Reprogramming the tumor immune microenvironment using engineered dual-drug loaded Salmonella.

Synergistic combinations of immunotherapeutic agents can improve the performance of anti-cancer therapies but may lead to immune-mediated adverse effects. These side-effects can be overcome by using a tumor-specific delivery system. Here, we report a method of targeted immunotherapy using an attenuated Salmonella typhimurium (SAM-FC) engineered to release dual payloads: cytolysin A (ClyA), a cytolytic anti-cancer agent, and Vibrio vulnificus flagellin B (FlaB), a potent inducer of anti-tumor innate immunity. Localized secretion of ClyA from SAM-FC induces immunogenic cancer cell death and promotes release of tumor-specific antigens and damage-associated molecular patterns, which establish long-term antitumor memory. Localized secretion of FlaB promotes phenotypic and functional remodeling of intratumoral macrophages that markedly inhibits tumor metastasis in mice bearing tumors of mouse and human origin. Both primary and metastatic tumors from bacteria-treated female mice are characterized by massive infiltration of anti-tumorigenic innate immune cells and activated tumor-specific effector/memory T cells; however, the percentage of immunosuppressive cells is low. Here, we show that SAM-FC induces functional reprogramming of the tumor immune microenvironment by activating both the innate and adaptive arms of the immune system and can be used for targeted delivery of multiple immunotherapeutic payloads for the establishment of potent and long-lasting antitumor immunity.

The cytochrome P4501A1 (CYP1A1) inhibitor bergamottin enhances host tolerance to multidrug-resistant Vibrio vulnificus infection.

PURPOSE: Vibrio vulnificus (V. Vulnificus) infection is characterized by rapid onset, aggressive progression, and challenging treatment. Bacterial resistance poses a significant challenge for clinical anti-infection treatment and is thus the subject of research. Enhancing host infection tolerance represents a novel infection prevention strategy to improve patient survival. Our team initially identified cytochrome P4501A1 (CYP1A1) as an important target owing to its negative modulation of the body's infection tolerance. This study explored the superior effects of the CYP1A1 inhibitor bergamottin compared to antibiotic combination therapy on the survival of mice infected with multidrug-resistant V. Vulnificus and the protection of their vital organs. METHODS: An increasing concentration gradient method was used to induce multidrug-resistant V. Vulnificus development. We established a lethal infection model in C57BL/6J male mice and evaluated the effect of bergamottin on mouse survival. A mild infection model was established in C57BL/6J male mice, and the serum levels of creatinine, urea nitrogen, aspartate aminotransferase, and alanine aminotransferase were determined using enzyme-linked immunosorbent assay to evaluate the effect of bergamottin on liver and kidney function. The morphological changes induced in the presence of bergamottin in mouse organs were evaluated by hematoxylin and eosin staining of liver and kidney tissues. The bacterial growth curve and organ load determination were used to evaluate whether bergamottin has a direct antibacterial effect on multidrug-resistant V. Vulnificus. Quantification of inflammatory factors in serum by enzyme-linked immunosorbent assay and the expression levels of inflammatory factors in liver and kidney tissues by real-time quantitative polymerase chain reaction were performed to evaluate the effect of bergamottin on inflammatory factor levels. Western blot analysis of IκBα, phosphorylated IκBα, p65, and phosphorylated p65 protein expression in liver and kidney tissues and in human hepatocellular carcinomas-2 and human kidney-2 cell lines was used to evaluate the effect of bergamottin on the nuclear factor kappa-B signaling pathway. One-way ANOVA and Kaplan-Meier analysis were used for statistical analysis. RESULTS: In mice infected with multidrug-resistant V. Vulnificus, bergamottin prolonged survival (p = 0.014), reduced the serum creatinine (p = 0.002), urea nitrogen (p = 0.030), aspartate aminotransferase (p = 0.029), and alanine aminotransferase (p = 0.003) levels, and protected the cellular morphology of liver and kidney tissues. Bergamottin inhibited interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α expression in serum (IL-1ß: p = 0.010, IL-6: p = 0.029, TNF-α: p = 0.025) and inhibited the protein expression of the inflammatory factors IL-1ß, IL-6, TNF-α in liver (IL-1ß: p = 0.010, IL-6: p = 0.011, TNF-α: p = 0.037) and kidney (IL-1ß: p = 0.016, IL-6: p = 0.011, TNF-α: p = 0.008) tissues. Bergamottin did not affect the proliferation of multidrug-resistant V. Vulnificus or the bacterial load in the mouse peritoneal lavage fluid (p = 0.225), liver (p = 0.186), or kidney (p = 0.637). CONCLUSION: Bergamottin enhances the tolerance of mice to multidrug-resistant V. Vulnificus infection. This study can serve as a reference and guide the development of novel clinical treatment strategies for V. Vulnificus.

Antibiotic resistance trends among Vibrio vulnificus and Vibrio parahaemolyticus isolated from the Chesapeake Bay, Maryland: a longitudinal study.

Antibiotics are often used to treat severe Vibrio infections, with third-generation cephalosporins and tetracyclines combined or fluoroquinolones alone being recommended by the US Centers for Disease Control and Prevention. Increases in antibiotic resistance of both environmental and clinical vibrios are of concern; however, limited longitudinal data have been generated among environmental isolates to inform how resistance patterns may be changing over time. Hence, we evaluated long-term trends in antibiotic resistance of vibrios isolated from Chesapeake Bay waters (Maryland) across two 3-year sampling periods (2009-2012 and 2019-2022). Vibrio parahaemolyticus (n = 134) and Vibrio vulnificus (n = 94) toxR-confirmed isolates were randomly selected from both sampling periods and tested for antimicrobial susceptibility against eight antibiotics using the Kirby-Bauer disk diffusion method. A high percentage (94%-96%) of V. parahaemolyticus isolates from both sampling periods were resistant to ampicillin and only 2%-6% of these isolates expressed intermediate resistance or resistance to third-generation cephalosporins, amikacin, tetracycline, and trimethoprim-sulfamethoxazole. Even lower percentages of resistant V. vulnificus isolates were observed and those were mostly recovered from 2009 to 2012, however, the presence of multiple virulence factors was observed. The frequency of multi-drug resistance was relatively low (6%-8%) but included resistance against antibiotics used to treat severe vibriosis in adults and children. All isolates were susceptible to ciprofloxacin, a fluoroquinolone, indicating its sustained efficacy as a first-line agent in the treatment of severe vibriosis. Overall, our data indicate that antibiotic resistance patterns among V. parahaemolyticus and V. vulnificus recovered from the lower Chesapeake Bay have remained relatively stable since 2009.IMPORTANCEVibrio spp. have historically been susceptible to most clinically relevant antibiotics; however, resistance and intermediate-resistance have been increasingly recorded in both environmental and clinical isolates. Our data showed that while the percentage of multi-drug resistance and resistance to antibiotics was relatively low and stable across time, some Vibrio isolates displayed resistance and intermediate resistance to antibiotics typically used to treat severe vibriosis (e.g., third-generation cephalosporins, tetracyclines, sulfamethoxazole-trimethoprim, and aminoglycosides). Also, given the high case fatality rates observed with Vibrio vulnificus infections, the presence of multiple virulence factors in the tested isolates is concerning. Nevertheless, the continued susceptibility of all tested isolates against ciprofloxacin, a fluoroquinolone, is indicative of its use as an effective first-line treatment of severe Vibrio spp. infections stemming from exposure to Chesapeake Bay waters or contaminated seafood ingestion.

Gallic acid promotes macrophage phagosome acidification and phagolysosome formation by activating NLRP3/mTOR signaling pathway.

INTRODUCTION: Gallic acid (GA) has a good therapeutic effect in bacteriological inhibition and plays a variety of functions in maintaining the stability of the immune system. The aim of the present study was to investigate the effect of GA on the bactericidal activity of macrophages against Vibrio vulnificus (Vv). METHODS: A cell counting kit-8 (CCK-8) assay was carried out to test the cytotoxicity of GA on J774A.1 cells. Concentration of proinflammatory cytokines in J774A.1 cells were evaluated by ELISA. The internalization and degradation of Vv in the phagosomes were observed by transmission electron microscopy (TEM). The phagosome acidification and phagolysosome formation were detected to evaluate the bacteria-clearing function of J774A.1 cells. The bactericidal activity of GA in vivo was also investigated by collecting the survival time of Vv infected mice and observing the inflammatory infiltration of organs. RESULTS: Our results demonstrated that GA at 50 µM significantly inhibited the proinflammatory cytokines levels, promoted phagosome acidification and phagolysosome formation in J774A.1 cells with Vv infection. This may be related to the activation of NLRP3/mTOR signaling pathway. Additionally, GA treatment improves the survival and bactericidal activity of mice infected with Vv. CONCLUSIONS: In summary, GA exerts bactericidal activity against Vv infection by regulating the formation and acidification of phagocytic lysosomes in macrophages.

Antibacterial Mechanism of Shikonin Against Vibrio vulnificus and Its Healing Potential on Infected Mice with Full-Thickness Excised Skin.

Shikonin has anticancer, anti-inflammatory, and wound healing activities. Vibrio vulnificus is an important marine foodborne pathogen with a high fatality rate and rapid pathogenesis that can infect humans through ingestion and wounds. In this study, the antibacterial activity and possible antibacterial mechanism of shikonin against V. vulnificus were investigated. In addition, the ability of shikonin to control V. vulnificus infection in both pathways was assessed by artificially contaminated oysters and full-thickness excised skin-infected mice. Shikonin treatment can cause abnormal cell membrane function, as evidenced by hyperpolarization of the cell membrane, significant decreased intracellular ATP concentration (p < 0.05), significant increased intracellular reactive oxygen species and malondialdehyde content (p < 0.05), decreased cell membrane integrity, and changes in cell morphology. Shikonin at 40 and 80 µg/mL reduced bacterial numbers in shikonin-contaminated oysters by 3.58 and 2.18 log colony-forming unit (CFU)/mL. Shikonin can promote wound healing in mice infected with V. vulnificus by promoting the formation of granulation tissue, hair follicles, and sebaceous glands, promoting epithelial cell regeneration and epidermal growth factor production. These findings suggest that shikonin has a strong inactivation effect on V. vulnificus and can be used in food production and wound healing to effectively control V. vulnificus and reduce the number of diseases associated with it.

New Atypical Manifestations and Prognostic Factors of Vibrio vulnificus Infection: a 10-Year Retrospective Study.

Vibrio vulnificus (V. vulnificus) infection is rare but potentially fatal. This study explored new atypical manifestations and prognostic factors of V. vulnificus-infected patients during hospitalization. We retrospectively reviewed the medical records of 33 patients diagnosed with V. vulnificus infection in Guangdong Province, China between 2010 and 2020. Multiple logistic regression and receiver operating characteristic (ROC) curve analyses were performed. The new atypical manifestations included cholangitis, urinary tract infection, and suppurative otitis media. Eleven of the 33 (33.3%) V. vulnificus-infected patients eventually died. Univariate analysis showed that patients with cardio-cerebrovascular diseases, lower platelet counts, and higher levels of C-reactive protein and procalcitonin (PCT) had statistically higher mortality. However, multivariate analysis showed that only the PCT level (P = 0.036) was statistically significant. In addition, the area under the ROC value estimate for PCT was 0.8816 (95% confidence interval (CI), 0.759-1.000; P = 0.0009). More than half of the patients with V. vulnificus infection died when PCT was > 20 ng/mL, while no patient died when PCT was ≤ 20 ng/mL. This study found new atypical manifestations of V. vulnificus infection. In addition, PCT was an effective and independent predictor of mortality in patients with V. vulnificus infection, allowing clinicians to conduct early risk stratification and determine the best therapeutic strategies.

Ethidium and propidium monoazide: comparison of potential toxicity on Vibrio sp. viability.

Vibrio sp., ubiquitous in the aquatic ecosystem, are bacteria of interest because of their involvement in human health, causing gastroenteritis after ingestion of seafood, as well as their role in vibriosis leading to severe losses in aquaculture production. Their ability to enter a viable but non-culturable (VBNC) state under stressful environmental conditions may lead to underestimation of the Vibrio population by traditional microbiological enumeration methods. As a result, using molecular methods in combination with EMA or PMA allows the detection of viable (VBNC and culturable viable) cells. In this study, the impact of the EMA and PMA was tested at different concentrations on the viability of several Vibrio species. We compared the toxicity of these two DNA-binding dyes to determine the best pretreatment to use with qPCR to discriminate between viable and dead Vibrio cells. Our results showed that EMA displayed lethal effects for each strain of V. cholerae and V. vulnificus tested. In contrast, the concentrations of PMA tested had no toxic effect on the viability of Vibrio cells studied. These results may help to achieve optimal PMA-qPCR methods to detect viable Vibrio sp. cells in food and environmental samples.

rpoB mutations conferring rifampicin-resistance affect growth, stress response and motility in Vibrio vulnificus.

Rifampicin is a broad-spectrum antibiotic that binds to the bacterial RNA polymerase (RNAP), compromising DNA transcription. Rifampicin resistance is common in several microorganisms and it is typically caused by point mutations in the gene encoding the ß subunit of RNA polymerase, rpoB. Different rpoB mutations are responsible for various levels of rifampicin resistance and for a range of secondary effects. rpoB mutations conferring rifampicin resistance have been shown to be responsible for severe effects on transcription, cell fitness, bacterial stress response and virulence. Such effects have never been investigated in the marine pathogen Vibrio vulnificus, even though rifampicin-resistant strains of V. vulnificus have been isolated previously. Moreover, spontaneous rifampicin-resistant strains of V. vulnificus have an important role in conjugation and mutagenesis protocols, with poor consideration of the effects of rpoB mutations. In this work, effects on growth, stress response and virulence of V. vulnificus were investigated using a set of nine spontaneous rifampicin-resistant derivatives of V. vulnificus CMCP6. Three different mutations (Q513K, S522L and H526Y) were identified with varying incidence rates. These three mutant types each showed high resistance to rifampicin [minimal inhibitory concentration (MIC) >800 µg ml-1], but different secondary effects. The strains carrying the mutation H526Y had a growth advantage in rich medium but had severely reduced salt stress tolerance in the presence of high NaCl concentrations as well as a significant reduction in ethanol stress resistance. Strains possessing the S522L mutation had reduced growth rate and overall biomass accumulation in rich medium. Furthermore, investigation of virulence characteristics demonstrated that all the rifampicin-resistant strains showed compromised motility when compared with the wild-type, but no major effects on exoenzyme production were observed. These findings reveal a wide range of secondary effects of rpoB mutations and indicate that rifampicin resistance is not an appropriate selectable marker for studies that aim to investigate phenotypic behaviour in this organism.

Antibacterial Activity of Honey/Chitosan Nanofibers Loaded with Capsaicin and Gold Nanoparticles for Wound Dressing.

This paper describes the preparation, characterization, and evaluation of honey/tripolyphosphate (TPP)/chitosan (HTCs) nanofibers loaded with capsaicin derived from the natural extract of hot pepper (Capsicum annuumL.) and loaded with gold nanoparticles (AuNPs) as biocompatible antimicrobial nanofibrous wound bandages in topical skin treatments. The capsaicin and AuNPs were packed within HTCs in HTCs-capsaicin, HTCs-AuNP, and HTCs-AuNPs/capsaicin nanofibrous mats. In vitro antibacterial testing against Pasteurella multocida, Klebsiella rhinoscleromatis,Staphylococcus pyogenes, and Vibrio vulnificus was conducted in comparison with difloxacin and chloramphenicol antibiotics. Cell viability and proliferation of the developed nanofibers were evaluated using an MTT assay. Finally, in vivo study of the wound-closure process was performed on New Zealand white rabbits. The results indicate that HTCs-capsaicin and HTCs-AuNPs are suitable in inhibiting bacterial growth compared with HTCs and HTCs-capsaicin/AuNP nanofibers and antibiotics (P < 0.01). The MTT assay demonstrates that the nanofibrous mats increased cell proliferation compared with the untreated control (P < 0.01). In vivo results show that the developed mats enhanced the wound-closure rate more effectively than the control samples. The novel nanofibrous wound dressings provide a relatively rapid and efficacious wound-healing ability, making the obtained nanofibers promising candidates for the development of improved bandage materials.

DIDS inhibits Vibrio vulnificus cytotoxicity by interfering with TolC-mediated RtxA1 toxin secretion.

Vibrio vulnificus (V. vulnificus) infection, frequently resulting in fatal septicemia, has become a growing health concern worldwide. The present study aimed to explore the potential agents that could protect against V. vulnificus cytotoxicity, and to analyze the possible underlying mechanisms. First, we observed that 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate (DIDS) significantly suppressed V. vulnificus cytotoxicity to host cells by using a lactate dehydrogenase (LDH) assay. DIDS did not exhibit any effect on host cell viability, bacterial growth, microbial adhesion and swarming motility. DIDS effectively lowered V. vulnificus RtxA1 toxin-induced calcium influx into host mitochondria and RtxA1 binding to host cells. To further elucidate the underlying mechanism, the synthesis and secretion of RtxA1 toxin were investigated by Western blotting. Intriguingly, DIDS selectively inhibited the secretion of RtxA1 toxin, but did not influence its synthesis. Consequently, the outer membrane portal TolC, a key conduit for RtxA1 export coupled with tripartite efflux pumps, was examined by RT-PCR and Western blotting. We found that DIDS significantly reduced the expression of TolCV1 protein at the transcriptional level. Taken together, these results suggest that DIDS is a promising new paradigm as an antimicrobial drug that targets TolC-mediated toxin.

A MARTX Toxin rtxA Gene Is Controlled by Host Environmental Signals through a CRP-Coordinated Regulatory Network in Vibrio vulnificus.

A multifunctional autoprocessing repeats-in-toxin (MARTX) toxin plays an essential role in the virulence of many pathogens, including a fulminating human pathogen Vibrio vulnificus H-NS and HlyU repress and derepress expression of the MARTX toxin gene rtxA in V. vulnificus, respectively. However, little is known about other regulatory proteins and environmental signals involved in rtxA regulation. In this study, we found that a leucine-responsive regulatory protein (Lrp) activates rtxA by binding directly and specifically to the rtxA promoter, P rtxA Phased hypersensitivity resulting from DNase I cleavage of the P rtxA regulatory region suggests that Lrp probably induces DNA bending in P rtxA Lrp activates P rtxA independently of H-NS and HlyU, and leucine inhibits Lrp binding to P rtxA and reduces the Lrp-mediated activation. Furthermore, a cyclic AMP receptor protein (CRP) represses P rtxA , and exogenous glucose relieves the CRP-mediated repression. Biochemical and mutational analyses demonstrated that CRP binds directly and specifically to the upstream region of P rtxA , which presumably alters the DNA conformation in P rtxA and thus represses rtxA Moreover, CRP represses expression of lrp and hlyU by binding directly to their upstream regions, forming coherent feed-forward loops with Lrp and HlyU. In conclusion, expression of rtxA is controlled by a regulatory network comprising CRP, Lrp, H-NS, and HlyU in response to changes in host environmental signals such as leucine and glucose. This collaborative regulation enables the elaborate expression of rtxA, thereby enhancing the fitness and pathogenesis of V. vulnificus during the course of infection.IMPORTANCE A MARTX toxin, RtxA, is an essential virulence factor of many pathogens, including Vibrio species. H-NS and HlyU repress and derepress, respectively, rtxA expression of a life-threatening pathogen, Vibrio vulnificus We found that Lrp directly activates rtxA independently of H-NS and HlyU, and leucine inhibits the Lrp-mediated activation of rtxA Furthermore, we demonstrated that CRP represses rtxA but derepresses in the presence of exogenous glucose. CRP represses rtxA not only directly by binding to upstream of rtxA but also indirectly by repressing lrp and hlyU This is the first report of a regulatory network comprising CRP, Lrp, H-NS, and HlyU, which coordinates the rtxA expression in response to environmental signals such as leucine and glucose during infection. This elaborate regulatory network will enhance the fitness of V. vulnificus and contribute to its successful infection within the host.

Recovery of antioxidant and antimicrobial peptides through the reutilization of Nile perch wastewater by biodegradation using two Bacillus species.

Nile perch wastewater was biodegraded using two Bacillus species to recover bioactive substances to enhance its reutilization value. The two Bacillus species successfully produced low-molecular-weight substances with a 47.8% degree of hydrolysis. The antioxidant activities of the Nile perch wastewater increased as the biodegradation proceeded, and the culture supernatant exhibited the highest DPPH (80.1%), ABTS (93.1%) and Fe2+ chelating (88.5%) antioxidant activities at 60 h. The antioxidant potential of the biodegraded Nile perch wastewater was found to be higher than those of other fish hydrolysates. Moreover, the biodegraded Nile perch wastewater exhibited effective antimicrobial activity against Vibrio vulnificus, exhibiting a minimal inhibitory concentration of 585 µg mL-1. Two-dimensional thin layer chromatography analysis revealed the specific amino acids responsible for the antioxidant activity, and molecular-weight cut-off ultrafiltration revealed that the <2-kDa fraction exhibited the highest antioxidant activity with the lowest IC50 values (0.43 and 0.22 mg mL-1 for DPPH and ABTS antioxidant activities, respectively). This is the first report of the reutilization of Nile perch wastewater as a natural antioxidant and antimicrobial ingredient for nutraceuticals.

Tetra-(p-tolyl)antimony(III)-Containing Heteropolytungstates, [{(p-tolyl)SbIII}4(A-α-XW9O34)2]n- (X = P, As, or Ge): Synthesis, Structure, and Study of Antibacterial and Antitumor Activity.

We have synthesized and structurally characterized three tetra-(p-tolyl)antimony(III)-containing heteropolytungstates, [{(p-tolyl)SbIII}4(A-α-XW9O34)2]n- [X = PV (1-P), AsV (1-As), or GeIV (1-Ge)], in aqueous solution using conventional, one-pot procedures. The polyanions 1-P, 1-As, and 1-Ge were fully characterized in the solid state and in solution and were shown to be soluble and stable in aqueous medium at pH 7. Biological studies demonstrated that all three polyanions possess significant antibacterial and antitumor activities. The minimum inhibitory concentrations of 1-P, 1-As, and 1-Ge were determined against four kinds of bacteria, including the two pathogenic bacteria strains, Vibrio parahaemolyticus and Vibrio vulnificus. The three novel polyanions also showed high cytotoxic potency in the human cell lines A549 (non-small cell lung cancer), CH1/PA-1 (ovarian teratocarcinoma), and SW480 (colon carcinoma).

The effects of temperature, pH, and iron on biofilm formation by clinical versus environmental strains of Vibrio vulnificus.

Due to the nature of Vibrio vulnificus infections (i.e., gastroenteritis and septicemia), only very few studies of a biofilm-associated form in this pathogen's life cycle have been conducted. We proposed that biofilm production by clinical strains of V. vulnificus would be higher than by environmental strains. Biofilm formation by clinical and environmental reference strains was tested under different temperatures (24, 30, and 37 °C), pH (5.5, 7.5, and 8.5) and iron concentrations (18, 30, 50, 100, and 200 µM). Biofilm production by clinical strains was consistently higher (p < 0.001) at 24 °C than by environmental strains. Higher biofilm production was observed at pH 5.5 by all strains. Growth rates were lowest at pH 5.5 for environmental strains but for clinical strains there were no differences at pH 5.5, 7.5, and 8.5, demonstrating a tolerance to acidic and alkaline conditions. There was a strong, direct correlation between iron concentration in the growth medium and biofilm production by all strains tested. The current study indicates that biofilm formation might be important for the survival of V. vulnificus in vivo as well as in the marine environment. With regard to temperature and pH, higher biofilm production appears to be a trait of clinical strains and could be considered a virulence determinant in V. vulnificus.

The cytochrome d oxidase complex regulated by fexA is an Achilles' heel in the in vivo survival of vibrio vulnificus.

Vibrio vulnificus is a halophilic estuarine bacterium causing severe opportunistic infections. To successfully establish an infection, V. vulnificus must adapt to redox fluctuations in vivo. In the present study, we show that deletion of V. vulnificus fexA gene caused hypersensitivity to acid and reactive oxygen species. The ΔfexA mutant exhibited severe in vivo survival defects. For deeper understanding the role of fexA gene on the successful V. vulnificus infection, we analyzed differentially expressed genes in ΔfexA mutant in comparison with wild type under aerobic, anaerobic or in vivo culture conditions by genome-scale DNA microarray analyses. Twenty-two genes were downregulated in the ΔfexA mutant under all three culture conditions. Among them, cydAB appeared to dominantly contribute to the defective phenotypes of the ΔfexA mutant. The fexA deletion induced compensatory point mutations in the cydAB promoter region over subcultures, suggesting essentiality. Those point mutations (PcydSMs) restored bacterial growth, motility, cytotoxicity ATP production and mouse lethality in the ΔfexA mutant. These results indicate that the cydAB operon, being regulated by FexA, plays a crucial role in V. vulnificus survival under redox-fluctuating in vivo conditions. The FexA-CydAB axis should serve an Achilles heel in the development of therapeutic regimens against V. vulnificus infection.

In Vitro Synergy and In Vivo Activity of Tigecycline-Ciprofloxacin Combination Therapy against Vibrio vulnificus Sepsis.

The mortality rate associated with Vibrio vulnificus sepsis remains high. An in vitro time-kill assay revealed synergism between tigecycline and ciprofloxacin. The survival rate was significantly higher in mice treated with tigecycline plus ciprofloxacin than in mice treated with cefotaxime plus minocycline. Thus, combination treatment with tigecycline-ciprofloxacin may be an effective novel antibiotic regimen for V. vulnificus sepsis.

Outcomes of Third-Generation Cephalosporin Plus Ciprofloxacin or Doxycycline Therapy in Patients with Vibrio vulnificus Septicemia: A Propensity Score-Matched Analysis.

BACKGROUND: Combination therapy with a third-generation cephalosporin (TGC) and a tetracycline analogue is recommended for Vibrio vulnificus infection. The combination of a TGC and ciprofloxacin has synergistic in vitro bactericidal activity against V. vulnificus. No clinical study has compared the standard regimen with TGC plus ciprofloxacin therapy for V. vulnificus infection. METHODS: Patients with a confirmed V. vulnificus infection at two medical centers in Korea from 1991 to 2016 were enrolled in this study. The patients were grouped according to the type of antibiotic administered. A retrospective propensity-score-matched case-control study of patients treated with TGC plus doxycycline or TGC plus ciprofloxacin was performed. The clinical characteristics and outcomes of the patients were analyzed. RESULTS: A total of 218 patients were confirmed to have V. vulnificus septicemia during the study, and the 30-day survival rate was 39% (85/218). The patients were classified into the following six treatment groups: TGC monotherapy (n = 82), TGC plus doxycycline therapy (n = 42), TGC plus ciprofloxacin therapy (n = 39), ciprofloxacin monotherapy (n = 14), other ß-lactam monotherapy (n = 10), and other (n = 31). The survival rates of these groups were as follows: TGC monotherapy (35%), TGC plus doxycycline (38%), TGC plus ciprofloxacin (54%), ciprofloxacin monotherapy (29%), other ß-lactam (20%), and other (39%). The 30-day survival rate showed no significant difference between the TGC plus doxycycline and TGC plus ciprofloxacin groups (log-rank test, P = 0.18). Among the 81 patients treated with TGC plus doxycycline or TGC plus ciprofloxacin, 12 per treatment group were selected by propensity-score matching. There was no significant difference in the baseline characteristics or the frequency of fasciotomy between the two groups. The 30-day survival rate showed no significant difference between the TGC plus doxycycline (50%) and TGC plus ciprofloxacin (67%) groups (log-rank test, P = 0.46). CONCLUSION: Our data suggest that the outcome of TGC plus ciprofloxacin therapy was comparable to that of TGC plus doxycycline therapy in patients with V. vulnificus septicemia.

Tropodithietic acid induces oxidative stress response, cell envelope biogenesis and iron uptake in Vibrio vulnificus.

The Roseobacter group is a widespread marine bacterial group, of which some species produce the broad-spectrum antibiotic tropodithietic acid (TDA). A mode of action for TDA has previously been proposed in Escherichia coli, but little is known about its effect on non-producing marine bacteria at in situ concentrations. The purpose of this study was to investigate how a sub-lethal level of TDA affects Vibrio vulnificus at different time points (30 and 60 min) using a transcriptomic approach. Exposure to TDA for as little as 30 min resulted in the differential expression of genes associated with cell regeneration, including the up-regulation of those involved in biogenesis of the cell envelope. Defence mechanisms including oxidative stress defence proteins and iron uptake systems were also up-regulated in response to TDA, while motility-related genes were down-regulated. Gene expression data and scanning electron microscopy imaging revealed a switch to a biofilm phenotype in the presence of TDA. Our study shows that a low concentration of this antibiotic triggers a defence response to reactive oxygen species and iron depletion in V. vulnificus, which indicates that the mode of action of TDA is likely more complex in this bacterium than what is known for E. coli.

Ability of Vibrio vulnificus isolated from fish of the Lagoa dos Patos estuary in south Brazil to form biofilms after sublethal stress and bacterial resistance to antibiotics and sanitizers.

The present study aimed to investigate the presence of Vibrio vulnificus in fish captured at the Lagoa dos Patos estuary (RS, Brazil), to establish a correlation between bacterial biofilm formation and sublethal stress, and to assess the resistance of the isolates to antimicrobials and sanitizers. A total of 217 isolates characteristic of Vibrio sp. were analyzed. Isolates were identified and subsequently their ability to form biofilm, the impact of exposure to sublethal stress on their biofilm formation ability, and their resistance to antimicrobial and to sodium hypochlorite and chlorine dioxide sanitizers were evaluated. V. vulnificus was isolated from the fish Paralichthys orbignyanus and Micropogonias furnieri. The bacterial isolates examined were able to form biofilms. Biofilm formation ability of these strains was decreased or inhibited after being exposed to sublethal stress. The isolates were resistant to most antimicrobials. The sanitizer concentrations necessary to eliminate V. vulnificus were higher than those usually used in the fishing industry.

Anti-bacterial effects of components from Sanguisorba officinalis L. on Vibrio vulnificus and their soluble epoxide hydrolase inhibitory activity.

Sanguisorba officinalis L. is a traditional herbal medicine, which is prevailingly applied to cure hemorrhoids, wounds and ulcers in Eastern Asian countries. The purpose of this study was to investigate the antibacterial and soluble epoxide hydrolase (sEH) inhibitory effects of the extracts and components from S. officinalis. The methanol extract was divided into ethyl acetate (EtOAc), n-butanol (n-BuOH), and water layers. In our screening procedure, the EtOAc and n-BuOH extracts and compounds (1-2) remarkably suppressed the growth of V. vulnificus in a dose-dependent manner. In addition, the EtOAc extract and compound 1 exhibited significant inhibitory effect on the V. vulnificus induced cytotoxicity on HeLa cells. Furthermore, compound 4 displayed an inhibition against sEH with an IC50 value of 7.0 ± 0.5 µM. A kinetic analysis demonstrated that the inhibitory effect of compound 4 was a mixed type, with an inhibitory constant (Ki) 0.22 ± 0.0 µM.