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.     

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.     

A Potential Epidemic Factor from the Bacteria, <Emphasis Type="Italic">Vibrio cholerae WO7</Emphasis>

Certain species of Vibrio cholerae have evolved mechanisms to become pathogenic to humans, with the potential to cause a severe life-threatening diarrheal disease, cholera. Cholera can emerge as explosive outbreaks in the human population. V. cholerae illness is produced primarily through the expression of a potent toxin (cholera toxin) within the human intestine. The present study has been carried out on a novel toxin purified from V. cholerae W07, an epidemic cholera strain devoid of cholera toxin gene (ctx). A modified method of purification improved purification fold as well as yield of this toxin. Heating was found to be the essential and sufficient condition for dissociation of the two subunits (58 kDa and 40 kDa) of this toxin (pI 5.2). The 40-kDa subunit of the purified toxin was identified as the carbohydrate binding subunit. This toxin was found to induce apoptosis in HEp-2 cells. Thus, the WO7 toxin seems to have potential importance in the pathogenesis of disease associated with Vibrio cholerae WO7.     

Transfer of cholera toxin genes from O1 to non‐O1/O139 strains by vibriophages from California coastal waters

Aims: Vibrio cholerae is an important bacterial pathogen that causes global cholera epidemic. Although they are commonly found in coastal waters around the world, most environmental isolates do not contain cholera toxin genes. This study investigates vibriophages in southern California coastal waters and their ability to transfer cholera toxin genes. Methods and Results: Lytic phages infecting V. cholerae were isolated from Newport Bay, California, between May and November, while none was found in winter. Some of the phage isolates can infect multiple environmental V. cholerae strains and El Tor strains. All phages contained double‐stranded DNA. Transduction experiments using kanamycin‐resistant gene marked CTXΦ demonstrated that some environmental vibriophages can transfer CTXΦ genes from O1 El Tor strain to environmental non‐O1/O139 V. cholerae via generalized transduction. Conclusions: Vibriophages are important components of the natural aquatic ecosystem. They play an important role in influencing the dynamics and evolution of V. cholerae in the environment. Significance and Impact of the Study: This study demonstrates the significance of vibriophages in the coastal environment and transduction as one of the mechanisms of pathogenicity evolution among environmental V. cholerae.     

Role of Temperate Bacteriophage 139 in Changing Cholera Toxin Production in Vibrio cholerae Classical Biovar

New data were obtained concerning cell sensitivity of pathogenic strains of cholera vibrions, which belong to the serogroup O1 of classical biovar, to the temperate bacteriophage 139, the native host of which is Vibrio cholerae O139. Molecular–genetic and biochemical studies showed that phage 139 integrated into the chromosome of strains V. cholerae O1 can change their toxigenic properties. A change in the production of cholera toxin (CT) in lysogens is associated both with an increase in the activity of the toxR regulatory gene and with an alteration of the structure of a chromosomal DNA region that contains a copy of the operon ctxAB encoding CT biosynthesis.     

Comparison of a Reversed Passive Latex Agglutination and a Polymerase Chain Reaction for Identification of Cholera Toxin Producing Vibrio cholerae O1

Production of cholera toxin (CT) in AKI medium and conservation of CT gene (ctx) of 49 strains of Vibrio cholerae O1 were compared by reversed passive latex agglutination (RPLA) and polymerase chain reaction (PCR). The production of CT agreed with conservation of the ctx in 48 out of the 49 strains. Ten strains were positive, and 38 strains were negative by both methods. Only one strain was negative in RPLA and positive in PCR. This suggested that the combination of AKI-SW and RPLA is comparable to PCR to identify CT-producing V. cholerae O1.     

Toxigenicity and toxin genes of Vibrio cholerae 01 isolated from an artificial aquatic environment

A simple experiment was carried out to examlne the effect of varlous physicochemical conditions on toxigenicity and toxin genes of Vibrio cholerae 01 lsolated from an artificial aquatic environment. All isolated strains, tested by tissue culture assay, DNA-DNA hybridization and ELISA, were cytotoxic to Vero cells, did not lose their toxin genes and continued to produce cholera toxin. These results are consistent with the hypothesis that V. cholerae can survive in the environment without losing potential pathogenicity.     

Novel insights into Haemagglutinin Protease (HAP) gene regulation in Vibrio cholerae

Quorum sensing is the phenomenon, whereby bacteria use signal molecules to communicate with each other. For example, to establish a successful infection, pathogenic bacteria become virulent only when they reach a certain local concentration in their host. Bassler and others have highlighted the surprising observation that quorum sensing seems to repress Vibrio cholerae virulence factor expression (e.g. cholera toxin), in contrast to what has been observed for virulence gene expression in other bacteria. Here, I present a novel insight that may clarify the way V. cholerae quorum‐sensing signals regulate its genes. Chironomids (Diptera; Chironomidae), which occur worldwide and are frequently the insect found most abundantly in fresh water bodies, are natural reservoirs of V. cholerae. Quorum‐sensing signals in V. cholerae up‐regulate the production of an extracellular enzyme, haemagglutinin protease (HAP), which degrades chironomid egg masses and prevents the eggs from hatching. HAP, therefore, is a virulence factor against chironomids. Indeed, in a survey carried out over the course of a year, V. cholerae and chironomids showed a pattern that mirrored the dynamics of predator‐prey populations. Globally, the numbers of chironomids are much larger than those of humans, so quorum‐sensing signals of V. cholerae and HAP gene regulation should be understood with regard to their role in chironomids rather than humans. Further research is needed to understand the role of cholera toxin in the environmental existence of V. cholerae.     

Molecular Epidemiology of Vibrio cholerae O1 Isolated from Sporadic Cholera Cases in Okinawa,Japan

In July 1994, 6 cholera cases due to Vibrio cholerae O1 El Tor Ogawa sporadically appeared in Okinawa. All 6 patients had no history of traveling abroad. In the period of this cholera outbreak, a strain of V. cholerae O1 El Tor Ogawa was detected from an imported fish at the Naha port quarantine station. The isolates were characterized to clarify whether or not, they belonged to a common clone. Phenotypes were identical except that one strain revealed cured Celebes and the others were original Celebes in kappa phage typing. The restriction fragment patterns of DNA of the isolates hybridized with an enzyme-labeled oligonucleotide probe for cholera toxin gene (ctx) were identical. Randomly amplified polymorphic DNA of the isolates were identical when a primer was used, but 2 patterns were seen when another primer was used. Pulsed-field gel electrophoresis of the chromosomal DNA digested with NotI restriction enzyme showed 3 patterns. The DNA fragment pattern of the strain isolated from the imported fish was different from the clinical isolates. These results suggested that there was no epidemiological relation among the strains of V. cholerae O1 isolated during this period.     

Virulence profile and clonal relationship among the <Emphasis Type="Italic">Vibrio cholerae</Emphasis> isolates from ground and surface water in a cholera endemic area during rainy season

All the V. cholerae non-O1, non-O139 isolates from ground and surface water samples collected during the rainy season (rainfall contributes significantly in the spread of cholera) contained ompW and a regulatory toxR gene, while many others possessed accessory cholera toxin (ace), hemolysin (hlyA) and outer membrane protein (ompU) genes. All the isolates lacked ctxAB, tcp, zot, rfbO1 and rfbO139 genes. The strains could be grouped into two main clusters colligating the isolates from ground water and surface water samples. The results suggest that surface water harbors various virulent V. cholerae strains that contaminate the ground water due to rain or poor hygienic practices, and result in the emergence of new toxigenic strains for cholera.     

The Autophagic Pathway: A Cell Survival Strategy Against the Bacterial Pore-Forming Toxin Vibrio Cholerae Cytolysin

Vibrio cholerae is the causative agent of cholera in humans. In addition to the critical virulence factors cholera toxin and toxin co-regulated pilus, V. cholerae secretes V. cholerae cytolysin (VCC), a pore-forming exotoxin able to induce cell lysis and extensive vacuolation. We have shown that this vacuolation is related to the activation of autophagy in response to VCC action. Furthermore, we found that the autophagic pathway was required to protect cells upon VCC intoxication. Based on additional data presented here, we propose a model aimed to explain the mechanism of cell protection. We postulate that VCC-induced autophagic vacuoles, which display features of multivesicular bodies and enclose the toxin, are implicated in cell defense through VCC degradation involving fusion with lysosomes.

Addendum to:

Protective Role of Autophagy Against Vibrio cholerae Cytolysin, a Pore-Forming Toxin from V. cholerae

M.G. Gutierrez, H.A. Saka, I. Chinen, F.C.M. Zoppino, T. Yoshimori, J.L. Bocco and M.I. Colombo

Proc Natl Acad Sci USA 2007; 104:1829-34     

Binding efficiencies of carbohydrate ligands with different genotypes of cholera toxin B: molecular modeling, dynamics and docking simulation studies

Vibrio cholerae produces cholera toxin (CT) that consists of two subunits, A and B, and is encoded by a filamentous phage CTXΦ. The A subunit carries enzymatic activity that ribosylates ADP, whereas the B subunit binds to monosialoganglioside (GM1) receptor in epithelial cells. Molecular analysis of toxigenic V. cholerae strains indicated the presence of multiple ctxB genotypes. In this study, we employed a comparative modeling approach to define the structural features of all known variants of ctxB found in O139 serogroup V. cholerae. Modeling, molecular dynamics and docking simulations studies suggested subtle variations in the binding ability of ctxB variants to carbohydrate ligands of GM1 (galactose, sialic acid and N-acetyl galactosamine). These findings throw light on the molecular efficiencies of pathogenic isolates of V. cholerae harboring natural variants of ctxB in causing the disease, thus suggesting the need to consider ctxB variations when designing vaccines against cholera.     

Evaluation of a highly discriminating multiplex multi-locus variable-number of tandem-repeats (MLVA) analysis for Vibrio cholerae

Vibrio cholerae is the etiological agent of cholera and may be used in bioterror actions due to the easiness of its dissemination, and the public fear for acquiring the cholera disease. A simple and highly discriminating method for connecting clinical and environmental isolates of V. cholerae is needed in microbial forensics. Twelve different loci containing variable numbers of tandem-repeats (VNTRs) were evaluated in which six loci were polymorphic. Two multiplex reactions containing PCR primers targeting these six VNTRs resulted in successful DNA amplification of 142 various environmental and clinical V. cholerae isolates. The genetic distribution inside the V. cholerae strain collection was used to evaluate the discriminating power (Simpsons Diversity Index = 0.99) of this new MLVA analysis, showing that the assay have a potential to differentiate between various strains, but also to identify those isolates which are collected from a common V. cholerae outbreak. This work has established a rapid and highly discriminating MLVA assay useful for track back analyses and/or forensic studies of V. cholerae infections.     

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.     

Prevalence of Cholera Toxin Genes (ctxA and zot) among Non-O1/O139 Vibrio cholerae Strains from Newport Bay, California

The examination of 137 non-O1/O139 Vibrio cholerae isolates from Newport Bay, California, indicated the presence of diverse genotypes and a temporal succession. Unexpectedly, the cholera toxin gene (ctxA) was found in 17% of the strains, of which one-third were also positive for the zot gene. This suggests that ctxA is prevalent in the region of nonepidemicity and is likely to have an environmental origin.     

Multiplex polymerase chain reaction-based assay for the specific detection of toxin-producing Vibrio cholerae in fish and fishery products

A multiplex polymerase chain reaction (MPCR)-based assay was developed for the simultaneous detection of Vibrios using the genus-specific RNA polymerase subunit A (rpoA) gene and specific detection of toxin-producing Vibrio cholerae strains using two sets of primer based on cholera toxin subunit A (ctxA) and repeat in toxin subunit A (RtxA)-producing genes. The MPCR method developed is applicable to both the simultaneous and the two-step detection of genus Vibrio total and toxigenic V. cholerae species. This assay was specific as no amplification occurred with the other bacterial pathogens tested. The sensitivity of the assay was tested by artificially spiking the shrimp homogenate with the toxigenic strain of V. cholerae (NICED 16582) in different dilutions. The developed MPCR assay could detect three cells of V. cholerae in 12 h pre-enrichment in APW. The proposed method is rapid, sensitive, and specific for the detection of Vibrio genus as well as toxin-producing V. cholerae strains in environmental samples.     

Evaluation of synthetic schemes to prepare immunogenic conjugates of Vibrio cholerae O139 capsular polysaccharide with chicken serum albumin

Vibrio cholerae serotype O139 is a new etiologic agent of epidemic cholera. There is no vaccine available against cholera caused by this serotype. V. cholerae O139 is an encapsulated bacterium, and its polysaccharide capsule is an essential virulent factor and likely protective antigen.This study evaluated several synthetic schemes for preparation of conjugates of V. cholerae O139 capsular polysaccharide (CPS) with chicken serum albumin as the carrier protein (CSA) using 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) or 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as activating agents. Four conjugates described here as representative of many experiments were synthesized in 2 steps: 1) preparation of adipic acid hydrazide derivative of CPS (CPS_AH) or of CSA (CSA_AH), and 2) binding of CPS_AH to CSA or of CPS to CSA_AH. Although all conjugates induced CPS antibodies, the conjugate prepared by EDC-mediated binding of CPS and CSA_AH (EDC:CPS-CSA_AH) was statistically significantly less immunogenic than the other three conjugates. Representative sera from mice injected with these three conjugates contained antibodies that mediated the lysis of V. cholerae O139 inoculum.Evaluation of the different synthetic schemes and reaction conditions in relation to the immunogenicity of the resultant conjugates provided the basis for the preparation of a V. cholerae O139 conjugate vaccine with a medically useful carrier protein such as diphtheria toxin mutant.     

Evolutionary perspective on the origin of Haitian cholera outbreak strain

Cholera epidemic has not been reported in Haiti for at least 100?years, although cholera has been present in Latin America since 1991. Surprisingly, the recent cholera epidemic in Haiti (October 2010) recorded more than 250,000 cases and 4000 deaths in the first 6?months and became one of the most explosive and deadly cholera outbreak in recent history. In the present study, we conducted genomic analyses of pathogenicity islands of three Haitian Vibrio cholerae strains and compared them with nine different V. cholerae O1 El Tor genomes. Although CIRS101 is evolutionarily most similar to the Haitian strains, our study also provides some important differences in the genetic organization of pathogenicity islands of Haitian strains with CIRS101. Evolutionary analysis suggests that unusual functional constraints have been imposed on the Haitian strains and we hypothesize that amino acid substitution is more deleterious in Haitian strains than in nonHaitian strains.     

Synthesis of cholera toxin B subunit gene: cloning and expression of a functional 6XHis-tagged protein in Escherichia coli

Cholera toxin B subunit (CTB) has been extensively studied as immunogen, adjuvant, and oral tolerance inductor depending on the antigen conjugated or coadministered. It has been already expressed in several bacterial and yeast systems. In this study, we synthesized a versatile gene coding a 6XHis-tagged CTB (359 bp). The sequence was designed according to codon usage of Escherichia coli, Lactobacillus casei, and Salmonella typhimurium. The gene assembly was based on a polymerase chain reaction, in which the polymerase extends DNA fragments from a pool of overlapping oligonucleotides. The synthetic gene was amplified, cloned, and expressed in E. coli in an insoluble form, reaching levels about 13 mg of purified active pentameric rCTB per liter of induced culture. Western blot and ELISA analyses showed that recombinant CTB is strongly and specifically recognized by polyclonal antibodies against the cholera toxin. The ability to form the functional pentamers was observed in cell culture by the inhibition of cholera toxin activity on Y1 adrenal cells in the presence of recombinant CTB. The 6XHis-tagged CTB provides a simple way to obtain functional CTB through Ni²⁺-charged resin after refolding and also free of possible CTA contaminants as in the case of CTB obtained from Vibrio cholerae cultures.     

Characterization of Vibrio cholerae O1 isolates responsible for cholera outbreaks in Kenya between 1975 and 2017

Kenya is endemic for cholera with different waves of outbreaks having been documented since 1971. In recent years, new variants of Vibrio cholerae O1 have emerged and have replaced most of the traditional El Tor biotype globally. These strains also appear to have increased virulence, and it is important to describe and document their phenotypic and genotypic traits. This study characterized 146 V. cholerae O1 isolates from cholera outbreaks that occurred in Kenya between 1975 and 2017. Our study reports that the 1975–1984 strains had typical classical or El Tor biotype characters. New variants of V. cholerae O1 having traits of both classical and El Tor biotypes were observed from 2007 with all strains isolated between 2015 and 2017 being sensitive to polymyxin B and carrying both classical and El Tor type ctxB. All strains were resistant to Phage IV and harbored rstR, rtxC, hlyA, rtxA and tcpA genes specific for El Tor biotype indicating that the strains had an El Tor backbone. Pulsed field gel electrophoresis (PFGE) genotyping differentiated the isolates into 14 pulsotypes. The clustering also corresponded with the year of isolation signifying that the cholera outbreaks occurred as separate waves of different genetic fingerprints exhibiting different genotypic and phenotypic characteristics. The emergence and prevalence of V. cholerae O1 strains carrying El Tor type and classical type ctxB in Kenya are reported. These strains have replaced the typical El Tor biotype in Kenya and are potentially more virulent and easily transmitted within the population.