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The evolution of epidemic Vibrio cholerae strains
Abstract
The emergence of the novel Vibrio cholerae strain, O139 Bengal, which caused a large epidemic in Southeast Asia, underlines the adaptability of pathogenic microorganisms. Recent studies reveal that horizontal transfer of cell-wall polysaccharide genes played a central role in the emergence of this strain and that its genesis may not be as unique as initially believed.
... El cólera representa la enfermedad más deshidratante que se conoce. Se presenta generalmente en forma epidémica o pandémica' y se adquiere por ingestión de agua o alimentos contaminados con materia fecal humana proveniente de individuos infectados (Madico, 1996;Mooi, 1997;McLaughlin, 1995). ...
... Una vez que la bacteria del cólera (Ebrio cholerae) llega al intestino delgado, se adhiere firmemente a las células epiteliales, en cuya superficie mucosa se reproduce rápidamente,"iberando la potente enterotoxina del cólera, también conocida como colerágeno (Madico, 1996;Mooi, 1997;McLaughlin, 1995;Roth, 1995;Salyers, 1994). ...
... CLAVE: ' = peptona al 1% estbril ajustada a pH aproximado de 8.6; '=Tiosulfato-citrato-bilis-sacarosa; '= las colonias sacarosa positiva son de color amarillo, ya que el TCBS contiene indicador azul de bromo-tirnol; = los rnicroorganisrnos se observan al microscopio, previa preparacibn de extensiones teñidas al ~ram;~=reaccibn en cadena de la polimerasa;6= reacciones de aglutinacibn con sueros anti-B y anti-C. Diagrama 3.Metodología implicada en la detección de V. cholerae en muestras clínicas(Madico, 1996;Mooi, 1997;McLaughlin, 1995; Faruque, 1997;Osawa, 1997;Falklind, 1996). ...
span>Sin lugar a dudas, los padecimientos bacterianos destacan por su frecuencia, gravedad y diversidad; algunos ejemplos representativos son los siguientes: a) las numerosas intoxicaciones alimentarias asociadas a la ingestión de toxinas liberadas en los alimentos y en las fórmulas lácteas por especies tales como Staphylococcus aureus y Bacillus cereus, b) las cotidianas enteritis y gastroenteritis debidas a Salmonella, Shigella, Campylobacter jejuni, Yersinia enterocolitica, Clostridium pefringenr y a diversos grupos de Escherichia coli...</span
... These infections usually lead to "vibriosis" a terminal hemorrhagic septicemia in marine and freshwater fishes (Thune et al. 1993;Okuda et al. 2001), as well as in humans (Stelma et al.. 1992). Other Vibrio species, like V. cholerae, have been extensively studied due to their broad range of host virulence (Mooi and Bik 1997). Evolution of virulent strains has been of importance to human health and control of epidemics; although only a few serotypes have been fully characterized, there are still continual outbreaks of disease due to newly formed serotypes that are closely related to well studied strains. ...
... One example is that of the O1 serotype of V. cholerae; the main subdivisions of this strain are the classical and El Tor biotypes. These strains were linked to several outbreaks between 1881 and 1961, and yet a third serotype (O139 or Bengal) was discovered to be the culprit of a subsequent outbreak in India in 1992 (Mooi and Bik 1997). DNA fingerprinting confirms that the O139 strain is similar to El Tor and classic strains, however there are major differences that exist in several virulence factors that cause differences in virulence phenotypes. ...
... As stated previously, genomic data has revealed that there is no strict congruence between phylogenetic relatedness and the physiological mechanisms that cause virulence. Several genes or gene families have been found that are responsible for transfer; cell-wall polysaccharide genes of several V. cholerae strains (El Tor, O139, O69, and O141) are associated with a mobile element (IS1358) that allows homologous recombination between lipopolysaccharide (LPS) gene clusters between different strains (Mooi and Bik 1997;Stroeher et al. 1995) Homologous gene clusters are also found in a number of other bacteria (not all pathogens), and may also play a role in mobilizing DNA directly by transposition to a phage (or plasmid) as well as in recombination. Both clinical and environmental isolates of V. cholerae have been found to contain the CTX phage, which encode the genes for cholera toxin (CT; Dalsgaard et al. 2001). ...
... Smith and Fratamico (1995) listed foodborne pathogens that have emerged within the past 20 years (Table 1). More recent additions include Salmonella typhirnurium DT104 (CDC 1997a), Cyclospora cuyetanensis (Herwaldt and Ackers 1997), Vibrio cholerae 0139 (Albert 1994;Mooi and Bik 1997) and non-0157 serotypes of enterohemorrhagic Escherichia coli (Johnson et al. 1996). ...
... Interestingly, in 1992, epidemic cholera due to serotype 0139 V. cholerae emerged and has spread through southeast Asia (Albert 1994). A toxin-producing V. cholerae probably acquired the 0139 antigen by horizontal gene transfer; the emergence of the new cholera toxinproducing serotype may have been facilitated by naturally acquired immunity to the 0 1 serotype (Mooi and Bik 1997). What is even more interesting, a new clone of El Tor 0 1 has displaced 0139 as the cholera-producing organism in Calcutta. ...
IntroductionFactors Contributing to Emergence or ReemergenceEscherichza Coli 0157:H7 as a Model for the Emergence of a Foodborne PathogenAnticipating the Next Emerging PathogenForecast for Pathogen EmergenceTwo-Tiered Approach to Addressing Research NeedsWars Avoided; Battles WonConclusions
References
... Traditional typing of bacterial pathogens by serological or other methods (antibiotic resistance, phage typing, etc.) may be relevant to disease treatment and to vaccine development but is increasingly unsuitable for modern epidemiology because it is based on variable phenotypes that are not necessarily related to genetic descent. Horizontal genetic exchange leads to the acquisition of genes encoding foreign lipopolysaccharides (LPS) (Reeves, 1993; Comstock et al., 1996; Davies et al., 1997; Mooi and Bik, 1997; Shepherd et al., 2000) or capsular polysaccharides (Kroll and Moxon, 1990; Swartley et al., 1997, 1998; Morona et al., 1999). Antibiotic resistance is also spread by horizontal genetic exchange (Dowson et al., 1994; Reichmann et al., 1997; Bootsma et al., 2000) as well as by mobile genetic elements (Hall and Collis, 1995). ...
... The extent of microevolution in V. cholerae has not yet been extensively investigated, although numerous hints exist that it occurs frequently (Mooi and Bik, 1997; Reidl and Mekalanos, 1995). The CTX0 phage containing the ctxA and ctxB genes that encode cholera toxin subunits has apparently been introduced from separate sources into El Tor and classical strains (Boyd et al., 2000). ...
This chapter presents a phylogenetic perspective of molecular bacterial epidemiology. It summarizes approaches that are informative from the phylogenetic viewpoint, and describes results from selected pathogenic species where the available information seems to be somewhat more complete. Horizontal genetic exchange leads to the acquisition of genes encoding foreign lipopolysaccharides (LPS) or capsular polysaccharides. Antibiotic resistance is also spread by horizontal genetic exchange as well as by mobile genetic elements. Selectively neutral markers that are not under selection are more likely to index the genetic relatedness between isolates and less likely to be imported by gene transfer than are serological markers. A classification scheme that is based on selectively neutral markers would reflect the population biology of the species under investigation and can be used for phylogenetic analyses. Analysis of the sequence variation of multiple genes encoding housekeeping enzymes is probably the most reliable method for the classification of clonal bacteria into genetically related groupings. Sequence variation in housekeeping genes is usually neutral, that is, not subject to natural selection, and therefore the differences between alleles of housekeeping genes are more likely to reflect the time since phylogenetic descent than are those of genes whose products are under selection.
... Smith and Fratamico (1995) listed foodborne pathogens that have emerged within the past 20 years (Table 1). More recent additions include Salmonella typhirnurium DT104 (CDC 1997a), Cyclospora cuyetanensis (Herwaldt and Ackers 1997), Vibrio cholerae 0139 (Albert 1994;Mooi and Bik 1997) and non-0157 serotypes of enterohemorrhagic Escherichia coli (Johnson et al. 1996). ...
... Interestingly, in 1992, epidemic cholera due to serotype 0139 V. cholerae emerged and has spread through southeast Asia (Albert 1994). A toxin-producing V. cholerae probably acquired the 0139 antigen by horizontal gene transfer; the emergence of the new cholera toxinproducing serotype may have been facilitated by naturally acquired immunity to the 0 1 serotype (Mooi and Bik 1997). What is even more interesting, a new clone of El Tor 0 1 has displaced 0139 as the cholera-producing organism in Calcutta. ...
Control of foodborne emerging or reemerging microbial pathogens has proven to be difficult. Even the Hazard Analysis Critical Control Point (HACCP) approach is intended to manage known hazards. We propose that each change occurring in the food chain – encompassing human, technological, and environmental factors – creates a new selection pressure that drives microbial adaptation and emergence potential. Escherichia coli O157:H7 is examined here, as a case study, to illustrate the multidimensional nature of pathogen emergence. While future emergence or reemergence events are expected, the fundamental questions of what, where, who, when, and how such events will unfold are unknown. Contingency planning can provide responses to probable hazard scenarios, with a goal of developing practical controls. Examples of potential microbial hazards and changes in the food chain are presented. Once a hazard, associated food, locale, and at-risk population are identified, critical acute research questions need to be answered. Longer term research will improve our ability to respond to the next inevitable emergence event. Such coordinated endeavors will permit rapid modification and deployment of a science-based hazard management system that will prevent or minimize human risks.
... Once it was shown that certain strains of V. cholerae O37 were closely related to classical V. cholerae O1 (discussed more deeply elsewhere in this review), the similarity of the sequence of DNA flanking the O-antigen locus in V. cholerae O1 and V. cholerae O37 led to the hypothesis that homologous recombination had caused the genes conferring an O1 genotype to have been exchanged with those encoding the O37 serogroup, causing seroconversion [49]. This was of particular relevance because similar recombination events had been suggested to have enabled seroconversion of V. cholerae O1 to O139 [13,49,67,68]. Subsequently, Blokesch and Schoolnik demonstrated that genomic DNA prepared from O37 serogroup strain ATCC 25872 could be used to transform naturally competent V. cholerae O1 and convert them to serogroup O37 [32]. ...
Between 1965 and 1968, outbreaks of cholera in Sudan and former Czechoslovakia provoked considerable public health concern. These still represent important historical events that need to be linked to the growing genomic evidence describing the aetiological agent of cholera, Vibrio cholerae. Whilst O1 serogroup V. cholerae are canonically associated with epidemic and pandemic cholera, these events were caused by a clone of toxigenic V. cholerae O37 that may be more globally distributed than just to Europe and North Africa. Understanding the biology of these non-O1 strains of V. cholerae is key to understanding how diseases like cholera continue to be globally important. In this article, we consolidate epidemiological, molecular and genomic descriptions of the bacteria responsible for these outbreaks. We attempt to resolve discrepancies in order to summarize the history and provenance of as many commonly used serogroup O37 strains as possible. Finally, we highlight the potential for whole-genome sequencing of V. cholerae O37 isolates from strain collections to shed light on the open questions that we identify.
... In addition to the pandemic-causing O1 serogroup, a new serogroup of V. cholerae, O139, emerged in Bangladesh and India in 1992 causing localized cholera outbreaks (Nair et al. 1994). The LPS of the O139 serogroup was very different from V. cholerae O1 and emerged due to the replacement of a 22 kb wbe locus that codes for the O-antigen polysaccharide in the O1 strains with a wbf region encoding the O139 antigen (Comstock et al. 1995;Mooi and Bik 1997;Stroeher et al. 1997). Interestingly, evidence suggests that the O139 wbf locus arose due to genetic rearrangements of DNA from several donors because parts of wbf have been identified in other non-O1 V. cholerae serotypes such as O22 (Stroeher et al. 1997;Yamasaki et al. 1999). ...
Cholera is a severe diarrheal disease caused by the aquatic bacterium Vibrio cholerae. Interestingly, to date, only one major clade has emerged to cause pandemic disease in humans: the clade that encompasses the strains from the O1 and O139 serogroups. In this chapter, we provide a comprehensive perspective on the virulence factors and mobile genetic elements (MGEs) associated with the emergence of pandemic V. cholerae strains and highlight novel findings such as specific genomic background or interactions between MGEs that explain their confined distribution. Finally, we discuss pandemic cholera dynamics contextualizing them within the evolution of the bacterium.Keywords
Vibrio cholerae
CholeraPathogen emergenceCholera pandemicsPathogen evolution
... In de loop van de 19e eeuw werd Nederland viermaal door een cholera-epidemie geteisterd, de eerste in 1832, de laatste in 1866. 2,3 Organisatorisch was het land in 1832 voorbereid dankzij choleracommissies en cholerahospitalen. Buiten het land was de pandemie al enige tijd aan de gang en men verwachtte terecht dat ook ons land zou worden getroffen. ...
Infectieziekten waren altijd in meer of mindere mate een onderdeel van het werk van huisartsen. De COVID-19-pandemie heeft de dagelijkse praktijk voor huisartsen echter blijvend veranderd. Hoe deden dokters hun werk tijdens eerdere pandemieën? Wat waren hun ervaringen en wat schreven ze daarover? Deze bijdrage schetst een beeld van tijden van cholera, pokken en de Spaanse griep.
... V. cholerae O139 most likely emerged through the homologous recombination at the gmhD gene and IS1358 that are present upstream and downstream of the wbe cluster conferring the O antigen phenotype 18 . This resulted in the replacement of a 22 kb wbe region of the O1 antigen with 35 kb wbf gene cluster encoding the O139 antigen 19 . Biosynthesis of the O139 antigen appeared to be similar to that in a strain of the O22 serogroup, which might have been the original donor 18 . ...
Cholera is a life-threatening infectious disease that remains an important public health issue in several low and middle-income countries. In 1992, a newly identified O139 Vibrio cholerae temporarily displaced the O1 serogroup. No study has been able to answer why the potential eighth cholera pandemic (8CP) causing V. cholerae O139 emerged so successfully and then died out. We conducted a genomic study, including 330 O139 isolates, covering emergence of the serogroup in 1992 through to 2015. We noted two key genomic evolutionary changes that may have been responsible for the disappearance of genetically distinct but temporally overlapping waves (A-C) of O139. Firstly, as the waves progressed, a switch from a homogenous toxin genotype in wave-A to heterogeneous genotypes. Secondly, a gradual loss of antimicrobial resistance (AMR) with the progression of waves. We hypothesize that these two changes contributed to the eventual epidemiological decline of O139. The O139 Vibrio cholerae serogroup emerged in the 1990s and spread rapidly but did not become globally dominant. Here, the authors describe the genomic epidemiology of this strain and identify changes in virulence and antimicrobial resistance characteristics that they hypothesise may have contributed to its decline.
... Its genes were compared with other somatic antigenic types known (which are O2-O138) as at then but it shows remarkably difference from all existing sero-group types (Faruque et al. 2003). It was later referred to as O139 V. cholerae which continues to cause epidemic, spreading from India through the entire Asia continent to other continents (Aydanian et al. 2011;Mooi and Bik, 1997;Manning et al. 1995;Nair et al. 1994). These novel characters observed amongst the O139 members actually change the myth of the Non-O1 involvement in epidemic and choleragenic potential. ...
Somatic antigen agglutinable type Vibrio cholerae of 1/139 (SAAT-Vc-1/139) members or O1/O139 Vibrio cholerae have been described by various investigators as choleragenic due to their increase pathogenic potential and production of choleragen. Reported cholera outbreak cases around the World have been associated with these choleragenic Vibrio cholerae with high case fatality involving various educational, human, governmental, animal and financial resources. These Vibrio members have shown genealogical and phylogenetic relationship with the somatic antigen non-agglutinable strains of 1/139 Vibrio cholerae (SANAS- Vc -1/139) or O1/O139 non-agglutinating Vibrio cholerae (O1/O139-NAG-Vc). The O1/O139-NAGVc members have been reported to be implicated in most cholera/cholera-like cases, sporadic cases, diarrhea, production of cholera toxin and transmitted via consumption and/or contact with contaminated water/seafood. Some reported cases of cholera outbreaks, sporadic cases and observed change in nature has also been tracable to these non-agglutinable Vibrio members (O1/O139-NAGVc) yet there is a sustained paucity of reports on the non-agglutinable V. cholerae members implication in cholera outbreak. The emergence of fulminating extraintestinal and systemic Vibriosis is another aspect of SANAS- Vc -1/139 involvement which has received low attention in terms of research driven interest. This review addresses the need to appraise and continue in research based studies on the somatic antigen non-serogroup agglutinable type-1/139 Vibrio cholerae members which are currently prevalent in water bodies, fruits/vegetables, foods and terrestrial environment. Our opinion is a summative of interest in integrated surveillance studies, management/control of cholera outbreaks as well as diarrhea and other disease related cases both in the rural, suburban and urban metropolis.
... Its genes were compared with other somatic antigenic types known (which are O2-O138) as at then but it shows remarkably difference from all existing sero-group types (Faruque et al. 2003). It was later referred to as O139 V. cholerae which continues to cause epidemic, spreading from India through the entire Asia continent to other continents (Aydanian et al. 2011;Mooi and Bik, 1997;Manning et al. 1995;Nair et al. 1994). These novel characters observed amongst the O139 members actually change the myth of the Non-O1 involvement in epidemic and choleragenic potential. ...
Somatic antigen agglutinable type-1/139 Vibrio cholerae (SAAT-1/139-Vc) members or O1/O139 V. cholerae have been described by various investigators as pathogenic due to their increasing virulence potential and production of choleragen. Reported cholera outbreak cases around the world have been associated with these choleragenic V. cholerae with high case fatality affecting various human and animals. These virulent Vibrio members have shown genealogical and phylogenetic relationship with the avirulent somatic antigen non-agglutinable strains of 1/139 V. cholerae (SANAS-1/139- Vc) or O1/O139 non-agglutinating V. cholerae (O1/O139-NAG-Vc). Reports on implication of O1/O139-NAGVc members in most sporadic cholera/cholera-like cases of diarrhea, production of cholera toxin and transmission via consumption and/or contact with contaminated water/seafood are currently on the rise. Some reported sporadic cases of cholera outbreaks and observed change in nature has also been tracable to these non-agglutinable Vibrio members (O1/O139-NAGVc) yet there is a sustained paucity of research interest on the non-agglutinable V. cholerae members. The emergence of fulminating extraintestinal and systemic vibriosis is another aspect of SANAS-1/139- Vc implication which has received low attention in terms of research driven interest. This review addresses the need to appraise and continually expand research based studies on the somatic antigen non-serogroup agglutinable type-1/139 V.cholerae members which are currently prevalent in studies of water bodies, fruits/vegetables, foods and terrestrial environment. Our opinion is amassed from interest in integrated surveillance studies, management/control of cholera outbreaks as well as diarrhea and other disease-related cases both in the rural, suburban and urban metropolis.
... The V. cholerae genome has been constantly evolving with the changes in the environmental conditions, and the V. cholerae's mobilome is majorly responsible for its adaptation (Banerjee et al., 2014). Horizontal gene transfer, gene-capturing systems termed integrons, and other mobile genetic elements encompassing the transposons, bacteriophages, conjugative plasmids, ICEs, and SXT elements contribute to most the gene adaptation by the bacteria (Mooi and Bik, 1997;Cho et al., 2010). With changes in genetic constituent, the bacteria has also developed drug resistance, and even multi-drug resistant strains have emerged. ...
Vibrio cholerae, a gram-negative bacterium that causes cholera, has already caused seven major pandemics across the world and infects roughly 1.3–4 million people every year. Cholera treatment primarily involves oral rehydration therapy supplemented with antibiotics. But recently, multidrug-resistant strains of V. cholerae have emerged. High genomic plasticity further enhances the pathogenesis of this human pathogen. Guanines in DNA or RNA assemble to form G-quadruplex (GQ) structures which have begun to be seen as potential drug targeting sites for different pathogenic bacteria and viruses. In this perspective, we carried out a genome-wide hunt in V. cholerae using a bio-informatics approach and observed ∼85 G-quadruplex forming motifs (VC-PGQs) in chromosome I and ∼45 putative G-quadruplexs (PGQs) in chromosome II. Ten putative G-quadruplex forming motifs (VC-PGQs) were selected on the basis of conservation throughout the genus and functional analysis displayed their location in the essential genes encoding bacterial proteins, for example, methyl-accepting chemotaxis protein, orotate phosphoribosyl transferase protein, amidase proteins, etc. The predicted VC-PGQs were validated using different bio-physical techniques, including Nuclear Magnetic Resonance spectroscopy, Circular Dichroism spectroscopy, and electrophoretic mobility shift assay, which demonstrated the formation of highly stable GQ structures in the bacteria. The interaction of these VC-PGQs with the known specific GQ ligand, TMPyP4, was analyzed using ITC and molecular dynamics studies that displayed the stabilization of the VC-PGQs by the GQ ligands and thus represents a potential therapeutic strategy against this enteric pathogen by inhibiting the PGQ harboring gene expression, thereby inhibiting the bacterial growth and virulence. In summary, this study reveals the presence of conserved GQ forming motifs in the V. cholerae genome that has the potential to be used to treat the multi-drug resistance problem of the notorious enteric pathogen.
... The significance of GlcNAc6-dependent natural transformation lies in the fact that GlcNAc6 makes this process more specific for Vibrios because relatively few other microbes can take up long chitin oligosaccharides compared to mono-and di-saccharides [27] and that ultimately leads to the acquisition of new features. As a consequence, novel strains of Vibrios could emerge with heightened ecological fitness and pathogenicity [28]. Moreover, in a nutrient-poor marine environment, the ability to uptake GlcNAc6 might provide them a competitive advantage over other microbial species. ...
Vibrios can degrade chitin surfaces to soluble N-acetyl glucosamine oligosaccharides (GlcNAcn) that can be utilized as a carbon source and also induce a state of natural genetic competence. In this study, we characterized chitin-dependent growth and natural competence in Vibrio parahaemolyticus and its regulation. We found that growth on chitin was regulated through chitin sensors ChiS (sensor histidine kinase) and TfoS (transmembrane transcriptional regulator) by predominantly controlling the expression of chitinase VPA0055 (ChiA2) in a TfoX-dependent manner. The reduced growth of ΔchiA2, ΔchiS and ΔtfoS mutants highlighted the critical role played by ChiA2 in chitin breakdown. This growth defect of ΔchiA2 mutant could be recovered when chitin oligosaccharides GlcNAc2 or GlcNAc6 were supplied instead of chitin. The ΔtfoS mutant was also able to grow on GlcNAc2 but the ΔchiS mutant could not, which indicates that GlcNAc2 catabolic operon is dependent on ChiS and independent of TfoS. However, the ΔtfoS mutant was unable to utilize GlcNAc6 because the periplasmic enzymes required for the breakdown of GlcNAc6 were found to be downregulated at the mRNA level. We also showed that natural competence can be induced only by GlcNAc6, not GlcNAc2, because the expression of competence genes was significantly higher in the presence of GlcNAc6 compared to GlcNAc2. Moreover, this might be an indication that GlcNAc2 and GlcNAc6 were detected by different receptors. Therefore, we speculate that GlcNAc2-dependent activation of ChiS and GlcNAc6-dependent activation of TfoS might be crucial for the induction of natural competence in V. parahaemolyticus through the upregulation of the master competence regulator TfoX.
... The gene differences observed in this study would likely change the synthesis and processing of both K-and O-antigen, but perhaps not the specific serotype itself. It has previously been described that glycosyltransferases contributing to LPS structure can vary within a clonal population and also that the O-antigen cluster is a product of genetic variation based on recombination (van der Woude and Bäumler, 2004;Popoff and Le Minor, 1985;Viret et al., 1993;Shepherd et al., 2000;Mooi and Bik, 1997). ...
Whole-genome sequencing has enabled detailed studies on bacterial evolution during infection, but there is limited knowledge on intraclonal variation. In this study, we sought to provide a snapshot of the intraclonal diversity of Escherichia coli as both commensal in the faecal environment and pathogen during urinary tract infection, respectively. This was performed by whole-genome sequencing and analyses of single nucleotide polymorphisms (SNPs) and gene-content variation in ten isolates belonging to the same clone and isolated from rectal swabs or urine samples. We identified only one clone in eight of the nine urines sampled (89 %). In both the commensal and pathogenic state, the within-host diversity was limited with intraclonal SNP diversity of 0-2 non-synonymous SNPs for each clone. The genetic diversity showed variation in gene content in a range of 2-15 genes in total for all clones, including genes positioned on plasmids, and in the K- and O-antigen cluster. The observed SNP- and gene variation shows that sampling of one colony would be enough for surveillance, outbreak investigations and clonal evolution. However, for studies of adaptation during or between colonization and infection, this variation is relevant to consider.
... Thus, the evidence suggests that the O8 LPS type has emerged by modification of the O-antigen component of the O1 LPS. O-antigen modification is a well-recognized phenomenon in Gram-negative bacteria and may be generated by horizontal gene transfer (43,44) or bacteriophage-mediated mechanisms (45,46). Indeed, horizontal gene transfer involving O-antigen biosynthesis gene clusters has contributed to LPS diversity in the Aeromonas hydrophila strains responsible for septicemia in catfish (47). ...
Unlabelled:
Yersinia ruckeri is the etiological agent of enteric redmouth (ERM) disease of farmed salmonids. Enteric redmouth disease is traditionally associated with rainbow trout (Oncorhynchus mykiss, Walbaum), but its incidence in Atlantic salmon (Salmo salar) is increasing. Yersinia ruckeri isolates recovered from diseased Atlantic salmon have been poorly characterized, and very little is known about the relationship of the isolates associated with these two species. Phenotypic approaches were used to characterize 109 Y. ruckeri isolates recovered over a 14-year period from infected Atlantic salmon in Scotland; 26 isolates from infected rainbow trout were also characterized. Biotyping, serotyping, and comparison of outer membrane protein profiles identified 19 Y. ruckeri clones associated with Atlantic salmon but only five associated with rainbow trout; none of the Atlantic salmon clones occurred in rainbow trout and vice versa These findings suggest that distinct subpopulations of Y. ruckeri are associated with each species. A new O serotype (designated O8) was identified in 56 biotype 1 Atlantic salmon isolates and was the most common serotype identified from 2006 to 2011 and in 2014, suggesting an increased prevalence during the time period sampled. Rainbow trout isolates were represented almost exclusively by the same biotype 2, serotype O1 clone that has been responsible for the majority of ERM outbreaks in this species within the United Kingdom since the 1980s. However, the identification of two biotype 2, serotype O8 isolates in rainbow trout suggests that vaccines containing serotypes O1 and O8 should be evaluated in both rainbow trout and Atlantic salmon for application in Scotland.
Importance:
Vaccination plays an important role in protecting Atlantic salmon against the bacterial pathogen Yersinia ruckeri, but, in recent years, there has been an increasing incidence of vaccine breakdown in salmon. This is largely because current vaccines are aimed at rainbow trout and are based on serotypes specific for this species. A wider range of serotypes is responsible for infection in Atlantic salmon, but very little is known about the diversity of these strains and their relationships to those recovered from rainbow trout. In the present study, we demonstrate that Y. ruckeri isolates recovered from diseased Atlantic salmon in Scotland are more diverse than those from rainbow trout; furthermore, isolates from the two species represent distinct subpopulations. In addition, a new O serotype was identified that is responsible for a significant proportion of the disease in Atlantic salmon. Our findings are likely to have important implications for the development of improved vaccines against Y. ruckeri.
... These adaptations can occur quite rapidly (50) and result both from the lateral transfer of DNA between organisms and from mutation or loss of pre-existing genes within organisms (51,52). Such changes have influenced the evolution of diseases such as cholera (53) and bacterial meningitis in humans (54). With the opportunity to amplify in the efficient but intensive farming of developed societies for over 100 years, MAP has probably undergone a similar but slower adaptive radiation and has made the intestine of animals and humans one of its natural habitats, acquiring an intermediate status between an environmental organism and a low grade pathogen. ...
Mycobacterium avium subspecies paratuberculosis (MAP) is a member of the M avium complex (MAC). It differs genetically from other MAC in having 14 to 18 copies of IS900 and a single cassette of DNA involved in the biosynthesis of surface carbohydrate. Unlike other MAC, MAP is a specific cause of chronic inflammation of the intestine in many animal species, including primates. The disease ranges from pluribacillary to paucimicrobial, with chronic granulomatous inflammation like leprosy in humans. MAP infection can persist for years without causing clinical disease. The herd prevalence of MAP infection in Western Europe and North America is reported in the range 21% to 54%. These subclinically infected animals shed MAP in their milk and onto pastures. MAP is more robust than tuberculosis, and the risk that is conveyed to human populations in retail milk and in domestic water supplies is high. MAP is harboured in the ileocolonic mucosa of a proportion of normal people and can be detected in a high proportion of full thickness samples of inflamed Crohn's disease gut by improved culture systems and IS900 polymerase chain reaction if the correct methods are used. MAP in Crohn's disease is present in a protease-resistant nonbacillary form, can evade immune recognition and probably causes an immune dysregulation. As with other MAC, MAP is resistant to most standard antituberculous drugs. Treatment of Crohn's disease with combinations of drugs more active against MAC such as rifabutin and clarithromycin can bring about a profound improvement and, in a few cases, apparent disease eradication. New drugs as well as effective MAP vaccines for animals and humans are needed. The problems caused by MAP constitute a public health issue of tragic proportions for which a range of remedial measures are urgently needed.
... The sxt element harbors several antibiotic resistance genes that provide selective advantages to the host and is present in almost all post-1990 V. cholerae O1 clinical isolates (Waldor et al., 1996). The third selective sweep is considered to have been triggered by replacement of V. cholerae O1 serogroup encoding genes with those for O139, resulting in an outbreak of cholera across the Indian subcontinent (Bik et al., 1995;Comstock et al., 1996;Mooi and Bik, 1997). The fourth, or most recent selective sweep, is proposed to have been initiated by replacement of the El Tor ctx allele with the classical ctx allele in an El Tor background (Raychoudhuri et al., 2009), presumed to be associated with the acute form of cholera (Nair et al., 2002). ...
Vibrio cholerae, the etiological agent of cholera, has been a scourge for centuries. Cholera remains a serious health threat for developing countries and has been responsible for millions of deaths globally over the past 200 years. Identification of V. cholerae has been accomplished using a variety of methods, ranging from phenotypic strategies to DNA based molecular typing and currently whole genomic approaches. This array of methods has been adopted in epidemiological investigations, either singly or in the aggregate, and more recently for evolutionary analyses of V. cholerae. Because the new technologies have been developed at an ever increasing pace, this review of the range of fingerprinting strategies, their relative advantages and limitations, and cholera case studies was undertaken. The task was challenging, considering the vast amount of the information available. To assist the study, key references representative of several areas of research are provided with the intent to provide readers with a comprehensive view of recent advances in the molecular epidemiology of V. cholerae. Suggestions for ways to obviate many of the current limitations of typing techniques are also provided. In summary, a comparative report has been prepared that includes the range from traditional typing to whole genomic strategies.
... However, O139 contains a distinct O antigen and has been shown to express a polysaccharide capsule (Johnson et al., 1994;Weintraub et al., 1994). Accumulating data suggest that serogroup O139 Bengal arose from a serogroup O1 biotype El Tor by deletion of the genes responsible for O1 antigen biosynthesis (Manning, Stroeher & Morona, 1994;Waldor & Mekalanos, 1994;Comstock et al., 1995) and acquired DNA from another non-pathogenic serogroup (Mooi & Bik, 1997). In a cholera-endemic area, a newly emergent non-O1 serogroup has a selective advantage because of the absence of preexisting immunity. ...
Vibrio cholerae, a member of the family Vibrionaceae, is a facultatively anaero-bic, Gram-negative, non-spore-forming curved rod, about 1.4–2.6 mm long, capable of respiratory and fermentative metabolism; it is well defined on the basis of biochemical tests and DNA homology studies (Baumann, Furniss & Lee, 1984). The bacterium is oxidase-positive, reduces nitrate, and is motile by means of a single, sheathed, polar flagellum. Growth of V. cholerae is stimulated by addition of 1% sodium chloride (NaCl). However, an important distinction from other Vibrio spp is the ability of V. cholerae to grow in nutrient broth without added NaCl. Differences in the sugar composition of the heat-stable surface somatic " O " antigen are the basis of the serological classification of V. cholerae first described by Gardner & Venkatraman (1935); currently the organism is classified into 206 " O " serogroups (Shimada et al., 1994; Yamai et al., 1997). Until recently, epidemic cholera was exclusively associated with V. cholerae strains of the O1 serogroup. All strains that were identified as V. cholerae on the basis of biochemical tests but that did not agglutinate with " O " antiserum were collectively referred to as non-O1 V. cholerae. The non-O1 strains are occasionally isolated from cases of diarrhoea (Ramamurthy et al., 1993a) and from a variety of extrain-testinal infections, from wounds, and from the ear, sputum, urine, and cere-brospinal fluid (Morris & Black, 1985). They are ubiquitous in estuarine environments, and infections due to these strains are commonly of environmental origin (Morris, 1990). The O1 serogroup exists as two biotypes, classical and El Tor; antigenic factors allow further differentiation into two major serotypes— Ogawa and Inaba. Strains of the Ogawa serotype are said to express the A and B antigens and a small amount of C antigen, whereas Inaba strains express only
... Nevertheless, the emergence of the novel Vibrio cholerae strain 0139 Bengal that caused a large cholera epidemic in Southeast Asia is thought to have occurred by natural horizontal transfer of genes regulating the biosynthesis of cell wall polysaccharide. 89 Considering the discussion on metabolic engineering above, it may indeed be possible to transfer complex gene systems which encode enzymes involved in biosynthetic pathways and have them expressed in bacteria. ...
Biotechnology is a very old technology spanning a period of over 5000 years. In the earliest phases, it was concerned primarily with fermentation processes leading to bread-making as well as to the production of alcoholic beverages and cheese. With the recognition that microorganisms and their products were responsible for such processes and the establishment of microbiology as a science towards the end of the ninteenth century, biotechnology indeed acquired a scientific character. At this point the term biotechnology encompassed:1,2 “the use of living organisms or enzymes in the technically regulated production of organic substances.” Biotechnology has since been revolutionized by molecular biology and genetic engineering, and any modern account has to take these developments into consideration.
... Vibrio cholerae is a highly motile non-invasive Gram-negative organism which colonizes the small intestine and produces a potent enterotoxin called cholera toxin (CT)-a major virulence determinant that causes massive intestinal fluid loss leading to profuse watery diarrheal syndrome associated with V. cholerae infection [1,2]. There are more than 200 serogroups of V. cholerae recognized on the basis of their lipo polysaccharide (LPS) O side chain antigenic structures [3]. There are two serogroups of V. cholerae O1 and O139 which can infect humans and cause epidemic and pandemic cholera. ...
Cholera remains a significant health problem in developing countries due to its ability to spread rapidly and kills a high proportion of those affected. The disease is produced by Vibrio cholerae that colonizes in the human intestine and causes inflammatory diarrheal diseases. The reactogenicity of vaccine strain causes a serious problem in clinical settings. Besides the study of organisms V. cholerae, a thorough understanding of the host response following V. cholerae infection is indispensable to combat the disease from newly emerging threats. Elucidation of molecular mechanisms of V. cholerae induced inflammatory response through Toll-like receptor (TLR) and nucleotide-binding oligomerization domain (NOD) will eventually help to design proper vaccine or drugs for appropriate targets.
... V. cholerae O1 and O139 are the major serogroups that cause outbreaks of cholera (Kaper et al., 1995). Studies have shown that V. cholerae O139 has most probably been derived from an El Tor O1 strain (Manning et al., 1994) or from a strain belonging to the non-O1 serogroup (Mooi and Bik, 1997) by the acquisition of new polysaccharide genes. The pathogenesis of cholera in humans is a complex process, involving a number of factors that help the pathogen to colonize the epithelium of the small intestine (Singh et al., 2002). ...
... The seventh and current pandemic, however, is caused by the El Tor variant and an additional, concurrent pandemic, sometimes referred to as the eighth pandemic, is caused by the O139 strain (Faruque et al., 1998). Upon further analysis of the O139 serotype, it was found that this strain likely arose from an O1 El Tor strain which picked up exogenous DNA, thus altering its O antigen (Mooi & Bik, 1997) This emphasizes how rapidly V. cholerae can adapt by means of horizontal gene transfer. For both pathogenic and non-pathogenic strains of V. cholerae, the ability to persist in aquatic environments requires unique strategies, including the extracellular secretion of proteins. ...
The Type II secretion (T2S) apparatus is required for the survival and fitness of Vibrio cholerae both in the human host as well as its natural aquatic environment due to its ability to secrete proteins such as cholera toxin and hydrolytic enzymes. This apparatus, which is widely distributed among Gram negative pathogens, is composed of at least 12 proteins and spans the entire cell envelope. The energy to support secretion through the T2S system is provided by an ATPase associated with this complex, EpsE. EpsE is homologous to many ATPases involved with other molecular trafficking machineries and is comprised of three distinct subdomains: the N-terminal domain (NTD), the C-terminal domain (CTD), and a tetracysteine coordinating zinc binding domain (CM). These studies demonstrate that the mechanism of ATP hydrolysis requires oligomerization of EpsE, which allows for the realignment of specific arginine residues from the NTD, CTD and the CTD of a neighboring subunit to form the active site. Replacement of any of these arginine residues in the active site or of residues within the subunit interface halts the activity of EpsE, suggesting conformational changes associated with the binding, hydrolysis and release of nucleotide must be precise. An additional arginine from the CM domain is also required to complete the active site, suggesting a role for all three domains in ATP hydrolysis and energizing the T2S machinery. Previous work has shown EpsE to interact with the T2S apparatus through its NTD with another T2S component, EpsL. The movement of the NTD in association with EpsL likely provides a means of transforming the energy from ATP hydrolysis into mechanical work to support T2S. This work provides further evidence that the CM domain may be involved in additional protein-protein contacts with either EpsL or another protein associated with the T2S machinery. It is conceivable that the CM domain may play a direct role in the regulation of ATPase activity as it is in prime position to connect the nucleotide binding pocket to other components of the T2S apparatus through protein-protein interactions.
... The V. cholerae O139 strains that emerged in 1992 were derived from an El Tor progenitor by O-antigen switching likely facilitated by bacteriophages as well as the acquisition of a novel CTXU and SXT constin (Bik et al., 1996;Davis and Waldor, 2000;Waldor et al., 1997) (Fig. 4). The O139 Bengal strain is similar to the O1 classical and El Tot strains, producing major V. cholerae virulence factors such as cholera toxin and the toxin coregulated pilus (TCP) (Mooi and Bik, 1997). However, there are important differences between the O139 and O1 strains (Stroeher, 1995). ...
... Among the mobile elements are pathogenic islands (described below) and the serotype: although often thought as a phylogenetic marker and by definition not subject to lateral gene transfer, the first evidence that it might be mobile came from the discovery of the O139 morph of the O1-O139 lineage. It has been shown by three groups that the O1 encoding genes were replaced by O139 encoding genes (Bik, Bunschoten et al. 1995;Comstock, Johnson et al. 1996;Mooi and Bik 1997). Subsequently, the O1 encoding genes were shown to transfer between SNP-defined lineages, and furthermore the "jump start" sequence (Hobbs and Reeves 1994) was identified as the junction point and shown to be similar to a DNA uptake sequence leading to the suggestion that serotype genes are mechanistically prone to being mobile (Gonzalez-Fraga, Pichel et al. 2008 Salim et al. 2013). ...
Cholera Cholera is still a major public health problem. The underlying bacterial pathogen Vibrio cholerae Vibrio cholerae (V. cholerae) is evolving and some of its mutations have set the stage for outbreaks outbreaks . After V. cholerae acquired the mobile elements VSP I & II, the El Tor pandemic began and spread across the tropics. The replacement of the O1 serotype encoding genes with the O139 encoding genes triggered an outbreak that swept across the Indian subcontinent. The sxt element generated a third selective sweep and most recently a fourth sweep was associated with the exchange of the El Tor ctx allele for a classical ctx allele in the El Tor background. In Kenya, variants of this fourth selective sweep have differentiated and become endemic residing in and emerging from environmental reservoirs. On a local level, studies in Bangladesh have revealed that outbreaks may arise from a nonrandom subset of the genetic lineages in the environment and as the population of the pathogen expands, many novel mutations may be found increasing the amount of genetic variation genetic variation , a phenomenon known as a founder flush. In Haiti, after the initial invasion and expansion of V. cholerae in 2010, a second outbreak occurred in the winter of 2011-2012 driven by natural selection of specific mutations.
... Interestingly, LPS molecules and components thereof have been classified as PAMPs (Pathogen Associated Molecular Patterns) in plant-pathogenic bacteria [84,85]. Despite the fact that it is a conserved element in animal and plant pathogens, variations in the LPS cluster have been associated with virulence and host range [86]. The LPS gene cluster in Xam CIO151 consists of fifteen genes located in two consecutive scaffolds, with a total length of 17.2 kb. ...
Xanthomonas axonopodis pv. manihotis (Xam) is the causal agent of bacterial blight of cassava, which is among the main components of human diet in Africa and South America. Current information about the molecular pathogenicity factors involved in the infection process of this organism is limited. Previous studies in other bacteria in this genus suggest that advanced draft genome sequences are valuable resources for molecular studies on their interaction with plants and could provide valuable tools for diagnostics and detection. Here we have generated the first manually annotated high-quality draft genome sequence of Xam strain CIO151. Its genomic structure is similar to that of other xanthomonads, especially Xanthomonas euvesicatoria and Xanthomonas citri pv. citri species. Several putative pathogenicity factors were identified, including type III effectors, cell wall-degrading enzymes and clusters encoding protein secretion systems. Specific characteristics in this genome include changes in the xanthomonadin cluster that could explain the lack of typical yellow color in all strains of this pathovar and the presence of 50 regions in the genome with atypical nucleotide composition. The genome sequence was used to predict and evaluate 22 variable number of tandem repeat (VNTR) loci that were subsequently demonstrated as polymorphic in representative Xam strains. Our results demonstrate that Xanthomonas axonopodis pv. manihotis strain CIO151 possesses ten clusters of pathogenicity factors conserved within the genus Xanthomonas. We report 126 genes that are potentially unique to Xam, as well as potential horizontal transfer events in the history of the genome. The relation of these regions with virulence and pathogenicity could explain several aspects of the biology of this pathogen, including its ability to colonize both vascular and non-vascular tissues of cassava plants. A set of 16 robust, polymorphic VNTR loci will be useful to develop a multi-locus VNTR analysis scheme for epidemiological surveillance of this disease.
... Phages may also play a role in the emergence of pathogenic clones and may be involved in territorialism between different strains of V. cholerae (5). For example, the emergence and dominance of V. cholerae O139 in Bangladesh and India during 1992 to 1993 may have involved phages both as a means of horizontal gene transfer and as a selective mechanism (17). ...
... Due to the abundance of the Vibrionaceae in a wide range of aquatic ecosystems, and their close association with various marine and freshwater plants and animals, natural competence in the Vibrionaceae is expected to have significant environmental, ecological and clinical implications. A previous study designed to mimic aquatic reservoirs demonstrated that, through chitin-induced natural transformation, a V. cholerae strain can acquire a gene cluster (Blokesch and Schoolnik, 2007), which effectively converts the recipient into a different serogroup (Bik et al., 1995, Mooi andBik 1997) that is known for its heightened fitness, virulence and central role in the 1992 cholera epidemic (Albert et al., 1997). V. cholerae strains lacking the cholera toxin (ctxAB) genes required to cause cholera, can also acquire DNA encoding ctxAB via chitin-induced natural competence (Udden et al., 2008). ...
Natural transformation is a major mechanism of horizontal gene transfer in bacteria. By incorporating exogenous DNA elements into chromosomes, bacteria are able to acquire new traits that can enhance their fitness in different environments. Within the past decade, numerous studies have revealed that natural transformation is prevalent among members of the Vibrionaceae, including the pathogen Vibrio cholerae. Four environmental factors, i) nutrient limitation, ii) availability of extracellular nucleosides, iii) high cell density, and iv) the presence of chitin, promote genetic competence and natural transformation in Vibrio cholerae by coordinating expression of the regulators CRP, CytR, HapR, and TfoX, respectively. Studies of other Vibrionaceae members highlight the general importance of natural transformation within this bacterial family.
... Genomic analysis indicates that our strains have swapped the Bordetellae O-antigen biosynthesis cluster for a different system. Intra-and inter-species transfer of O-antigen genes has been described in different bacteria [27,28,29], including B. bronchiseptica [30]. Importantly, O-antigen loss in B. petrii 3 is not due to a complete deletion or disruption of the LPS biosynthesis locus. ...
The first described, environmentally isolated, Bordetella petrii was shown to undergo massive genomic rearrangements in vitro. More recently, B. petrii was isolated from clinical samples associated with jaw, ear bone, cystic fibrosis and chronic pulmonary disease. However, the in vivo consequences of B. petrii genome plasticity and its pathogenicity remain obscure. B. petrii was identified from four sequential respiratory samples and a post-mortem spleen sample of a woman presenting with bronchiectasis and cavitary lung disease associated with nontuberculous mycobacterial infection. Strains were compared genetically, phenotypically and by antibody recognition from the patient and from inoculated mice. The successive B. petrii strains exhibited differences in growth, antibiotic susceptibility and recognition by the patient's antibodies. Antibodies from mice inoculated with these strains recapitulated the specificity and strain dependent response that was seen with the patient's serum. Finally, we characterize one strain that was poorly recognized by the patient's antibodies, due to a defect in the lipopolysaccharide O-antigen, and identify a mutation associated with this phenotype. We propose that B. petrii is remarkably adaptable in vivo, providing a possible connection between immune response and bacterial evasion and supporting infection persistence.
... Subsequent to the genesis of the O139 serogroup, the rfb region has been analyzed in great detail to understand the origin of the O139 serogroup. Extensive studies have shown that V. cholerae O139 has most probably been derived from an El Tor O1 strain [8] or from a strain belonging to the non-O1 serogroup [9] by the acquisition of new polysaccharide genes and the subsequent or simultaneous deletion of the V. cholerae O1 rfb genes. Hybridization analysis using several DNA fragments derived from O139 rfb genes as probes has shown unique regions in the rfb gene which are speci¢c for the O139 and O1 serogroups [10]. ...
A multiplex polymerase chain reaction assay was developed for concurrent detection of rfb sequences specific for the O1 and the O139 serogroups of Vibrio cholerae and for ctxA specific sequences. The multiplex PCR assay was found to be highly specific and sensitive and was capable of detecting 65 cfu and 200 cfu per assay of V. cholerae O1 and O139, respectively. Evaluation of the multiplex PCR assay using 121 stool samples from patients admitted to the Infectious Diseases Hospital, Calcutta, showed the assay to be 100% sensitive and 95.2% specific when the culture method was taken as the standard. In addition to the 38 PCR positive stool samples, an additional four samples which were negative by culture method but positive by PCR assay belonged to the O139 serogroup. All the 38 stool samples positive for either O1 or O139 serogroup by PCR assay were also positive for the ctxA amplicon indicating that the O1 and O139 V. cholerae strains have the genetic potential of producing cholera toxin.
... Acquisition of virulence factors mediated by transduction has been reported in Vibrio cholerae [26] and Streptococcus pyogenes [27]. In V. cholerae, it has been suggested that transduction contributes to the evolution of epidemic strains [28]. Evidence for the assimilation of foreign DNA by B. cepacia has been presented by citing the signi¢cantly lower G+C content of insertion sequences when compared with native DNA [29]. ...
Burkholderia cepacia is now recognised as a life-threatening pathogen among several groups of immunocompromised patients. In this context, the proposed large-scale use of these bacteria in agriculture has increased the need for a better understanding of the genetics of the species forming the B. cepacia complex. Until now, little information has been available on the bacteriophages of the B. cepacia complex. Transducing phages, named NS1 and NS2, were derived from the lysogenic B. cepacia strains ATCC 29424 and ATCC 17616. The frequency of transduction per phage particle ranged from 1.0×10−8 to 7.0×10−6 depending on the phage and recipient strain used. The host range of NS1 and NS2 differed but in each case included environmental and clinical isolates, and strains belonging to several species and genomovars of the B. cepacia complex. The host range of both phages also included Pseudomonas aeruginosa. Some B. cepacia complex isolates were sensitive to the well-characterised P. aeruginosa transducing phages, B3, F116L and G101. The lytic activity of NS1 and NS2 was inhibited by B. cepacia lipopolysaccharide suggesting that this moiety is a binding site for both phages. The molecular size of the NS1 and NS2 genomes was approximately 48 kb.
The bacterium Vibrio cholerae is a natural inhabitant of aquatic ecosystems across the planet. V. cholerae serogroups O1 and O139 are responsible for cholera outbreaks in developing countries accounting for 3–5 million infections worldwide and 28.800–130.000 deaths per year according to World Health Organization. In contrast, V. cholerae serogroups other than O1 and O139, also designated as V. cholerae non‐O1/O139 (NOVC), are not associated with epidemic cholera but can cause other illnesses that may range in severity from mild (e.g. gastroenteritis, otitis, etc.) to life‐threatening (e.g. necrotizing fasciitis). Although generally neglected, NOVC related infections are on the rise and represent one of the most striking examples of emerging human diseases linked to climate change. NOVC strains are also believed to potentially contribute to the emergence of new pathogenic strains including strains with epidemic potential as a direct consequence of genetic exchange mechanisms such as horizontal gene transfer and genetic recombination. Besides general features concerning the biology and ecology of NOVC strains and their associated diseases this review aims to highlight most relevant aspects related to the emergence and potential threat posed by NOVC strains under a rapidly changing environmental and climatic scenario. This article is protected by copyright. All rights reserved.
Cholera is an endemic disease in many regions of Asia including, Pakistan. Vibrio cholerae, the causative agent of cholera, is considered as one of the best adapted bacteria due to its ability to withstand severe environmental stresses. The V. cholerae genome is very plastic with many gene additions and deletions. In this study, we sought to understand the diversity of V. cholerae genes in two Pakistani subclades [e.g. Pakistani subclade I (PSC I) and Pakistani subclade II (PSC II)]. We have analyzed 44 PSC I and 56 PSC II strains, respectively. By analyzing our data, it was concluded that subclade group 2 (PSC II) has 2967 core genes repositories, while the PSC 1 group has just 1062 core genes. It was observed that the pangenome in the PSC II group is open while the pan-genome in PSC I are closed. It was also noted that the number of accessory genes (n = 2500) is higher in the PSC I group compared to the PSC II group (n = 550). Furthermore, analysis extended to the study of unique gene profiles suggested that all strains of the PSC II group have unique genes. One strain among the PSC II group had a high number of unique genes (n = 2612). However, in the PSC I group, only a few strains had unique genes with a maximum of 86 unique genes being found in a single strain. Core phylogeny of PSC I indicated that just three groups initially arose from a single common ancestor. At the same time, a complex pattern of evolution was found in the PSC II phylogenetic tree based on core gene information. This comparative genomic analysis has revealed ‘waves’ of V. cholerae evolution and information on its transmission and ability to modify its genetic content to survive in different environmental conditions. Here, we have investigated how the versatility of V. cholerae, a bacterium that persists across different habitats, is reflected in its genome. The data generated during the study should be extremely beneficial in defining the evolutionary relationship as well as diversity between V. cholerae subclades. It will also benefit epidemiological studies and the design of better treatment strategies for controlling epidemics.
The new epidemic serovar O139 of Vibrio cholerae has emerged from the pandemic serovar O1 biotype El Tor through the replacement of a 22-kbp DNA region by a 40-kbp O139-specific DNA fragment. This O139-specific DNA fragment contains an insertion sequence that was described previously (U. H. Stroeher, K. E. Jedani, B. K. Dredge, R. Morona, M. H. Brown, L. E. Karageorgos, J. M. Albert, and P. A. Manning, Proc. Natl. Acad. Sci. USA 92:10374–10378, 1995) and designated IS 1358 O139 . We studied the distribution of the IS 1358 element in strains from various serovars by Southern analysis. Its presence was detected in strains from serovars O1, O2, O22, O139, and O155 but not in strains from serovars O15, O39, and O141. Furthermore, IS 1358 was present in multiple copies in strains from serovars O2, O22, and O155. We cloned and sequenced four copies of IS 1358 from V. cholerae O22 and one copy from V. cholerae O155. A comparison of their nucleotide sequences with those of O1 and O139 showed that they were almost identical. We constructed a transposon consisting of a kanamycin resistance gene flanked by two directly oriented copies of IS 1358 to study the functionality of this element. Transposition of this element from a nonmobilizable plasmid onto the conjugative plasmid pOX38-Gen was detected in an Escherichia coli recA donor at a frequency of 1.2 × 10 ⁻⁸ . Sequence analysis revealed that IS 1358 duplicates 10 bp at its insertion site.
Presented is the review of literarary and authors` data on the ways of emergence of Vibrio cholerae eltor strains, atypical as regards their virulence properties. The enhancement of virulence of cholera eltor agent at present was shown to be associated with acquisition of Vibrio cholerae cholerae CTXφ prophage as a result of horizontal gene transfer. Another possible way of formation of strains with higher virulence is described - alteration of cholera toxin resident genes regulation due to rearrangement of CTXφ prophage genome by the introduced transposon. Also considered are evidences on origination of ctxA- tcpA+ strains and their enhanced viability in aquatic environment as compared with isogenic toxinogenic clones. Presented are the prospects of the development of new PCR test for detection of cholera eltor agent with altered virulence in monitoring investigations.
Die Biologie beschreibt die Strukturen und Funktionen der Organismen sowie die Wechselwirkung der Organismen miteinander und mit der Umwelt. Da der Aufbau der Organismen chemischer Natur ist, handelt es sich bei lebenden Organismen um regulierte Reaktionen von Molekülen. Die Regelungen, die die Lebensprozesse steuern, sind hoch komplex. Dieses Kapitel stellt zunächst einige Grundlagen der Biologie vor (Teilkapitel 4.1), es muss durchgearbeitet werden, damit Sie die im weiteren Teil behandelte Problematik der biologischen Waffen für die Biosicherheit nachvollziehen können.
Die Biologie beschreibt die Strukturen und Funktionen der Organismen sowie die Wechselwirkung der Organismen miteinander und mit der Umwelt. Da der Aufbau der Organismen chemischer Natur ist, handelt es sich bei lebenden Organismen um regulierte Reaktionen von Molekülen. Die Regelungen, die die Lebensprozesse steuern, sind hoch komplex. Dieses Kapitel stellt zunächst einige Grundlagen der Biologie vor (Teilkapitel 4.1), es muss durchgearbeitet werden, damit Sie die im weiteren Teil behandelte Problematik der biologischen Waffen nachvollziehen können. Es berichtet über die Zelle als Grundeinheit aller Lebewesen und gibt einen Einblick in die Prozesse der DNA- und Proteinbiosynthese. In Teilkapitel 4.2 werden potentielle biologische Waffen und ihre Eigenschaften dargestellt. Ferner werden die Methoden zur Herstellung und Ausbringung biologischer Agenzien beschrieben, um zu zeigen, dass die Konstruktion einer biologischen Waffe nicht so leicht ist, wie häufig behauptet wird. In den Teilkapiteln 4.3 bis 4.5 werden die Stärken und Schwächen der B-Waffen-Konvention dargestellt. In diesem Zusammenhang wird die Relevanz der Fortschritte in der Biotechnologie für die B-Waffen-Konvention diskutiert. Hierzu werden Dual-Use-Aspekte der biomedizinischen Forschung anhand von konkreten Beispielen aus der Fachliteratur erläutert; dieses Teilkapitel 4.6 kann beim ersten Lesen überflogen werden. Die Aufgaben sollten alle bearbeitet werden.
This chapter includes a general introduction to the bacterium Vibrio cholerae and to the disease cholera, including a brief discussion of the major virulence factors. It also analyzes the mechanistic aspects of cholera infection, with particular emphasis on the cholera toxin, the most important virulence factor in the pathophysiology of disease. Infection with Vibrio cholerae leading to the disease cholera continues to threaten large portions of the world's population. The disease process is not very complicated, owing to the facts that the disease occurs at the mucosal surface, with no invasion by the microbe into deeper tissue, and that disease symptoms are primarily due to the action of a single molecule, the cholera toxin. Within this simple system of mucosal pathogenesis, extremely interesting mechanisms are uncovered at practically every level of study, and this chapter outlines many of those mechanisms.
Cholera is an epidemic life threatening disease caused by V. cholerae. The main symptoms of this acute disease are vomiting, profuse watery diarrhea, and severe dehydration. The infection is transmitted by fecal-oral mechanism, food consumption and V. cholerae contaminated water (10,15). Often outbreaks are related to wars or natural disasters like hurricanes and floods when water and food sources become contaminated with V. cholerae in areas with bad living conditions and poor sanitation. Although cholera is a rare disease eradicated in developed countries it is still common in some parts of the world like Latin America, Africa, India and Asia. Its potential to cause large outbreaks and pandemics with lethal exit creates the need of a better epidemic prevention and an early response (25, 27). In the last decades the cholera is not common disease in our country but the increasing number of refugees from the Middle East and Africa makes it potential threat for Bulgarian population and requests the health authorities to react in case of possible outbreak.
To investigate the molecular basis of the emergence of Aeromonas hydrophila responsible for an epidemic outbreak of motile aeromonad septicemia of catfish in the Southeastern United States, we sequenced 11 A. hydrophila isolates that includes five reference and six recent epidemic isolates. Comparative genomics revealed that recent epidemic A. hydrophila isolates are highly clonal, whereas reference isolates are greatly diverse. We identified 55 epidemic-associated genetic regions with 313 predicted genes that are present in epidemic isolates but absent from reference isolates and 35% of these regions are located within genomic islands, suggesting their acquisition through lateral gene transfer. The epidemic-associated regions encode predicted prophage elements, pathogenicity islands, metabolic islands, fitness islands and genes of unknown functions, and 34 of the genes encoded in these regions were predicted as virulence factors. We found two pilus biogenesis gene clusters encoded within predicted pathogenicity islands. A functional metabolic island that encodes a complete pathway for myo-inositol catabolism was evident by the ability of epidemic A. hydrophila isolates to use myo-inositol as a sole carbon source. Testing of A. hydrophila field isolates found a consistent correlation between myo-inositol utilization as a sole carbon source and the presence of an epidemic-specific genetic marker. All epidemic isolates and one reference isolate shared a novel O-antigen cluster. Altogether we identified four different O-antigen biosynthesis gene clusters within the 11 sequenced A. hydrophila genomes. Our study reveals new insights into the evolutionary changes that have resulted in the emergence of recent epidemic A. hydrophila strains.
Cholera is a highly contagious diarrheal disease caused by the Gram negative bacterium, Vibrio cholerae. This pathogen lives as a saprophyte in aquatic environments of estuaries, where it can parasite the Zooplankton. Humans are infected by ingestion of contaminated food or water, and by direct inter-human contact. Cholera is associated with poverty, malnutrition and over-population. The surprising plasticity of the genome of V. cholerae is illustrated by the emergence of new strains belonging to serovars O1 and O139, probably favored by the environment (pollution, climatic changes…), and continuous interhuman passages in overcrowded endemic areas. The emergence of cholera epidemics appears therefore as the consequence of complex interactions between the pathogen, a susceptible population and the environment.
Vibrio cholerae and V. anguillarum are recognized as aquatic-borne human and fish pathogens, respectively. Based upon analyses of several genes and the presence of novel genetic elements it seems that these two species are very closely related. Studies in this laboratory have identified an association of IS1358 with rfb and capsule loci in these two species. The most recent findings suggest that IS1358 is associated with the rfb region in V. cholerae O1 and O139 and in V. anguillarum O1 and O2. In addition, the rfb region in both V. cholerae serogroups and in V. anguillarum O1 is limited at one end by gmhD. These features make it feasible to envisage a mechanism by which the evolution of new rfb genes is taking place involving IS1358 and the region around gmhD. Furthermore, it is possible to envisage that there is or has been an exchange of genetic material between these species leading to new rfb/capsule regions. This review examines the genetics and biosynthesis of the O-antigen and capsule of V. cholerae O1 and O139, as well as the V. anguillarum serogroup O1 and the role of IS1358. Throughout this review we have used the new nomenclature for rfb genes proposed by Reeves et al. (1996).
Vibrio cholerae and V. anguillarum are recognized as aquatic-borne human and fish pathogens, respectively. Based upon analyses of several genes and the presence of novel genetic elements it seems that these two species are very closely related. Studies in this laboratory have identified an association of IS1358 with rfb and capsule loci in these two species. The most recent findings suggest that IS1358 is associated with the rfb region in V. cholerae O1 and O139 and in V. anguillarum O1 and O2. In addition, the rfb region in both V. cholerae serogroups and in V. anguillarum O1 is limited at one end by gmhD. These features make it feasible to envisage a mechanism by which the evolution of new rfb genes is taking place involving IS1358 and the region around gmhD. Furthermore, it is possible to envisage that there is or has been an exchange of genetic material between these species leading to new rfb/capsule regions. This review examines the genetics and biosynthesis of the O-antigen and capsule of V. cholerae O1 and O139, as well as the V. anguillarum serogroup O1 and the role of IS1358. Throughout this review we have used the new nomenclature for rfb genes proposed by Reeves et al. (1996).
In the last decades, we have experienced a revolution in biotechnology that is still on the rise and has not yet reached its
peak. With the use of these modern technologies, the mechanisms regulating pathogenic processes of infectious agents can be
elucidated more readily and precisely, which could lead to the development of more effective therapeutics and diagnostic reagents.
This research is clearly essential. However, the possible misuse of biotechnology for the development and production of biological
weapons is an actuality that can not be ignored. The latest reports of the accidental creation of a “killer” mousepox virus
underscores this danger. Preventive arms control criteria emphasize the need for monitoring developments early in the process,
that is, at the level of research. Because technology in this area is advancing at a dramatic pace, the task is a large one,
and a concerted effort in close monitoring is needed. Activities in connection with genomics and proteomics should be monitored
closely. As more and more information about gene sequences and functions becomes available, the possibility that sequences
distinguishing populations may be identified increases. At present, clear distinctions in human populations have not been
determined and the possibility of developing ethnic weapons seems remote. However, plant and animal populations are highly
vunerable to genotype-specific weapons, and concerns about genetic marking of human populations to make them more vunerable
are real.
Vibrio cholerae elaborates three types of polysaccharide structures: lipopolysaccharide (LPS), a component of which is the O-polysaccharide or O-antigen, capsular polysaccharide (CPS) or K-antigen, and “rugose” polysaccharide also known as exopolysaccharide (EPS) or Vibrio polysaccharide (VPS). The major protective antigen for V. cholerae is the O-antigen. A strain typing scheme based on the somatic O-antigen has been in use for a number of years. There are 206 serogroups identified so far and of these only O1 and O139 are known to cause epidemic/pandemic cholera, although a handful of non-O1/non-O139 strains are known to possess the major virulence factors. The O-antigen diversity is due to the number and composition of monosaccharide components, linkages, addition of non-sugar moieties, modal length of the polysaccharide chain, and biosynthesis mechanisms. The genetic basis of this diversity is just beginning to be understood with the sequencing of a number of gene clusters that encode O-polysaccharide (OPS)/capsule structures. In this review, we summarize our current knowledge on the biochemical composition and structure of some of the O-polysaccharides, genes involved in their biosynthesis, and touch upon the role of horizontal gene transfer in creating this diversity and possible mechanisms that may be operative in this process. We highlight the fact that the distinction between OPS and CPS seems to be less evident in V. cholerae than in other species since the genes encoding these structures are shared and map in the same region of the genome. We also describe our current understanding of the genetics and regulation of EPS/VPS synthesis and its role in biofilm formation and environmental survival of V. cholerae.
Several studies have shown that the emergence of the O139 serogroup of Vibrio cholerae is a result of horizontal gene transfer of a fragment of DNA from a serogroup other than O1 into the region responsible for O-antigen biosynthesis of the seventh pandemic V. cholerae O1 biotype El Tor strain. In this study, we show that the gene cluster responsible for O-antigen biosynthesis of the O139 serogroup of V. cholerae is closely related to those of O22. When DNA fragments derived from O139 O-antigen biosynthesis gene region were used as probes, the entire O139 O-antigen biosynthesis gene region could be divided into five classes, designated as I–V based on the reactivity pattern of the probes against reference strains of V. cholerae representing serogroups O1–O193. Class IV was specific to O139 serogroup, while classes I–III and class V were homologous to varying extents to some of the non-O1, non-O139 serogroups. Interestingly, the regions other than class IV were also conserved in the O22 serogroup. Long and accurate PCR was employed to determine if a simple deletion or substitution was involved to account for the difference in class IV between O139 and O22. A product of approx. 15 kb was amplified when O139 DNA was used as the template, while a product of approx. 12.5 kb was amplified when O22 DNA was used as the template, indicating that substitution but not deletion could account for the difference in the region between O22 and O139 serogroups. In order to precisely compare between the genes responsible for O-antigen biosynthesis of O139 and O22, the region responsible for O-antigen biosynthesis of O22 serogroup was cloned and analyzed. In concurrence with the results of the hybridization test, all regions were well conserved in O22 and O139 serogroups, although wbfA and the five or six genes comprising class IV in O22 and O139 serogroups, respectively, were exceptions. Again the genes in class IV in O22 were confirmed to be specific to O22 among the 155 ‘O’ serogroups of V. cholerae. These data suggest that the gene clusters responsible for O139 O-antigen biosynthesis are most similar to those of O22 and genes within class IV of O139, and O22 defines the unique O antigen of O139 or O22.
In 1992 a new Vibrio cholerae strain, designated V. cholerae O139 Bengal, emerged which has been responsible for large outbreaks of cholera in India and Bangladesh. Previously, we have shown that this strain arose from a V. cholerae O1 strain by the acquisition of novel DNA. Sequence analysis revealed that the novel DNA is flanked by two genes, rfaD and rfbQRS, which are also found in O1 strains. The mosaic structure of rfaDvco139 indicated that it was one of the regions involved in recombination between donor and acceptor DNA. However, sequence divergence between the O1 and O139 rfbQRS genes indicated that the second recombination site between donor and O1-acceptor DNA is probably located downstream of rfbDvco139. The DNA region between rfaDvco139 and rfbQRSvco139, designated otn, contained seven open reading frames (ORFs). Two ORFs, otnA and otnB, showed homology with genes involved in cell-wall polysaccharide synthesis. Mutations in otnA and otnB indicated that they are required for capsule synthesis but not lipopolysaccharide synthesis. The otn DNA is also found inV. cholerae O69 and O141 strains, and the organization of this DNA was essentially identical to that in the O139 strain. However, sequence divergence of the otnAB genes indicated that the O139 otn DNA region was not derived from the O69 or O141 strains. No antigenic relationship was found between the different V. cholerae serotypes carrying otn DNA, so the genes determining the antigenic specificity of the O antigen or capsule must be located outside the otn DNA. The O139 otn DNA contained a JUMPstart sequence, which is associated with polysaccharide-synthetic genes in several bacterial species. Furthermore, a repeat motif was observed in extragenic regions. A number of observations suggest that these sequences may facilitate gene flow between V. cholerae strains and the assembly of clusters of functionally related genes.
Vibrio cholerae O1 exists as two major serotypes, Inaba and Ogawa, which are associated with the O antigen of the lipopolysaccharide and are capable of unequal reciprocal interconversion. The 20-kilobase rfb regions encoding O-antigen biosynthesis in strains 569B (Inaba) and O17 (Ogawa) have been cloned in Escherichia coli K-12 and the nucleotide sequences have been determined. Besides several base substitutions and a small deletion in the 569B sequence relative to O17, there is a single nucleotide change resulting in a TGA stop codon within the gene for the 32-kDa RfbT protein. We have demonstrated that rfbT is responsible for serotype conversion (Inaba to Ogawa). The construction of a specific rfbT mutation in the Ogawa strain O17, and the ability of the gene from O17 to complement Inaba strains to Ogawa, confirmed rfbT as the gene required for the Ogawa serotype. By Southern hybridization and sequencing of PCR products of a number of strains, we have shown that the changes observed in one Inaba strain (569B) are not conserved in other Inaba strains. This may explain why some Inaba strains are able to convert to Ogawa whereas others are not. The protein encoded by rfbT has been identified and expressed in E. coli K-12 using a phage T7 expression system. Amino-terminal analysis of partially purified protein has identified the translational start of the protein. Primer extension studies have enabled the 5' end of the mRNA to be defined. It exists as a separate transcript from the rest of the rfb region, and the distinctive G + C content of rfbT suggests that it has been acquired from a non-Vibrio source.
The recent emergence of a pathogenic new non-O1 serotype (O139) of Vibrio cholerae has led to numerous studies in an attempt to identify the origins of this new strain. Our studies indicate that O139 strains have clear differences in the surface polysaccharides when compared with O1 strains: the lipopolysaccharide can be described as semi-rough. Southern hybridization with the O1 rfb region demonstrates that O139 strains no longer contain any of the rfb genes required for the synthesis of the O1 O-antigen or its modification and also lack at least 6 kb of additional contiguous DNA. However, O139 strains have retained rfaD and have a single open reading frame closely related to three small open reading frames of the O1 rfb region. This region is closely related to the H-repeat of Escherichia coli and to the transposases of a number of insertion sequence elements and has all the features of an insertion sequence element that has been designated VcIS1. Transposon insertion mutants defective in O139 O-antigen (and capsule) biosynthesis map to the same fragment as VcIS1. Preliminary sequence data of complementing clones indicate that this DNA encodes a galactosyl-transferase and other enzymes for the utilization of galactose in polysaccharide biosynthesis. We propose a mechanism by which both the Ogawa serotype of O1 strains and the O139 serotype strains may have evolved.
Vibrio cholerae O139 Bengal, although closely related to V. cholerae O1 El Tor, produces a polysaccharide capsule and has a distinct O antigen. We have identified a chromosomal region of at least 11 kb, as defined by three TnphoA mutations, that is required for the expression of both polysaccharides. Electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis show that these TnphoA mutants have lost the abilities both to express capsule and to produce lipopolysaccharide beyond the core oligosaccharide. Reactivity with O139 typing serum and resistance to serum are also lost in the mutants. DNA probes for this region do not hybridize with O1 V. cholerae but do react with other vibrios, implying that the region was recently acquired.
Vibrio cholerae O139 organisms isolated from different parts of India and from Bangladesh were characterised with respect to their haemagglutination (HA) activity, plasmid content, cholera toxin (CT) production, cell surface protein and lipopolysaccharide (LPS) profiles, and antigenic properties. Of 28 V. cholerae O139 isolates tested, 14 (50%) were shown to agglutinate chicken erythrocytes; the HA activity was sensitive to D-mannose 0.1%. In parallel experiments, 12 (92.3%) of 13 V. cholerae O1 (El Tor) and 12 (75%) of 16 non-O1, non-O139 strains agglutinated chicken erythrocytes. Plasmid analysis of 32 O139 isolates showed that 12 (37.5%) carried one or more plasmids of 35.8-2.6 MDa. Plasmids were not detected in any of the V. cholerae O1 strains, although plasmids were demonstrable in 35% of the non-O1, non-O139 strains tested. V. cholerae O139 isolates showed an ability to produce CT that depended on media composition and other cultural conditions. A comparison of envelope and outer-membrane protein profiles between O1 and O139 isolates failed to show any significant differences. LPS analysis of O139 isolates revealed that these organisms were devoid of long "O" side-chain polysaccharides. Some of the non-O1, non-O139 strains also showed similar LPS profiles whereas others showed the presence of long repetitive "O" side-chain polysaccharides similar to those seen in O1 organisms. An antiserum raised against V. cholerae O1 strain O395 did not show any significant reactivity towards O139 and non-O1, non-O139 strains although it reacted with other O1 strains. Furthermore, the anti-O1 serum induced marked protection against challenge with an O1 strain but not with an O139 strain in passive protection experiments.(ABSTRACT TRUNCATED AT 250 WORDS)
Only Vibrio cholerae strains of serotype O1 are known to cause epidemics, while non-O1 strains are associated with sporadic cases of cholera. It was therefore unexpected that the recent cholera epidemic in Asia was caused by a non-O1 strain with the serotype O139. We provide evidence that O139 arose from a strain closely related to the causative agent of the present cholera pandemic, V. cholerae O1 El Tor, by acquisition of novel DNA which was inserted into, and replaced part of, the O antigen gene cluster of the recipient strain. Part of the novel DNA was sequenced and two open reading frames (otnA and otnB) were observed, the products of which showed homology to proteins involved in capsule and O antigen synthesis, respectively. This suggests that the otnAB DNA determines the distinct antigenic properties of the O139 cell surface. The otnAB DNA was not detected in O1 strains, but was present in two non-O1 V. cholerae strains with serotypes O69 and O141. In the O69 and O139 strains the otnAB genes were located proximate to the putative insertion sequence (IS) element rfbQRS, which is associated with O antigen synthesis genes in O1 strains, and may have played a role in the insertion of the otnAB DNA in the recipient chromosome. Our results suggest that the O139 strain arose by horizontal gene transfer between a non-O1 and an O1 strain. The acquired DNA has altered the antigenic properties of the recipient O1 strain, providing a selective advantage in a region where a large part of the population is immune to O1 strains.(ABSTRACT TRUNCATED AT 250 WORDS)
Vibrio cholerae serogroup O139 emerged on the Indian subcontinent in October 1992 to become the first non-O1 V. cholerae serogroup documented to cause epidemic cholera. Although related to V. cholerae El Tor O1 strains, O139 strains have unique surface structures that include a capsular surface layer and lipopolysaccharide (LPS). Immunoblot analysis of either whole-cell lysates or LPS preparations revealed three electrophoretic forms of the O139 antigen: two slowly migrating forms and one rapidly migrating form that appeared identical to O139 LPS. All three forms of the antigen shared an epitope defined by an O139-specific monoclonal antibody. A serum-sensitive nonencapsulated mutant was isolated that lacks only the slow migrating forms. The slow migrating forms did not stain with silver whereas the rapidly migrating form did, suggesting that the former might constitute highly polymerized O-antigen side-chain molecules that were not covalently bound to core polysaccharide and lipid A (an "O-antigen capsule"). A single transposon insertion resulted in the loss of immunoreactivity of both the LPS and the O-antigen capsule, implying that there are genes common to the biosynthesis of both these macromolecules. The O139 LPS and O-antigen capsule were both important for colonization of the small intestine of the newborn mouse and for serum resistance, demonstrating that both of these forms of the O139 serogroup antigen are virulence factors.
Although Vibrio cholerae O139 synonym Bengal strains, from the current epidemics in India and Bangladesh, are closely related to seventh-pandemic strains, as shown by multilocus enzyme electrophoresis, Bengal strains are encapsulated and portions of the O1 antigen biosynthetic complex genes found in O1 strains are altered or lacking. Encapsulated Bengal strains showed resistance to killing by normal human serum. The presence of the capsule suggests the potential for bloodstream invasion in susceptible hosts and has profound implications for vaccine development.
The complete sequences of the RhsB and RhsC elements of Escherichia coli K-12 have been determined. These sequence data reveal a new repeated sequence, called H-rpt (Hinc repeat), which is distinct from the Rhs core repetition that is found in all five Rhs elements. H-rpt is found in RhsB, RhsC, and RhsE. Characterization of H-rpt supports the view that the Rhs elements are composite structures assembled from components with very different evolutionary histories and that their incorporation into the E. coli genome is relatively recent. In each case, H-rpt is found downstream from the Rhs core and is separated from the core by a segment of DNA that is unique to the individual element. The H-rpt's of RhsB and RhsE are very similar, diverging by only 2.1%. They are 1,291 bp in length, and each contains an 1,134-bp open reading frame (ORF). RhsC has three tandem copies of H-rpt, all of which appear defective in that they are large deletions and/or have the reading frame interrupted. Features of H-rpt are analogous to features typical of insertion sequences; however, no associated transposition activity has been detected. A 291-bp fragment of H-rpt is found near min 5 of the E. coli K-12 map and is not associated with any Rhs core homology. The complete core sequences of RhsB and RhsC have been compared with that of RhsA. As anticipated, the three core sequences are closely related, all having identical lengths of 3,714 bp each. Like RhsA, the RhsB and RhsC cores constitute single ORFs that begin with the first core base. In each case, the core ORF extends beyond the core into the unique sequence. Of the three cores, RhsB and RhsA are the most similar, showing only 0.9% sequence divergence, while RhsB and RhsC are the least similar, diverging by 2.9%. All three cores conserve the 28 repetitions of a peptide motif noted originally for RhsA. A secondary structure is proposed for this motif, and the possibility of its having an extracellular binding function is discussed. RhsB contains one additional unique ORF, and RhsC contains two additional unique ORFs. One of these ORFs includes a signal peptide that is functional when fused to TnphoA.
Sixty-nine strains of Vibrio cholerae O1 isolated at different times were analysed to investigate if there were any differences among the O1 strains isolated before, during and after the advent of the O139 serogroup. Of the 69 O1 strains examined, 68 belonged to the Ogawa serotype while one belonged to the Inaba serotype. With the exception of one strain all other strains of V. cholerae O1 belonged to the eltor biotype. A single O1 strain isolated before the emergence of the O139 serogroup could not be classified as either eltor or classical biotype because it was resistant to both classical and eltor specific bacteriophages. Marked variations in the susceptibility to antibiotics of V. cholerae O1 isolated during the different periods were observed. In addition, strains of V. cholerae isolated after the epidemic of serogroup O139 in Calcutta showed an expanding R-type with resistance to a variety of drugs as compared to the O1 strains isolated before the advent of the O139 serogroup. From this study, it is clear that there is a substantial mobility in genetic elements of V. cholerae O1 which necessitates a continuous monitoring to keep abreast of the changing traits of the etiologic agent of cholera.
Vibrio cholerae, the causative agent of cholera, requires two coordinately regulated factors for full virulence: cholera toxin (CT), a potent
enterotoxin, and toxin-coregulated pili (TCP), surface organelles required for intestinal colonization. The structural genes
for CT are shown here to be encoded by a filamentous bacteriophage (designated CTXΦ), which is related to coliphage M13. The
CTXΦ genome chromosomally integrated or replicated as a plasmid. CTXΦ used TCP as its receptor and infected V. cholerae cells within the gastrointestinal tracts of mice more efficiently than under laboratory conditions. Thus, the emergence of
toxigenic V. cholerae involves horizontal gene transfer that may depend on in vivo gene expression.
At the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B) Dhaka we studied the trends in cholera for the period January 1992 to May 1995. Vibrio cholerae O139 Bengal emerged as a second aetiologic agent of cholera in Dhaka in January 1993. In 1993, the majority of cholera cases was due to V. cholerae O139, with V. cholerae O1 accounting for a small proportion of cases. During the latter part of the study period (Jan 1994-May 1995), V. cholerae O1 re-emerged as the predominant cholera strain. The predominant age group affected in endemic cholera due to V. cholerae O1 was children 2-9 years old, and the organism was isolated from more females than from males at all ages. In contrast, cholera due to V. cholerae O139 caused disease mostly in adults 15 years and older, which indicated that this organism was new in this population. As with V. cholerae O1, V. cholerae O139 was isolated from more females than males. The initial rapid emergence and predominance of V. cholerae O139 was considered possibly to herald the start of the eighth pandemic of cholera. However, just after a year, the prevalence of V. cholerae O139 decreased dramatically with V. cholerae O1 resuming the role of the dominant cholera strain. The factor(s) contributing to the dramatic decline in prevalence of V. cholerae O139 is not well understood.
A novel Vibrio cholerae insertion sequence element, designated IS1004, was characterized and used for DNA fingerprinting of Vibrio spp. IS1004 comprises 628 bp and contains an open reading frame whose product shows a large degree of sequence identity with the IS200-encoded transposase. IS1004 was present in one to eight copies in most of the V. cholerae strains analyzed. The IS1004-generated fingerprints of epidemic V. cholerae strains with serotype O1 were closely related, although it was possible to distinguish between the two biotypes, classical and El Tor. Non-O1 serotype strains generally showed heterogeneous patterns unrelated to those of the epidemic O1 strains. Several strains were observed with identical or related fingerprint patterns but expressed different serotypes. Conversely, strains with different fingerprint patterns but identical serotypes were also found. These observations indicate that the gene clusters coding for distinct O antigens may be transferred horizontally between V. cholerae strains. Two examples of non-O1 strains with a fingerprint resembling that of epidemic O1 strains were found; they were the O139 Bengal strain and an O37 strain. The O139 Bengal strain is closely related to the El Tor biotype. The O37 strain was responsible for a large cholera outbreak in Sudan in 1968 and was classified as a noncholera vibrio. Our study, however, shows that the O37 Sudan strain is genetically closely related to classical O1 strains. Similar to O139 Bengal, O37 Sudan lacked most of the O1 antigen cluster but did contain flanking genes. Thus, O37 Sudan represents a second example of an epidemic V. cholerae strain carrying non-O1 antigens. This study underlines the importance of genotypic methods for the differentiation of V. cholerae strains and for recognition of strains with epidemic potential.
Vibrio cholerae O139 is the first non-O1 serogroup of V. cholerae to give rise to epidemic cholera. Apparently, this new serogroup arose from an El Tor O1 strain of V cholerae, but V. cholerae O139 is distinguishable from V. cholerae El Tor O1 by virtue of its novel antigenic structure and also its characteristic pattern of resistances to the antibiotics sulfamethoxazole, trimethoprim, streptomycin, and furazolidone. We found that the first three of these antibiotic resistances are carried on an approximately 62-kb self-transmissible, chromosomally integrating genetic element which we have termed the SXT element. This novel conjugative transposon-like element could be conjugally transferred from V. cholerae O139 to V cholerae O1 and Escherichia coli strains, where it integrated into the recipient chromosomes in a site-specific manner independent of recA. To study the potential virulence properties of the SXT element as well as to improve upon the live attenuated O139 vaccine strain Bengal-2, a large internal deletion in the SXT element was crossed on to the Bengal-2 chromosome. The resulting strain, Bengal-2.SXT(s), is sensitive to sulfamethoxazole and trimethoprim and colonizes the intestines of suckling mice as well as wild-type strains do, suggesting that the SXT element does not encode a colonization factor. Derivatives of Bengal-2.SXT(s) are predicted to be safe, antibiotic-sensitive, live attenuated vaccines for cholera due to the O139 serogroup.
This study presents results of a surveillance on cholera conducted with hospitalized patients admitted to the Infectious Diseases Hospital, Calcutta, India, from January 1993 to December 1995. The O139 serogroup of Vibrio cholerae dominated in 1993 but was replaced by O1 as the dominant serogroup in 1994 and 1995. The isolation rate of V. cholerae non-O1 non-O139 did not exceed 4.9% throughout the study period, while the isolation rate of the O139 serogroup in 1994 and 1995 was below 9%. No temporal clustering of any non-O1 non-O139 serogroup was observed. With the exception of 1 strain, none of the 64 strains belonging to the non-O1 non-O139 serogroup hybridized with ctx, zot, and ace gene probes, while 97.3 and 97.7% of the O139 and O1 strains, respectively, hybridized with all the three probes. Multiplex PCR studies revealed that all the O1 strains belonged to the EIT or biotype. There was a progressive increase in the cytotoxic response on CHO and HeLa cells evoked by culture supernatants of strains of V. cholerae non-O1 non-O139 isolated during 1994 and 1995 compared with the response evoked by those isolated in 1993. Dramatic shifts in patterns of resistance to antibiotics between strains of V. cholerae belonging to different serogroups and within strains of a serogroup isolated during different time periods were observed. There was a discernible increase in the incidence of multidrug-resistant strains of V. cholerae O1 isolated in 1994 and 1995 compared with that in 1993. On the basis of the results of this study, we predict the possibility of newer variants of V. cholerae emerging in the future.
Several new species of the genus Vibrio have been recognized in recent years thanks to improved laboratory techniques, which have allowed the isolation and differentiation of members of the genus Vibrio and allied organisms. Epidemiology and pathogenesis of these new species have drawn the attention of epidemiologists and basic scientists alike. Some of these species, such as V. vulnificus, V. alginolyticus, V. damsela, and V. metschnikovii, do not cause diarrhea but cause certain extraintestinal lesions. For example, V. vulnificus has been implicated in fulminating septicemia and a rapidly progressing cellulitis; V. alginolyticus and V. damsela, in superficial skin and ear infections; and V. metschnikovii (an opportunistic pathogen), in peritonitis and bacteremia. This chapter will focus on the current status of knowledge on the epidemiology and pathogenicity of vibrios other than V. cholerae 01 and related organisms which cause diarrhea in humans.
Public health authorities would like to have at their disposal a safe, practical, inexpensive, and highly effective vaccine to provide long-term protection against cholera. The Diarrheal Diseases Control Programme of the World Health Organization has targeted the development of an improved vaccine against cholera as a high priority.1 Such a vaccine to combat cholera is not yet in the public health armamentarium but, as shown in this chapter, several promising candidates are on the horizon.KeywordsCholera ToxinOral VaccineOral ImmunizationVibrio CholeraeCombination VaccineThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
Improved Vibrio cholerae donors were constructed by introducing the ampicillin transposon, Tn1, into both the conjugative plasmid, P, and the bacterial chromosome to provide "portable regions of homology." The resulting Tfr (Transposon-facilitated recombination) donors transferred genes at high frequency from origins specified by the chromosomally inserted Tn1 copies. Tn1 was transposed into the chromosome from a deleted P::Tn1 vector, which was eliminated from the cells by superinfection with a thermosensitive P::Tn9 (chloramphenicol) mutant plasmid. After eliminating the thermosensitive plasmid, the chromosomally resistant isolates were converted into donors with a P::Tn1 conjugative plasmid. Tfr donors were also obtained by isolating Tn1 insertion mutations in a gene for thymine biosynthesis. Chromosomal sites of Tn1 relative to bacterial genes were determined by measuring gene transfer frequencies and genetic linkage. In one case, linkage of the amp gene to the chromosomal genes that defined its location was demonstrated. Chromosomal transfer by Tfr donors was reversed by isolating P::Tn1 plasmids that contained Tn1 inserted in the opposite orientation.
Fourteen mutants have been identified in which the frequency of spontaneous mutations in mitochondrial DNA is increased. As well as increasing the frequency of mutations to resistance to erythromycin, oligomycin and spiramycin, all the mutants also show changes in the frequency of spontaneous petite induction. None of the mutants has any effect on the frequency of spontaneous nuclear mutations. Nine of the mutants are in one complementation group and five are in another. The phenotype of both groups is caused by a single nuclear mutation.
The DNA sequences of the asd genes from 45 isolates of Vibrio cholerae (19 clinical O1 isolates, 2 environmental nontoxigenic O1 isolates, and 24 isolates with different non-O1 antigens) were determined. No differences were found within either sixth- or seventh-pandemic isolates; however, variation was found between the two forms and among the non-O1 isolates. O139 isolates had sequences identical to those of seventh-pandemic isolates. Phylogenetic trees with Vibrio mimicus as the outgroup suggest that the sixth-pandemic, seventh-pandemic, and U.S. Gulf isolates are three clones that have evolved independently from different lineages of environmental, nontoxigenic, non-O1 V. cholerae isolates. There is evidence for horizontal transfer of O antigen, since isolates with nearly identical asd sequences had different O antigens, and isolates with the O1 antigen did not cluster together but were found in different lineages. We also found evidence for recombination events within the asd gene of V. cholerae. V. cholerae may have a higher level of genetic exchange and a lower level of clonality than species such as Salmonella enterica and Escherichia coli.
Genetic variation and molecular evolution within the seventh-pandemic clone of Vibrio cholerae O1 and its relationship to other V. cholerae isolates were examined by studying 58 clinical isolates that were epidemiologically unassociated and isolated from patients in different countries over 62 years (1931 to 1993). The sample consisted of 45 isolates from the seventh cholera pandemic (1961 to the present), 3 from the sixth pandemic, 3 from sporadic El Tor outbreaks prior to the seventh pandemic, 2 from the U.S. Gulf Coast, and 5 O139 Bengal isolates. Ribotyping detected 11 polymorphic restriction sites within the seventh-pandemic isolates and showed major differences in ribotypes in comparison with sixth- and pre-seventh-pandemic isolates. O139 isolates were very similar to isolates from the start of the seventh pandemic, differing at only two sites. The majority of seventh-pandemic isolates fall into two groups, the first present from 1961 to the present and found only in Asia and the second arising in 1966 and spreading worldwide. Both groups underwent change over time, allowing a provisional estimate for the nucleotide substitution rate within the seventh pandemic clone.
The Salmonella enterica O antigen is a highly variable surface polysaccharide composed of a repeated oligosaccharide (the O unit). The O unit produced by serogroup D2 has structural features in common with those of groups D1 and E1, and hybridization studies had previously suggested that the D2 rfb gene cluster responsible for O-unit biosynthesis is indeed a hybrid of the two. In this study, the rfb gene cluster was cloned from a group D2 strain of S. enterica sv. Strasbourg. Mapping, hybridization, and DNA sequencing showed that the organization of the D2 rfb genes is similar to that of group D1, with the alpha-mannosyl transferase gene rfbU replaced by rfbO, the E1-specific beta-mannosyl transferase gene. The E1-specific polymerase gene (rfc) has also been acquired. Interestingly, the D1-like and E1-like rfb regions are separated by an additional sequence closely related to an element (Hinc repeat [H-rpt]) associated with the Rhs loci of Escherichia coli. The H-rpt resembles an insertion sequence and possibly mediated the intraspecific recombination events which produced the group D2 rfb gene organization.
A newly described Vibrio cholerae serogroup--O139 Bengal, the causative agent of the recent large epidemics of cholera-like disease in the Indian subcontinent and neighbouring countries--possesses a high molecular weight capsular polysaccharide (CPS) that can be visualized by electron microscopy and in composition differs from the lipopolysaccharide (LPS). The CPS and LPS can be separated from each other by a two-step extraction procedure, a phenol-water extraction in order to extract all polysaccharides from the bacterial suspension followed by a phenol-chloroform-petroleum ether (PCP) extraction. The CPS is mainly composed of 3,6-dideoxyhexose (abequose or colitose), quinovosamine and glucosamine. The LPS of the O139 Bengal strain appears to possess a short polysaccharide which contains glucose, galactose, glucosamine and heptose. Both the LPS and CPS are immunogenic. They react in an enzyme immunoassay with rabbit antibodies generated against whole heat-killed bacteria. By analogy with other capsulated bacteria, the possession of a capsule may confer increased virulence of O139 Bengal.
The tetragonal paracrystalline surface protein array (A-layer) of the fish pathogenic bacterium Aeromonas salmonicida is a virulence factor and bacteria which are unable to produce A-layer are attenuated in their ability to kill fish. Ten independent mutants of Aeromonas salmonicida which were unable to produce A-layer were isolated by growth at 30 degrees C. These mutants displayed either reduced synthesis of the A-layer subunit, synthesis of a truncated subunit, or complete loss of the ability to produce the subunit protein. Restriction mapping and analysis by polymerase chain reaction showed that the mutations had resulted from insertion of two different insertion sequence (IS) elements into different sites in the A-layer subunit gene (vapA) and its promoter. Sequence comparisons indicated that ISAS1 is unique among reported IS elements. It is 1223 bp long with imperfect terminal inverted repeats of 22 bp and insertion resulted in a duplicated 8 bp target sequence in vapA. ISAS1 expressed a 42,000 molecular weight (M(r)) protein in mini-cells. ISAS2 was 1084 bp long, expressed proteins of M(r) 38,000 and 39,000 in vitro, had imperfect 29 bp terminal inverted repeats and had duplicated a 3 bp target sequence. Sequence comparisons indicated that ISAS2 was also unique to A. salmonicida; however, the proteins encoded by ISAS2 showed strong homology to the putative transposases encoded by the IS30 family of IS elements. Southern analyses showed that both ISAS1 and ISAS2 were restricted to A. salmonicida strains A449 and A450 where they were present in low copy number. The ability of these two IS elements to mutate the ability of A. salmonicida to produce its paracrystalline surface array provides a novel method for the attenuation of virulence.
A large cluster of virulence genes encoding proteins involved in Vibrio cholerae accessory colonization factor (ACF) expression and toxin-coregulated pilus (TCP) biogenesis is flanked by sequences that resemble bacteriophage attachment (att) half-sites. Adjacent to the attL-like site is a gene (int) that encodes a protein related to the integrase family of site-specific recombinases. The putative vibrio integrase appears to be most closely related to the Escherichia coli cryptic prophage (CP4-57) integrase protein (52% identity, 73% similarity). Genomic analysis of numerous V. cholerae strains (O1, non-O1 and O139) revealed that only vibrios capable of causing epidemic Asiatic cholera possess the TCP-ACF colonization gene cluster in association with the integrase. The fact that the integrase gene is absent in avirulent strains suggests that epidemic strains of V. cholerae obtained the TCP-ACF colonization gene cluster via horizontal transfer.
Vibrio cholerae serogroup O139 Bengal is the first documented serogroup other than O1 to cause epidemic cholera. The O139 Bengal strains are very similar to V. cholerae serogroup O1 biotype El Tor strains. The major differences between the two serogroups are that O139 Bengal contains a distinct O antigen and produces a polysaccharide capsule. We previously described three TnphoA mutants of O139 strain AI1837 which abolish both O antigen and capsule production. These TnphoA insertions were mapped to a 21.5 kb EcoRI fragment of the O139 chromosome. We describe here the cloning and mapping of this 21.5 kb EcoRI fragment and it was shown to complement each of the mutants in trans to produce O antigen and capsule. The EcoRI fragment contains 13 kb of DNA that is specific to O139 and 8.5 kb of DNA that is common to O1 and O139. Sequence analysis of the 13 kb of O139-specific DNA revealed that it contains 11 open reading frames all of which are transcribed in the same direction. Eight of the 11 open reading frames are homologous to sugar biosynthesis genes from other organisms. Using extended polymerase chain reactions, we show that the extent of the DNA region in O139 that is not present in O1 is approximately 35 kb. The site of insertion of this O139-specific DNA in the O1 chromosome was mapped to the rfbO1 region. We also demonstrate that O139 Bengal strain AI1837 contains a deletion of 22 kb that in serogroup O1 strains contains the rfb region. Therefore, O139 Bengal probably arose from an O1 strain that had undergone genetic rearrangements including deletion of the O1 rfb region and acquisition of a 35 kb region of DNA which encodes O139 surface polysaccharide.
The presence of a temperate phage was demonstrated in a strain of Vibrio cholerae O139 isolated from a patient. Spontaneous variants with translucent colonies had lost this phage. The loss of the phage was associated with increased hydrophobicity, indicating the loss of the capsule. These clones were sensitive to serum bactericidal activity, showed decreased expression of such presumed virulence factors as proteases, motility and mannose-sensitive pili. Furthermore, excision of the phage made the strain dependent on purines for growth.
Gene nomenclature for bacterial surface polysaccharides is complicated by the large number of structures and genes. We propose a scheme applicable to all species that distinguishes different classes of genes, provides a single name for all genes of a given function and greatly facilitates comparative studies.
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