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Polyphasic taxonomy of the genus vibrio: numerical taxonomy of Vibrio cholerae, Vibrio parahaemolyticus, and related Vibrio species.

A set of 86 bacterial cultures, including 30 strains of Vibrio cholerae, 35 strains of V. parahaemolyticus, and 21 representative strains of Pseudomonas, Spirillum, Achromobacter, Arthrobacter, and marine Vibrio species were tested for a total of 200 characteristics. Morphological, physiological, and biochemical characteristics were included in the analysis. Overall deoxyribonucleic acid (DNA) base compositions and ultrastructure, under the electron microscope, were also examined. The taxonomic data were analyzed by computer by using numerical taxonomy programs designed to sort and cluster strains related phenetically. The V. cholerae strains formed an homogeneous cluster, sharing overall S values of >/=75%. Two strains, V. cholerae NCTC 30 and NCTC 8042, did not fall into the V. cholerae species group when tested by the hypothetical median organism calculation. No separation of "classic" V. cholerae, El Tor vibrios, and nonagglutinable vibrios was observed. These all fell into a single, relatively homogeneous, V. cholerae species cluster. V. parahaemolyticus strains, excepting 5144, 5146, and 5162, designated members of the species V. alginolyticus, clustered at S >/=80%. Characteristics uniformly present in all the Vibrio species examined are given, as are also characteristics and frequency of occurrence for V. cholerae and V. parahaemolyticus. The clusters formed in the numerical taxonomy analyses revealed similar overall DNA base compositions, with the range for the Vibrio species of 40 to 48% guanine plus cytosine. Generic level of relationship of V. cholerae and V. parahaemolyticus is considered dubious. Intra- and intergroup relationships obtained from the numerical taxonomy studies showed highly significant correlation with DNA/DNA reassociation data.

Alcaligenes↗

The ability of two different Vibrio spp. bacteriophages to infect Vibrio harveyi, Vibrio cholerae and Vibrio mimicus.

AIMS: To determine the host range of the Vibrio harveyi myovirus-like bacteriophage (VHML) and the cholera toxin conversion bacteriophage (CTX Phi) within a range of Vibrio cholerae and V. mimicus and V. harveyi, V. cholerae and V. mimicus isolates respectively. METHODS AND RESULTS: Three V. harveyi, eight V. cholerae and five V. mimicus isolates were incubated with VHML and CTX Phi. Polymerase chain reaction (PCR) was used to determine the presence of VHML and CTX Phi in infected isolates. We demonstrated that it was possible to infect one isolate of V. cholerae (isolate ACM #2773/ATCC #14035) with VHML. This isolate successfully incorporated VHML into its genome as evident by positive PCR amplification of the sequence coding part of the tail sheath of VHML. Attempts to infect all other V. cholerae and V. mimicus isolates with VHML were unsuccessful. Attempts to infect V. cholerae non-01, V. harveyi and V. mimicus isolates with CTX Phi were unsuccessful. CONCLUSIONS: Bacteriophage infection is limited by bacteriophage-exclusion systems operating within bacterial strains and these systems appear to be highly selective. One system may allow the co-existence of one bacteriophage while excluding another. VHML appears to have a narrow host range which may be related to a common receptor protein in such strains. The lack of the vibrio pathogenicity island bacteriophage (VPI Phi) in the isolates used in this study may explain why infections with CTX Phi were unsuccessful. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study has demonstrated that Vibrio spp. bacteriophages may infect other Vibrio spp.

Bacteriophages↗

Seasonal distribution of facultatively enteropathogenic vibrios (Vibrio cholerae, Vibrio mimicus, Vibrio parahaemolyticus) in the freshwater of the Elbe River at Hamburg.

Between June 1981 and December 1982 the incidence of Vibrio cholerae, V. mimicus and V. parahaemolyticus was determined at two sampling sites on the Elbe River at Hamburg. A total of 183 strains was isolated from 147 water samples. Of these, 107 belonged to non-01 V. cholerae (ten strains producing a cholera-like enterotoxin); 33 were identified as V. mimicus, including two enterotoxin producers; 42 strains were Kanagawa-negative cultures of V. parahaemolyticus; and one was V. fluvialis. The highest incidence was observed from June to September with about 10(2) organisms/l. Halophilic vibrios, less than five organisms/l, were detectable during the period June/July to October. The vibrio incidence was not influenced by the numbers of aerobic heterotrophic bacteria, coliforms or faecal bacteria. In general water temperature correlated with the seasonal variation. Thus, a temperature rise over 10 degrees to 20 degrees C was followed by a distinct increase in vibrio numbers. Of 14 chemical parameters only chloride concentration might have had an influence on the seasonal variation. It is concluded that the three Vibrio species are indigenous organisms of the Elbe River.

Enterotoxins↗

CLASSIFICATION OF VIBRIO CHOLERAE (VIBRIO COMMA), INCLUDING EL TOR VIBRIOS, BY INFRASUBSPECIFIC CHARACTERISTICS.

Feeley, John C. (National Institutes of Health, Bethesda, Md.). Classification of Vibrio cholerae (Vibrio comma), including El Tor vibrios, by infrasubspecific characteristics. J. Bacteriol. 89:665-670. 1965.-A study of the properties of 220 serotype O group I vibrios indicated striking similarity in most of their properties. However, by using four tests often applied in the identification of the El Tor vibrio, five types were identified and characterized as follows: type 1 strains, phage IV-sensitive, nonhemolytic by tube and plate methods, unable to agglutinate chicken red cells (CCA), and Voges-Proskauer (VP) negative or weakly positive at 22 C; type 2, same as type 1 except for CCA; type 3, phage IV-resistant, CCA and VP usually positive, and strongly hemolytic by tube and plate methods; type 4, same as type 3, except hemolytic only by plate method unless culture has undergone pronounced rugose variation; and Type 5, same as type 3, except stably nonhemolytic. Type 1 and type 3 strains possess the characteristics usually ascribed to classic cholera vibrios and El Tor vibrios, respectively. Geographical and chronological distribution of the types is discussed. The thesis is presented that it is invalid to recognize two species, V. cholerae and V. eltor, because alleged differences are infrasubspecific. Recognition of a single species, V. cholerae, consisting of several types, is recommended.

Bacteriophages↗

Vibrio cholerae, Vibrio parahaemolyticus, and other vibrios: occurrence and distribution in Chesapeake Bay.

Vibrio cholerae was isolated at several locations in Chesapeake Bay in fall 1976 and spring 1977. Strains induced fluid accumulation in rabbit ileal loops and positive activity in Y-1 adrenal cells. Vibrio cholerae, Vibrio parahaemolyticus, and related vibrios show a spatial and temporal distribution characteristic of Vibrio species in an estuary. The Vibrio cholerae strains isolated from Chesapeake Bay represent serotypes other than O-group I--that is, so-called nonagglutinable vibrios--and are not recognized as a serious epidemic threat, although they have caused cholera-like diarrhea sporadically.

Maryland↗

Vibrio limimangrovi sp. nov., Vibrio phycocola sp. nov. and Vibrio sediminis sp. nov., isolated from coastal habitats.

Four marine bacterial strains of the genus Vibrio, designated WJH972T, RC27, FJH11T, and HA2012T, were isolated from coastal habitats in China. Phenotypic and physiological characterization showed that all four strains are Gram-stain-negative, facultatively anaerobic, short rod-shaped cells, and motile. The 16S rRNA gene sequence similarities between the isolates and their closest relatives were 96.5% - 98.2%, above the genus-level threshold but below the species delineation cutoff. Genomic analysis revealed that strains WJH972T and RC27 shared an average nucleotide identity (ANI) of 98.7% and a digital DNA-DNA hybridization (dDDH) value of 89.9%, confirming their conspecificity. In contrast, the ANI and dDDH values between the four strains and known Vibrio species ranged from 78.6% to 85.8% and from 13.7% to 59.3%, respectively, both falling below the thresholds for species delineation. The biosynthetic gene clusters support bacterial potentials for stress tolerance and antibiotic synthesis, consistent with strain FJH11T's resistance to all 20 antibiotics and other strains' sensitivity to chloramphenicol and penicillin. Based on these polyphasic taxonomic evidence above, three novel species are thus proposed: Vibrio limimangrovi sp. nov. (type strain FJH11T), Vibrio phycocola sp. nov. (type strain WJH972T), and Vibrio sediminis sp. nov. (type strain HA2012T). This study provides novel insights into the systematics, metabolic diversity, antibiotic resistance, and ecological distribution of the genus Vibrio.

Vibrio↗

Vibrio kanaloae sp. nov., Vibrio pomeroyi sp. nov. and Vibrio chagasii sp. nov., from sea water and marine animals.

The taxonomic position of the fluorescent amplified fragment length polymorphism fingerprinting groups A46 (five isolates), A51 (six isolates), A52 (five isolates) and A53 (seven isolates) obtained in a previous study were further analysed through a polyphasic approach. The 23 isolates were phylogenetically related to Vibrio splendidus, but DNA-DNA hybridization experiments proved that they belong to three novel species. Chemotaxonomic and phenotypic analyses further disclosed several features that differentiate between the 23 isolates and known Vibrio species. The names Vibrio kanaloae sp. nov. (type strain LMG 20539(T) = CAIM 485(T); EMBL accession no. AJ316193; G + C content 44.7 mol%), Vibrio pomeroyi sp. nov. (type strain LMG 20537(T) = CAIM 578(T); EMBL accession no. AJ491290; G +C content 44.1 mol%) and Vibrio chagasii sp. nov. (type strain LMG 21353(T) = CAIM 431(T); EMBL accession no. AJ316199; G + C content 44.6 mol%) are respectively proposed to encompass the five isolates of A46, the six isolates of A51 and the 12 isolates of A52/A53. The three novel species can be distinguished from known Vibrio species by several phenotypic features, including utilization and fermentation of various carbon sources, beta-galactosidase activity and fatty acid content (particularly of 12 : 0, 14: 0, 14 : 0 iso and 16 : 0 iso).

Animals↗

A collagenase-targeted multiplex PCR assay for identification of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus.

A multiplex PCR assay using three collagenase-targeted primer pairs for the species-specific detection of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus was developed. The results highlight the species specificity of the three primer sets designed. Because of the increasing importance of Vibrio spp. in human foodborne diseases, molecular approaches for routine microbial screening and monitoring of clinical, environmental, and food samples also have become more important. The results of this study indicate that the gene coding for collagenase should be used as an alternative molecular target to discriminate among the three Vibrio species.

DNA Primers↗

Identification and characterization of the sodA genes encoding manganese superoxide dismutases in Vibrio parahaemolyticus, Vibrio mimicus, and Vibrio vulnificus.

Sequencing of Fur titration assay-positive clones obtained from genomic DNA libraries of Vibrio parahaemolyticus, V. mimicus and V. vulnificus revealed open reading frames encoding proteins of 202, 205 and 202 amino acid residues, respectively. Each open reading frame was preceded by a predicted Fur box which overlaps a likely promoter with similarity to the -10 and -35 consensus sequence of Escherichia coli. The deduced amino acid sequences shared considerable homology with bacterial Mn-containing superoxide dismutases (MnSODs). Consistent with this, these Vibrio strains produced proteins with SOD activity resistant to inhibition by H2O2 and KCN only when grown under iron-limiting conditions. Primer extension analysis of the total RNA from these vibrios revealed iron-repressible expression of the genes. Furthermore, when grown under iron-limiting conditions, E. coli carrying a plasmid with each cloned gene overexpressed protein with the same electrophoretic mobility and insensitivity of SOD activity to H2O2 and KCN. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by N-terminal amino acid sequencing revealed that proteins (MnSODs) having N-terminal amino acid sequences consistent with those deduced from the corresponding genes were present in cell lysates of the vibrios grown under these iron-limited conditions. These results demonstrate that the genes cloned in this study are sodA homologs encoding MnSODs, whose expression is regulated by the iron status of the growth medium. PCR using a primer set based on the V. parahaemolyticus sodA sequence revealed the presence of homologous genes in certain other Vibrio species.

Amino Acid Sequence↗

BIOCHEMICAL CHARACTERISTICS OF VIBRIO SPUTORUM AND RELATIONSHIP TO VIBRIO BUBULUS AND VIBRIO FETUS.

Loesche, Walter J. (Forsyth Dental Center and Harvard School of Dental Medicine, Boston, Mass.), Ronald J. Gibbons, and Sigmund S. Socransky. Biochemical characteristics of Vibrio sputorum and relationship to Vibrio bubulus and Vibrio fetus. J. Bacteriol. 89:1109-1116. 1965.-Twenty-seven strains of Vibrio sputorum were isolated from human gingival debris. All strains reduced nitrate beyond nitrite, and all produced H(2)S. None produced catalase, indole, or acetylmethylcarbinol. Carbohydrates were not fermented, and acid end products or volatile neutral products could not be detected. Motility was observed infrequently, although 14-hr cultures usually had a few cells which displayed darting motility. Shadowed electron microscopic preparations demonstrated that most cells in a given culture were nonflagellated, although some cells with a single terminal or subterminal flagellum were always observed. V. sputorum was demonstrated not to be a strict anaerobe as previously described, but rather a microaerophile. Addition of nitrate to culture media markedly enhanced growth. V. sputorum was found to be closely related to the animal microaerophilic vibrios, V. bubulus and V. fetus. V. sputorum differed from V. fetus by being capable of anaerobic growth, producing H(2)S, and failing to produce catalase or to grow in 10% O(2). Antigens of V. sputorum cross-reacted in low titer with antisera to V. fetus serotypes I, II, III, and V. V. sputorum differed from V. bubulus by being inhibited by 3.5% NaCl and by qualitative differences in growth in the presence of 1% glycine and 10% O(2). These differences do not appear sufficient to recognize two distinct species, and it is proposed that both species be recognized as V. sputorum, which has priority. Strains unable to grow in 3.5% NaCl would be considered V. sputorum var. sputorum, whereas strains capable of growth in 3.5% NaCl would be V. sputorum var. bubulum.

Animals↗

Coagglutination of Vibrio cholerae, Vibrio mimicus, and Vibrio vulnificus with anti-flagellar monoclonal antibody.

Monoclonal antibodies (MAbs) with serological activity for purified flagellar (H) core protein prepared from Vibrio cholerae were identified by enzyme-linked immunosorbent assay. Four of these MAbs reacted with the flagella of V. cholerae and V. mimicus exclusively, while eight MAbs reacted with at least 1 of 30 heterologous Vibrio species tested by enzyme-linked immunosorbent assay or coagglutination. It appears that V. cholerae and V. mimicus express similar, if not identical, H determinants unique to these two Vibrio species. Staphylococcus aureus cells or latex beads armed with the four species-specific MAbs coagglutinated each of 47 isolates identified bacteriologically as V. cholerae or V. mimicus from among 103 Vibrio isolates tested. One coagglutination reagent armed with anti-V. vulnificus H MAb exhibited species specificity in that only V. vulnificus cells were coagglutinated from among the 31 Vibrio species examined. This reagent coagglutinated 20 isolates identified bacteriologically as V. vulnificus in a serological survey. MAb coagglutination reagents offer a rapid, specific, and economical alternative to the classical bacteriological approach to identify the human pathogens V. cholerae, V. mimicus, and V. vulnificus.

Agglutination↗

Vibrio neptunius sp. nov., Vibrio brasiliensis sp. nov. and Vibrio xuii sp. nov., isolated from the marine aquaculture environment (bivalves, fish, rotifers and shrimps).

The fluorescent amplified fragment length polymorphism (FAFLP) groups A5 (21 isolates), A8 (6 isolates) and A23 (3 isolates) distinguished in an earlier paper (Thompson et al., Syst Appl Microbiol 24, 520-538, 2001) were examined in more depth. These three groups were phylogenetically related to Vibrio tubiashii, but DNA-DNA hybridization experiments proved that the three AFLP groups are in fact novel species. Chemotaxonomic and phenotypic analyses further revealed several differences among the 30 isolates and known Vibrio species. It is proposed to accommodate these isolates in three novel species, namely Vibrio neptunius (type strain LMG 20536T; EMBL accession no. AJ316171; G +C content of the type strain 46.0 mol%), Vibrio brasiliensis (type strain LMG 20546T; EMBL accession no. AJ316172; G + C content of the type strain 45.9 mol%) and Vibrio xuii (type strain LMG 21346T; EMBL accession no. AJ316181; G +C content of the type strain 46.6 mol%). These species can be differentiated on the basis of phenotypic features, including fatty acid composition (particularly 14:0 iso, 14:0 iso 3-OH, 16:0 iso, 16:0, 17:0 and 17:1 omega8c), enzyme activities and utilization and fermentation of various carbon sources.

Animals↗

Genetic transformation of Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio cholerae non O-1 with plasmid DNA by electroporation.

An electroporation procedure for the plasmid-mediated transformation of the genus Vibrio was performed, as part of an effort to develop recombinant DNA techniques for genetic manipulation of the genus Vibrio. Vibrio parahaemolyticus, V. alginolyticus, and V. cholerae non O-1 (9 different strains) were transformed with 3 vector plasmids (pACYC184, pHSG398, and pBR325). The efficiency of transformation was highly dependent on three parameters: the concentration of plasmid DNA; the strength of the electric field; and the combination of plasmid DNA and recipient strain. The drug-resistance genes on the vector plasmid were expressed in the Vibrio strains.

DNA, Bacterial↗

Characterization of Vibrio viscosus and Vibrio wodanis isolated at different geographical locations: a proposal for reclassification of Vibrio viscosus as Moritella viscosa comb. nov.

Vibrio viscosus and Vibrio wodanis are recently described species of psychrotropic bacteria that have been found associated with a disease called 'winter ulcer', affecting salmonid fish reared in saline water in Norway, Iceland and recently in Scotland. V. viscosus and V. wodanis strains initially isolated from fish in Iceland and Norway were subjected to characterization using biochemical tests, SDS-PAGE of whole-cell proteins and a novel DNA fingerprinting method, amplified fragment length polymorphism (AFLP). The V. viscosus strains isolated from diseased fish grouped into homogeneous subgroups according to geographical origin and challenge experiments revealed that representatives of these groups are virulent. The results revealed that the V. wodanis strains are heterogeneous genotypically and phenotypically. Sequencing of almost complete 16S rRNA genes of V. viscosus and V. wodanis revealed that V. viscosus showed a 99.1% sequence similarity to Moritella marina and V. wodanis showed a 98.8% sequence similarity to Vibrio logei CIP 103204. A reclassification of Vibrio viscosus as Moritella viscosa comb. nov. is proposed.

Animals↗

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

AIMS: 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. METHODS: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.

China↗

White shrimp (Litopenaeus vannamei) recombinant lysozyme has antibacterial activity against Gram negative bacteria: Vibrio alginolyticus, Vibrio parahemolyticus and Vibrio cholerae.

C-type lysozyme has been described as an antibacterial component of the shrimp innate defence system. We determined quantitatively the antibacterial activity of white shrimp (Litopenaeus vannamei) recombinant lysozyme against three Gram negative bacteria: Vibrio alginolyticus, Vibrio parahemolyticus and Vibrio cholerae, using a turbidimetric assay with live bacteria and differential bacterial viable count after interaction with the protein. In conclusion, the antibacterial activity of recombinant shrimp lysozyme against Vibrio sp. is at least equal to the values against the Gram positive M. luteus and more active against the shrimp pathogens V. alginolyticus and V. parahemolyticus.

Animals↗

Comparison of hemolysins of Vibrio cholerae non-O1 and Vibrio hollisae with thermostable direct hemolysin of Vibrio parahaemolyticus.

Hemolysin (Vh-rTDH) produced by Vibrio hollisae and hemolysin (NAG-rTDH) produced by Vibrio cholerae non-O1 were characterized and compared with hemolysin (Vp-TDH) produced by Vibrio parahaemolyticus. These three hemolysins are each composed of two subunits and have similar, but not identical, molecular weights. The amino acid compositions of Vp-TDH and NAG-rTDH are similar, but are different from that of Vh-rTDH. The three hemolysins showed similar lethal toxicities to mice. The effects of temperature on hemolysis and the time dependencies of hemolysis by the three hemolysins were similar. The three were concluded to be immunologically related, but not identical, and to have common and also unique antigenic determinants.

Amino Acids↗