Cholerae El Tor Research Articles

Plasticity of Regulation of Mannitol Phosphotransferase System Operon by CRP-cAMP Complex in Vibrio cholerae

ObjectiveThe complex of the cyclic AMP receptor protein (CRP) and cAMP is an important transcriptional regulator of numerous genes in prokaryotes. The transport of mannitol through the phosphotransferase systems (PTS) is regulated by the CRP-cAMP complex. The aim of the study is to investigate how the CRP-cAMP complex acting on the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype. MethodsThe crp mutant strain was generated by homologous recombination to assess the need of CRP to activate the mannitol PTS operon of V. cholerae El Tor. Electrophoretic mobility shift assays (EMSA) and the reporter plasmid pBBRlux were used to confirm the role that the CRP-cAMP complex playing on the mannitol PTS operon mtl. ResultsIn this study, we confirmed that CRP is strictly needed for the activation of the mtl operon. We further experimentally identified five CRP binding sites within the promoter region upstream of the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype and found that these sites display different affinities for CRP and provide different contributions to the activation of the operon. ConclusionThe five binding sites collectively confer the strong activation of mannitol transfer by CRP in V. cholerae, indicating an elaborate and subtle CRP activation mechanism.

Diagnosis of Vibrio cholerae O1 Infection in Africa

Isolation of Vibrio cholerae O1 is necessary for cholera outbreak confirmation. Rapid diagnostic testing of fecal specimens, based on lipopolysaccharide detection of V. cholerae O1 or O139, may assist in early outbreak detection and surveillance. Cary-Blair transport medium is recommended for specimen transport. Filter paper, although used in epidemics, needs evaluation against rectal swab specimens. Fecal specimens are subcultured onto selective and nonselective media, including 5% blood agar and TCBS agar, for detection of V. cholerae O1 or O139. Suspicious, oxidase-positive isolates are serotyped in monovalent antisera. Antimicrobial-susceptibility testing is performed to detect resistance. Molecular characterization supports phenotypic identification and outbreak investigations. The presence of genes encoding cholera toxin, lipopolysaccharide, and El Tor biotype traits can be confirmed. Standardized pulsed-field gel electrophoresis analysis facilitates strain comparison. Quality management ensures reliability of results through validation and verification of functional laboratory equipment; quality control of testing procedures, laboratory reagents, and consumables; and participation in proficiency-testing schemes.

Vibrio choleraeO1 El Tor and O139 Bengal Strains CarryingctxBET, Bangladesh

<i>Vibrio cholerae</i>O1 El Tor and O139 Bengal Strains Carrying<i>ctxB</i>^ET, Bangladesh

A Study on the Geophylogeny of Clinical and Environmental Vibrio cholerae in Kenya

Cholera remains a significant public health challenge in many sub-Saharan countries including Kenya. We have performed a combination of phylogenetic and phenotypic analysis based on whole genome DNA sequences derived from 40 environmental and 57 clinical V. cholerae from different regions of Kenya isolated between 2005 and 2010. Some environmental and all clinical isolates mapped back onto wave three of the monophyletic seventh pandemic V. cholerae El Tor phylogeny but other environmental isolates were phylogenetically very distinct. Thus, the genomes of the Kenyan V. cholerae O1 El Tor isolates are clonally related to other El Tor V. cholerae isolated elsewhere in the world and similarly harbour antibiotic resistance-associated STX elements. Further, the Kenyan O1 El Tor isolates fall into two distinct clades that may have entered Kenya independently.

Genetic Characteristics and Relatedness of ImportedVibrio choleraeO1 Biotype El Tor in Korea

Background Cholera is a representative water-borne disease that is caused by V. cholera ctx (+). V. cholera El Tor was previously the primary pathogen, but after the seventh pandemic outbreak, it was replaced by a V. cholera El Tor variant with a classical pheno-type and genotype. In this study, we investigated the genotypic and phenotypic characteristics of imported V. cholerae El Tor in Korea. Methods Forty-nine V. cholerae O1 El Tor strains isolated from 2004 to 2011 were used in this study. Polymerase chain reaction amplification of the ctxB and rstR genes was used for biotype determination. An antimicrobial susceptibility test was performed for phenotypic analysis, and pulse field gel electropho-resis (PFGE) was used for analysis of genetic relatedness. Results Classical ctxB genes were found in all of the isolates, while classical, El Tor, and combined rstRgenes were found. Twenty strains showed antimicrobial resistance against streptomycin, sulfamethoxazole/ trimethoprim, nalidixic acid, and ciprofloxacin. Based on PFGE, all isolates were grouped as cluster B. The country of origin and resistance pattern were highly related, although the time of influx and serogroup were not. Conclusion Isolates of V. cholera El Tor imported since 2004 were hybrids of V. cholera El Tor, which has the classical ctxB gene and is considered to be a CTX prophage. The SXT element plays an important role in antimicrobial resistance. PFGE patterns, which can be used for analysis of imported V. cholera, revealed the relatedness of the resistant isolates.

Serotyping, Antibiotic Susceptibility Pattern and Detection of hlyA Gene Among Cholera Patients in Iran

Background: Cholera is a severe diarrheal illness caused by Vibrio cholerae, which produces a virulence factor named El Tor hemolysin encoded by the hlyA gene. Objectives: This study meant to analyze the phenotypic characteristics and presence of hlyA gene in V. cholerae isolated from patients in Iran. The hlyA gene which codes for hemolysin, plays an essential role in manifestation of cholera ,and could be used to diagnose pathogenic V. cholerae El Tor O1 strains. Patients and Methods: One hundred stool samples from the patients with cholera during 2002-2003 were collected from Tehran, Kashan, Kermanshah and Ahvaz cities, which were subject to diagnostic tests. Serotyping, and antibiotic susceptibility tests were applied and polymerase chain reaction (PCR) was also used to detect the hlyA gene. Results: The group specific antisera identified the isolates as Ogawa, Inaba, Hikojima and NAG (Non-agglutinable) in 74%, 3%, 0% and 23% of the isolates, respectively. Antibiotic susceptibility test showed that all of the strains were sensitive to ciprofloxacin, gentamycin and doxycyclin but the isolates showed resistance to sulfamethoxazole/trimethoprim (74%), erythromycin (64%) and tetracycline (50%). V. cholerae El Tor isolates were 100% positive for hlyA gene, but hemolysis phenotype characteristics were found in 95% of the cases. Conclusions: The results indicated that Ogawa serotype was identified as the dominant serotype which revealed multiple antibiotic resistances to sulfamethoxazole/trimethoprim, oxytetracycline, erythromycin, tetracycline and chloramphenicol. The presence of hlyA gene in nonhemolytic strains of V. cholerae O1 biotype El Tor indicated that some factors prevent expression of the hemolysin gene.

The evolution of the pathogen and the clinical and epidemiological features of the recent cholera (el tor)

In a comparative perspective studied cholera outbreak in the Caucasus due to typical toxigenic and genetically modified (hybrid) El Tor variant strains have been studied. Revealed features of the genetic structure of the genome, factors and ways of transmission of the causative agent of modern cholera El tor should be considered when improving the program of epidemiological supervision in terms of enhancing antiepidemic and prevention measures in cholera, the causative factor of which are of hybrid variants of Vibrio cholerae El tor.

THE INFLUENCE OF SOME FACTORS ON THE FORMATION OF BIOFILM BY TOXIGENIC AND NON-TOXIGENIC VIBRIO CHOLERAE EL TOR STRAINS

The early stages of biofilm formation on polystyrene surface were studied in 14 Vibrio cholerae El Tor strains, all of which carried genes of vps cluster, mshA, and hapR genes. 13 Vibrio cholerae El Tor strains were shown to be capable of adsorption on the walls of microtiter plate wells and of ring formation at the "polystyrene-medium" interface. One of the Vibrio cholerae El Tor strains tested was defective in this ability. The use of different media - modified M1 medium and 1 per cent peptone - failed to reveal sufficient influence of culture medium composition on the ability of Vibrio cholerae to form multilayer biofilm at the phase boundary "polystyrene-medium". Temperature reduction from 37°C to 25°C also didn t result in statistically significant enhancement of monolayer formation in both media. Quantitative evaluation of biofilm formation showed that this ability was clearly marked only in two toxigenic and two non-toxigenic strains of Vibrio cholerae El Tor. Monolayer comparison in ELISA demonstrated that only two toxigenic and two non-toxigenic strains possessed the ability for exopolysaccharide production.

Genetic characteristics of drug-resistant Vibrio cholerae O1 causing endemic cholera in Dhaka, 2006–2011

Vibrio cholerae O1 biotype El Tor (ET), causing the seventh cholera pandemic, was recently replaced in Bangladesh by an altered ET possessing ctxB of the Classical (CL) biotype, which caused the first six cholera pandemics. In the present study, V. cholerae O1 strains associated with endemic cholera in Dhaka between 2006 and 2011 were analysed for major phenotypic and genetic characteristics. Of 54 representative V. cholerae isolates tested, all were phenotypically ET and showed uniform resistance to trimethoprim/sulfamethoxazole (SXT) and furazolidone (FR). Resistance to tetracycline (TE) and erythromycin (E) showed temporal fluctuation, varying from year to year, while all isolates were susceptible to gentamicin (CN) and ciprofloxacin (CIP). Year-wise data revealed erythromycin resistance to be 33.3 % in 2006 and 11 % in 2011, while tetracycline resistance accounted for 33, 78, 0, 100 and 27 % in 2006, 2007, 2008, 2009 and 2010, respectively; interestingly, all isolates tested were sensitive to TE in 2011, as observed in 2008. All V. cholerae isolates tested possessed genetic elements such as SXT, ctxAB, tcpA(ET), rstR(ET) and rtxC; none had IntlI (Integron I). Double mismatch amplification mutation assay (DMAMA)-PCR followed by DNA sequencing and analysis of the ctxB gene revealed a point mutation at position 58 (C→A), which has resulted in an amino acid substitution from histidine (H) to asparagine (N) at position 20 (genotype 7) since 2008. Although the multi-resistant strains having tetracycline resistance showed minor genetic divergence, V. cholerae strains were clonal, as determined by a PFGE (NotI)-based dendrogram. This study shows 2008-2010 to be the time of transition from ctxB genotype 1 to genotype 7 in V. cholerae ET causing endemic cholera in Dhaka, Bangladesh.

Amino acid addition to Vibrio cholerae LPS establishes a link between surface remodeling in Gram-positive and Gram-negative bacteria

Historically, the O1 El Tor and classical biotypes of Vibrio cholerae have been differentiated by their resistance to the antimicrobial peptide polymyxin B. However, the molecular mechanisms associated with this phenotypic distinction have remained a mystery for 50 y. Both gram-negative and gram-positive bacteria modify their cell wall components with amine-containing substituents to reduce the net negative charge of the bacterial surface, thereby promoting cationic antimicrobial peptide resistance. In the present study, we demonstrate that V. cholerae modify the lipid A anchor of LPS with glycine and diglycine residues. This previously uncharacterized lipid A modification confers polymyxin resistance in V. cholerae El Tor, requiring three V. cholerae proteins: Vc1577 (AlmG), Vc1578 (AlmF), and Vc1579 (AlmE). Interestingly, the protein machinery required for glycine addition is reminiscent of the gram-positive system responsible for D-alanylation of teichoic acids. Such machinery was not thought to be used by gram-negative organisms. V. cholerae O1 El Tor mutants lacking genes involved in transferring glycine to LPS showed a 100-fold increase in sensitivity to polymyxin B. This work reveals a unique lipid A modification and demonstrates a charge-based remodeling strategy shared between gram-positive and gram-negative organisms.

Type III Effector VopC Mediates Invasion for Vibrio Species

Vibrio spp. are associated with infections caused by contaminated food and water. A type III secretion system (T3SS2) is a shared feature of all clinical isolates of V. parahaemolyticus and some V. cholerae strains. Despite its being responsible for enterotoxicity, no molecular mechanism has been determined for the T3SS2-dependent pathogenicity. Here, we show that although Vibrio spp. are typically thought of as extracellular pathogens, the T3SS2 of Vibrio mediates host cell invasion, vacuole formation, and replication of intracellular bacteria. The catalytically active effector VopC is critical for Vibrio T3SS2-mediated invasion. There are other marine bacteria encoding VopC homologs associated with a T3SS; therefore, we predict that these bacteria are also likely to use T3SS-mediated invasion as part of their pathogenesis mechanisms. These findings suggest a new molecular paradigm for Vibrio pathogenicity and modify our view of the roles of T3SS effectors that are translocated during infection.

Molecular-genetic analysis of Vibrio cholerae El Tor strains of epidemic risk isolated in Siberian and Far East regions of Russia

PCR detection of biovar specificity and pathogenicity determinants has been performed in order to analyze the structural components of the genome of V. cholerae El Tor strains (n = 90) isolated during epidemic outbreaks in Siberia and the Far East, and the nucleotide sequence of the ctxB gene and the structure of ctxAB gene promoter region have been determined. As a result, toxicogenic strains V. cholerae El Tor were divided into two groups: the first group contained strains isolated at the initial stages of the seventh pandemia (in the 1970s), and they had the genotype ctxB3+rstREl+rstRCl-rstC+TCL+tbr4; all El Tor vibrios posing an epidemic risk isolated in the 1990s were characterized as atypical variants due to ctxB gene harboring of the classical genotype (ctxB) in their genome and were placed in the second group. The second group fell into three genotypes according to the set of tested genetic markers (ctxB, rstR, rstC, TLC, and tbr) and affect certain territories. The established variability of the genome structure of Vibrio El Tor atypical variants may serve as a marker for molecular and epidemiological analysis of cholera entry pathways and distribution and is useful for speculating on the most likely directions of pathogen evolution.

Cholera in the Early XXI Century: Global Prognosis

The prognosis for cholera on 2011 proved true: the tendency of morbidity growth remained in the last decade (2002-2011) worldwide. Imported cholera cases caused large outbreaks and epidemics in the Caribbean countries and Asia. Formation of endemic foci in a number of countries of Asia and Africa took place, where favorable conditions for epidemics development (environmental and social conditions, military conflicts, economical and political instability) existed. Wide spread of genetically altered V. cholerae eltor and V. cholerae O139 variants with epidemic and pandemic potential was observed. All the events mentioned above predetermine the unfavorable prognosis for cholera on a global level.

Genome variability in the altered variants of Vibrio cholerae biovar El Tor isolated in Russia

Molecular-genetic analysis of 56 clinical strains of the Vibrio cholerae biovar El Tor isolated during the period 1965–2010 in Russia and neighboring countries was carried out. Thirty-one isolates (55.3%) bearing the gene ctxB in the genome of the prophage CTXφ in cholera vibrios of the classical biovar were found. These strains are altered variants of the cholera El Tor pathogen. The nucleotide sequence of their genes ctxB and rstR included in the prophage CTXϕ, which are related with the production of the cholera toxin, was determined. It was found that altered variants bear different alleles of these genes (ctxB1 or ctxB7; rstREl and/or rstRClass) and belong to four gene types: ctxB1, rstRClass; ctxB1, rstREl; ctxB1, rstRClass/rstREl; and ctxB7, rstRClass/rstREl. It was shown that these gene variants produce 42 to 48 times more cholera toxin than typical strains. Isolates in which the pathogenicity island VSP-2 contains a long deletion were found among altered variants. The study data are evidence of the genomic variability of altered pathogen variants of the seventh cholera pandemic and require the development of new gene-diagnostic testing systems to improve the efficiency of epidemiological control of cholera distribution.

Functional characterization of VC1929 of Vibrio cholerae El Tor: role in mannose-sensitive haemagglutination, virulence and utilization of sialic acid

The nonadhesive mutant CD11 of Vibrio cholerae El Tor, defective in expression of mannose-sensitive haemagglutinin, lacks a protein when compared with its parent strain. Determination of the amino acid sequence revealed the identity of the protein as the product of VC1929, which is annotated to encode a protein, DctP, involved in the transport of C₄-dicarboxylates. We cloned the dctP gene in pUC19 vector and expressed it in mutant CD11. Expression of DctP in the resulting complemented strain restored virulence, adhesive and colonizing capabilities, mannose-sensitive haemagglutination (MSHA) and ability to grow in medium containing sialic acid as a sole carbon source. The mutation in CD11 was caused by insertion of an adenine nucleotide in the reading frame of dctP. Recombinant purified DctP protein showed MSHA of human red blood cells, and protected rabbits against infection by V. cholerae. The protein was localized in membrane and cell wall fractions. The mutant, recombinant CD11 expressing DctP and parent strains were grown in M9 minimal medium in the presence of various carbohydrates (glucose, malate, fumarate, succinate or N-acetylneuraminic acid). The mutant was unable to grow in minimal medium containing N-acetylneuraminic acid (sialic acid) as the sole carbon source whereas the recombinant and parent strains utilized all the sugars tested. It is concluded that DctP is a mannose-sensitive haemagglutinin and a virulence factor and is involved in the utilization of sialic acid.

Construction of the PCR Test-System for the Detection of &lt;i&gt;Vibrio cholerae&lt;/i&gt; O1 Toxigenic Strains, for Indication of Their Biovar and for Differentiation between Typical and Altered El Tor-Vibrio Strains

Worked out is the multilocus PCR test-system which makes it possible to identify Vibrio cholerae O1 strains on the basis of rfb gene detection, to determine their biovar - either Classical or El Tor by testing cas3 or rtxC genes respectively, and at the same time to differentiate them into typical (which carry ctxB Eltor gene) and genetically altered (which carry ctxB Class gene ) variants. Efficacy and specificity of the test-system is demonstrated by the analysis of 64 natural V. cholerae strains of various serogroups and biovars, and 13 strains of enterobacteria and other species of Vibrio genus. Relation of the studied V. cholerae El Tor isolates to the typical or altered vibrio variants is proved out by ctxB gene sequencing.

Proteomic Analysis of the Vibrio cholerae Type II Secretome Reveals New Proteins, Including Three Related Serine Proteases

The type II secretion (T2S) system is responsible for extracellular secretion of a broad range of proteins, including toxins and degradative enzymes that play important roles in the pathogenesis and life cycle of many gram-negative bacteria. In Vibrio cholerae, the etiological agent of cholera, the T2S machinery transports cholera toxin, which induces profuse watery diarrhea, a hallmark of this life-threatening disease. Besides cholera toxin, four other proteins have been shown to be transported by the T2S machinery, including hemagglutinin protease, chitinase, GbpA, and lipase. Here, for the first time, we have applied proteomic approaches, including isotope tagging for relative and absolute quantification coupled with multidimensional liquid chromatography and tandem mass spectrometry, to perform an unbiased and comprehensive analysis of proteins secreted by the T2S apparatus of the V. cholerae El Tor strain N16961 under standard laboratory growth conditions. This analysis identified 16 new putative T2S substrates, including sialidase, several proteins participating in chitin utilization, two aminopeptidases, TagA-related protein, cytolysin, RbmC, three hypothetical proteins encoded by VCA0583, VCA0738, and VC2298, and three serine proteases VesA, VesB, and VesC. Focusing on the initial characterization of VesA, VesB, and VesC, we have confirmed enzymatic activities and T2S-dependent transport for each of these proteases. In addition, analysis of single, double, and triple protease knock-out strains indicated that VesA is the primary protease responsible for processing the A subunit of cholera toxin during in vitro growth of the V. cholerae strain N16961.

Waterborne cholera outbreak following Cyclone Aila in Sundarban area of West Bengal, India, 2009

Following Cyclone Aila, a block of Sundarban area, West Bengal, India, reported an increased number of diarrhoea cases at the end of May 2009. This study was performed to identify the agent and source of the outbreak as well as to propose control measures. The outbreak is described by time, place and person. A matched case–control study was conducted and rectal swabs and water specimens were collected. In total, 1076 probable case patients and 14 deaths (attack rate 44/10 000) were identified. Vibrio cholerae El Tor Ogawa was isolated from two of five probable case patients’ stool specimens. The outbreak started in the fourth week of May, with two peaks in the second and fourth weeks of June, and lasted until August 2009. Compared with controls, cases were more likely to drink non-chlorinated piped water [matched odds ratio (MOR) = 16, 95% CI 4.9–51; population attributable fraction 58%) and were less likely to drink chlorine-treated water (MOR = 0.06, 95% CI 0.02–0.18). Villagers broke the water pipelines near their houses for easy access to water. Piped water specimens and stored drinking water were positive for faecal contamination. Contaminated drinking water was the probable source of the cholera outbreak. Repairing the pipelines, chlorination at household level and educating villagers regarding the danger of breached water pipe connections controlled the outbreak.

Altered Variants of Cholera Agent Isolated in the Territory of the Russian Federation

Carried out was the retrospective genetic analysis of cholera vibrio strains which caused the outbreaks and sporadic cases of cholera in the territory of the Russian Federation from 1993 to 2006. It was elucidated that beginning from 1993 the disease had been caused by the altered strains of V. cholerae El Tor containing ctxB gene of classical type, encoding B subunit of cholera toxin, in their CTXφ prophage genome. In addition, identified were two strains whose prophage genome contained classical type rstR gene.

Altered Variants of Cholera Agent Isolated in the Territory of the Russian Federation

Carried out was the retrospective genetic analysis of cholera vibrio strains which caused the outbreaks and sporadic cases of cholera in the territory of the Russian Federation from 1993 to 2006. It was elucidated that beginning from 1993 the disease had been caused by the altered strains of V. cholerae El Tor containing ctxB gene of classical type, encoding B subunit of cholera toxin, in their CTXφ prophage genome. In addition, identified were two strains whose prophage genome contained classical type rstR gene.