Vibrio cholerae O139 produces a protease which is indistinguishable from the haemagglutinin/protease of Vibrio cholerae O1 and non-O1

Abstract Haemaglutinin/protease (HA/P) is one of the virulence factors of Vibrio cholerae O1 and pathogenic strains of V. cholerae non-O1. In this study, we examined protease activity of a new serogroup of Vibrio cholerae recently designated as O139 synonym Bengal. The protease activity was produced by all eight isolates of V. cholerae O139 from Bangladeshi patients. Purification and partial characterization of the protease from V. cholerae O139 demonstrated the purified protease (O139-P) was indistinguishable from that previously reported for HA/P of V. cholerae non-O1 (NAG-HA/P) and V. cholerae O1 (Vc-HA/P). These results prove that V. cholerae O139 produces a protease belonging to solHA/P, and suggest that the protease is another virulence factor found in newly emerged V. cholerae O139, as in V. cholerae O1.     

Demonstration of a plasmid-borne gene encoding a thermostable direct hemolysin in Vibrio cholerae non-O1 strains

Of 15 Vibrio cholerae non-O1 strains, 2 produced a hemolysin termed NAG-rTDH, which is very similar to the thermostable direct hemolysin of V. parahaemolyticus. These two strains contained DNA sequences which are homologous to a DNA probe for the V. parahaemolyticus thermostable direct hemolysin gene. A probe-positive 9-kilobase HindIII fragment was cloned from a plasmid of a V. cholerae non-O1 strain into plasmid pBR322, and the resulting Escherichia coli clones produced intracellular NAG-rTDH.     

Demonstration of a plasmid-borne gene encoding a thermostable direct hemolysin in Vibrio cholerae non-O1 strains.

Of 15 Vibrio cholerae non-O1 strains, 2 produced a hemolysin termed NAG-rTDH, which is very similar to the thermostable direct hemolysin of V. parahaemolyticus. These two strains contained DNA sequences which are homologous to a DNA probe for the V. parahaemolyticus thermostable direct hemolysin gene. A probe-positive 9-kilobase HindIII fragment was cloned from a plasmid of a V. cholerae non-O1 strain into plasmid pBR322, and the resulting Escherichia coli clones produced intracellular NAG-rTDH.     

Production and partial purification of a fluid-accumulating factor of non-O1 Vibrio cholerae

A fluid-accumulating factor (FAF in the ligated rabbit ileal loop test) from a strain of non-O1 Vibrio cholerae not producing cholera toxin-like enterotoxin (CTLT) was partially purified by ammonium sulfate precipitation, gel filtration with Sephadex G-100, and DEAE cellulose column chromatography. The preparation thus obtained showed collagenolytic, cytolytic, necrotic, and hemorrhagic activities, but was not lethal to mice nor hemolytic to sheep erythrocytes. Desquamation of epithelial cells, inflammatory edema, and hemorrhage were observed in sections of rabbit intestine after inoculation of partially purified FAF (PPFAF). Biological and enzymatic activities of FAF were completely neutralized with anti-PPFAF rabbit serum. More than 70% of non-O1 V. cholerae strains from human diarrheal feces produced FAF in the shake culture of heart infusion broth (Difco). A fluid-accumulating factor immunologically similar to FAF of non-O1 V. cholerae was also produced by V. mimicus strains isolated from human diarrheal feces. These results indicate that the FAF produced by CTLT-negative non-O1 V. cholerae strains is an entity closely related to a cytolytic and hemorrhagic substance or the like, and that this FAF may play a role in the enteropathogenicity of CTLT-negative strains.     

Variation in epitopes of the B subunit of Vibrio cholerae non-O1 and Vibrio mimicus cholera toxins.

Monoclonal antibodies reacting with the B subunit of Vibrio cholerae O1 strain 569B cholera toxin (CT-B) were used to identify unique and common epitopes of V. cholerae non-O1 and Vibrio mimicus CT-B. Vibrio cholerae non-O1 strains produced CT-B showing three monoclonal antibody reaction patterns (epitypes), which corresponded with epitypes described previously for V. cholerae O1 classical biotype CT-B (CT1), El Tor biotype CT-B (CT2), and a unique V. cholerae non-O1 CT-B (CT3), which lacked an epitope located in or near the GM1 ganglioside binding site of 569B CT-B. Vibrio mimicus CT-B was immunologically indistinguishable from 569B CT-B. These and previous results define six epitopes on 569B CT-B, and a fourth epitope in or near the GM1 ganglioside binding site.     

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.     

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.     

Distribution of Vibrio cholerae virulence genes among different Vibrio species isolated in Sardinia, Italy

The members of the genus Vibrio include harmless aquatic strains as well as strains capable of causing epidemics of cholera. Diarrhoea caused by Vibrio cholerae is attributed to cholerae enterotoxin (CT) codified by the ctx operon and regulated by a number of virulence genes such as toxT, toxR and toxS. Fifty-two Vibrio strains were isolated from different aquatic environments in and around Sardinia and searched by PCR for the presence of ctxA, zot, ace, toxR, toxS, toxT, tcpA and vpi virulence genes in the genomes of the isolates. The toxR operon was found in 27 Vibrio alginolyticus strains out of 42 analysed, in three out of four V. cholerae non-O1 strains and in three Vibrio parahaemolyticus isolates. A positive amplification for the virulence pathogenic island (vpi) was produced by five V. alginolyticus strains. Finally, the ace expected amplification fragment was found in two V. alginolyticus isolates whereas the amplification with zot primers produced the expected fragment in one V. alginolyticus isolate. Differentiation of these strains with a PCR fingerprinting technique revealed no association between the presence of virulence genes and a particular fingerprinting pattern. Although most Vibrio species are considered non-pathogenic or only potentially harmful to humans, the finding of V. cholerae virulence genes in other members of the genus Vibrio, and the recent reports of the creation and evolution of pandemic strains of V. cholerae, may give a new perspective to the significance of these results.     

Sensitivity of Vibrio and Aeromonas to antibiotics]

The antibiotic sensitivity of 696 cultures belonging to the family Vibrionaceae (V. cholerae O1, V. cholerae non-O1, V. albensis, V. parahaemolyticus, V. alginolyticus and Aeromonas spp.) was studied and general regularities of the antibiotic sensitivity were shown: a high sensitivity to broad-spectrum antibiotics (tetracycline and chloramphenicol) and a low sensitivity to ++beta lactams (carbenicillin and ampicillin). The comparative examinations revealed similarity in the antibioticograms of V. cholerae O1 (el Tor++), V. cholerae non-O1 and V. albensis, especially the latter two groups, as well as the tested halophilic Vibrio cultures by the range of the MICs, Mo, Me and the nature of the antibiotic resistance. Cultures of V. cholerae and luminescent Vibrio tended to preserve a high sensitivity. High resistance levels were noted in the halophilic Vibrio and Aeromonas cultures. No significant differences in the sensitivity of the strains of various origin (from man and environmental objects) were detected. However, several more resistant strains were isolated from the environmental objects.     

Characterization of a Vibrio cholerae phage isolated from the coastal water of Peru

A Vibrio cholerae bacteriophage, family Myoviridae, was isolated from seawater collected from the coastal water of Lima, Peru. Genome size was estimated to be 29 kbp. The temperate phage was specific to V. cholerae and infected 12/13 V. cholerae O1 strains and half of the four non-O1/non-O139 strains tested in this study. Vibrio cholerae O139 strains were resistant to infection and highest infection rates were obtained in low nutrient media amended with NaCl or prepared using seawater as diluent.     

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.     

Haemolysin genes of Vibrio cholerae: presence of homologous DNA in non-haemolytic O1 and haemolytic non-O1 strains

Abstract The structural gene for the haemolysin and two accessory genes from a Vibrio cholerae O1 El Tor strain have previously been cloned in Escherichia coli K-12 to give the plasmid pPM431. This plasmid has been used as a probe with a variety of O1 and non-O1 Vibrio cholerae strains to examine by Southern DNA hybridisations for the presence of homologous DNA. Such experiments show that the DNA homologous to that present in pPM431 is present in all of the 20 strains examined, whether they were haemolytic or non-haemolytic, implying that the genes were present but not expressed in non-haemolytic strains. Using a variety of restriction enzymes to cut the chromosomal DNA of different V. cholerae strains and probing with pPM431, it was possible to distinguish O1 and non-O1 strains, as well as haemolytic or non-haemolytic strains. This variability between hly⁺ and hly⁻ may be indicative of a change in the regulatory region of the haemolysin genes. The results also imply a high degree of homology of the haemolysin of O1 and non-O1 strains.     

Distribution of genes for virulence and ecological fitness among diverse Vibrio cholerae population in a cholera endemic area: tracking the evolution of pathogenic strains

The pathogenic strains of Vibrio cholerae that cause acute enteric infections in humans are derived from environmental nonpathogenic strains. To track the evolution of pathogenic V. cholerae and identify potential precursors of new pathogenic strains, we analyzed 324 environmental or clinical V. cholerae isolates for the presence of diverse genes involved in virulence or ecological fitness. Of 251 environmental non-O1, non-O139 strains tested, 10 (3.9%) carried the toxin coregulated pilus (TCP) pathogenicity island encoding TCPs, and the CTX prophage encoding cholera toxin, whereas another 10 isolates carried the TCP island alone, and were susceptible to transduction with CTX phage. Most V. cholerae O1 and O139 strains carried these two major virulence determinants, as well as the Vibrio seventh pandemic islands (VSP-1 and VSP-2), whereas 23 (9.1%) non-O1, non-O139 strains carried several VSP island genes, but none carried a complete VSP island. Conversely, 30 (11.9%) non-O1, non-O139 strains carried type III secretion system (TTSS) genes, but none of 63 V. cholerae O1 or O139 strains tested were positive for TTSS. Thus, the distribution of major virulence genes in the non-O1, non-O139 serogroups of V. cholerae is largely different from that of the O1 or O139 serogroups. However, the prevalence of putative accessory virulence genes (mshA, hlyA, and RTX) was similar in all strains, with the mshA being most prevalent (98.8%) followed by RTX genes (96.2%) and hlyA (94.6%), supporting more recent assumptions that these genes imparts increased environmental fitness. Since all pathogenic strains retain these genes, the epidemiological success of the strains presumably depends on their environmental persistence in addition to the ability to produce major virulence factors. Potential precursors of new pathogenic strains would thus require to assemble a combination of genes for both ecological fitness and virulence to attain epidemiological predominance.     

Comparative PCR-based fingerprinting of Vibrio cholerae isolated in Malaysia

Vibrio cholerae, the causative agent of cholera, is endemic in many parts of the world, especially in countries poor in resources. Molecular subtyping of V. cholerae is useful to trace the regional spread of a clone or multidrug-resistant strains during outbreaks of cholera. Current available PCR-based fingerprinting methods such as Random Amplified Polymorphic DNA (RAPD)-PCR, Enterobacterial Repetitive Intergenic Consensus Sequence (ERIC)-PCR, and Repetitive Extragenic Palindromic (REP)-PCR were used to subtype V. cholerae. However, there are problems for inter-laboratory comparison as these PCR methods have their own limitations especially when different PCR methods have been used for molecular typing. In this study, a Vibrio cholerae Repeats-PCR (VCR-PCR) approach which targets the genetic polymorphism of the integron island of Vibrios was used and compared with other PCR-based fingerprinting methods in subtyping. Forty-three V. cholerae of different serogroups from various sources were tested. The PCR-fingerprinting approaches were evaluated on typeability, reproducibility, stability and discriminatory power. Overall, Malaysian non-O1/non-O139 V. cholerae were more diverse than O1 strains. Four non-O1/non-O139 strains were closely related with O1 strains. The O139 strain in this study shared similarity with strains of both O1 and non-O1/non-O139 serogroups. ERIC-PCR was the most discriminative approach (D value = 0.996). VCR-PCR was useful in discriminating non-O1/non-O139 strains. RAPD-PCR and REP-PCR were less suitable for efficient subtyping purposes as they were not reproducible and lacked stability. The combination of the ERIC-PCR and VCR-PCR may overcome the inadequacy of any one approach and hence provide more informative data.     

Ecology of Vibrio species, including Vibrio cholerae, in natural waters of Kent, England

The distribution of Vibrio species in water from two sites in Kent was studied between 1978 and 1980. They were counted by a most probable number technique using alkaline peptone water for enrichment followed by plating onto thiosulphate citrate bile salt sucrose agar, or by direct plating of water onto the same agar. In a freshwater stream both upstream and downstream from a human sewage works outfall V. metschnikovii was the predominant Vibrio. Vibrio anguillarum was isolated sporadically. Non-O1 serovars of V. cholerae occurred only twice. At the other site in a ditch containing static, brackish water, non-O1 V. cholerae (highest number 400 cfu/ml) was observed as present only from May to November. Vibrio anguillarum was isolated throughout the sampling period. The presence of non-O1 V. cholerae in both sites was not dependent on the input of human sewage. The hypothesis that non-toxigenic V. cholerae can survive and multiply in water was tested in the ditch by the use of submersible chambers constructed of polycarbonate membranes and Plexiglass. The seasonal incidence of non-O1 V. cholerae in the brackish water site could be explained by the multiplication of the organism when the water temperature exceeded 9°C. It was concluded that strains of V. cholerae that are unable to produce cholera toxin are indigenous to static brackish water environments and the possibility that this applies to toxigenic strains as well should be investigated.     

Homology between the deoxyribonucleic acid of fertility factor P and Vibrio cholerae chromosomal deoxyribonucleic acid.

The deoxyribonucleic acid (DNA) of the Vibrio cholerae fertility factor P was isolated by the dye-buoyant density method and hybridized to V. cholerae chromosomal DNA. The DNA of this fertility plasmid had between 35 to 40% homology with the V. cholerae chromosomal DNA. Little or no homology was detected between the P factor DNA and DNA of the Escherichia coli sex factor F.     

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.     

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.     

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.