Vulnificus Infection Research Articles

Invitro and invivo antibacterial activity of tigecycline against Vibrio vulnificus.

The aim of this study is to investigate the role of tigecycline in Vibrio vulnificus infection. Eight randomly selected clinical V. vulnificus isolates were studied to obtain the minimal inhibitory concentrations (MICs) of minocycline, cefotaxime, and tigecycline, and the time-kill curves of tigecycline alone or in combination with other drugs. A peritonitis mouse model was used for the evaluation of the therapeutic efficacy of tigecycline alone or cefotaxime in combination with minocycline or tigecycline. The MIC of minocycline, cefotaxime, and tigecycline for eight clinical V. vulnificus isolates was 0.06-0.12μg/mL, 0.03-0.06μg/mL, and 0.03-0.06μg/mL, respectively. In time-killing studies, at the concentration of 1×MIC, the inhibitory effect of tigecycline persisted for 24hours in five of eight isolates. With 2×MIC and trough level, the inhibitory effect was noted in all isolates for 24hours. With the combination of minocycline plus cefotaxime and tigecycline plus cefotaxime at 1/2×MIC, the bactericidal effect was noted in 25% and 62.5% of eight isolates and synergism in 50% and 75% of isolates. With a low (1.25×105CFU/mL) inoculum, all infected mice survived with tigecycline alone, tigecycline plus cefotaxime, or minocycline plus cefotaxime on the 14th day. At the inoculum of 1.25×106CFU, the survival rate was 33.3% on the 14th day in the tigecycline plus cefotaxime-treated group, but none of the mice treated by tigecycline alone or minocycline plus cefotaxime survived (33.3% vs. 0%, p=0.01 by Fisher's exact test). Our invitro combination and animal studies indicate that tigecycline could be an option for the treatment of invasive V. vulnificus infections.

Gyrodactylus anguillae and Vibrio vulnificus infections affecting cultured eel, Anguilla anguilla

Gyrodactylus anguillae and Vibrio vulnificus are pathogens of critical significance in anguilliculture. Both agents were involved in mortalities affecting earthen pond cultured European eel, Anguilla anguilla, in Egypt during the period extended from June to September 2014. Moribund eels showed excessive slimy mucus and haemorrhages on the external body surface. All investigated eels were parasitized with the monogenean, Gyrodactylus anguillae. Majority of specimens 93.3 % were concomitantly found to be infected with V. vulnificus. No other parasitic or bacterial infections were noticed in the investigated fish samples. V. vulnificus was detected in Gyrodactylus anguillae homogenates collected from diseased fish. Bacterial isolates were confirmed by polymerase chain reaction (PCR). G. anguillae were demonstrated in skin and gills histopathological sections. Varieties of circulatory, proliferative as well as degenerative changes were noticed in haemopoietic tissues. It was concluded that G. anguillae presumably enhanced eels vulnerability to V. vulnificus infections through providing portals of entry as well as acting as mechanical vectors.

Electrotransfer of the tilapia piscidin 3 and tilapia piscidin 4 genes into skeletal muscle enhances the antibacterial and immunomodulatory functions of Oreochromis niloticus.

Tilapia piscidin 3 (TP3) and tilapia piscidin 4 (TP4) are antimicrobial peptides recently isolated from Oreochromis niloticus. We previously showed that synthetic TP3 and TP4 possessed antimicrobial activities. Here, we analyzed the bactericidal abilities and immunomodulatory properties of these AMPs following the electroporation of pCMV-GFP-TP3 or pCMV-GFP-TP4 plasmid into tilapia (O. niloticus) muscle and subsequent infection with Vibrio vulnificus or Streptococcus agalactiae. Prior overexpression of TP3 or TP4 in tilapia muscle tissues efficiently reduced bacterial numbers at 24 and 48 h after V. vulnificus infection and reduced bacterial numbers at 24 h after S. agalactiae infection compared to numbers in controls expressing pCMV-GFP (EGFP). Electroporation of pCMV-EGFP-TP3 (TP3) or pCMV-EGFP-TP4 (TP4) significantly increased expression of several immune-related genes in muscle (IL-1β (12 h, TP3), IL-8 (12 h, TP3), TGFβ (3 h, TP4), and IκB (48 h, TP3, TP4)) and decreased the expression of TLR5 (12 h and 24 h, TP3) after V. vulnificus infection. Following S. agalactiae infection, expression of the following genes was significantly decreased in muscle: IL-1β (12 h, TP3), IL-8 (12 h, TP3, TP4), TLR5 (3 h-24 h, TP3, TP4), TGFβ (3 h, TP4; 24 h, TP3, TP4), and IκB (3 h, TP3). These data suggest that TP3 and TP4 exert antimicrobial effects after overexpression in the O. niloticus muscle, and also play important roles in the regulation of immune-related gene expression.

Transgenic expression of omega-3 PUFA synthesis genes improves zebrafish survival during Vibrio vulnificus infection.

BackgroundHighly desaturated n-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are synthesized by desaturases and elongase. They exert hepatoprotective effects to prevent alcoholic fatty liver syndrome or cholestatic liver injury. However, it is unclear how n-3 PUFAs improve immune function in liver. Vibrio vulnificus, a gram-negative bacterial pathogen, causes high mortality of aquaculture fishes upon infection. Humans can become infected with V. vulnificus through open wounds or by eating raw seafood, and such infections may result in systemic septicemia. Moreover, patients with liver diseases are vulnerable to infection, and are more likely than healthy persons to present with liver inflammation following infection. This study quantified n-3 PUFAs and their anti-bacterial effects in Fadsd6 and Elvol5a transgenic zebrafish.ResultsTwo transgenic zebrafish strains with strong liver specific expression of Fadsd6 and Elvol5a (driven by the zebrafish Fabp10 promoter) were established using the Tol2 system. Synthesis of n-3 PUFAs in these strains were increased by 2.5-fold as compared to wild type (Wt) fish. The survival rate in 24 h following challenge with V. vulnificus was 20 % in Wt, but 70 % in the transgenic strains. In addition, the bacteria counts in transgenic fish strains were significantly decreased. The expression levels of pro-inflammatory genes, such as TNF-α, IL-1β, and NF-κB, were suppressed between 9 and 12 h after challenge. This study confirms the anti-bacterial function of n-3 PUFAs in a transgenic zebrafish model.ConclusionsFadsd6 and Elvol5a transgenic zebrafish are more resistant to V. vulnificus infection, and enhance survival by diminishing the attendant inflammatory response.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-015-0208-1) contains supplementary material, which is available to authorized users.

Safety and vaccine efficacy of an attenuated Vibrio vulnificus strain with deletions in major cytotoxin genes.

Vibrio vulnificus is a human pathogen causing a rapidly progressing fatal septicemia. We have previously reported that a V. vulnificus large toxin RtxA1 causes programmed necrotic cell death through calcium-mediated mitochondrial dysfunction. Here we developed a live attenuated vaccine strain (CMM781) having deletions in three genes encoding major virulence factors: RTX cytotoxin (rtxA1), hemolysin/cytolysin (vvhA) and metalloprotease (vvpE) of a clinical isolate strain CMCP6. The CMM781 strain showed significant attenuation in cytotoxicity and mouse lethality. The safety of CMM781 was also confirmed by measuring the transepithelial electric resistance of Caco-2 cell monolayers. Intragastric immunization of mice with the live attenuated V. vulnificus strain resulted in induction of systemic and mucosal antibodies specific to the pathogen. Moreover, the vaccinated mice were protected from challenges with high doses of the virulent strain through various injection routes. These results suggest that CMM781 appears to be a safe and effective vaccine candidate that would provide significant protection against V. vulnificus infection.

Vibrio vulnificus VvpE Stimulates IL-1β Production by the Hypomethylation of the IL-1β Promoter and NF-κB Activation via Lipid Raft-Dependent ANXA2 Recruitment and Reactive Oxygen Species Signaling in Intestinal Epithelial Cells.

An inflammatory response is a hallmark of necrosis evoked by bacterial pathogens. Vibrio vulnificus, VvpE, is an elastase that is responsible for tissue necrosis and inflammation; however, the molecular mechanism by which it regulates host cell death has not been characterized. In the present study, we investigate the cellular mechanism of VvpE with regard to host cell death and the inflammatory response of human intestinal epithelial (INT-407) cells. The recombinant protein (r)VvpE (50 pg/ml) caused cytotoxicity mainly via necrosis coupled with IL-1β production. The necrotic cell death induced by rVvpE is highly susceptible to the knockdown of annexin A (ANXA)2 and the sequestration of membrane cholesterol. We found that rVvpE induces the recruitment of NADPH oxidase 2 and neutrophil cytosolic factor 1 into membrane lipid rafts coupled with ANXA2 to facilitate the production of reactive oxygen species (ROS). The bacterial signaling of rVvpE through ROS production is uniquely mediated by the phosphorylation of redox-sensitive transcription factor NF-κB. The silencing of NF-κB inhibited IL-1β production during necrosis. rVvpE induced hypomethylation and region-specific transcriptional occupancy by NF-κB in the IL-1β promoter and has the ability to induce pyroptosis via NOD-, LRR-, and pyrin domain-containing 3 inflammasome. In a mouse model of V. vulnificus infection, the mutation of the vvpE gene from V. vulnificus negated the proinflammatory responses and maintained the physiological levels of the proliferation and migration of enterocytes. These results demonstrate that VvpE induces the hypomethylation of the IL-1β promoter and the transcriptional regulation of NF-κB through lipid raft-dependent ANXA2 recruitment and ROS signaling to promote IL-1β production in intestinal epithelial cells.

To notify or not to notify: decision aid for policy makers on whether to make an infectious disease mandatorily notifiable.

Mandatory notification can be a useful tool to support infectious disease prevention and control. Guidelines are needed to help policymakers decide whether mandatory notification of an infectious disease is appropriate. We developed a decision aid, based on a range of criteria previously used in the Netherlands or in other regions to help decide whether to make a disease notifiable. Criteria were categorised as being effective, feasible and necessary with regard to the relevance of mandatory notification. Expert panels piloted the decision aid. Here we illustrate its use for three diseases (Vibrio vulnificus infection, chronic Q fever and dengue fever) for which mandatory notification was requested. For dengue fever, the expert panel advised mandatory notification; for V. vulnificus infection and chronic Q fever, the expert panel concluded that mandatory notification was not (yet) justified. Use of the decision aid led to a structured, transparent decision making process and a thorough assessment of the advantages and disadvantages of mandatory notification of these diseases. It also helped identify knowledge gaps that required further research before a decision could be made. We therefore recommend use of this aid for public health policy making.

An Enriched European Eel Transcriptome Sheds Light upon Host-Pathogen Interactions with Vibrio vulnificus.

Infectious diseases are one of the principal bottlenecks for the European eel recovery. The aim of this study was to develop a new molecular tool to be used in host-pathogen interaction experiments in the eel. To this end, we first stimulated adult eels with different pathogen-associated molecular patterns (PAMPs), extracted RNA from the immune-related tissues and sequenced the transcriptome. We obtained more than 2x106 reads that were assembled and annotated into 45,067 new descriptions with a notable representation of novel transcripts related with pathogen recognition, signal transduction and the immune response. Then, we designed a DNA-microarray that was used to analyze the early immune response against Vibrio vulnificus, a septicemic pathogen that uses the gills as the portal of entry into the blood, as well as the role of the main toxin of this species (RtxA13) on this early interaction. The gill transcriptomic profiles obtained after bath infecting eels with the wild type strain or with a mutant deficient in rtxA13 were analyzed and compared. Results demonstrate that eels react rapidly and locally against the pathogen and that this immune-response is rtxA13-dependent as transcripts related with cell destruction were highly up-regulated only in the gills from eels infected with the wild-type strain. Furthermore, significant differences in the immune response against the wild type and the mutant strain also suggest that host survival after V. vulnificus infection could depend on an efficient local phagocytic activity. Finally, we also found evidence of the presence of an interbranchial lymphoid tissue in European eel gills although further experiments will be necessary to identify such tissue.

Characterization of Prohibitin 1 as a Host Partner of Vibrio vulnificus RtxA1 Toxin.

RtxA1 toxin, which results in cytoskeletal rearrangement, contact cytotoxicity, hemolysis, tissue invasion, and lethality in mice, is the most potent cytotoxic virulence factor of Vibrio vulnificus. Bioinformatics analysis of rtxA1 predicted 4 functional domains that presumably performed discrete functions during host cell killing. V. vulnificus RtxA1 has a unique domain designated as RtxA1-D2, corresponding to amino acids 1951-2574, which is absent in Vibrio cholerae multifunctional-autoprocessing repeats-in-toxin, suggesting that this domain confers specific biological functions to V. vulnificus RtxA1. HeLa cells expressing green fluorescent protein-RtxA1-D2 became round and lost their viability. A yeast 2-hybrid system identified prohibitin (PHB) 1 as the host partner of RtxA1-D2. The specific interaction of RtxA1-D2 with PHB1 was confirmed by performing immunoprecipitation. Interestingly, V. vulnificus RtxA1 up-regulated PHB1 expression on the cytoplasmic membrane of host cells. Extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways were confirmed as being important in the up-regulation of PHB1 by using inhibitors. Down-regulation of PHB1 by small interfering RNAs decreased the cytotoxicity of RtxA1-D2 against HeLa cells. The pretreatment of an anti-PHB1 antibody impaired the cytotoxicity of V. vulnificus RtxA1. These results suggest that the involvement PHB1 in the RtxA1 cytotoxicity has significant implications for the pathogenesis of V. vulnificus infections.

Antibiotic use for Vibrio infections: important insights from surveillance data

BackgroundThere is a paucity of data on the in vivo efficacy of antibiotics for lethal Vibrio species. Analyses of long-term surveillance datasets may provide insights into use of antibiotics to decrease mortality.MethodsThe United States Centers for Disease Control and Prevention (CDC) Cholera and Other Vibrio Illness Surveillance (COVIS) dataset from 1990 to 2010, with 8056 records, was analysed to ascertain trends in antibiotics use and mortality.ResultsTwo-thirds of patients (5243) were prescribed antibiotics - quinolones (56.1 %), cephalosporins (24.1 %), tetracyclines (23.5 %), and penicillins (15.4 %). Considering all Vibrio species, the only class of antibiotic associated with reduced odds of mortality was quinolone (odds ratio 0.56, 95 % CI 0.46-0.67). Patients with V. vulnificus treated according to CDC recommendations had lower mortality (quinolone alone: 16.7 %, 95 % CI 10.2-26.1; tetracycline plus cephalosporin: 21.7 %, 16.8-27.5; no antibiotic: 51.1 %, 45.6-56.7; each p < 0.001). Cephalosporin alone was associated with higher mortality (36.8 %, 28.2-46.3). For V. cholerae non-O1, non-O139, mortality rates were lower for quinolone (0 %, 0–2.0) or tetracycline (4.3 %, 1.2-14.5) compared to no antibiotic (9.3 %, 6.4-13.3). For all Vibrio species, mortality rates increased with number of antibiotics in the treatment regimen (p < 0.001). Treatment regimens that included quinolone were associated with lower mortality rates regardless of the number of antibiotics used. The main clinical syndromes of patients with V. vulnificus infection were septicaemia (53.1 %) and wound infections (30.6 %). Mortality among V. vulnificus patients with septicaemia was significantly higher than for other clinical syndromes (p < 0.001). In a multivariate regression model, mortality in cases with V. vulnificus was associated with presence of pre-existing conditions (ORs ranged from 4.52 to 10.30), septicaemia (OR 2.64, 95 % CI 1.92-3.63) and no antibiotic treatment (OR 7.89, 95 % CI 3.94-15.80).ConclusionIn view of the lack of randomized control trials, surveillance data may inform treatment decisions for potentially lethal Vibriosis. Considering all Vibrio species, use of quinolones is associated with lower mortality and penicillin alone is not particularly effective. For the most lethal species, V. vulnificus, treatment that includes either quinolone or tetracycline is associated with lower mortality than cephalosporin alone. We recommend treating patients who present with a clinical syndrome suggestive of V. vulnificus infection with a treatment regimen that includes a quinolone.

Vibrio vulnificus VvpE inhibits mucin 2 expression by hypermethylation via lipid raft-mediated ROS signaling in intestinal epithelial cells.

Mucin is an important physical barrier against enteric pathogens. VvpE is an elastase encoded by Gram-negative bacterium Vibrio vulnificus; however, the functional role of VvpE in intestinal mucin (Muc) production is yet to be elucidated. The recombinant protein (r) VvpE significantly reduced the level of Muc2 in human mucus-secreting HT29-MTX cells. The repression of Muc2 induced by rVvpE was highly susceptible to the knockdown of intelectin-1b (ITLN) and sequestration of cholesterol by methyl-β-cyclodextrin. We found that rVvpE induces the recruitment of NADPH oxidase 2 and neutrophil cytosolic factor 1 into the membrane lipid rafts coupled with ITLN to facilitate the production of reactive oxygen species (ROS). The bacterial signaling of rVvpE through ROS production is uniquely mediated by the phosphorylation of ERK, which was downregulated by the silencing of the PKCδ. Moreover, rVvpE induced region-specific methylation in the Muc2 promoter to promote the transcriptional repression of Muc2. In two mouse models of V. vulnificus infection, the mutation of the vvpE gene from V. vulnificus exhibited an increased survival rate and maintained the level of Muc2 expression in intestine. These results demonstrate that VvpE inhibits Muc2 expression by hypermethylation via lipid raft-mediated ROS signaling in the intestinal epithelial cells.

Analysis of Vibrio vulnificus Infection Risk When Consuming Depurated Raw Oysters

A beta Poisson dose-response model for Vibrio vulnificus food poisoning cases leading to septicemia was used to evaluate the effect of depuration at 15°C on the estimated health risk associated with raw oyster consumption. Statistical variability sources included V. vulnificus level at harvest, time and temperature during harvest and transportation to processing plants, decimal reductions (SV) observed during experimental circulation depuration treatments, refrigerated storage time before consumption, oyster size, and number of oysters per consumption event. Although reaching nondetectable V. vulnificus levels (<30 most probable number per gram) throughout the year and a 3.52 SV were estimated not possible at the 95% confidence level, depuration for 1, 2, 3, and 4 days would reduce the warm season (June through September) risk from 2,669 cases to 558, 93, 38, and 47 cases per 100 million consumption events, respectively. At the 95% confidence level, 47 and 16 h of depuration would reduce the warm and transition season (April through May and October through November) risk, respectively, to 100 cases per 100 million consumption events, which is assumed to be an acceptable risk; 1 case per 100 million events would be the risk when consuming untreated raw oysters in the cold season (December through March).

Identification of Novel Inflammatory Cytokines and Contribution of Keratinocyte-Derived Chemokine to Inflammation in Response to Vibrio vulnificus Infection in Mice.

Currently, only tumor necrosis factor alpha (TNF-α) and interleukin family cytokines have been found to be elicited in Vibrio vulnificus (V. vulnificus)-infected animal models and humans. However, multiple other cytokines are also involved in the immune and inflammatory responses to foreign microorganism infection. Antibody array technology, unlike traditional enzyme-linked immunosorbent assay (ELISA), is able to detect multiple cytokines at one time. Therefore, in this study, we examined the proinflammatory cytokine profile in the serum and liver homogenate samples of bacterial-infected mice using antibody array technology. We identified nine novel cytokines in response to V. vulnificus infection in mice. We found that keratinocyte-derived chemokine (KC) was the most elevated cytokine and demonstrated that KC played a very important role in the V. vulnificus infection-elicited inflammatory response in mice, as evidenced by the fact that the blocking of KC by anti-KC antibody reduced hepatic injury in vivo and that KC induced by V. vulnificus infection in AML-12 cells chemoattracted neutrophils. Our findings implicate that KC may serve as a novel diagnostic biomarker and a possible therapeutic target for V. vulnificus infection.

PH level as a marker for predicting death among patients with Vibrio vulnificus infection, South Korea, 2000-2011.

Vibrio vulnificus infection can progress to necrotizing fasciitis and death. To improve the likelihood of patient survival, an early prognosis of patient outcome is clinically important for emergency/trauma department doctors. To identify an accurate and simple predictor for death among V. vulnificus-infected persons, we reviewed clinical data for 34 patients at a hospital in South Korea during 2000-2011; of the patients, 16 (47%) died and 18 (53%) survived. For nonsurvivors, median time from hospital admission to death was 15 h (range 4-70). For predicting death, the areas under the receiver operating characteristic curves of the Acute Physiology and Chronic Health Evaluation (APACHE) II score and initial pH were 0.746 and 0.972, respectively (p = 0.005). An optimal cutoff pH of <7.35 had a sensitivity of 100% and specificity of 83%. Compared with the APACHE II score, the initial arterial blood pH level in V. vulnificus-infected patients was a more accurate predictive marker for death.

Vibrio vulnificus detected in the spleen leads to fatal outcome in a mouse oral infection model

Vibrio vulnificus causes rapid disseminating septicemia by oral infection in infected individuals who have an underlying disease, especially chronic liver diseases. Although the elucidation of specific risk factors for V. vulnificus infection in patients with liver diseases is of urgent importance, no appropriate experimental animal model that mimics the liver diseases in this bacterial infection has been available so far. To discover these risk factors, we generated a liver disordered mouse by performing bile duct ligation (BDL). Hepatitis developed in the BDL mice; however, this did not affect mortality in mice after orogastric administration of V. vulnificus, suggesting that the liver disorders caused by the BDL were not risk factors for V. vulnificus septicemia. When the dead and surviving mice were compared, V. vulnificus could be detected from the spleen only in the dead group. Furthermore, significantly higher numbers of V. vulnificus were detected from the intestines in the dead group than in the surviving group ( P < 0.001). These findings suggested that proliferation of the challenge inoculum in the intestine was needed for the oral infection with V. vulnificus, and that the elimination of V. vulnificus in the liver and/or spleen plays a critical role in survival of the host.

Signature-tagged mutagenesis of Vibrio vulnificus.

Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged mutagenesis (STM) was applied to identify the virulence genes of V. vulnificus. Using STM, 6,480 mutants in total were constructed and divided into 81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool was intraperitoneally injected into iron-overloaded mice, and in vivo surviving mutants were collected from blood samples from the heart (OUTPUT pools). From the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes, were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than 1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000. Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase, UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a transposon, they were protected against V. vulnificus infection. In this study, we demonstrated that the STM method can be used to search for the virulence genes of V. vulnificus.

Necrotizing fasciitis and sepsis caused by Vibrio vulnificus and Klebsiella pneumoniae in diabetic patients.

Vibrio vulnificus related necrotizing fasciitis is a fatal, rapidly progressive soft-tissue infection. Necrotizing fasciitis caused by Klebsiella pneumoniae is rare, which is indistinguishable from V. vulnificus infection in the emergency room. The purpose of this study was to compare the clinical characteristics and outcome between these two pathogens in diabetic patients. Thirty diabetic patients were retrospectively reviewed over an 8-year period. Necrotizing fasciitis caused by V. vulnificus was found in 19 patients and by K. pneumoniae in 11 patients. The demographic, clinical, and laboratory characteristics, and the outcome between diabetic patients with V. vulnificus and K. pneumoniae infections were compared. Two patients in the V. vulnificus group (10.5%) and three patients in the K. pneumoniae group (27.3%) died. Fourteen patients in the V. vulnificus group (73.6%) had a history of exposure to seawater or raw seafood, and eight patients in the K. pneumoniae group (72.8%) had abrasions or chronic ulcers over the site of infection. We found that the time interval between onset of illness and presentation to the hospital was significantly shorter in the V. vulnificus group than in the K. pneumoniae group (2.47 days vs. 5.45 days, p < 0.001). The exposure history and the time from exposure to hospital presentation with severe sepsis syndromes should alert clinicians to distinguish between necrotizing soft-tissue infections with V. vulnificus (contact with seawater or raw seafood) and K. pneumoniae (abrasions or chronic ulcers) in diabetic patients. Infection with V. vulnificus progresses more rapidly than infection with K. pneumoniae during the initial clinical course.

Zebrafish fed on recombinant Artemia expressing epinecidin-1 exhibit increased survival and altered expression of immunomodulatory genes upon Vibrio vulnificus infection

Artemia has been used extensively in aquaculture as fodder for larval fish, shrimp, and shellfish. Epinecidin-1, an antimicrobial peptide, was isolated from grouper (Epinephelus coioides) in 2005. Epinecidin-1 has been previously reported to possess antimicrobial activity against several Gram-positive and Gram-negative bacterial species, including Staphylococcus coagulase, Pseudomonas aeruginosa, Streptococcus pyogenes, and Vibrio vulnificus. In this study, we used electroporation to introduce plasmid DNA encoding a green fluorescent protein (EGFP)-epinecidin-1 fusion protein under the control of the cytomegalovirus (CMV) promoter into decapsulated Artemia cysts. Optimization of various properties (including cyst weight (0.2 g), plasmid concentration (50 μg/100 μl), and pulse voltage (150 V), length (10 ms), and number (2)) resulted in a hatching rate of 41.15%, a transfection efficiency of 49.81%, and a fluorescence intensity (A.U.) of 47.46. The expression of EGFP-epinecidin-1 was first detected by quantitative RT-PCR at 120 h post-electroporation, and protein was identified by Western blot at the same time. Furthermore, the EGFP-epinecidin-1 protein inhibited V. vulnificus (204) growth, as demonstrated by zone of inhibition studies. Zebrafish fed on transgenic Artemia expressing CMV-gfp-epi combined with commercial fodder were more resistant to infection by V. vulnificus (204): survival rate was enhanced by over 70% at 7, 14, and 21 days post-infection, and bacterial numbers in the liver and intestine were reduced. In addition, feeding of transgenic Artemia to zebrafish affected the immunomodulatory response to V. vulnificus (204) infection; expression of immune-responsive genes, including hepcidin and defbl2, was altered, as shown by qPCR. These findings suggest that feeding transgenic Artemia expressing CMV-gfp-epi to larval fish has antimicrobial effects, without the drawbacks of introducing drug residues or inducing bacterial drug resistance.

Emergency treatment of 16 patients with necrotizing fasciitis caused by Vibrio vulnificus infection complicated with septic shock

Emergency treatment of 16 patients with necrotizing fasciitis caused by Vibrio vulnificus infection complicated with septic shock

Dietary supplementation of Bacillus subtilis and fructooligosaccharide enhance the growth, non-specific immunity of juvenile ovate pompano, Trachinotus ovatus and its disease resistance against Vibrio vulnificus

A feeding trial was conducted to investigate the effects of dietary administration of probiotic Bacillus subtilis and prebiotic fructooligosaccharide (FOS) on growth performance, immune responses and disease resistance of juvenile ovate pompano, Trachinotus ovatus. One thousand six hundred and twenty individuals (initial body weight: 10.32 ± 0.46 g, mean ± S.E) were fed nine practical diets according to a 3 × 3 factorial design: the basal diet as the control diet supplemented with three levels of B. subtilis (0, 1.05 × 107 or 5.62 × 107 CFU g−1 diet), crossed with 0, 0.2% or 0.4% FOS. After an 8-week feeding experimental period, six fish per cage were sampled for immunity determination. Then 18 fish of each cage left were challenged by Vibrio vulnificus. The results showed that fish fed with 5.62 × 107 CFU B. subtilis g−1 in combination with 0.2% FOS produced the highest specific growth rate, and were significantly higher than the groups fed with 0 and 0.2% FOS without B. subtilis supplementation (P < 0.05). Feed efficiency ratio significantly increased with the increasing doses of dietary FOS without B. subtilis added (P < 0.05). The immune assay showed that fish fed with the control diet produced the lowest respiratory burst activity and was significantly different from the groups fed the diets containing 0.2% FOS at each B. subtilis level and containing 0.4% FOS single (P < 0.05). Phagocytic activity was significantly decreased with the increasing doses of dietary B. subtilis at 0.4% FOS level (P < 0.05). Alternative complement pathway activity of the fish fed with 0.2% FOS single was significantly lower than those fed with 5.62 × 107 CFU B. subtilis g−1 diet supplemented at each FOS level (P < 0.05). Fish fed with the control diet had the lowest lysozyme activity, and were significantly different from those fed with 0.2 or 0.4% FOS at 1.05 and 5.62 × 107 CFU B. subtilis g−1 diet level. Moreover, fish fed with diets supplemented with 0.2% and 0.4% FOS at each B. subtilis level had notably lower cumulative mortality after 10 days following V. vulnificus infection (P < 0.05). Under the experimental conditions, dietary B. subtilis and FOS had a significant interaction on enhancing the immune responses and disease resistance of juvenile ovate pompano (P < 0.05).