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Approximately 200 O-serogroups of Vibrio cholerae have already been identified; however, only 2 serogroups, O1 and O139, are strongly related to pandemic cholera. The study of non-O1 and non-O139 strains has hitherto been limited. Nevertheless, there are other clinically and epidemiologically important serogroups causing outbreaks with cholera-like disease. Here, we report a comprehensive genome analysis of the whole set of V. cholerae O-serogroup reference strains to provide an overview of this important bacterial pathogen. It revealed structural diversity of the O-antigen biosynthesis gene clusters located at specific loci on chromosome 1 and 16 pairs of strains with almost identical O-antigen biosynthetic gene clusters but differing in serological patterns. This might be due to the presence of O-antigen biosynthesis-related genes at secondary loci on chromosome 2.

Vibrio spp. are natural inhabitants of marine and estuarine environments. Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus are the major infectious agents for humans. Their densities are affected by environmental factors such as water temperature and salinity. The detailed contribution of each factor still remains to be elucidated. Here we conducted multi-coastal study in a 21-month period to examine relationships between environmental factors and V. cholerae, V. parahaemolyticus and V. vulnificus densities in sea surface water in eight coastal sites of four prefectures in Japan. Vibrio densities were measured by a most-probable-number with PCR method which is highly sensitive and quantitative (3/100 ml of detection limit). Vibrio densities were analyzed with environmental factors including water temperature, salinity, total dissolved substance, and pH, and their quadratics. A linear regression model suited best for prediction of V. cholerae density. A novel double-quadratic model suited best for the prediction of V. parahaemolyticus and V. vulnificus densities.

Twelve Vibrio cholerae isolates with genes for a type III secretion system (T3SS) were detected among 110 environmental and 14 clinical isolates. T3SS-related genes were distributed among the various serogroups and pulsed-field gel electrophoresis of NotI-digested genomes showed genetic diversity in these strains. However, the restriction fragment length polymorphism profiles of the T3SS-related genes had similar patterns. Additionally, naturally competent T3SS-negative V. cholerae incorporated the ca. 47 kb gene cluster of T3SS, which had been integrated into a site on the chromosome by recombination. Therefore, it is suggested that horizontal gene transfer of T3SS-related genes occurs among V. cholerae in natural ecosystems., (© 2013 The Societies and Wiley Publishing Asia Pty Ltd.)

The tfoX (also called sxy) gene product is the central regulator of DNA uptake in the naturally competent bacteria Haemophilus influenzae and Vibrio cholerae. However, the mechanisms regulating tfoX gene expression in both organisms are poorly understood. Our previous studies revealed that in V. cholerae, chitin disaccharide (GlcNAc)₂ is needed to activate the transcription and translation of V. cholerae tfoX (tfoX(VC)) to induce natural competence. In this study, we screened a multicopy library of V. cholerae DNA fragments necessary for translational regulation of tfoX(VC). A clone carrying the VC2078-VC2079 intergenic region, designated tfoR, increased the expression of a tfoX(VC)::lacZ translational fusion constructed in Escherichia coli. Using a tfoX(VC)::lacZ reporter system in V. cholerae, we confirmed that tfoR positively regulated tfoX(VC) expression at the translational level. Deletion of tfoR abolished competence for exogenous DNA even when (GlcNAc)₂ was provided. The introduction of a plasmid clone carrying the tfoR(+) gene into the tfoR deletion mutant complemented the competence deficiency. We also found that the tfoR gene encodes a 102-nucleotide small RNA (sRNA), which was transcriptionally activated in the presence of (GlcNAc)₂. Finally, we showed that this sRNA activated translation from tfoX(VC) mRNA in a highly purified in vitro translation system. Taking these results together, we propose that in the presence of (GlcNAc)₂, TfoR sRNA is expressed to activate the translation of tfoX(VC), which leads to the induction of natural competence.

A pathogenic marine bacterium Vibrio cholerae shows natural competence for genetic transformation in the presence of chitin, a polymer of N-acetylglucosamine (GlcNAc). In this study, we extensively analyzed the regulatory mechanisms of tfoX(VC), encoding an activator protein for the chitin-induced competence. Using a chromosomal tfoX(VC)-lacZ reporter system, we showed that a disaccharide of chitin, (GlcNAc)(2), at least was needed to activate both the transcription and translation of tfoX(VC). This activation was moderate at the transcriptional level but was strong at the translational level. We also identified two sequence elements, one for transcription and another for translation. The transcriptional control element (TCE) included a 34-bp potential transcriptional operator overlapped by the tfoX(VC) promoter, while the translational control element (TLE) consisted of a 42-bp sequence located within the 5'-untranslated region. Deletion of either TCE or TLE still resulted in (GlcNAc)(2)-dependent competence for exogenous DNA. However, the deletion in both elements induced competence for transformation at high efficiency regardless of the presence or absence of (GlcNAc)(2). These results suggested the dual activation of tfoX(VC) expression to be essential to induce competence. The highly transformable strain created here should aid the study of natural competence in V. cholerae., (Copyright 2010 Elsevier B.V. All rights reserved.)

We determined the genotype of cholera toxin by amplifying and sequencing the B-subunit in a sequential collection of 90 strains of Vibrio cholerae O139 isolated over the past 13 years since its first description in 1992. Representative strains isolated during 1993-1997 harboured ctxB of El Tor type (genotype 3). Twenty-six strains isolated during 1999, 2001, 2005 and three strains isolated in 1998, 2000 and 2002 were identified to belong to new ctxB genotypes 4 and 5, respectively. Genotype 5 was similar to genotype 1 except at position 28 (D-->A). The genotype 6 was similar to genotype 4 except at position 34 (H-->P). The implication of switch in terms of function of the toxin and its impact on human disease is unclear. How this change has influenced their prevalence relative to that of V. cholerae O1 in human infection is also not clear. The other common virulence gene clusters including the Vibrio pathogenicity island-1, Vibrio seventh pandemic island (VSP)-I and VSP-II of V. cholerae O139 did not show any remarkable difference from that of the O1 El Tor strains. Overall, the majority of the O139 strains tested in this study were similar to the El Tor strains but had altered ctxB genotype. This change and the impact that it causes to the epidemiology of cholera caused by O139 should be closely monitored.

The lambda Red-based recombination system is very useful for genetic manipulation of some Gram-negative bacteria. Here we report simple procedures for the inactivation and modification of genes of interest on Vibrio cholerae chromosome using this recombination technique. For this purpose, a polymerase chain reaction (PCR) fragment carrying an antibiotic resistance cassette flanked by regions homologous to the target locus was electroporated into recipient V. cholerae strains expressing a highly proficient lambda Red recombination system. Two PCR procedures were tested to generate an amplification product carrying an antibiotic resistance cassette flanked by short (50 or 100 nt) or long (1000 nt) homologous extensions, which allowed successful disruption of four chromosomal loci (ctxB, toxT, lacZ, and recA). Our results suggest that 100-nt homology between the PCR product and the target gene is sufficient to stimulate the lambda Red-dependent recombination. To increase recombination efficiency, however, the PCR procedure should be used to generate a product with 1000-nt homologous extensions. Furthermore, we applied this gene replacement method to create lacZ reporter fusion to the target gene. Transcriptional fusion to the V. cholerae ctxA gene was constructed using a PCR product that contains the 100-nt homologous extension to ctxA on each side of the lacZ::cat cassette, and was shown to respond appropriately to a null mutation in the regulatory gene, toxT. Use of the techniques presented here should prompt rapid and efficient mutagenesis/modification of V. cholerae chromosomal genes.

Cholera toxin (CT)-producing Vibrio cholerae O1 and O139 cause acute diarrheal disease and are proven etiological agents of cholera epidemics and pandemics. On the other hand, V. cholerae non-O1/non-O139 are designated as non-agglutinable (NAG) vibrios and are not associated with epidemic cholera. The majority of NAG vibrios do not possess the gene for CT (ctx). In this study, we isolated three NAG strains (strains No. 1, 2, and 3) with ctx from pond water in Kolkata, India, and examined their pathogenic properties. The enterotoxicity of the three NAG strains in vivo was examined using the rabbit ileal intestinal loop test. Strain No. 1 induced the accumulation of fluid in the loop, and the volume of fluid was reduced by simultaneous administration of anti-CT antiserum into the loop. The volume of fluid in the loop caused by strains No. 2 and 3 was small and undetectable, respectively. Then, we cultured these three strains in liquid medium in vitro at two temperatures, 25°C and 37°C, and examined the amount of CT accumulated in the culture supernatant. CT was accumulated in the culture supernatant of strain No.1 when the strain was cultured at 25°C, but that was low when cultured at 37°C. The CT amount accumulated in the culture supernatants of the No. 2 and No. 3 strains was extremely low at both temperature under culture conditions examined. In order to clarify the virulence properties of these strains, genome sequences of the three strains were analyzed. The analysis showed that there was no noticeable difference among three isolates both in the genes for virulence factors and regulatory genes of ctx. However, vibrio seventh pandemic island-II (VSP-II) was retained in strain No. 1, but not in strains No. 2 or 3. Furthermore, it was revealed that the genotype of the B subunit of CT in strain No. 1 was type 1 and those of strains No. 2 and 3 were type 8. Histopathological examination showed the disappearance of villi in intestinal tissue exposed to strain No. 1. In addition, fluid accumulated in the loop due to the action of strain No. 1 had hemolytic activity. This indicated that strain No. 1 may possesses virulence factors to induce severe syndrome when the strain infects humans, and that some strains of NAG vibrio inhabiting pond water in Kolkata have already acquired virulence, which can cause illness in humans. There is a possibility that these virulent NAG vibrios, which have acquired genes encoding factors involved in virulence of V. cholerae O1, may emerge in various parts of the world and cause epidemics in the future. [ABSTRACT FROM AUTHOR]

Non‐O1/non‐O139 nontoxigenic Vibrio cholerae associated with cholera‐like diarrhea has been reported in Kolkata, India. However, the property involved in the pathogenicity of these strains has remained unclear. The character of 25 non‐O1/non‐O139 nontoxigenic V. cholerae isolated during 8 years from 2007 to 2014 in Kolkata was examined. Determination of the serogroup showed that the serogroups O6, O10, O35, O36, O39, and O70 were represented by two strains in each serogroup, and the remaining isolates belonged to different serogroups. To clarify the character of antibiotic resistance of these isolates, an antibiotic resistance test and the gene analysis were performed. According to antimicrobial drug susceptibility testing, 13 strains were classified as drug resistant. Among them, 10 strains were quinolone resistant and 6 of the 13 strains were resistant to more than three antibiotics. To define the genetic background of the antibiotic character of these strains, whole‐genome sequences of these strains were determined. From the analysis of these sequences, it becomes clear that all quinolone resistance isolates have mutations in quinolone resistance‐determining regions. Further research on the genome sequence showed that four strains possess Class 1 integrons in their genomes, and that three of the four integrons are found to be located in their genomic islands. These genomic islands are novel types. This indicates that various integrons containing drug resistance genes are spreading among V. cholerae non‐O1/non‐O139 strains through the action of newly generated genomic islands. [ABSTRACT FROM AUTHOR]

We performed whole-genome sequencing of Vibrio cholerae O1 isolates from Laos, Thailand, and Vietnam, where cholera outbreaks occurred, to determine their genetic lineages. Core genome phylogenetic analysis revealed that the isolates located in same lineage without regional clusters, which suggests that closely related strains circulated in Southeast Asia. [ABSTRACT FROM AUTHOR]

Cholera is an acute diarrheal disease and a major public health problem in many developing countries in Asia, Africa, and Latin America. Since the Bay of Bengal is considered the epicenter for the seventh cholera pandemic, it is important to understand the genetic dynamism of Vibrio cholerae from Kolkata, as a representative of the Bengal region. We analyzed whole genome sequence data of V. cholerae O1 isolated from cholera patients in Kolkata, India, from 2007 to 2014 and identified the heterogeneous genomic region in these strains. In addition, we carried out a phylogenetic analysis based on the whole genome single nucleotide polymorphisms to determine the genetic lineage of strains in Kolkata. This analysis revealed the heterogeneity of the Vibrio seventh pandemic island (VSP)-II in Kolkata strains. The ctxB genotype was also heterogeneous and was highly related to VSP-II types. In addition, phylogenetic analysis revealed the shifts in predominant strains in Kolkata. Two distinct lineages, 1 and 2, were found between 2007 and 2010. However, the proportion changed markedly in 2010 and lineage 2 strains were predominant thereafter. Lineage 2 can be divided into four sublineages, I, II, III and IV. The results of this study indicate that lineages 1 and 2-I were concurrently prevalent between 2007 and 2009, and lineage 2-III observed in 2010, followed by the predominance of lineage 2-IV in 2011 and continued until 2014. Our findings demonstrate that the epidemic of cholera in Kolkata was caused by several distinct strains that have been constantly changing within the genetic lineages of V. cholerae O1 in recent years. [ABSTRACT FROM AUTHOR]

A loop-mediated isothermal amplification assay was developed. It was designed for recognizing Vibrio cholerae O1/O139, where atpA , rfbN , and wfbR genes were adopted. The assay specifically detected the target with sensitivities of 5–67 copies per reaction in 1 h. The assay will aid rapid detection of the cholera bacterium. • Multiple LAMP-based methods for rapid detection of cholera-causing agents were developed. • Combination of these LAMP methods can be applied in molecular diagnosis or detection of V. cholerae O1 or O139. [ABSTRACT FROM AUTHOR]

Background. From 2003 through 2007, Vibrio cholerae serogroup O75 strains possessing the cholera toxin gene were isolated from 6 patients with severe diarrhea, including 3 in Georgia, 2 in Alabama, and 1 in South Carolina. These reports represent the first identification of V. cholerae O75 as a cause of illness in the United States. V. cholerae O75 was isolated from a water sample collected from a pond in Louisiana in 2004. Subsequently, 3 V. cholerae isolates from Louisiana (2 from patients with diarrhea in 2000 and 1 from a water sample collected in 1978) that had been previously reported as serogroup O141 were also discovered to be serogroup O75. Results. All 8 patients who were infected with V. cholerae O75 were adults who became ill after consuming seafood; 2 had eaten raw oysters traced back to the Gulf Coast of the United States. All 10 isolates possessed the cholera toxin gene and were susceptible to 10 antimicrobials. One clinical isolate and 1 environmental (water) isolate had the same pulsed-field gel electrophoresis pattern; 4 clinical isolates shared a common pulsed-field gel electrophoresis pattern. Conclusions. The occurrence of these cases over many years and the concurrent identification of V. cholerae O75 in water from a Gulf Coast state suggest that these strains may survive for long periods in this environment. The patients' exposure histories suggest that infection can be acquired from consumption of raw oysters from the Gulf Coast. Clinicians and public health authorities should be vigilant for the occurrence of new toxigenic serogroups of V. cholerae that are capable of causing severe diarrhea. [ABSTRACT FROM AUTHOR]

Abstract: A novel genotyping method for toxigenic Vibrio cholerae O1 El Tor and O139 was developed. The method was designed to amplify DNA sequences “sandwiched“ between any given pair of repetitive sequences, “V. cholera repeats (VCR)”, in highly polymorphic “integron island” of ca. 125 kb in the small chromosome of toxigenic V. cholerae so that the resultant PCR amplicons would present with a strain-specific electrophoretic pattern. The VCR-targeted PCR assay (VCR-PCR) for 37 strains of toxigenic V. cholerae O1 El Tor and O139 revealed that the O1 strains isolated before 1990 showed distinct clonality whereas those isolated after 1990 could be separated into two clones, one consisting of strains isolated from South American countries and another of those from other countries. By contrast, O139 strains were genotypically homogenous regardless of the geographic origin or time of isolation. VCR-PCR therefore would be a robust but rapid method for genotypic differentiation of toxigenic V. cholerae O1 El Tor and O139 strains and to recognize strains with epidemic potential. [Copyright &y& Elsevier]

Cholera epidemics have been recorded periodically in Vietnam during the seventh cholera pandemic. Since cholera is a water-borne disease, systematic monitoring of environmental waters for Vibrio cholerae presence is important for predicting and preventing cholera epidemics. We conducted monitoring, isolation, and genetic characterization of V. cholerae strains in Nam Dinh province of Northern Vietnam from Jul 2013 to Feb 2015. In this study, four V. cholerae O1 strains were detected and isolated from 110 analyzed water samples (3.6%); however, none of them carried the cholera toxin gene, ctxA , in their genomes. Whole genome sequencing and phylogenetic analysis revealed that the four O1 isolates were separated into two independent clusters, and one of them diverged from a common ancestor with pandemic strains. The analysis of pathogenicity islands (CTX prophage, VPI-I, VPI-II, VSP-I, and VSP-II) indicated that one strain (VNND_2014Jun_6SS) harbored an unknown prophage-like sequence with high homology to vibriophage KSF-1 phi and VCY phi, identified from Bangladesh and the USA, respectively, while the other three strains carried tcpA gene with a distinct sequence demonstrating a separate clonal lineage. These results suggest that the aquatic environment can harbor highly divergent V. cholera strains and serve as a reservoir for multiple V. cholerae virulence-associated genes which may be exchanged via mobile genetic elements. Therefore, continuous monitoring and genetic characterization of V. cholerae strains in the environment should contribute to the early detection of the sources of infection and prevention of cholera outbreaks as well as to understanding the natural ecology and evolution of V. cholerae . [ABSTRACT FROM AUTHOR]