Cholerae O1 El Tor Strains Research Articles

Comparative analysis of the structure of regulatory genes of Vibrio cholerae serotype О1 biotype El Tor strains

Introduction. The expression of the ctxAB and tcpA-F genes encoding the main pathogenicity factors of the Vibrio cholerae is controlled by regulatory genes. The structure of these genes has not been fully studied in the pathogen strains isolated during different periods of the current pandemic. The aim of the study was a comparative analysis of the structure of regulatory genes of V. cholerae O1 biovar El Tor strains isolated on the territory of the Russian Federation and neighboring countries during the seventh cholera pandemic. Materials and methods. The nucleotide sequences of the complete genomes of 29 toxigenic strains isolated from 1970 to 2023 were analyzed. The analysis was carried out using BioEdit v7.2.6.1 software and Blast tool. Results. The analysis of ten regulatory genes (toxT, aphA, aphB, hns, hapR, vieA, luxO, luxT, carS, carR) was carried out. Almost all strains were found to have a thymine insertion in the hapR gene at position 219. The exception was V. cholerae strain M3208 (Tambov, 2023), which had an insertion of five nucleotides in this gene. Mutations of the luxO gene with an unknown effect were detected in 44.8% of the studied strains. In 46.7% and 33.3% of the studied genetic variants carrying the ctxB1 allele, non-synonymous substitutions were detected in the hns (G319A) and vieA (C235T) genes, respectively. All genetic variants with the ctxB7 allele have mutations in both the hns and vieA genes. Three genetic variants with the ctxB7 allele, imported to the Russian Federation in recent years, contain an altered structure of the carR gene (G265A). Conclusion. The structure of genes (toxT, aphA, aphB, carS, luxT, hapR) of V. cholerae O1 El Tor strains remains unchanged for the majority of the studied isolates. At the same time, variability in the hns (G319A), vieA (C235T) and carR (G256A) genes was detected. Mutations in these genes can be used as genetic markers of modern V. cholerae O1 El Tor genetic variants.

Vibriocidal efficacy of Bifidobacterium bifidum and Lactobacillus acidophilus cell-free supernatants encapsulated in chitosan nanoparticles against multi-drug resistant Vibrio cholerae O1 El Tor

BackgroundCholera is a diarrheal disease recognized for being caused by toxin-producing Vibrio (V.) cholerae. This study aims to assess the vibriocidal and immunomodulatory properties of derived cell-free supernatants (CFSs) of Bifidobacterium (B.) bifidum and Lactobacillus (L.) acidophilus encapsulated in chitosan nanoparticles (CFSb-CsNPs and CFSa-CsNPs) against clinical multi-drug resistance (MDR) isolates of V. cholerae O1 El Tor.MethodsWe synthesized CFSb-CsNPs and CFSa-CsNPs using the ionic gelation technique. The newly nanostructures were characterized for size, surface zeta potential, morphology, encapsulation efficacy (EE), stability in different pH values and temperatures, release profile, and in vitro cytotoxicity. The antimicrobial and antibiofilm effects of the obtained nanocomposites on clinical MDR isolates (N = 5) of V. cholerae E1 Tor O1 were investigated by microbroth dilution assay and crystal violet staining, respectively. We conducted quantitative real-time PCR (qRT-PCR) to analyze the relative gene expressions of Bap, Rbmc, CTXAB, and TCP in response to CFSb-CsNPs and CFSa-CsNPs. Additionally, the immunomodulatory effects of formulated structures on the expression of TLR2 and TLR4 genes in human colorectal adenocarcinoma cells (Caco-2) were studied.ResultsNano-characterization analyses indicated that CFSb-CsNPs and CFSa-CsNPs exhibit spherical shapes under scanning electron microscopy (SEM) imaging, with mean diameters of 98.16 ± 0.763 nm and 83.90 ± 0.854 nm, respectively. Both types of nanoparticles possess positive surface charges. The EE% of CFSb-CsNPs was 77 ± 4.28%, whereas that of CFSa-CsNPs was 62.5 ± 7.33%. Chitosan (Cs) encapsulation leads to increased stability of CFSs in simulated pH conditions of the gastrointestinal tract in which the release rates for CFSb-CsNPs and CFSa-CsNPs were reached at 58.00 ± 1.24% and 55.01 ± 1.73%, respectively at pH = 7.4. The synergistic vibriocidal effects observed from the co-administration of both CFSb-CsNPs and CFSa-CsNPs, as evidenced by a fractional inhibitory concentration (FIC) index of 0.57, resulting in a significantly lower MIC of 2.5 ± 0.05 mg/mL (p < 0.0001) compare to individual administration. The combined antibacterial effect of CFSb-CsNPs and CFSa-CsNPs on Bap (0.14 ± 0.05), Rbmc (0.24 ± 0.01), CTXAB (0.30 ± 0.09), and TCP (0.38 ± 0.01) gene expression was significant (p < 0.001). Furthermore, co-administration of CFSb-CsNPs and CFSa-CsNPs also demonstrated the potency of suppressing TLR 2/4 (0.20 ± 0.01 and 0.12 ± 0.02, respectively) gene expression (p = 0.0019) and reduced Caco-2 cells attached bacteria to 526,000 ± 51,46 colony-forming units/mL (11.19%) (p < 0.0001).ConclusionOur study revealed that encapsulating CFSs within CsNPs enhances their vibriocidal activity by improving stability and enabling a controlled release mechanism at the site of interaction between the host and bacteria. Additionally, the simultaneous use of CFSb-CsNPs and CFSa-CsNPs exhibited superior vibriocidal potency against MDR V. cholerae O1 El Tor strains, indicating these combinations as a potential new approach against MDR bacteria.

Elevated concentrations of polymyxin B elicit a biofilm-specific resistance mechanism in Vibrio cholerae

Vibrio cholerae can form biofilms in the aquatic environment and in the human intestine, facilitating the release of hyper-infectious aggregates. Due to the increasing antibiotic resistance, alternatives need to be found. One of these alternatives is antimicrobial peptides, including polymyxin B (PmB). In this study, we first investigated the resistance of V. cholerae O1 El Tor strain A1552 to various antimicrobials under aerobic and anaerobic conditions. An increased resistance to PmB is observed in anaerobiosis, with a 3-fold increase in the dose required for 50 % growth inhibition. We then studied the impact of the PmB on the formation and the degradation of V. cholerae biofilms to PmB. Our results show that PmB affects more efficiently biofilm formation under anaerobic conditions. On the other hand, preformed biofilms are susceptible to degradation by PmB at concentrations close to the minimal inhibitory concentration. At higher concentrations, we observe an opacification of the biofilm structures within 20 min post-treatment, suggesting a densification of the structure. This densification does not seem to result from the overexpression of matrix genes but rather from DNA release through massive cell lysis, likely forming a protective shield that limits the penetration of the PmB into the biofilm.

Analysis of Antiphage Systems in Vibrio Cholerae O1 El Tor Biotype Strains

Introduction: Cholera lytic phages contribute to the genetic diversity and evolution of Vibrio cholerae. To protect against the phages, the pathogen has acquired various resistance mechanisms. Objective: To identify antiphage systems located on mobile genetic elements in V. cholerae serogroup O1 El Tor biotype strains. Materials and methods: Nucleotide sequences of complete genomes of 77 toxigenic V. cholerae O1 El Tor strains imported to the Russian Federation and neighboring countries in 1970–2014 were analyzed using the Blast NCBI GenBank algorithm and REALPHY online tool. Results: We observed that the examined strains contained two types of anti-phage systems in hotspot 5 of the ICE SXT element: BREX, common for ICE VchBan9, and BREX with Abi typical of ICE VchInd5. We established a direct relationship between the presence of the PLE4 antiphage island and the kappa phage. V. cholerae O1 El Tor strains containing PLE4, except for one isolate, have BREX ICE VchBan9 and are grouped into a separate cluster in phylogenetic analysis. Strains with ICE VchInd5 lacking PLE4 and kappa phage also form a separate group. Conclusions: The data obtained on the presence of antiphage systems in previously imported strains of V. cholerae O1 biotype El Tor expand knowledge of their genetic organization. The study of the structure of antiphage genes of hotspot 5 of the ICE SXT element makes it possible to reveal genetic differences between closely related strains of V. cholerae O1 biotype El Tor and to determine the type of ICE SXT element.

Sequential outbreaks due to mixed ctxB alleles of Vibrio cholerae O1 in Mayurbhanj district of Odisha, India.

Cholera is a significant threat causing outbreaks/epidemics with high morbidity and mortality in coastal and tribal districts of Odisha. A sequential cholera outbreak reported from four places in Mayurbhanj district of Odisha during June to July 2009 was investigated. Rectal swabs from diarrhea patients were analyzed for the identification, antibiogram profiles and detection of ctxB genotypes by double mismatch amplification mutation (DMAMA) polymerase chain reaction (PCR) assays and sequenced. The different virulent and drug resistant genes were detected by multiplex PCR assays. The clonality analysis on selected strains was done by pulse field gel electrophoresis (PFGE). Bacteriological analysis of rectal swabs revealed the presence of V. cholerae O1 Ogawa biotype El Tor which were resistant to co-trimoxazole, chloramphenicol, streptomycin, ampicillin, nalidixic acid, erythromycin, furazolidone and polymyxin B. DMAMA-PCR assay revealed that the cholera outbreak in Mayurbhanj district was due to both ctxB1 and ctxB7 alleles of V. cholerae O1 El Tor strains. All the V. cholerae O1 strains were positive for all virulence genes. The multiplex PCR assay on V. cholerae O1 strains revealed the presence of antibiotic resistance genes like dfrA1 (100%), intSXT (100%), sulII (62.5%) and StrB (62.5%). PFGE results on V. cholerae O1 strains exhibited two different pulsotypes with 92% similarity. This outbreak was a transition phase where both ctxB genotypes were prevalent after which the ctxB7 genotype gradually became dominant in Odisha. Therefore, close monitoring and continuous surveillance on diarrheal disorders is essential to prevent the future diarrheal outbreaks in this region.

Molecular characterization of Vibrio cholerae O1 El Tor strains in Malaysia revealed genetically diverse variant lineages.

Vibrio cholerae O1 El Tor variants have been the major causative agents of cholera worldwide since their emergence in the 2000s. Cholera remains endemic in some regions in Malaysia. Therefore, we aimed to investigate the genetic characteristics of the V. cholerae O1 El Tor strains associated with outbreaks and sporadic cases to elucidate the molecular evolution among the strains circulating in this region. A total of 45 V. cholerae O1 El Tor strains isolated between 1991 and 2011 were examined. All strains were subjected to phenotypic characterization, and molecular characterization including detection of virulence genes and CTX prophage (CTXΦ) by Polymerase Chain Reaction (PCR) and genotyping by Pulsed-Field Gel Electrophoresis (PFGE). All strains were phenotypically confirmed as El Tor biotype and were mostly Ogawa serotype (96%). Antimicrobial susceptibility testing showed that the outbreak strains isolated in 1991 (Sabah) and 2009 (Terengganu) were all multidrug-resistant while the sporadic strains were resistant to erythromycin and furazolidone only. All strains (n=45) were resistant to erythromycin. The virulence genes ctxA, ctxB, ompW, rfb, rtxC, tcpA, tcpI, rstR, zot and hlyA were present in all strains. The outbreak strains isolated in 1991 harboured El Tor cholera toxin gene (ctxB3) while sporadic strains from 2004 to 2011 harboured classical ctxB1. Four distinctive CTXΦ arrays were identified among the El Tor variants, one of which co-occurred with El Tor strains during the 2009 outbreak in Terengganu. PFGE analysis revealed that a genetically diverse El Tor variants population persisted in Sabah. The co-existence of multiple El Tor variants together with the prototypic El Tor strains suggested a multiclonal emergence of V. cholerae O1 El Tor variants in this region.

Characteristics of the Epidemiological Situation on Cholera in the World and in the Russian Federation in 2020 and Forecast for 2021

Aim of the work – to assess the epidemiological situation on cholera in 2020 and to make a forecast for 2021 based on the monitoring data and analysis of morbidity around the world for the period of 2011–2020. During the period between 2011 and 2020, 4 413 988 cases of cholera were recorded in 97 countries of the world with a general trend towards a decrease in the incidence (coefficient of accuracy of approximation R2 – 0.5705). However, due to the continuing epidemic manifestations of cholera in the endemic countries of Asia, Africa and America, the epidemiological situation on cholera on these continents was characterized as unfavorable in 2020. The emergence of a new “post-Haitian” lineage was observed among epidemically hazardous strains of Vibrio cholerae O1. In 2020, no epidemically dangerous strains of V. cholerae O1, O139 were isolated from humans on the territory of the Russian Federation. 25 non-toxigenic V. cholerae O1 El Tor strains were isolated from environmental objects, eight out of which (ctxA-, tcpA+), according to PCR-INDEL typing and SNP analysis of sequences, belonged to the clonal complex. The results of the analysis of biological properties and phylogenetic relations between the isolated non-toxigenic strains provided the basis for considering the epidemiological situation on cholera in Russia in 2020 as a stable one and a similar forecast of its development in 2021. At the same time, the possibility of importation of this infection from endemic countries cannot be ruled out, as well as the need to carry out a complex of differentiated anti-epidemic (preventive) measures within the framework of the state sanitary-epidemiological surveillance in order to localize and eliminate the imported focus and avoid the spread of the infection.

Genome Sequence of a Pathogenic Vibrio cholerae O1 El Tor Strain Defective for the Entire Vibrio Pathogenicity Island 1, Isolated in Eastern Democratic Republic of the Congo.

We report here a complete genome sequence of a Vibrio cholerae O1 El Tor (Inaba; sequence type 515 [ST515]) strain isolated from a cholera patient in North Kivu Province, Democratic Republic of the Congo (DRC), which showed a complete deletion (∼80 kb) of the Vibrio pathogenicity island 1.

Spatial Visualization of Data on the Antibiotic Resistance of Vibrio cholerae Strains Isolated in Russia

Aim. The aim was to develop an integrated online and updated geographic information system (GIS) for the systematiza tion and analysis of information on V. cholerae El Tor antibiotic resistance.Material and methods. The method of serial dilutions in a dense nutrient medium (MUK 4.2.2495-09) was used to determine the sensitivity/resistance to 22 antibacterial preparations of V. cholerae O1 El Tor strains isolated from people and from aquatic environmental objects in Russia in 2005–2016. The development of the Internet version of the GIS was carried out using programming languages HTML, JavaScript and PHP and cartographic data obtained from the corporation Rostelecom (Russia) and the community OpenStreetMap.Results. A comparative analysis of the antibiotic resistance of the strains by regions of the Russian Federation and by isolation time showed an increase of strains resistant to streptomycin, ampicillin, rifampicin and furazolidone, the appearance of cultures resistant to nalidixic acid and ceftriaxone, reduced resistance to trimethoprim/sulfamethoxazole in 2012–2016 compared to 2005–2009. In the Stavropol Krai, the isolated V. cholerae O1 El Tor were characterized by resistance to furazolidone (33.3%) and trimethoprim/sulfamethoxazole (100%); in the Primorsky Krai — to ampicillin, streptomycin, rifampicin (7%), furazolidone (43%), trimethoprim/sulfamethoxazole (100%); in the Irkutsk region and Kalmykia, to ampicillin (8.3% and 11%), furazolidone and trimethoprim/sulfamethoxazole (11% and 89%).Conclusion. The GIS developed allows to collect and analyze information on the antibiotic resistance of V. cholerae O1 El Tor, and to select the strains for given properties.

Spatial Visualization of Data on the Antibiotic Resistance of Vibrio cholerae Strains Isolated in Russia

Aim. The aim was to develop an integrated online and updated geographic information system (GIS) for the systematiza tion and analysis of information onV. choleraeEl Tor antibiotic resistance.Material and methods. The method of serial dilutions in a dense nutrient medium (MUK 4.2.2495-09) was used to determine the sensitivity/resistance to 22 antibacterial preparations of V. cholerae O1 El Tor strains isolated from people and from aquatic environmental objects in Russia in 2005–2016. The development of the Internet version of the GIS was carried out using programming languages HTML, JavaScript and PHP and cartographic data obtained from the corporation Rostelecom (Russia) and the community OpenStreetMap.Results. A comparative analysis of the antibiotic resistance of the strains by regions of the Russian Federation and by isolation time showed an increase of strains resistant to streptomycin, ampicillin, rifampicin and furazolidone, the appearance of cultures resistant to nalidixic acid and ceftriaxone, reduced resistance to trimethoprim/sulfamethoxazole in 2012–2016 compared to 2005–2009. In the Stavropol Krai, the isolatedV. choleraeO1 El Tor were characterized by resistance to furazolidone (33.3%) and trimethoprim/sulfamethoxazole (100%); in the Primorsky Krai — to ampicillin, streptomycin, rifampicin (7%), furazolidone (43%), trimethoprim/sulfamethoxazole (100%); in the Irkutsk region and Kalmykia, to ampicillin (8.3% and 11%), furazolidone and trimethoprim/sulfamethoxazole (11% and 89%).Conclusion. The GIS developed allows to collect and analyze information on the antibiotic resistance ofV. choleraeO1 El Tor, and to select the strains for given properties.

Genetic Diversity of Vibrio cholerae О1 El Tor during Epidemic Complications in the Siberian and Far East Regions

The epidemic of cholera in Siberia and the Russian Far East during the seventh pandemic was recorded as sporadic cases of introduction of the infection into the territory or severe local outbreaks associated with the infection’s importation in the 1970s and 1990s. The genetic diversity and relationship of 83 V. cholerae O1 El Tor strains isolated from the region with epidemic complications were studied on the basis of the multilocus analysis of the variable number tandem repeats at five loci: VcA, VcB, VcC, VcD, and VcG. The VcB locus was characterized by the maximum polymorphism in the sample of the studied strains, while the VcC locus displayed the minimum polymorphism. Cluster analysis revealed the differentiation of V. cholerae strains during epidemic complications into seven clusters depending on the structure of the pathogenic determinants (typical and atypical El Tor vibrios), as well as on the time and direction of the pathogen importation. In some outbreaks in the Far East in 1999, the heterogeneity of the etiological agent populations was shown (five genotypes were detected among the outbreak strains from Yuzhno-Sakhalinsk, and six genotypes were determined among the strains from Primorskii krai). At the same time, the predominant MLVA profiles and their single locus variants were identified. The absence of a strict connection between the territory and the genotypes of V. cholerae outbreak strains (one strain from Yuzhno-Sakhalinsk showed the identity of the allele profile with the isolates from Vladivostok), as well as no correspondence of the V. cholerae genotype to a specific infection focus, was shown. According to the data of the MLVA typing, the V. cholerae O1 El Tor strains isolated from the Siberian and Far Eastern regions (Russia) with epidemic complications were characterized by the heterogeneity of their genetic organization, which agrees with the period and direction of their importation to the territory. During sporadic outbreaks of cholera, the clonality of the etiological agent was revealed. Closely related subclones, the emergence of which could result from the presence of the causative agent in the environment or passaging through a susceptible organism, were identified.

Long-Read-Based Genome Sequences of Pandemic and Environmental Vibrio cholerae Strains.

The bacterium Vibrio cholerae exhibits two distinct lifestyles, one as an aquatic bacterium and the other as the etiological agent of the pandemic human disease cholera. Here, we report closed genome sequences of two seventh pandemic V. cholerae O1 El Tor strains, A1552 and N16961, and the environmental strain Sa5Y.

Vibrio cholerae accessory colonisation factor AcfC: a chemotactic protein with a role in hyperinfectivity

Vibrio cholerae O1 El Tor is an aquatic Gram-negative bacterium responsible for the current seventh pandemic of the diarrheal disease, cholera. A previous whole-genome analysis on V. cholerae O1 El Tor strains from the 2010 epidemic in Pakistan showed that all strains contained the V. cholerae pathogenicity island-1 and the accessory colonisation gene acfC (VC_0841). Here we show that acfC possess an open reading frame of 770 bp encoding a protein with a predicted size of 28 kDa, which shares high amino acid similarity with two adhesion proteins found in other enteropathogens, including Paa in serotype O45 porcine enteropathogenic Escherichia coli and PEB3 in Campylobacter jejuni. Using a defined acfC deletion mutant, we studied the specific role of AcfC in V. cholerae O1 El Tor environmental survival, colonisation and virulence in two infection model systems (Galleria mellonella and infant rabbits). Our results indicate that AcfC might be a periplasmic sulfate-binding protein that affects chemotaxis towards mucin and bacterial infectivity in the infant rabbit model of cholera. Overall, our findings suggest that AcfC contributes to the chemotactic response of WT V. cholerae and plays an important role in defining the overall distribution of the organism within the intestine.

ANTIBIOTIC RESISTANCE OF VIBRIO CHOLERAE O1 EL TOR ISOLATED ON THE TERRITORY OF THE RUSSIAN FEDERATION IN 2006-2015

Aim of the study. The analysis of antibiotic resistance and molecular mechanisms of the persistence of Vibrio cholerae О1 El Tor strains, isolated from patients and environmental objects on the territory of the Russian Federation in 2006-2015. Material and Methods. The susceptibility of 34 V. cholerae El Tor strains to 13 antibacterial drugs was determined by the method of serial dilutions in a rich culture medium (Instructional Guidelines (МUК) 4.2.2495-09). Results. The strains showed resistance to 1-5 antibiotics (levomicetin, streptomycin, nalidixic acid, furazolidone, trimethoprim/sulfamethoxazole) and harbored SXT-element with antibiotic resistance genes. The emergence of quinolone resistance in V. cholerae O1 El Tor strains with accompanying multiple drug resistance limits the choice for drugs for etiotropic therapy and aggravates the possibility of unfavourable cholera outcome.

A novel phase variant of the cholera pathogen shows stress-adaptive cryptic transcriptomic signatures.

BackgroundIn a process known as phase variation, the marine bacterium and cholera pathogen Vibrio cholerae alternately expresses smooth or rugose colonial phenotypes, the latter being associated with advanced biofilm architecture and greater resistance to ecological stress. To define phase variation at the transcriptomic level in pandemic V. cholerae O1 El Tor strain N16961, we compared the RNA-seq-derived transcriptomes among the smooth parent N16961, its rugose derivative (N16961R) and a smooth form obtained directly from the rugose at high frequencies consistent with phase variation (N16961SD).ResultsDifferentially regulated genes which clustered into co-expression groups were identified for specific cellular functions, including acetate metabolism, gluconeogenesis, and anaerobic respiration, suggesting an important link between these processes and biofilm formation in this species. Principal component analysis separated the transcriptome of N16961SD from the other phase variants. Although N16961SD was defective in biofilm formation, transcription of its biofilm-related vps and rbm gene clusters was nevertheless elevated as judged by both RNA-seq and RT-qPCR analyses. This transcriptome signature was shared with N16961R, as were others involving two-component signal transduction, chemotaxis, and c-di-GMP synthesis functions.ConclusionsPrecise turnarounds in gene expression did not accompany reversible phase transitions (i.e., smooth to rugose to smooth) in the cholera pathogen. Transcriptomic signatures consisting of up-regulated genes involved in biofilm formation, environmental sensing and persistence, chemotaxis, and signal transduction, which were shared by N16961R and N16961SD variants, may implicate a stress adaptation in the pathogen that facilitates transition of the N16961SD smooth form back to rugosity should environmental conditions dictate.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3233-x) contains supplementary material, which is available to authorized users.

Genomic profile of antibiotic resistant, classical ctxB positive Vibrio cholerae O1 biotype El Tor isolated in 2003 and 2005 from Puri, India: A retrospective study

Objectives: To examine eight strains of Vibrio cholerae O1 isolated in 2003 and 2005 from Puri, India, for antibiotic susceptibility, presence of virulence and regulatory genes, cholera toxin (CT) production, CTX arrangement and genomic profiles. Materials and Methods: Bacterial strains were tested for antibiotic susceptibility using disc diffusion assay. Polymerase chain reaction determined the presence of antibiotic resistance, virulence and regulatory genes. To determine the type of cholera toxin subunit B (ctxB), nucleotide sequencing was performed. Southern hybridisation determined the number and arrangement of CTXΦ. Ribotyping and pulsed-field gel electrophoresis (PFGE) were used to determine the genomic profile of isolates. Results: All the eight strains, except one strain, showed resistant to nalidixic acid, sulphamethoxazole, streptomycin and trimethoprim and possessed the sullI, strB, dfrA1 and intSXT genes. All the strains carried the toxin-co-regulated pilus pathogenicity island, the CTX genetic element, the repeat in toxin and produced CT. Restriction fragment length polymorphism (RFLP) analysis showed that V. cholerae O1 possess a single copy of the CTX element flanked by tandemly arranged RS element. Nucleotide sequencing of the ctxB gene showed the presence of classical ctxB. RFLP analysis of conserved rRNA gene showed two ribotype patterns. PFGE analysis also showed at least three PFGE patterns, irrespective of year of isolations, indicating the genomic relatedness among them. Conclusion: Overall, these data suggest that classical ctxB-positive V. cholerae O1 El Tor strains that appeared in 2003 continue to cause infection in 2005 in Puri, India, and belong to identical ribotype(s) and/or pulsotype(s). There is need to continuous monitor the emergence of variant of El Tor because it will improve our understanding of the evolution of new clones of variant of V. cholerae.

Sequences of a co-existing SXT element, a chromosomal integron (CI) and an IncA/C plasmid and their roles in multidrug resistance in a Vibrio cholerae O1 El Tor strain

The ongoing seventh cholera pandemic is attributed to Vibrio cholerae O1 El Tor biotype strains. Although antibiotic therapy ameliorates symptoms in patients and reduces pathogen transfer to the environment, multidrug resistance remains a major clinical threat. An O1 El Tor strain isolated from a patient in 1998 was intermediate or resistant to 13 antibiotics and could potentially produce extended-spectrum β-lactamase (ESBL), which is very rare in O1 strains. Using genome sequencing, three relevant genetic elements were identified in this strain: a hybrid SXT element (ICEVchCHN1307); a new IncA/C plasmid (pVC1307); and a chromosomal integron. Twenty antibiotic resistance genes were located on them, including blaTEM-1, blaCTX-M-14 and phenotypically silenced tetRA genes. These data elucidate the role of individual genetic components in antibiotic resistance and the accumulation of drug resistance genes in V. cholerae.

Phenotypic Analysis Reveals that the 2010 Haiti Cholera Epidemic Is Linked to a Hypervirulent Strain.

Vibrio cholerae O1 El Tor strains have been responsible for pandemic cholera since 1961. These strains have evolved over time, spreading globally in three separate waves. Wave 3 is caused by altered El Tor (AET) variant strains, which include the strain with the signature ctxB7 allele that was introduced in 2010 into Haiti, where it caused a devastating epidemic. In this study, we used phenotypic analysis to compare an early isolate from the Haiti epidemic to wave 1 El Tor isolates commonly used for research. It is demonstrated that the Haiti isolate has increased production of cholera toxin (CT) and hemolysin, increased motility, and a reduced ability to form biofilms. This strain also outcompetes common wave 1 El Tor isolates for colonization of infant mice, indicating that it has increased virulence. Monitoring of CT production and motility in additional wave 3 isolates revealed that this phenotypic variation likely evolved over time rather than in a single genetic event. Analysis of available whole-genome sequences and phylogenetic analyses suggested that increased virulence arose from positive selection for mutations found in known and putative regulatory genes, including hns and vieA, diguanylate cyclase genes, and genes belonging to the lysR and gntR regulatory families. Overall, the studies presented here revealed that V. cholerae virulence potential can evolve and that the currently prevalent wave 3 AET strains are both phenotypically distinct from and more virulent than many El Tor isolates.

Circulation of a Quorum-Sensing-Impaired Variant of Vibrio cholerae Strain C6706 Masks Important Phenotypes.

Vibrio cholerae, the causative agent of cholera, is a model organism for studying virulence regulation, biofilm formation, horizontal gene transfer, and the cell-to-cell communication known as quorum sensing (QS). As in any research field, discrepancies between data from diverse laboratories are sometimes observed for V.cholerae. Such discrepancies are often caused by the use of diverse patient or environmental isolates. In this study, we investigated the inability of a few laboratories to reproduce high levels of natural transformation, a mode of horizontal gene transfer that is specifically induced on chitinous surfaces. This irreproducibility was mostly related to one specific isolate of V.cholerae: the O1 El Tor C6706 strain. C6706 was previously described as QS proficient, an important prerequisite for the induction of natural competence for transformation. To elucidate the underlying problem, we collected seven isolates of the same C6706 strain from different research laboratories in North America and Europe and compared their phenotypes. Importantly, we observed a split response with respect to QS-related gene expression, including chitin-induced natural competence and type VI secretion (T6S). While approximately half of the strains behaved as reported for several other O1 El Tor pandemic isolates that are commonly studied in the laboratory, the other half were significantly impaired in QS-related expression patterns. This impairment was caused by a mutation in a QS-related gene (luxO). We conclude that the circulation of such QS-impaired wild-type strains is responsible for masking several important phenotypes of V.cholerae, including natural competence for transformation and T6S. IMPORTANCE Phenotypic diversity between laboratory-domesticated bacterial strains is a common problem and often results in the failed reproduction of published data. However, researchers rarely compare such strains to elucidate the underlying mutation(s). In this study, we tested one of the best-studied V.cholerae isolates, O1 El Tor strain C6706 (a patient isolate from Peru), with respect to two main phenotypes: natural competence for transformation and type VI secretion. We recently demonstrated that the two phenotypes are coregulated and specifically induced upon the growth of pandemic V.cholerae O1 El Tor strains on chitinous surfaces. We provide evidence that of seven C6706 strains collected from different laboratories, four were impaired in the tested phenotypes due to a mutation in a QS gene. Collectively, our data indicate that the circulation of such a mutated wild-type strain of C6706 might have had important consequences for QS-related data.

Targeting aphA : a new high-throughput screening assay identifies compounds that reduce prime virulence factors of Vibrio cholerae.

A high-throughput screening (HTS) assay was developed for identifying compounds with inhibitory effect on aphA, one of the key regulators positively controlling Vibrio cholerae pathogenesis. An inhibitory effect on aphA was expected to lead to attenuation in the secretion of the major pathogenicity factors of V. cholerae, cholera toxin and toxin co-regulated pilus. The plasmid construct pAKSB was developed with a kanamycin resistance (KmR) gene under the control of the aphA -like promoter for conferring a KmR phenotype under aphA -expressing conditions. The HTS assay was performed to identify compounds with inhibitory effect on the growth of O139 V. cholerae MO10 carrying the construct pAKSB in growth medium containing Km (30 g ml-1), but not in its absence. Of 20 338 compounds screened, six compounds were identified to inhibit the pAKSB-induced KmR phenotype and these compounds caused transcriptional inhibition of aphA in V. cholerae O139 strain MO10 as well as variant V. cholerae O1 El Tor strain NM06-058. Of the three most active substances, compound 53760866 showed lowest half-maximal cytotoxicity in a eukaryotic cell viability assay and was characterized further. Compound 53760866 caused reduction in cholera toxin secretion and expression of TcpA in vitro. The in vitro virulence attenuation corroborated well in a suckling mouse model in vivo, which showed reduction of colonization by V. cholerae NM06-058 when co-administered with 53760866. The screening method and the compounds may lead to new preventive strategies for cholera by reducing the pathogenicity of V. cholerae .