Distribution of serogroups of Vibrio cholerae non-O1 non-O139 with specific reference to their ability to produce cholera toxin, and addition of novel serogroups

A total of 1898 strains of Vibrio cholerae non-O1 non-O139, which had been collected worldwide for the past 3 year period of 1994-1996, were serogrouped. The strains were also examined for presence of cholera toxin (CT) gene (ctx) and NAG-ST gene, and strains which carried to ctx were further analyzed for their ability to produce CT. In addition, attempts were made to establish novel serogroups for those serologically untypable strains. Of those examined, 1,774 strains of V. cholerae non-O1 non-O139 was classified into 128 known serogroups while 50 strains were found to belong to R type, and the rest of the 74 strains could not be serotyped. Distribution of the serogroups did not seem to correspond to either the strains geographic distribution or sources of isolation. Of those serologically untypable strains, 38 novel serogroups (O156-O193) were established and added to our reference of V. cholerae antigenic schema. It was also found that antisera raised against many V. cholerae strains included R antibodies. This indicates that any V. cholerae antisera for diagnostic purpose should be absorbed with the reference R strains, CA385, before use. There were luminescence producing strains among those sucrose and VP reaction negative strains. Subsequent DNA/DNA homology analysis revealed that they were identified as V. cholerae. This points to a possibility that strains tentatively identified as Vibrio mimicus by conventional biochemical tests may have included luminescent strains of V. cholerae. It is thus highly recommended that strains in question should be tested for the luminescence production in order to differentiate V. cholerae from V. mimicus. Of those 1989 strains examined, 37 strains (ca. 2%) were found to produce CT. Interestingly, CT producing strains were prevalent in serogroup O141; 10 strains out of 16 strains (63%) were positive for CT. The evidence calls for a caution to possible occurrence of cholera-like diarrhea caused by V. cholerae O141 in the future.     

Rapid diagnosis of cholera caused by Vibrio cholerae O139

Hybridomas secreting specific monoclonal antibodies (MAbs) to Vibrio cholerae serogroup O139 were produced. Six monoclones (hybridomas) secreting MAbs specific only to lipopolysaccharide of V. cholerae O139 strains and which did not cross-react to 137 strains of other enteric microorganisms were obtained. These clones were designated 12F5-G11, 12F5-G2, 15F5-H5, 5B9-F8, 14C9-D2, and 6D2-D8. The immunoglobulin (Ig) heavy chain isotypes secreted by these clones were IgG2b, IgG2b, IgG2b, IgM, IgG2b, and IgG3, respectively. Clone 12F5-G11 was selected for mass production of MAb, which was used as a detection reagent in the antigen detection assay for diagnosis of cholera caused by V. cholerae O139, and this assay was compared to the conventional bacterial isolation method. Five batches of rectal swab cultures in alkaline-peptone water were collected from 6,497 patients with watery diarrhea. These were 6,310 patients admitted to Bamrasnaradura Infectious Diseases Hospital, 16 patients from Krung Thon Hospital, 78 patients from Bangkok Children's Hospital, 19 patients from Karen refugee camps, and 74 Indian patients from the National Institute of Cholera and Enteric Diseases, Calcutta, India. The V. cholerae O139 isolations from the rectal swab cultures and the antigen detection assays (i.e., the MAb-based dot-blot ELISA) were performed by different persons of different laboratories, and the results were revealed after all specimens had been tested. Of the 6,497 samples tested, the dot-blot ELISA correctly identified 42 of 42 V. cholerae O139-positive samples and gave a result of positive for three samples which were culture negative for V. cholerae O139. The diagnostic sensitivity, specificity, and efficacy of the dot-blot ELISA were 100, 99.95, and 99.26%, respectively. The ELISA is easy to perform and relatively inexpensive. It can test multiple samples at a single time, does not require special equipment, and does not produce great quantities of contaminated waste. Most of all, it reduces the diagnostic time from at least 2 days for the bacterial isolation to less than 90 min. The assay is recommended as a rapid screening test of cholera cases caused by V. cholerae O139.     

A number of Vibrio cholerae non-O1 isolated from aquatic environments

To estimated existence of Vibrio cholerae non-O1 in aquatic environments, the organisms isolated from river, estuary and sea water. V. cholerae non-O1 isolated form midstream and estuary water could be counted from 1.6 to 2400 CFU/100 ml by the membrane filtrated method (MF). V. cholerae non-O1 existed in midstream water more than in estuary water. However, the isolated organisms from estuary rate by MF (37.5%) was lower than it by alkaline peptone enrichment medium method (AP) (75.0%), as a result of halophilic bacteria grow an selected medium of MF. And the number of V. cholerae non-O1 isolated from aquatic environment did not correlate environmental parameters. The number of V. cholerae non-O1 isolated from river water varied, it suggested that the organism collectively adhere a floating matter. V. cholerae non-O1 was not detected in 500 ml sea water by AP and MF method. These results conclude that V. cholerae non-O1 exist in river water more than in sea.     

Purification and characterization of pili of a Vibrio cholerae non-O1 strain

Pili of the Vibrio cholerae non-O1 strain V10 were purified and characterized. The V10 pili were physicochemically and immunologically different from those of the previously reported V. cholerae non-O1 strain S7, although the pili of the two strains had homologous N-terminal amino acid sequences. V10 plus antigen was detected only in V. cholerae non-O1 strains.     

Structural studies on the short-chain lipopolysaccharide of Vibrio cholerae O139 Bengal

A Vibrio cholerae O139 strain, MO10-T4, lacking capsular polysaccharide, produces a short-chain lipopolysaccharide (LPS), similar to enterobacterial SR strains. It was studied by acidic and alkaline degradation, dephosphorylation, sugar and methylation analysis, high-performance anion-exchange chromatography, one- and two-dimensional 1H-, 13C-, and 31P-NMR spectroscopy, and electrospray ionization mass spectrometry. The following structure was proposed for the core region of the LPS: [structure: see text] where PEtn stands for 2-aminoethyl phosphate, Fru for fructose, Hep for L-glycero-D-manno-heptose, and Kdo for 3-deoxy-D-manno-octulosonic acid; unless otherwise stated, the monosaccharide residues are D and present in the pyranose form. An O-acetyl group is present on a secondary position, tentatively O4 of the alpha-linked glucosyl group. Some LPS species contain an additional putative fructose residue whose location remains unknown. An O139-negative mutant strain, Bengal-2R, derived from V. cholerae O139, has also been investigated and shown to produce an O-antigen-lacking LPS similar to those from enterobacterial R strains, some of the LPS species containing the same core region as the strain MO10-T4 LPS and the other lacking the lateral heptose residue. The carbohydrate backbone core structure is the same for the V. cholerae O139 and V. cholerae O1 LPS, thus confirming the close relation between these bacteria; however, the 2-aminoethyl phosphate, the O-acetyl group, and the second fructose residue have not been reported for the O1 LPS. In the V. cholerae O139 strain MO10-T4 LPS, a short O-side chain is attached at position 3 of the 7-substituted heptose residue and has the same structure as one repeating unit of the V. cholerae O139 capsular polysaccharide. Some details of the structure proposed are at variance with those recently published for another V. cholerae O139 strain [Cox, A. D., Brisson, J.-R., Varma, V. & Perry, M. B. (1996) Carbohydr. Res. 290, 43-58; Cox, A. D. & Perry, M. B. (1996) Carbohydr. Res. 290, 59-65.]     

Morphological and physical characterization of the capsular layer of Vibrio cholerae O139

Low-speed isokinetic exercise has been recommended to exert a maximal contraction and produce greater muscle torque than high-speed exercise in young adults. The purpose of this study was to compare the effectiveness of low- and high-speed isokinetic exercise programs for increasing muscle torque in young and elderly people. Twenty healthy elderly and 20 young subjects participated. The elderly subjects were divided into two groups. One group performed high-speed (300 degrees/s) isokinetic exercise training three times a week for the dominant-side knee extensor and low-speed (60 degrees/s) exercise for the non-dominant side for 6 weeks. The other group was trained using the reverse exercise regime. The training program for the young subjects was the same as that for the elderly groups. All subjects had their knee extensor torque evaluated with an isokinetic test before and at 2-week intervals during the training program. For young and elderly groups, both high- and low-speed isokinetic exercise training increased extensor torque in low- and high-speed tests. For the young group, low-speed exercise effectively improved muscle torque at low and high speeds. The improvement in slow muscle torque was significantly greater than that in fast muscle torque. For the elderly subjects, high-speed isokinetic exercise produced the greatest muscle torque at high speed in the first 2 weeks of training, and demonstrated a sharp increase in muscle torque in the final 2 weeks. Low-speed exercise frequently caused knee stress and the inability of some elder subjects to continue the exercises with maximal effort. Our findings indicate that high-speed exercise may be more appropriate for the elderly, and low-speed exercise may be more appropriate for younger people.     

Sporadic diarrhea caused by Vibrio cholerae non-O1 and characteristics of the isolates

In 1996, we examined five domestic and eight imported cases of sporadic diarrhea caused by Vibrio cholerae non-O1 in Tokyo. The domestic cases occurred during the summer, from June to September, while the imported cases were seen throughout the year. The major clinical symptoms of the patients were watery diarrhea (100%) with an average frequency of 5.5 times/day, abdominal pain (77%), vomiting (31%) and fever (15%). A total of 13 strains isolated from these 13 cases had the typical biochemical characteristics of Vibrio cholerae, and were classified into 11 kinds of serovars (O2, O5, O8, O9, O12, O14, O27, O51, O88, O97, and O161). All strains produced hemolysin, and two strains produced NAG-ST, while no strain produced cholera toxin.     

Emergence of a new clone of toxigenic Vibrio cholerae O1 biotype El Tor displacing V. cholerae O139 Bengal in Bangladesh

C-X-C chemokines are potent chemoattractants that are believed to mediate neutrophilic inflammation in several organs. Recent studies suggest a role for C-X-C chemokines in the pathogenesis of neutrophilic hepatitis but do not prove causation. We investigated the biological consequences of hepatic chemokine production in vivo by transiently overexpressing cytokine-induced neutrophil chemoattractant (CINC), a member of the C-X-C chemokine family, in intact rats. Rats were injected intraportally with a replication-defective recombinant adenovirus containing the CINC complementary DNA (cDNA). Within 4 days, treated animals had high levels of CINC in both liver tissue and plasma Rats overexpressing CINC exhibited an eightfold increase in circulating neutrophils; they also developed severe hepatic injury, characterized by a 6- to 25-fold increase in plasma transaminases and marked hepatic inflammation on biopsy. Liver disease in CINC-producing rats correlated positively with the number of neutrophils sequestered in the hepatic parenchyma. Tissue injury was attributed directly to chemokine overproduction, because control rats infected with adenoviruses lacking the CINC cDNA did not produce CINC and developed only minor hepatic abnormalities. These experiments provide direct evidence that C-X-C chemokines, when expressed in sufficient quantity in the liver in vivo, induce neutrophil recruitment and tissue invasion and provoke severe liver injury. The data suggest that C-X-C chemokines have important pathogenic potential in both clinical and experimental liver disease.     

Isolation and characterization of rugose form of Vibrio cholerae O139 strain MO10

An extracellular exopolysaccharide (slime) is produced by Vibrio cholerae O139 MO10 in response to nutrient starvation. The presence of this slime layer on the cell surface and its subsequent release have been shown to be associated with biofilm formation and the change from a normal smooth colony morphology to a rugose one. An immunoelectron microscopic examination demonstrated that there is an epitope common to the exopolysaccharide antigen of V. cholerae O1 and that of O139 MO10.     

Rapid detection of Vibrio cholerae O139 Bengal from stool specimens by PCR

In a previous study using pure bacterial cultures in a PCR assay, a primer pair corresponding to a unique chromosomal region of Vibrio cholerae O139 Bengal generated an amplicon from only V. cholerae O139 Bengal. PCR with the same primer pair was used to screen 180 diarrheal stool specimens. All the 67 V. cholerae O139 culture-positive stool specimens were positive by PCR, and the remaining specimens, which contained either other recognized enteric pathogens or no pathogens, were all negative by PCR.     

Induction of the lysogenic phage encoding cholera toxin in naturally occurring strains of toxigenic Vibrio cholerae O1 and O139

In toxigenic Vibrio cholerae, the CTX genetic element which carries the genes for cholera toxin (CT) is the genome of a lysogenic bacteriophage (CTXPhi). Clinical and environmental strains of V. cholerae O1 or O139 and stools that were culture positive for cholera were analyzed to study the induction and transmission of CTXPhi. To our knowledge, this is the first report of the examination of CTXPhi in clinical materials and in naturally occurring strains. DNA probe analysis revealed that 4.25% (6 of 141) of the isolated V. cholerae strains spontaneously produced a detectable level of extracellular CTXPhi particles in the culture supernatants whereas another 34.04% (48 of 141) produced CTXPhi particles when induced with mitomycin C. CTXPhi isolated from 10 clinical or environmental strains infected a CT-negative recipient strain, CVD103, both inside the intestines of infant mice and under laboratory conditions. All culture-positive stools analyzed were negative for the presence of CTXPhi both in the DNA probe assay and by in vivo assay for the infection of the recipient strain in infant mice. These results suggested that naturally occurring strains of toxigenic V. cholerae are inducible lysogens of CTXPhi but that cholera pathogenesis in humans is not associated with the excretion of CTXPhi particles in stools, indicating that induction of the phage may not occur efficiently inside the human intestine. However, in view of the efficient transmission of the phage under conditions conducive to the expression of toxin-coregulated pili, it appears that propagation of CTXPhi in the natural habitat may involve both environmental and host factors.     

Note: characterization of Vibrio cholerae O139 Bengal isolated from water in Malaysia

Four Vibrio cholerae O139 Bengal strains isolated from surface water were characterized by antibiotic resistance, plasmid profile, presence of cholera toxin gene and random amplification of polymorphic DNA (RAPD) analysis. All four strains exhibit multiple resistance towards the antibiotics tested with a multiple antibiotic resistance index of 0.5-0.66, and harboured a 2.0 MDa non-conjugative plasmid. The Vibrio cholerae O139 Bengal were positive for the cholera toxin gene. Antibiotyping and random amplification of polymorphic DNA analysis with four primers proved to be useful in discriminating the isolates. RAPD proved to be more sensitive. These results reveal that there is significant genetic diversity among the Vibrio cholerae O139 Bengal strains studied.     

Phenotypic and genotypic biotyping of environmental strains of Vibrio cholerae non-O1 isolated in Italy

The purpose of this study was to characterize strains of Vibrio cholerae non-O1 isolated in Italy from different sources by biochemical and serological assays, antibiotic susceptibility testing, and molecular biotyping. Serotyping and genomic analysis by pulsed-field gel electrophoresis proved to be useful in discriminating the isolates. The data obtained show a wide heterogeneity at the genomic level, and in keeping with this, the serogrouping classification provided evidence of a high variability of the investigated strains. In addition, none of the strains tested produced cholera-like toxins.     

The Vibrio cholerae mannose-sensitive hemagglutinin is the receptor for a filamentous bacteriophage from V. cholerae O139

We previously isolated from a 1994 isolate of Vibrio cholerae O139 a filamentous lysogenic bacteriophage, choleraphage 493, which inhibits pre-O139 but not post-O139 El Tor biotype V. cholerae strains in plaque assays. We investigated the role of the mannose-sensitive hemagglutinin (MSHA) type IV pilus as a receptor in phage 493 infection. Spontaneous, Tn5 insertion, and mshA deletion mutants are resistant to 493 infection. Susceptibility is restored by mshA complementation of deletion mutants. Additionally, the 493 phage titer is reduced by adsorption with MSHA-positive strains but not with a DeltamshA1 strain. Monoclonal antibody against MSHA inhibits plaque formation. We conclude that MSHA is the receptor for phage 493. The emergence and decline of O139 in India and Bangladesh are correlated with the susceptibility and resistance of El Tor strains to 493. However, mshA gene sequences of post-O139 strains are identical to those of susceptible pre-O139 isolates, indicating that phage resistance of El Tor is not due to a change in mshA. Classical biotype strains are (with rare exceptions) hemagglutinin negative and resistant to 493 in plaque assays. Nevertheless, they express the mshA pilin gene. They can be infected with 493 and produce low levels of phage DNA, like post-O139 El Tor strains. Resistance to 493 in plaque assays is thus not equivalent to resistance to infection. The ability of filamentous phages, such as 493, to transfer large amounts of DNA provides them, additionally, with the potential for quantum leaps in both identity and pathogenicity, such as the conversion of El Tor to O139.     

Septicemia due to a non-0:1, non-0:139 Vibrio cholerae

We describe a patient with a non-0:1, non-0:139 Vibrio cholerae septicemia associated with ecythema gangrenosa-like skin lesions. The patient acquired the infection in Puerto Rico. Given the high fatality rate, it is important for the medical community to consider the diagnosis in high risk patients with exposures in Puerto Rico tropical waters.     

Application of ribotyping for differentiating Vibrio cholerae non-O1 isolated from shrimp farms in Thailand

A collection of 143 Vibrio cholerae non-O1 strains isolated from shrimp farms in Thailand were characterized and grouped by ribotyping. Sixty-four ribotypes were distinguished following digestion of chromosomal DNA with the restriction enzyme BglI, and the reproducibility of the method was 100%. There was no correlation between specific ribotype distributions and the locations of the shrimp farms. Ribotype similarity was examined by cluster analysis, and two main groups with 10 and 54 ribotypes, respectively, were found. Correlation between ribotype and O-antigen expression was shown to exist among those isolates tested. Ribotyping appears to be a suitable method for differentiating environmental V. cholerae non-O1 strains, and comparison of ribotype patterns showed a high degree of genetic divergence within V. cholerae non-O1.     

Resurgent Vibrio cholerae O139: rearrangement of cholera toxin genetic elements and amplification of rrn operon

PURPOSE: The objective of this work is to prepare microspheres by the emulsion-solvent evaporation process using MPOE-PLA copolymers as the matrix material and/or the surfactant. This preparation was studied in order to avoid the use of PVA as the surfactant in the process. METHODS: Two series of MPOE-PLA copolymers were synthesised. The first, with a long and constant length PLA chain (45,000 g.mol(-1), was used as the matrix material, the second, with short PLA chains (< or = 2.200 g.mol(-1)), and different HLB as the surfactant. Microspheres were prepared by the "simple" and "double" emulsion methods. The steric stabilization effect of the copolymers was investigated using confocal microscopy and X-ray photoelectron spectroscopy (XPS). RESULTS: Confocal microscopy and XPS analysis showed that the microspheres prepared using MPOE5K-PLA0.5K as the surfactant and MPOE-PLA45K copolymers as the matrix material had an MPOE coating present at the microsphere surface. This hydrophilic layer ensures steric stabilization of the particles. CONCLUSIONS: MPOE-PLA copolymers can be used for the preparation of particles instead of PVA and their use can be extended whenever a biocompatible and bioeliminable surfactant is required for biological or medical applications.     

Isolation of Vibrio cholerae O139 from the drinking water supply during an epidemic of cholera

In mid-1994, the public water supply was investigated in a medium-sized town in south India during an epidemic of cholera due to Vibrio cholerae O139. Vibrio cholerae O139 was isolated from the public water supply including one of the wells supplying the town, the central overhead tank, and domestic taps connected to the public supply. Following chlorination, the organism was no longer isolated from the water supply and the epidemic subsided. This demonstration of V. cholerae O139 in the drinking water supply of a town underlines the need for adequate treatment of the water supply.