Draft Whole-Genome Sequence of a New Variant of Vibrio cholerae O1 El Tor Strain Isolated from a Cholera Patient in Russia.
ABSTRACT: Draft whole-genome sequencing of the Vibrio cholerae ?1 El Tor clinical strain L3226, isolated in Moscow in 2010, was carried out. Various mutations in the virulence-associated mobile elements were determined in its genome that differentiated this strain from the reference V. cholerae ?1 El Tor strain N16961.
Project description:Cholera outbreaks in subSaharan African countries are caused by strains of the El Tor biotype of toxigenic Vibrio cholerae O1. The El Tor biotype is the causative agent of the current seventh cholera pandemic, whereas the classical biotype, which was associated with the sixth pandemic, is now extinct. Besides other genetic differences the CTX prophages encoding cholera toxin in the two biotypes of V. cholerae O1 have distinct repressor (rstR) genes. However, recent incidences of cholera in Mozambique were caused by an El Tor biotype V. cholerae O1 strain that, unusually, carries a classical type (CTX(class)) prophage. We conducted genomic analysis of the Mozambique strain and its CTX prophage together with chromosomal phage integration sites to understand the origin of this atypical strain and its evolutionary relationship with the true seventh pandemic strain. These analyses showed that the Mozambique strain carries two copies of CTX(class) prophage located on the small chromosome in a tandem array that allows excision of the prophage, but the excised phage genome was deficient in replication and did not produce CTX(class) virion. Comparative genomic microarray analysis revealed that the strain shares most of its genes with the typical El Tor strain N16961 but did not carry the TLC gene cluster, and RS1 sequence, adjacent to the CTX prophage. Our data are consistent with the Mozambique strain's having evolved from a progenitor similar to the seventh pandemic strain, involving multiple recombination events and suggest a model for origination of El Tor strains carrying the classical CTX prophage.
Project description:Pandemic V. cholerae strains in the O1 serogroup have 2 biotypes: classical and El Tor. The classical biotype strains of the sixth pandemic, which encode the classical type cholera toxin (CT), have been replaced by El Tor biotype strains of the seventh pandemic. The prototype El Tor strains that produce biotype-specific cholera toxin are being replaced by atypical El Tor variants that harbor classical cholera toxin. Atypical El Tor strains are categorized into 2 groups, Wave 2 and Wave 3 strains, based on genomic variations and the CTX phage that they harbor. Whole-genome analysis of V. cholerae strains in the seventh cholera pandemic has demonstrated gradual changes in the genome of prototype and atypical El Tor strains, indicating that atypical strains arose from the prototype strains by replacing the CTX phages. We examined the molecular mechanisms that effected the emergence of El Tor strains with classical cholera toxin-carrying phage. We isolated an intermediary V. cholerae strain that carried two different CTX phages that encode El Tor and classical cholera toxin, respectively. We show here that the intermediary strain can be converted into various Wave 2 strains and can act as the source of the novel mosaic CTX phages. These results imply that the Wave 2 and Wave 3 strains may have been generated from such intermediary strains in nature. Prototype El Tor strains can become Wave 3 strains by excision of CTX-1 and re-equipping with the new CTX phages. Our data suggest that inter-chromosomal recombination between 2 types of CTX phages is possible when a host bacterial cell is infected by multiple CTX phages. Our study also provides molecular insights into population changes in V. cholerae in the absence of significant changes to the genome but by replacement of the CTX prophage that they harbor.
Project description:Vibrio cholerae O1 has two biotypes, El Tor and Classical, and the latter is now presumed to be extinct in nature. Under carbohydrate-rich growth conditions, El Tor biotype strains produce the neutral fermentation end product 2,3-butanediol (2,3-BD), which prevents accumulation of organic acids from mixed acid fermentation and thus avoids a lethal decrease in the medium pH, while the Classical biotype strains fail to do the same. In this study, we investigated the inhibitory effect of 2,3-BD on the production of two proinflammatory biomarkers, intreleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-?), in human intestinal epithelial HT29 and alveolar epithelial A549 cells. Cell-free culture supernatants of El Tor strain N16961 grown in LB supplemented with 1% glucose induced a negligible amount of IL-8 or TNF-?, while the Classical O395 strain induced much higher levels of these proinflammatory cytokines. On the other hand, three mutant strains constructed from the N16961 strain with defects in the constitutive 2,3-BD pathway were also able to induce high levels of cytokines. When HT29 and A549 cells were treated with bacterial flagella, known proinflammatory cytokine inducers, and chemically synthesized 2,3-BD at various concentrations, a dose-dependent decrease in IL-8 and TNF-? production was observed, demonstrating the suppressive effect of 2,3-BD on the production of proinflammatory cytokines in epithelial cells. Upon cotreatment with extraneous 2,3-BD, elevated levels of I?B?, the inhibitor of the NF-?B pathway, were detected in both HT29 and A549 cells. Furthermore, treatments containing 2,3-BD elicited lower levels of NF-?B-responsive luciferase activity, demonstrating that the reduced cytokine production is likely through the inhibition of the NF-?B pathway. These results reveal a novel and potential role of 2,3-BD as an immune modulator that might have conferred a superior pathogenic potential of the El Tor over the Classical biotype.
Project description:<i>Vibrio cholerae</i> is a global health threat and a model enteric pathogen that causes the human disease cholera. Here, we report the complete genome sequence of the seventh-pandemic <i>V. cholerae</i> O1 El Tor strain C6706.
Project description:Vibrio cholerae O139 is a recently identified non-O1 V. cholerae strain responsible for outbreaks of epidemic cholera in India, Bangladesh, and Thailand in the past 2 years. Other workers have demonstrated the presence of the cholera toxin genetic element in V. cholerae O139, unlike the situation for other non-O1 V. cholerae strains. We sought to compare further this strain with strains of V. cholerae O1, classical and El Tor biotypes, by classic microbiologic methods, Southern blot analysis for restriction fragment length polymorphisms with probes for iron-regulated genes of V. cholerae O1, and comparisons of outer membrane protein profiles. Our results were similar for V. cholerae O139 and the El Tor biotype of V. cholerae O1, with the exception of the constitutive expression in V. cholerae O139 of OmpS, an outer membrane protein that was maltose inducible in comparison strains of V. cholerae O1.
Project description:Antimicrobial peptides play an important role in host defense against Vibrio cholerae Generally, the V. cholerae O1 classical biotype is polymyxin B (PB) sensitive and El Tor is relatively resistant. Detection of classical biotype traits like the production of classical cholera toxin and PB sensitivity in El Tor strains has been reported in recent years, including in the devastating Yemen cholera outbreak during 2016-2018. To investigate the factor(s) responsible for the shift in the trend of sensitivity to PB, we studied the two-component system encoded by carRS, regulating the lipid A modification of El Tor vibrios, and found that only carR contains a single nucleotide polymorphism (SNP) in recently emerged PB-sensitive strains. We designated the two alleles present in PB-resistant and -sensitive strains carR r and carR s alleles, respectively, and replaced the carR s allele of a sensitive strain with the carR r allele, using an allelic-exchange approach. The sensitive strain then became resistant. The PB-resistant strain N16961 was made susceptible to PB in a similar fashion. Our in silico CarR protein models suggested that the D89N substitution in the more stable CarRs protein brings the two structural domains of CarR closer, constricting the DNA binding cleft. This probably reduces the expression of the carR-regulated almEFG operon, inducing PB susceptibility. Expression of almEFG in PB-sensitive strains was found to be downregulated under natural culturing conditions. In addition, the expression of carR and almEG decreased in all strains with increased concentrations of extracellular Ca2+ but increased with a rise in pH. The downregulation of almEFG in CarRs strains confirmed that the G265A mutation is responsible for the emergence of PB-sensitive El Tor strains.
Project description:Vibrio cholerae is a Gram-negative waterborne human pathogen and the causative agent of cholera. Here, we present the complete genome sequence of the seventh pandemic O1 biovar El Tor Inaba strain A1552 isolated in 1992. This clinical strain has served as an important model strain for studying cholera pathogenicity traits.
Project description:Vibrio cholerae O1 serogroup strains have been classified into classical and El Tor biotypes. Cholera, a life-threatening diarrheal disease, can be caused by either biotype through the cholera toxin (CT) that they produce. To increase our knowledge of the pathogenicity of bacteria, we must understand the toxigenicity of bacteria. CT production by classical biotype strains in simple single-phase cell cultures has been established; however, special culture media and growth conditions that are not appropriate for mass production of CT are required to facilitate CT production in El Tor biotype strains. In this report, we produced CT in El Tor biotype strains using simple media and single-phase culture conditions. A single point mutation in ToxT, a transcriptional activator of toxin co-regulated pilus (TCP) and CT, enabled the El Tor biotype strains to produce CT in similar quantities as classical biotype strains in single-phase laboratory culture conditions. CT production capacity varied between El Tor biotype strains. Wave 2 and 3 atypical El Tor strains tended to produce more CT than prototype Wave 1 strains. Wave 2 and 3 strains lack neutral fermentation; however, the capacity for neutral fermentation was not associated with significant differences in CT production by El Tor biotype strains. The Wave 3 strain that caused the 2010 cholera outbreak in Haiti produced CT only when neutral fermentation was abolished. The disparity in CT production between the seventh cholera pandemic strains highlight the differences in virulence between strains and the cause of population changes in V. cholerae.
Project description:The 2 biotypes of Vibrio cholerae O1 serogroup strains-classical and El Tor-use glucose in distinct ways. Classical biotype strains perform organic acid-producing fermentation and eventually lose viability due to the self-induced creation of an acidic environment, whereas El Tor biotype strains use an alternative neutral fermentation pathway, which confers them with survival advantages. However, we report that the neutral fermentation pathway has only been recruited in prototype Wave 1 El Tor biotype strains, which have not been isolated since the mid-1990s. Current Wave 2 and Wave 3 atypical El Tor strains contain a single-base deletion in a gene that directs bacteria toward neutral fermentation, resulting in the loss of neutral fermentation and an appearance that is similar to classical biotype strains. Moreover, when sufficient glucose was supplied, Wave 1 El Tor strains maintained their use of acid-producing fermentation, in parallel with neutral fermentation, and thus lost viability in the late stationary phase. The global replacement of Wave 1 El Tor strains by Wave 2 and 3 atypical El Tor strains implies that the acidic fermentation pathway may not be disadvantageous to V. cholerae. The characteristics that we have reported might improve oral rehydration in the treatment of cholera.
Project description: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.