Differences in virulence genes in Vibrio cholerae eltor strains isolated from different sources in Turkmenistan territory

Polymerase chain reaction (PCR) detected the presence of various genes associated with virulence in genome of strains V. cholerae eltor isolated in Turkmenistan territory during epidemic and epidemic-free perios. It was found that a complete set of virulence genes (ctxA+, tcpA+ and toxR+) contained strains isolated from patients, carriers and environment only in cholera epidemics. Strains isolated from the environment in the period free of epidemics did not contain ctxA and tcpA in 78.2% of cases, but 5.2% of the strains carried a complete set of virulence genes. There were also nontoxigenic strains containing genes tcpA and toxR. Such strains were isolated from the environment (16.6%) and vibrion carriers (42.9%). Isolated were also strains V.cholerae eltor carrying bacteriophage CTX phi with incomplete set of virulence genes and having genotype ctxA-, ace+ and zot+. Almost all the strains ctxA-, tcpA+ carry attRS1-site in genome. This shows that such strains may transform into toxigenic as a result of infection with bacteriophage CTX phi.     

Environment and virulence factors of Vibrio cholerae strains isolated in Argentina

AIMS: To determine the presence of Vibrio cholerae in different areas of Argentina in three sample types, to determine the composition of planktonic communities in areas at which this pathogen was detected and to characterize the virulence properties and antimicrobial resistance of the recovered environmental isolates. METHODS AND RESULTS: Water and plankton samples were collected in marine, brackish and freshwater environments. Vibrio cholerae non-O1, non-O139 was isolated in 36.1% of the samples analysed. The micro-organism was detected in freshwater but not in marine or brackish samples. No relationship was found between isolation of V. cholerae and presence of any species of plankton. All the isolates presented very similar virulence profiles by PCR, lacking ctxA and tcpA El Tor and containing hlyA (98.7%), rtxA (99.0%), toxR (98.7%) and stn-sto (1.9%). Resistance to ampicillin was found in both Tucumán (21%) and Buenos Aires isolates (45%). CONCLUSIONS: We identified two geographic areas in Argentina where V. cholerae was present: freshwaters of the rivers from Tucumán and the Río de la Plata. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of V. cholerae strains in the environment, carrying both virulence factors and resistance to antimicrobial agents, highlight the need for a continuous and active surveillance of this pathogen.     

Expression of Vibrio cholerae virulence genes in response to environmental signals

Vibrio cholerae, the causative agent of Asiatic cholera, is a gram-negative motile bacterial species acquired via oral ingestion of contaminated food or water sources. The O1 serogroup of V. cholerae is responsible for pandemic cholera and is divided into two biotypes, classical and El Tor (Butterton and Calderwood, 1995; Mekalanos, 1985). The El Tor biotype is responsible for the current cholera pandemic. In the absence of disease, the vibrio life cycle consists of a free-swimming phase in marine and estuarine environments in association with zooplankton, crustaceans, insects, and water plants. Vibrios interact with various surfaces found in the environment to generate biofilms which may promote survival (Watnick etaL, 1999). Within the host the motile vibrios must evade the innate host defense mechanisms, penetrate the mucus layer covering the intestinal villi, adhere to and colonize the epithelial surface of the small intestine, assume a non-motile phase, replicate and cause disease by secreting numerous exoproteins at the site of infection (Oliver and Kaper, 1997). The voluminous diarrhea associated with cholera infection leads to the dissemination of the vibrios back into a watery environment and thus a continuation of the environmental phase of the life cycle. The host phase of the vibrio life cycle is only possible through the action of a group of virulence genes (ToxR-regulon) controlled by a complex and incompletely understood regulatory cascade. The ToxR regulon colonization and toxin genes are coordinately expressed in response to specific host signals that have yet to be completely defined (Skorupsky and Taylor 1997). Although little is known regarding the host signals that impact the ToxR regulatory cascade, it is clear that these intraintestinal signals play an important role in maximizing the ability of the vibrios to survive and multiply within the host. Key to understanding the complex events involved in the pathogenesis of V. cholerae will be elucidating the intraintestinal signaling molecules that trigger the expression of vibrio virulence genes. Understanding the molecular basis of this host-parasite interaction will provide important information with respect to how pathogenic bacteria establish infection and provide insights leading to novel methods for treating and/or preventing bacterial infections. This review will summarize what is known regarding host signaling and the complex ToxR regulatory system employed by V. cholerae to coordinate virulence gene expression within the host.     

Distribution of Vibrio cholerae O1 antigen biosynthesis genes among O139 and other non-O1 serogroups of Vibrio cholerae

The organization and distribution of the genes responsible for O antigen biosynthesis in various serogroups of Vibrio cholerae were investigated using several DNA probes derived from various regions of the genes responsible for O1 antigen biosynthesis. Based on the reactivity pattern of the probes against the various serogroups, the cluster of genes responsible for the O1 antigen biosynthesis could be broadly divided into six groups, designated as class 1-6. The class 3 cluster of genes corresponding to gmd to wbeO, wbeT and a part of wbeU was specific for only the O1 serogroup. The other cluster of genes (class 1, 2, 4-6) reacted with other serogroups of V. cholerae. These data indicate that serotype conversion in V. cholerae does not depend on a simple mutational event but may involve horizontal gene transfer not only between V. cholerae strains but also between V. cholerae and species other than V. cholerae.     

Prevalence of major virulence genes among various Vibrio cholerae El-TOR strains for evaluating their epidemiological significance

Specific oligonucleotide primers were chosen for identifying the fragments of the four major virulence genes of V. cholerae eltor (ctxA, tcpA, toxR, and hap) using the polymerase chain reaction (PCR). In order to estimate the efficiency of complex PCR testing of V. cholerae for evaluation of their epidemiological significance, a collection of 80 V. cholerae eltor strains with known virulence was selected, whose most important specific features had been studied previously. The hap was appropriate species-specific gene making it possible to detect V. cholerae strains regardless of their virulence. The most complete and objective data for evaluating the epidemic significance can be obtained by detecting the presence of three virulence genes (ctxA, tcpA, and toxR) in their chromosome. The prevalence of the above four genes in various V. cholerae strains isolated from the environment during epidemic and non-epidemic periods was studied.     

Vibrio virulence genes in fishes collected from estuarine waters in Italy

Aims: The aim of this study was to investigate the presence of Vibrio vulnificus and potentially pathogenic strains of Vibrio parahaemolyticus in mullets collected from estuarine environment in Italy. Methods and Results: Two hundred and ninety‐five mullets were analysed by culture using the selective medium thiosulfate citrate bile salt sucrose agar, during a monitoring period of 2 years (2008–2009). Presumptive Vibrio colonies were initially identified by using biochemical tests, and strains identified as V. parahaemolyticus and V. vulnificus were subsequently examined by PCR for the presence of species‐specific and virulence genes (toxR, trh, tdh and vvh). V. parahaemolyticus was found in 55% (162/295) of fishes and V. vulnificus in 1% (3/295) with a higher presence in summer months. The trh+/tdh? strains were detected in 16% (47/295) of samples and only one strain resulted trh+/tdh+. One of the V. parahaemolyticus trh+ strains isolated belonged to the O1:KUT (K untypeable), a serotype recently associated to gastroenteritis in Italy. Conclusions: This is the first report demonstrating a high percentage of potential pathogenic V. parahaemolyticus trh+ strains in estuarine fishes of the Mediterranean area. Significance and Impact of the Study: These findings indicate the potential human health risk associated with the presence of pathogenic Vibrio spp. in wild fishes.     

Regulation and temporal expression patterns of Vibrio cholerae virulence genes during infection

The temporal expression patterns of the critical Vibrio cholerae virulence genes, tcpA and ctxA, were determined during infection using a recombinase reporter. TcpA was induced biphasically in two temporally and spatially separable events in the small intestine, whereas ctxA was induced monophasically only after, and remarkably, dependent upon, tcpA expression; however, this dependence was not observed during in vitro growth. The requirements of the virulence regulators, ToxR, TcpP, and ToxT, for expression of tcpA and ctxA were determined and were found to differ significantly during infection versus during growth in vitro. These results illustrate the importance of examining virulence gene expression in the context of bona fide host-pathogen interactions.     

Distribution of genes for virulence and ecological fitness among diverse Vibrio cholerae population in a cholera endemic area: tracking the evolution of pathogenic strains

The pathogenic strains of Vibrio cholerae that cause acute enteric infections in humans are derived from environmental nonpathogenic strains. To track the evolution of pathogenic V. cholerae and identify potential precursors of new pathogenic strains, we analyzed 324 environmental or clinical V. cholerae isolates for the presence of diverse genes involved in virulence or ecological fitness. Of 251 environmental non-O1, non-O139 strains tested, 10 (3.9%) carried the toxin coregulated pilus (TCP) pathogenicity island encoding TCPs, and the CTX prophage encoding cholera toxin, whereas another 10 isolates carried the TCP island alone, and were susceptible to transduction with CTX phage. Most V. cholerae O1 and O139 strains carried these two major virulence determinants, as well as the Vibrio seventh pandemic islands (VSP-1 and VSP-2), whereas 23 (9.1%) non-O1, non-O139 strains carried several VSP island genes, but none carried a complete VSP island. Conversely, 30 (11.9%) non-O1, non-O139 strains carried type III secretion system (TTSS) genes, but none of 63 V. cholerae O1 or O139 strains tested were positive for TTSS. Thus, the distribution of major virulence genes in the non-O1, non-O139 serogroups of V. cholerae is largely different from that of the O1 or O139 serogroups. However, the prevalence of putative accessory virulence genes (mshA, hlyA, and RTX) was similar in all strains, with the mshA being most prevalent (98.8%) followed by RTX genes (96.2%) and hlyA (94.6%), supporting more recent assumptions that these genes imparts increased environmental fitness. Since all pathogenic strains retain these genes, the epidemiological success of the strains presumably depends on their environmental persistence in addition to the ability to produce major virulence factors. Potential precursors of new pathogenic strains would thus require to assemble a combination of genes for both ecological fitness and virulence to attain epidemiological predominance.     

Groups of the species Vibrio cholerae having different epidemic importance

Subdivision of V. cholerae 01 into toxins on the basis of the whole complex of signs characteristic of this species does not make it possible to judge on their epidemic importance and to use the data on identification of V. cholerae for solving practical problems. Classification of V. cholerae by their capacity for producing toxin (choleragen and hemolysin of type 1, subtype beta) removes these difficulties.     

Biotype-specific tcpA genes in Vibrio cholerae

Abstract The tcpA gene, encoding the structural subunit of the toxin-coregulated pilus, has been isolated from a variety of clinical isolates of Vibrio cholerae , and the nucleotide sequence determined. Strict biotype-specific conservation within both the coding and putative regulatory regions was observed, with important differences between the El Tor and classical biotypes. V. cholerae O139 Bengal strains appear to have El Tor-type tcpA genes. Environmental O1 and non-O1 isolates have sequences that bind an E1 Tor-specific tcpA DNA probe and that are weakly and variably amplified by tcpA -specific polymerase chain reaction primers, under conditions of reduced stringency. The data presented allow the selection of primer pairs to help distinguish between clinical and environmental isolates, and to distinguish El Tor (and Bengal) biotypes from classical biotypes from classical biotypes of V. cholerae . While the role of TcpA in cholera vaccine preparations remains unclear, the data strongly suggest that TcpA-containing vaccines directed at O1 strains need include only the two forms of TcpA, and that such vaccines directed at (O139) Bengal strains should include the TcpA of El Tor biotype.     

TolC affects virulence gene expression in Vibrio cholerae

A Vibrio cholerae tolC mutant showed increased toxT expression in M9 medium, but not in the presence of four amino acids that induce cholera toxin production, and in LB with high osmolarity but not high pH or temperature. TolC did not affect expression of other regulatory genes in the ToxR regulon.     

Filamentous phages linked to virulence of Vibrio cholerae

The pathogenicity of Vibrio cholerae depends upon its production of two key virulence factors: the toxin co-regulated pilus (TCP), a colonization factor, and cholera toxin, an exotoxin. Genes encoding both virulence factors were introduced into V. cholerae by horizontal gene transfer. The toxin genes are contained within the genome of CTXphi, an integrated filamentous phage identified in 1996. In the past few years, it has been shown that CTXphi relies on novel processes for phage DNA integration, replication and secretion. In addition, expression of CTXphi genes--including the toxin genes--and transmission of CTXphi were recently found to be promoted by the antirepressor RstC, which is encoded within RS1, a newly described satellite phage of CTXphi. The genetic island that encodes TCP has also been described as a filamentous phage; however, these sequences are unlike the genome of any previously characterized filamentous phage.     

The epidemiological importance of Vibrio cholerae isolated from different ecological systems

The analysis of the data on the isolation of V. cholerae from different ecological systems indicates that V. eltor do not constantly inhibit the rivers and sea at the territory under control. Hemolytically active V. cholerae without the vct gene, found to be faintly virulent and avirulent when studied on suckling rabbits used as a model and when evaluated by the complex method, show no tendency towards epidemic spread in the presence of conditions for the realization of the transmission of vibrios by the water route.     

Identification of virulence factors in Vibrio cholerae isolated from Iraq during the 2007-2009 outbreak

Thousands of people were infected with Vibrio cholerae during the outbreak in Iraq in 2007-2009. Vibrio cholerae was shown to be variable in its content of virulence determinants and in its antibiotic sensitivity. This study was designed to isolate and characterize clinical and environmental V.?cholerae isolates and to determine antibiotic sensitivity, enzyme and toxin production, and the presence of virulence genes. Eighty clinical and five environmental bacterial isolates were collected and diagnosed by subjecting them to microscopic, biochemical, serological, and molecular analysis. The results revealed that 55% of clinical isolates belonged to the Inaba serotype, 32.5% to the Ogawa serotypes, and 12.5% to the Non-O1 serotype. All environmental V.?cholerae isolates belonged to the Non-O1 serotype. All environmental isolates were sensitive to all examined antimicrobial agents, while all clinical isolates showed a high sensitivity (100%) to ampicillin, gentamicin, cephalothin, tetracycline, erythromycin, and ciprofloxacin, and a high resistance (97.5%) to co-trimoxazole, nalidixic acid, and chloramphenicol. It was found that all V.?cholerae (O1) isolates were resistant to the Vibrio static O129 and all Non-O1 V.?cholerae isolates were sensitive to the Vibrio static O129. All clinical and environmental isolates produced hemolysin (100%) and lecithinase (100%), while they showed various production rates of protease (90% of clinical and 60% of environmental) and lipase (50% of clinical and 20% of environmental). The ompW gene was amplified in all the clinical and environmental V.?cholerae isolates, but not in other related and nonrelated bacteria. Multiplex PCR analysis showed that the toxR gene was amplified in all clinical and environmental isolates, while ctxA, ctxB, tcpA genes were amplified only in clinical (O1) isolates. This study indicates the differences in the production of some enzymes and toxins and in the content of virulence genes between clinical and environmental isolates in Iraq during the outbreak (2007-2009).     

Detection of heat-stable enterotoxin genes among Australian Vibrio cholerae O1 strains

Abstract DNA probes derived from the heat-stable enterotoxin gene of Vibrio cholerae non-O1 ( stn ), and the cholera toxin gene (etc), were used to screen 199 strains of V. cholerae O1, which were isolated within Australia from 1977–1986. 13 environmental strains isolated from the riverine environment in Southeast Queensland in 1980 and 1981, hybridized with the stn and ctx DNA probes. The concentrated supernatant of 6 of these strains elicited fluid accumulation in the infant mouse assay both before and after heating at 100 °C for 5 min. Genetic relationships among the 13 stn ⁺ strains were studied by a comparison of the rRNA-RFLPs (ribotyping) and by Southern blot analysis with a stn gene probe. The results indicate that there is a clonal relationship among the Australian stn ⁺ strains and that there is an environmental reservoir of stn genes among Australian V. cholerae O1 isolates.     

Genetics of stress adaptation and virulence in toxigenic Vibrio cholerae

Vibrio cholerae, a Gram-negative bacterium belonging to the gamma-subdivision of the family Proteobacteriaceae is the etiologic agent of cholera, a devastating diarrheal disease which occurs frequently as epidemics. Any bacterial species encountering a broad spectrum of environments during the course of its life cycle is likely to develop complex regulatory systems and stress adaptation mechanisms to best survive in each environment encountered. Toxigenic V. cholerae, which has evolved from environmental nonpathogenic V. cholerae by acquisition of virulence genes, represents a paradigm for this process in that this organism naturally exists in an aquatic environment but infects human beings and cause cholera. The V. cholerae genome, which is comprised of two independent circular mega-replicons, carries the genetic determinants for the bacterium to survive both in an aquatic environment as well as in the human intestinal environment. Pathogenesis of V. cholerae involves coordinated expression of different sets of virulence associated genes, and the synergistic action of their gene products. Although the acquisition of major virulence genes and association between V. cholerae and its human host appears to be recent, and reflects a simple pathogenic strategy, the establishment of a productive infection involves the expression of many more genes that are crucial for survival and adaptation of the bacterium in the host, as well as for its onward transmission and epidemic spread. While a few of the virulence gene clusters involved directly with cholera pathogenesis have been characterized, the potential exists for identification of yet new genes which may influence the stress adaptation, pathogenesis, and epidemiological characteristics of V. cholerae. Coevolution of bacteria and mobile genetic elements (plasmids, transposons, pathogenicity islands, and phages) can determine environmental survival and pathogenic interactions between bacteria and their hosts. Besides horizontal gene transfer mediated by genetic elements and phages, the evolution of pathogenic V. cholerae involves a combination of selection mechanisms both in the host and in the environment. The occurrence of periodic epidemics of cholera in endemic areas appear to enhance this process.