Development of an enzyme-labeled oligonucleotide probe for the cholera toxin gene

An alkaline phosphatase-conjugated 30-mer oligonucleotide probe was developed to detect the cholera toxin gene (ctx) in Vibrio cholerae O1. For rapid identification, V. cholerae O1 was grown on selective agar (thiosulfate-citrate-bile salts agar) or in alkaline peptone water and organisms were transferred directly to nylon membranes. Lysis of cells, denaturation of DNA, neutralization, and hybridization were carried out on the membrane. These procedures required only 3 h for completion. The results of the hybridization test with 88 clinical and 20 environmental isolates agreed almost exactly with the results of the immunological tests (anti-cholera toxin antibody-sensitized latex agglutination tests). The specificity of the probe was also tested with strains of enterotoxigenic Escherichia coli, V. cholerae non-O1, and Vibrio mimicus.     

Vitek system antimicrobial susceptibility testing of O1, O139, and non-O1 Vibrio cholerae

Vibrio cholerae causes epidemic diarrhea throughout the world. Fluid replacement is the primary therapy for cholera; however, high mortality rates often necessitate the use of antibiotics. V. cholerae, like most bacteria, has developed resistance to some antibiotics. In the early 1990s a new serotype strain, Bengal 0139, began a new wave of cholera epidemics. Bengal isolates showed unique trends in antimicrobial resistance. Many clinical laboratories use automated antibiotic susceptibility testing for V. cholerae. It is important to know if automated susceptibility test results for V. cholerae coincide with reported trends in antibiotic susceptibility. In the present study, we used the Vitek automated susceptibility system to determine the susceptibilities of 79 V. cholerae O1 isolates, 100 O139 isolates, and 112 non-O1 isolates. Vitek susceptibilities for V. cholerae showed a good correlation with preestablished epidemiological data. Although the new O139 serogroup showed a trend of increased resistance to trimethoprim-sulfamethoxazole and nitrofurantoin, it was more susceptible to ampicillin than previous serogroup O1 and non-O1 strains. Regardless of serogroup, > or = 98% of the V. cholerae isolates tested were susceptible to most antibiotics tested by us. It is important to continue susceptibility testing of all new isolates of V. cholerae because of emerging resistant strains. However, V. cholerae remains susceptible to most of the available antibiotics.     

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.     

Rearrangements in the genomes of Vibrio cholerae strains belonging to different serovars and biovars

The intron-encoded enzyme I-CeuI provides an excellent tool for rapidly examining the organization of genomes of related species of bacteria. Vibrio cholerae strains belonging to serovars O1 and O139 have 9 I-ceuI sites in their genomes, and V. cholerae strains belonging to serovars non-O1 and non-O139 have 10 I-ceuI sites in their genomes. This information can be used as a criterion to differentiate O1 strains from non-O1 and non-O139 strains. To our knowledge, intraspecies variation in the number of rrn operons has not been reported in any other organism. Our data revealed extensive restriction fragment length polymorphism based on a comparison of the I-ceuI digestion profiles of strains belonging to different serovars and biovars. From the analysis of partial digestion products, I-CeuI macrorestriction maps of several classical, El Tor, and O139 strains were constructed. While the linkage maps are conserved within biovars, linkage maps vary substantially between biovars.     

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.     

Infections due to non-O1 Vibrio cholerae in southern Taiwan: predominance in cirrhotic patients

Although Taiwan is not an area where cholera is endemic, from October 1988 to October 1997 30 episodes of non-O1, non-O139 Vibrio cholerae infection were noted at the National Cheng Kung University Hospital in Taiwan. Infections generally occurred in hot seasons, and two episodes were concomitant with Vibrio vulnificus infection. Three major clinical presentations were found: bacteremia with concurrent spontaneous bacterial peritonitis or invasive soft-tissue infections that occurred solely in cirrhotic patients; self-limited acute febrile gastroenteritis that occurred in patients with no underlying medical disease; and necrotizing fasciitis or cellulitis that often resulted from a wound on extremities. Other manifestations included fatal pneumonitis in a drowned man and acute pyosalpinx. The differential diagnosis of invasive infections in cirrhotic patients should include infections due to non-O1 V. cholerae or V. vulnificus, and a third-generation cephalosporin and a tetracycline analogue or a fluoroquinolone alone is recommended for treatment of severe vibrio infections.     

Use of representational difference analysis to identify genomic differences between pathogenic strains of Vibrio cholerae

Representational difference analysis (RDA) is a recently developed technique used for amplifying genetic differences between two closely related genomes. We compared RDA and a modified version of RDA to examine genomic differences between the two Vibrio cholerae serogroups that cause epidemic cholera, O1 and O139, and between the two biotypes of the O1 serogroup. With both techniques, we recovered several sequences known to be found only in V. cholerae O139 but absent in its presumed progenitor, V. cholerae O1 El Tor. A greater number of unique fragments were generated in comparing the two V. cholerae O1 biotypes, consistent with the probable greater genetic differences between the two biotypes.     

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.     

Structural and functional characterization of IS1358 from Vibrio cholerae

The new epidemic serovar O139 of Vibrio cholerae has emerged from the pandemic serovar O1 biotype El Tor through the replacement of a 22-kbp DNA region by a 40-kbp O139-specific DNA fragment. This O139-specific DNA fragment contains an insertion sequence that was described previously (U. H. Stroeher, K. E. Jedani, B. K. Dredge, R. Morona, M. H. Brown, L. E. Karageorgos, J. M. Albert, and P. A. Manning, Proc. Natl. Acad. Sci. USA 92:10374-10378, 1995) and designated IS1358O139. We studied the distribution of the IS1358 element in strains from various serovars by Southern analysis. Its presence was detected in strains from serovars O1, O2, O22, O139, and O155 but not in strains from serovars O15, O39, and O141. Furthermore, IS1358 was present in multiple copies in strains from serovars O2, O22, and O155. We cloned and sequenced four copies of IS1358 from V. cholerae O22 and one copy from V. cholerae O155. A comparison of their nucleotide sequences with those of O1 and O139 showed that they were almost identical. We constructed a transposon consisting of a kanamycin resistance gene flanked by two directly oriented copies of IS1358 to study the functionality of this element. Transposition of this element from a nonmobilizable plasmid onto the conjugative plasmid pOX38-Gen was detected in an Escherichia coli recA donor at a frequency of 1.2 x 10(-8). Sequence analysis revealed that IS1358 duplicates 10 bp at its insertion site.     

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.     

The effect of epidermal growth factor in human granulosa cells varies with follicle size

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.     

Potentially pathogenic vibrios associated with mussels from a tropical region on the Atlantic coast of Brazil

Mussels (Perna perna) harvested on the coast of Ubatuba, in three different stations in the State of S?o Paulo, Brazil, were examined for Vibrio spp. over a 1 year period. The ranges of most probable number (MPN 100 g-1) were: Vibrio alginolyticus (< 3-24,000), V. parahaemolyticus (< 3-24,000), V. fluvialis (< 3-1100), V. cholerae non-O1 (< 3-23), V. furnissii (< 3-30), V. mimicus (< 3-9) and V. vulnificus (< 3-3). The highest incidence was observed for V. alginolyticus (92-100%), followed by V. parahaemolyticus (67-92%), V. fluvialis (34-67%), V. vulnificus (8-17%), V. furnissii (0-17%), V. mimicus (0-17%) and V. cholerae non-O1 (0-8%). Tests for virulence factors were positive in 34.1% of the vibrios in the rabbit ileal loop and 31.7% in the Dean test. Positive results in the Kanagawa test were obtained with 0.51% of V. parahaemolyticus strains. The mean values (MPN 100 g-1) of faecal coliforms in mussels from the three regions varied from 1100 to 44,000, and seawater collected at the same stations gave average values for faecal coliforms in the range 18-3300 MPN 100 ml-1. These results highlight the potential risks of food poisoning associated with raw or undercooked seafood.     

Isolation of sucrose late-fermenting and nonfermenting variants of Vibrio cholerae O139 Bengal: implications for diagnosis of cholera

The sucrose-containing selective medium thiosulfate-citrate-bile salt-sucrose agar missed a sucrose nonfermenting and four sucrose late-fermenting variant strains of Vibrio cholerae O139 Bengal from diarrheal stools. These strains were, however, correctly identified as V. cholerae O139 on a sucrose-deficient selective medium, taurocholate-tellurite-gelatin agar.     

Molecular characterization of Vibrio cholerae O1 biotype El Tor strains isolated between 1992 and 1995 in Calcutta, India: evidence for the emergence of a new clone of the El Tor biotype

Sixty-one clinical strains of Vibrio cholerae O1 El Tor isolated in Calcutta before, during, and after the V. cholerae O139 Bengal outbreak were examined to see if the O1 strains of the post-O139 period were different from those in existence before. Comparison of the restriction fragment length polymorphism of the rRNA genes (ribotyping) and the CTX genetic element revealed that all "before" strains except 1 belonged to a single known ribotype, whereas all "after" strains except 2 belonged to a hitherto undescribed ribotype. Also, 23 of 25 "before" strains harbored two or more copies of CTX in tandem and also a "free" RS1 element away from CTX, whereas 19 of 21 "after" strains had a single copy of CTX and no free RS1 element. CTX occupied different chromosomal locations in "before" and "after" strains. These studies clearly showed that El Tor O1 strains, which displaced V. cholerae O139 in Calcutta, belonged to a new clone and suggested that there is a continuous genetic reassortment among El Tor strains of V. cholerae O1.     

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.     

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.]     

Molecular characterization of Vibrio cholerae O1 strains isolated in Romania

A collection of 89 Vibrio cholerae O1 strains, isolated in Romania between 1977 and 1994, and 6 strains from the Republic of Moldavia, was characterized by ribotyping, toxin gene restriction pattern (toxinogenotype) and distribution of cholera toxin gene (ctx), accessory toxin gene (ace) and zonula occludens toxin gene (zot). After Bg/I endonuclease restriction of chromosomal DNA, a total of 18 ribotypes and 21 toxinogenotypes were distinguished. Deletions in the core region of the toxin gene cassette were found in 20% of strains; however, with the exception of one strain, all the isolates contained the ctx gene. Used in association, the three methods of molecular typing provided an accurate characterization of V. cholerae O1 isolates.     

Genetic diversity and population structure of Vibrio cholerae

Multilocus enzyme electrophoresis (MLEE) of 397 Vibrio cholerae isolates, including 143 serogroup reference strains and 244 strains from Mexico and Guatemala, identified 279 electrophoretic types (ETs) distributed in two major divisions (I and II). Linkage disequilibrium was demonstrated in both divisions and in subdivision Ic of division I but not in subdivision Ia, which includes 76% of the ETs. Despite this evidence of relatively frequent recombination, clonal lineages may persist for periods of time measured in at least decades. In addition to the pandemic clones of serogroups O1 and O139, which form a tight cluster of four ETs in subdivision Ia, MLEE analysis identified numerous apparent clonal lineages of non-O1 strains with intercontinental distributions. A clone of serogroup O37 that demonstrated epidemic potential in the 1960s is closely related to the pandemic O1/O139 clones, but the nontoxigenic O1 Inaba El Tor reference strain is not. A strain of serogroup O22, which has been identified as the most likely donor of exogenous rfb region DNA to the O1 progenitor of the O139 clone, is distantly related to the O1/O139 clones. The close evolutionary relationships of the O1, O139, and O37 epidemic clones indicates that new cholera clones are likely to arise by the modification of a lineage that is already epidemic or is closely related to such a clone.     

Epidemiology and transmission of V. cholerae O1 and V. cholerae O139 infections in Delhi in 1993

In 1993, rectal swabs from clinically suspected cases of cholera admitted to the Infectious Diseases Hospital (IDH), Delhi were examined for Vibrio cholerae O1 and O139. Epidemiological data of 396 cholera cases were collected before the patients' discharge from IDH. Of the 1528 laboratory-confirmed cholera cases, 46% and 54% were caused by serotype O1 and O139 respectively. Both serotypes appeared and disappeared simultaneously, and peaked during the same time of the year. However, the two serotypes affected persons of different age groups; about 65% of the O1 cases occurred in children aged less than 10 years, whereas this age group accounted for 40% of the cases due to V. cholerae O139. Although there were some focal outbreaks due to serotype O139, both serotypes had almost similar geographical distributions. Important risk factors for transmission of cholera were almost equally prevalent in the majority of both types of cholera cases. Since the seasonality, geographical distribution, and risk factors for transmission were similar for both serotypes, the study indicates that the preventive and control measures are also likely to be similar. The study also shows that the emergence of V. cholerae O139 in 1993 did not affect the incidence, seasonality, and epidemiology of endemic V. cholerae O1 E1 Tor strains in Delhi.     

Immune mechanisms and protective antigens of Vibrio cholerae serogroup O139 as a basis for vaccine development

We have characterized 11 isolates of Vibrio cholerae O139 Bengal with regard to properties deemed to be relevant for development of a vaccine against O139 cholera. For most strains two colony variants, A and B, which are nonhemolytic and hemolytic, respectively, were detected on blood agar. The A and B variants were associated with high- and low-level production of soluble hemagglutinin-protease, respectively. However, on Luria-Bertani agar both types formed opaque colonies, which has been shown to be associated with capsule formation. Interestingly, under the stationary tube-shaken flask culture conditions in yeast extract-peptone water medium which were used to stimulate the production of cholera toxin (CT) and toxin-coregulated pili, B variants constitutively produced CT and TcpA, two ToxR-regulated proteins, at 28 and 37 degrees C, whereas the production of these proteins by A variants was downregulated at the higher temperature. One of the strains, 4260B, having a well-exposed O antigen and capsule and the capacity to produce large amounts of TcpA, CT, and mannose-sensitive hemagglutinin pili but minimal amounts of the proteolytic soluble hemagglutinin, was selected to produce antibacterial antisera and as a challenge strain in protection studies using the rabbit ileal loop model. Rabbit antisera to live, heat-killed, or formalin-killed O139 vibrios or to purified O139 lipopoly-saccharide (LPS) as well as monoclonal antibodies (MAbs) to O139 LPS agglutinated all O139 isolates. However, when A and B variants of strain 4260 were tested for sensitivity to vibriocidal activity of these antibody preparations, only the B variant was killed. All of the antisera against live or killed O139 vibrios conferred passive protection against fluid accumulation induced by the challenge strain. The protective effects of the antisera were correlated to anti-LPS antibody titers rather than to titers against whole bacteria that had been grown for toxin-coregulated pilus expression. This protection was considerably higher than that conferred by antisera to classical, EI Tor, or recombinantly produced (classical) CT or CTB. Furthermore, MAbs to O139 LPS and CTB-CT exhibited a strong synergistic protection against O139 challenge irrespective of the level of sensitivity of challenge strains to O139 LPS MAbs in vibriocidal assays in vitro.