Clonal relationship among Vibrio cholerae O1 El Tor strains isolated in Somalia

One hundred and three Vibrio cholerae O1 strains, selected to represent the cholera outbreaks which occurred in Somalia in 1998-1999, were characterized by random amplified polymorphic DNA patterns, ribotyping, and antimicrobial susceptibility. All strains showed a unique amplified DNA pattern and 2 closely related ribotypes (B5a and B8a), among which B5a was the more frequently identified. Ninety-one strains were resistant to ampicillin, chloramphenicol, spectinomycin, streptomycin, sulfamethoxazole, and trimethoprim, conferred, except for spectinomycin, by a conjugative plasmid IncC. These findings indicated that the group of strains active in Somalia in the late 1990s had a clonal origin.     

Purification and characterization of a hemolysin produced by Vibrio cholerae biotype El Tor: another toxic substance produced by cholera vibrios.

A thermolabile direct hemolysin from an El Tor cholera vibrio strain has been isolated and partially characterized as a simple protein of ca. 20,000 molecular weight. In addition to its hemolytic activity, the hemolysin is cytotoxic, cardiotoxic, and rapidly lethal. In these respects it resembles the thermostable direct hemolysin/cytotoxin/cardiotoxin/lethal toxin of Vibrio parahaemolyticus and certain other bacterial hemolysins, although there are other significant differences. Because identical diseases are produced by both hemolytic and nonhemolytic cholera vibrios, the El Tor hemolysin may be presumed to be pathogenetically irrelevant. These observations raise the question of "When is a toxic substance also a toxin?"     

Genomic variability of vibrio cholerae El Tor biovariant strains

The authors performed comparative analysis of the genomes of 145 clinical and environmental isolates of Vibrio cholerae El Tor biovariants using single locus and multiplex PCR. The study found that clinical strains isolated from patients with cholera formed a genetically homogenous group, where bacterial chromosome contained all the tested virulence genes, situated on mobile genetic elements that had been acquired by the pathogen at various stages of its evolution. Strains isolated from water ecosystems during interepidemic period were heterogeneous and formed three groups: a small number of virulent strains; non-toxigenic vibrio strains that, in the process of reductional variation in their new econiche, had only managed to maintain individual virulence genes; non-pathogenic "water" vibrios, whose chromosome contained only the genes from its core part, mobile genetic elements being optionally represented only by the persistence island. Molecular typing established genetic relations among V. cholerae strains under study.     

Cholera Toxin Production by the El Tor Variant of Vibrio cholerae O1 Compared to Prototype El Tor and Classical Biotypes

Vibrio cholerae O1 El Tor variant strains produced much more cholera toxin than did prototype El Tor strains. The amount of cholera toxin produced by El Tor variant strains both in vitro and in vivo was more or less equivalent to that produced by classical strains.Vibrio cholerae O1 is classified into classical and El Tor biotypes. Among other genetic, biochemical, and physiological differences, each biotype has unique gene sequences encoding cholera toxin B subunit (CTB), that is, classical ctxB and El Tor ctxB. Besides these two prototype biotypes of V. cholerae O1, Nair et al. (9) in 2002 in Bangladesh isolated strains that possess phenotypic properties of both classical and El Tor biotypes carrying classical ctxB. The same group also isolated El Tor strains that had classical ctxB (10). For these new types of strains of V. cholerae O1, we have recently proposed the designations of hybrid and El Tor variants, respectively (13). Subsequent to the isolation of the El Tor variant in Bangladesh by Nair et al. (10), El Tor variant strains were isolated from several countries and areas in Asia and Africa (1, 11, 15-18). In Kolkata, India, we showed that El Tor variant strains appeared in 1990 and that a complete replacement of prototype El Tor strains by El Tor variant strains has occurred since 1995 (14).In this study, we investigated the amount of cholera toxin (CT) produced both in vitro and in vivo by V. cholerae O1 El Tor variant strains isolated in Kolkata during a period from 1996 to 2007. It was found that El Tor variant strains produced a much larger amount of CT than did prototype El Tor strains and that the amount of CT produced by El Tor variant strains was more or less equivalent to that produced by classical strains.V. cholerae O1 strains used in this study are listed in Table ​Table1.1. AKI (3) and Syncase medium (2) were used for culturing the test strains. The rationale for selecting these media was that AKI preferentially supports the production of El Tor CT (3) while Syncase medium is reported to be the best medium supporting the production of CT by the classical biotype (2). Measurement of CT concentration produced by V. cholerae O1 strains was carried out as follows. Each strain was cultured either in AKI medium at 37°C for 20 h without shaking or in Syncase medium at 37°C for 20 h with shaking, and the optical density of the culture was measured at 600 nm (OD₆₀₀). After centrifugation, the supernatants were collected and the concentration of CT (ng/ml/OD₆₀₀) in the samples was measured by bead enzyme-linked immunosorbent assay (ELISA). The method of the bead ELISA employed was essentially that described by Oku et al. (12). In brief, a polystyrene bead (6.5 mm in diameter) was coated with anti-CT IgG and used as a solid phase. The coated bead was first incubated with the sample and then incubated with anti-CT IgG [F(ab′)]-horseradish peroxidase conjugate. Peroxidase activity was determined colorimetrically with 3,3′,5,5′-tetramethylbenzidine as the substrate. The absorbance at 450 nm (OD₄₅₀) was linear between 0 and 0.5, representing CT concentrations of 0 to 20 ng/ml. The sample prepared as described above (the supernatant of the culture of the strain) was appropriately diluted so that the OD₄₅₀ fell in the range of 0.1 to 0.5, and the amount of CT produced by the strain was expressed as ng/ml/OD₆₀₀.

TABLE 1.

V. cholerae O1 strains used

Identification of a mannose-binding pilus on Vibrio cholerae El Tor.

The mannose-sensitive hemagglutinin (MSHA) that is associated with Vibrio cholerae strains of El Tor biotype is identified as a pilus composed of subunits with a molecular mass of approximately 17 kDa. In immunoelectron microscopy, a monoclonal antibody against MSHA that inhibited El Tor vibrio-mediated mannose-sensitive agglutination of chicken erythrocytes or El Tor bacterial binding to mannose-coated agarose beads, bound specifically to repetitive subunits along typical fimbriae extending from the surface of El Tor vibrios. No such pili were seen on the surface of MSHA negative classical vibrios, although non-surface exposed fimbrial subunits could be demonstrated in these bacteria by immunoblotting techniques.     

Cholera between 1991 and 1997 in Mexico Was Associated with Infection by Classical,El Tor,and El Tor Variants of Vibrio cholerae

Vibrio cholerae O1 biotype El Tor (ET), the cause of the current 7th pandemic, has recently been replaced in Asia and Africa by an altered ET biotype possessing cholera toxin (CTX) of the classical (CL) biotype that originally caused the first six pandemics before becoming extinct in the 1980s. Until recently, the ET prototype was the biotype circulating in Peru; a detailed understanding of the evolutionary trend of V. cholerae causing endemic cholera in Latin America is lacking. The present retrospective microbiological, molecular, and phylogenetic study of V. cholerae isolates recovered in Mexico (n = 91; 1983 to 1997) shows the existence of the pre-1991 CL biotype and the ET and CL biotypes together with the altered ET biotype in both epidemic and endemic cholera between 1991 and 1997. According to sero- and biotyping data, the altered ET, which has shown predominance in Mexico since 1991, emerged locally from ET and CL progenitors that were found coexisting until 1997. In Latin America, ET and CL variants shared a variable number of phenotypic markers, while the altered ET strains had genes encoding the CL CTX (CTX^CL) prophage, ctxB^CL and rstR^CL, in addition to resident rstR^ET, as the underlying regional signature. The distinct regional fingerprints for ET in Mexico and Peru and their divergence from ET in Asia and Africa, as confirmed by subclustering patterns in a pulsed-field gel electrophoresis (NotI)-based dendrogram, suggest that the Mexico epidemic in 1991 may have been a local event and not an extension of the epidemics occurring in Asia and South America. Finally, the CL biotype reservoir in Mexico is unprecedented and must have contributed to the changing epidemiology of global cholera in ways that need to be understood.In 1991, when cholera reemerged after being absent from Latin America for about a century, millions of people were affected, with nearly 9,000 dying in 1993 alone (13). Following its first appearance along the coast of Peru in January 1991, cholera rapidly spread to all countries in Latin America except Uruguay, reaching Mexico in June of the same year (13, 19, 25, 32). Since then, there has been a great interest in understanding the source and transmission of cholera in Latin America. Limited genetic analysis of Vibrio cholerae O1 biotype El Tor (ET) strains identified from the epidemic showed that they have a unique signature, distinguishing them from 7th pandemic ET strains (25). However, later studies showed the clonal nature of the bacterium, suggesting that the Latin American epidemic was an extension of the 7th pandemic caused by ET strains from the Western Hemisphere (34).V. cholerae O1 and O139 are the two serogroups known to cause cholera. V. cholerae O1 has two biotypes, classical (CL) and ET, which differ in specific phenotypic traits (hemolysis of sheep erythrocytes, agglutination of chicken erythrocytes, sensitivity to polymyxin B [PMB], phage susceptibilities, and Voges-Proskauer [VP] test results) (15) and genotypic traits (ctxB, acfB, tcpA, and rstR). In addition, the CL and ET biotype strains differ in two major genomic regions, namely, the Vibrio seventh pandemic pathogenicity island I (VSP-I) and VSP-II, that are unique to the 7th pandemic ET biotype (10). Biotype CL caused the first six of the seven cholera pandemics recorded between 1817 and 1923 (26), with five of these affecting the American continents. Ever since the first cases were detected in the Americas in the 1830s, endemic cholera continued to be prevalent until 1895 (18).Historically, cholera has been endemic in Asia for centuries (26), with Asia being at the center of each of the seven cholera pandemics (10, 26). Although the ET biotype was first reported in 1905 and the 6th pandemic, caused by the CL biotype, lasted until 1923, it was not until the early 1960s that the ET biotype displaced the CL biotype in Asia and became the causal agent of the 7th pandemic (33). The CL biotype maintained a low profile in its last recognized niche in the coastal ecosystem of the Bay of Bengal, before it disappeared as a causal agent of clinical disease in the 1980s (33).Over the past few years, the ET biotype causing Asiatic cholera has shown remarkable changes in its phenotypic and genetic characteristics (23). Recent molecular analysis of ET strains causing acute watery diarrhea in Bangladesh shows them to be hybrids because they possess phenotypic and genotypic traits of the CL biotype against an ET background (23). Subsequent retrospective studies showed that all of the O1 ET strains isolated in Bangladesh since 2001 have been hybrids of both the CL and ET biotypes, while those isolated before 2001 contained all the attributes of the 7th pandemic V. cholerae O1 ET strains (22). Such genetic changes among ET strains causing cholera in Latin America were also evident from the changing serotypes, electrophoretic types, ribotypes, and pulsed-field gel electrophoresis (PFGE) types (3, 9, 11, 27). While the ET prototype has been completely replaced by an altered ET in Asia and Africa (29), recent data show the nature of the ET prototype (7th pandemic) of V. cholerae O1 isolated in Peru between 1991 and 2003 (24). The Peruvian ET strains that seem to be closely linked clonally to the Asian and African ET prototype strains were shown to have a distinct region in VSP-II that differentiates them from the ET prototype strains isolated in other continents (24). To better understand the dynamics of the cholera epidemic in Latin America, the present study analyzed 91 V. cholerae O1 strains isolated from both clinical and environmental sources in Mexico between 1983 and 1997. V. cholerae O1 ET strains from Peru (1991 to 1999), Bangladesh (1985 to 2007), and Zambia (1996 to 2004) were also included for comparative purposes.     

Characterization of Vibrio cholerae El Tor cytolysin as an oligomerizing pore-forming toxin

V. cholerae El Tor cytolysin is a secreted, water-soluble protein of M _r 60,000 that may be relevant to the pathogenesis of acute diarrhea. In this communication, we demonstrate that the toxin binds to and oligomerizes in target membranes to form SDS-stable aggregates of M _r 200000–250000 that generate small transmembrane pores. Pores formed in erythrocytes were approximately 0.7 nm in size, as demonstrated by osmotic protection experiments. Binding was shown to occur in a temperature-independent manner preceding the temperature-dependent oligomerization step. Pores were also shown to be formed in L929 and HEp-2 cells, human fibroblasts and keratinocytes, albeit with highly varying efficacy. At neutral pH and in the presence of serum, human fibroblasts were able to repair a limited number of lesions. The collective data identify V. cholerae El Tor cytolysin as an oligomerizing toxin that damages cells by creating small transmembrane pores.     

Identification of a mannose-binding pilus on Vibrio cholerae El Tor

The mannose-sensitive hemagglutinin (MSHA) that is associated with Vibrio cholerae strains of El Tor biotype is identified as a pilus composed of subunits with a molecular mass of approximately 17 kDa. In immunoelectron microscopy, a monoclonal antibody against MSHA that inhibited El Tor vibrio-mediated mannose-sensitive agglutination of chicken erythrocytes or El Tor bacterial binding to mannose-coated agarose beads, bound specifically to repetitive subunits along typical fimbriae extending from the surface of El Tor vibrios. No such pili were seen on the surface of MSHA negative classical vibrios, although non-surface exposed fimbrial subunits could be demonstrated in these bacteria by immunoblotting techniques.     

Determination of relationships among non-toxigenic Vibrio cholerae O1 biotype El Tor strains from housekeeping gene sequences and ribotype patterns

Sequencing of three housekeeping genes, mdh, dnaE and recA, and ribotyping for seven non-toxigenic Vibrio cholerae O1 strains isolated from different geographic sources indicate a phylogenetic relationship among the strains. Results of MLST and ribotyping indicate a clear difference between three toxigenic strains (N16961, O395, and 569B) and three non-toxigenic strains from India (GS1, GS2, and GW87) and one Guam strain (X392), the latter of which were similar in both MLST and ribotyping, while two other non-toxigenic strains from the USA and India (2740-80 and OR69) appeared to be more closely related to toxigenic strains than to non-toxigenic strains, although this was not supported by ribotyping. These results provide clues to the emergence of toxigenic strains from a non-toxigenic progenitor by acquisition of virulence gene clusters. Results of split decomposition analysis suggest that widespread recombination occurs among the three housekeeping genes and that recombination plays an important role in the emergence of toxigenic strains of V. cholerae O1.     

2,3-butanediol synthesis and the emergence of the Vibrio cholerae El Tor biotype

Vibrio cholerae is an aquatic bacterium that causes the severe diarrheal disease cholera. V. cholerae strains of the O1 serogroup exist as two biotypes, classical and El Tor. Toxigenic strains of the El Tor biotype emerged to cause the seventh pandemic of cholera in 1961 and subsequently displaced strains of the classical biotype both in the environment and as a cause of cholera within a decade. The factors that drove emergence of the El Tor biotype and the displacement of the classical biotype are unknown. Here, we show a unique difference in carbohydrate metabolism between these two biotypes. When grown with added carbohydrates, classical biotype strains generated a sharp decrease in medium pH, resulting in loss of viability. However, growth of El Tor biotype strain N16961 was enhanced due to its ability to produce 2,3-butanediol, a neutral fermentation end product, and suppress the accumulation of organic acids. An N16961 mutant (SSY01) defective in 2,3-butanediol synthesis showed the same defect in growth that classical biotype strains show in media rich in carbohydrates. Importantly, the SSY01 mutant was attenuated in its ability to colonize the intestines of infant mice, suggesting that host carbohydrates may be available to V. cholerae within the intestinal environment. Similarly, the SSY01 mutant failed to develop biofilms when utilizing N-acetyl-D-glucosamine as a carbon source. Because growth on N-acetyl-D-glucosamine likely reflects the ability of a strain to grow on chitin in certain aquatic environments, we conclude that the strains of classical biotype are likely defective compared to those of El Tor in growth in any environmental niche that is rich in chitin and/or other metabolizable carbohydrates. We propose that the ability to metabolize sugars without production of acid by-products might account for the improved evolutionary fitness of the V. cholerae El Tor biotype compared to that of the classical biotype both as a global cause of cholera and as an environmental organism.     

Mechanism of membrane damage by El Tor hemolysin of Vibrio cholerae O1.

El Tor hemolysin (ETH; molecular mass, 65 kDa) derived from Vibrio cholerae O1 spontaneously assembled oligomeric aggregates on the membranes of rabbit erythrocyte ghosts and liposomes. Membrane-associated oligomers were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting into two to nine bands with apparent molecular masses of 170 to 350 kDa. ETH assembled oligomers on a liposomal membrane consisting of phosphatidylcholine and cholesterol, but not on a membrane of phosphatidylcholine alone. Cholesterol could be replaced with diosgenin or ergosterol but not with 5alpha-cholestane-3-one, suggesting that sterol is essential for the oligomerization. The treatment of carboxyfluorescein-encapsulated liposomes with ETH caused a rapid release of carboxyfluorescein into the medium. Because dextrin 20 (molecular mass, 900 Da) osmotically protected ETH-mediated hemolysis, this hemolysis is likely to be caused by pore formation on the membrane. The pore size(s) estimated from osmotic protection assays was in the range of 1.2 to 1.6 nm. The pore formed on a rabbit erythrocyte membrane was confirmed morphologically by electron microscopy. Thus, we provide evidence that ETH damages the target by the assembly of hemolysin oligomers and pore formation on the membrane.     

Cholera due to altered El Tor strains of Vibrio cholerae O1 in Bangladesh

We determined the types of cholera toxin (CT) produced by a collection of 185 Vibrio cholerae O1 strains isolated in Bangladesh over the past 45 years. All of the El Tor strains of V. cholerae O1 isolated since 2001 produced CT of the classical biotype, while those isolated before 2001 produced CT of the El Tor biotype.     

Clonal relationship among Vibrio cholerae O1 El Tor strains causing the largest cholera epidemic in Kenya in the late 1990s

Eighty Vibrio cholerae O1 strains selected to represent the 1998-to-1999 history of the largest cholera epidemic in Kenya were characterized by ribotyping, antimicrobial susceptibility, and random amplified polymorphic DNA patterns. Except for 19 strains from 4 local outbreaks in North Eastern Province along the Somalia border, the other 61 strains from 25 outbreaks occurring in districts scattered around the country were all ribotype B27 and resistant to chloramphenicol, spectinomycin, streptomycin, sulfamethoxazole, and trimethoprim. The 61 strains showed similar and specific amplified DNA patterns. These findings indicate that the predominant strains that caused the Kenyan epidemic had a clonal origin and suggest that ribotype B27 strains, which first appeared in West Africa in 1994, have had a rapid spread to eastern Africa.     

The role of toxin-coregulated pili in the pathogenesis of Vibrio cholerae O1 El Tor

Studies in the infant mouse cholera model have evaluated the significance of toxin-coregulated pili (TCP) in the pathogenesis of Vibrio cholerae strains of El Tor biotype. Four El Tor strains—two which produce TCP during in vitro growth and two which do not—were mutated by the insertion of an antibiotic-resistance cartridge into the tcpA gene (encoding the pilin monomer). The resulting mutants were otherwise indistinguishable from wild-type and in particular were unaltered in their sensitivity to antibody-dependent, complement-mediated bacteriolysis. All were dramatically attenuated and showed a marked impairment in terms of in vivo persistence in mixed-infection competition experiments. Virulence was restored by provision of a functional tcp operon in trans, confirming that the pathogenic potential of El Tor strains is critically dependent upon product(s) of this operon.     

Construction and characterization of a nonproliferative El Tor cholera vaccine candidate derived from strain 638

In recent clinical assays, our cholera vaccine candidate strain, Vibrio cholerae 638 El Tor Ogawa, was well tolerated and immunogenic in Cuban volunteers. In this work we describe the construction of 638T, a thymidine auxotrophic version of improved environmental biosafety. In so doing, the thyA gene from V. cholerae was cloned, sequenced, mutated in vitro, and used to replace the wild-type allele. Except for its dependence on thymidine for growth in minimal medium, 638T is essentially indistinguishable from 638 in the rate of growth and morphology in complete medium. The two strains showed equivalent phenotypes with regard to motility, expression of the celA marker, colonization capacity in the infant mouse cholera model, and immunogenicity in the adult rabbit cholera model. However, the ability of this new strain to survive environmental starvation was limited with respect to that of 638. Taken together, these results suggest that this live, attenuated, but nonproliferative strain is a new, promising cholera vaccine candidate.     

Genetic mapping of the tox-1000 locus of Vibrio cholerae El Tor strain RJ1

The results of a genetic cross between a Vibrio cholerae RJ1 donor and a V. cholerae 3083-2 recipient suggest that the map position of tox-1000 is between his and trp.