Characterization of Vibrio cholerae O1 El Tor biotype variant clinical isolates from Bangladesh and Haiti, including a molecular genetic analysis of virulence genes

Vibrio cholerae serogroup O1, the causative agent of the diarrheal disease cholera, is divided into two biotypes: classical and El Tor. Both biotypes produce the major virulence factors toxin-coregulated pilus (TCP) and cholera toxin (CT). Although possessing genotypic and phenotypic differences, El Tor biotype strains displaying classical biotype traits have been reported and subsequently were dubbed El Tor variants. Of particular interest are reports of El Tor variants that produce various levels of CT, including levels typical of classical biotype strains. Here, we report the characterization of 10 clinical isolates from the International Centre for Diarrhoeal Disease Research, Bangladesh, and a representative strain from the 2010 Haiti cholera outbreak. We observed that all 11 strains produced increased CT (2- to 10-fold) compared to that of wild-type El Tor strains under in vitro inducing conditions, but they possessed various TcpA and ToxT expression profiles. Particularly, El Tor variant MQ1795, which produced the highest level of CT and very high levels of TcpA and ToxT, demonstrated hypervirulence compared to the virulence of El Tor wild-type strains in the infant mouse cholera model. Additional genotypic and phenotypic tests were conducted to characterize the variants, including an assessment of biotype-distinguishing characteristics. Notably, the sequencing of ctxB in some El Tor variants revealed two copies of classical ctxB, one per chromosome, contrary to previous reports that located ctxAB only on the large chromosome of El Tor biotype strains.     

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.     

Vibrio cholerae classical biotype strains reveal distinct signatures in Mexico

Vibrio cholerae O1 classical (CL) biotype caused the fifth and sixth pandemics, and probably the earlier cholera pandemics, before the El Tor (ET) biotype initiated the seventh pandemic in Asia in the 1970s by completely displacing the CL biotype. Although the CL biotype was thought to be extinct in Asia and although it had never been reported from Latin America, V. cholerae CL and ET biotypes, including a hybrid ET, were found associated with areas of cholera endemicity in Mexico between 1991 and 1997. In this study, CL biotype strains isolated from areas of cholera endemicity in Mexico between 1983 and 1997 were characterized in terms of major phenotypic and genetic traits and compared with CL biotype strains isolated in Bangladesh between 1962 and 1989. According to sero- and biotyping data, all V. cholerae strains tested had the major phenotypic and genotypic characteristics specific for the CL biotype. Antibiograms revealed the majority of the Bangladeshi strains to be resistant to trimethoprim-sulfamethoxazole, furazolidone, ampicillin, and gentamicin, while the Mexican strains were sensitive to all of these drugs, as well as to ciprofloxacin, erythromycin, and tetracycline. Pulsed-field gel electrophoresis (PFGE) of NotI-digested genomic DNA revealed characteristic banding patterns for all of the CL biotype strains although the Mexican strains differed from the Bangladeshi strains in 1 to 2 DNA bands. The difference was subtle but consistent, as confirmed by the subclustering patterns in the PFGE-based dendrogram, and can serve as a regional signature, suggesting the pre-1991 existence and evolution of the CL biotype strains in the Americas, independent from Asia.     

Emergence of a new clone of toxigenic Vibrio cholerae O1 biotype El Tor displacing V. cholerae O139 Bengal in Bangladesh.

The emergence of Vibrio cholerae O139 Bengal in 1993, its rapid spread in an epidemic form, in which it replaced existing strains of V. cholerae O1 during 1992 and 1993, and the subsequent reemergence of V. cholerae O1 of the El Tor biotype in Bangladesh since 1994 have raised questions regarding the origin of the reemerged El Tor vibrios. We studied 50 El Tor vibrio strains isolated in Bangladesh and four other countries in Asia and Africa before the emergence of V. cholerae O139 and 32 strains isolated in Bangladesh during and after the epidemic caused by V. cholerae O139 and 32 strains isolated in Bangladesh during and after the epidemic caused by V. cholerae O139 to determine whether the reemerged El Tor vibrios were genetically different from the El Tor vibrios which existed before the emergence of V. cholerae O139. Analysis of restriction fragment length polymorphisms in genes for conserved rRNA, cholera toxin (ctxA), and zonula occludens toxin (zot) or in DNA sequences flanking the genes showed that the El Tor strains isolated before the emergence of V. cholerae O139 belonged to four different ribotypes and four different ctx genotypes. Of 32 El Tor strains isolated after the emergence of O139 vibrios, 30 strains (93.7%) including all the clinical isolates belonged to a single new ribotype and a distinctly different ctx genotype. These results provide evidence that the reemerged El Tor strains represent a new clone of El Tor vibrios distinctly different from the earlier clones of El Tor vibrios which were replaced by the O139 vibrios. Further analysis showed that all the strains carried the structural and regulatory genes for toxin-coregulated pilus (tcpA, tcpI, and toxR). All strains of the new clone produced cholera toxin (CT) in vitro, as assayed by the GM1-dependent enzyme-linked immunosorbent assay, and the level of CT production was comparable to that of previous epidemic isolates of El Tor vibrios. Further studies are required to assess the epidemic potential of the newly emerged clone of V. cholerae O1 and to understand the mechanism of emergence of new clones of toxigenic V. cholerae.     

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.     

Analysis of expression of toxin-coregulated pili in classical and El Tor Vibrio cholerae O1 in vitro and in vivo.

The expression of toxin-coregulated pili (TCP) and their structural subunit TcpA was compared in 20 strains of Vibrio cholerae of the classical and El Tor biotypes. Bacteria were isolated from the intestines of rabbits with experimental cholera and compared with the same strains grown under optimal TCP expression conditions in vitro. Immunoblotting revealed that TcpA production was induced in both biotypes after vibrios entered the intestinal milieu; TcpA-negative inocula gave rise to TcpA-positive vibrios after multiplication in the gut. The levels of TcpA expressed during growth in the intestine were, for most strains, comparable to those attained under optimal growth conditions in vitro. Of 11 classical strains tested, 10 expressed TCP antigen on the bacterial surface at levels comparable to or exceeding those seen after growth in vitro as determined by an inhibition enzyme-linked immunosorbent assay. In contrast, only one of the nine El Tor strains studied produced detectable amounts of TCP surface antigen in vivo and no fimbriae or surface antigen reacting with anti-TCP serum was found on El Tor vibrios from human cholera stools. Distinct TCP fimbriae were observed by immunoelectron microscopy on classical-biotype vibrios grown either in rabbit intestines or in vitro but were not detected on El Tor vibrios. The results show that TCP is expressed on V. cholerae O1 of the classical biotype but not on V. cholerae O1 of the El Tor biotype in the intestines of rabbits with experimental cholera infection.     

Clonal diversity of Vibrio cholerae O1 evidenced by rRNA gene restriction patterns

The rRNA gene restriction patterns of 89 Vibrio cholerae O1 isolates from different geographic origins were studied. The probe was Escherichia coli 16 + 23S rRNA labelled with "ECL Gene detection system". A total of 17 rRNA gene restriction patterns were observed after BglI cleavage. Four patterns (B1 to B4) were only given by biotype cholerae (14 strains studied). Thirteen patterns (B5 to B17) were only given by biotype El Tor (75 strains studied). There was no correlation between serotypes and rRNA gene restriction patterns. This study provides arguments that (1) strains of biotypes cholerae and El Tor are different clones, (2) a cholera pandemic is not a single world-wide epidemic (due to a single clone) but rather a simultaneous occurrence of several epidemics (several clones involved), and (3) epidemic waves of biotype El Tor could be due to the emergence of new clones.     

Genomic relatedness of the new Matlab variants of Vibrio cholerae O1 to the classical and El Tor biotypes as determined by pulsed-field gel electrophoresis

The genomes of the recently described Matlab variants of Vibrio cholerae O1 that are hybrids between classical and El Tor biotypes were compared with those of El Tor and classical biotypes by the use of pulsed-field gel electrophoresis. Dendrograms constructed using the unweighted-pair group method using average linkages generated from NotI restriction patterns of whole-chromosomal DNA grouped these strains into two major clusters that were found to be similar but not identical to those of either of the biotypes. Strains that clustered with the classical biotype appear to have been derived from the classical strains, which are thought to be extinct.     

Molecular analysis of toxigenic Vibrio cholerae O139 Bengal strains isolated in Bangladesh between 1993 and 1996: evidence for emergence of a new clone of the Bengal vibrios.

Vibrio cholerae O139 Bengal emerged in 1992 and rapidly spread in an epidemic form, in which it replaced existing strains of V. cholerae O1 in Bangladesh during 1992 and 1993. The subsequent emergence of a new clone of V. cholerae O1 of the El Tor biotype that transiently displaced the O139 vibrios during 1994 to 1995 and the recent reemergence of V. cholerae O139 and its coexistence with the El Tor vibrios demonstrated temporal changes in the epidemiology of cholera in Bangladesh. We studied clonal diversity among V. cholerae O139 strains isolated from cholera patients and environmental surface water since their first appearance until their transient disappearance in 1994 as well as the O139 strains that reemerged during 1995 to 1996 and were isolated in the capital Dhaka and four rural districts of Bangladesh to investigate the origin of the reemerged strains. Analysis of restriction fragment length polymorphisms in genes for conserved rRNA and cholera toxin (CT) (ctxA) or in DNA sequences flanking these genes revealed four different ribotypes and four different ctx genotypes among the 93 strains of V. cholerae O139 studied. Ribotypes I and II and ctx genotypes A through C were shared by strains isolated from the epidemic outbreak during 1992 and 1993 in Bangladesh and India, ribotype III was represented by a single CT-negative O139 strain from Argentina, and 16 of 27 (59.2%) of the reemerged strains isolated during 1995 and 1996 belonged to a new ribotype of O139 vibrios designated ribotype IV. All 16 strains belonging to ribotype IV also belonged to a new ctx genotype (genotype 4). These results provide evidence for the emergence of a new clone of toxigenic V. cholerae O139 in Bangladesh. Further analysis of the rfb gene cluster by PCR revealed the absence of a large region of the O1-specific rfb operon and the presence of an O139-specific genomic region in all O139 strains. The PCR amplicon corresponding to the rfaD gene of a CT-negative O139 strain from Argentina was smaller in length than those of the toxigenic O139 strains but was identical to those of seven non-O1 and non-O139 strains. All O139 strains except the CT-negative strain carried structural and regulatory genes for CT and toxin-coregulated pili (ctxA, tcpA, tcpI, and toxR). These results suggest that the O139 Bengal strains possibly emerged from an El Tor strain but that the CT-negative non-Bengal O139 strain might have emerged from a non-O1, non-O139 strain. Thus, strains belonging to the O139 serogroup may have emerged from similar serotype-specific genetic changes in more than one progenitor strain of V. cholerae.     

Strong biotype and serotype cross-protective antibacterial and antitoxic immunity in rabbits after cholera infection

We studied whether immunity evoked by infection with classical or El Tor V. cholerae 01 organisms in rabbits (the RITARD model) also gives protection against cholera caused by V. cholerae of heterologous biotype as well as serotype, and whether such protection is antibacterial and/or antitoxic. A primary infection with a classical Ogawa or El Tor Inaba strain resulted in intestinal colonization and diarrheal disease in a dose-related manner though the El Tor strain was more virulent. As few as 10(3) El Tor organisms gave disease in more than 90% of the rabbits as compared to 10(9) classical organisms (ED90); the El Tor strain also gave rise to diarrhea with earlier onset and of greater severity and longer duration. The primary infection induced strong protective immunity against later challenge with either the homologous or the heterologous strain in doses that corresponded to 1,000 x ED90. Protection was associated with marked inhibition of colonization, and when rabbits convalescing from cholera infection were challenged with graded doses of bacteria or purified toxin in ligated intestinal loops significant antibacterial as well as antitoxic immunity was evident. Titer rises in serum vibriocidal and anti-lipopolysaccharide antibodies were similar after infection with either strain, whilst antitoxin titer rises were more marked after El Tor infection. During infection V. cholerae 01 organisms seem to express protective antigens that stimulate immunity which extends across both biotype and serotype barriers.     

Comparison of antibiogram, virulence genes, ribotypes and DNA fingerprints of Vibrio cholerae of matching serogroups isolated from hospitalised diarrhoea cases and from the environment during 1997-1998 in Calcutta, India

This study identified 17 matching serogroups of Vibrio cholerae belonging to serogroups other than O1 and O139 isolated from human cases and from the environment during a concurrent clinical and environmental study conducted in Calcutta, a cholera endemic area. Isolates within these matching serogroups were compared by various phenotypic and genotypic traits to determine if the environment was the source of the organisms associated with the disease. Clinical strains of V. cholerae were resistant to a greater number of drugs and exhibited multi-drug resistance compared with their environmental counterparts. Except for the presence of the genes for the El Tor haemolysin and the regulatory element ToxR in most of the strains of V. cholerae examined, non-O1, non-O139 V. cholerae strains lacked most of the other known virulence traits associated with toxigenic V. cholerae O1 or O139. Restriction fragment-length polymorphism of virulence-associated genes, ribotypes and DNA fingerprints of strains of matched serogroups showed considerable diversity, although some gene polymorphisms and ribotypes of a few strains of different serogroups were similar. It is concluded that despite sharing the same serogroup, environmental and clinical isolates were genetically heterogeneous and were of different lineages.     

In vitro and in vivo cholera toxin production by classical and El Tor isolates of Vibrio cholerae

A comparative study was carried out on the in vitro production of cholera toxin by 19 Vibrio cholerae El Tor isolates from patients with cholera in South Africa, one El Tor isolate from a patient in Malawi (a country approximately 1000 km north-northeast of South Africa), 6 El Tor and 12 classical type isolates from patients in Bangladesh, and 5 culture collection classical strains. Identical phage types and indistinguishable toxigenicities among the South African and Malawi V. cholerae, representing isolations obtained over a 10-year period, indicated that essentially a single strain was involved in the cholera of these regions. Similarly, phage typing and toxin profiles indicated that the 12 classical and 6 El Tor V. cholerae cultures in Bangladesh, all isolated in November 1983, represented just two strains. As assessed by titrations in Y-1 mouse adrenal and Chinese hamster ovary cell lines, the general order of toxigenicities was Bangladesh and culture collection classical greater than Bangladesh El Tor greater than southern African El Tor. The African isolates consistently gave rise to very low titers. Their relative reluctance to produce the toxin in vitro compared with the culture collection classical strains, particularly strain 569B, was confirmed by rocket electrophoresis. In somewhat of a contrast, maximum in vivo titers in rice water stools from cholera patients in South Africa and from both classical and El Tor type cholera patients in Bangladesh were essentially equal. It is postulated that under the continuous culture conditions that occur in vivo, cholera toxin concentrations can accumulate to a maximum level, depending on the rate of purging by the diarrheal fluid rather than the toxigenicity of the infecting stain. The relevance of these findings to the relative severities of classical and El Tor types of cholera is discussed.     

Emergence of Vibrio cholerae O1 biotype El Tor serotype Inaba from the prevailing O1 Ogawa serotype strains in India

The toxigenic Inaba serotype of Vibrio cholerae O1 biotype El Tor reappeared in India in 1998 and 1999, almost 10 years after its last dominance in Calcutta in 1989. Extensive molecular characterization by ribotyping, restriction fragment length polymorphism, and pulsed-field gel electrophoresis indicated that recent Inaba strains are remarkably different from the earlier Inaba strains but are very similar to the prevailing V. cholerae O1 Ogawa El Tor biotype strains. The antibiograms of the Inaba strains were also similar to those of the recent V. cholerae Ogawa strains. These V. cholerae O1 Inaba strains appear to have evolved from the currently prevailing Ogawa strains and are likely to dominate in the coming years.     

Evolutionary genetic analysis of the emergence of epidemic Vibrio cholerae isolates on the basis of comparative nucleotide sequence analysis and multilocus virulence gene profiles

Vibrio cholerae, the causative agent of cholera, is a natural inhabitant of the aquatic ecosystem. We examined a unique collection of V. cholerae clinical and environmental isolates of widespread geographic distribution recovered over a 60-year period to determine their evolutionary genetic relationships based on analysis of two housekeeping genes, malate dehydrogenase (mdh) and a chaperonin (groEL). In addition, the phylogenetic distribution of 12 regions associated with virulence was determined. Comparative sequence analysis of mdh revealed that all V. cholerae O1 and O139 serogroup isolates belonged to the same clonal lineage. Single-strand conformational polymorphism (SSCP) analysis of these O1 and O139 strains at groEL confirmed the presence of an epidemic clonal complex. Of the 12 virulence regions examined, only three regions, Vibrio seventh pandemic island 1 (VSP-I), VSP-II, and RS1, were absent from all classical V. cholerae isolates. Most V. cholerae El Tor biotype and O139 serogroup isolates examined encoded all 12 virulence regions assayed. Outside of V. cholerae O1/O139 serogroup isolates, only one strain, VO7, contained VSP-I. Two V. cholerae El Tor isolates, GP155 and 2164-78, lacked both VSP-I and VSP-II, and one El Tor isolate, GP43, lacked VSP-II. Five non-O1/non-O139 serogroup isolates had an mdh sequence identical to that of the epidemic O1 and O139 strains. These isolates, similar to classical strains, lack both VSP-I and VSP-II. Four of the 12 virulence regions examined were found to be present in all isolates: hlyA, pilE, MSHA and RTX. Among non-O1/non-O139 isolates, however, the occurrence of the additional eight regions was considerably lower. The evolutionary relationships and multilocus virulence gene profiles of V. cholerae natural isolates indicate that consecutive pandemic strains arose from a common O1 serogroup progenitor through the successive acquisition of new virulence regions.     

Comparative proteomic analysis of Vibrio cholerae O139, O22, O155 and El Tor biotype epidemic strains

Cholera ,as one of the most severe epidemic dis-eases inthe world,can occurin a short time ,andspread quicklyto a wide region.Until 1992 ,onlyV.choleraeserogroup O1 was recognized as thecause of epidemic cholera . However in October1992 ,a major outbreak of a cholera-like illnesscaused by a newserogroupstrain ofV.choleraee-mergedinIndia and Bangladesh.In contrast to allpreviously reported non-O1 strains , which induceonly sporadic cases of human diarrhea without epi-demic potential ,the new…     

Protection against Vibrio cholerae El Tor infection by specific antibodies against mannose-binding hemagglutinin pili.

Both specific polyclonal antiserum and monoclonal antibodies against mannose-binding hemagglutinin fimbriae of Vibrio cholerae (mannose-sensitive hemagglutinin [MSHA]) were shown to protect against experimental cholera caused by vibrios of the El Tor biotype in the infant mouse and in the rabbit intestinal loop models. MSHA-specific Fab immunoglobulin fragments were also protective. No protective effect was observed against challenge with V. cholerae O1 of the classical biotype. These results suggest that MSHA pili play an important role in the pathogenesis of cholera caused by the El Tor biotype of V. cholerae and that induction of intestinal anti-MSHA immunity may be a worthwhile additional objective in the development of oral cholera vaccines.     

Vibrio cholerae serogroup O1 in northeast Thailand

Strains of Vibrio cholerae serogroup O1 biotype El Tor that are susceptible to Mukerjee cholera bacteriophage group IV (S. Mukerjee, Bull. W.H.O. 28:333-336, 1963) were found. Cholera vibrios isolated from epidemics in northeast Thailand were characterized, and 57 of 60 strains isolated in 1986 were susceptible to cholera phage IV. However, all 113 strains isolated in 1988 were not susceptible to the phage. All isolates in both epidemics revealed behaviors typical of El Tor vibrios, except phage IV susceptibility in the 57 strains. Although the plaques of phage IV were generally translucent, plaques on some isolates looked transparent, just like those on classical vibrios. The organisms grown in the plaques were lysogenized. If this kind of strain is frequently isolated, the biotype of V. cholerae O1 should be reconsidered.     

Vibrio cholerae non-O1 serogroup associated with cholera gravis genetically and physiologically resembles O1 E1 Tor cholera strains.

Until recently, only Vibrio cholerae strains of the O1 serogroup have been associated with epidemic cholera. In December 1992, an outbreak of cholera gravis in Vellore, India, was attributed to a new serogroup of V. cholerae recently designated O139. Serogroup O139 cholera has since spread to 13 countries and has reached pandemic proportions. Serogroup O139 cholera evades immunity to O1 cholera and is not detected by the standard O1 antigen test. Understanding the origins of O139 cholera and determining the relatedness of O139 to O1 cholera are necessary to device strategies for detecting, reporting, and controlling this new pandemic. In order to determine the origins of this novel cholera serogroup, O139 was analyzed for virulence genes, for virulence proteins and their regulation, and for its genomic background. We found that O139 and O1 V. cholera strains of the E1 Tor biotype possess highly homologous virulence genes encoding cholera toxin and toxin-coregulated pili and that the regulation of virulence protein expression likewise was indistinguishable between O139 and O1. Pulsed-field gel electrophoresis (PFGE) revealed the restriction digest pattern of O139 strains to be closely related to that of O1 serogroup E1 Tor biotype cholera strains from the Indian subcontinent. However, PFGE showed minor differences among individual O139 cholera isolates, suggesting that O139 V. cholerae is evolving.