, a component of the sodium-type flagellar motor.

Journal of bacteriology 176 (14)
PMID : 8021208  :   DOI  :   10.1128/jb.176.14.4219-4225.1994     PMC  :   PMC205632    
Abstract >>
Energy to power the rotation of bacterial flagella can be derived from the proton or sodium transmembrane potential. Until now, genes encoding a bacterial sodium-type flagellar motor have not been defined. A gene, motY, encoding one component of the sodium-type flagellar motor of Vibrio parahaemolyticus was cloned by complementation of a Mot- mutant strain. Sequencing revealed an open reading frame of 879 nucleotides in which a transposon conferring a motility defect mapped. Overexpression of motY in Escherichia coli allowed identification of a product 33 kDa in apparent size on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This size correlated well with the predicted molecular mass of 33,385 Da. Unlike mot genes identified in other bacteria, localized transposon mutagenesis suggested that the locus was not an extended region containing multiple genes required for swimming motility. Sequencing upstream and downstream of motY confirmed that the gene maps alone and placed it within a locus homologous to the E. coli rnt locus. Although data bank searches failed to reveal significant similarity to known motility components, the carboxyl terminus of MotY showed extensive homology to a number of outer membrane proteins known to interact with peptidoglycan, including OmpA and peptidoglycan-associated lipoproteins. To a limited extent, this domain could also be identified in the Bacillus subtilis MotB protein. This finding suggests that MotY plays the role of a stator in the sodium flagellar motor, stabilizing the force-generating unit through direct interaction with the cell wall.
KeywordMeSH Terms
120. Gildemeister  OS, Zhu  BC, Laine  RA,     ( 1994 )

Chitovibrin: a chitin-binding lectin from Vibrio parahemolyticus.

Glycoconjugate journal 11 (6)
PMID : 7696854  :  
Abstract >>
A novel 134 kDa, calcium-independent chitin-binding lectin, 'chitovibrin', is secreted by the marine bacterium Vibrio parahemolyticus, inducible with chitin or chitin-oligomers. Chitovibrin shows no apparent enzymatic activity but exhibits a strong affinity for chitin and chito-oligomers > dp9. The protein has an isoelectric pH of 3.6, shows thermal tolerance, binds chitin with an optimum at pH 6 and is active in 0-4 M NaCl. Chitovibrin appears to be completely different from other reported Vibrio lectins and may function to bind V. parahemolyticus to chitin substrates, or to capture or sequester chito-oligomers. It may be a member of a large group of recently described proteins in Vibrios related to a complex chitinoclastic (chitinivorous) system.
KeywordMeSH Terms
Bacterial Proteins
121. Inoue  T, Matsuzaki  S, Tanaka  S,     ( 1995 )

A 26-kDa outer membrane protein, OmpK, common to Vibrio species is the receptor for a broad-host-range vibriophage, KVP40.

FEMS microbiology letters 125 (1)
PMID : 7867914  :   DOI  :   10.1111/j.1574-6968.1995.tb07342.x    
Abstract >>
KVP40 is a broad-host-range vibriophage forming plaques on strains of at least eight Vibrio and one Photobacterium species. A spontaneous KVP40-resistant mutant, R4000, derived from Vibrio parahaemolyticus 1010 lacked a 26-kDa outer membrane protein designated OmpK. KVP40 was inactivated by outer membrane and OmpK prepared from 1010, but not by outer membrane from R4000. These results strongly suggest that OmpK is the receptor for KVP40. Immunoblotting analyses using an anti-OmpK rabbit serum revealed that OmpK or its homologs of molecular masses 25-29 kDa were distributed widely among Vibrio and Photobacterium strains including those naturally resistant to KVP40.
KeywordMeSH Terms
122. McCarter  LL,     ( 1995 )

Genetic and molecular characterization of the polar flagellum of Vibrio parahaemolyticus.

Journal of bacteriology 177 (6)
PMID : 7883718  :   DOI  :   10.1128/jb.177.6.1595-1609.1995     PMC  :   PMC176778    
Abstract >>
Vibrio parahaemolyticus possesses two alternate flagellar systems adapted for movement under different circumstances. A single polar flagellum propels the bacterium in liquid (swimming), while multiple lateral flagella move the bacterium over surfaces (swarming). Energy to rotate the polar flagellum is derived from the sodium membrane potential, whereas lateral flagella are powered by the proton motive force. Lateral flagella are arranged peritrichously, and the unsheathed filaments are polymerized from a single flagellin. The polar flagellum is synthesized constitutively, but lateral flagella are produced only under conditions in which the polar flagellum is not functional, e.g., on surfaces. This work initiates characterization of the sheathed, polar flagellum. Four genes encoding flagellins were cloned and found to map in two loci. These genes, as well as three genes encoding proteins resembling HAPs (hook-associated proteins), were sequenced. A potential consensus polar flagellar promoter was identified by using upstream sequences from seven polar genes. It resembled the enterobacterial sigma 28 consensus promoter. Three of the four flagellin genes were expressed in Escherichia coli, and expression was dependent on the product of the fliA gene encoding sigma 28. The fourth flagellin gene may be different regulated. It was not expressed in E. coli, and inspection of upstream sequence revealed a potential sigma 54 consensus promoter. Mutants with single and multiple defects in flagellin genes were constructed in order to determine assembly rules for filament polymerization. HAP mutants displayed new phenotypes, which were different from those of Salmonella typhimurium and most probably were the result of the filament being sheathed.
KeywordMeSH Terms
123.     ( 1994 )

Properties and sequence of the NhaA Na+/H+ antiporter of Vibrio parahaemolyticus.

Journal of biochemistry 116 (5)
PMID : 7896730  :   DOI  :   10.1093/oxfordjournals.jbchem.a124624    
Abstract >>
A gene encoding an Na+/H+ antiporter was cloned from chromosomal DNA of the slightly halophilic marine bacterium Vibrio parahaemolyticus. The host was an Escherichia coli mutant that lacked both of the two major Na+/H+ antiporters, NhaA and NhaB. Untransformed mutant cells were unable to grow in the presence of 0.6 M NaCl or 0.1 M LiCl, but Na+ and Li+ were non-toxic to cells transformed with a plasmid carrying the antiporter gene. Membrane vesicles prepared from the original E. coli mutant did not show any detectable Na+/H+ (and Li+/H+) antiport activity. However, we observed high Na+/H+ (and Li+/H+) antiport activity in membrane vesicles prepared from the transformed cells. The activity increased greatly when the pH of the assay medium was increased from 7.0 and 8.5. This property is very similar to that of the NhaA Na+/H+ antiporter of E. coli. Drastic decreases in Km values for Li+ and Na+ were observed with membrane vesicles prepared from the transformed cells compared with those observed with V. parahaemolyticus vesicles. The amino acid sequence deduced from the nucleotide sequence of the cloned gene showed high homology (59% identity and 87% similarity) with the NhaA Na+/H+ antiporter of E. coli. Thus, we conclude that the gene we cloned and sequenced is the nhaA of V. parahaemolyticus. We also found that several regions of the NhaA protein showed sequence similarity with transport proteins from some other organisms. Such regions seem to be important for Na+ recognition, transport or amiloride binding.
KeywordMeSH Terms
124. Nagayama  K, Yamamoto  K, Mitawani  T, Honda  T,     ( 1995 )

Characterisation of a haemolysin related to Vp-TDH produced by a Kanagawa phenomenon-negative clinical isolate of Vibrio parahaemolyticus.

Journal of medical microbiology 42 (2)
PMID : 7869352  :   DOI  :   10.1099/00222615-42-2-83    
Abstract >>
The production of a family of haemolysins--thermostable direct haemolysin (Vp-TDH), Vp-TDH-related haemolysin (Vp-TRH) and Vp-TDH/I--has been reported in clinical isolates of Vibrio parahaemolyticus. This paper describes a fourth type of haemolysin--Vp-TDH/II--produced by a Kanagawa phenomenon-negative clinical isolate of V. parahaemolyticus (O13:K, untypable). Vp-TDH/II was purified by ammonium sulphate precipitation and successive filtrations on DEAE-cellulose, hydroxyapatite, Sepharose 4B and Mono Q columns. Vp-TDH/II was biophysicochemically and immunologically similar to, but not identical to Vp-TDH, Vp-TRH and Vp-TDH/I. Vp-TDH/II stimulated vascular permeability in rabbit skin and was lethal to mice. Purified Vp-TDH/II and viable cells of the Vp-TDH/II-producing strain both induced fluid accumulation in ligated rabbit intestine. The plasmid-determined structural gene for Vp-TDH/II was cloned and the nucleotide sequence determined. The deduced amino acid sequence of Vp-TDH/II differed from those of Vp-TDH, Vp-TRH and Vp-TDH/I.
KeywordMeSH Terms
125. Taniguchi  H, Hirano  H, Kubomura  S, Higashi  K, Mizuguchi  Y,     ( 1986 )

Comparison of the nucleotide sequences of the genes for the thermostable direct hemolysin and the thermolabile hemolysin from Vibrio parahaemolyticus.

Microbial pathogenesis 1 (5)
PMID : 3508495  :  
Abstract >>
The nucleotide sequences of genes encoding the thermostable direct (TSD) hemolysin and the thermolabile (TL) hemolysin of Vibrio parahaemolyticus were determined. From the nucleotide sequence of the TSD hemolysin gene, it was revealed that the preprotein and the mature protein consisted of 189 amino acids and 165 amino acids, and that the molecular weights were 21.1 kDa or 18.5 kDa, respectively. Our data regarding TSD hemolysin were in complete agreement with previously published data. From the nucleotide sequence of the TL hemolysin gene, it was revealed that the preprotein and the mature protein consisted of 418 amino acids and 398 amino acids, and that the molecular weights were 47.5 kDa and 45.3 kDa, respectively. The GC content of the TSD hemolysin gene was 35.6%, while that of the TL hemolysin gene was 47.6% which is almost the same as that of V. parahaemolyticus genome. Maxicell analysis revealed that the molecular weights of the proteins encoded by the TSD hemolysin gene were 22.0 and 19.5 kDa, and that of the protein encoded by the TL hemolysin gene was 45.5 kDa, and that the promoters of these two hemolysin genes of V. parahaemolyticus were functional in Escherichia coli.
KeywordMeSH Terms
126. Lång  H, Ferenci  T,     ( 1995 )

Sequence alignment and structural modelling of the LamB glycoporin family.

Biochemical and biophysical research communications 208 (3)
PMID : 7702622  :   DOI  :   10.1006/bbrc.1995.1423    
Abstract >>
lamB gene segments were obtained from Yersinia enterocolitica and Vibrio parahaemolyticus by the PCR and the DNA sequence determined. The deduced polypeptide sequences showed high similarity to six other LamB-related proteins and all contained typical signature sequences present in all members of the family but not other proteins. The aligned amino acid sequences permitted derivation of a model of LamB folding across the bacterial outer membrane using an approach successfully applied in the identification of structural features in other porins (Ferenci,T. (1994) Mol. Microbiol. 14:188-189). The alignment-based model differs from previous LamB structure predictions and is also more complex than that found for OmpF-related porins; more than 16 conserved stretches of amino acid sequence potentially corresponded to membrane-spanning segments.
KeywordMeSH Terms
Protein Folding
Protein Structure, Secondary
127. Pang  R, Xie  T, Wu  Q, Li  Y, Lei  T, Zhang  J, Ding  Y, Wang  J, Xue  L, Chen  M, Wei  X, Zhang  Y, Zhang  S, Yang  X,     ( 2019 )

Comparative Genomic Analysis Reveals the Potential Risk of Vibrio parahaemolyticus Isolated From Ready-To-Eat Foods in China.

Frontiers in microbiology 10 (N/A)
PMID : 30792709  :   DOI  :   10.3389/fmicb.2019.00186     PMC  :   PMC6374323    
Abstract >>
Vibrio parahaemolyticus is a major foodborne pathogen associated with the consumption of aquatic products. The presence of this bacterium in ready-to-eat (RTE) foods has recently been reported. However, the genomic features and potential risks of V. parahaemolyticus isolated from RTE foods are poorly understood. To help understand the genome-wide characteristics of RTE food isolates, the complete genomes of 27 RTE food isolates were sequenced and compared to those of 20 clinical and 19 other environmental (e.g., water and aquatic product source) isolates using a comparative genomics approach. Analysis revealed that V. parahaemolyticus RTE food isolates had higher numbers of genes on average and possessed more accessory genes than isolates from other sources. Most RTE food isolates were positive for some known virulence-associated genes and pathogenicity islands (PAIs), and some of these isolates were genetically homologous to clinical isolates. Genome-wide association analysis revealed 79 accessory genes and 78 missense single-nucleotide polymorphisms that affected 11 protein-coding genes were significantly associated with RTE food sources. These genes were mostly involved in defense mechanisms and energy production and conversion according to functional annotation in the COG database. KEGG Pathway analysis showed that these genes mainly affected the biofilm formation of V. parahaemolyticus, and subsequent experiments confirmed that nearly all RTE food isolates possessed the ability to form biofilm. The biofilm formation can facilitate the persistence of V. parahaemolyticus in RTE foods, and the presence of virulence-associated genes poses a pathogenic potential to humans. Our findings highlight the potential risk of V. parahaemolyticus in Chinese RTE foods and illustrate the genomic basis for the persistence of these isolates. This study will aid in re-evaluating the food safety threats conferred by this bacterium.
KeywordMeSH Terms
Vibrio parahaemolyticus
biofilm
genomics
potential risk
ready-to-eat foods
128. Zhang  Y, Zheng  Z, Chan  EW, Dong  N, Xia  X, Chen  S,     ( 2018 )

Molecular Characterization of qnrVC Genes and Their Novel Alleles in Vibrio spp. Isolated from Food Products in China.

Antimicrobial agents and chemotherapy 62 (7)
PMID : 29661884  :   DOI  :   10.1128/AAC.00529-18     PMC  :   PMC6021619    
Abstract >>
This study reports the prevalences of qnrVC genes in 74 ciprofloxacin-resistant Vibrio sp. isolates. Two novel functional qnrVC alleles, qnrVC8 and qnrVC9, sharing 98% and 99% nucleotide similarity with qnrVC6 and qnrVC7, respectively, were identified. Our findings suggested that carriage of qnrVC alleles, together with target mutations in gyrA and parC genes, may contribute to the development of fluoroquinolone resistance in Vibrio species, posing a serious threat to public health.
KeywordMeSH Terms
Vibrio
ciprofloxacin resistance
qnrVC
Vibrio
ciprofloxacin resistance
qnrVC
129. Dong  X, Bi  D, Wang  H, Zou  P, Xie  G, Wan  X, Yang  Q, Zhu  Y, Chen  M, Guo  C, Liu  Z, Wang  W, Huang  J,     ( 2017 )

pirABvp -Bearing Vibrio parahaemolyticus and Vibrio campbellii Pathogens Isolated from the Same AHPND-Affected Pond Possess Highly Similar Pathogenic Plasmids.

Frontiers in microbiology 8 (N/A)
PMID : 29051747  :   DOI  :   10.3389/fmicb.2017.01859     PMC  :   PMC5633605    
Abstract >>
Acute hepatopancreatic necrosis disease (AHPND) is a severe shrimp disease originally shown to be caused by virulent strains of Vibrio parahaemolyticus (VPAHPND). Rare cases of AHPND caused by Vibrio species other than V. parahaemolyticus were reported. We compared an AHPND-causing V. campbellii (VCAHPND) and a VPAHPND isolate from the same AHPND-affected pond. Both strains are positive for the virulence genes pirABvp . Immersion challenge test with Litopenaeus vannamei indicated the two strains possessed similar pathogenicity. Complete genome comparison showed that the pirABvp -bearing plasmids in the two strains were highly homologous, and they both shared high homologies with plasmid pVA1, the reported pirABvp -bearing plasmid. Conjugation and DNA-uptake genes were found on the pVA1-type plasmids and the host chromosomes, respectively, which may facilitate the dissemination of pirABvp . Novel variations likely driven by ISVal1 in the genetic contexts of the pirABvp genes were found in the two strains. Moreover, the VCAHPND isolate additionally contains multiple antibiotic resistance genes, which may bring difficulties to control its future outbreak. The dissemination of the pirABvp in non-parahaemolyticus Vibrio also rises the concern of missing detection in industrial settings since the isolation method currently used mainly targeting V. parahaemolyticus. This study provides timely information for better understanding of the causes of AHPND and molecular epidemiology of pirABvp and also appeals for precautions to encounter the dissemination of the hazardous genes.
KeywordMeSH Terms
Vibrio campbellii
Vibrio parahaemolyticus
acute hepatopancreatic necrosis disease
comparative genomics
plasmid
130.     ( 2013 )

Diversity of Vibrio spp. isolated at ambient environmental temperature in the Eastern English Channel as determined by pyrH sequencing.

Journal of applied microbiology 114 (6)
PMID : 23473469  :   DOI  :   10.1111/jam.12181    
Abstract >>
To describe the diversity of the culturable mesophilic and potentially pathogenic vibrios isolated at 22 and 37�XC on TCBS medium, in September 2009 from seawater and surface sediments. q-PCR assays previously selected for the identification of bacterial strains isolated at 37�XC were used in combination with the partial sequencing of two housekeeping genes, pyrH and toxR, to identify 315 strains isolated at 22�XC. The great majority of the 37�XC strains was identified by q-PCR assays, (five of the six species) with the predominance of Vibrio alginolyticus (85�P9%) and V. harveyi (10�P7%). The human pathogens V. parahaemolyticus and V. cholerae were rarely detected (two strains each). The 22�XC strains were successfully identified by the phylogeny analysis of pyrH and toxR genes, revealing 20 Vibrio species, with the predominance of the clam pathogen V. celticus (36�P8%). The Splendidus and the Harveyi groups represented the main Vibrio group at 22�XC (80%) and 37�XC (99�P5%), respectively. The combination of q-PCR assays and the sequencing of pyrH and toxR genes highlighted two different Vibrio communities at 22 and 37�XC both dominated by pathogenic species for marine organisms. The sequencing of the pyrH gene revealed to be a valuable tool to identify environmental Vibrio spp. strains isolated at 22�XC, as 92�P3% of them were identified in this study.
KeywordMeSH Terms
131. Phiwsaiya  K, Charoensapsri  W, Taengphu  S, Dong  HT, Sangsuriya  P, Nguyen  GTT, Pham  HQ, Amparyup  P, Sritunyalucksana  K, Taengchaiyaphum  S, Chaivisuthangkura  P, Longyant  S, Sithigorngul  P, Senapin  S,     ( 2017 )

A Natural Vibrio parahaemolyticus �GpirA Vp pirB Vp+ Mutant Kills Shrimp but Produces neither Pir Vp Toxins nor Acute Hepatopancreatic Necrosis Disease Lesions.

Applied and environmental microbiology 83 (16)
PMID : 28576761  :   DOI  :   10.1128/AEM.00680-17     PMC  :   PMC5541212    
Abstract >>
Acute hepatopancreatic necrosis disease (AHPND) of shrimp is caused by Vibrio parahaemolyticus isolates (VPAHPND isolates) that harbor a pVA plasmid encoding toxins PirA Vp and PirB Vp These are released from VPAHPND isolates that colonize the shrimp stomach and produce pathognomonic AHPND lesions (massive sloughing of hepatopancreatic tubule epithelial cells). PCR results indicated that V. parahaemolyticus isolate XN87 lacked pirA Vp but carried pirB Vp Unexpectedly, Western blot analysis of proteins from the culture broth of XN87 revealed the absence of both toxins, and the lack of PirB Vp was further confirmed by enzyme-linked immunosorbent assay. However, shrimp immersion challenge with XN87 resulted in 47% mortality without AHPND lesions. Instead, lesions consisted of collapsed hepatopancreatic tubule epithelia. In contrast, control shrimp challenged with typical VPAHPND isolate 5HP gave 90% mortality, accompanied by AHPND lesions. Sequence analysis revealed that the pVA plasmid of XN87 contained a mutated pirA Vp gene interrupted by the out-of-frame insertion of a transposon gene fragment. The upstream region and the beginning of the original pirA Vp gene remained intact, but the insertion caused a 2-base reading frameshift in the remainder of the pirA Vp gene sequence and in the downstream pirB Vp gene sequence. Reverse transcription-PCR and sequencing of 5HP revealed a bicistronic pirAB Vp mRNA transcript that was not produced by XN87, explaining the absence of both toxins in its culture broth. However, the virulence of XN87 revealed that some V. parahaemolyticus isolates carrying mutant pVA plasmids that produce no Pir Vp toxins can cause mortality in shrimp in ponds experiencing an outbreak of early mortality syndrome (EMS) but may not have been previously recognized to be AHPND related because they did not cause pathognomonic AHPND lesions.IMPORTANCE Shrimp acute hepatopancreatic necrosis disease (AHPND) is caused by Vibrio parahaemolyticus isolates (VPAHPND isolates) that harbor the pVA1 plasmid encoding toxins PirA Vp and PirB Vp The toxins are produced in the shrimp stomach but cause death by massive sloughing of hepatopancreatic tubule epithelial cells (pathognomonic AHPND lesions). V. parahaemolyticus isolate XN87 harbors a mutant pVA plasmid that produces no Pir toxins and does not cause AHPND lesions but still causes ?50% shrimp mortality. Such isolates may cause a portion of the mortality in ponds experiencing an outbreak of EMS that is not ascribed to VPAHPND Thus, they pose to shrimp farmers an additional threat that would be missed by current testing for VPAHPND Moribund shrimp from ponds experiencing an outbreak of EMS that exhibit collapsed hepatopancreatic tubule epithelial cells can serve as indicators for the possible presence of such isolates, which can then be confirmed by additional PCR tests for the presence of a pVA plasmid.
KeywordMeSH Terms
AHPND
EMS
Penaeus vannamei
Pir toxin
Vibrio parahaemolyticus
shrimp
132. Sakata  J, Yonekita  T, Kawatsu  K,     ( 2018 )

Development of a rapid immunochromatographic assay to detect contamination of raw oysters with enteropathogenic Vibrio parahaemolyticus.

International journal of food microbiology 264 (N/A)
PMID : 29080422  :   DOI  :   10.1016/j.ijfoodmicro.2017.10.016    
Abstract >>
Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are major virulence factors of enteropathogenic Vibrio parahaemolyticus. TDH and TRH are bacterial exotoxins, and their presence in culture medium serves as a specific marker for detecting this significant pathogen. Here, we developed and evaluated an immunochromatographic assay (TDH/TRH-ICA) to simultaneously or individually detect TDH and TRH. The TDH/TRH-ICA detected TDH in all broth cultures of 47 V. parahaemolyticus strains carrying tdh. The genes encoding TRH are classified as variants trh1 and trh2, and TRH was detected in all broth cultures of 25 V. parahaemolyticus strains carrying trh1 and certain proportion (5/31) of broth cultures of V. parahaemolyticus strains carrying trh2. In contrast, TDH and TRH were not detected in broth cultures of 12 non-enteropathogenic V. parahaemolyticus strains without tdh and trh. It was difficult to detect TRH2 using the TDH/TRH-ICA. However, TRH2 may not serve as a suitable marker for detecting enteropathogenic V. parahaemolyticus, and evidence indicates that TRH2 may not contribute to enteropathogenesis. Further, a screening method using a combination of TDH/TRH-ICA and SPP medium supplemented with 1.5% NaCl (modified-SPP medium) detected oyster samples artificially spiked with 1.1-22 colony-forming units of enteropathogenic V. parahaemolyticus per 25g of oysters within approximately 8.5h, including the enrichment culture. The assay may serve as a method that facilitates the rapid and easy detection of raw oysters contaminated with enteropathogenic V. parahaemolyticus.
KeywordMeSH Terms
Immunochromatographic assay
Monoclonal antibody
TDH
TRH
V. parahaemolyticus
133. Xu  F, Gonzalez-Escalona  N, Drees  KP, Sebra  RP, Cooper  VS, Jones  SH, Whistler  CA,     ( 2017 )

Parallel Evolution of Two Clades of an Atlantic-Endemic Pathogenic Lineage of Vibrio parahaemolyticus by Independent Acquisition of Related Pathogenicity Islands.

Applied and environmental microbiology 83 (18)
PMID : 28687650  :   DOI  :   10.1128/AEM.01168-17     PMC  :   PMC5583489    
Abstract >>
Shellfish-transmitted Vibrio parahaemolyticus infections have recently increased from locations with historically low disease incidence, such as the Northeast United States. This change coincided with a bacterial population shift toward human-pathogenic variants occurring in part through the introduction of several Pacific native lineages (ST36, ST43, and ST636) to nearshore areas off the Atlantic coast of the Northeast United States. Concomitantly, ST631 emerged as a major endemic pathogen. Phylogenetic trees of clinical and environmental isolates indicated that two clades diverged from a common ST631 ancestor, and in each of these clades, a human-pathogenic variant evolved independently through acquisition of distinct Vibrio pathogenicity islands (VPaI). These VPaI differ from each other and bear little resemblance to hemolysin-containing VPaI from isolates of the pandemic clonal complex. Clade I ST631 isolates either harbored no hemolysins or contained a chromosome I-inserted island we call VPaI�] that encodes a type 3 secretion system (T3SS2�]) typical of Trh hemolysin producers. The more clinically prevalent and clonal ST631 clade II had an island we call VPaI�^ that encodes both tdh and trh and that was inserted in chromosome II. VPaI�^ was derived from VPaI�] but with some additional acquired elements in common with VPaI carried by pandemic isolates, exemplifying the mosaic nature of pathogenicity islands. Genomics comparisons and amplicon assays identified VPaI�^-type islands containing tdh inserted adjacent to the ure cluster in the three introduced Pacific and most other emergent lineages that collectively cause 67% of infections in the Northeast United States as of 2016.IMPORTANCE The availability of three different hemolysin genotypes in the ST631 lineage provided a unique opportunity to employ genome comparisons to further our understanding of the processes underlying pathogen evolution. The fact that two different pathogenic clades arose in parallel from the same potentially benign lineage by independent VPaI acquisition is surprising considering the historically low prevalence of community members harboring VPaI in waters along the Northeast U.S. coast that could serve as the source of this material. This illustrates a possible predisposition of some lineages to not only acquire foreign DNA but also become human pathogens. Whereas the underlying cause for the expansion of V. parahaemolyticus lineages harboring VPaI�^ along the U.S. Atlantic coast and spread of this element to multiple lineages that underlies disease emergence is not known, this work underscores the need to define the environment factors that favor bacteria harboring VPaI in locations of emergent disease.
KeywordMeSH Terms
HGT
emerging pathogen
genomics
molecular epidemiology
pathogen evolution
pathogenicity islands
type III secretion systems
vibrio
whole-genome phylogeny
134.     ( 2013 )

Population structure of clinical and environmental Vibrio parahaemolyticus from the Pacific Northwest coast of the United States.

PloS one 8 (2)
PMID : 23409028  :   DOI  :   10.1371/journal.pone.0055726     PMC  :   PMC3567088    
Abstract >>
Vibrio parahaemolyticus is a common marine bacterium and a leading cause of seafood-borne bacterial gastroenteritis worldwide. Although this bacterium has been the subject of much research, the population structure of cold-water populations remains largely undescribed. We present a broad phylogenetic analysis of clinical and environmental V. parahaemolyticus originating largely from the Pacific Northwest coast of the United States. Repetitive extragenic palindromic PCR (REP-PCR) separated 167 isolates into 39 groups and subsequent multilocus sequence typing (MLST) separated a subset of 77 isolates into 24 sequence types. The Pacific Northwest population exhibited a semi-clonal structure attributed to an environmental clade (ST3, N = 17 isolates) clonally related to the pandemic O3:K6 complex and a clinical clade (ST36, N = 20 isolates) genetically related to a regionally endemic O4:K12 complex. Further, the identification of at least five additional clinical sequence types (i.e., ST43, 50, 65, 135 and 417) demonstrates that V. parahaemolyticus gastroenteritis in the Pacific Northwest is polyphyletic in nature. Recombination was evident as a significant source of genetic diversity and in particular, the recA and dtdS alleles showed strong support for frequent recombination. Although pandemic-related illnesses were not documented during the study, the environmental occurrence of the pandemic clone may present a significant threat to human health and warrants continued monitoring. It is evident that V. parahaemolyticus population structure in the Pacific Northwest is semi-clonal and it would appear that multiple sequence types are contributing to the burden of disease in this region.
KeywordMeSH Terms
135.     ( 1990 )

Duplication and variation of the thermostable direct haemolysin (tdh) gene in Vibrio parahaemolyticus.

Molecular microbiology 4 (1)
PMID : 2319944  :   DOI  :   10.1111/j.1365-2958.1990.tb02017.x    
Abstract >>
The relationship between phenotypic variation and nucleotide sequence variation of the gene encoding Vibrio parahaemolyticus thermostable direct haemolysin (tdh gene) was examined. Strains showing a typical haemolysin-positive phenotype carried two chromosomal gene copies (designated tdh1 and tdh2) while tdh-gene-positive strains showing a weakly positive or negative haemolysin phenotype possessed only a single chromosomal gene copy. Both gene copies from a typical haemolysin-positive strain were cloned and sequenced and possessed 97.2% homology. Comparison of the amino acid sequence predicted from the nucleotide sequence with the protein sequence determined by Edman degradation as well as construction of a tdh1-deficient yet haemolytic strain of V. parahaemolyticus suggest that the tdh2 locus is primarily responsible for the haemolytic phenotype. Two other tdh gene copies were cloned from a phenotypically negative strain which was unusual in that it contained one gene copy on a plasmid (designated tdh4) in addition to a single copy on the chromosome (tdh3). Both tdh3 and tdh4 were expressed in Escherichia coli and TDHs with haemolytic activity were produced. These gene copies were sequenced and shared 96.7% homology with the tdh1 gene. The V. parahaemolyticus strain carrying tdh3 and tdh4 gene copies did not produce detectable amount of tdh-specific RNA transcript. It seems, therefore, that differences in the transcriptional control are primarily responsible for the differences seen in haemolytic phenotype.
KeywordMeSH Terms
Genes, Bacterial
Genetic Variation
Multigene Family
136.     ( 2012 )

Development of O-serogroup specific PCR assay for detection and identification of Vibrio parahaemolyticus.

International journal of food microbiology 159 (2)
PMID : 23072697  :   DOI  :   10.1016/j.ijfoodmicro.2012.08.012    
Abstract >>
Vibrio parahaemolyticus is a human pathogen that is widely disseminated in estuarine, marine and coastal environments throughout the world, and is recognized as the leading cause of food-borne illness worldwide. V. parahaemolyticus infections have been characterized by causal associations with multiple, diverse serotypes. To date, 13 O-serogroups have been recognized in V. parahaemolyticus, although only the O-serogroup genetic determinants (OGDs) of serogroups O3 and O4 have been sequenced. In this study, the OGDs of the remaining 11 serogroups were identified. A PCR assay based on O-serogroup specific genes was developed for the identification and detection of all 13 V. parahaemolyticus O-serogroups and tested against 41 target strains and 21 strains of other bacterial species. A double-blind test including 105 environmental specimens was also performed. The developed method was shown to distinguish all V. parahaemolyticus O-serogroups effectively with the only exception of O3 and O13. The method was found to be highly specific and reproducible, with detection sensitivity of 1ng of genomic DNA, and it was demonstrated that V. parahaemolyticus at the level of 10(4)CFU/ml in mock water specimens and the enrichment culture of samples inoculated with at the level of 1CFU/ml were detected. As few as 2 to 18CFU (initial inoculum) of V. parahaemolyticus were detectable in a 1g oyster sample after enrichment using this PCR method. The molecular protocol developed in this study for identification of all V. parahaemolyticus serogroups is therefore suitable for rapid detection and identification of V. parahaemolyticus pathogens from clinical and environmental samples, with the potential for application in epidemiologic investigations and other food safety applications.
KeywordMeSH Terms
Food Contamination
137.     ( 2013 )

Partial characterization of an exopolysaccharide secreted by a marine bacterium, Vibrio neocaledonicus sp. nov., from New Caledonia.

Journal of applied microbiology 114 (6)
PMID : 23480553  :   DOI  :   10.1111/jam.12184    
Abstract >>
Exopolysaccharides (EPS) are industrially valuable molecules with numerous useful properties. This study describes the techniques used for the identification of a novel Vibrio bacterium and preliminary characterization of its EPS. Bioprospection in marine intertidal areas of New Caledonia followed by screening for EPS producing brought to selection of the isolate NC470. Phylogenetic analysis (biochemical tests, gene sequencing and DNA-DNA relatedness) permitted to identify NC470 as a new member of the Vibrio genus. The EPS was produced in batch fermentation, purified using the ultrafiltration process and analysed by colorimetry, Fourier Transform Infrared spectroscopy, gas chromatography, Nuclear Magnetic Resonance and HPLC-size exclusion chromatography. This EPS exhibits a high N-acetyl-hexosamines and uronic acid content with a low amount of neutral sugar. The molecular mass was 672 �� 10(3) Da. These data are relevant for possible technological exploitation. We propose the name Vibrio neocaledonicus sp. nov for this isolate NC470, producing an EPS with an unusual sugar composition. Comparison with other known polymers permitted to select applications for this polymer. This study contributes to evaluate the marine biodiversity of New Caledonia. It also highlights the biotechnological potential of New Caledonia marine bacteria.
KeywordMeSH Terms
138.     ( 2013 )

High frequency of virulence factor genes tdh, trh, and tlh in Vibrio parahaemolyticus strains isolated from a pristine estuary.

Applied and environmental microbiology 79 (7)
PMID : 23354697  :   DOI  :   10.1128/AEM.03792-12     PMC  :   PMC3623233    
Abstract >>
Virulence factor genes encoding the thermostable direct hemolysin (tdh) and the thermostable direct hemolysin-related hemolysin (trh) are strongly correlated with virulence of the emergent human pathogen Vibrio parahaemolyticus. The gene encoding the thermolabile hemolysin (tlh) is also considered a signature molecular marker for the species. These genes are typically reported in very low percentages (1 to 2%) of nonclinical strains. V. parahaemolyticus strains were isolated from various niches within a pristine estuary (North Inlet, SC) and were screened for these genes using both newly designed PCR primers and more commonly used primers. DNA sequences of tdh and trh were recovered from 48% and 8.3%, respectively, of these North Inlet strains. The recovery of pathogenic V. parahaemolyticus strains in such high proportions from an estuarine ecosystem that is virtually free of anthropogenic influences indicates the potential for additional, perhaps environmental roles of the tdh and trh genes.
KeywordMeSH Terms
Estuaries
Water Microbiology
139.     ( 1998 )

Note: molecular cloning of chitinase genes from Vibrio anguillarum and V. parahaemolyticus.

Journal of applied microbiology 84 (6)
PMID : 9717305  :  
Abstract >>
Chitinase genes from Vibrio anguillarum KV9001 and V. parahaemolyticus ATCC17802 were cloned into Escherichia coli. Open reading frames of chitinase genes from V. anguillarum (vac) and V. parahaemolyticus (vpc) are 1755 bp and 1890 bp, respectively. The deduced amino acid sequences of these genes have 71.6% identity. There are two consensus sequence regions in the VAC and VPC proteins. The vac gene was highly prevalent in V. anguillarum, and the DNA probe of the vac gene hybridized to V. alginolyticus and Beneckea proteolytica DNA. The DNA probe of the upc gene hybridized to V. alginolyticus, V. harveyi and V. ordalii DNA.
KeywordMeSH Terms
Cloning, Molecular
140.     ( 1998 )

OpaR, a homolog of Vibrio harveyi LuxR, controls opacity of Vibrio parahaemolyticus.

Journal of bacteriology 180 (12)
PMID : 9620967  :   PMC  :   PMC107818    
Abstract >>
Vibrio parahaemolyticus is an organism well adapted to communal life on surfaces. When grown on a surface or in a viscous layer, the bacterium induces a large gene system and differentiates to swarmer cells capable of movement over and colonization of surfaces. V. parahaemolyticus displays additional phenotypic versatility manifested as variable colony morphology, switching between translucent and opaque colony types. Although not itself luminescent, V. parahaemolyticus produces autoinducer molecules capable of inducing luminescence in Vibrio harveyi. To examine the role of quorum signaling in the lifestyles of V. parahaemolyticus, the functional homolog of the gene encoding the V. harveyi autoinducer-controlled transcriptional regulatory protein LuxR was cloned. Sequence analysis of the clone predicted an open reading frame with a deduced product 96% identical to LuxR. Introduction of the clone carrying the luxR-like locus into V. parahaemolyticus dramatically affected colony morphology, converting a translucent strain to an opaque one. When the coding sequence for the luxR homolog was placed under the control of the Ptac promoter, conversion to the opaque phenotype became inducible by isopropyl-beta-D-thiogalactopyranoside. Allelic disruption of the luxR-like gene on the chromosome of an opaque strain produced a translucent strain proficient in swarming ability. Primer extension mapping demonstrated opaR transcription in opaque but not translucent cell types. It is postulated that this gene, which has been named opaR, encodes a transcription factor controlling cell type. The underlying genetic basis for opaque-translucent variation may be the consequence of a genomic alteration detected in the opaR locus of opaque and translucent strains.
KeywordMeSH Terms
141.     ( 1998 )

A new Na+/H+ antiporter, NhaD, of Vibrio parahaemolyticus.

Biochimica et biophysica acta 1369 (2)
PMID : 9518619  :   DOI  :   10.1016/s0005-2736(97)00223-x    
Abstract >>
A gene encoding an Na+/H+ antiporter was cloned from chromosomal DNA of Vibrio parahaemolyticus, a slightly halophilic bacterium, and expressed in Escherichia coli cells. The gene enabled mutant E. coli cells, which were unable to grow in the presence of 10 mM LiCl (or 0.2 M NaCl) because of the lack of major Na+(Li+)/H+ antiporters, to grow under such conditions. We detected Na+/H+ antiport activity due to the gene in membrane vesicles prepared from E. coli cells that harbored the plasmid carrying the gene. Li+ was also a substrate for this antiporter. Activity of this antiporter was pH-dependent with highest activity at pH 8.5 to 9 and no activity at 7.0 to 7.5. Restriction mapping and a Southern blot analysis revealed that the cloned gene was different from the nhaA and the nhaB of V. parahaemolyticus. We designated the gene nhaD. The gene was sequenced, and the amino acid sequence of the NhaD protein was deduced. The NhaD is a unique Na+/H+ antiporter with respect to the primary structure compared with known Na+/H+ antiporters.
KeywordMeSH Terms
142.     ( 1997 )

Evidence for genetic linkage between the ure and trh genes in Vibrio parahaemolyticus.

Journal of medical microbiology 46 (8)
PMID : 9511811  :   DOI  :   10.1099/00222615-46-8-639    
Abstract >>
Although V. parahaemolyticus does not generally produce urease, several studies have reported urease-positive V. parahaemolyticus isolates from clinical sources. Recently, studies have shown a complete coincidence between the urease-producing phenotype of V. parahaemolyticus strains and the possession of the thermostable direct haemolysin (TDH)-related haemolysin (TRH) gene (trh). TRH, like TDH, is considered to be an important virulence factor in the pathogenesis of V. parahaemolyticus gastroenteritis. The present study attempted to identify the gene ure encoding urease in V. parahaemolyticus to clarify the relationship between urease production and possession of trh. The polymerase chain reaction with mixed oligonucleotide primers targeted for conserved sequences of reported ure genes from other species was used to prepare a DNA probe to detect the V. parahaemolyticus ure gene. Colony hybridisation with this ure probe demonstrated that all the ure-positive strains produced urease. Considering the coincidence between production of urease and possession of trh in V. parahaemolyticus, it was concluded that the presence or absence of the ure gene is completely coincident with that of the trh gene in V. parahaemolyticus strains. Furthermore, the relative location of ure and trh on V. parahaemolyticus chromosomal DNA was analysed by pulsed-field gel electrophoresis. The results showed that, in all the strains examined, ure and trh were detected on the same NotI fragment, showing that the two genes localise within a relatively small portion of the chromosome DNA. These results suggest that the ure and trh genes are genetically linked in V. parahaemolyticus strains.
KeywordMeSH Terms

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