Molecular Analysis of Vibrio cholerae O1, O139, non-O1, and non-O139 Strains: Clonal Relationships between Clinical and Environmental Isolates

A total of 26 strains of Vibrio cholerae, including members of the O1, O139, and non-O1, non-O139 serogroups from both clinical and environmental sources, were examined for the presence of genes encoding cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), and outer membrane protein (ompU), for genomic organization, and for the presence of the regulatory protein genes tcpI and toxR in order to determine relationships between epidemic serotypes and sources of isolation. While 22 of the 26 strains were hemolytic on 5% sheep blood nutrient agar, all strains were PCR positive for hlyA, the hemolysin gene. When multiplex PCR was used, all serogroup O1 and O139 strains were positive for tcpA, ompU, and tcpI. All O1 and O139 strains except one O1 strain and one O139 strain were positive for the ctxA, zot, and ace genes. Also, O1 strain VO3 was negative for the zot gene. All of the non-O1, non-O139 strains were negative for the ctxA, zot, ace, tcpA, and tcpI genes, and all of the non-O1, non-O139 strains except strain VO26 were negative for ompU. All of the strains except non-O1, non-O139 strain VO22 were PCR positive for the gene encoding the central regulatory protein, toxR. All V. cholerae strains were negative for the NAG-specific st gene. Of the nine non-ctx-producing strains of V. cholerae, only one, non-O1, non-O139 strain VO24, caused fluid accumulation in the rabbit ileal loop assay. The other eight strains, including an O1 strain, an O139 strain, and six non-O1, non-O139 strains, regardless of the source of isolation, caused fluid accumulation after two to five serial passages through the rabbit gut. Culture filtrates of all non-cholera-toxigenic strains grown in AKI media also caused fluid accumulation, suggesting that a new toxin was produced in AKI medium by these strains. Studies of clonality performed by using enterobacterial repetitive intergenic consensus sequence PCR, Box element PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) collectively indicated that the V. cholerae O1 and O139 strains had a clonal origin, whereas the non-O1, non-O139 strains belonged to different clones. The clinical isolates closely resembled environmental isolates in their genomic patterns. Overall, there was an excellent correlation among the results of the PCR, AFLP, and PFGE analyses, and individual strains derived from clinical and environmental sources produced similar fingerprint patterns. From the results of this study, we concluded that the non-cholera-toxin-producing strains of V. cholerae, whether of clinical or environmental origin, possess the ability to produce a new secretogenic toxin that is entirely different from the toxin produced by toxigenic V. cholerae O1 and O139 strains. We also concluded that the aquatic environment is a reservoir for V. cholerae O1, O139, non-O1, and non-O139 serogroup strains.     

Genetic characterization of toxigenic Vibrio cholerae non-O1/non-O139 strains, isolated in the Middle Asia

Here, we report on the characterization of 22 clinical toxigenic V. cholerae non-O1/non-O139 strains isolated in the Middle Asia (Uzbekistan) in 1971–1990. PCR analysis has revealed that these strains contain the main virulence genes such as ctxA, zot, ace (CTXφ); rstC (RS1φ); tcpA, toxT, aldA (pathogenicity island VPI), but they lack both pandemic islands VSP-I and VSP-II specific to epidemic strains of O1 serogroup of El Tor biotype and O139 serogroup. Only two of the twenty two toxigenic strains have tcpA gene of El Tor type, one strain has tcpA gene of classical type, while nineteen other strains carry a new variant of this gene, designated as tcpA ^uzb. Nucleotide sequences analysis of virulence genes in toxigenic V. cholerae non-O1/non-O139 strains from Uzbekistan showed that they differ significantly from the sequences of these genes in epidemic O1 and O139 strain indicating that they belong to a separate line of evolution of virulent V. cholerae strains. For the first time it is shown that V. cholerae non-O1/non-O139 toxigenic strains of different serogroups may belong to the same clone.     

Pili of a Vibrio cholerae O139

The pili of a strain of Vibrio cholerae O139 were purified and characterized. They were morphologically, electrophoretically and immunologically indistinguishable from the pili with 16 kDa subunit protein of V. cholerae O1. All 22 strains of V. cholerae O139 examined possessed the pili. The pili were different in hemagglutination inhibition pattern from V. cholerae O1 16K pili.     

Crystal structure of the Vibrio cholerae colonization factor TcpF and identification of a functional immunogenic site

Vibrio cholerae relies on two main virulence factors—toxin-coregulated pilus (TCP) and cholera toxin—to cause the gastrointestinal disease cholera. TCP is a type IV pilus that mediates bacterial autoagglutination and colonization of the intestine. TCP is encoded by the tcp operon, which also encodes TcpF, a protein of unknown function that is secreted by V. cholerae in a TCP-dependent manner. Although TcpF is not required for TCP biogenesis, a tcpF mutant has a colonization defect in the infant mouse cholera model that is as severe as a pilus mutant. Furthermore, TcpF antisera protect against V. cholerae infection. TcpF has no apparent sequence homology to any known protein. Here, we report the de novo X-ray crystal structure of TcpF and the identification of an epitope that is critical for its function as a colonization factor. A monoclonal antibody recognizing this epitope is protective against V. cholerae challenge and adds to the protection provided by an anti-TcpA antibody. These data suggest that TcpF has a novel function in V. cholerae colonization and define a region crucial for this function.     

Serological Reactivity of Humans to a Vibrio cholerae Common Antigen

Over the course of seven pandemics, Vibrio cholerae serotypes have varied. In 1992 the appearance of a new serotype, O139 Bengal, began the eighth cholera pandemic. Several new O139 antigens have been identified, yet a common V. cholerae antigen has not been described. In this study, a monoclonal antibody specific against an 18.7-kDa outer membrane antigen reacted in dotblot analysis with 292 epidemiologically diverse V. cholerae isolates including O1, non-O1, and O139 serotypes. Serum collected from volunteers experimentally challenged with V. cholerae O139, and rabbit antisera to V. cholerae O1, were reactive with the 18.7-kDa antigen by Western immunoblot. This is the first report that the 18.7-kDa antigen is present in V. cholerae O139. Received: 11 August 1997 / Accepted: 22 September 1997     

Interaction of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> non-O1/non-O139 with Copepods,Cladocerans and Competing Bacteria in the Large Alkaline Lake Neusiedler See,Austria

Vibrio cholerae is a human pathogen and natural inhabitant of aquatic environments. Serogroups O1/O139 have been associated with epidemic cholera, while non-O1/non-O139 serogroups usually cause human disease other than classical cholera. V. cholerae non-O1/non-O139 from the Neusiedler See, a large Central European lake, have caused ear and wound infections, including one case of fatal septicaemia. Recent investigations demonstrated rapid planktonic growth of V. cholerae non-O1/non-O139 and correlation with zooplankton biomass. The aim of this study was to elucidate the interaction of autochthonous V. cholerae with two dominant crustacean zooplankton species in the lake and investigate the influence of the natural bacterial community on this interaction. An existing data set was evaluated for statistical relationships between zooplankton species and V. cholerae and co-culture experiments were performed in the laboratory. A new fluorescence in situ hybridisation protocol was applied for quantification of V. cholerae non-O1/non-O139 cells, which significantly reduced analysis time. The experiments clearly demonstrated a significant relationship of autochthonous V. cholerae non-O1/non-O139 with cladocerans by promoting growth of V. cholerae non-O1/non-O139 in the water and on the surfaces of the cladocerans. In contrast, copepods had a negative effect on the growth of V. cholerae non-O1/non-O139 via competing bacteria from their surfaces. Thus, beside other known factors, biofilm formation by V. cholerae on crustacean zooplankton appears to be zooplankton taxon specific and may be controlled by the natural bacterial community.     

Genotypes Associated with Virulence in Environmental Isolates of Vibrio cholerae

Vibrio cholerae is an autochthonous inhabitant of riverine and estuarine environments and also is a facultative pathogen for humans. Genotyping can be useful in assessing the risk of contracting cholera, intestinal, or extraintestinal infections via drinking water and/or seafood. In this study, environmental isolates of V. cholerae were examined for the presence of ctxA, hlyA, ompU, stn/sto, tcpA, tcpI, toxR, and zot genes, using multiplex PCR. Based on tcpA and hlyA gene comparisons, the strains could be grouped into Classical and El Tor biotypes. The toxR, hlyA, and ompU genes were present in 100, 98.6, and 87.0% of the V. cholerae isolates, respectively. The CTX genetic element and toxin-coregulated pilus El Tor (tcpA ET) gene were present in all toxigenic V. cholerae O1 and V. cholerae O139 strains examined in this study. Three of four nontoxigenic V. cholerae O1 strains contained tcpA ET. Interestingly, among the isolates of V. cholerae non-O1/non-O139, two had tcpA Classical, nine contained tcpA El Tor, three showed homology with both biotype genes, and four carried the ctxA gene. The stn/sto genes were present in 28.2% of the non-O1/non-O139 strains, in 10.5% of the toxigenic V. cholerae O1, and in 14.3% of the O139 serogroups. Except for stn/sto genes, all of the other genes studied occurred with high frequency in toxigenic V. cholerae O1 and O139 strains. Based on results of this study, surveillance of non-O1/non-O139 V. cholerae in the aquatic environment, combined with genotype monitoring using ctxA, stn/sto, and tcpA ET genes, could be valuable in human health risk assessment.     

Structural characterization of CFA/III and Longus type IVb pili from enterotoxigenic Escherichia coli

The type IV pili are helical filaments found on many Gram-negative pathogenic bacteria, with multiple diverse roles in pathogenesis, including microcolony formation, adhesion, and twitching motility. Many pathogenic enterotoxigenic Escherichia coli (ETEC) isolates express one of two type IV pili belonging to the type IVb subclass: CFA/III or Longus. Here we show a direct correlation between CFA/III expression and ETEC aggregation, suggesting that these pili, like the Vibrio cholerae toxin-coregulated pili (TCP), mediate microcolony formation. We report a 1.26-Å resolution crystal structure of CofA, the major pilin subunit from CFA/III. CofA is very similar in structure to V. cholerae TcpA but possesses a 10-amino-acid insertion that replaces part of the α2-helix with an irregular loop containing a 3₁₀-helix. Homology modeling suggests a very similar structure for the Longus LngA pilin. A model for the CFA/III pilus filament was generated using the TCP electron microscopy reconstruction as a template. The unique 3₁₀-helix insert fits perfectly within the gap between CofA globular domains. This insert, together with differences in surface-exposed residues, produces a filament that is smoother and more negatively charged than TCP. To explore the specificity of the type IV pilus assembly apparatus, CofA was expressed heterologously in V. cholerae by replacing the tcpA gene with that of cofA within the tcp operon. Although CofA was synthesized and processed by V. cholerae, no CFA/III filaments were detected, suggesting that the components of the type IVb pilus assembly system are highly specific to their pilin substrates.     

Structure and secretion of CofJ,a putative colonization factor of enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) colonize the human gut, causing severe cholera‐like diarrhoea. ETEC utilize a diverse array of pili and fimbriae for host colonization, including the Type IVb pilus CFA/III. The CFA/III pilus machinery is encoded on the cof operon, which is similar in gene sequence and synteny to the tcp operon that encodes another Type IVb pilus, the Vibrio cholerae toxin co‐regulated pilus (TCP). Both pilus operons possess a syntenic gene encoding a protein of unknown function. In V. cholerae, this protein, TcpF, is a critical colonization factor secreted by the TCP apparatus. Here we show that the corresponding ETEC protein, CofJ, is a soluble protein secreted via the CFA/III apparatus. We present a 2.6 Å resolution crystal structure of CofJ, revealing a large β‐sandwich protein that bears no sequence or structural homology to TcpF. CofJ has a cluster of exposed hydrophobic side‐chains at one end and structural homology to the pore‐forming proteins perfringolysin O and α‐haemolysin. CofJ binds to lipid vesicles and epithelial cells, suggesting a role in membrane attachment during ETEC colonization.     

Overexpression of the tcp Gene Cluster Using the T7 RNA Polymerase/Promoter System and Natural Transformation-Mediated Genetic Engineering of Vibrio cholerae

The human pathogen and aquatic bacterium Vibrio cholerae belongs to the group of naturally competent bacteria. This developmental program allows the bacterium to take up free DNA from its surrounding followed by a homologous recombination event, which allows integration of the transforming DNA into the chromosome. Taking advantage of this phenomenon we genetically engineered V. cholerae using natural transformation and FLP recombination. More precisely, we adapted the T7 RNA polymerase/promoter system in this organism allowing expression of genes in a T7 RNA polymerase-dependent manner. We naturally transformed V. cholerae by adding a T7-specific promoter sequence upstream the toxin-coregulated pilus (tcp) gene cluster. In a V. cholerae strain, which concomitantly produced the T7 RNA polymerase, this genetic manipulation resulted in the overexpression of downstream genes. The phenotypes of the strain were also in line with the successful production of TCP pili. This provides a proof-of-principle that the T7 RNA polymerase/promoter system is functional in V. cholerae and that genetic engineering of this organism by natural transformation is a straightforward and efficient approach.     

Genomic characterization of antibiotic resistance-encoding genes in clinical isolates of Vibrio cholerae non-O1/non-O139 strains from Kolkata,India: generation of novel types of genomic islands containing plural antibiotic resistance genes

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

Molecular analysis of Vibrio cholerae O1, O139, non-O1, and non-O139 strains: clonal relationships between clinical and environmental isolates

A total of 26 strains of Vibrio cholerae, including members of the O1, O139, and non-O1, non-O139 serogroups from both clinical and environmental sources, were examined for the presence of genes encoding cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), and outer membrane protein (ompU), for genomic organization, and for the presence of the regulatory protein genes tcpI and toxR in order to determine relationships between epidemic serotypes and sources of isolation. While 22 of the 26 strains were hemolytic on 5% sheep blood nutrient agar, all strains were PCR positive for hlyA, the hemolysin gene. When multiplex PCR was used, all serogroup O1 and O139 strains were positive for tcpA, ompU, and tcpI. All O1 and O139 strains except one O1 strain and one O139 strain were positive for the ctxA, zot, and ace genes. Also, O1 strain VO3 was negative for the zot gene. All of the non-O1, non-O139 strains were negative for the ctxA, zot, ace, tcpA, and tcpI genes, and all of the non-O1, non-O139 strains except strain VO26 were negative for ompU. All of the strains except non-O1, non-O139 strain VO22 were PCR positive for the gene encoding the central regulatory protein, toxR. All V. cholerae strains were negative for the NAG-specific st gene. Of the nine non-ctx-producing strains of V. cholerae, only one, non-O1, non-O139 strain VO24, caused fluid accumulation in the rabbit ileal loop assay. The other eight strains, including an O1 strain, an O139 strain, and six non-O1, non-O139 strains, regardless of the source of isolation, caused fluid accumulation after two to five serial passages through the rabbit gut. Culture filtrates of all non-cholera-toxigenic strains grown in AKI media also caused fluid accumulation, suggesting that a new toxin was produced in AKI medium by these strains. Studies of clonality performed by using enterobacterial repetitive intergenic consensus sequence PCR, Box element PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) collectively indicated that the V. cholerae O1 and O139 strains had a clonal origin, whereas the non-O1, non-O139 strains belonged to different clones. The clinical isolates closely resembled environmental isolates in their genomic patterns. Overall, there was an excellent correlation among the results of the PCR, AFLP, and PFGE analyses, and individual strains derived from clinical and environmental sources produced similar fingerprint patterns. From the results of this study, we concluded that the non-cholera-toxin-producing strains of V. cholerae, whether of clinical or environmental origin, possess the ability to produce a new secretogenic toxin that is entirely different from the toxin produced by toxigenic V. cholerae O1 and O139 strains. We also concluded that the aquatic environment is a reservoir for V. cholerae O1, O139, non-O1, and non-O139 serogroup strains.     

Effects of Polyamines on Vibrio cholerae Virulence Properties

Vibrio cholerae is the causative agent of the severe enteric disease cholera. To cause cholera the bacterium must be able to synthesize both cholera toxin (CT) and toxin-coregulated pilus (TCP) which mediates autoagglutination and is required for colonization of the small intestine. Only a few environmental signals have been shown to regulate V. cholerae virulence gene expression. Polyamines, which are ubiquitous in nature, and have been implicated in regulating virulence gene expression in other bacteria, have not been extensively studied for their effect on V. cholerae virulence properties. The objective of this study was to test the effect of several polyamines that are abundant in the human intestine on V. cholerae virulence properties. All of the polyamines tested inhibited autoagglutination of V. cholerae O1 classical strain in a concentration dependent manner. Putrescine and cadaverine decreased the synthesis of the major pilin subunit, TcpA, spermidine increased its production, and spermine had no effect. Putrescine and spermidine led to a decrease and increase, respectively, on the relative abundance of TCP found on the cell surface. Spermine led to a small reduction in cholera toxin synthesis whereas none of the other polyamines had an effect. The polyamines did not affect pili bundling morphology, but caused a small reduction in CTXφ transduction, indicating that the TCP present on the cell surface may not be fully functional. We hypothesize the inhibition of autoagglutination is likely to be caused by the positively charged amine groups on the polyamines electrostatically disrupting the pili-pili interactions which mediate autoagglutination. Our results implicate that polyamines may have a protective function against colonization of the small intestine by V. cholerae.     

Ribotyping and TCP gene cluster analysis of environmental and clinical Vibrio cholerae strains isolated in Iran

The aim of this study was to investigate the presence of TCP gene clusters among clinical and environmental Vibrio cholerae isolates and to explore the genetic relatedness of isolates using ribotyping technique. A total of 50 V. cholerae strains (30 clinical and 20 environmental) were included in this study. Three clinical isolates were negative for TCP cluster genes while the cluster was absent in all of the environmental strains. Ribotyping of rRNA genes with BglI produced 18 different ribotype patterns, three of which belonged to clinical O1 serotype isolates. The remaining 15 ribotypes belonged to clinical non-O1, non-O139 serogroups (two patterns) and environmental non-O1, non-O139 serogroups (13 patterns). Clinical V. cholerae O1 strains from 2004 through 2006 and several environmental non-O1, non-O139 V. cholerae strains from 2006 showed 67.3 % similarity and fell within one single gene cluster. Ribotyping analysis made it possible to further comprehend the close originality of clinical isolates as very little changes have been occurred within rRNA genes of different genotypes of V. cholerae strains through years. In conclusion, ribotyping analysis of environmental V. cholerae isolates showed a substantial genomic diversity supporting the fact that genetic changes within bacterial genome occurs during years in the environment, while only little changes may arise within the genome of clinical isolates.     

Secretion of TcpF by the Vibrio cholerae Toxin-Coregulated Pilus Biogenesis Apparatus Requires an N-Terminal Determinant

Type IV pili are important for microcolony formation, biofilm formation, twitching motility, and attachment. We and others have shown that type IV pili are important for protein secretion across the outer membrane, similar to type II secretion systems. This study explored the relationship between protein secretion and pilus formation in Vibrio cholerae. The toxin-coregulated pilus (TCP), a type IV pilus required for V. cholerae pathogenesis, is necessary for the secretion of the colonization factor TcpF (T. J. Kirn, N. Bose, and R. K. Taylor, Mol. Microbiol. 49:81–92, 2003). This phenomenon is not unique to V. cholerae; secreted virulence factors that are dependent on the presence of components of the type IV pilus biogenesis apparatus for secretion have been reported with Dichelobacter nodosus (R. M. Kennan, O. P. Dhungyel, R. J. Whittington, J. R. Egerton, and J. I. Rood, J. Bacteriol. 183:4451–4458, 2001) and Francisella tularensis (A. J. Hager et al., Mol. Microbiol. 62:227–237, 2006). Using site-directed mutagenesis, we demonstrated that the secretion of TcpF is dependent on the presence of selected amino acid R groups at position five. We were unable to find other secretion determinants, suggesting that Y5 is the major secretion determinant within TcpF. We also report that proteins secreted in a type IV pilus biogenesis apparatus-dependent manner have a YXS motif within the first 15 amino acids following the Sec cleavage site. The YXS motif is not present in proteins secreted by type II secretion systems, indicating that this is unique to type IV pilus-mediated secretion. Moreover, we show that TcpF interacts with the pilin TcpA, suggesting that these proteins are secreted by the type IV pilus biogenesis system. These data provide a starting point for understanding how type IV pili can mediate secretion of virulence factors important for bacterial pathogenesis.     

Genetic Diversity of Clinical and Environmental Isolates of Vibrio cholerae Determined by Amplified Fragment Length Polymorphism Fingerprinting

Vibrio cholerae, the causative agent of major epidemics of diarrheal disease in Bangladesh, South America, Southeastern Asia, and Africa, was isolated from clinical samples and from aquatic environments during and between epidemics over the past 20 years. To determine the evolutionary relationships and molecular diversity of these strains, in order to understand sources, origin, and epidemiology, a novel DNA fingerprinting technique, amplified fragment length polymorphism (AFLP), was employed. Two sets of restriction enzyme-primer combinations were tested for fingerprinting of V. cholerae serogroup O1, O139, and non-O1, O139 isolates. Amplification of HindIII- and TaqI-digested genomic DNA produced 30 to 50 bands for each strain. However, this combination, although capable of separating environmental isolates of O1 and non-O1 strains, was unable to distinguish between O1 and O139 clinical strains. This result confirmed that clinical O1 and O139 strains are genetically closely related. On the other hand, AFLP analyses of restriction enzyme ApaI- and TaqI-digested genomic DNA yielded 20 to 30 bands for each strain, but were able to separate O1 from O139 strains. Of the 74 strains examined with the latter combination, 26 serogroup O1 strains showed identical banding patterns and were represented by the O1 El Tor strain of the seventh pandemic. A second group, represented by O139 Bengal, included 12 strains of O139 clinical isolates, with 7 from Thailand, 3 from Bangladesh, and 2 from India. Interestingly, an O1 clinical isolate from Africa also grouped with the O139 clinical isolates. Eight clinical O1 isolates from Mexico grouped separately from the O1 El Tor of the seventh pandemic, suggesting an independent origin of these isolates. Identical fingerprints were observed between an O1 environmental isolate from a river in Chile and an O1 clinical strain from Kenya, both isolated more than 10 years apart. Both strains were distinct from the O1 seventh pandemic strain. Two O139 clinical isolates from Africa clustered with environmental non-O1 isolates, independent of other O139 strains included in the study. These results suggest that although a single clone of pathogenic V. cholerae appears responsible for many cases of cholera in Asia, Africa, and Latin America during the seventh pandemic, other cases of clinical cholera were caused by toxigenic V. cholerae strains that appear to have been derived locally from environmental O1 or non-O1 strains.     

Indigenous Vibrio cholerae strains from a non-endemic region are pathogenic

Of the 200+ serogroups of Vibrio cholerae, only O1 or O139 strains are reported to cause cholera, and mostly in endemic regions. Cholera outbreaks elsewhere are considered to be via importation of pathogenic strains. Using established animal models, we show that diverse V. cholerae strains indigenous to a non-endemic environment (Sydney, Australia), including non-O1/O139 serogroup strains, are able to both colonize the intestine and result in fluid accumulation despite lacking virulence factors believed to be important. Most strains lacked the type three secretion system considered a mediator of diarrhoea in non-O1/O13 V. cholerae. Multi-locus sequence typing (MLST) showed that the Sydney isolates did not form a single clade and were distinct from O1/O139 toxigenic strains. There was no correlation between genetic relatedness and the profile of virulence-associated factors. Current analyses of diseases mediated by V. cholerae focus on endemic regions, with only those strains that possess particular virulence factors considered pathogenic. Our data suggest that factors other than those previously well described are of potential importance in influencing disease outbreaks.     

Occurrence of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> non-O1 in three wastewater treatment plants in Agadir (Morocco)

A total of 21 isolates of Vibrio cholerae non-O1 strains were isolated from three wastewater treatment plants in Agadir, Morocco. The isolates were analyzed by biochemical analysis, antibiogram, pulsed-field gel electrophoresis and the MALDI-TOF patterns of their protein masses were compared. Over 67% of isolates were susceptible to antimicrobial agents tested and 14% proved resistant to both trimethoprim-sulfamethoxazole and nalidixic acid. Typing by pulsed-field gel electrophoresis with NotI digestion revealed that the V. cholerae non-O1 strains from Agadir (Morocco) have a lower level of genetic homogeneity, the restriction patterns of whole-chromosomal DNA grouped the V. cholerae O1 and V. alginolyticus strains into a separate cluster from V. metschnikovii and V. cholerae non-O1 isolates. Furthermore, to gain additional analytical accuracy and reliability in the analysis we used dendrogram based on MALDI-TOF spectral patterns generated by the BioTyper 1.1™ software. All m/z signatures of all strains tested indicate that the mass spectral data contained sufficient information to distinguish between strains of V. cholerae.     

Epidemic isolates of Vibrio cholerae 0139 express antigenically distinct types of colonization pili

Abstract Vibrio cholerae belonging to the recently described serogroup 0139, which are responsible for the current cholera epidemics in India and Bangladesh, were shown to express pilus-like structures partially cross-reacting with the toxin-coregulated pilus of V. cholerae strain (0395) belonging to the 01 serogroup and classical biotype. The 0139 pili were composed of 20 kDa subunit proteins which were antigenically related to the 20 kDa pilus protein of another diarrhoeagenic non-01 V. cholerae strain (serogroup 034) isolated earlier. The pili described in this study were found to be involved in the intestinal colonization process and, therefore, may contribute towards the virulence of the 0139 epidemic isolates.