Therapeutic potential of otilonium bromide against Vibrio vulnificus.

New drugs are urgently required for the treatment of infections due to an increasing number of new strains of diseases-causing pathogens and antibiotic-resistant bacteria. A library of drugs approved by Food and Drug Administration was screened for efficacy against Vibrio vulnificus using antimicrobial assays. We found that otilonium bromide showed potent antimicrobial activity against V.vulnificus and had a synergistic effect in combination with antibiotics. Field emission transmission electron microscope images revealed that otilonium bromide caused cell division defects in V.vulnificus. Moreover, it significantly inhibited V.vulnificus swarming motility and adhesion to host cells at concentrations lower than the minimum inhibitory concentration. To investigate its inhibitory action mechanisms, we examined the effect of otilonium bromide on the expression levels of several proteins crucial for V.vulnificus growth, motility, and adhesion. It decreased the protein expression levels of cAMP receptor protein and flagellin B, but not HlyU or OmpU. In addition, otilonium bromide significantly decreased the expression levels of outer membrane protein TolCV1, thus inhibiting RtxA1 toxin secretion and substantially reducing V.vulnificus cytotoxicity to host cells. Collectively, these findings suggest that otilonium bromide may be considered as a promising candidate for treating V.vulnificus infections.

Nanosphere Loaded with Curcumin Inhibits the Gastrointestinal Cell Death Signaling Pathway Induced by the Foodborne Pathogen Vibrio vulnificus.

Curcumin, a hydrophobic polyphenol of turmeric, has a variety of biological functions as a herbal supplement, but its poor gastric absorption rate is one of the major factors limiting its oral bioavailability. In the present study, we have investigated the functional role of a nanosphere loaded with curcumin (CN) during host cell death elicited by the Gram-negative bacterium V. vulnificus in human gastrointestinal epithelial HT-29 cells and an ileal-ligated mouse model. The recombinant protein (r) VvhA produced by V. vulnificus significantly reduced the viability of HT-29 cells. The cytotoxic effect of rVvhA was restored upon a treatment with CN (100 ng/mL), which had shown 1000-fold higher anti-apoptotic efficacy than curcumin. CN inhibited the phosphorylation of c-Src and PKC mediated by intracellular ROS responsible for the distinctive activation of the MAPKs in rVvhA-treated HT-29 cells. Interestingly, CN significantly restored the expression of Bax, Bcl-2, and cleaved caspase-3 as regulated by the phosphorylation of NF-κB. In mouse models of V. vulnificus infection, treatment with CN had a blocking effect that elevated the levels of TUNEL-positive DNA fragmentation and apoptosis-related proteins. These results indicate that CN is a functional agent that manipulates the V. vulnificus VvhA signaling pathway responsible for gastrointestinal cell death.

Vibrio vulnificus meningoencephalitis in a patient with thalassemia and a splenectomy.

Vibrio vulnificus usually causes wound infection, gastroenteritis, and septicemia. However, it is a rare conditional pathogen causing meningoencephalitis. We report a case of a young, immunocompromised man presenting with severe sepsis after exposure to sea water and consumption of seafood. The patient subsequently developed meningoencephalitis, and Vibrio vulnificus was isolated from his blood culture. The sequence was confirmed by Next-generation sequencing of a sample of cerebrospinal fluid, as well as from a bacteria culture. After the pathogen was detected, the patient was treated with ceftriaxone, doxycycline, and moxifloxacin for 6 weeks, which controlled his infection. In this case, we acquired his clinical and dynamic MRI presentations, which were never reported. Physicians should consider Vibrio vulnificus infections when they see a similar clinical course, brain CT and MRI findings, susceptibility factors and recent seafood ingestion or exposure to seawater. Due to high mortality, the early diagnosis and treatment of Vibrio vulnificus infections are crucial. Next-generation sequencing was found to be useful for diagnosis.

Identification and Validation of an Antivirulence Agent Targeting HlyU-Regulated Virulence in Vibrio vulnificus.

Antimicrobial resistance (AMR) in pathogens is the result of indiscriminate use of antibiotics and consequent metabolic/genetic modulation to evolve survival strategies and clonal-selection in AMR strains. As an alternative to antibiotic treatment, antivirulence strategies are being developed, not only to combat bacterial pathogenesis, but also to avoid emerging antibiotic resistance. Vibrio vulnificus is a foodborne pathogen that causes gastroenteritis, necrotizing wound infections, and sepsis with a high rate of mortality. Here, we developed an inhibitor-screening reporter platform to target HlyU, a master transcriptional regulator of virulence factors in V. vulnificus by assessing rtxA1 transcription under its control. The inhibitor-screening platform includes wild type and ΔhlyU mutant strains of V. vulnificus harboring the reporter construct P rtxA1::luxCDABE for desired luminescence signal detection and control background luminescence, respectively. Using the inhibitor-screening platform, we identified a small molecule, fursultiamine hydrochloride (FTH), that inhibits the transcription of the highly invasive repeat-in-toxin (rtxA1) and hemolysin (vvhA) along with other HlyU regulated virulence genes. FTH has no cytotoxic effects on either host cells or pathogen at the tested concentrations. FTH rescues host cells from the necrotic cell-death induced by RtxA1 and decreases the hemolytic activity under in vitro conditions. The most important point is that FTH treatment does not induce the antivirulence resistance. Current study validated the antivirulence strategy targeting the HlyU virulence transcription factor and toxin-network of V. vulnificus and demonstrated that FTH, exhibits a potential to inhibit the pathogenesis of deadly, opportunistic human pathogen, V. vulnificus without inducing AMR.

In vitro and in vivo antibacterial activity of tigecycline against Vibrio vulnificus.

BACKGROUND/PURPOSE: The aim of this study is to investigate the role of tigecycline in Vibrio vulnificus infection. METHODS: 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. RESULTS: The MIC of minocycline, cefotaxime, and tigecycline for eight clinical V. vulnificus isolates was 0.06-0.12 µg/mL, 0.03-0.06 µg/mL, and 0.03-0.06 µg/mL, respectively. In time-killing studies, at the concentration of 1 × MIC, the inhibitory effect of tigecycline persisted for 24 hours in five of eight isolates. With 2 × MIC and trough level, the inhibitory effect was noted in all isolates for 24 hours. 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 × 105 CFU/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 × 106 CFU, 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). CONCLUSION: Our in vitro combination and animal studies indicate that tigecycline could be an option for the treatment of invasive V. vulnificus infections.

Efficacy of Ceftriaxone, Cefepime, Doxycycline, Ciprofloxacin, and Combination Therapy for Vibrio vulnificus Foodborne Septicemia.

Foodborne Vibrio vulnificus infections are associated with higher rates of sepsis and mortality than wound infections; however, antibiotic efficacy studies have not been performed in foodborne infection models. The efficacies of ceftriaxone, cefepime, doxycycline, ciprofloxacin, and combination therapy were assessed in V. vulnificus intestinal infection in mice in order to model foodborne infections. In accordance with prior studies of cefotaxime, cefepime was synergistic with doxycycline and ciprofloxacin in vitro; combination therapy significantly decreased bacterial growth, by ≥2 log10 units, from that with antibiotic monotherapy (P < 0.01). In vivo, survival rates in the ceftriaxone (50%), doxycycline (79%), and ciprofloxacin (80%) groups were significantly higher than those in the control group (0%) (P < 0.0001). Survival was significantly higher with ceftriaxone-doxycycline (91%) or ceftriaxone-ciprofloxacin (100%) therapy than with ceftriaxone (50%) (P ≤ 0.05). Survival with cefepime-doxycycline (96%) or cefepime-ciprofloxacin (90%) therapy was significantly higher than that with cefepime alone (20%) (P < 0.001). There was no difference in survival between the combination therapy groups. Thus, we conclude that combination therapy was the most effective treatment for foodborne V. vulnificus septicemia. In a septic patient with a recent ingestion of raw seafood, cefepime in combination with doxycycline or ciprofloxacin should be initiated for coverage of resistant Gram-negative organisms and V. vulnificus pending a microbiological diagnosis. Once a diagnosis of foodborne V. vulnificus septicemia is established, treatment can safely transition to ceftriaxone in combination with doxycycline or ciprofloxacin.

Transcriptome analysis of the effect of polyunsaturated fatty acids against Vibrio vulnificus infection in Oreochromis niloticus.

Vibrio vulnificus infection causes severe economic losses in Oreochromis niloticus aquaculture by inducing pro-inflammatory cytokines, that lead to inflammation and mortality. Omega-3 fatty acids, such as Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA), have been reported for their anti-inflammatory and antibacterial abilities in murine and zebrafish models. However, the anti-inflammatory and antibacterial functions of DHA and EPA in commercial aquaculture organisms such as Oreochromis niloticus remain unknown. The present study demonstrates antibacterial function and transcriptional modulation of inflammation-associated genes by DHA and EPA in Vibrio vulnificus infection in Oreochromis niloticus fish models. The administration of EPA or DHA improved the Oreochromis niloticus survival rate against Vibrio vulnificus infection. The induction of proinflammatory cytokines, Interleukin (IL)-1ß, IL-6, Tumor necrosis factor (TNF)-α, and Toll-like receptor (TLR)-2 by Vibrio vulnificus was suppressed in fish that were administered DHA. Bacterial membrane disruption and the killing of Vibrio vulnificus by EPA and DHA was observed using SEM, TEM, and cytoplasm leakage studies. In silico analysis of the transcription profile in Ingenuity Pathway Analysis software showed that DHA may enhance anti-Vibrio vulnificus activity in Oreochromis niloticus via the activation of peroxisome proliferator-activated receptor α (PPARα) to inhibit nuclear factor kappa B and suppress hepatocyte nuclear factor 4 α (HNF4α). In summary, the results of the present study demonstrated that DHA and EPA reduce the severity of Vibrio vulnificus infection and increase the survival rate of Oreochromis niloticus.

Vibrio Vulnificus Necrotizing Fasciitis Associated with Acupuncture.

Necrotizing fasciitis is a severe life-threatening infection of the deep subcutaneous tissues and fascia. Infection with Vibrio vulnificus, a halophilic Gram-negative bacillus found worldwide in warm coastal waters, can lead to severe complications, particularly among patients with chronic liver diseases. We herein present an unusual case of necrotizing fasciitis caused by V. vulnificus triggered by acupuncture needle insertion. The patient, who suffered from diabetes mellitus and nonalcoholic fatty liver disease and worked at a fish hatchery, denied any injury prior to acupuncture. This is the first ever reported case of V. vulnificus infection triggered by acupuncture needle insertion, clearly emphasizing the potential hazards of the prolonged survival of V. vulnificus on the skin. The potential infectious complications of acupuncture needle insertion are discussed.

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 × 10(7) or 5.62 × 10(7) 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 × 10(7) 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 × 10(7) 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 × 10(7) 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).

Role of periplasmic binding proteins, FatB and VatD, in the vulnibactin utilization system of Vibrio vulnificus M2799.

Vibrio vulnificus, an opportunistic marine bacterium that causes a serious, often fatal, infection in humans, requires iron for its pathogenesis. This bacterium uses iron from the environment via the vulnibactin-mediated-iron-uptake system. In this study, we constructed the deletion mutants of the genes encoding the proteins involved in the vulnibactin-mediated-iron-uptake system, isochorismate synthase (ICS), vulnibactin utilization protein (VuuB), periplasmic ferric-vulnibactin binding protein (FatB), and ferric-vulnibactin receptor protein (VuuA). The Δics and ΔvuuA mutants were unable to grow under low-iron concentration conditions compared with the isogenic wild-type, indicating that the involvement of ICS in the vulnibactin biosynthesis pathway and uptake of ferric-vulnibactin through the VuuA receptor protein are essential for V. vulnificus M2799 growth under low-iron concentration conditions. Similar growth impairment was also observed in ΔfatB, with growth recovery of this mutant observed 6 h after the beginning of the culture. These results indicate that there must be other periplasmic ferric-vulnibactin binding proteins in V. vulnificus M2799 that complement the defective fatB gene. Complementary growth studies confirmed that VatD protein, which functions as a periplasmic ferric-aerobactin binding protein, was found to participate in the ferric-vulnibactin uptake system in the absence of FatB. Furthermore, the expression of ics, vuuB, fatB, vuuA, and vatD genes was found to be regulated by iron and the ferric uptake regulator.

Protective Effect of Ginsan Against Vibrio vulnificus Infection

Ginsan, a botanic polysaccharide extracted from Panax ginseng, has recently been reported to modulate mucosal immune response. In this study, we investigated the protective effect of Ginsan against fatal Vibrio vulnificus mucosal infection. A lethal dose of V. vulnificus (1.0 x 106 CFU/mouse) was nasally inoculated to mice. The bacterial count in the nasal associated lymphoid tissue (NALT) of the mouse was significantly reduced in the Ginsan-treated group. The Ginsan-treated group showed improved survival compared to the control group (100% vs 18%). To elucidate the effect of Ginsan on modulating host immune response, cytokine mRNA expressions involved in mediating inflammation were determined by semiquantitative RT-PCR in the NALTs of the infected mice. Most of the cytokine mRNAs were similarly expressed as the control group. However, COX-1 mRNA expression level was higher in Ginsan-treated group compared to the control group. The protective effect of Ginsan was antagonized by treating with a specific COX-1 inhibitor, SC-560. Thus, these data suggest that the protective effect of Ginsan against V. vulnificus infection is partly mediated by modulating COX-1 expression.

The Extraction of Vibrio vulnificus Lipopolysaccharide by the Phenol-Chloroform-Petroleum Ether Method and Biological Activity of the LPS / 대한피부과학회지

BACKGROUND: Vibrio vulnificus is a pathogenic, marine, halophilic, gram-negative bacillus which causes fulminant infecticn in humans through the ingestion of raw seafood or skin wounds. V. vulnificus produces seveal kinds of virulent factors including cytolysin, endotoxin, exoenzymes, and siderphores. Among these, the lipopolysaccharide(LPS) of V. vulnificus has recently been purified, but the biological activity of this endotoxin is not well clarified as yet. OBJECTIVE: The purpose of this study was to extract LPS from Vibrio vulnificus and to test the biological activity of extracted LPS for the elucidation of the role in V. vulnificas septicemia. METHODS: V. vulnificus LPS was extracted by the Phenol Chloroform-Petroleurn ether(PCP) method. The biological activity of LPS was evaluated with a limulus amebocyte lysate assay and by assessment of lethality to ICR mice. RESULTS: Five hundreds mg of LPS were extracted from 10g of dried V. vulnificus. Lirnulus amebocyte lysate formed a hard gel in response to the extracted LPS. This LPS showed low level of protein contaminatior in SDS-PAGE electophoresis and spectrophotometry. A High dose of LPS(200 mg/ml body weight) was lethal to mice. CONCLUSION: The PCP extraction yield relatively large amounts of LPS from V. vnlnificus. with out significant protein contamination and e xtrated LPS has endotoxin activity. This extrated LPS can be used for further studies such as making antibody or characterizing pathogenic roles in the V. vulnificus infection.