Broth medium for enrichment of Vibrio fluvialis from the environment.

A medium was designed for the enrichment and enumeration of Vibrio fluvialis from environmental samples. The medium contains 1% peptone plus 4% sodium chloride and 5 micrograms of novobiocin per ml, pH 8.5. This V. fluvialis enrichment medium (FEM) was tested, in comparison with alkaline peptone (AP), in field samplings. A total of 177 samples (estuarine waters and sediment, sewage, and crabs) collected over a 14-month period were examined with FEM and with AP broth. Results showed that FEM was more effective than AP in detecting V. fluvialis, particularly from water and sewage samples with low salinities (less than 6%). The best recovery of V. fluvialis occurred when both enrichment media were used simultaneously.     

Vibrio fluvialis andV. furnissii serotyping scheme for international use

A combination of two serotyping systems forVibrio fluvialis andV. furnissii, which were developed independently at the National Institute of Health (Tokyo) and Tokyo Metropolitan Research Laboratory of Public Health (Tokyo), was established as a single serotyping scheme comprising 35 O-antigen groups for international use.     

Sugar composition of the polysaccharide portion of lipopolysaccharides of Vibrio fluvialis, Vibrio vulnificus, and Vibrio mimicus

A chemotaxonomic study was carried out on Vibrio fluvialis and V. vulnificus on the basis of the sugar composition of the polysaccharide portion of their lipopolysaccharides (LPS). A previously developed rapid method of preparing samples for compositional sugar analysis was employed. Nineteen O-serogroups of V. fluvialis were divided into 14 chemotypes while seven O-serogroups of V. vulnifucus were divided also into seven chemotypes since the polysaccharide portion of LPS of each serogroup has a different sugar composition from that of the other serogroups. Close similarities in the sugar composition of the same portion were demonstrated between serologically cross-reacting non-O1 group V. cholerae and V. fluvialis, and non-O1 V. cholerae and V. mimicus.     

Structure of the repeating chain of the O-specific polysaccharide of Vibrio fluvialis

O-Specific polysaccharide chain of the Vibrio fluvialis lipopolysaccharide is built up of pentasaccharide repeating units, containing one N-acetyl-D-glucosamine and four L-rhamnose residues. The structure of the polysaccharide was elucidated using two-dimensional correlation 1H-NMR-spectroscopy, 13C-NMR-spectroscopy and nuclear Overhauser effect and confirmed by methylation analysis and selective cleavage of N-acetylglucosamine residues by the N-deacetylation-deamination method which yielded linear L-rhamnan representing the backbone of the polysaccharide. Thus, the repeating unit of the O-specific polysaccharide has the following structure: (formula; see text)     

Use of enrichment culture for directed evolution of the Vibrio fluvialis JS17 omega-transaminase, which is resistant to product inhibition by aliphatic ketones

A novel high-throughput screening method that overcame product inhibition was used to isolate a mutant omega-transaminase from Vibrio fluvialis JS17. An enzyme library was generated using error-prone PCR mutagenesis and then enriched on minimal medium containing 2-aminoheptane as the sole nitrogen source and 2-butanone as an inhibitory ketone. An identified mutant enzyme, omega-TAmla, showed significantly reduced product inhibition by aliphatic ketone. The product inhibition constants of the mutant with 2-butanone and 2-heptanone were 6- and 4.5-fold higher than those of the wild type, respectively. Using omega-TAmla (50 U/ml) overexpressed in Escherichia coli BL21, 150 mM 2-aminoheptane was successfully resolved to (R)-2-aminoheptane (enantiomeric excess, >99%) with 53% conversion with an enantioselectivity of >100.     

Enhanced Broth Media for Selective Growth of Vibrio vulnificus

Rapid detection of Vibrio vulnificus can be enhanced by optimizing the components of enrichment broth. PNC (5% peptone, 1% NaCl, and 0.08% cellobiose [pH 8.0]) enhanced the growth of V. vulnificus compared to alkaline peptone broth. PNCC (PNC with 1.0 to 4.1 U of colistin methanesulfonate per ml) increased the growth of low levels of V. vulnificus while suppressing non-target bacteria.     

Structure of the O-specific polysaccharide of Vibrio fluvialis serovar 3]

O-Specific polysaccharide composed of L-rhamnose and 2-acetamido-2-deoxy-D-mannose was obtained on mild acid degradation of the V. fluvialis lipopolysaccharide. On the basis of the 13C-NMR data and methylation studies, the following structure was suggested for the polysaccharide repeating unit: ----4)-alpha-L-Rhap-(1----3)-beta-D-ManpNAc-(1---- This structure was confirmed by calculations using known glycosidation effects on 13C chemical shifts.     

The isolation of Vibrio fluvialis on the territory of the USSR]

For the first time V. fluvialis strains were detected on the territory of the USSR. The taxonomic position of these vibrios was determined by their nucleotide DNA composition (the content of guanine + cytosine was 49.3-51.0 mole%) and the characteristic features of their phenotype. The individual features of the strains consisted in their capacity for agglutination with cholera antisera, groups 01 and Inaba, in diagnostic dilutions in the presence of differences in genomes and phenotypes with cholera vibrios. Molecular hybridization DNA-DNA also gave no confirmation of their relationship to cholera vibrios (23-26% homology). The comparative study of V. fluvialis strains from the USSR and other countries by a broader set of their phenotypical signs confirmed their identity.     

Optimal enrichment time for isolation of Vibrio parahaemolyticus from seafood.

The growth of Vibrio parahaemolyticus in a liquid medium was compared with that of human fecal flora and estuarine flora. No marked differences were noted between growth at 25 and 37 degrees C for V. parahaemolyticus. However, the marine organisms were strongly inhibited when incubated at 37 degrees C. Incubation for 8 h in an enrichment broth yielded V. parahaemolyticus growth, even with a small inoculum, whereas the marine and fecal floras were inhibited. Therefore, enrichment for 8 h at 37 degrees C appears to be optimal for isolation of V. parahaemolyticus, permitting more rapid results in seafood analysis.     

Purification,characterization and molecular cloning of Vibrio fluvialis hemolysin

Hemolysin of Vibrio fluvialis (VFH) was purified from culture supernatants by ammonium sulfate precipitation and successive column chromatographies on DEAE-cellulose and Mono-Q. N-terminal amino acid sequences of the purified VFH were determined. The purified protein exhibited hemolytic activity on many mammalian erythrocytes with rabbit erythrocytes being the most sensitive to VFH. Activity of the native VFH was inhibited by the addition of Zn2+, Ni2+, Cd2+ and Cu2+ ions at low concentrations. Pores formed on rabbit erythrocytes were approximately 2.8-3.7 nm in diameter, as demonstrated by osmotic protection assay. Nucleotide sequence analysis of the vfh gene revealed an open reading frame (ORF) consisting of 2200 bp which encodes a protein of 740 amino acids with a molecular weight of 82 kDa. Molecular weight of the purified VFH was estimated to be 79 kDa by SDS-PAGE and N-terminal amino acid sequence revealed that the 82 kDa prehemolysin is synthesized in the cytoplasm and is then secreted into the extracellular environment as the 79 kDa mature hemolysin after cleavage of 25 N-terminal amino acids. Deletion of 70 amino acids from the C-terminus exhibited a smaller hemolytic activity, while deletion of 148 C-terminal amino acids prevented hemolytic activity.     

Medium-dependent production of extracellular enterotoxins by non-O-1 Vibrio cholerae, Vibrio mimicus, and Vibrio fluvialis.

Fluid accumulation at 4 h in the intestines of suckling mice enabled us to distinguish non-O-1 Vibrio cholerae, V. mimicus, and V. fluvialis clinical isolates from environmental isolates. Enterotoxin production was culture medium dependent. Filtrates of cultures grown in tryptic soy broth without glucose but with added 0.5% NaCl did not exhibit marked enterotoxin activity in the assay. Culture filtrates of all clinical strains grown in brain heart infusion broth supplemented with 0.5% NaCl induced large amounts of fluid accumulation in mouse intestines. However, most environmental strains grown in brain heart infusion broth amended as described above were unable to induce fluid accumulation. The enterotoxin present in culture filtrates lost activity at 56 degrees C and appeared to be distinct from previously described virulence factors, including the well-described cholera toxin. The new enterotoxin could represent an important virulence mechanism common to all three species.     

水产品中河流弧菌分离株OmpU基因的克隆测序与生物信息学分析

目的 对河流弧菌(Vibrio fluvialis)水产品分离株OmpU基因进行克隆测序和生物信息学分析,为建立该菌的检测方法和研制疫苗奠定基础.方法 从市售水产品中分离细菌,采用表型和分子鉴定方法确定其种属,并测定其致病性和药物敏感性.根据弧菌属OmpU基因的序列特点,设计引物扩增河流弧菌OmpU基因,将其克隆到T载体上,筛选重组质粒并对其进行序列测定及生物信息学分析.结果 从水产品中分离的2个菌株(Vf1和Vt2)经鉴定确认为河流弧菌,它们均具有致病性,对15种测试抗菌药物敏感.2株河流弧菌OmpU基因全长分别为1 044和1 005 bp,含有1个开放性阅读框,分别编码由348和335个氨基酸组成的OmpU蛋白,该蛋白N端前22个氨基酸为信号肽.序列比对结果显示2株河流弧菌OmpU基因的核苷酸及其推导的氨基酸序列相似性分别为82.6％和81.2％.OmpU蛋白序列在6种弧菌种内和种间的相似性分别为81.4％ ～ 99.2％和71.2％ ～78.1％.表位预测结果显示河流弧菌OmpU蛋白的B细胞线性表位主要集中在第24 ～ 28、45～ 53、113～116、153～156、215～ 221和242～254位氨基酸区域,6种弧菌具有1个共同的抗原表位基序KDG-A-D-S.结论 OmpU蛋白是弧菌属中较为保守的一类功能蛋白,共同的抗原表位基序有望成为检测多种弧菌的靶标和研制多表位疫苗的靶位.     

Simple procedure for rapid identification of Vibrio cholerae from the aquatic environment

Biochemical tests commonly used to screen for Vibrio cholerae in environmental samples were evaluated, and we found that a combination of alkaline peptone enrichment followed by streaking on thiosulfate citrate bile salts sucrose agar and testing for arginine dihydrolase activity and esculin hydrolysis was an effective rapid technique to screen for aquatic environmental V. cholerae. This technique provided 100% sensitivity and > or =70% specificity.     

Infaunal burrows are enrichment zones for Vibrio parahaemolyticus

Vibrio parahaemolyticus, a species that includes strains known to be pathogenic in humans, and other Vibrionaceae are common, naturally occurring bacteria in coastal environments. Understanding the ecology and transport of these organisms within estuarine systems is fundamental to predicting outbreaks of pathogenic strains. Infaunal burrows serve as conduits for increased transport of tidal waters and V. parahaemolyticus cells by providing large open channels from the sediment to salt marsh tidal creeks. An extensive seasonal study was conducted at the North Inlet Estuary in Georgetown, SC, to quantify Vibrionaceae and specifically V. parahaemolyticus bacteria in tidal water, fiddler crab (Uca pugilator, Uca pugnax) burrow water, and interstitial pore water. Numbers of V. parahaemolyticus bacteria were significantly higher within burrow waters (4,875 CFU ml(-1)) than in creek water (193 CFU ml(-1)) and interstitial pore water (128 CFU ml(-1)), demonstrating that infaunal burrows are sites of V. parahaemolyticus enrichment. A strong seasonal trend of increased abundances of Vibrionaceae and V. parahaemolyticus organisms during the warmer months of May through September was observed. Multilocus sequence typing (MLST) analysis of isolates presumed to be V. parahaemolyticus from creek water, pore water, and burrow water identified substantial strain-level genetic variability among V. parahaemolyticus bacteria. Analysis of carbon substrate utilization capabilities of organisms presumed to be V. parahaemolyticus also indicated physiological diversity within this clade, which helps to explain the broad distribution of these strains within the estuary. These burrows are "hot spots" of Vibrionaceae and V. parahaemolyticus cell numbers and strain diversity and represent an important microhabitat.     

Lipopolysaccharide composition and virulence properties of clinical and environmental strains of Vibrio fluvialis and Vibrio mimicus.

Vibrio mimicus strains W-26768 (stool isolate) and N-1301 (environmental isolate) and Vibrio fluvialis strains AA-18239 (stool isolate) and M-940 (environmental isolate) were studied for virulence properties and lipopolysaccharide composition. All four strains were hydrophobic, produced cytotoxin, adhered to HeLa cells and showed mannose-sensitive agglutination of guinea pig erythrocyte. The strains were negative for enterotoxin production and were mostly susceptible to the common antibiotics. The environmental and clinical isolates of both species were antigenically unrelated to each other. Lipopolysaccharide antigen analysis showed that O-antigen polysaccharides of two strains of V. fluvialis and two strains of V. mimicus differed with respect to the sugar components. Only LPS from V. mimicus W-26768 showed the presence of an unusual sugar, 3,6-dideoxy-3-acetamido-hexose. The sugar compositions of these V. fluvialis and V. mimicus strains differed from those of previously reported Japanese isolates. These differences probably reflect differences in the serogroup of strains.     

Vibrio fluvialis: a new serogroup (19) possessing the Inaba factor antigen of Vibrio cholerae O1

A serogroup of Vibrio fluvialis possessing the C (Inaba) antigen but not the B (Ogawa) nor A antigen of V. cholerae O1 is described. The O-antigen of this serogroup was identical with that of bioserogroup 1875-variant of a marine Vibrio species. As the O-antigen of this serogroup was not agglutinated by any of O-antisera for the 18 serogroups of V. fluvialis already recognized, it was designated O-serogroup 19 of this species.     

Changes in membrane functions during short-term starvation of Vibrio fluvialis strain NCTC 11328

The marine bacterium Vibrio fluvialis strain NCTC 11328 responded to starvation conditions by forming ultramicrocells of dwarf bacteria. The viability of starved cells began to decrease after 2–3 days. During this time the respiratory potential of the bacteria decreased by four- or fivefold, most probably as a result of a decrease in the specific activity of NADH and succinate dehydrogenases. Although respiratory potential in starving cells was lower than in growing cells, bacteria starved for 1 or 2 days maintained a proton motive force that was slightly larger than that of growing bacteria. Starved bacteria contained substantial concentrations of ATP although the UTP and GTP concentrations were much lower in starved than in growing cells. Two or three proteins that were not present in membranes of growing cells, were evident in the membranes of starved bacteria.Abbreviations MMS modified Morita's salts - MMSGC modified Morita's salts plus 20 mM glucose and 0.1% (w/v) casamino acids - MMST modified Morita's salts buffered with 50 mM tricine, (pH 8.5) - NM broth nutrient modified Morita's salts - CFU colony-forming unit - TPP tetraphenylphosphonium - STM 0.1 M tricine, (pH 8.0) plus 0.25 M sucrose and 0.02 M magnesium acetate - DCPIP dichlorophenolindophenol - CCCP carbonyl cyanidem-chlorophenylhydrazone - PMF proton motive force     

New selective and differential medium for Vibrio cholerae and Vibrio vulnificus

Thiosulfate-citrate-bile salts-sucrose agar has been routinely used for the isolation of pathogenic vibrios, although its selectivity for Vibrio cholerae and Vibrio vulnificus is inadequate. Therefore, a new plating medium, cellobiose-polymyxin B-colistin agar, was developed for the isolation of these two species. Cellobiose-polymyxin B-colistin agar demonstrated a significant advantage over other media designed for the isolation or differentiation of vibrios: of both the 136 strains representing 19 Vibrio species and the marine isolates of the genera Pseudomonas, Flavobacterium, and Photobacterium, only V. vulnificus and V. cholerae were able to grow. Furthermore, the fermentation of cellobiose by V. vulnificus allowed for the easy differentiation of these two species. This medium offers significant potential as a selective and differential medium for these two pathogenic vibrios.     

Use of colistin-polymyxin B-cellobiose agar for isolation of Vibrio vulnificus from the environment.

Colistin-polymyxin B-cellobiose agar was employed for the isolation of Vibrio vulnificus from shellfish. Isolates were examined phenotypically and with a gene probe and monoclonal antibody specific for V. vulnificus. Results indicated that colistin-polymyxin B-cellobiose agar is superior to both sodium dodecyl sulfate-polymyxin B-sucrose agar and thiosulfate-citrate-bile salts-sucrose agar in its ability to select and differentiate this species from background vibrios.