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.     

Uptake and retention of Vibrio cholerae O1 in the Eastern oyster, Crassostrea virginica.

Vibrio cholerae 01, the causative agent of cholera, is known to persist in estuarine environments as endogenous microflora. The recent introduction of V. cholerae 01 into estuaries of the North and South American continents has stimulated the need to determine the effect of controlled purification on reducing this pathogen in edible molluscan shellfish. Experiments defined parameters for the uptake and retention of V. cholerae 01 in tissues of Crassostrea virginica, and these parameters were compared with those for Escherichia coli and Salmonella tallahassee, bacteria which are usually eliminated from moderately contaminated shellfish within 48 h. Oysters accumulated greater concentrations of V. cholerae 01 than E. coli and S. tallahassee. When V. cholerae 01 was exposed to controlled purification at 15, 19 and 25 degrees C over 48 h, it persisted in oysters at markedly higher levels than E. coli and S. tallahassee. The concentration of a V. cholerae 01-specific agglutinin did not positively correlate with the uptake or retention of V. cholerae 01. These data show that state and federally approved controlled purification techniques are not effective at reducing V. cholerae 01 in oysters.     

Development and evaluation of a rapid, simple, sensitive, monoclonal antibody-based co-agglutination test for direct detection of Vibrio cholerae 01

Cholera epidemics caused by Vibrio cholerae 01 continue to represent a major public health concern in many developing countries. A rapid and simple test kit for the detection of V. cholerae 01 has been developed. The kit, CholeraScreen is a monoclonal antibody-based, co-agglutination test and is used directly with stool specimens. It does not include culturing the specimen and is performed without the need for sophisticated laboratory equipment. Specificity of the test was demonstrated, using 118 reference cultures, to which cross-reactions were not observed. Preliminary results of field trials carried out in Guatemala and Bangladesh demonstrated that the test is equally sensitive as conventional culture methods in detecting V. cholerae and, in many cases, more sensitive. The CholeraScreen test is simple, specific, and does not require culturing procedures, making it suitable for direct detection of cells of V. cholerae in clinical specimens, even in the field. Also, the test requires less than five minutes to complete.     

Diversity of DNA sequences among Vibrio cholerae O1 and non-O1 isolates detected by whole-cell repetitive element sequence-based polymerase chain reaction

Vibrio cholerae strains isolated from patient, food and environmental sources in Taiwan and reference V. cholerae strains were examined by repetitive element sequence-based PCR (rep-PCR). Specimens from broth cultures were used directly in the PCR mixture with three different primers. The PCR fingerprinting profiles of toxigenic 01 isolates were not only homogeneous with primers from enterobacterial repetitive intergenic consensus (ERIC) sequences, but also allowed the differentiation from non-toxigenic O1 and non-O1 strains. Toxigenic 01 strains were further differentiated into El Tor and classical biotypes with primers designed from ERIC-related sequences of V. cholerae. Primers from the other V. cholerae repetitive DNA sequences, VCR, separated toxigenic El Tor strains into six groups and a unique pattern was also obtained in 16 isolates from imported cases of cholera and imported seafood. The results indicated that rep-PCR can be used to identify and differentiate different toxigenic 01, non-toxigenic 01 and non-O1 V. cholerae isolates.     

Enterobacterial repetitive intergenic consensus sequences and the PCR to generate fingerprints of genomic DNAs from Vibrio cholerae O1, O139, and non-O1 strains.

Enterobacterial repetitive intergenic consensus (ERIC) sequence polymorphism was studied in Vibrio Cholerae strains isolated before and after the cholera epidemic in Brazil (in 1991), along with epidemic strains from Peru, Mexico, and India, by PCR. A total of 17 fingerprint patterns (FPs) were detected in the V. cholerae strains examined; 96.7% of the toxigenic V. cholerae O1 strains and 100% of the O139 serogroup strains were found to belong to the same FP group comprising four fragments (FP1). The nontoxigenic V. cholerae O1 also yielded four fragments but constituted a different FP group (FP2). A total of 15 different patterns were observed among the V. cholerae non-O1 strains. Two patterns were observed most frequently for V. cholerae non-01 strains, 25% of which have FP3, with five fragments, and 16.7% of which have FP4, with two fragments. Three fragments, 1.75, 0.79, and 0.5 kb, were found to be common to both toxigenic and nontoxigenic V. cholerae O1 strains as well as to group FP3, containing V. cholerae non-O1 strains. Two fragments of group FP3, 1.3 and 1.0 kb, were present in FP1 and FP2 respectively. The 0.5-kb fragment was common to all strains and serogroups of V. cholerae analyzed. It is concluded from the results of this study, based on DNA FPs of environmental isolates, that it is possible to detect an emerging virulent strain in a cholera-endemic region. ERIC-PCR constitutes a powerful tool for determination of the virulence potential of V. cholerae O1 strains isolated in surveillance programs and for molecular epidemiological investigations.     

Effect of various biophysicochemical conditions on toxigenicity of Vibrio cholerae 01 during survival with a green alga, Rhizoclonium fontanum, in an artificial aquatic environment

Toxigenic and nontoxigenic strains of Vibrio cholerae 01 occur in the natural aquatic environment. It is not clear whether V. cholerae 01 lose toxigenicity and become nontoxigenic during survival in the aquatic environment as a result of the effect of various biophysicochemical conditions (e.g., sunlight, pH, temperature, competition with other bacteria for nutrients, etc.). Five toxigenic strains were exposed to artificial aquatic environments in the presence of a filamentous green alga. Rhizoclonium fontanum, and recovered after different time intervals (0 and 0.5 h, 3, 6, 9, and 15 days). This experimental system was exposed to sunlight and the V. cholerae 01 were in competition for nutrients with resident bacterial flora from R. fontanum. The toxigenicity of Vibrio cholerae 01 that were recovered at different time intervals was assessed by tissue culture assay using Vero cells. The toxigenicity of recovered strains was compared with that of the parent strains. The results demonstrated that toxigenic V. cholerae 01 are unlikely to lose their toxigenicity in aquatic environments as a result of the effects of various biophysicochemical conditions. These results are consistent with the hypothesis of environmental reservoirs of V. cholerae.     

Molecular cloning of the plasmids of Vibrio cholerae 01 and the incidence of related plasmids in clinical isolates and other Vibrio species

Abstract We describe the subcloning of plasmids present in Vibrio cholerae 01 strain V58: the P factor, the large cryptic plasmid (lcp) and small cryptic plasmid (scp). Strains harbouring fragments of the P sex factor and the lcp were examined for plasmid encoded proteins by Coomassie blue staining and analysis in Escherichia coli K-12 minicells.
The distribution of these three plasmids in a variety of Vibrio species has been examined using some of the cloned fragments as DNA probes. Most recent clinical isolates of V. cholerae were found to contain the scp. None of the strains contained the lcp. The P factor was only detected in one clinical isolate in addition to the scp. While plasmids appear to be generally uncommon among V. cholerae , and do not appear to differentiate biovars, the presence of plasmids may be a useful epidemiological adjunct.     

Distribution of the zot (zonula occludens toxin) gene among strains of Vibrio cholerae 01 and non-01

Abstract The distribution of the zot gene that encodes the zonula occludens toxin, a newly described toxin of Vibrio cholerae , among clinical, environmental and food isolates of V. cholerae 01 and non-01 was investigated. Both the zot gene and the ctx gene that encode cholera toxin were found in 247 of 257 clinical strains and 62 of 415 environmental or food isolates of V. cholerae 01. The zot gene, but not the ctx gene was found in 37 strains (one clinical strain and 36 environmental or food isolates). In addition, two of 31 clinical strains and six of 98 environmental or food isolates of V. cholerae non-01 possessed both the zot gene and the ctx gene. These results demonstrated the predominantly concurrent occurrence of the zot gene and ctx genes among strains of V. cholerae 01 which suggests a possible synergistic role of ZOT in the causation of acute dehydrating diarrhea produced by V. cholerae 01.     

Ecology of Vibrio cholerae non-O1 and Salmonella spp. and role of zooplankton in their seasonal distribution in Fukuyama coastal waters, Japan

Seasonal variation of human pathogens such as Vibrio Cholerae non-01 and Salmonella spp. in Fukuyama coastal waters and the role of zooplankton in their distribution were studies for a period of 1 year. Comparison of two established methods, viz., the elevated temperature method and the two-step enrichment method of enumerating V. cholerae, showed that the former is superior in the recoveries of V. cholerae non-01. Isolation of this pathogen on a wider range of salinities (0.4 to 32.5%) revealed that these organisms are apparently an autochthonous component of the aquatic environment. Temperature appears to be the most crucial element in governing the distribution of V. cholerae non-01. Among the 69 isolates serotyped, 22 different serovars were identified, while one isolate failed to react with any of the known Louisiana State University antisera tested. Zooplankton samples did not harbor more V. Cholerae non-01 than the water column did. Better isolation of an allochthonous pathogen, viz., Salmonella spp., was noticed from the water samples when swabs were employed. Of the 251 isolates serotyped, 18 serotypes with three variants of Salmonella spp. were identified. A high amount of nutrients in the water column increased the survival rate of these pathogens in saline waters as evidenced by a higher incidence of various serotypes in polluted Fukuyama port than in clean marine waters. Salmonella spp. association between V. cholerae non-01 of Salmonella spp. with zooplankton could be noticed as influencing their seasonal distribution.     

Ecology of Vibrio cholerae non-O1 and Salmonella spp. and role of zooplankton in their seasonal distribution in Fukuyama coastal waters, Japan.

Seasonal variation of human pathogens such as Vibrio Cholerae non-01 and Salmonella spp. in Fukuyama coastal waters and the role of zooplankton in their distribution were studies for a period of 1 year. Comparison of two established methods, viz., the elevated temperature method and the two-step enrichment method of enumerating V. cholerae, showed that the former is superior in the recoveries of V. cholerae non-01. Isolation of this pathogen on a wider range of salinities (0.4 to 32.5%) revealed that these organisms are apparently an autochthonous component of the aquatic environment. Temperature appears to be the most crucial element in governing the distribution of V. cholerae non-01. Among the 69 isolates serotyped, 22 different serovars were identified, while one isolate failed to react with any of the known Louisiana State University antisera tested. Zooplankton samples did not harbor more V. Cholerae non-01 than the water column did. Better isolation of an allochthonous pathogen, viz., Salmonella spp., was noticed from the water samples when swabs were employed. Of the 251 isolates serotyped, 18 serotypes with three variants of Salmonella spp. were identified. A high amount of nutrients in the water column increased the survival rate of these pathogens in saline waters as evidenced by a higher incidence of various serotypes in polluted Fukuyama port than in clean marine waters. Salmonella spp. association between V. cholerae non-01 of Salmonella spp. with zooplankton could be noticed as influencing their seasonal distribution.     

The incidence of Vibrio cholerae in water, animals and birds in Kent, England

Between 1976 and 1979 several surveys were carried out in Kent, England, to establish the incidence of Vibrio cholerae in the aquatic environment. Vibrio cholerae occurred sporadically in all types of water during the summer but only in very low numbers in water containing <5 mmol Na⁺/litre. Highest numbers of up to 700 colony-forming units/ml appeared regularly in static brackish water containing 25–200 mmol Na⁺/litre. They were not introduced by sewage contamination of the water and there was no correlation between the counts of Escherichia coli and V. cholerae. A wide range of serovars including O1 was isolated. Vibrio cholerae was not isolated from sheep faeces but was detected in 6% of cloacal swabs taken from gulls caught at times when V. cholerae could not be isolated from water. It was concluded that: the presence of these organisms in the environment in Kent does not present any significant risk to health; aquatic birds may be vectors of V. cholerae; V. cholerae occurs naturally in static brackish water.     

Isolation of Vibrio cholerae from aquatic birds in Colorado and Utah

Vibrio cholerae was isolated from cloacal swabs and freshly voided feces collected from 20 species of aquatic birds in Colorado and Utah during 1986 and 1987. About 17% (198 of 1,131) fecal specimens collected from July 1986 through August 1987 contained the organism. Both O1 and non-O1 V. cholerae strains were isolated from the fecal specimens. Isolates from eight birds (representing five species) agglutinated in O group 1 antiserum. Supernatants of broth cultures from three isolates which typed as V. cholerae O1 serotype Ogawa gave reactions typical of cholera toxin when tested on Y-1 mouse adrenal cell cultures. Several serovars of non-O1 V. cholerae were isolated from the fecal specimens; serovar 22 was the most prevalent type. All non-O1 isolates were cytotoxic to Y-1 mouse adrenal cells. Only non-O1 V. cholerae was detected in water samples collected from the habitat of the birds. The results of this study suggest that aquatic birds serve as carriers and disseminate V. cholerae over a wide area.     

Isolation of Vibrio cholerae from aquatic birds in Colorado and Utah.

Vibrio cholerae was isolated from cloacal swabs and freshly voided feces collected from 20 species of aquatic birds in Colorado and Utah during 1986 and 1987. About 17% (198 of 1,131) fecal specimens collected from July 1986 through August 1987 contained the organism. Both O1 and non-O1 V. cholerae strains were isolated from the fecal specimens. Isolates from eight birds (representing five species) agglutinated in O group 1 antiserum. Supernatants of broth cultures from three isolates which typed as V. cholerae O1 serotype Ogawa gave reactions typical of cholera toxin when tested on Y-1 mouse adrenal cell cultures. Several serovars of non-O1 V. cholerae were isolated from the fecal specimens; serovar 22 was the most prevalent type. All non-O1 isolates were cytotoxic to Y-1 mouse adrenal cells. Only non-O1 V. cholerae was detected in water samples collected from the habitat of the birds. The results of this study suggest that aquatic birds serve as carriers and disseminate V. cholerae over a wide area.     

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.     

Direct detection of Vibrio cholerae and ctxA in Peruvian coastal water and plankton by PCR

Seawater and plankton samples were collected over a period of 17 months from November 1998 to March 2000 along the coast of Peru. Total DNA was extracted from water and from plankton grouped by size into two fractions (64 micro m to 202 micro m and >202 micro m). All samples were assayed for Vibrio cholerae, V. cholerae O1, V. cholerae O139, and ctxA by PCR. Of 50 samples collected and tested, 33 (66.0%) were positive for V. cholerae in at least one of the three fractions. Of these, 62.5% (n = 32) contained V. cholerae O1; ctxA was detected in 25% (n = 20) of the V. cholerae O1-positive samples. None were positive for V. cholerae O139. Thus, PCR was successfully employed in detecting toxigenic V. cholerae directly in seawater and plankton samples and provides evidence for an environmental reservoir for this pathogen in Peruvian coastal waters.     

01群与非01群0139型霍乱弧菌毒力的对比研究

非０１群０１３９型霍乱弧菌是近年引起南亚次大陆霍乱流行的新型病原体,将其与０１群霍乱弧菌的毒力特性进行对比研究对于了解其特性及研制相关的菌苗具有重要意义。本文报告了４株０１３９型霍乱弧菌与０１群霍乱弧菌菌株的对比测定结果。发现０１３９型霍乱弧菌与０１群霍乱弧菌有所不同,呈不透明的菌落形态,光学显微镜及电子显微镜检显示有荚膜的表型,在体内具有较高的繁殖能力并产生肠毒素,体内侵袭试验结果表明所有４株０     

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.     

Comparative Trial of Tetracycline,Chloramphenicol, and Trimethoprim Sulphamethoxazole in Eradication of Vibrio cholera El Tor

A comparison of tetracycline, chloramphenicol, and trimethoprim/sulphamethoxazole showed that all hasten the eradication of Vibrio cholerae from the stools of patients with cholera.A four-day period of tetracycline or trimethoprim/ sulphamethoxazole was adequate for eradicating V. cholerae from the stools of all patients, but three days, as suggested by the W.H.O. Expert Committee, was not. Four days of chloramphenicol therapy was sufficient for most patients, but a minority required up to seven days'' therapy.Purging produced reappearance of V. cholerae in the stools of one-eighth of the patients who had had three successive daily negative stool cultures; such patients are a potential danger to the population.     

TaqMan PCR for detection of Vibrio cholerae O1, O139, non-O1, and non-O139 in pure cultures, raw oysters, and synthetic seawater

Vibrio cholerae is recognized as a leading human waterborne pathogen. Traditional diagnostic testing for Vibrio is not always reliable, because this bacterium can enter a viable but nonculturable state. Therefore, nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing. In this article, a TaqMan PCR assay is presented for quantitative detection of V. cholerae in pure cultures, oysters, and synthetic seawater. Primers and probe were designed from the nonclassical hemolysin (hlyA) sequence of V. cholerae strains. This probe was applied to DNA from 60 bacterial strains comprising 21 genera. The TaqMan PCR assay was positive for all of the strains of V. cholerae tested and negative for all other species of Vibrio tested. In addition, none of the other genera tested was amplified with the TaqMan primers and probe used in this study. The results of the TaqMan PCR with raw oysters and spiked with V. cholerae serotypes O1 and O139 were comparable to those of pure cultures. The sensitivity of the assay was in the range of 6 to 8 CFU g(-1) and 10 CFU ml(-1) in spiked raw oyster and synthetic seawater samples, respectively. The total assay could be completed in 3 h. Quantification of the Vibrio cells was linear over at least 6 log units. The TaqMan probe and primer set developed in this study can be used as a rapid screening tool for the presence of V. cholerae in oysters and seawater without prior isolation and characterization of the bacteria by traditional microbiological methods.