Reassessment of the prevalence of heat-stable enterotoxin (NAG-ST) among environmental Vibrio cholerae non-O1 strains isolated from Calcutta, India, by using a NAG-ST DNA probe.

A collection of 521 environmental isolates of Vibrio cholerae which were previously examined by the suckling mouse assay and found to be negative for the heat-stable enterotoxin NAG-ST were reassessed by a recently developed DNA probe for NAG-ST. A total of 12 (2.3%) of the isolates hybridized with the NAG-ST probe. By using a cholera toxin (CT) DNA probe, the CT gene was detected in six of the strains in the collection, although none of the isolates of V. cholerae non-O1 hybridized with both of the toxin probes. All of the NAG-ST and CT probe-positive strains were hemolysin positive. Thirty-fold-concentrated supernatants of the three representative NAG-ST DNA probe-positive V. cholerae non-O1 strains gave positive fluid accumulation ratios in the suckling mouse assay even after heating (100 degrees C for 5 min) and also inhibited the binding of a NAG-ST monoclonal antibody to the bound NAG-ST in a competitive enzyme-linked immunosorbent assay (ELISA). Likewise, all six CT probe-positive V. cholerae non-O1 strains produced in vitro CT when examined by the CT bead ELISA. HindIII digest patterns of chromosomal DNA from the representative NAG-ST gene-positive strains were visually indistinguishable. Between the groups of NAG-ST probe-positive strains examined, there was a variation in the hybridizable fragments, with one group of strains exhibiting a hybridizable fragment similar to that of the NRT 36 reference strain; a smaller HindIII fragment hybridized with the NAG-ST probe in the other group of strains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Toxin profiles of Vibrio cholerae non-O1 from environmental sources in Calcutta, India.

A collection of Vibrio cholerae non-O1 isolated from the aquatic environs of Calcutta, a cholera-hyperendemic area, were examined for the production of cholera toxin (CT), Shiga-like toxins (Vero toxins), heat-stable enterotoxin, and hemolysins. Two (0.5%) V. cholerae non-O1 isolates produced CT. The DNA from both these isolates also hybridized with a DNA probe containing sequences encoding the A subunit of CT. None of the strains produced Shiga-like toxins or heat-stable enterotoxin. Hemolytic activity was observed in 89.7% of the strains, of which 36.1% exhibited biological activity in the suckling mouse. However, none of them produced a hemolysin that cross-reacted with the thermostable direct hemolysin of Vibrio parahaemolyticus. It appears from this study that a small percentage of environmental V. cholerae non-O1 strains do possess the potential for causing cholera-like diarrhea.     

Toxin profiles of Vibrio cholerae non-O1 from environmental sources in Calcutta, India

A collection of Vibrio cholerae non-O1 isolated from the aquatic environs of Calcutta, a cholera-hyperendemic area, were examined for the production of cholera toxin (CT), Shiga-like toxins (Vero toxins), heat-stable enterotoxin, and hemolysins. Two (0.5%) V. cholerae non-O1 isolates produced CT. The DNA from both these isolates also hybridized with a DNA probe containing sequences encoding the A subunit of CT. None of the strains produced Shiga-like toxins or heat-stable enterotoxin. Hemolytic activity was observed in 89.7% of the strains, of which 36.1% exhibited biological activity in the suckling mouse. However, none of them produced a hemolysin that cross-reacted with the thermostable direct hemolysin of Vibrio parahaemolyticus. It appears from this study that a small percentage of environmental V. cholerae non-O1 strains do possess the potential for causing cholera-like diarrhea.     

Detection of heat-stable enterotoxin genes among Australian Vibrio cholerae O1 strains

Abstract DNA probes derived from the heat-stable enterotoxin gene of Vibrio cholerae non-O1 ( stn ), and the cholera toxin gene (etc), were used to screen 199 strains of V. cholerae O1, which were isolated within Australia from 1977–1986. 13 environmental strains isolated from the riverine environment in Southeast Queensland in 1980 and 1981, hybridized with the stn and ctx DNA probes. The concentrated supernatant of 6 of these strains elicited fluid accumulation in the infant mouse assay both before and after heating at 100 °C for 5 min. Genetic relationships among the 13 stn ⁺ strains were studied by a comparison of the rRNA-RFLPs (ribotyping) and by Southern blot analysis with a stn gene probe. The results indicate that there is a clonal relationship among the Australian stn ⁺ strains and that there is an environmental reservoir of stn genes among Australian V. cholerae O1 isolates.     

R plasmids in environmental Vibrio cholerae non-O1 strains.

The occurrence of drug resistance and its plasmid-mediated transferability was investigated in 140 environmental strains of Vibrio cholerae non-O1 and 6 strains of Vibrio cholerae, both O1 and non-O1, of clinical origin. Of the 146 strains tested, 93% were resistant to at least one drug and 74% were resistant to two or more antibiotics. The O1 strains were susceptible to all antibiotics used. A total of 26 of 28 selected resistant wild strains carried R plasmids that were transferable by intraspecific and intergeneric matings. The most common transmissible R factor determined resistance to ampicillin, amoxicillin, and sulfanilamide (30%), followed by resistance to ampicillin and amoxicillin (13%) and resistance to ampicillin, amoxicillin, phosphomycin, and sulfanilamide (9%). Comparison of the three methods of plasmid analysis showed that the method of Birnboim and Doly (Nucleic Acids Res. 7:1513-1523, 1979) without EDTA and lysozyme was optimal for isolation of both large and small plasmids in environmental V. cholerae strains. Most strains harbored more than one plasmid, and the molecular sizes ranged from 1.1 to 74.8 megadaltons. The plasmids of high molecular size (around 74 megadaltons) were responsible for the resistance pattern transferred and were maintained with high stability in the hosts.     

Vibrio cholerae (non-O1) isolated from California coastal waters.

Nineteen strains of Vibrio cholerae non-O1 were isolated from five separate marine sites along the Santa Cruz County coast. This environmental study was initiated after a human case of non-O1 cholera-like diarrhea was acquired endemically.     

Vibrio cholerae non-O1 isolated from ayu fish (Plecoglossus altivelis) in Japan.

A fish pathogen, Vibrio cholerae non-O1, was isolated from diseased ayu fish (Plecoglossus altivelis) collected from rivers in eight prefectural districts of Japan. This organism was found to have biochemical characteristics similar to those of V. cholerae non-O1, except that our isolates were negative for ornithine decarboxylase. Antiserum against an ayu isolate did not agglutinate with the majority of environmental V. cholerae non-O1 isolates, but a major O antigen was common among the ayu isolates. All strains were hemolytic to sheep erythrocytes, and oral administration of culture supernatants induced fluid accumulation in suckling mice. However, the crude toxin was not lethal to adult mice, and no cholera toxin-like enterotoxins were detected.     

Vibrio cholerae non-O1 isolated from ayu fish (Plecoglossus altivelis) in Japan.

A fish pathogen, Vibrio cholerae non-O1, was isolated from diseased ayu fish (Plecoglossus altivelis) collected from rivers in eight prefectural districts of Japan. This organism was found to have biochemical characteristics similar to those of V. cholerae non-O1, except that our isolates were negative for ornithine decarboxylase. Antiserum against an ayu isolate did not agglutinate with the majority of environmental V. cholerae non-O1 isolates, but a major O antigen was common among the ayu isolates. All strains were hemolytic to sheep erythrocytes, and oral administration of culture supernatants induced fluid accumulation in suckling mice. However, the crude toxin was not lethal to adult mice, and no cholera toxin-like enterotoxins were detected.     

Genetic characterization of toxigenic Vibrio cholerae non-O1/non-O139 strains, isolated in the Middle Asia

Here, we report on the characterization of 22 clinical toxigenic V. cholerae non-O1/non-O139 strains isolated in the Middle Asia (Uzbekistan) in 1971–1990. PCR analysis has revealed that these strains contain the main virulence genes such as ctxA, zot, ace (CTXφ); rstC (RS1φ); tcpA, toxT, aldA (pathogenicity island VPI), but they lack both pandemic islands VSP-I and VSP-II specific to epidemic strains of O1 serogroup of El Tor biotype and O139 serogroup. Only two of the twenty two toxigenic strains have tcpA gene of El Tor type, one strain has tcpA gene of classical type, while nineteen other strains carry a new variant of this gene, designated as tcpA ^uzb. Nucleotide sequences analysis of virulence genes in toxigenic V. cholerae non-O1/non-O139 strains from Uzbekistan showed that they differ significantly from the sequences of these genes in epidemic O1 and O139 strain indicating that they belong to a separate line of evolution of virulent V. cholerae strains. For the first time it is shown that V. cholerae non-O1/non-O139 toxigenic strains of different serogroups may belong to the same clone.     

Vibrio cholerae non-O1, non-O139 strains isolated before 1992 from Varanasi, India are multiple drug resistant, contain intSXT, dfr18 and aadA5 genes

In this study, we report the presence of the SXT element and Class I integron in Vibrio cholerae non-O1, non-O139 strains isolated from Varanasi, India. Isolates were resistant to cotrimoxazole, trimethoprim and/or streptomycin, furazolidone and ampicillin. None contained plasmids. Polymerase chain reaction (PCR) and DNA sequencing revealed the presence of antibiotic resistance gene cassettes, aadA1 , aadA2 , aadA5 and dfrA15 , in the Class I integron and SXT, an integrative conjugative element containing dfr18 , sulII and strAB , in three and six of the isolates respectively. Conjugation experiments, followed by PCR analysis of transconjugants, provided evidence for the transferable nature of int SXT and associated antibiotic resistance gene cassettes. This is the first report of the occurrence of SXT ICE, dfr18 , sulII , strAB and aadA5 genes in environmental V. cholerae non-O1, non-O139 strains from Varanasi, India, that had been isolated before 1992.     

Cholera enterotoxin production in Vibrio cholerae O1 strains isolated from the environment and from humans in Japan.

Vibrio cholerae O1 strains isolated from various sources in Japan over the years 1977 through 1987 were examined to confirm the presence or absence of the cholera enterotoxin (CT) gene and production of CT and to determine the kappa-phage type. The CT gene was detected in none of 225 isolates from natural waters but was present in all of the 10 isolates from environmental waters implicated in domestic cholera cases, in 64 strains (26.6%) of the 241 isolates from imported seafoods, in 43 strains (95.6%) of the 45 isolates from domestic cholera cases, and in 119 strains (93.7%) of the 127 isolates from imported cholera cases. The results suggest that the CT gene-positive strains of V. cholerae O1 have been imported into Japan through seafoods and/or by travelers. Sporadic cholera cases have resulted in contamination of the surrounding environment, but the CT gene-positive strains may not have persisted in natural waters to serve as a reservoir for epidemic cholera. The commercially available VET-RPLA kit (a latex agglutination kit for immunological detection of CT) detected production of CT in all of the CT gene-positive strains, indicating that there was no silent CT gene in the test strains. There was a strong correlation between the kappa-phage type and the presence or absence of the CT gene, suggesting a significant clonal difference between CT gene-positive and -negative strains. Five CT gene-negative strains isolated from imported cholera cases (travelers with mild diarrhea) induced a considerable amount of fluid accumulation in rabbit and/or suckling mouse intestines, indicating production of an enterotoxic factor(s) other than CT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholera enterotoxin production in Vibrio cholerae O1 strains isolated from the environment and from humans in Japan.

Vibrio cholerae O1 strains isolated from various sources in Japan over the years 1977 through 1987 were examined to confirm the presence or absence of the cholera enterotoxin (CT) gene and production of CT and to determine the kappa-phage type. The CT gene was detected in none of 225 isolates from natural waters but was present in all of the 10 isolates from environmental waters implicated in domestic cholera cases, in 64 strains (26.6%) of the 241 isolates from imported seafoods, in 43 strains (95.6%) of the 45 isolates from domestic cholera cases, and in 119 strains (93.7%) of the 127 isolates from imported cholera cases. The results suggest that the CT gene-positive strains of V. cholerae O1 have been imported into Japan through seafoods and/or by travelers. Sporadic cholera cases have resulted in contamination of the surrounding environment, but the CT gene-positive strains may not have persisted in natural waters to serve as a reservoir for epidemic cholera. The commercially available VET-RPLA kit (a latex agglutination kit for immunological detection of CT) detected production of CT in all of the CT gene-positive strains, indicating that there was no silent CT gene in the test strains. There was a strong correlation between the kappa-phage type and the presence or absence of the CT gene, suggesting a significant clonal difference between CT gene-positive and -negative strains. Five CT gene-negative strains isolated from imported cholera cases (travelers with mild diarrhea) induced a considerable amount of fluid accumulation in rabbit and/or suckling mouse intestines, indicating production of an enterotoxic factor(s) other than CT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prevalence of cholera toxin genes (ctxA and zot) among non-O1/O139 Vibrio cholerae strains from Newport Bay, California

The examination of 137 non-O1/O139 Vibrio cholerae isolates from Newport Bay, California, indicated the presence of diverse genotypes and a temporal succession. Unexpectedly, the cholera toxin gene (ctxA) was found in 17% of the strains, of which one-third were also positive for the zot gene. This suggests that ctxA is prevalent in the region of nonepidemicity and is likely to have an environmental origin.     

Molecular analysis of Vibrio cholerae O1, O139, non-O1, and non-O139 strains: clonal relationships between clinical and environmental isolates

A total of 26 strains of Vibrio cholerae, including members of the O1, O139, and non-O1, non-O139 serogroups from both clinical and environmental sources, were examined for the presence of genes encoding cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), and outer membrane protein (ompU), for genomic organization, and for the presence of the regulatory protein genes tcpI and toxR in order to determine relationships between epidemic serotypes and sources of isolation. While 22 of the 26 strains were hemolytic on 5% sheep blood nutrient agar, all strains were PCR positive for hlyA, the hemolysin gene. When multiplex PCR was used, all serogroup O1 and O139 strains were positive for tcpA, ompU, and tcpI. All O1 and O139 strains except one O1 strain and one O139 strain were positive for the ctxA, zot, and ace genes. Also, O1 strain VO3 was negative for the zot gene. All of the non-O1, non-O139 strains were negative for the ctxA, zot, ace, tcpA, and tcpI genes, and all of the non-O1, non-O139 strains except strain VO26 were negative for ompU. All of the strains except non-O1, non-O139 strain VO22 were PCR positive for the gene encoding the central regulatory protein, toxR. All V. cholerae strains were negative for the NAG-specific st gene. Of the nine non-ctx-producing strains of V. cholerae, only one, non-O1, non-O139 strain VO24, caused fluid accumulation in the rabbit ileal loop assay. The other eight strains, including an O1 strain, an O139 strain, and six non-O1, non-O139 strains, regardless of the source of isolation, caused fluid accumulation after two to five serial passages through the rabbit gut. Culture filtrates of all non-cholera-toxigenic strains grown in AKI media also caused fluid accumulation, suggesting that a new toxin was produced in AKI medium by these strains. Studies of clonality performed by using enterobacterial repetitive intergenic consensus sequence PCR, Box element PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) collectively indicated that the V. cholerae O1 and O139 strains had a clonal origin, whereas the non-O1, non-O139 strains belonged to different clones. The clinical isolates closely resembled environmental isolates in their genomic patterns. Overall, there was an excellent correlation among the results of the PCR, AFLP, and PFGE analyses, and individual strains derived from clinical and environmental sources produced similar fingerprint patterns. From the results of this study, we concluded that the non-cholera-toxin-producing strains of V. cholerae, whether of clinical or environmental origin, possess the ability to produce a new secretogenic toxin that is entirely different from the toxin produced by toxigenic V. cholerae O1 and O139 strains. We also concluded that the aquatic environment is a reservoir for V. cholerae O1, O139, non-O1, and non-O139 serogroup strains.     

Surface and virulence properties of environmental Vibrio cholerae non-O1 from Albufera Lake (Valencia, Spain).

A total of 140 environmental Vibrio cholerae non-O1 isolates, together with several culture collection strains from both environmental and clinical sources, were studied in relation to hemagglutination, surface hydrophobicity, and the enzymatic, hemolytic, cytotoxic, and enterotoxic activities of their extracellular products. A total of 78 and 62% of the strains produced hemagglutinins and exohemagglutinins, respectively. Four different hemagglutinating and two exohemagglutinating activities were found by using eight sugars in the inhibition assays. Cell-bound mannose-sensitive hemagglutination was detected mainly in chicken blood, whereas fucose-sensitive hemagglutination was recorded only in human blood. Cell-bound hemagglutinin resistant to all sugars tested was the only one related to surface hydrophobicity. The surface properties varied along the growth curves. The non-O1 strains displayed strong enzymatic and hemolytic activities, except for esculin hydrolysis. Of 26 non-O1 isolates selected for cytotoxin and enterotoxin production, 23 showed a wide spectrum of cytotoxic effects on cell lines of poikilothermic and homoiothermic species, but they were weakly enterotoxigenic in the infant mouse test. All extracellular products of cytotoxic strains were proteolytic, lipolytic, and hemolytic, and a high percentage produced hemagglutination of chicken blood. The cytotoxic factors in the non-O1 strains analyzed were not R plasmid mediated.     

Surface and virulence properties of environmental Vibrio cholerae non-O1 from Albufera Lake (Valencia, Spain).

A total of 140 environmental Vibrio cholerae non-O1 isolates, together with several culture collection strains from both environmental and clinical sources, were studied in relation to hemagglutination, surface hydrophobicity, and the enzymatic, hemolytic, cytotoxic, and enterotoxic activities of their extracellular products. A total of 78 and 62% of the strains produced hemagglutinins and exohemagglutinins, respectively. Four different hemagglutinating and two exohemagglutinating activities were found by using eight sugars in the inhibition assays. Cell-bound mannose-sensitive hemagglutination was detected mainly in chicken blood, whereas fucose-sensitive hemagglutination was recorded only in human blood. Cell-bound hemagglutinin resistant to all sugars tested was the only one related to surface hydrophobicity. The surface properties varied along the growth curves. The non-O1 strains displayed strong enzymatic and hemolytic activities, except for esculin hydrolysis. Of 26 non-O1 isolates selected for cytotoxin and enterotoxin production, 23 showed a wide spectrum of cytotoxic effects on cell lines of poikilothermic and homoiothermic species, but they were weakly enterotoxigenic in the infant mouse test. All extracellular products of cytotoxic strains were proteolytic, lipolytic, and hemolytic, and a high percentage produced hemagglutination of chicken blood. The cytotoxic factors in the non-O1 strains analyzed were not R plasmid mediated.     

Characterization of the antiseptic-resistance gene qacEΔ1 isolated from clinical and environmental isolates of Vibrio parahaemolyticus and Vibrio cholerae non-O1

The nucleotide sequence and mechanism of action were examined on the antiseptic-resistance gene qacE delta 1 that had been isolated from Pseudomonas aeruginosa, Vibrio parahaemolyticus and Vibrio cholerae non-O1. The nucleotide sequences of qacE delta 1 genes isolated from environmental isolates of V. cholerae non-O1 and V. parahaemolyticus differed by one base from that of the gene from P. aeruginosa. Escherichia coli C600 that harbored qacE delta 1 genes from several strains of Vibrio spp. exhibited low-level resistance to intercalating dyes. The resistance of E. coli cells with these genes to intercalating dyes, such as ethidium bromide, was mediated by an efflux system. Moreover, the activity of QacE delta 1 was inhibited in the presence of calcium channel blockers but not of calmodulin inhibitors. These results indicate that the qacE delta 1 gene can be function in E. coli and that the gene mediates resistance in a similar manner to the antiseptic-resistance gene smr.