Vulnificus Virulence Research Articles

Pathogenic mechanism of Vibrio vulnificus infection.

Vibrio vulnificus is a fatal, opportunistic human pathogen transmitted through the consumption of raw/undercooked seafood or direct contact. V. vulnificus infection progresses rapidly and has severe consequences; some cases may require amputation or result in death. Growing evidence suggests that V. vulnificus virulence factors and regulators play a large role in disease progression, involving host resistance, cellular damage, iron acquisition, virulence regulation and host immune responses. Its disease mechanism remains largely undefined. Further evaluation of pathogenic mechanisms is important for selecting appropriate measures to prevent and treat V. vulnificus infection. In this review, the possible pathogenesis of V. vulnificus infection is described to provide a reference for treatment and prevention.

The role of Vibrio vulnificus virulence factors and regulators in its infection-induced sepsis.

Due to the development of Marine aquaculture, infections caused by Vibrio vulnificus are common all over the world. Symptoms of V. vulnificus infection vary from gastrointestinal illness to septicemia. After infection with V. vulnificus, some patients showed gastrointestinal symptoms, including vomiting, fever, diarrhea, and so on. Others appeared wound infection at the site of contact with bacteria, and even developed sepsis. Once it develops into sepsis, the prognosis of patients is very poor. However, its underlying pathogenic mechanism remains largely undetermined. Growing evidence shows that it can induce primary septicemia mainly via essential virulence factors and regulators. Therefore, it is important to identify the factors that play roles in sepsis. In this review, we systematically expounded the role of V. vulnificus virulence factors and regulators in its infection-induced sepsis in order to provide useful information for the treatment and prevention of V. vulnificus.

Surface hypothermia predicts murine mortality in the intragastric Vibrio vulnificus infection model

BackgroundThe Gram-negative bacterium Vibrio vulnificus can cause severe disease in humans who consume undercooked, contaminated seafood. To study food-borne V. vulnificus disease in the laboratory, mouse virulence studies predominantly use death as the primary experimental endpoint because behaviorally based moribund status does not consistently predict lethality. This study assessed ventral surface temperature (VST) and its association with mouse survival during V. vulnificus virulence studies as an efficacious, humane alternative.MethodsVST of mice intragastrically inoculated with V. vulnificus was measured every 2-h for 24 h and data for minimal VST analyzed for prediction of lethal outcome.ResultsIn contrast to the relatively stable VST of mock-infected control animals, mice infected with V. vulnificus exhibited hypothermia with minima occurring 8 to 12 h post-inoculation. The minimum VST of mice that proceeded to death was significantly lower than that of surviving mice. VST ≤ 23.5 °C was predictive of subsequent death with a sensitivity of 68% and specificity of 95%.ConclusionsUse of VST ≤ 23.5 °C as an experimental endpoint during V. vulnificus infection has potential to reduce suffering of nearly 70% of mice for a mean of 10 h per mouse, without compromising experimental efficacy. Temperature cutoff of 23.5 °C exhibited 93% positive and 77% negative predictive value. For future V. vulnificus virulence studies requiring only binary comparison (e.g., LD50 assays), we find that VST can be applied as a humane endpoint. However, use of VST is not recommended when detailed survival kinetics are desired.

A silkworm infection model to investigate Vibrio vulnificus virulence genes.

The halophilic marine bacterium, Vibrio vulnificus, occasionally causes fatal septicemia in immunocompromised patients. Mice are commonly used as experimental animals to investigate the virulence of V. vulnificus, however, a large number of mice are generally required for bioassays. The present study examined whether the invertebrate species, silkworms, can be used instead of mice to investigate V. vulnificus virulence. When the silkworms were inoculated with 1.2x107colony forming units of V. vulnificus OPU1‑Rf, a virulent strain of V.vulnificus, all injected silkworms died within 48h, however, those injected with culture filtrate or diluent did not. This silkworm infection model was then used to isolate attenuated V. vulnificus mutants from 1,016transposon‑inserted mutants. Consequently, a harmless mutant, SW998, was isolated. In this strain, the transposon was inserted into the rtxA gene, which is a known V. vulnificus virulence gene. In conclusion, the present study demonstrated that silkworms are useful animals for investigating the virulence of V. vulnificus.

Detection of vvhA gene of Vibrio vulnificus and trh virulence genes of Vibrio parahaemolyticus from samples of crustaceans’ species from the Black Sea using a PCR-based method

Detection of vvhA gene of Vibrio vulnificus and trh virulence genes of Vibrio parahaemolyticus from samples of crustaceans’ species from the Black Sea using a PCR-based method

Modeling and forecasting the distribution of Vibrio vulnificus in Chesapeake Bay.

To construct statistical models to predict the presence, abundance and potential virulence of Vibrio vulnificus in surface waters of Chesapeake Bay for implementation in ecological forecasting systems. We evaluated and applied previously published qPCR assays to water samples (n = 1636) collected from Chesapeake Bay from 2007-2010 in conjunction with State water quality monitoring programmes. A variety of statistical techniques were used in concert to identify water quality parameters associated with V. vulnificus presence, abundance and virulence markers in the interest of developing strong predictive models for use in regional oceanographic modeling systems. A suite of models are provided to represent the best model fit and alternatives using environmental variables that allow them to be put to immediate use in current ecological forecasting efforts. Environmental parameters such as temperature, salinity and turbidity are capable of accurately predicting abundance and distribution of V. vulnificus in Chesapeake Bay. Forcing these empirical models with output from ocean modeling systems allows for spatially explicit forecasts for up to 48 h in the future. This study uses one of the largest data sets compiled to model Vibrio in an estuary, enhances our understanding of environmental correlates with abundance, distribution and presence of potentially virulent strains and offers a method to forecast these pathogens that may be replicated in other regions.

Quorum sensing-dependent metalloprotease VvpE is important in the virulence of Vibrio vulnificus to invertebrates

Vibrio vulnificus, a Gram-negative bacterium, is an opportunistic human pathogen responsible for fatal septicemia caused by contaminated sea foods in eastern Asia. Quorum sensing (QS) is a cell-density dependent gene regulation mechanism that controls the expression of many virulence genes in various bacteria and V. vulnificus has been also suggested to express their virulence genes through the QS system. In this study, we investigated the role of QS system and QS-regulated exoproteases in the virulence of V. vulnificus using several invertebrate host models, Tenebrio molitor, an insect, Caenorhabditis elegans, a nematode, and brine shrimp (Artemia), an aquatic crustacean. When the culture supernatant of smcR (major QS regulator of V. vulnificus) mutant was injected to T. molitor larvae, it failed to induce the melanization of T. molitor larvae, while the culture supernatant of luxO (upstream negative regulator of smcR) mutant more strongly induced the melanization than wild type. These results demonstrated that QS system of V. vulnificus is crucial for virulence to T. molitor larvae. Among several QS-dependently expressed exoproteases of V. vulnificus, vvpE encoding a metalloprotease was mainly responsible for the melanization of T. molitor larvae, in that the culture supernatant of vvpE mutant failed to induce the melanization. This result was confirmed using the C. elegans and Artemia salina model systems, in which the vvpE mutant strains were attenuated in killing C. elegans and A. salina, compared with wild type, indicating that VvpE is important in the infection of V. vulnificus. In conclusion, we suggest that QS system and a QS-dependent exoprotease, VvpE are crucial for the V. vulnificus virulence to invertebrates.

Presence of virulence markers in environmental Vibrio vulnificus strains

This work aims to demonstrate the presence of several genes and factors associated with virulence in strains isolated from the environment at Pueblo Viejo Lagoon, State of Veracruz, Mexico. In this study, we investigated the production of V. vulnificus virulence factors, as cytolysin (haemolysin), RTX toxin, metalloprotease, siderophores, capsular polysaccharide, adhesion structures (like type IV pili), and polar and lateral flagella, involved in swimming and swarming (or, at least, the presence of genes encoding some of them) in 40 strains of V. vulnificus isolated from water and food. The results indicate that strains of environmental origin possess potential virulence characteristics. Caution should be exercised when consuming raw shellfish (especially by those more susceptible risk groups). This is the first work focused on the evaluation of V. vulnificus virulence factors in Mexico.

PilF polymorphism-based real-time PCR to distinguish Vibrio vulnificus strains of human health relevance

The Gram-negative bacterium Vibrio vulnificus is a common inhabitant of estuarine environments. Globally, V. vulnificus is a significant foodborne pathogen capable of causing necrotizing wound infections and primary septicemia, and is a leading cause of seafood-related mortality. Unfortunately, molecular methods for the detection and enumeration of pathogenic V. vulnificus are hampered by the genetically diverse nature of this pathogen, the range of different biotypes capable of infecting humans and aquatic animals, and the fact that V. vulnificus contains pathogenic as well as non-pathogenic variants. Here we report an alternative approach utilizing the development of a real-time PCR assay for the detection of pathogenic V. vulnificus strains based on a polymorphism in pilF, a gene previously indicated to be associated with human pathogenicity. Compared to human serum reactivity, the real-time PCR assay successfully detected pathogenic strains in 46 out of 47 analysed V. vulnificus isolates (97.9%). The method is also rapid, sensitive, and more importantly can be reliably utilised on biotype 2 and 3 strains, unlike other current methods for V. vulnificus virulence differentiation.

FatalVibrio vulnificusInfection Associated with Eating Raw Oysters, New Caledonia

To the Editor: The bacterium Vibrio vulnificus is a marine flora saprophyte that can cause necrotic skin infection and septicemia in humans who eat shellfish. Symptoms of septicemia (mortality rate >50%) have been described mostly in Florida and Japan among persons who ate raw filter-feeding shellfish when seawater temperatures are >20°C (1). V. vulnificus–related septicemia introduced through the digestive system appears within 7 days after ingestion (2). Clinical signs and symptoms include fever, collapse, and metastatic necrotic skin lesions. We report 3 patients from New Caledonia who died after V. vulnificus infection, which they probably acquired by eating contaminated oysters. These patients were hospitalized during February–May 2008 at Noumea Hospital (Noumea, New Caledonia). Patient 1 was a 51-year-old man with fever, muscle pains, bleeding gums, and a history of alcohol abuse; within 48 hours after symptom onset, he died of septic shock, with diffuse ecchymoses and purpura. Patient 2 was a 67-year-old woman with no known concurrent conditions who was admitted to the hospital with chills, diarrhea, and vomiting; septic shock developed, with painful erythematous plaques on the lower limbs becoming foamy, confluent, and necrotic. Patient 3 was a 74-year-old woman with untreated lupus who was hospitalized with lower-limb edema, hypotension, hypothermia, and erythematous skin lesions. All 3 patients received cephalosporins but died of multiple organ failure within 12 hours after hospital admission. Peripheral blood aerobic–anaerobic samples were taken from all patients, stored in BacT/Alert FA vials (bioMerieux, Marcy-l’Etoile, France), and incubated in the BacT/Alert 3D system (bioMerieux). Curved mobile gram-negative bacilli were isolated from blood samples cultured on conventional media without additional salt within 24 h after incubation at 37°C in a 5% CO2-enriched atmosphere. V. vulnificus was identified through the Vitek2 system (bioMerieux) and confirmed by using the Api 20E system (bioMerieux). Strains were sent to the Centre National de Reference des Vibrions et du Cholera, (Institut Pasteur, Paris, France), which by PCR confirmed the gene encoding virulence-associated hemolysin, a species-specific marker (3). Molecular typing by pulsed-field gel electrophoresis was performed to assess possible clonality of the strains. Several studies have shown the genomic diversity among environmental and clinical V. vulnificus isolates. The use of genotyping methods has identified >100 V. vulnificus strains in a single oyster (4) and notable heterogeneity among clinical isolates from multiple patients, even if a unique pathogenic strain causes the infection in each patient. Thus, V. vulnificus infections within a large population at risk may result from rare events controlled more by the host than by the bacterial strain (5). Pulsed-field gel electrophoresis genotype analysis enabled us to divide the strains into 2 groups. One group included the isolate from patient 1, and the other group included isolates from patients 2 and 3, which despite having slightly different NotI and SfiI patterns reflecting genetic rearrangement, clearly belonged to a single clone. Isolation of strains with such a high degree of homogeneity is not common, raising the question of the existence of V. vulnificus clones that are particularly virulent or adapted to humans. Currently, however, reliable markers for determining V. vulnificus virulence do not exist. Thus, no genotyping system is likely to be useful for rapidly identifying strains that affect public health (6). V. vulnificus–related analysis requires the assumption that all strains are virulent. Epidemiologic information collected from patients’ families indicated recent consumption of raw oysters. Two of the 3 cases occurred within a short time frame and were associated with eating local oysters harvested on the west coast of New Caledonia. The literature mentions few cases of V. vulnificus infection in the South Pacific. Cases described were isolated, rarely fatal, and involved infection through the skin (7–10). The V. vulnificus infections we report may be related to the emergence of a new clone or to changes in the climate or environmental conditions. New Caledonia experienced unusual weather conditions during the first half of 2008 (heavy rains and exceptionally high temperatures). These specific conditions may have favored higher sea surface temperatures, lower salinity, increased turbidity, and subsequent multiplication of V. vulnificus in seawater. A range of projects were implemented to train practitioners to recognize potential V. vulnificus infections. Local health authorities issued criteria for defining suspected cases of V. vulnificus infection and recommendations for early medical care of patients with clinical symptoms. Methods of detecting the bacterium in human and animal health laboratories were improved, particularly by the systematic use of selective media in the event of suspected clinical V. vulnificus infection and standardized reporting of V. vulnificus isolation. Preventive measures, such as improving microbial surveillance and warning consumers about risks associated with eating raw seafood, are essential to help reduce the risk for V. vulnificus–induced illness.

Roles of Flagellar Hook-Associated Proteins in Vibrio vulnificus Motility and Virulence

The bacterial flagellar structure can be divided into the basal body, the hook and the filament. Three minor components called hook associated proteins (HAP1, HAP2 and HAP3) form a junction between the hook and the filament (HAP1 and HAP3) and a capping structure at the distal end of flagellar filament (HAP2). Vibrio vulnificus is a halophilic pathogenic bacterium that is locomotive by means of a polar flagellum. From a V. vulnificus genome sequencing project, we obtained sequences of V. vulnificus flgK (Vv-flgK), flgL (Vv-flgL), and flaH (Vv-flaH) genes that encode HAP1, HAP3, and HAP2, respectively. To investigate roles of the HAP proteins, deletion mutants of the Vv-flgK, Vv-flgL and Vv-flaH were constructed. Electron microscopic analysis showed that the Vv-flgK or Vv-flgL mutant did not produce an intact polar flagellum while the Vv-flaH mutant produced a fragile flagellar structure. Western blot analysis against a major polar flagellin proposed that the null HAP1 and HAP3 mutations resulted in a failure of normal flagellar assembly since flagellins produced by the mutants were secreted out in the culture supernatants without long flagellar filaments. Motility was completely abolished by a single mutation in HAP1 or HAP3, and the HAP2 mutant showed a decreased motility. Also each of the mutants showed an impaired cytotoxicity and adherence to HeLa cell compared with the isogenic wild type strain. LD?? increased by 10- and 11-fold in the V. vulnificus HAP3 and HAP2 mutant, respectively. These results suggest that the HAP proteins play important roles in polar flagellation and the virulence of V. vulnificus.

Suppression and inactivation ofVibrio vulnificushemolysin in cirrhotic ascites, a humanex vivoexperimental system

To elucidate the mechanisms underlying the in vivo suppression and inactivation of Vibrio vulnificus hemolysin (VvhA), we used cirrhotic ascites fluid as a human ex vivo experimental system. VvhA expression was suppressed in proportion to the amount of cirrhotic ascites. The expression of vvhA in undiluted cirrhotic ascites could be suppressed further by the addition of glucose, a constituent of cirrhotic ascites. VvhA was readily inactivated in the presence of cirrhotic ascites by a cholesterol-mediated oligomerization and interaction with an undefined constituent(s) of cirrhotic ascites. These results indicate that the expression of vvhA can be suppressed and that any VvhA produced is inactivated by the constituents of cirrhotic ascites. Our results suggest that only a very small portion of the VvhA that is produced in human body fluids may actually contribute to the pathogenesis of V. vulnificus septicemia. It is suggested that cirrhotic ascites could be used as a human ex vivo experimental system for the studies on the in vivo expression and the significance of V. vulnificus virulence factors.

Effect of thecrpmutation on the utilization of transferrin-bound iron byVibrio vulnificus

Cyclic AMP-cAMP receptor protein (CRP) complex plays an essential role in the global regulation of Vibrio vulnificus virulence. We found that growth retardation of V. vulnificus caused by mutation of the crp gene encoding CRP was exacerbated under iron-limited conditions. Accordingly, we investigated the effect of crp mutation on the expression of the vulnibactin-mediated iron-uptake system and the ability of V. vulnificus to utilize transferrin-bound iron, and thus to grow in cirrhotic ascites, a human ex vivo system. The production of vulnibactin was suppressed, and the transcription of the vis and vuuA genes, which encode an enzyme required for vulnibactin synthesis and vulnibactin receptor protein, was also suppressed in the crp mutant. Moreover, the crp mutant could not utilize transferrin-bound iron, and its growth was severely suppressed both on transferrin-bound iron and in cirrhotic ascites. All the defects in the crp mutant were recovered by the in trans complementation of the wild-type crp gene. Putative CRP-binding sequences were found in the regulatory regions of the fur, vis and vuuA genes. These results indicate that crp mutation attenuates the ability to grow on transferrin-bound iron and in a human body fluid by down-regulating the vulnibactin-mediated iron-uptake system.

Essential role of an adenylate cyclase in regulating Vibrio vulnificus virulence

Vibrio vulnificus, a halophilic estuarine bacterium, causes a fatal septicemia and necrotizing wound infection. To investigate the role of cAMP in V. vulnificus virulence regulation, an in-frame deletion mutant of the cya gene encoding adenylate cyclase was constructed. The cya null mutation resulted in a pleiotropic change of virulence phenotypes. The production of hemolysin and protease, the motility, and the cytotoxicity were decreased by the cya mutation. The defects in the cya mutant were functionally complemented in trans by a plasmid carrying the wild type cya allele. The V. vulnificus cya mutant exhibited a 100-fold increase in LD 50 to mice. The result indicates that cAMP plays an essential role in the global regulation of V. vulnificus virulence.

Regulation of metalloprotease gene expression in Vibrio vulnificus by a Vibrio harveyi LuxR homologue.

Expression of the Vibrio vulnificus metalloprotease gene, vvp, was turned up rapidly when bacterial growth reached the late log phase. A similar pattern of expression has been found in the metalloprotease gene of Vibrio cholerae, and this has been shown to be regulated by a Vibrio harveyi LuxR-like transcriptional activator. To find out whether a LuxR homologue exists in V. vulnificus, a gene library of this organism was screened by colony hybridization using a probe derived from a sequence that is conserved in various luxR-like genes of vibrios. A gene containing a 618-bp open reading frame was identified and found to be identical to the smcR gene of V. vulnificus reported previously. An isogenic SmcR-deficient (RD) mutant was further constructed by an in vivo allelic exchange technique. This mutant exhibited an extremely low level of vvp transcription compared with that of the parent strain. On the other hand, the cytolysin gene, vvhA, was expressed at a higher level in the RD mutant than in the parent strain during the log phase of growth. These data suggested that SmcR might not only be a positive regulator of the protease gene but might also be involved in negative regulation of the cytolysin gene. Virulence of the RD mutant in either normal or iron-overloaded mice challenged by intraperitoneal injection was comparable to that of the parent strain, indicating that SmcR is not required for V. vulnificus virulence in mice.

Role of catechol siderophore synthesis in Vibrio vulnificus virulence.

We isolated a Vibrio vulnificus TnphoA mutant that was unable to produce catechol siderophores or to acquire iron from transferrin. This mutant showed reduced virulence in an infant mouse model. The TnphoA insertion was in an open reading frame designated venB. The venB gene cloned on a plasmid restored catechol production to the mutant. The deduced amino acid sequence of venB is 41% identical to the enzyme isochorismatase of Escherichia coli (EntB), an enzyme involved in the biosynthesis of the catechol siderophore enterobactin.

Immunogenicity of Vibrio vulnificus capsular polysaccharides and polysaccharide-protein conjugates

Opaque colony morphology has been correlated to Vibrio vulnificus virulence. However, the number of capsular serotypes expressed by virulent isolates is unknown. In an effort to produce anticapsule sera, capsular polysaccharide (CPS) from three opaque V. vulnificus strains was purified and characterized. Purified CPSs were acidic and contained considerable amounts of hexosamine and trace quantities of protein and nucleic acid. CPS purified from strain C7184 was poorly immunogenic for rabbits and mice, since repeated injection produced little detectable anticapsular antibody. To improve immunogenicity, CPS-protein conjugates were prepared from adipic acid hydrazide derivatives of CPS purified from each strain and carbodiimide as a coupling reagent. The immunogenicity of C7184 CPS was enhanced by conjugation to keyhole limpet hemocyanin, since injection into mice elicited production of anticapsular antibodies, the level of which was dependent on the dose and time since initial immunization. Injection of rabbits with CPS-protein conjugates also produced anticapsular antibodies. Staphylococcus aureus cells armed with each of the three anticapsular antibodies coagglutinated only the homologous opaque strain, indicating the existence of at least three capsular types. Further screening of 32 opaque and translucent V. vulnificus isolates revealed only three cross-reacting strains. These results suggest the presence of numerous V. vulnificus capsular types.