Structural organization of cholera toxin gene and its expression in an environmental non-pathogenic strain ofVibrio cholerae

Non-pathogenic, environmental strain ofVibrio cholerae, ELTOR Ogawa EW6 carries a copy of the cholera toxin gene in its chromosome. Restriction enzyme digestion followed by Southern blot analysis revealed that the structure of the cholera toxin gene in this organism is different from that found in the virulent strains. The xbaI site which has been found to be conserved in the cholera toxin of the virulent strains examined so far, is absent here. Results of the RNA dot blot analysis indicated that the cholera toxin gene in EW6 is transcribed much less efficiently compared to the cholera toxin gene present in the virulent strainVibrio cholerae classical Inaba 569B.     

Structural organization of the transfer RNA operon I ofVibrio cholerae: Differences between classical and El Tor strains

Nine major transfer RNA (tRNA) gene clusters were analysed in variousVibrio cholerae strains. Of these, only the tRNA operon I was found to differ significantly inV. cholerae classical (sixth pandemic) and El Tor (seventh pandemic) strains. Amongst the sixteen tRNA genes contained in this operon, genes for tRNA Gln3 (CAA) and tRNA Leu6 (CUA) were absent in classical strains as compared to El Tor strains. The observation strongly supported the view that the above two pandemic strains constitute two different clones.     

Molecular phylogenetic analysis ofVibrio cholerae O1 El Tor strains isolated before, during and after the O139 outbreak based on the intergenomic heterogeneity of the 16S-23S rRNA intergenic spacer regions

We have cloned, sequenced and analysed all the five classes of the intergenic (16S-23S rRNA) spacer region (ISR) associated with the eightrrn operons (rrna-rrnh) ofVibrio cholerae serogroup O1 El Tor strains isolated before, during and after the O139 outbreak. ISR classes ‘a’ and ‘g’ were found to be invariant, ISR-B (ISRb and ISRe) exhibited very little variation, whereas ISR-C (ISRc, ISRd, and ISRf) and ISRh showed the maximum variation. Phylogenetic analysis conducted with all three ISR classes (ISR-B, ISR-C and ISRh) showed that the pre-O139 serogroup and post-O139 serogroup O1 El Tor strains arose out of two independent clones, which was congruent with the observation made by earlier workers suggesting that analyses of ISR-C and ISR-h, instead of all five ISR classes, could be successfully used to study phylogeny in this organism.     

Seasonality and toxigenicity ofVibrio cholerae non-01 isolated from different components of pond ecosystems of Dhaka City,Bangladesh

Diarrhoea due toVibrio cholerae non-01 is common in Bangladesh. Four hundred and eighty samples, including plants, water, phytoplankton and sediment, were collected from five ponds in Dhaka every 15 days for one year.V. cholerae non-01 was isolated from 181 (38%) of the samples. Two peaks were evident: one in April and the other in August/September. Forty-three (23%) of the 181 isolates were examined for toxigenicity and 19% were cytotoxic to Y1 adrenal cells. This study provides evidence of the likely infectious nature of some ponds and may have relevance to the epidemiology of diarrhoea caused byV. cholerae non-01 in Bangladesh.     

Viable but non-culturable Vibrio cholerae O1 revert to a cultivable state in the human intestine

Vibrio cholerae O1 can enter a state in which they remain viable but are non-culturable. Presumably, such bacteria can be pathogenic if they retain the capacity to proliferate in the human intestine following ingestion. Two groups of volunteeers were given inocula containing viable but non-culturable V. cholerae O1 of the attenuated vaccine strain CVD 101 (viable CVD 101 organisms readily colonize the human intestine). Volunteers in one of the two groups excreted viable CVD 101, demonstrating that, in the environment of the human intestine, previously non-culturable vibrios can regain the capacity to multiply. These observations support the proposition that viable but non-culturable bacterial enteropathogens may pose a potential threat to health.     

Cholera toxin gene polymerase chain reaction for detection of non-culturable Vibrio cholerae O1

Cholera enterotoxin is a major antigenic determinant for virulence of Vibrio cholerae O1 which can enter into a viable but non-culturable (N-C) state, not detectable by conventional culture methods, yet remain capable of producing enterotoxin and potentially pathogenic. PCR was applied in the current study to detect the chilera toxin (ctx) gene of N-C cells, thus eliminating the necessity of culture. Sets of oligonucleotide primers were designed, based on the ctxAB operon of V. cholerae O1, to detect the presence of the ctx gene. DNA from both culturable and N-C cells of V. cholerae O1 was amplified by PCR using sets of primers flanking 302-, 564- and 777-bp fragments of the ctx gene. The PCR method employed was capable of detecting the ctx gene in N-C V. cholerae in aquatic microcosms and in diarrheal stool samples from three patients who had distinct clinical symptoms of cholera but were culture-negative for V. cholerae O1 and non-O1 and enterotoxigenic Escherichia coli. Forty cycles of a two-step reaction (30 s each at 94 and 60°C) were optimal and more time efficient than a three-step PCR described previously. The procedure, from the point of heating microcosms or broth culture samples to observation on gels, requires < 4 h to complete.J.A.K. Hasan, A. Huq, M.A.R. Chowdhury, and R.R. Colwell are with the Department of Microbiology, University of Maryland, College Park, MD, USA. M. Shahabuddin is with the National Institute of Health. Bethesda, MD, USA. L. Loomis is with New Horizons Diagnostics Corporation, Columbia, MD, USA.     

Analysis of lolB gene sequence and its use in the development of a PCR assay for the detection of Vibrio cholerae

A PCR assay has been developed based on a lolB (hemM) gene, which was found to be highly conserved among the Vibrio cholerae species but non-conserved among the other enteric bacteria. The lolB PCR detected all O1, O139 and non-O1/non-O139 serogroup and biotypes of V. cholerae. The analytical specificity of this assay was 100% while the analytical sensitivity was 10 pg/microL and 10(3) CFU/mL at DNA and bacterial level respectively. The diagnostic sensitivity and specificity was 98.5% and 100% respectively.     

Induction of SOS like responses by nitrofurantoin in Vibrio cholerae el tor cells

Treatment of Vibrio cholerae el tor strain SLH22(J) with nitrofurantoin induced dose-dependent prophage kappa, the maximum induction being 6-fold the spontaneous induction level. UV-inactivated kappa phages were Weigle reactivated, the maximum Weigle factor being 1.8 and 2.0 respectively in nitrofurantoin and UV pretreated el tor strain H218 Sm^r. Nitrofurantoin treatment also caused significant filamentation of the el tor strain H218 Sm^r and mutation of these cells from ampicillin sensitivity to ampicillin resistance. The levels of the four SOS-like responses induced by this drug were low but significant.     

Antimicrobial susceptibility of non-O1 Vibrio cholerae isolated from wastewater stabilization ponds in Marrakesh,Morocco

The 365 strains of Vibrio cholerae, isolated in Marrakesh from raw sewage and stabilization pond effluent, were all identified as non-O1 Vibrio cholerae. When tested for their susceptibilities to ampicillin, amoxicillin, cephalothin, streptomycin, novobiocin, chloramphenicol, nalidixic acid and trimethoprim-sulphamethoxazole, 13% of the strains from raw sewage and 20% of those from stabilization pond effluent were found to be resistant to one or more of the antibiotics. There were no significant differences, in terms of drug resistance, between isolates from the new sewage and those from the ponds' effluent.The authors are with the Université Cadi Ayyad, Faculté des Sciences Semialia, Département de Biologie, Laboratoire de Microbiologie, BP S/15, Marrakesh, Morocco     

Regions of the cloned Vibrio cholerae rfb genes needed to determine the Ogawa form of the O-antigen.

Summary The O-antigen of the lipopolysaccharides of Vibrio cholerae 01 can exist in two forms termed Inaba and Ogawa. We used a complementation system to demonstrate that the Ogawa phenotype is dominant over the Inaba phenotype. By using a set of deletions affecting the Ogawa rfb genes, we identified two regions which are needed to confer the Ogawa phenotype. In vitro mutagenesis of the cloned Ogawa rfb genes resulted in the isolation of variants with the Inaba phenotype. The results are interpreted with respect to previous studies demonstrating interconversion between the two forms of the V. cholerae O-antigen.     

Variable Tandem Repeat VcA of Vibrio cholerae

Computer analysis revealed seven potential variable-number tandem-repeat (VNTR) loci in the Vibrio cholerae genome. Specific primers were designed to amplify locus VcA located on chromosome 2 and containing a TGCTGT repeat. The locus was found in all tested strains from aV. cholerae strain collection, the repeat number varying from 3 to 23. In total, 14 VcA alleles were observed. The VcA locus was proposed as a marker for the molecular typing of V. cholerae strains.     

Functional role of a conserved aspartic acid residue in the motor of the Na-driven flagellum from Vibrio cholerae

The flagellar motor consists of a rotor and a stator and couples the flux of cations (H⁺ or Na⁺) to the generation of the torque necessary to drive flagellum rotation. The inner membrane proteins PomA and PomB are stator components of the Na⁺-driven flagellar motor from Vibrio cholerae. Affinity-tagged variants of PomA and PomB were co-expressed in trans in the non-motile V. cholerae pomAB deletion strain to study the role of the conserved D23 in the transmembrane helix of PomB. At pH 9, the D23E variant restored motility to 100% of that observed with wild type PomB, whereas the D23N variant resulted in a non-motile phenotype, indicating that a carboxylic group at position 23 in PomB is important for flagellum rotation. Motility tests at decreasing pH revealed a pronounced decline of flagellar function with a motor complex containing the PomB-D23E variant. It is suggested that the protonation state of the glutamate residue at position 23 determines the performance of the flagellar motor by altering the affinity of Na⁺ to PomB. The conserved aspartate residue in the transmembrane helix of PomB and its H⁺-dependent homologs might act as a ligand for the coupling cation in the flagellar motor.     

Short communication: Detection of non-culturable Vibrio cholerae O139, by PCR and fluorescent antibody methods,in laboratory microcosms

Non-culturable Vibrio cholerae O139 was detected in microcosms by PCR and fluorescent-antibody (FA) techniques. When survival of V. cholerae O139 in microcosms was assessed by viable counting on culture media, the vibrio became non-culturable after 44 days and remained non-culturable for an additional 7 weeks.     

Synthesis of the conjugation ready, downstream disaccharide fragment of the O-PS of Vibrio cholerae O:139

The linker-equipped disaccharide, 8-amino-3,6-dioxaoctyl 2,6-dideoxy-2-acetamido-3-O-β-d-galactopyranosyluronate-β-d-glucopyranoside (10), was synthesized in eight steps from acetobromogalactose and ethyl 4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-1-thio-β-d-glucopyranoside. The hydroxyl group present at C-4^II in the last intermediate, 8-azido-3,6-dioxaoctyl 4-O-benzyl-6-bromo-2,6-dideoxy-2-trichloroacetamido-3-O-(benzyl 2,3-di-O-benzyl-β-d-galactopyranosyluronate)-β-d-glucopyranoside (9), is positioned to allow further build-up of the molecule and, eventually, construction of the complete hexasaccharide. Global deprotection (9→10) was done in one step by catalytic hydrogenolysis over palladium-on-charcoal.     

Detection of Viable Toxigenic Vibrio cholerae from Environmental Water Sources by Direct Cell Duplex PCR Assay

Summary Environmental monitoring is important to enable effective resource management and public health protection as well as rapid and accurate identification of Vibrio cholerae in drinking-water sources. Traditional methods employed in identification are laborious, time-consuming and practically not viable for screening of a large number of samples. In this study, a direct cell duplex PCR assay for the detection of viable toxigenic V. cholerae in environmental water samples was developed. In the PCR assay, two gene sequences were amplified together, one of outer membrane protein (ompW), which is species-specific and another of cholera toxin (ctxAB). The detection limit of duplex PCR was 5 × 10⁴ V. cholerae/reaction. Different environmental water samples were artificially spiked with V. cholerae O1 cells and filtered through a 0.22 μm membrane, and the filters enriched in alkaline peptone water for 6 h and then used directly in the duplex PCR assay. The PCR procedure coupled with enrichment could detect as few as 1.2 c.f.u./ml in ground water, 1.2 × 10² c.f.u. ml⁻¹ in sewer water and 1.2 × 10³c.f.u. ml⁻¹ in tap water. The assay was successfully applied directly for screening of environmental potable water samples collected from a cholera-affected area. The proposed method is simple and can be used for environmental monitoring of toxigenic as well as non-toxigenic V. cholerae.     

Response and tolerance of toxigenic Vibro cholerae O1 to cold temperatures

Survival and tolerance at cold temperatures, the differentially expressed cellular proteins, and cholera toxin (CTX) production were evaluated in Vibrio cholerae O1. Rapid loss of culturability and change to distinct coccoid morphology occurred when cultures of V. cholerae O1 were exposed to 5°C directly from 35°C. Also, cultures of V. cholerae first exposed to 15°C for 2 h and then maintained at 5°C failed to exhibit an adaptive response, instead a rapid loss of viable plate count was noticed. Results from Western blot experiments revealed the absence of a major cold shock protein, CS7.4. Also, a decreased level of CTX was noticed in V. cholerae O1 cultures exposed to 5 or 15°C after first being exposed to 15°C for 2 h, followed by transfer to 5°C. Reduced expression of CTX at cold temperatures, compared to the cultures maintained at 35°C, may be a result of decreased cellular metabolic activity. When V. cholerae O1 cultures were exposed to 15°C for 2 h, elevated expressions of 8, 26 and 194 kDa, and decreased expression of 28 and 183 kDa proteins occurred. It is suggested that these differentially expressed cold-responsive proteins are involved in regulating culturability and conversion to a coccoid cell morphology in V. cholerae O1.     

Detection and molecular characterization of Vibrio cholerae O1 Inaba biotype El Tor strain in Kerala, S. India

The resurgence of enteric pathogen Vibrio cholerae, the causative organism of epidemic cholera, remains a major health problem in many developing countries. The outbreaks of cholera follow a seasonal pattern in regions of endemicity. The southern Indian state of Kerala is endemic to cholera. A V. cholerae strain isolated from the stool sample of a patient in Piravam, Kerala, South India, was analysed. However, this case occurred at a time not associated with cholera outbreaks, leading to concern among the State health officials. We compared the virulence potential of the isolate with that of the standard or reference strains, that have been widely used as positive control. The isolate was identified as V. cholerae O1 biotype El Tor serotype Inaba. The resistance pattern of the isolate to common antibiotics was examined and it was found to be multi-drug resistant in nature. The strain was analysed for the presence of the CTX genetic element, which encodes genes for cholera toxin and other important regulatory genes. It was found to be positive for all the genes tested. In Kerala, most of the cholera outbreaks have been reported to be caused by V. cholerae O1 El Tor belonging to Ogawa serotype. Interestingly, the V. cholerae strain isolated from this case has been found to be of Inaba serotype, which is rarely reported.     

Construction of shuttle vectors for cloning inVibrio cholerae andEscherichia coli

Starting from a naturally occurring cryptic plasmid pVC540 ofVibrio cholerae non-OI. strain 1095, a number of plasmid vectors have been constructed for cloning genes inVibrio cholerae by introducing antibiotic resistance markers containing a set of unique cloning sites. The constructs pVC810 and pVE920 have the origins of bothVibrio cholerae andEscherichia coli replicons and are stable in both organisms in the absence of selective pressure. These plasmids can serve as shuttle vectors betweenEscherichia coli andVibrio cholerae. The plasmid vectors reported here along with the demonstration of transformation inVibrio cholerae by plasmid DNA will facilitate genetic analysis of this important human pathogen.     

Detection of cholera (ctx) and zonula occludens (zot) toxin genes in Vibrio cholerae O1, O139 and non-O1 strains

Vibrio cholerae O1 and V. cholerae non-O1 strains isolated from environmental samples collected in São Paulo, Brazil, during cholera epidemics and pre-epidemic periods were examined for the presence of toxin genes. V. cholerae O1 strains isolated from clinical samples in Peru and Mexico, and V. cholerae O139 strains from India were also examined for the presence of ctx (cholera toxin gene) and zot (zonula occludens toxin gene) by polymerase chain reaction (PCR). A modified DNA-extraction method applied in this study yielded satisfactory recovery of genomic DNA from vibrios. Results showed that strains of V. cholerae O1 isolated during the preepidemic period were ctx ^-/zot ^- whereas strains isolated during the epidemic were ctx ⁺/zot ⁺. All V. cholerae non-O1 strains tested in the study were ctx ^-/zot ^-, whereas all V. cholerae O139 strains were ctx ⁺/zot ⁺. Rapid detection of the virulence genes (ctx and zot) can be achieved by PCR and this can serve as an important tool in the epidemiology and surveillance of V. cholerae.