Lipopolysaccharide Isolated from a New O-Antigenic Form (O13) of Vibrio parahaemolyticus

The chemical properties of a lipopolysaccharide (LPS) isolated from a new O-antigenic form (O13) of Vibrio parahaemolyticus were investigated. The LPS contained glucose, galactose, L -glycero-D -manno-heptose and glucosamine. 2-Keto-3-deoxy-octonate (KDO) was not detected in the LPS by the periodate-thiobarbituric acid test (Weissbach's reaction) under conventional hydrolysis conditions. Instead, phosphorylated KDO (X1 and X2) was found in its strong-acid hydrolysate. This sugar composition was identical to that of V. parahaemolyticus O3, O5 and O11 LPS, indicating that, based on the sugar composition, O13 LPS belongs to Chemotype III to which O3, O5 and O11 belong. In addition, structural study demonstrated the presence of KDO 4-phosphate in its inner-core region.     

Siderophore production of Vibrio parahaemolyticus strains from different sources.

Vibrio parahaemolyticus strains isolated from different sources were assayed for their ability to produce a siderophore, vibrioferrin, under iron-limited growth conditions. The mean value +/- standard error of mean (microM vibrioferrin in spent culture supernatant/optical density at 660 nm) was 832.3 +/- 66.9 for clinical isolates (n=44), which was significantly higher (P<0.01) than those for food isolates (461.0 +/- 66.5; n=37) and coastal isolates (378.8 +/- 37.2; n=26). This suggests that greater productivity of vibrioferrin by clinical isolates may be associated with a selective advantage for survival and proliferation under conditions of iron-limitation such as in the intestine [corrected].     

Emergence and serovar transition of Vibrio parahaemolyticus pandemic strains isolated during a diarrhea outbreak in Vietnam between 1997 and 1999

We characterized 523 Vibrio parahaemolyticus strains isolated during a survey of diarrhea patients in Khanh Hoa province, Vietnam between 1997 and 1999. Forty-nine percent of the strains were judged to belong to the pandemic strains that emerged around 1996 and spread to many countries. These strains were positive in the GS-PCR assay and carried the tdh gene. The ORF8 of the f237 phage genome, a possible marker of the pandemic clone, was absent in 10% of these strains. Eleven O: K serovars were detected among the pandemic strains and the strains representing all 11 serovars of pandemic strains were shown to be closely related regardless of the ORF8 genotype using arbitrarily primed PCR and pulsed field gel electrophoresis analyses. It was clear that a transition of major serovars occurred among the pandemic strains represented by the emergence of O3: K6 in 1997, O4: K68 in 1998, and O1: K25 in 1998 and 1999.     

Release of the Outer Membrane Vesicles from Vibrio cholerae and Vibrio parahaemolyticus

We found numerous small vesicles released from the cell by thin sectioning of the plate culture of Vibrio cholerae and V. parahaemolyticus fixed with the freeze-substitution technique. From the broth media of exponentially growing bacteria we could collect the vesicles by the centrifugation but not enough without fixation. The vesicles are encompassed with a membrane structure similar to the outer membrane of these bacteria. The anti-O (Inaba) serum reacted with the surface of the vesicles and the inside of the vesicle are generally filled with an electron-dense mass.     

Purification of a serine protease of Vibrio parahaemolyticus and its characterization

A 50 kDa protease designated as VPP1 was purified from the culture supernatant of a clinical strain of Vibrio parahaemolyticus by ammonium sulfate fractionation, Sephacryl S-200 HR gel filtration and Fractogel EMD TMAE 650 ion-exchange chromatography. VPP1 was inhibited by EDTA, EGTA and serine protease inhibitors, suggesting that it is a calcium-dependent serine protease. N-terminal amino acid sequence of VPP1 was quite similar to that of V. metschnikovii protease and antibody against VPP1 inhibited the activity of V. metschnikovii protease, suggesting the similarity of the two proteases. It was demonstrated that VPP1 or its related protease widely distribute in not only V. parahaemolyticus but also V. alginolyticus.     

Ribotyping of Vibrio parahaemolyticus isolates obtained from food poisoning outbreaks in Taiwan.

Vibrio parahaemolyticus is a prevalent food-borne pathogen in Taiwan, Japan and other Asian countries. This work presents a novel ribotyping method for the molecular epidemiological examination of this pathogen. Genomic DNA was fragmented by HindIII digestion and hybridized with cDNA probe for Escherichia coli 16S and 23S RNA genes. A total of 121 isolates obtained from outbreaks during 1992 and 1994 in Taiwan were characterized by this ribotyping method. Four to seventeen restricted fragments were visualized in these isolates. After hierarchical cluster analysis, these isolates were grouped into thirty different ribotypes. In addition, A3, A7, E3 and F1 were the major ribotypes, consisting of 22.3, 13.2, 9.1, and 8.3% of the isolates, respectively. A, E, F, G and B were the major groups, consisting of 46.2, 14.0, 9.1, 6.7, and 6.7% of the isolates, respectively. The discriminatory ability of this ribotyping method, as determined by Simpson's index of diversity, was 0.93, which closely resembled that of a previously reported pulsed-field gel electrophoresis method.     

Cloning and Sequencing of the Vibrio parahaemolyticus fur Gene

A ferric uptake regulatory gene (fur) was cloned from Vibrio parahaemolyticus WP1 by a polymerase chain reaction-based technique followed by functional complementation of a fur mutation in Escherichia coli. A sequence analysis showed that, at the amino acid level, the V. parahaemolyticus Fur protein is 81% identical with the Fur protein from E. coli and over 90% identical with those of the Vibrio species.     

PCR-based identification of pandemic group Vibrio parahaemolyticus with a novel group-specific primer pair

A PCR-based assay to identify pandemic group Vibrio parahaemolyticus has been developed. The assay employs an oligonucleotide primer pair derived from the group-specific sequence of an arbitrarily primed-PCR fragment, which is located in the genome encoding a "hypothetical protein," approximately 80% homologous to the Mn2+ and Fe2+ transporter of the NRAMP family of V. vulnificus. The assay distinguished the pandemic group from other V. parahaemolyticus strains by yielding a 235-bp specific amplicon, and can be a useful diagnostic tool for identification of pandemic group strains.     

Virulence properties of rough and smooth strains of Vibrio cholerae O1

A comparative study was carried out to see the differences in pathogenicity of rough and smooth strains. A total of 10 strains including 5 each of rough and smooth strains of Vibrio cholerae O1 were tested and found positive for toxin production by enzyme-linked immunosorbent assay (ELISA) in Richardson's and AKI media. All the smooth and rough strains, except one, showed a titre of 1: 10 and 1: 100 in Richardson's and AKI media, respectively. Both types of strains produced enterotoxin in rabbit ileal loop (RIL). The differences in multiplication abilities of smooth and rough strains in RIL were statistically significant (P <0.05). However, these differences in multiplying abilities did not influence the adherence potential or enterotoxin production as there was no significant difference (P >0.05) between these properties. This study demonstrated that the rough strains are equally pathogenic and as important as smooth strains.     

A Filamentous Phage of Vibrio parahaemolyticus O3:K6 Isolated in Laos

A filamentous phage, ‘lvpf5’, of Vibrio parahaemolyticus O3:K6 strain LVP5 was isolated and characterized. The host range was not restricted to serotype O3:K6, but 7 of 99 V. parahaemolyticus strains with a variety of serotypes were susceptible to the phage. The phage was inactivated by heating at 80 C for 10 min and by treating with chloroform. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the phage exhibited a 3.8 kDa protein. The amino-terminal amino acid sequence of the coat protein was determined as AEGGAADPFEAIDLLGVATL. The phage genome consisted of a single-stranded DNA molecule. The activity of the phages was inhibited by anti-Na2 pili antibody.     

大连市两种来源的副溶血性弧菌分子分型

目的 分析食品来源、患者来源及2种来源的副溶血性弧菌之间的PFGE图谱的关系,从分子流行病学角度探讨2种来源的副溶血性弧菌的关联。 方法 收集患者和食品2种来源的副溶血性弧菌178株,经限制性内切酶SfiI酶切,用脉冲场凝胶电泳方法进行电泳,凝胶成像仪获得电泳图谱,利用BioNumerics软件对图谱进行聚类分析。 结果 食品来源的96株菌,有13株降解,83株菌被限制性内切酶SfiI酶切出83个PFGE types(PT),聚类分析发现各菌株间相似系数为55.6%～97.4%,按带型相似系数为85%标准划分为1~67共67个克隆群。患者来源的82株菌,有5株降解,77株菌被限制性内切酶SfiI酶切出46个PFGE types(PT),聚类分析发现各菌株间相似系数为64.1%～100.0%,按带型相似系数为85.0%标准划分为A～O群共15个克隆群。将2种来源的共160株副溶血性弧菌的PFGE图谱进行聚类分析,各菌株间相似系数为41.7%～100.0%,按带型相似系数为85.0%标准划分为79个克隆群。 结论 多数食品来源菌株间相似系数较低,患者来源菌株间相似系数高,而食品和患者来源菌株间相似系数较低,只有少数食品来源与患者来源菌株相似系数较高。     

Siderophore-Mediated Utilization of Iron Bound to Transferrin by Vibrio parahaemolyticus

Vibrio parahaemolyticus produces a structurally novel type of siderophore, termed vibrioferrin, in response to iron-limitation. This study was performed to examine whether vibrioferrin can assimilate iron from human iron-binding proteins for growth. Comparison of the growth rates between V. parahaemolyticus AQ 3354 and its spontaneously arising, vibrioferrin-deficient mutant revealed that vibrioferrin was able to sequester iron from 30% iron-saturated human transferrin for growth, but not from human lactoferrin even if fully saturated with iron. In both strains, iron limitation induced two high-molecular-weight outer membrane proteins with apparent molecular masses of approximately 78 and 83 kDa. Since only the outer membrane fraction including these proteins showed a binding capacity to ferric vibrioferrin complex, either of them may function as its cell surface receptor. These results suggested that the organism might utilize such a source of host iron through the action of vibrioferrin during in vivo survival and proliferation, although its importance in pathogenesis is unknown.     

Cytotoxicity and enterotoxicity of the thermostable direct hemolysin-deletion mutants of Vibrio parahaemolyticus

The thermostable direct hemolysin (TDH) has been proposed to be a major virulence factor of Vibrio parahaemolyticus. We have recently completed the genome sequence of a TDH-producing V. parahaemolyticus strain, RIMD2210633. In this study, we constructed tdh-deletion mutants from the sequenced strain by homologous recombination and analyzed their phenotypes. Although the deletion of both copies of tdh completely abolished the hemolytic activity of the wild-type strain, the deletion did not affect the cytotoxicity to HeLa cells. Enterotoxicity, assayed by the rabbit ileal loop test, was lowered by tdh deletion, but the mutant still showed partial fluid accumulation in rabbit intestine. These results indicate that the cytotoxicity and enterotoxicity of TDH-producing V. parahaemolyticus are not explained by TDH alone, and suggest that an unknown virulence factor(s) could be involved in these pathogenic activities.     

Analysis of seawaters for the recovery of culturable Vibrio parahaemolyticus and some other vibrios

We investigated the recovery of dormant and injured cells along with the normally culturable cells of Vibrio species with special emphasis on V. parahaemolyticus using both selective and non-selective media at moderate (20 C) and standard (37 C) culture temperatures from a bay water environment. Culture temperatures (20 or 37 C) did not affect the recovery of V. parahaemolyticus but did for other vibrios. We observed similar seasonality of V parahaemolyticus as in most other environmental studies. V. parahaemolyticus and other Vibrio species were recovered in higher numbers by a replica plating method compared to most probable number (MPN) and direct TCBS (thiosulfate citrate bile-salt sucrose) agar counts. Even with the replica plating method, however, vibrios number goes down to a minimum level and V. parahaemolyticus was undetectable during the cool temperature period of the year, although total bacterial cells and CFU on nutrient agar (with 2% NaCl) did not vary so much during the study period.     

Utilization of Iron Sources and Its Possible Roles in the Pathogenesis of Vibrio parahaemolyticus

Vibrio parahaemolyticus is an important enteropathogen in Japan, Taiwan and other coastal regions. The influence of the regulation of iron on the pathogenesis of this pathogen has not been well characterized. The growth of pathogenic and non-pathogenic strains of V. parahaemolyticus on iron-limited agar plates was stimulated by ferritin, lactoferrin and transferrin at 30 μM , and also by hemin, hemoglobin and ferric ammonium citrate at 100 μM . Spontaneous iron-utilizing mutant strains (mutants) were derived from a clinical strain, ST550. Compared with the parent strain, lowered virulence was demonstrated for these mutants, as assayed by adult mouse and suckling mouse models. The in vivo growth and enterotoxigenicity of these mutants were also lower in the suckling mice. Adherence of the mutants to excised mouse intestine was lower as demonstrated by scanning electron microscopy. The iron-regulated outer membrane protein profile also changed in selected mutants. These results indicate that iron-regulated outer membrane proteins and other unknown factors associated with iron utilization may have profound influences, besides iron acquisition, on the pathogenesis of V. parahaemolyticus.     

Molecular typing of Vibrio parahaemolyticus strains isolated from the Philippines by PCR-based methods

AIM: The main aim of the present study was to use three PCR-based techniques for the analysis of genetic variability among Vibrio parahaemolyticus strains isolated from the Philippines. METHODS AND RESULTS: Seventeen strains of V. parahaemolyticus isolated from shrimps (Penaeus monodon) and from the environments where these shrimps are being cultivated were analysed by random amplified polymorphic DNA PCR (RAPD-PCR), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) and repetitive extragenic palindromic PCR (REP-PCR). The results of this work have demonstrated genetic variability within the V. parahaemolyticus strains that were isolated from the Philippines. In addition, RAPD, ERIC and REP-PCR are suitable rapid typing methods for V. parahaemolyticus. All three methods have good discriminative ability and can be used as a rapid means of comparing V. parahaemolyticus strains for epidemiological investigation. Based on the results of this study, we could say that REP-PCR is inferior to RAPD and ERIC-PCR owing to the fact that it is less reproducible. Moreover, the REP-PCR analysis yielded a relatively small number of products. This may suggests that the REP sequences may not be widely distributed in the V. parahaemolyticus genome. CONCLUSIONS: Genetic variability within V. parahaemolyticus strains isolated in the Philippines has been demonstrated. The presence of ERIC and REP sequences in the genome of this bacterial species was confirmed. SIGNIFICANCE AND IMPACT OF THE STUDY: The RAPD, ERIC and REP-PCR techniques are useful methods for molecular typing of V. parahaemolyticus strains. To our knowledge this is the first study of this kind carried out on V. parahaemolyticus strains isolated from the Philippines.     

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

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSIONS: The new O3:K6 and new O3:K6-like strains were highly similar in virulence and in several other phenotypical and genotypical traits. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrated the spread and divergence of the pandemic and related clone of V. parahaemolyticus with similar virulence.     

Characterization of the carbohydrate backbone of Vibrio parahaemolyticus O6 lipopolysaccharides

Structural characterization studies have been carried out on the carbohydrate backbone of Vibrio parahaemolyticus serotype O6 lipopolysaccharides (LPS). The carbohydrate backbone isolated from O6 LPS by sequential derivatization, that is, dephosphorylation, O-deacylation, pyridylamination, N-deacylation and N-acetylation, is a nonasaccharide consisting of 3 mol of D-glucosamine (GlcN) (of which one is pyridylaminated), 2 mol of L-glycero-D-manno-heptose (Hep), and 1 mol each of D-galactose (Gal), D-glucose (Glc), D-glucuronic acid (GlcA) and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). Structural analyses by nuclear magnetic resonance spectroscopy and fast-atom bombardment mass spectrometry demonstrated that the carbohydrate backbone is β-Galp-(1→2)-α-Hepp-(1→3)-α-Hepp-(1→5)-α-Kdop-(2→6)-β-GlcpNAc-(1→6)-GlcNAc-PA, in which the 3-substituted α-Hepp is further substituted by β-GlcpNAc-(1→4)-β-Glcp at position 4 and by β-GlcpA at position 2. In native O6 LPS, an additional 1 mol of D-galacturonic acid, which is liberated by dephosphorylation in hydrofluoric acid, is present at an unknown position. A previous study by the present authors reported that, of 13 O-serotype LPS of V. parahaemolyticus, the only LPS from which Kdo was detected was from O6 LPS after mild acid hydrolysis. In the present study, we have demonstrated that only 1 mol of Kdo is present at the lipid A proximal position, a component which is common to the LPS in all serotypes of the bacterium, and that there is no additional Kdo in the carbohydrate backbone of O6 LPS. ELISA and ELISA inhibition analysis using antisera against O6 and Salmonella enterica Minnesota R595 and LPS of both strains further revealed that Kdo is not involved as an antigenic determinant of O6 LPS.     

A Chemotaxonomic Study of Vibrio fluvialis Based on the Sugar Composition of the Polysaccharide Portion of the Lipopolysaccharides

A chemotaxonomic study was carried out with a new serotyping scheme comprising 35 O-antigen groups of Vibrio fluvialis on the basis of the sugar composition of the polysaccharide portion of their lipopolysaccharide (LPS). A previously developed rapid method of preparing samples for compositional sugar analysis was employed. The 35 O-antigen groups were divided into 21 chemotypes. It is noted that a rarely occurring component sugar of gram-negative bacterial LPS, D -glycero-D -manno-heptose, and two kinds of uronic acids, i.e., galacturonic acid of a weakly bound type and glucuronic acid of a strongly bound type, were found in common in all the 21 chemotypes. A characteristic sugar component of gram-negative bacterial LPS, 2-keto-3-deoxyoctonate (KDO), was not detectable in any of the 21 chemotypes. Instead, three kinds of “KDO-like substances” were found, one in each of three chemotypes (III, XI and XVII). They were strongly positive in Weissbach's periodate-thiobarbituric acid test for KDO, but definitely not identical to it in high-voltage paper electrophoresis (HVPE); the substance present in chemotype XI was indicated by HVPE to be 3-deoxy-D -threo-hexulosonic acid which is a sugar constituent of Vibrio parahaemolyticus O7 and O12 LPS.