Examination of diverse toxin-coregulated pilus-positive Vibrio cholerae strains fails to demonstrate evidence for Vibrio pathogenicity island phage

The major virulence factors of toxigenic Vibrio cholerae are cholera toxin, which is encoded by a lysogenic filamentous bacteriophage (CTXPhi), and toxin-coregulated pilus (TCP), an essential colonization factor that is also the receptor for CTXPhi. The genes involved in the biosynthesis of TCP reside in a pathogenicity island, which has been reported to correspond to the genome of another filamentous phage (designated VPIPhi) and to encode functions necessary for the production of infectious VPIPhi particles. We examined 46 V. cholerae strains having diverse origins and carrying different genetic variants of the TCP island for the production of the VPIPhi and CTXPhi in different culture conditions, including induction of prophages with mitomycin C and UV irradiation. Although 9 of 10 V. cholerae O139 strains and 12 of 15 toxigenic El Tor strains tested produced extracellular CTXPhi, none of the 46 TCP-positive strains produced detectable VPIPhi in repeated assays, which detected as few as 10 particles of a control CTX phage per ml. These results contradict the previous report regarding VPIPhi-mediated horizontal transfer of the TCP genes and suggest that the TCP island is unable to support the production of phage particles. Further studies are necessary to understand the mechanism of horizontal transfer of the TCP island.     

Sunlight-induced propagation of the lysogenic phage encoding cholera toxin

In toxigenic Vibrio cholerae, the cholera enterotoxin (CT) is encoded by CTXPhi, a lysogenic bacteriophage. The propagation of this filamentous phage can result in the origination of new toxigenic strains. To understand the nature of possible environmental factors associated with the propagation of CTXPhi, we examined the effects of temperature, pH, salinity, and exposure to direct sunlight on the induction of the CTX prophage and studied the transmission of the phage to potential recipient strains. Exposure of cultures of CTXPhi lysogens to direct sunlight resulted in approximately 10,000-fold increases in phage titers. Variation in temperature, pH, or salinity of the culture did not have a substantial effect on the induction of the prophage, but these factors influenced the stability of CTXPhi particles. Exposure of mixed cultures of CTXPhi lysogens and potential recipient strains to sunlight significantly increased both the in vitro and in vivo (in rabbit ileal loops) transduction of the recipient strains by CTXPhi. Included in these transduction experiments were two environmental nontoxigenic (CTXPhi(-)) strains of V. cholerae O139. These two O139 strains were transduced at high efficiency by CTXPhi, and the phage genome integrated into the O139 host chromosome. The resulting CTXPhi lysogens produced biologically active CT both in vitro and in rabbit ileal loops. This finding suggests a possible mechanism explaining the origination of toxigenic V. cholerae O139 strains from nontoxigenic progenitors. This study indicates that sunlight is a significant inducer of the CTX prophage and suggests that sunlight-induced transmission of CTXPhi may constitute part of a natural mechanism for the origination of new toxigenic strains of V. cholerae.     

Molecular characterization of a new ribotype of Vibrio cholerae O139 Bengal associated with an outbreak of cholera in Bangladesh

Vibrio cholerae O139 Bengal initially appeared in the southern coastal region of Bangladesh and spread northward, causing explosive epidemics during 1992 and 1993. The resurgence of V. cholerae O139 during 1995 after its transient displacement by a new clone of El Tor vibrios demonstrated rapid changes in the epidemiology of cholera in Bangladesh. A recent outbreak of cholera in two north-central districts of Bangladesh caused by V. cholerae O139 led us to analyze strains collected from the outbreak and compare them with V. cholerae O139 strains isolated from other regions of Bangladesh and neighboring India to investigate their origins. Analysis of restriction fragment length polymorphisms in genes for conserved rRNA (ribotype) revealed that the recently isolated V. cholerae O139 strains belonged to a new ribotype which was distinct from previously described ribotypes of toxigenic V. cholerae O139. All strains carried the genes for toxin-coregulated pili (tcpA and tcpI) and accessory colonization factor (acfB), the regulatory gene toxR, and multiple copies of the lysogenic phage genome encoding cholera toxin (CTXPhi) and belonged to a previously described ctxA genotype. Comparative analysis of the rfb gene cluster by PCR revealed the absence of a large region of the O1-specific rfb operon downstream of the rfaD gene and the presence of an O139-specific genomic region in all O139 strains. Southern hybridization analysis of the O139-specific genomic region also produced identical restriction patterns in strains belonging to the new ribotype and those of previously described ribotypes. These results suggested that the new ribotype of Bengal vibrios possibly originated from an existing strain of V. cholerae O139 by genetic changes in the rRNA operons. In contrast to previously isolated O139 strains which mostly had resistance to trimethoprim, sulfamethoxazole, and streptomycin encoded by a transposon (SXT element), 68.6% of the toxigenic strains analyzed in the present study, including all strains belonging to the new ribotype, were susceptible to these antibiotics. Molecular analysis of the SXT element revealed possible deletion of a 3.6-kb region of the SXT element in strains which were susceptible to the antibiotics. Thus, V. cholerae O139 strains in Bangladesh are also undergoing considerable reassortments in genetic elements encoding antimicrobial resistance.     

Lysogenic conversion of environmental Vibrio mimicus strains by CTXPhi.

The filamentous bacteriophage CTXPhi, which encodes cholera toxin (CT) in toxigenic Vibrio cholerae, is known to propagate by infecting susceptible strains of V. cholerae by using the toxin coregulated pilus (TCP) as its receptor and thereby causing the origination of new strains of toxigenic V. cholerae from nontoxigenic progenitors. Besides V. cholerae, Vibrio mimicus strains which are normally TCP negative have also been shown to occasionally produce CT and cause diarrhea in humans. We analyzed nontoxigenic V. mimicus strains isolated from surface waters in Bangladesh for susceptibility and lysogenic conversion by CTXPhi and studied the expression of CT in the lysogens by using genetically marked derivatives of the phage. Of 27 V. mimicus strains analyzed, which were all negative for genes encoding TCP but positive for the regulatory gene toxR, 2 strains (7.4%) were infected by CTX-KmPhi, derived from strain SM44(P27459 ctx::km), and the phage genome integrated into the host chromosome, forming stable lysogens. The lysogens spontaneously produced infectious phage particles in the supernatant fluids of the culture, and high titers of the phage could be achieved when the lysogens were induced with mitomycin C. This is the first demonstration of lysogenic conversion of V. mimicus strains by CTXPhi. When a genetically marked derivative of the replicative form of the CTXPhi genome carrying a functional ctxAB operon, pMSF9.2, was introduced into nontoxigenic V. mimicus strains, the plasmid integrated into the host genome and the strains produced CT both in vitro and inside the intestines of adult rabbits and caused mild-to-severe diarrhea in rabbits. This suggested that in the natural habitat infection of nontoxigenic V. mimicus strains by wild-type CTXPhi may lead to the origination of toxigenic V. mimicus strains which are capable of producing biologically active CT. The results of this study also supported the existence of a TCP-independent mechanism for infection by CTXPhi and showed that at least one species of Vibrio other than V. cholerae may contribute to the propagation of the phage.     

Molecular epidemiology of Vibrio cholerae O139 in China: polymorphism of ribotypes and CTX elements

Vibrio cholerae O139, the second etiological serogroup of cholera, triggered the first outbreak of O139 cholera in China in 1993. To analyze the clone polymorphism of O139 isolates in China, 117 strains of V. cholerae O139, isolated from different areas in China between 1993 and 1999, were selected to characterize the phylogenetic relationships by molecular techniques. Analysis of restriction fragment length polymorphism in the conserved 16S rRNA gene revealed seven different ribotypes within the 117 strains. Among these strains, there were eight that lacked the cholera toxin gene (ctxAB), zot, and the repetitive sequence (RS); these eight strains belonged to three individual ribotypes. Our results suggested that V. cholerae O139 strains in China had clone diversity in phylogeny. The results of our hybridization patterns for CTX genetic elements (ctxAB, zot, and RS) showed that CTXPhi genomes in most V. cholerae O139 strains had two or more copies and had extensive restriction patterns even for the strains which belong to the same ribotype. For 22 (20.1%) strains, the copies of ctxAB were different from those of zot, suggesting that a ctxAB-negative CTXPhi genome may exist in O139 strains. This ctxAB-negative CTXPhi genome may coexist with the intact CTXPhi genome in a strain. In addition, the dendrogram for I-CeuI-generated pulsed-field gel electrophoresis patterns showed that V. cholerae serogroup O139 has a closer relationship with one strain of serogroup O22 than with the strains of serogroup O1. The results of this study showed the clonal diversity and the distribution of O139 strains in China, suggesting multiple origins of the O139 cholera epidemic or sporadic events.     

Pathogenic potential of environmental Vibrio cholerae strains carrying genetic variants of the toxin-coregulated pilus pathogenicity island

The major virulence factors of toxigenic Vibrio cholerae are cholera toxin (CT), which is encoded by a lysogenic bacteriophage (CTXPhi), and toxin-coregulated pilus (TCP), an essential colonization factor which is also the receptor for CTXPhi. The genes for the biosynthesis of TCP are part of a larger genetic element known as the TCP pathogenicity island. To assess their pathogenic potential, we analyzed environmental strains of V. cholerae carrying genetic variants of the TCP pathogenicity island for colonization of infant mice, susceptibility to CTXPhi, and diarrheagenicity in adult rabbits. Analysis of 14 environmental strains, including 3 strains carrying a new allele of the tcpA gene, 9 strains carrying a new allele of the toxT gene, and 2 strains carrying conventional tcpA and toxT genes, showed that all strains colonized infant mice with various efficiencies in competition with a control El Tor biotype strain of V. cholerae O1. Five of the 14 strains were susceptible to CTXPhi, and these transductants produced CT and caused diarrhea in adult rabbits. These results suggested that the new alleles of the tcpA and toxT genes found in environmental strains of V. cholerae encode biologically active gene products. Detection of functional homologs of the TCP island genes in environmental strains may have implications for understanding the origin and evolution of virulence genes of V. cholerae.     

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.     

霍乱弧菌O139某些生物学特性及毒力基因检测

Ｏ１３９霍乱弧菌是１９９２年发现的新型霍乱弧菌，其毒力强，危害严重。对中国、印度、孟加拉国分离的２３株Ｏ１３９菌株的部分生物学特征检查表明：对弧菌抑制剂Ｏ／１２９均具有抗性、溶原菌、山梨醇慢发酵、非溶血性。核酸分子杂交显示，所有被检Ｏ１３９菌株都具有主要的霍乱弧菌毒素基因：ｃｔｘ，ｚｏｔ，ａｃｅ和ＲＳ１序列。ＰＣＲ检测ｃｔｘ基因与Ｏ１群流行珠具有相同的扩增产物，ｔｃｐＡ基因扩增表现为与埃尔托型霍乱弧菌相似的扩增产物。兔肠段结扎测毒表明，Ｏ１３９霍乱弧菌为强毒株。因此Ｏ１３９菌与Ｏ１群霍乱弧菌流行株具有共同的毒力特征。     

Clinical and Environmental Isolates of Vibrio cholerae Serogroup O141 Carry the CTX Phage and the Genes Encoding the Toxin-Coregulated Pili

We report sporadic cases of a severe gastroenteritis associated with Vibrio cholerae serogroup O141. Like O1 and O139 serogroup strains of V. cholerae isolated from cholera cases, the O141 clinical isolates carry DNA sequences that hybridize to cholera toxin (CT) gene probes. The CT genes of O1 and O139 strains are carried by a filamentous bacteriophage (termed CTX phage) which is known to use toxin-coregulated pili (TCP) as its receptor. In an effort to understand the mechanism of emergence of toxigenic O141 V. cholerae, we probed a collection of O141 clinical and environmental isolates for genes involved in TCP production, toxigenicity, virulence regulation, and other phylogenetic markers. The collection included strains isolated between 1964 and 1995 from diverse geographical locations, including eight countries and five U.S. states. Information collected about the clinical and environmental sources of these isolates suggests that they had no epidemiological association. All clinical O141 isolates hybridized to probes specific for genes encoding CT (ctx), zonula occludens toxin (zot), repetitive sequence 1 (RS1), RTX toxin (rtxA), the major subunit of TCP (tcpA), and the essential regulatory gene that controls expression of both CT and TCP (toxR). In contrast, all but one of the nonclinical O141 isolates were negative for ctx, zot, RS1, and tcpA, although these strains were positive for rtxA and toxR. The one toxigenic environmental O141 isolate was also positive for tcpA. Ribotyping and CT typing showed that the O141 clinical isolates were indistinguishable or closely related, while a toxigenic water isolate from Louisiana showed a distantly related ribotype. Nonclinical O141 isolates displayed a variety of unrelated ribotypes. These data support a model for emergence of toxigenic O141 that involves acquisition of the CTX phage sometime after these strains had acquired the pathogenicity island encoding TCP. The clonal nature of toxigenic O141 strains isolated from diverse geographical locations suggests that the emergence is a rare event but that once it occurs, toxigenic O141 strains are capable of regional and perhaps even global dissemination. This study stresses the importance of monitoring V. cholerae non-O1, non-O139 serogroup strains for their virulence gene content as a means of assessing their epidemic potential.     

Construction and evaluation of a safe, live, oral Vibrio cholerae vaccine candidate, IEM108

IEM101, a Vibrio cholerae O1 El Tor Ogawa strain naturally deficient in CTXPhi, was previously selected as a live cholera vaccine candidate. To make a better and safer vaccine that can induce protective immunity against both the bacteria and cholera toxin (CT), a new vaccine candidate, IEM108, was constructed by introducing a ctxB gene and an El Tor-derived rstR gene into IEM101. The ctxB gene codes for the protective antigen CTB subunit, and the rstR gene mediates phage immunity. The stable expression of the two genes was managed by a chromosome-plasmid lethal balanced system based on the housekeeping gene thyA. Immunization studies indicate that IEM108 generates good immune responses against both the bacteria and CT. After a single-dose intraintestinal vaccination with 10(9) CFU of IEM108, both anti-CTB immunoglobulin G and vibriocidal antibodies were detected in the immunized-rabbit sera. However, only vibriocidal antibodies are detected in rabbits immunized with IEM101. In addition, IEM108 but not IEM101 conferred full protection against the challenges of four wild-type toxigenic strains of V. cholerae O1 and 4 micro g of CT protein in a rabbit model. By introducing the rstR gene, the frequency of conjugative transfer of a recombinant El Tor-derived RS2 suicidal plasmid to IEM108 was decreased 100-fold compared to that for IEM101. This indicated that the El Tor-derived rstR cloned in IEM108 was fully functional and could effectively inhibit the El Tor-derived CTXPhi from infecting IEM108. Our results demonstrate that IEM108 is an efficient and safe live oral cholera vaccine candidate that induces antibacterial and antitoxic immunity and CTXPhi phage immunity.     

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.     

Infectious CTXPhi and the vibrio pathogenicity island prophage in Vibrio mimicus: evidence for recent horizontal transfer between V. mimicus and V. cholerae

Vibrio mimicus differs from Vibrio cholerae in a number of genotypic and phenotypic traits but like V. cholerae can give rise to diarrheal disease. We examined clinical isolates of V. mimicus for the presence of CTXPhi, the lysogenic filamentous bacteriophage that carries the cholera toxin genes in epidemic V. cholerae strains. Four V. mimicus isolates were found to contain complete copies of CTXPhi. Southern blot analyses revealed that V. mimicus strain PT5 contains two CTX prophages integrated at different sites within the V. mimicus genome whereas V. mimicus strains PT48, 523-80, and 9583 each contain tandemly arranged copies of CTXPhi. We detected the replicative form of CTXPhi, pCTX, in all four of these V. mimicus isolates. The CTX prophage in strain PT5 was found to produce infectious CTXPhi particles. The nucleotide sequences of CTXPhi genes orfU and zot from V. mimicus strain PT5 and V. cholerae strain N16961 were identical, indicating contemporary horizontal transfer of CTXPhi between these two species. The receptor for CTXPhi, the toxin-coregulated pilus, which is encoded by another lysogenic filamentous bacteriophage, VPIPhi, was also present in the CTXPhi-positive V. mimicus isolates. The nucleotide sequences of VPIPhi genes aldA and toxT from V. mimicus strain PT5 and V. cholerae N16961 were identical, suggesting recent horizontal transfer of this phage between V. mimicus and V. cholerae. In V. mimicus, the vibrio pathogenicity island prophage was integrated in the same chromosomal attachment site as in V. cholerae. These results suggest that V. mimicus may be a significant reservoir for both CTXPhi and VPIPhi and may play an important role in the emergence of new toxigenic V. cholerae isolates.     

Vibriophage VcA-3 as an epidemic strain marker for the U.S. Gulf Coast Vibrio cholerae O1 clone.

Toxigenic and nontoxigenic Vibrio cholerae O1, El Tor biotype strains, which are endemic to the U.S. Gulf Coast, can be lysogenic for bacteriophage VcA-3. To evaluate the presence of VcA-3 as an indicator of toxigenicity and as an epidemic strain marker, phage production and the presence of phage and cholera toxin genes were assayed in 98 strains of V. cholerae O1 (35 U.S. and 63 foreign strains). By using a HindIII chromosomal digest for Southern blot analysis, 39 of the study strains hybridized with the VcA-3 probe in 10 banding patterns. The 15 toxigenic and 6 of the 20 nontoxigenic U.S. isolates gave four VcA-3-related patterns. Among the foreign isolates, 12 of 12 toxigenic classical biotype strains, 1 of 43 toxigenic El Tor biotype strains, and 3 of 8 nontoxigenic atypical strains gave six patterns that were clearly distinct from that of VcA-3. Compared with Southern blot analysis, the phage production assay had a sensitivity of 1.0 and a specificity of 0.48, while the colony hybridization assay had a sensitivity of 1.0 and a specificity of 0.77 for identification of VcA-3. Neither assay reliably identified the toxigenic Gulf Coast clone. The presence of VcA-3, as defined by Southern blot analysis, always separated toxigenic U.S. from foreign isolates and often from nontoxigenic U.S. isolates of V. cholerae O1.     

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.     

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.     

El Tor and Calcutta CTXPhi precursors coexisting with intact CTXPhi copies in Vibrio cholerae O139 isolates

Restriction fragment length polymorphism analyses of the array of CTXPhi prophages in strains CRC262 and CRC266 of Vibrio cholerae O139 revealed the presence of copies of complete CTXPhi and pre-CTXPhi prophages coexisting at a single chromosomal locus in each strain. Restriction pattern and comparative nucleotide sequence analysis revealed pre-CTXPhi precursors of both the El Tor and Calcutta lineages. Thus, we hypothesize that two precursor variants independently acquired cholera toxin genes and gave rise to the current El Tor and Calcutta CTXPhi prophages. We discuss the implications of these results in terms of the evolution and origin of the current diversity of CTXPhi prophages.     

Molecular characterization of a new variant of toxin-coregulated pilus protein (TcpA) in a toxigenic non-O1/Non-O139 strain of Vibrio cholerae

A toxigenic non-O1/non-O139 strain of Vibrio cholerae (10259) was found to contain a new variant of the toxin-coregulated pilus (TCP) protein gene (tcpA) as determined by PCR and Southern hybridization experiments. Nucleotide sequence analysis data of the new tcpA gene in strain 10259 (O53) showed it to be about 74 and 72% identical to those of O1 classical and El Tor biotype strains, respectively. The predicted amino acid sequence of the 10259 TcpA protein shared about 81 and 78% identity with the corresponding sequences of classical and El Tor TcpA strains, respectively. An antiserum raised against the TCP of a classical strain, O395, although it recognized the TcpA protein of strain 10259 in an immunoblotting experiment, exhibited considerably less protection against 10259 challenge compared to that observed against the parent strain. Incidentally, the tcpA sequences of two other toxigenic non-O1/non-O139 strains (V2 and S7, both belonging to the serogroup O37) were determined to be almost identical to that of classical tcpA. Further, tcpA of another toxigenic non-O1/non-O139 strain V315-1 (O nontypeable) was closely related to that of El Tor tcpA. Analysis of these results with those already available in the literature suggests that there are at least four major variants of the tcpA gene in V. cholerae which probably evolved in parallel from a common ancestral gene. Existence of highly conserved as well as hypervariable regions within the sequence of the TcpA protein would also predict that such evolution is under the control of considerable selection pressure.     

Molecular characterisation of rough variants of Vibrio cholerae isolated from hospitalised patients with diarrhoea

Seven rough isolates of Vibrio cholerae isolated as the sole infecting agent from patients with cholera-like diarrhoea were examined for the presence of the regulatory element toxR and certain virulence-associated genes of the CTX genetic element and V. cholerae pathogenicity island (VPI). Multiplex PCR analysis with wb-specific genes of either O1 or O139 origin showed that six of the seven isolates produced an O1 wb-specific amplicon and the remaining isolate produced an O139-specific amplicon. Analysis of lipopolysaccharide profiles of smooth variants of V. cholerae revealed the presence of long repeated units of 'O' polysaccharide side chains but all the rough variants appeared to be devoid of the latter and possessed only core oligosaccharide. PCR amplification with primers specific to the ctxA, ctxB, tcpA, tagA, int, aldA, toxT, LJ, RJ and toxR genes revealed that six of the seven rough isolates were positive for these genes. One isolate was found to be negative for tagA and RJ, indicating the presence of an altered VPI. Each of these isolates showed media-dependent expression of cholera toxin (CT) and produced more toxin than the reference V. cholerae O1 El Tor strain VC20 or O139 strain SG24 under comparable conditions. Studies on the clonality of these isolates by the analysis of rRNA genes indicated their relatedness to strains of V. cholerae O1 El Tor or O139, isolated during the same time period.     

Comparative and genetic analyses of the putative Vibrio cholerae lipopolysaccharide core oligosaccharide biosynthesis (wav) gene cluster

We identified five different putative wav gene cluster types, which are responsible for the synthesis of the core oligosaccharide (OS) region of Vibrio cholerae lipopolysaccharide. Preliminary evidence that the genes encoded by this cluster are involved in core OS biosynthesis came from analysis of the recently released O1 El Tor V. cholerae genome sequence and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of O1 El Tor mutant strains defective in three genes (waaF, waaL, and wavB). Investigations of 38 different V. cholerae strains by Southern blotting, PCR, and sequencing analyses showed that the O1 El Tor wav gene cluster type is prevalent among clinical isolates of different serogroups associated with cholera and environmental O1 strains. In contrast, we found differences in the wav gene contents of 19 unrelated non-O1, non-O139 environmental and human isolates not associated with cholera. These strains contained four new wav gene cluster types that differ from each other in distinct gene loci, providing evidence for horizontal transfer of wav genes and for limited structural diversity of the core OS among V. cholerae isolates. Our results show genetic diversity in the core OS biosynthesis gene cluster and predominance of the type 1 wav gene locus in strains associated with clinical cholera, suggesting that a specific core OS structure could contribute to V. cholerae virulence.     

Development and evaluation of a phage typing scheme for Vibrio cholerae O139

The scenario of cholera that existed previously changed in 1992 and 1993 with the emergence of toxigenic Vibrio cholerae O139 in India. The genesis of the new serogroup formed the impetus to search for O139 phages in and around the country. A total of five newly isolated phages lytic to V. cholerae O139 strains were used for the development of this phage typing scheme. These phages differed from each other and also differed from the existing O1 phages in their lytic patterns, morphologies, restriction endonuclease digestion profiles, and immunological criteria. With this scheme, 500 V. cholerae O139 strains were evaluated for their phage types, and almost all strains were found to be typeable. The strains clustered into 10 different phage types, of which type 1 (38.2%) was the dominant type, followed by type 2 (22.4%) and type 3 (18%). Additionally, a comparative study of phage types in 1993 and 1994 versus those from 1996 to 1998 for O139 strains showed a higher percentage of phage type 1 (40.5%), followed by type 3 (18.8%) during the period between 1993 and 1994, whereas phage type 2 (32. 1%) was the next major type during the period from 1996 to 1998. This scheme comprising five newly isolated phages would be another useful tool in the study of the epidemiology of cholera caused by V. cholerae O139.