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 and toxigenicity of Vibrio cholerae in a freshwater lake during the epidemic of cholera caused by serogroup O139 Bengal in Calcutta, India

Abstract The extent of contamination of a freshwater lake with Vibrio cholerae 0139 Bengal and the toxigenicity of all the V. cholerae isolates recovered during the period of the study were examined during and after an explosive outbreak of 0139 cholera in Calcutta. Strains biochemically characterized as V. cholerae could be isolated throughout the period of study examined from the freshwater lake samples. Most probable number of V. cholerae belonging to the 0139 serogroup in surface waters was 3 to 4 per 100 ml during major part of the study but isolation of this serogroup from sediment and plankton samples was infrequent. Of the total of 150 strains recovered, 23 (15.3%) agglutinated with the 0139 antiserum while the remaining belonged to the non-O1 non-O139 serogroups. None of the strains agglutinated with the O1 antiserum. All the 23 strains of V. cholerae O139 produced cholera toxin while 7.9% of the 127 non-O1 non-O139 strains also produced cholera toxin. Resistance to ampilicillin, furazolidone and streptomycin was encountered among strains belonging to both V. cholerae O139 and V. cholerae non-O1 non-O139 strains, but the percentage of resistant strains in the former was much higher than in the latter. During this cholera epidemic, possibly due to the introduction of large numbers of toxigenic V. cholerae such as the O139 serogroup, there was an increase in the number of toxigenic vibrios among the innocuous aquatic residents. This presumably occured through genetic exchange and, if substantiated, could play an important role in the re-emergence of epidemics.     

Substrate Degradation and Pressure Tolerance of Free-Living and Attached Bacterial Populations in the Intestines of Shallow-Water Fish

This report compares recovery of non-O1 Vibrio cholerae strains from seven California coastal sites during the winter and summer of 1983. A total of 41 identified and 27 presumptive nn-O1 V. cholerae strains were recovered from six of seven coastal sites in the summer. A 5-to 56-fold increase in the numbers of organisms isolated from different sites occurred in the summer months, when water temperatures were 1.9 to 5.1 degrees C higher. At the three sites where the highest levels of non-O1 V. cholerae were found, pollution, as measured by the total number of coliforms, exceeded the legal limit (less than 1,000 coliforms per 100 ml.).     

Substrate degradation and pressure tolerance of free-living and attached bacterial populations in the intestines of shallow-water fish

This report compares recovery of non-O1 Vibrio cholerae strains from seven California coastal sites during the winter and summer of 1983. A total of 41 identified and 27 presumptive nn-O1 V. cholerae strains were recovered from six of seven coastal sites in the summer. A 5-to 56-fold increase in the numbers of organisms isolated from different sites occurred in the summer months, when water temperatures were 1.9 to 5.1 degrees C higher. At the three sites where the highest levels of non-O1 V. cholerae were found, pollution, as measured by the total number of coliforms, exceeded the legal limit (less than 1,000 coliforms per 100 ml.).     

Lecithinase activity of Vibrio cholerae and of vibrios not agglutinated by O-cholera serum

The lecithinase activities of 187 V. cholerae strains differing in their virulence and 70 cultures of NAG vibrios of different origin varied in all experimental groups. The level of lecithinase activity in the groups of V. cholerae strains having low virulence, or no virulence at all, was considerably higher than in virulent strains. NAG vibrios isolated from diarrhea patients and from samples of lake and pond water did not differ in the activity of this enzyme.     

Occurrence and distribution of halophilic vibrios in subtropical coastal waters of Hong Kong.

The summer occurrence and distribution of halophilic vibrios in the subtropical coastal waters of Hong Kong were investigated. The density of vibrios in six sample sites ranged from 90 to 6,700 per ml, which made up 0.41 to 40% of the total bacterial populations of these sample sites. The sucrose-positive vibrios were found to be much more common (88% of total vibrios) than the sucrose-negative ones. A total of 48 strains belonging to six Vibrio species were fully characterized. Among these, Vibrio alginolyticus was the most frequently isolated, followed by V. parahaemolyticus, V. harveyi, V. vulnificus, V. campbellii, and V. fluvialis. The finding that eight of the nine strains of V. harveyi showed a positive Kanagawa reaction warrants further study.     

Occurrence and distribution of halophilic vibrios in subtropical coastal waters of Hong Kong

The summer occurrence and distribution of halophilic vibrios in the subtropical coastal waters of Hong Kong were investigated. The density of vibrios in six sample sites ranged from 90 to 6,700 per ml, which made up 0.41 to 40% of the total bacterial populations of these sample sites. The sucrose-positive vibrios were found to be much more common (88% of total vibrios) than the sucrose-negative ones. A total of 48 strains belonging to six Vibrio species were fully characterized. Among these, Vibrio alginolyticus was the most frequently isolated, followed by V. parahaemolyticus, V. harveyi, V. vulnificus, V. campbellii, and V. fluvialis. The finding that eight of the nine strains of V. harveyi showed a positive Kanagawa reaction warrants further study.     

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.     

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.     

Temporal and spatial variability in culturable pathogenic Vibrio spp. in Lake Pontchartrain, Louisiana, following hurricanes Katrina and Rita

We investigated the abundance, distribution, and virulence gene content of Vibrio cholerae, V. parahaemolyticus, and V. vulnificus in the waters of southern Lake Pontchartrain in Louisiana on four occasions from October 2005 to September 2006, using selective cultivation and molecular assays. The three targeted pathogenic vibrios were generally below the detection level in January 2006, when the water was cold (13°C), and most abundant in September 2006, when the lake water was warmest (30°C). The maximum values for these species were higher than reported previously for the lake by severalfold to orders of magnitude. The only variable consistently correlated with total vibrio abundance within a single sampling was distance from shore (P = 0.000). Multiple linear regression of the entire data set revealed that distance from shore, temperature, and turbidity together explained 82.1% of the variability in total vibrio CFU. The log-transformed mean abundance of V. vulnificus CFU in the lake was significantly correlated with temperature (P = 0.014), but not salinity (P = 0.625). Virulence-associated genes of V. cholerae (ctx) and V. parahaemolyticus (trh and tdh) were not detected in any isolates of these species (n = 128 and n = 20, respectively). In contrast, 16S rRNA typing of V. vulnificus (n = 298) revealed the presence of both environmental (type A) and clinical (type B) strains. The percentage of the B-type V. vulnificus was significantly higher in the lake in October 2005 (35.8% of the total) than at other sampling times (P ≤ 0.004), consistent with the view that these strains represent distinct ecotypes.     

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.     

Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays

This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50 degrees C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 10(2) to 10(3) CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers.     

Method of DNA extraction and application of multiplex polymerase chain reaction to detect toxigenic Vibrio cholerae O1 and O139 from aquatic ecosystems

Vibrio cholerae is a free-living bacterium found in water and in association with plankton. V. cholerae non-O1/non-O139 strains are frequently isolated from aquatic ecosystems worldwide. Less frequently isolated are V. cholerae O1 and V. cholerae O139, the aetiological agents of cholera. These strains have two main virulence-associated factors, cholera toxin (CT) and toxin co-regulated pilus (TCP). By extracting total DNA from aquatic samples, the presence of pathogenic strains can be determined quickly and used to improve a microbiological risk assessment for cholera in coastal areas. Some methods suggested for DNA extraction from water samples are not applicable to all water types. We describe here a method for DNA extraction from coastal water and a multiplex polymerase chain reaction (PCR) for O1 and O139 serogroups. DNA extraction was successfully accomplished from 117 sea water samples collected from coastal areas of Perú, Brazil and the USA. DNA concentration in all samples varied from 20 ng to 480 micro g micro l-1. The sensitivity of the DNA extraction method was 100 V. cholerae cells in 250 ml of water. The specificity of multiplex O1/O139 PCR was investigated by analysing 120 strains of V. cholerae, Vibrio and other Bacteria species. All V. cholerae O1 and O139 tested were positive. For cholera surveillance of aquatic environments and ballast water, total DNA extraction, followed by V. cholerae PCR, and O1/O139 serogroup and tcpA/ctxA genes by multiplex PCR offers an efficient system, permitting risk analysis for cholera in coastal areas.     

Evaluation of polysaccharide antigens of Vibrio cholerae O139 of different origin by monoclonal antibodies

The epitope composition of O-polysaccharides in the lipopolysaccharide (LPS) of V. cholerae, serogroup O139, isolated from clinical material and water of surface reservoirs was analyzed with the use of monoclonal antibodies. The analysis demonstrated that these O-polysaccharides were similar in their structure and chemical composition. In LPS of V. cholerae O139 clinical strains O-polysaccharide determinants occurred more often. Among V. cholerae isolated from water strains on whose surface individual epitopes of O-polysaccharide occurred less frequently or were absent appeared to be more numerous. A decrease in the concentration of microbial cells in the process of their testing by immunological methods led to increased percent of negative reactions with specific antibodies. Some V. cholerae O139 strains isolated from water were similar in the epitope composition of their O-polysaccharide and binding activity to cultures isolated from humans. As indicated by the results of these studies, cholera vibrios Bengal and vibrios isolated from river water on the territory of Russia had quantitative differences due to a higher level of the production of O-polysaccharide determinants and their occurrence in V. cholerae of serogroup O139.     

Evaluation of selective media for isolation and enumeration of vibrios from estuarine waters

We have compared the effectiveness of four media developed in the last years together with the medium GSTC(glucose-salt-tellurite-crystal violet), devised in our laboratory, for the recovery of vibrios from estuarine waters. In addition, a number of reference Vibrio and non-Vibrio strains have been tested for growth on the five media. TCBS and GSTC were the most selective media for reference strains of Vibrio spp. However, when the media were tested with samples of water from three different sites of an estuary, only TCBS was effective enough to recover vibrios inhibiting the growth of non-Vibrio populations. We also report here the usefulness of TCBS for isolation of the fish pathogen V. anguillarum, since a total of 81 strains isolated from diseased fish and water in various parts of the world grew on this medium. In conclusion, we consider the TCBS as the best medium to isolate Vibrio species pathogenic for humans and fish, and for recovery of vibrios from estuarine waters.     

Occurrence of pathogenic vibrios in coastal areas of France

AIMS: This study was carried out to investigate the occurrence of potentially pathogenic species of Vibrio in French marine and estuarine environments. METHODS AND RESULTS: 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. CONCLUSIONS: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.     

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.     

A bioserogroup of marine vibrios possessing somatic antigen factors in common with Vibrio cholerae O1

A bioserogroup of halophilic vibrios, tentatively labelled bioserogroup 1875, strongly agglutinated by O1 antiserum of Vibrio cholerae is described. Cross-agglutination and agglutinin-absorption tests showed that these vibrios had antigens identical with the Ogawa and Inaba factors of V. cholerae O1, although they possessed their own specific antigen distinctive from the A factor that is the specific major antigen of the latter species. In addition, quantitative antigen variation similar to that of the Ogawa-to-Inaba type with V. cholerae O1 was recognized in this halophilic group. They were isolated from estuarine water and are considered to inhabit coastal aquatic environments. Phenotypically, however, this group is not identifiable with any of the species already recognized in the genus Vibrio .     

A bioserogroup of marine vibrios possessing somatic antigen factors in common with Vibrio cholerae O1

A bioserogroup of halophilic vibrios, tentatively labelled bioserogroup 1875, strongly agglutinated by O1 antiserum of Vibrio cholerae is described. Cross-agglutination and agglutinin-absorption tests showed that these vibrios had antigens identical with the Ogawa and Inaba factors of V. cholerae O1, although they possessed their own specific antigen distinctive from the A factor that is the specific major antigen of the latter species. In addition, quantitative antigen variation similar to that of the Ogawa-to-Inaba type with V. cholerae O1 was recognized in this halophilic group. They were isolated from estuarine water and are considered to inhabit coastal aquatic environments. Phenotypically, however, this group is not identifiable with any of the species already recognized in the genus Vibrio.