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.     

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.     

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.     

Pseudo-outbreak of candidaemia with Candida parapsilosis

Vibrio cholerae was isolated from 1008 of 3496 stool samples (28.8%) examined in Tamil Nadu State, India, between November 1992 and December 1995. During November and December 1992, 363 of the 370 isolates serotyped (98%) were V. cholerae O139 (Bengal). The epidemic predominantly affected adults (91%; 597/656). Both V. cholerae O1 and O139 serotypes were sometimes isolated in the same locality from different individuals. From January 1993 onwards, the rate of isolation of V. cholerae O139 declined, and in 1995 V. cholerae E1 Tor (serotype O1) was isolated from most of the cases (85.6%; 131/153). V. cholerae E1 Tor has clearly not been replaced by serotype O139, but can survive during inter-epidemic periods and reappear at an opportune moment. The decline of serotype O139 may be due to the development of immunity as a result of repeated exposure.     

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.     

Identification of the Vibrio cholerae type 4 prepilin peptidase required for cholera toxin secretion and pilus formation

Cholera toxin secretion is dependent upon the extracellular protein secretion apparatus encoded by the eps gene locus of Vibrio cholerae. Although the eps gene locus encodes several type four prepilin-like proteins, the peptidase responsible for processing these proteins has not been identified. This report describes the identification of a prepilin peptidase from the V. cholerae genomic database by virtue of its homology with the PilD prepilin peptidase of Pseudomonas aeruginosa. Plasmid disruption or deletion of this peptidase gene in either EI Tor or classical V. cholerae O1 biotype strains results in a dramatic decrease in cholera toxin secretion. In the case of the EI Tor biotype mutants, surface expression of the type 4 pilus responsible for mannose-sensitive haemagglutination is abolished. The cloned V. cholerae peptidase processes either EpsI or MshA preproteins when co-expressed in E. coli. Mutation of the V. cholerae peptidase gene also results in a defect in virulence and decreased levels of OmpU. The V. cholerae peptidase gene sequence shows 80% homology with the Vibrio vulnificus VvpD type 4 prepilin peptidase required for pilus assembly and cytolysin secretion in V. vulnificus. Accordingly, the V. cholerae type 4 prepilin peptidase required for pilus assembly and cholera toxin secretion has been designated VcpD.     

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.     

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.     

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.     

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.     

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.     

The cellular fatty acid composition of bacteria in the family Vibrionaceae

It is shown that strains of Vibrio cholerae of serovar O1, biovar eltor, subtype Ogawa, museum strains V. cholerae of serovar O1 and NAG-vibrios (isolated from various sources: sea, river and sewage water, canal water and people) possess identical composition of cell fatty acids with prevailing hexadecenoic, hexadecanoic and octadecenoic acids. Being identical, fatty acid profiles of V. parahaemolyticus and V. alginolyticus, are close to that of V. cholerae differing from the latter mainly by the higher content of dodecanoic acid. Similarity of Aeromonas sp. and Vibrio strains in the fatty acid composition proves phylogenetic relation-ship of these bacteria. Fatty acid composition of Plesiomonas shigelloides cells characterized by the presence of methylenhexadecanoic acid as well as by similarity with Vibrio and Aeromonas by the content of most fatty acids confirms a supposition of R. R. Colwell on the intermediate status of genus Plesiomonas between the families Enterobacteriaceae and Vibrionaceae. Independent of the growth medium, the strains Vibrio. Aeromonas and Plesiomonas preserved a fatty-acid profile, inherent in them, with variations mainly in the content of fatty acids with the odd number of carbon atoms. Allowing for relative stability of fatty acid composition and its peculiarity in certain taxonomic groups of the studied bacteria, the above test may be used as additional objective criterion to identify the representatives of Vibrionaceae family.     

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.     

Molecular evidence that a distinct Vibrio cholerae O1 biotype El Tor strain in Calcutta may have spread to the African continent

We present molecular evidence that a distinct genotype of Vibrio cholerae O1 which appeared in Calcutta, India, in September 1993 and which is characterized by a unique ribotype that is not found in the standardized ribotyping scheme of V. cholerae and that shows a specific pulsed-field gel electrophoresis profile may have spread to the west African country of Guinea-Bissau where it was responsible for an epidemic of cholera which began in October 1994 and continued into 1996.     

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.     

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.     

rfb mutations in Vibrio cholerae do not affect surface production of toxin-coregulated pili but still inhibit intestinal colonization

The toxin-coregulated pilus (TCP) of Vibrio cholerae is essential for colonization. It was recently reported that rfb mutations in V. cholerae 569B cause the translocation arrest of the structural subunit of TCP, raising the possibility that the colonization defects of lipopolysaccharide mutants are due to effects on TCP biogenesis. However, an rfbB gene disruption in either V. cholerae O395 or 569B has no apparent effect on surface TCP production as assessed by immunoelectron microscopy and CTX phage transduction, and an rfbD::Tn5lac mutant of O395 also shows no defect in TCP expression. We conclude that the colonization defect associated with rfb mutations is unrelated to defects in TCP assembly.     

Unique organization of the CTX genetic element in Vibrio cholerae O139 strains which reemerged in Calcutta, India, in September 1996

We studied the restriction fragment length polymorphism of the rRNA gene and CTX genetic element in Vibrio cholerae O139 Bengal, which resurged in Calcutta in September 1996 after a gap of 32 months. While the strains from this resurgence were indistinguishable from the earlier strains by ribotyping, the structure of the CTX genetic element present in the current O139 strains was found to be unconventional.     

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.]     

Taking care of the elderly: the unmentioned abuses

A total of 10,427 diarrhoeal stool specimens were cultured for Vibrio cholerae between 1992 and 1997. The isolation rates were 2%, 2.6%, 6.7%, 7.08%, 0.9% and 2.6% in the years from 1992 to 1997 respectively. Till 1992, Vibrio cholerae 01 ogawa was the predominant strain. In 1993, 81.3% of the isolates were of 0139 Bengal strain and the rest were V. cholerae 01. From 1994 to 1997, V. cholerae 01 ogawa was the predominant strain and there were no isolation of 0139 strain. The predominant phage type in 1992 and 1993 were T2 and T27 thereafter. Most Vibrio cholerae strains were sensitive to tetracycline, gentamycin, netromycin, norfloxacin and furazolidine. Strains were resistant to cotrimaxozole till 1996, but were 100% sensitive in 1997. Strains were sensitive to chloramphenicol till 1993 but acquired resistance thereafter.