The Zymovars of Vibrio cholerae: multilocus enzyme electrophoresis of Vibrio cholerae

Zymovars analysis also known as multilocus enzyme electrophoresis is applied here to investigate the genetic variation of Vibrio cholerae strains and characterise strains or group of strains of medical and epidemiological interest. Fourteen loci were analyzed in 171 strains of non-O1 non-O139, 32 classical and 61 El Tor from America, Africa, Europe and Asia. The mean genetic diversity was 0.339. It is shown that the same O antigen (both O1 and non-O1) may be present in several genetically diverse (different zymovars) strains. Conversely the same zymovar may contain more than one serogroup. It is confirmed that the South American epidemic strain differs from the 7th pandemic El Tor strain in locus LAP (leucyl leucyl aminopeptidase). Here it is shown that this rare allele is present in 1 V. mimicus and 4 non-O1 V. cholerae. Non toxigenic O1 strains from South India epidemic share zymovar 14A with the epidemic El Tor from the 7th pandemic, while another group have diverse zymovars. The sucrose negative epidemic strains isolated in French Guiana and Brazil have the same zymovar of the current American epidemic V. cholerae.     

Molecular analyses of a putative CTXphi precursor and evidence for independent acquisition of distinct CTX(phi)s by toxigenic Vibrio cholerae

The genes encoding cholera toxin (ctxA and ctxB) are encoded in the genome of CTXphi, a filamentous phage that infects Vibrio cholerae. To study the evolutionary history of CTXphi, we examined genome diversity in CTX(phi)s derived from a variety of epidemic and nonepidemic Vibrio sp. natural isolates. Among these were three V. cholerae strains that contained CTX prophage sequences but not the ctxA and ctxB genes. These prophages each gave rise to a plasmid form whose genomic organization was very similar to that of the CTXphi replicative form, with the exception of missing ctxAB. Sequence analysis of these three plasmids revealed that they lacked the upstream control region normally found 5' of ctxA, as well as the ctxAB promoter region and coding sequences. These findings are consistent with the hypothesis that a CTXphi precursor that lacked ctxAB simultaneously acquired the toxin genes and their regulatory sequences. To assess the evolutionary relationships among additional CTX(phi)s, two CTXphi-encoded genes, orfU and zot, were sequenced from 13 V. cholerae and 4 V. mimicus isolates. Comparative nucleotide sequence analyses revealed that the CTX(phi)s derived from classical and El Tor V. cholerae isolates comprise two distinct lineages within otherwise nearly identical chromosomal backgrounds (based on mdh sequences). These findings suggest that nontoxigenic precursors of the two V. cholerae O1 biotypes independently acquired distinct CTX(phi)s.     

Whole genome PCR scanning reveals the syntenic genome structure of toxigenic Vibrio cholerae strains in the O1/O139 population

Vibrio cholerae is commonly found in estuarine water systems. Toxigenic O1 and O139 V. cholerae strains have caused cholera epidemics and pandemics, whereas the nontoxigenic strains within these serogroups only occasionally lead to disease. To understand the differences in the genome and clonality between the toxigenic and nontoxigenic strains of V. cholerae serogroups O1 and O139, we employed a whole genome PCR scanning (WGPScanning) method, an rrn operon-mediated fragment rearrangement analysis and comparative genomic hybridization (CGH) to analyze the genome structure of different strains. WGPScanning in conjunction with CGH revealed that the genomic contents of the toxigenic strains were conservative, except for a few indels located mainly in mobile elements. Minor nucleotide variation in orthologous genes appeared to be the major difference between the toxigenic strains. rrn operon-mediated rearrangements were infrequent in El Tor toxigenic strains tested using I-CeuI digested pulsed-field gel electrophoresis (PFGE) analysis and PCR analysis based on flanking sequence of rrn operons. Using these methods, we found that the genomic structures of toxigenic El Tor and O139 strains were syntenic. The nontoxigenic strains exhibited more extensive sequence variations, but toxin coregulated pilus positive (TCP+) strains had a similar structure. TCP+ nontoxigenic strains could be subdivided into multiple lineages according to the TCP type, suggesting the existence of complex intermediates in the evolution of toxigenic strains. The data indicate that toxigenic O1 El Tor and O139 strains were derived from a single lineage of intermediates from complex clones in the environment. The nontoxigenic strains with non-El Tor type TCP may yet evolve into new epidemic clones after attaining toxigenic attributes.     

Genetic diversity of toxigenic and nontoxigenic Vibrio cholerae serogroups O1 and O139 revealed by array-based comparative genomic hybridization

Toxigenic serogroups O1 and O139 of Vibrio cholerae may cause cholera epidemics or pandemics. Nontoxigenic strains within these serogroups also exist in the environment, and also some may cause sporadic cases of disease. Herein, we investigate the genomic diversity among toxigenic and nontoxigenic O1 and O139 strains by comparative genomic microarray hybridization with the genome of El Tor strain N16961 as a base. Conservation of the toxigenic O1 El Tor and O139 strains is found as previously reported, whereas accumulation of genome changes was documented in toxigenic El Tor strains isolated within the 40 years of the seventh pandemic. High phylogenetic diversity in nontoxigenic O1 and O139 strains is observed, and most of the genes absent from nontoxigenic strains are clustered together in the N16961 genome. By comparing these toxigenic and nontoxigenic strains, we observed that the small chromosome of V. cholerae is quite conservative and stable, outside of the superintegron region. In contrast to the general stability of the genome, the superintegron demonstrates pronounced divergence among toxigenic and nontoxigenic strains. Additionally, sequence variation in virulence-related genes is found in nontoxigenic El Tor strains, and we speculate that these intermediate strains may have pathogenic potential should they acquire CTX prophage alleles and other gene clusters. This genome-wide comparison of toxigenic and nontoxigenic V. cholerae strains may promote understanding of clonal differentiation of V. cholerae and contribute to an understanding of the origins and clonal selection of epidemic strains.     

Construction of cholera toxin B subunit-producing Vibrio cholerae strains using the Mariner-FRT transposon delivery system

The most widely used oral whole-cell-recombinant B subunit cholera vaccine contains the nontoxic cholera toxin B subunit (CTXB) and either heat- or formalin-killed Vibrio cholerae O1 strains. Vibrio cholerae O1 strains in the vaccine provide antibacterial immunity, and CTXB contributes to the vaccine's efficacy by stimulating production of anti-CTXB antibody. Various attempts have been made to increase CTXB production. In this study, the mariner-FRT transposon delivery system developed by Chiang and Mekalanos was used to place the ctxB gene under the control of a strong chromosomal promoter in a nontoxigenic V. cholerae El Tor strain, M7922. The expression level of CTXB in transposon insertion mutant clones was screened by ganglioside-dependent enzyme-linked immunosorbent assay. Among CTXB-producing V. cholerae clones that were isolated, M7922-C1 produced the highest amount of CTXB (3.17+/-1.69 microg mL(-1)). M7922-C1 harbors a single insertion of ctxB into VC0972, which encodes a putative porin protein. Although the level of CTXB expression in this strain was not exceptionally high, this study indicates the possibility of using this delivery system to construct vaccine strains that overexpress specific antigens.     

Genome sequence of the human pathogen Vibrio cholerae Amazonia

Vibrio cholerae O1 Amazonia is a pathogen that was isolated from cholera-like diarrhea cases in at least two countries, Brazil and Ghana. Based on multilocus sequence analysis, this lineage belongs to a distinct profile compared to strains from El Tor and classical biotypes. The genomic analysis revealed that it contains Vibrio pathogenicity island 2 and a set of genes related to pathogenesis and fitness, such as the type VI secretion system, present in choleragenic V. cholerae strains.     

Hemolytic Vibrio cholerae O1 that is sensitive to Mukerjee's cholera phage IV and the phage produced by the hemolytic vibrio lysogenized with the infection of Mukerjee's cholera phage IV

Strains of hemolytic Vibrio cholerae O1 (El Tor vibrio) which are sensitive to Mukerjee's cholera phage group IV were isolated from cholera patients in North-East Thailand in 1986. Plaques of the phage on these hemolytic V. cholerae O1 were usually translucent but almost transparent on some strains, just like the plaques on non-hemolytic V. cholerae O1 (classical vibrio). These hemolytic V. cholerae O1 were lysogenized with the infection of cholera phage IV, and the lysogenized strains produced phage different from cholera phage IV. These hemolytic strains were classified into Cured type in prophage typing of V. cholerae O1, El Tor, because they were also lysogenized with Kappa phage and were hemolytic. When Cured-type V. cholerae O1, El Tor previously isolated in various countries were examined for the sensitivity to cholera phage IV, some of the isolates were sensitive.     

不同温度条件下霍乱弧菌生存能力的实验研究

目的通过在不同温度条件下对霍乱弧菌生存能力的观察,探索大连市水域中霍乱弧菌在流行＂间歇期＂如何存活和越冬。方法在自然海水和湖水中分别接种本市分离出的埃尔托型霍乱弧菌流行株小川1b型（大E940014）和埃尔托型霍乱弧菌非流行株小川32l（大E940011）,在1～2℃、5～6℃及10～12℃3个温度条件下检测霍乱弧菌生存能力。结果流行株（小川1b型）在1～2℃海水存活26 d,5～6℃存活28 d,10～12℃存活46 d。可见,10～12℃生存时间明显高于1～2℃或5～6℃（P〈0.05）。结论两类菌株的生存对环境温度均很敏感,温度越低死亡越快。在自然水体中,非流行株生存时间略长于流行株;而在除菌水体中两类菌存活能力基本一致。     

CTX prophages in classical biotype Vibrio cholerae: functional phage genes but dysfunctional phage genomes

CTXphi is a filamentous, lysogenic bacteriophage whose genome encodes cholera toxin, the primary virulence factor produced by Vibrio cholerae. CTX prophages in O1 El Tor and O139 strains of V. cholerae are found within arrays of genetically related elements integrated at a single locus within the V. cholerae large chromosome. The prophages of O1 El Tor and O139 strains generally yield infectious CTXphi. In contrast, O1 classical strains of V. cholerae do not produce CTXphi, although they produce cholera toxin and they contain CTX prophages integrated at two sites. We have identified the second site of CTX prophage integration in O1 classical strains and characterized the classical prophage arrays genetically and functionally. The genes of classical prophages encode functional forms of all of the proteins needed for production of CTXphi. Classical CTX prophages are present either as solitary prophages or as arrays of two truncated, fused prophages. RS1, a genetic element that is closely related to CTXphi and is often interspersed with CTX prophages in El Tor strains, was not detected in classical V. cholerae. Our model for CTXphi production predicts that the CTX prophage arrangements in classical strains will not yield extrachromosomal CTX DNA and thus will not yield virions, and our experimental results confirm this prediction. Thus, failure of O1 classical strains of V. cholerae to produce CTXphi is due to overall deficiencies in the structures of the arrays of classical prophages, rather than to mutations affecting individual CTX prophage genes.     

Genome size and restriction fragment length polymorphism analysis of Vibrio cholerae strains belonging to different serovars and biotypes

Abstract The genome size of Vibrio cholerae has been determined by pulsed field gel electrophoresis following digestion of chromosomal DNA with endonucleases. The genome size of all the classical strains examined was about 3000 kb and that of El Tor biotype was 2500 kb. The Not I and S fi I digestion patterns of the genomes of several V. cholerae straimns belonging to different serovars and biotypes showed distinct restriction fragment length polymorphism (RFLP). RFLP analysis together with the genome size can be used to differentiate strains of different serovars and biotypes of V. cholerae .     

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.     

Detection and differentiation of the gene for toxin co-regulated pili (tcpA) in Vibrio cholerae non-O1 using the polymerase chain reaction

Abstract The polymerase chain reaction has been used to differentiate the gene which encodes the toxin co-regulated pili ( tcpA ) of the El Tor and classical biotypes of Vibrio cholerae O1. The same PCR primers were applied to strains belonging to non-O1 serogroups that produced cholera toxin. The size of fragment amplified was either identical to the tcpA of biotype El Tor (471 bp) or to the tcpA of biotype classical (617 bp). All strains belonging to the novel epidemic serogroup O139 generated a 471-bp fragment identical to El Tor tcpA . The present study suggests that there may be an association between non-O1 serogroup and tcpA type.     

Antimicrobial susceptibility and molecular characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> from cholera outbreaks in Chennai

The genotype and antibiotic resistance pattern of the toxigenic Vibrio cholerae strains associated with cholera outbreaks vary frequently. Fifty-one V. cholerae strains isolated from cholera outbreaks in Chennai (2002–2005) were screened for the presence of virulence and regulatory genes by multiplex polymerase chain reaction (PCR) assay. Genotyping of the isolates was done by VC1 primers derived from enterobacterial repetitive intergenic consensus (ERIC)-related sequence in V. cholerae. All the isolates possessed toxigenic genes, such as ctxA, ctxB, tcpA, ace, ompU, toxR and zot. Two different El Tor genotypes and one O139 genotype could be delineated by VC1-PCR. One of the El Tor genotypes was similar to the El Tor strains isolated from Bhind district and Delhi during 2004. Antibiotic susceptibility testing revealed greater variability among the isolates tested. All the isolates were found to be susceptible to norfloxacin, ciprofloxacin and tetracycline. Thiry-three per cent of the isolates were found to be resistant to more than 4 antibiotics and could be termed as multiple antibiotic resistant. Coexistence of O139 serogroup along with the El Tor biotype could be identified among the strains recovered during the period 2002–2004. The O139 isolates were found to be more susceptible to the antibiotics tested when compared to the El Tor isolates.     

Structure and arrangement of the cholera toxin genes in Vibrio cholerae O139

Abstract The sequence of the ctxB gene encoding the B subunit of cholera toxin has been determined for a strain of Vibrio cholerae of the novel O139 serotype associated with recent outbreaks of severe cholera throughout South-East Asia and found to be identical to the ctxB gene in V. cholerae O1 of the E1 Tor biotype. Analyses by Southern hybridization and PCR showed that all strains of the O139 serotype V. cholerae tested carried cholera toxin genes and other gene associated with a virulence cassette DNA region at two loci identical or homologous to those identified in the Classical rather than the E1 Tor biotype of V. cholerae serotype O1 although these loci in O139 could reside on restriction fragments of variable size.     

Variation in epitopes of the B subunit of El Tor and classical biotype Vibrio cholerae O1 cholera toxin

Variation in epitopes of the B subunit of cholera toxin (CT-B) produced by strains of El Tor and classical biotype Vibrio cholerae O1 was examined using monoclonal antibodies prepared to V. cholerae 569B CT. CT-B epitopes were markedly conserved for V. cholerae classical biotypes. In contrast, epitope variation was observed for El Tor biotypes, which produced both a classical-like CT-B and a unique CT-B lacking at least one epitope common to 569B CT-B. The missing epitope was located outside the GM1 ganglioside-binding site. From results of the study reported here, genetic divergence is exhibited in the El Tor biotype CT-B versus classical CT-B. Furthermore, at least five unique epitopes of V. cholerae 569B CT-B can be defined.     

Vibrio cholerae O1 strains are facultative intracellular bacteria, able to survive and multiply symbiotically inside the aquatic free-living amoeba Acanthamoeba castellanii

Vibrio cholerae species are extracellular, waterborne, gram-negative bacteria that are overwhelmed by predators in aquatic environments. The unencapsulated serogroup V. cholerae O1 and encapsulated V. cholerae O139 cause epidemic and pandemic outbreaks of cholera. It has recently been shown that the aquatic and free-living amoeba Acanthamoeba castellanii is not a predator to V. cholerae O139; rather, V. cholerae O139 has shown an intracellular compatibility with this host. The aim of this study was to examine the ability of V. cholerae O1 classical and El Tor strains to grow and survive in A. castellanii. The interaction between A. castellanii and V. cholerae O1 strains was studied by means of amoeba cell counts and viable counts of the bacteria in the absence or presence of amoebae. The viable count of intracellularly growing bacteria was estimated by utilizing gentamicin assay. Confocal microscopy and electron microscopy were used to determine the intracellular localization of V. cholerae in A. castellanii. The results showed that V. cholerae O1 classical and El Tor strains grew and survived intracellularly in the cytoplasm of trophozoites, and that the bacteria were also found in the cysts of A. castellanii. The interaction showed a facultative intracellular behaviour of V. cholerae O1 classical and El Tor strains and a possible role of A. castellanii as an environmental host of V. cholerae species.     

Phenotypic and genetic characterization of Vibrio cholerae O1 clinical isolates collected through national antimicrobial resistance surveillance network in Nepal

Cholera occurs in sporadic cases and outbreaks in Nepal each year. Vibrio cholerae O1 (n = 522) isolated during 2007-2010 from diarrheal patients at 10 different hospital laboratories in Nepal were characterized. Biochemical and serologic identifications showed that all the isolates belonged to serogroup O1, El Tor biotype. Except 72 isolates of Inaba serotype isolated in the year 2007, all the remaining isolates were of Ogawa serotype. All isolates were resistant to nalidixic acid and furazolidone. Resistance to tetracycline, ciprofloxacin, erythromycin and co-trimoxazole were 21, 4, 16 and 90 % respectively. Seventy-seven of these isolates were selected for further characterization for ctxB gene and MLVA typing. Two different variants of classical type cholera toxin were observed. Ogawa strains from 2007 and 2010-Western Nepal outbreak harbored CTX-3 type cholera toxin, whereas Inaba serotypes in 2007 and the remaining Ogawa serotypes in 2008-2010 harbored CTX 3b-type toxin. MLVA analysis showed circulation of four different groups of altered V. cholerae O1 El Tor strains. Two different profiles were seen among 2007 Inaba (9, 3, 6, x, x) and Ogawa (10, 7, 6, x, x) isolates. The MLVA profile of 2008 and 2009 Ogawa isolates were similar to those of Inaba strains of 2007. Isolates from 2010 also showed three different MLVA profiles; profile 9, 3, 6, x, x in 3 isolates, 11, 7, 6, x, x among 2010 Western Nepal outbreak strains and profile 8, 3, 6, x, x among isolates from Butwal and Kathmandu.     

Genetic analysis of mutations affecting the synthesis of somatic O-antigen of Vibrio cholerae

The mutations oagM and oagR affecting the synthesis of somatic O-antigen have been localized on the chromosome of Vibrio cholerae El Jor and classic biotypes by conjugational crosses between different donor and recipient strains. The mutations are localized in the vicinity of the arg marker in both classic and El Tor biotypes of Vibrio cholerae.     

Genotypes associated with virulence in environmental isolates of Vibrio cholerae

Vibrio cholerae is an autochthonous inhabitant of riverine and estuarine environments and also is a facultative pathogen for humans. Genotyping can be useful in assessing the risk of contracting cholera, intestinal, or extraintestinal infections via drinking water and/or seafood. In this study, environmental isolates of V. cholerae were examined for the presence of ctxA, hlyA, ompU, stn/sto, tcpA, tcpI, toxR, and zot genes, using multiplex PCR. Based on tcpA and hlyA gene comparisons, the strains could be grouped into Classical and El Tor biotypes. The toxR, hlyA, and ompU genes were present in 100, 98.6, and 87.0% of the V. cholerae isolates, respectively. The CTX genetic element and toxin-coregulated pilus El Tor (tcpA ET) gene were present in all toxigenic V. cholerae O1 and V. cholerae O139 strains examined in this study. Three of four nontoxigenic V. cholerae O1 strains contained tcpA ET. Interestingly, among the isolates of V. cholerae non-O1/non-O139, two had tcpA Classical, nine contained tcpA El Tor, three showed homology with both biotype genes, and four carried the ctxA gene. The stn/sto genes were present in 28.2% of the non-O1/non-O139 strains, in 10.5% of the toxigenic V. cholerae O1, and in 14.3% of the O139 serogroups. Except for stn/sto genes, all of the other genes studied occurred with high frequency in toxigenic V. cholerae O1 and O139 strains. Based on results of this study, surveillance of non-O1/non-O139 V. cholerae in the aquatic environment, combined with genotype monitoring using ctxA, stn/sto, and tcpA ET genes, could be valuable in human health risk assessment.