Characterization of toxigenic vibrios isolated from the freshwater environment of Hiroshima, Japan.

The occurrence and characterization of toxigenic vibrios in surface water and sediment samples of the fresh water environment of the Ohta River were studied. The membrane filter, pad preenrichment technique, followed by the placement of membranes onto thiosulfate citrate-bile salt-sucrose agar, was used for the enumeration of total vibrios. Qualitative examination of pathogenic vibrios was also attempted. In addition, a survey was conducted to determine the incidence of Clostridium botulinum in sediment samples of the Ohta River and the Hiroshima coast. In the identification of 361 strains, 12 species of Vibrio and two species of Listonella were observed. Non-01 Vibrio cholerae was prevalent among the members of the genus Vibrio. Vibrio parahaemolyticus (serotype 04:K34), isolated in fresh water, is significant and suggests that some still unknown conditions promote the survival of these organisms in fresh water. An estimated 132 strains were hemolytic by a simple agar method, and further characterization revealed that 82% of the hemolytic vibrios (107 strains) produced various toxins. About 71% (93 strains) elaborated cytotoxin, 55% (72 strains) produced hemolysin, and 44% (58 strains) responded for both cytotoxin and hemolysin in the crude toxin extracts. All the non-01 V. cholerae showed cytotoxic activity, and the virulent strains of Vibrio fluvialis and Vibrio spp. showed cytotonic responses in RK-13 cells. Of 36 sediment samples tested, 10 harbored C. botulinum spores (28%) and were isolated invariably in all the regions of the Hiroshima coast and in the Ohta River, except the upper region of the Ohta River.     

The isolation of monoclonal antibodies to the hemolysin of noncholerigenic vibrios]

The results of the work on obtaining monoclonal antibodies (McAb) to hemolysin of noncholerigenic vibrios and the study of hemolysin preparations and Vibrio cholerae strains are presented. As established with the use of McAb, hemolysin preparations proved to be complex compounds containing, in addition to hemolytic protein, some lipopolysaccharide determinants, which had not been detected with the use of traditional biochemical tests. The study revealed that V. cholerae non-01 and V. cholerae eltor hemolysins have common antigenic determinants, which was confirmed by the data of literature on the homogeneity of these substances. McAb obtained in this study interacted with V. cholerae avirulent hemolytic strains, not interacting with V. cholerae classical strains.     

Occurrence and distribution of Vibrio spp., Listonella spp., and Clostridium botulinum in the Seto Inland Sea of Japan.

The distribution of Vibrio species in samples of surface water, bottom water (water 2 m above the sediment), and sediment from the Seto Inland Sea was studied. A simple technique using a membrane filter and short preenrichment in alkaline peptone water was developed to resuscitate the injured cells, followed by plating them onto TCBS agar. In addition, a survey was conducted to determine the incidence of Clostridium botulinum in sediment samples. Large populations of heterotrophs were found in surface water, whereas large numbers of total vibrios were found in bottom water. In samples from various water sampling regions, high counts of all bacterial populations were found in the inner regions having little exchange of seawater when compared with those of the open region of the inland sea. In the identification of 463 isolates, 23 Vibrio spp. and 2 Listonella spp. were observed. V. harveyi was prevalent among the members of the Vibrio genus. Vibrio species were categorized into six groups; an estimated 20% of these species were in the so-called "pathogenic to humans" group. In addition, a significant proportion of this group was hemolytic and found in the Bisan Seto region. V. vulnificus, V. fluvialis, and V. cholerae non-O1 predominated in the constricted area of the inland sea, which is eutrophic as a result of riverine influence. It was concluded that salinity indirectly governs the distribution of total vibrios and analysis of variance revealed that all bacterial populations were distributed homogeneously and the variance values were found to be significant in some water sampling regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence and distribution of Vibrio spp., Listonella spp., and Clostridium botulinum in the Seto Inland Sea of Japan

The distribution of Vibrio species in samples of surface water, bottom water (water 2 m above the sediment), and sediment from the Seto Inland Sea was studied. A simple technique using a membrane filter and short preenrichment in alkaline peptone water was developed to resuscitate the injured cells, followed by plating them onto TCBS agar. In addition, a survey was conducted to determine the incidence of Clostridium botulinum in sediment samples. Large populations of heterotrophs were found in surface water, whereas large numbers of total vibrios were found in bottom water. In samples from various water sampling regions, high counts of all bacterial populations were found in the inner regions having little exchange of seawater when compared with those of the open region of the inland sea. In the identification of 463 isolates, 23 Vibrio spp. and 2 Listonella spp. were observed. V. harveyi was prevalent among the members of the Vibrio genus. Vibrio species were categorized into six groups; an estimated 20% of these species were in the so-called "pathogenic to humans" group. In addition, a significant proportion of this group was hemolytic and found in the Bisan Seto region. V. vulnificus, V. fluvialis, and V. cholerae non-O1 predominated in the constricted area of the inland sea, which is eutrophic as a result of riverine influence. It was concluded that salinity indirectly governs the distribution of total vibrios and analysis of variance revealed that all bacterial populations were distributed homogeneously and the variance values were found to be significant in some water sampling regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning of the hemolysin determinant from Vibrio cholerae El Tor

Vibrio cholerae El Tor RV79 is phenotypically nonhemolytic; however, strongly hemolytic convertants are occasionally observed on blood agar plates. We have cloned DNA sequences corresponding to the hemolysin determinant from RV79 (Hly+) in the lambda L47.1 and pBR322 vectors. A 2.3-kilobase fragment of V. cholerae DNA was found to be necessary for hemolytic activity. This cloned DNA sequence was used as a probe in Southern blot hybridization analysis of chromosomal restriction digests of a variety of El Tor and classical biotype V. cholerae strains. In all cases, DNA fragments with the same electrophoretic mobilities hybridized to the Hly probe. The results presented demonstrate that the cloned hemolysin determinant is the hly locus. By using mutator vibriophage VcA-3 insertion to promote high-frequency transfer, the hly locus was mapped between arg and ilv on the V. cholerae RV79 chromosome.     

Isolation of Vibrio cholerae no-01 in the Gulf of Nicoya, Costa Rica]

In a bacteriological study on samples of bivalves, mud and surface waters from the Gulf of Nicoya, Costa Rica, 18 strains of non-01 Vibrio cholerae and 50 of V. mimicus were isolated. The samples were enriched in alkaline peptone water, and streaked on MacConkey and inositol-brilliant green bile agars. Biochemical and serological tests were used for their identification. Both species were isolated from all sampling sites (Lepanto, Jicaral and Puntarenas) with either of the two agar media, even though these were not specific for vibrios.     

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.     

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.     

Cloning and partial characterization of a novel hemolysin gene of Vibrio tubiashii and the development of a PCR-based detection assay

Vibrio tubiashii expresses virulence factors, such as a vulnificolysin-like hemolysin or cytolysin and a zinc metalloprotease, similar to those of other pathogenic vibrios. In this study, we report the cloning of a novel hemolysin gene of V.?tubiashii in Escherichia coli . A V.?tubiashii gene library was screened for hemolytic activity on sheep blood agar. Three hemolytic clones pGem:hly1, pGem:hly2, and pGem:hly3 were sequenced, and the sequences showed a strong homology to the ribA gene coding for guanosine triphosphate cyclohydrolase II (GCH II), required for riboflavin biosynthesis and reported to be responsible for hemolytic activity in Helicobacter pylori . The plasmids pGem:hly1 and pGem:hly3 when introduced into E. coli BSV18 (ribA18::Tn5) were able to restore growth of strain BSV18 in a medium without riboflavin and also produced hemolytic activity on blood agar. PCR primers based on the cloned hly-ribA sequence were tested using 23 different Vibrio strains representing 10 different species. Amplification of ribA gene locus only occurred with V.?tubiashii strains. In summary, our results indicate that we have cloned a ribA homolog of V.?tubiashii that imparts hemolytic activity to E.?coli clones, and primers based on this gene locus might be useful as a species-specific identification tool for V.?tubiashii.     

An aberrant hemolysin of Vibrio cholerae non-O1.

An aberrant hemolysin produced by a Vibrio cholerae non-O1 strain N037 (N037-hly) was purified and characterized. N037-Hly was antigenically very similar to El Tor hemolysin but differed in molecular weight (48,000 vs. 60,000), interaction with glucose, and hemolytic activity. Of 100 V. cholerae non-O1 strains other than the N037 strain examined, none produced this aberrant hemolysin. The N-terminal amino acid sequence of N037-hly was highly homologous to that of El Tor hemolysin.     

Incidence of Vibrio cholerae and related vibrios in a coastal lagoon and seawater influenced by lake discharges along an annual cycle

Most probable numbers of Vibrio cholerae and related vibrios were determined in Albufera Lake, Valencia, Spain, and in coastal waters under the influence of the lake discharges over the course of an annual cycle. The influence of temperature, kind of water, and characteristics of the different sampling sites on the numbers of vibrios recovered was evaluated. Maximum recovery of vibrios reached 10(3)/ml in both types of waters analyzed. V. cholerae numbers reached 10(3)/ml in the lake and 10(2) in one of the coastal sites. Frequently during the warm season, all vibrios isolated were identified as V. cholerae. Occasionally, no V. cholerae was recovered. The recovery of vibrios was significantly influenced by the temperature of the water and the type of water analyzed. Most of the V. cholerae isolates were included in Heiberg groups I and II, and nearly 50% of the strains used chitin as sole carbon source. Indole was not produced by 100% of the strains. All strains tested were non-O1 serovars.     

Incidence of Vibrio cholerae and related vibrios in a coastal lagoon and seawater influenced by lake discharges along an annual cycle.

Most probable numbers of Vibrio cholerae and related vibrios were determined in Albufera Lake, Valencia, Spain, and in coastal waters under the influence of the lake discharges over the course of an annual cycle. The influence of temperature, kind of water, and characteristics of the different sampling sites on the numbers of vibrios recovered was evaluated. Maximum recovery of vibrios reached 10(3)/ml in both types of waters analyzed. V. cholerae numbers reached 10(3)/ml in the lake and 10(2) in one of the coastal sites. Frequently during the warm season, all vibrios isolated were identified as V. cholerae. Occasionally, no V. cholerae was recovered. The recovery of vibrios was significantly influenced by the temperature of the water and the type of water analyzed. Most of the V. cholerae isolates were included in Heiberg groups I and II, and nearly 50% of the strains used chitin as sole carbon source. Indole was not produced by 100% of the strains. All strains tested were non-O1 serovars.     

A note on colony-blot double-stain method for isolation of Vibrio cholerae serotype 01 from specimens heavily contaminated with non-01 Vibrio cholerae

A colony-blot double-stain method was developed to identify individual colonies of Vibrio cholerae serotype 01 (pandemic strain) in mixed bacterial cultures on solid media. The colonies are transferred from agar to nitrocellulose membranes for an enzyme-linked immunosorbent assay (ELISA). Colonies of 01 vibrios bind the enzyme-linked antibodies and appear as brown dots on the membranes; pale black dots develop at the site of replicated colonies of other bacteria as a result of the activity of endogenous oxidase-like enzymes and serve as reference points. The results indicate that the colony-blot double-stain method is useful for the isolation of colonies of V. cholerae serotype 01 in specimens that are heavily contaminated with non-01 vibrios.     

High Prevalence of Vibrio cholerae Non-O1 Carrying Heat-Stable-Enterotoxin-Encoding Genes among Vibrio Isolates from a Temperate-Climate River Basin of Central Italy

Vibrio spp. of clinical interest from the Arno River basin (Tuscany, Italy) were investigated in this study. Vibrios were isolated from 70% of water samples. Vibrio cholerae non-O1 was the most prevalent species (82% of isolates), followed by Vibrio mimicus (10%) and Vibrio metschnikovii (8%). Recovery of vibrios was correlated with temperature, pH, and various indicators of municipal pollution. None of the 150 Vibrio isolates carried ctx-related genomic sequences, whereas 18 (14.6%) of the 123 V. cholerae non-O1 isolates and 1 (6.7%) of the 15 V. mimicus isolates carried sto alleles. These findings indicate that considerable circulation of sto-positive vibrios may occur in temperate-climate freshwater environments.     

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.     

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 10² organisms/1. Halophilic vibrios, less than five organisms/1, 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° to 20°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.     

Effect of environmental factors on expression and activity of chitinase genes of vibrios with special reference to Vibrio cholerae

AIMS: The aim of this study was to investigate the distribution and inducibility of chitinase genes in vibrios and the effect of environmental factors on the expression level and activity of chitinase genes in Vibrio cholerae strains. METHODS AND RESULTS: Chitin agar plate assays showed that V. cholerae strains were more chitinolytic than non-cholerae vibrios. All of the identified or putative chitinase genes were expressed in V. cholerae (four strains) but not in non-cholerae vibrios (seven species/strains) under standard laboratory growth conditions. In non-cholerae vibrios, these genes were induced by chitin, its monomer N-acetyl-d-glucosamine and on exposure to rabbit intestine, while in V. cholerae strains, these genes showed significant variation in expression levels. To study the effects of environmental factors on the expression and activity of chitinase genes in V. cholerae, bacteria were cultured in different pH, temperature, sodium chloride and nutrients. RT-PCR analysis showed that lower temperatures and higher pH, salinity and nutrition favoured expression of these genes, while their activity increased under higher nutrition content and salinity. CONCLUSIONS: Chitinase genes are distributed in all the relatively small number of strains studied here, and biotic and abiotic factors have significant role in the induction, expression level and activity of this gene family in vibrios. SIGNIFICANCE AND IMPACT OF THE STUDY: Chitinases have important applications especially in recycling of chitin. Vibrios can be used as chitinolytic agents, using suitable culture conditions that maximize the expression and activity of these genes.     

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

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.     

Retrospective VNTR-analysis of genotypes of Vibrio cholerae 01 strains isolated, during the 7th cholera pandemic, in Rostov Region

Antiplague Research Institute, Rostov-on-Don, Russia Retrospective multi-locus VNTR-analysis was made for 166 Vibrio cholerae strains isolated, 1967-2001, in Rostov Region from clinical samples (82 strains) and from water samples (84 strains). On the basis of cluster analysis of heterogeneous identification strain genotypes, 45 variations of individual strains were shared between 11 separate clusters, among which the F cluster vibrios were predominant. Having emerged, 1970, in the region, they were widely spread during the 1973-1975 cholera pandemic and were registered, among the isolated strains, till 1992 indicating the possibility of long persistence of V. cholerae 01 in the natural aquatic environment. Presumably, the ecosystem specificity contributed to the long-term vibrio persistence.