Cholerae El Tor Research Articles

The Vibrio cholerae VarS/VarA two-component system controls the expression of virulence proteins through ToxT regulation

Although the conditions for inducing virulence protein expression in vitro are different, both classical and El Tor biotypes of Vibrio cholerae have been reported to regulate the expression of virulence proteins such as cholera toxin (CT) and toxin-coregulated pili (Tcp) through the ToxR/S/T system. The transcription activator ToxR responds to environmental stimuli such as pH and temperature and activates the second transcriptional regulator ToxT, which upregulates expression of virulence proteins. In addition to the ToxR/S/T signalling system, V. cholerae has been proposed to utilize another two-component system VarS/VarA to modulate expression of virulence genes. Previous study has shown that VarA of the VarS/VarA system is involved in the regulation of virulence proteins in the classical V. cholerae O395 strain; however, no further analysis was performed concerning VarS. In this study, we constructed varS mutants derived from the classical O395 and El Tor C6706 strains and demonstrated that VarS is also involved in the expression of the virulence proteins CT and Tcp from the V. cholerae classical and El Tor strains. This expression is through regulation of ToxT expression in response to environmental changes due to different toxin-inducing conditions.

The phenotypic and molecular biological characteristics of Vibrio cholerae El Tor strains isolated from environmental water objects in Rostov-on-Don in 2003-2008

The pheno- and genotypic properties of Vibrio cholerae El Tor cultures identified during monitoring for V. cholerae in the aquatic ecosystems of Rostov-on-Don in the period 2003-2008 were studied in the extended range. In addition to known laboratory diagnostic techniques for cholera, the authors used a volume agglomeration test with antilipase diagnosticum, as well as polymerase chain reaction using specific primers to detect 30 nucleotide sequences of the genes associated with V. cholerae pathogenicity. The range of pheno- and genotypic characteristics of study V. cholerae O1 cultures was extended. There was evidence for the volume agglomeration test with a polymerase antilipase agent as a complementary test for toxicogenicity. The Vibrio cholerae O1 strains isolated from the aquatic environment were shown to confer a real and potential risk in the low cholera incidence area of Rostov-on-Don. The findings may be used to improve methodic approaches to monitoring environmental objects for Vibrio cholerae in the optimization of epidemiological surveillance of cholera at the regional level.

Multidrug resistant Vibrio cholerae O1 El Tor carrying classical ctxB allele involved in a cholera outbreak in South Western India

Cholera is a fatal diarrheal disease characterized with enormous fluid loss through stools. A total of 41 Vibrio cholerae isolates collected from a recent cholera outbreak in Solapur, South Western India were found to belong to serogroup O1, biotype El Tor and serotype Ogawa. Molecular analysis revealed the prevalence of different toxigenic and pathogenic genes in the isolates. All the isolates harboured rstRElTor allele indicating the presence of CTXΦElTor. However, cholera toxin (ctxB) gene sequencing and a ctxB allele specific PCR of the isolates confirmed the presence of ctxB of classical biotype. The antibiogram profile revealed the resistance for several antibiotics including nalidixic acid, polymyxin B, streptomycin, sulfamethoxazole, trimethoprim, rifampicin and vibriostatic agent 2,4-diamino-6,7-diisopropylpteridine (O/129). All the isolates were PCR positive for class 1 integron and SXT elements also. Fingerprinting analysis revealed the clonal relationship among the outbreak isolates. The results suggested the involvement of multidrug resistant V. cholerae El Tor biotype isolates having ctxB gene of classical biotype in the cholera outbreak.

The evolution of the Vibrio cholerae genome during the modern period

A review of the modern data concerning the phenotypical and genetic peculiarities of the recently emerged highly virulent variants of V. cholerae El-Tor biovar (the etiological agent of the current cholera pandemic) is presented. The molecular-genetic mechanisms that are considered to be a basis for V. cholerae genome evolution during the modern period are discussed. The transposon role in the increased expression of cholera toxin genes, which are constituents of the CTXφ prophage genome, is addressed. The need to work out new gene-diagnostic test-systems for the identification of natural altered variants of the cholera etiological agent is shown.

Vibrio cholerae O1 Avirulent Strains - Producers of Protective O1 Antigen: Obtaining and Peculiarities

Among 15 Vibrio cholerae El Tor O1 virulent strains of Inaba and Ogawa serovars grown in sterile river water, 6 strains were found to lose spontaneously the core region of CTXφ prophage including structural genes ( ctxAB ) of cholera toxin. It was determined that the obtained ctxA - mutants did not produce cholera toxin and did not cause specific choleragenic reaction in the intra-intestinally inoculated suckling rabbits suggesting that they belonged to the III d pathogenicity group. Analysis of the population structure of two selected non-toxigenic strains M569A and 5/65A revealed clones with high level of O1 antigen of Inaba and Ogawa serovars production. The obtained avirulent strains can be used as producers of above-noted protective antigens both in production of chemical cholera vaccine and in constructing of diagnostic immunoenzyme test-systems.

Vibrio cholerae El Tor TcpA crystal structure and mechanism for pilus‐mediated microcolony formation

Type IV pili (T4P) are critical to virulence for Vibrio cholerae and other bacterial pathogens. Among their diverse functions, T4P mediate microcolony formation, which protects the bacteria from host defences and concentrates secreted toxins. The T4P of the two V. cholerae O1 disease biotypes, classical and El Tor, share 81% identity in their TcpA subunits, yet these filaments differ in their interaction patterns as assessed by electron microscopy. To understand the molecular basis for pilus-mediated microcolony formation, we solved a 1.5 A resolution crystal structure of N-terminally truncated El Tor TcpA and compared it with that of classical TcpA. Residues that differ between the two pilins are located on surface-exposed regions of the TcpA subunits. By iteratively changing these non-conserved amino acids in classical TcpA to their respective residues in El Tor TcpA, we identified residues that profoundly affect pilus:pilus interaction patterns and bacterial aggregation. These residues lie on either the protruding d-region of the TcpA subunit or in a cavity between pilin subunits in the pilus filament. Our results support a model whereby pili interact via intercalation of surface protrusions on one filament into depressions between subunits on adjacent filaments as a means to hold V. cholerae cells together in microcolonies.

In Vitro Production of Cholera Toxin by Vibrio cholerae El Tor Strains from Human Beings and the Environment

In Vitro Production of Cholera Toxin by Vibrio cholerae El Tor Strains from Human Beings and the Environment

Transfer of cholera toxin genes from O1 to non‐O1/O139 strains by vibriophages from California coastal waters

Vibrio cholerae is an important bacterial pathogen that causes global cholera epidemic. Although they are commonly found in coastal waters around the world, most environmental isolates do not contain cholera toxin genes. This study investigates vibriophages in southern California coastal waters and their ability to transfer cholera toxin genes. Lytic phages infecting V. cholerae were isolated from Newport Bay, California, between May and November, while none was found in winter. Some of the phage isolates can infect multiple environmental V. cholerae strains and El Tor strains. All phages contained double-stranded DNA. Transduction experiments using kanamycin-resistant gene marked CTXPhi demonstrated that some environmental vibriophages can transfer CTXPhi genes from O1 El Tor strain to environmental non-O1/O139 V. cholerae via generalized transduction. Vibriophages are important components of the natural aquatic ecosystem. They play an important role in influencing the dynamics and evolution of V. cholerae in the environment. This study demonstrates the significance of vibriophages in the coastal environment and transduction as one of the mechanisms of pathogenicity evolution among environmental V. cholerae.

Sequence analysis of Vibrio cholerae orfU and zot from pre‐CTXΦ and CTXΦ reveals multiple origin of pre‐CTXΦ and CTXΦ

A multiplex PCR was developed to detect pre-CTXΦ and CTXΦ in Vibrio cholerae. A total of 115 V. cholerae were tested, of which 42 V. cholerae O1 and 18 V. cholerae O139 contained CTXΦ. Six V. cholerae O139 contained only pre-CTXΦ and three V. cholerae O1 and 23 V. cholerae O139 contained both pre-CTXΦ and CTXΦ. None of the V. cholerae non-O1 and non-O139 that were tested had pre-CTXΦ or CTXΦ. Results of Restriction Fragment Length Polymorphism (RFLP) analysis revealed the V. cholerae isolates possessed single or multiple copies of pre-CTXΦ and CTXΦ, always proceeded by a tandemly arranged RS1 element. Comparative nucleotide sequence analyses of the core region genes, orfU and zot, of 15 V. cholerae showed pre-CTX(ET) Φ and CTX(ET) Φ lineage with V. cholerae El Tor and pre-CTX(Class) Φ, pre-CTX(Calc) Φ, and CTX(Calc) Φ with classical V. cholerae O1 and O139. Two distinct types of ctxB were detected in V. cholerae O139. Multi-locus Sequence Typing (MLST) of seven V. cholerae housekeeping genes indicated clonal origin, irrespective of the presence of pre-CTXΦ and/or CTXΦ.

Survey of epidemiology and bacteriology features of cholera in Iran

Objective To determine epidemiology and antimicrobial susceptibility patterns of Vibrio (V.) cholerae O1 biotype EL Tor in summer outbreak of 2008 in Iran. Methods Stool samples were collected from patients suspected to have cholera admitted to hospitals and clinics. Specimens examined by conventional bacteriological methods. All isolates were sent to cholera reference laboratory for further confirmation, stereotyping and susceptibility testing. Results A total of 220 patients were diagnosed as cholera. All cases confirmed by Iranian reference health laboratory. One hundred ninety nine of 220 V. cholerae serotypes were Inaba and 21 serotypes were Ogawa. All cases were reported from thirteen provinces. The majorities of cases were from Tehran, Qum and Zahedan provinces with 56, 26 and 25 cases respectively. 24(11%) of patients were under 15 years old and 196 (89%) of patients were older than 15 years.149 (68%) of patients were male and 71 (32 %) were female. 129(59%) of patients had Iranian nationality,79 (36.5%) were from Afghanistan and, 12 (5%) were from Pakistan. All isolates were resistant to co-trimoxazole, nalidixic acid, furazolidone, and intermediate to chloramphenicol and were susceptible to tetracycline, ciprofloxacin, and erythromycin. Conclusion Our study reveals that in recent outbreak caused by V. cholerae EL Tor serotype Inaba is the predominant serotype. All isolates are resistant to cotrimoxazole, nalidix acid and furazolidon.

An Outbreak of Cholera Associated with an Unprotected Well in Parbatia, Orissa, Eastern India

In November 2003, an outbreak (41 cases; attack rate-4.3%; no deaths) of severe diarrhoea was reported from a village in Orissa, eastern India. Thirteen of these cases were hospitalized. A matched case-control study was conducted to identify the possible exposure variables. Since all wells were heavily chlorinated immediately after the outbreak, water samples were not tested. The cases were managed symptomatically. Descriptive epidemiology suggested clustering of cases around one public well. Vibrio cholerae El Tor O1, serotype Ogawa was isolated from four of six rectal swabs. The water from the public well was associated with the outbreak (matched odds ratio: 12; 95% confidence interval 1.2-44.1). On the basis of these conclusions, access to the well was barred immediately, and it was protected. This investigation highlighted the broader use of field epidemiology methods to implement public-health actions guided by epidemiologic data to control a cholera epidemic.

Comparative Immunologic Activities of Polyclonal Antibodies Induced by Variably-Constituted Vibrio cholerae Ol (El Tor, Ogawa) Whole Cell Antigens

Three types of Vibrio cholerae (El tor, Ogawa) whole cell antigens constituted with liquid paraffin adjuvant (LPA), Freund’s incomplete adjuvant (FIA) and physiological saline (SA) were used for polyclonal antisera production in adult rabbits. Antisera were compared for serologic reactivities with homologous and heterologous whole cell antigens. Antisera were compared for non-specific agglutination with non-vibrio organisms and vibriocidal antibody titres against homologous Vibrio cholerae whole cell suspension in saline. The LPA and SA antisera showed cross-reactivities with Escherichia coli at titres of 20 and V. cholerae were 1280 each, while the titre of SA antiserum was 640. LPA and FIA antisera had vibriocidal titres of > 1280, while the titre of SA antiserum was 320. When cross reactive LPA and SA antisera were absorbed with E. coli , reactivity titres with homologous antigens were 640 each for LPA and SA. FIA had superior immunogenicity compared with LPA and SA, and agglutinins by FIA were more specific. Liquid paraffin appears to be a suitable low cost adjuvant for raising high quality antisera in rabbits. However, precautions should be taken to improve specificity by absorbing cross-reacting antibodies.

Presence of dfr6 Gene Cassette in Superintegron of Non-O1/Non-O139 Strain of Vibrio cholerae

Integrons, the DNA elements capable of capturing small mobile elements or gene cassettes, play a major role in the spread of antibiotic resistance in gram-negative bacteria. They are generally divided into two major groups: the multiresistance integrons (MRI), which carry mobile genetic elements, and the chromosomal superintegrons (SI). Gene cassettes carried by MRI encode resistance against antibiotics and are located in either chromosomes or plasmids. Based on the divergence of integrase genes, five classes of MRI have been reported (6). The SI identified in the genomes of the pathogenic Vibrio species sequenced to date have been found to contain a large number of gene cassettes, ranging from 72 to more than 200 (3, 7). The cassettes of the Vibrio cholerae SI were demonstrated to be substrates for the class 1 integrons of MRI (5, 8). Two class 1 integron gene cassettes, CARB4 and dfr6, were found to contain attC sites similar to those of V. cholerae repeats (VCRs) (5). Hence, it was predicted that such genes were recruited from SI. The strain identified (V. cholerae non-O1/non-O139, A444) was isolated from Vembanad Lake in the Allapuzha district of Kerala, India. The antimicrobial susceptibility test was done by using commercially available discs (Himedia). The presence of the SXT element and class 1 integrons was tested by PCR as described previously (1, 9). Shotgun cloning was performed by digesting genomic DNA with BfuC1 (NEB) and ligating the fragments with the BamHI (NEB)-digested pUC19 vector. The chemically competent Escherichia coli JM109 cells were transformed with a ligation mixture. The transformants were selected at 37°C on LB agar with ampicillin (100 mg/liter) and trimethoprim (TMP) (50 mg/liter). The plasmids from TMP-resistant clones were isolated and sequenced using a pUC/M13 universal pair of primers specific for the pUC series of vector. The sequencing was performed with an ABI Prism BigDye terminator kit using an ABI Prism 3100 DNA sequencer (Applied Biosystems). The nucleotide and deduced protein sequences were analyzed with BioEdit software (version 7.0.9.0; T. Hall, http://www.mbio.ncsu.edu/BioEdit/bioedit.html) and the BLAST search engine. V. cholerae strain A444 was resistant to ampicillin, polymixin B, TMP, cotrimoxazole, streptomycin, and furazolidone. The SXT element and class 1 integrons were shown to carry several genes encoding resistance to TMP, sulfamethoxazole, streptomycin, and spectinomycin in clinical and environmental isolates of V. cholerae (2, 9), but neither the element nor the integrons could be detected in A444. The sequencing analysis revealed that the size of the insert in the pUC19 vector is 1,708 bp. The BLAST analysis of the sequence showed the presence of intIA, dfr6, hypothetical proteins, and VCR. The dfr6 gene and one of the hypothetical proteins were flanked by VCR, and the arrangement resembled typical SI structure. The dfr6 (474 bp) obtained showed 96% similarity to the dfr6 gene reported from the class 1 integron in V. cholerae O1 (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AB200915,term_id:58219327,term_text:AB200915}}AB200915). Further, internal primers designed to amplify a 1,062-bp region from the cloned fragment in the pUC19 vector yielded a similar amplicon when PCR was done using genomic DNA as a template. The location of cassettes encoding hypothetical proteins was found within the SI of V. cholerae El Tor strain N16961 chromosome 2. Recently, novel dfr gene cassettes in the chromosomal integrons of environmental Vibrio splendidus were identified (4). Thus, the gene cassettes of SI in the Vibrionaceae family may constitute a reservoir of antibiotic resistance genes, such as dfr6, blaCARB-7, and blaCARB-9. To our knowledge, this is the first report of a demonstration of the dfr6 gene in SI of V. cholerae. Further studies are required to identify other gene cassettes in SI from non-O1/non-O139 strains of V. cholerae. This study provides evidence to support the in vivo capture of VCR cassettes by class 1 integrons.

Proteins involved in difference of sorbitol fermentation rates of the toxigenic and nontoxigenic Vibrio choleraeEl Tor strains revealed by comparative proteome analysis

BackgroundThe nontoxigenic V. cholerae El Tor strains ferment sorbitol faster than the toxigenic strains, hence fast-fermenting and slow-fermenting strains are defined by sorbitol fermentation test. This test has been used for more than 40 years in cholera surveillance and strain analysis in China. Understanding of the mechanisms of sorbitol metabolism of the toxigenic and nontoxigenic strains may help to explore the genome and metabolism divergence in these strains. Here we used comparative proteomic analysis to find the proteins which may be involved in such metabolic difference.ResultsWe found the production of formate and lactic acid in the sorbitol fermentation medium of the nontoxigenic strain was earlier than of the toxigenic strain. We compared the protein expression profiles of the toxigenic strain N16961 and nontoxigenic strain JS32 cultured in sorbitol fermentation medium, by using fructose fermentation medium as the control. Seventy-three differential protein spots were found and further identified by MALDI-MS. The difference of product of fructose-specific IIA/FPR component gene and mannitol-1-P dehydrogenase, may be involved in the difference of sorbitol transportation and dehydrogenation in the sorbitol fast- and slow-fermenting strains. The difference of the relative transcription levels of pyruvate formate-lyase to pyruvate dehydrogenase between the toxigenic and nontoxigenic strains may be also responsible for the time and ability difference of formate production between these strains.ConclusionMultiple factors involved in different metabolism steps may affect the sorbitol fermentation in the toxigenic and nontoxigenic strains of V. cholerae El Tor.

Bicarbonate InducesVibrio choleraeVirulence Gene Expression by Enhancing ToxT Activity

Vibrio cholerae is a gram-negative bacterium that is the causative agent of cholera, a severe diarrheal illness. The two biotypes of V. cholerae O1 capable of causing cholera, classical and El Tor, require different in vitro growth conditions for induction of virulence gene expression. Growth under the inducing conditions or infection of a host initiates a complex regulatory cascade that results in production of ToxT, a regulatory protein that directly activates transcription of the genes encoding cholera toxin (CT), toxin-coregulated pilus (TCP), and other virulence genes. Previous studies have shown that sodium bicarbonate induces CT expression in the V. cholerae El Tor biotype. However, the mechanism for bicarbonate-mediated CT induction has not been defined. In this study, we demonstrate that bicarbonate stimulates virulence gene expression by enhancing ToxT activity. Both the classical and El Tor biotypes produce inactive ToxT protein when they are cultured statically in the absence of bicarbonate. Addition of bicarbonate to the culture medium does not affect ToxT production but causes a significant increase in CT and TCP expression in both biotypes. Ethoxyzolamide, a potent carbonic anhydrase inhibitor, inhibits bicarbonate-mediated virulence induction, suggesting that conversion of CO(2) into bicarbonate by carbonic anhydrase plays a role in virulence induction. Thus, bicarbonate is the first positive effector for ToxT activity to be identified. Given that bicarbonate is present at high concentration in the upper small intestine where V. cholerae colonizes, bicarbonate is likely an important chemical stimulus that V. cholerae senses and that induces virulence during the natural course of infection.

Cholera outbreak secondary to contaminated pipe water in an urban area, West Bengal, India, 2006

Outbreaks of cholera are common in West Bengal. In April 2006, Garulia municipality reported a cluster of diarrhea cases. We investigated this cluster to identify the etiological agent, source of transmission and propose control measures. We defined a case of diarrhea as occurrence of > or =3 loose/watery stools a day among the residents of Garulia since April 2006. We searched for cases of diarrhea in health care facilities and health camp. We conducted a gender- and age-matched case-control study to identify risk factors. We inspected the sanitation and water supply system. We collected rectal swabs from diarrhea patients and water specimens from the affected areas for laboratory investigation. Two hundred and ninety-eight cases of diarrhea were reported to various health care facilities (attack rate: 3.5/1000, no deaths). The attack rate was highest among children (6.4/1000). Vibrio cholerae El Tor O1 Inaba was isolated from two of 7 rectal swabs. The outbreak started on 10 April 2006, peaked on 26 April and lasted till 6 May. Cases clustered in an area distal to leaking water pipelines. Drinking municipal water exclusively was significantly associated with the illness (OR 13, 95% CI=6.5-27). Eight of the 12 water specimens from the affected area had fecal contamination and poor chlorine content. This outbreak was due to a contaminated municipal piped water supply and V. cholera 01 Inaba was possibly the causative organism.

Two sequential outbreaks in two villages illustrate the various modes of transmission of cholera

We investigated two sequential outbreaks of severe diarrhoea in two neighbouring villages of Orissa, in 2005. We conducted descriptive and matched case-control studies. The attack rates were 5.6% (n=62) and 5.2% (n=51), respectively, in the first and second villages. One death was reported in the second village (case fatality 2%). We identified that consumption of milk products prepared in the household of the index case [matched odds ratio (mOR) 5.7, 95% confidence interval (CI) 1.7-30] in the first village, and drinking well water in the second village were associated with the illness (mOR 4.7, 95% CI 1.6-19). We isolated Vibrio cholerae El Tor O1 Ogawa from stool samples from both the villages. Mishandling of milk products led to a cholera outbreak in the first village, which led to sewerage contamination of a well and another outbreak in the second village. Environmental contamination should be expected and prevented during cholera outbreaks.

Isolation of Vibrio cholerae El Tor Serotype Inaba in 2006 and the most common phage type of V. cholerae in Mumbai

Sir, A total of 168 strains of Vibrio cholerae were isolated and tested over a period of 3 years (2004-2006). The strains were identified by standard methods(1) and were identified using a slide agglutination test with Vibrio cholerae O1 antisera and biotyped using a polymyxin B sensitivity test. The non-agglutinating strains were tested with V. cholerae O-139 antisera. Of 168 strains, 96 were isolated in 2004, 39 were isolated in 2005, and 33 were isolated in 2006. Eighty-three V. cholerae O1 isolates were sent to the National Institute of Cholera and Enteric Diseases (NICED) in Kolkata for serotyping and phagetyping. All the V. cholerae O1 isolates were of El Tor biotype. Among the two non-agglutinating vibrios isolated in 2005, both did not agglutinate with O-139 antisera. V. cholerae El Tor serotype Inaba was found only in 2006. The isolates of 2004 and 2005 were of the Ogawa serotype. In a previous study in the same institute, all isolates detected over a period of 5 years (1996-2000) were of the Ogawa serotype.(2) From 2001 to 2005, all isolates were V. cholerae El Tor Ogawa (unpublished). We had isolated serotype Inaba in 2006 for the first time. A shift in the occurrence of Ogawa and Inaba serotypes in a given area are thought to be a consequence of the genetic reversal that occurs in-vivo and in-vitro and is possibly mediated by the immune pressure in the population.(3) It appears that as an alternate to the Ogawa serotype, Inaba have appeared to aid the persistence of cholera and thus perpetuate the spread of Vibrio cholerae El Tor. All the isolates during 2004 to 2006 were Basu and Mukherjee phage type 2. Turbadkar et al.(4) from Mumbai have reported Ogawa serotypes in 2004 and all belonged to phage type 4. In the new phage typing scheme, out of 26 isolates sent to NICED in 2004, 14 were T26 and 12 were T27. Out of 39 isolates sent in 2005, 33 were T27 and 6 were other phage types (T13, T21, T22, T25, T23, or T15). In 2006, out of 18 isolates sent to NICED, 17 were T27 and only 1 was T26. Therefore, the most common phage type in this part of Mumbai is T27 (74.7%), followed by T26 (18.1%). In the study by Turbadkar et al.,(4) the majority belonged to phage type 27 (97.5%), which is in accordance with the present study. The 168 isolates of V.cholerae showed maximum sensitivity to amikacin (92.3%), followed by cefotaxime (89.9%). Tetracycline sensitivity was 91.1% followed by norfloxacin (86.3%). In the previous study during 1996-2000, tetracycline sensitivity was 39.6% and norfloxacin sensitivity was 46.2%.(2) Therefore, tetracycline and norfloxacin sensitivity have increased over the years. Co-trimoxazole and nalidixic acid susceptibility was only 2.4% and 1.2%, respectively in the present study. A decrease in sensitivity to nalidixic acid was observed in the present study (13.6% in previous study).(2)

Molecular characterisation of Vibrio cholerae O1 strains carrying an SXT/R391-like element from cholera outbreaks in Kenya: 1994-2007.

BackgroundOver the last decade, cholera outbreaks in parts of Kenya have become common. Although a number of recent studies describe the epidemiology of cholera in Kenya, there is paucity of information concerning the diversity and occurrence of mobile genetic elements in Vibrio cholerae strains implicated in these outbreaks. A total of 65 Vibrio cholerae O1 El Tor serotype Inaba isolated between 1994 and 2007 from various outbreaks in Kenya were investigated for mobile genetic elements including integrons, transposons, the integrating conjugative elements (ICEs), conjugative plasmids and for their genotypic relatedness.ResultsAll the strains were haemolytic on 5% sheep blood and positive for the Vibrio cholerae El Tor-specific haemolysin toxin gene (hylA) by PCR. They all contained strB, sulII, floR and the dfrA1 genes encoding resistance to streptomycin, sulfamethoxazole, chloramphenicol and trimethoprim respectively. These genes, together with an ICE belonging to the SXT/R391 family were transferable to the rifampicin-resistant E. coli C600 en bloc. All the strains were negative for integron class 1, 2 and 3 and for transposase gene of transposon Tn7 but were positive for integron class 4 and the trpM gene of transposon Tn21. No plasmids were isolated from any of the 65 strains. All the strains were also positive for all V. cholera El Tor pathogenic genes except the NAG- specific heat-stable toxin (st) gene. None of the strains were positive for virulence genes associated with the V. cholerae classical biotype. All the strains were positive for El Tor-specific CTXphi bacteriophage rstrR repressor gene (CTXETΦ) but negative for the Classical, Calcutta, and the Environmental repressor types. Pulse Field Gel Electrophoresis (PFGE) showed that regardless of the year of isolation, all the strains bearing the SXT element were clonally related.ConclusionsThis study demonstrates that the V. cholerae O1 strains carrying an SXT/R391-like element implicated in recent cholera outbreaks in Kenya has not changed significantly between 1994 and 2007 and are clonally related.

Anaerobic growth promotes synthesis of colonization factors encoded at the Vibrio pathogenicity island in Vibrio cholerae El Tor

Pathogenesis of the facultative anaerobe Vibrio cholerae takes place at the gut under low oxygen concentrations. To identify proteins which change their expression level in response to oxygen availability, proteomes of V. cholerae El Tor C7258 grown in aerobiosis, microaerobiosis and anaerobiosis were compared by two-dimensional electrophoresis. Twenty-six differentially expressed proteins were identified which are involved in several processes including iron acquisition, alanine metabolism, purine synthesis, energy metabolism and stress response. Moreover, two proteins implicated in exopolysaccharide synthesis and biofilm formation were produced at higher levels under microaerobiosis and anaerobiosis, which suggests a role of oxygen deprivation in biofilm development in V. cholerae. In addition, six proteins encoded at the Vibrio pathogenicity island attained the highest expression levels under anaerobiosis, and five of them are required for colonization: three correspond to toxin-coregulated pilus biogenesis components, one to soluble colonization factor TcpF and one to accessory colonization factor A. Thus, anaerobiosis promotes synthesis of colonization factors in V. cholerae El Tor, suggesting that it may be a key in vivo signal for early stages of the pathogenic process of V. cholerae.