Effect of capsular polysaccharide phase variation on biofilm formation, motility and gene expression in Vibrio vulnificus.

Vibrio vulnificus, a significant marine pathogen, undergoes opaque (Op)-translucent (Tr) colony switching based on whether capsular polysaccharide (CPS) is produced. CPS phase variation is sometime accompanied by genetic variation or down-regulation of particular genes, such as wzb. In addition, CPS prevents biofilm formation and is important to the virulence of V. vulnificus. However, the extent to which there is a difference in gene expression between Tr and Op colonies and the impact of CPS phase variation on other behaviors of V. vulnificus remain unknown. In this work, the data have shown that CPS phase variation of V. vulnificus is affected by incubation time. Tr and Op strains exhibited similar growth rates. However, Tr strains had enhanced biofilm formation capacities but reduced swimming motility compared to Op strains. The RNA-seq assay revealed 488 differentially expressed genes, with 214 downregulated and 274 upregulated genes, between Tr and Op colonies. Genes associated with Tad pili and CPS were downregulated, whereas those involved in flagellum were upregulated, in Tr colonies compared with Op colonies. In addition, 9 putative c-di-GMP metabolism-associated genes and 28 genes encoding putative regulators were significantly differentially expressed, suggesting that CPS phase variation is probably strictly regulated in V. vulnificus. Moreover, 8 genes encoding putative porins were also differentially expressed between the two phenotypic colonies, indicating that bacterial outer membrane was remodeled during CPS phase variation. In brief, this work highlighted the gene expression profiles associated with CPS phase variation, but more studies should be performed to disclose the intrinsic mechanisms in the future.

In vivo examination of pathogenicity and virulence in environmentally isolated Vibrio vulnificus.

Human exposure to Vibrio vulnificus, a gram-negative, halophilic environmental pathogen, is increasing. Despite this, the mechanisms of its pathogenicity and virulence remain largely unknown. Each year, hundreds of infections related to V. vulnificus occur, leading to hospitalization in 92% of cases and a mortality rate of 35%. The infection is severe, typically contracted through the consumption of contaminated food or exposure of an open wound to contaminated water. This can result in necrotizing fasciitis and the need for amputation of the infected tissue. Although several genes (rtxA1, vvpE, and vvhA) have been implicated in the pathogenicity of this organism, a defined mechanism has not been discovered. In this study, we examine environmentally isolated V. vulnificus strains using a zebrafish model (Danio rerio) to investigate their virulence capabilities. We found significant variation in virulence between individual strains. The commonly used marker gene of disease-causing strains, vcgC, did not accurately predict the more virulent strains. Notably, the least virulent strain in the study, V. vulnificus Sept WR1-BW6, which tested positive for vcgC, vvhA, and rtxA1, did not cause severe disease in the fish and was the only strain that did not result in any mortality. Our study demonstrates that virulence varies greatly among different environmental strains and cannot be accurately predicted based solely on genotype.

The role of MNK1-mTORC1 pathway in modulating macrophage responses to Vibrio vulnificus infection.

Vibrio vulnificus (Vv) is known to cause life-threatening infections, particularly septicemia. These patients often exhibit elevated levels of pro-inflammatory cytokines. While it is established that mitogen-activated protein kinase (MAPK)-interacting kinase (MNK) contributes to the production of pro-inflammatory cytokines, the role of MNK in macrophages during Vv infection remains unclear. In this study, we investigate the impact of MNK on macrophages. We demonstrate that the inhibition of MNK in J774A.1 cells, when treated with lipopolysaccharide or Vv, resulted in decreased production of tumor necrosis factor alpha and interleukin-6, without affecting their transcription. Interestingly, treatment with MNK inhibitor CGP57380 led to enhanced phosphorylation of MNK1 but decreased phosphorylation of eIF4E. Moreover, MNK1 knockout cells exhibited an increased capacity for phagocytosis and clearance of Vv, with more acidic phagosomes than the parental cells. Notably, CGP57380 did not impact phagocytosis, bacterial clearance, or phagosome acidification in Vv-infected J774A.1 cells. Considering the reported association between MNK and mammalian target of rapamycin complex 1 (mTORC1) activation, we investigated the mTORC1 signaling in MNK1 knockout cells infected with Vv. Our results revealed that attenuation of the mTORC1 signaling in these cells and treatment with the mTORC1 inhibitor rapamycin significantly enhanced bacterial clearance in J774A.1 cells following Vv infection. In summary, our findings suggest that MNK promotes the Vv-induced cytokine production in J774A.1 cells without affecting their transcription levels. MNK1 appears to impair the phagocytosis, bacterial clearance, and phagosome acidification in Vv-infected J774A.1 cells through the MNK1-mTORC1 signaling pathway rather than the MNK1-eIF4E signaling pathway. Our findings highlight the importance of the MNK1-mTORC1 pathway in modulating macrophage responses to Vv infection. IMPORTANCE: Mitogen-activated protein kinase (MAPK)-interacting kinase (MNK) plays a role in promoting the production of tumor necrosis factor alpha and interleukin-6 in macrophages during Vibrio vulnificus (Vv) infection. Inhibition or knockout of MNK1 in J774A.1 cells resulted in reduced cytokine production without affecting their transcription levels. MNK1 also impairs phagocytosis, bacterial clearance, and phagosome acidification in Vv-infected cells through the MNK1-mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. The findings highlight the importance of the MNK1-mTORC1 pathway in modulating macrophage responses to Vv infection.

Regulatory role of VvsB protein on serine protease activity of VvsA in Vibrio vulnificus.

BACKGROUND: Vibrio vulnificus NCIMB2137, a Gram-negative, metalloprotease negative estuarine strain was isolated from a diseased eel. A 45 kDa chymotrypsin-like alkaline serine protease known as VvsA has been recently reported as one of the major virulence factor responsible for the pathogenesis of this strain. The vvsA gene along with a downstream gene vvsB, whose function is still unknown constitute an operon designated as vvsAB. OBJECTIVE: This study examines the contribution of VvsB to the functionality of VvsA. METHOD: In this study, VvsB was individually expressed using Rapid Translation System (RTS system), followed by an analysis of its role in regulating the serine protease activity of VvsA. RESULT: The proteolytic activity of VvsA increased upon the addition of purified VvsB to the culture supernatant of V. vulnificus. However, the attempts of protein expression using an E. coli system revealed a noteworthy observation that protein expression from the vvsA gene exhibited higher protease activity compared to that from the vvsAB gene within the cytoplasmic fraction. These findings suggest an intricate interplay between VvsB and VvsA, where VvsB potentially interacts with VvsA inside the bacterium and suppress the proteolytic activity. While outside the bacterial milieu, VvsB appears to stimulate the activation of inactive VvsA. CONCLUSION: The findings suggest that Vibrio vulnificus regulates VvsA activity through the action of VvsB, both intracellularly and extracellularly, to ensure its survival.

Mobile-CRISPRi protocol optimization for Vibrionaceae.

Mobile clustered regularly interspaced palindromic repeats interference (Mobile-CRISPRi) is an established method for bacterial gene expression knockdown. The deactivated Cas9 protein and guide RNA are isopropyl ß-D-1-thiogalactopyranoside inducible, and all components are integrated into the chromosome via Tn7 transposition. Here, we optimized methods specific for applying Mobile-CRISPRi in multiple Vibrio species.

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.

Vibrio vulnificus infection from insect bites in Shanghai: a case report.

Background: Infection with Vibrio vulnificus is associated with high rates of amputation and mortality. Alterations in the global climate have heightened the risk of atypical infections caused by this pathogen. Case presentation: In the case report we describe, a 75-year-old man residing in a coastal city contracted Vibrio vulnificus secondary to an insect bite. Discussion and conclusion: This case underscores the importance for clinicians of recognizing that early administration of appropriate antibiotics in patients with non-traditional routes of Vibrio vulnificus infection can significantly reduce rates of amputation and mortality.

Vibrio MARTX toxin processing and degradation of cellular Rab GTPases by the cytotoxic effector Makes Caterpillars Floppy.

Vibrio vulnificus causes life-threatening wound and gastrointestinal infections, mediated primarily by the production of a Multifunctional-Autoprocessing Repeats-In-Toxin (MARTX) toxin. The most commonly present MARTX effector domain, the Makes Caterpillars Floppy-like (MCF) toxin, is a cysteine protease stimulated by host adenosine diphosphate (ADP) ribosylation factors (ARFs) to autoprocess. Here, we show processed MCF then binds and cleaves host Ras-related proteins in brain (Rab) guanosine triphosphatases within their C-terminal tails resulting in Rab degradation. We demonstrate MCF binds Rabs at the same interface occupied by ARFs. Moreover, we show MCF preferentially binds to ARF1 prior to autoprocessing and is active to cleave Rabs only subsequent to autoprocessing. We then use structure prediction algorithms to demonstrate that structural composition, rather than sequence, determines Rab target specificity. We further determine a crystal structure of aMCF as a swapped dimer, revealing an alternative conformation we suggest represents the open, activated state of MCF with reorganized active site residues. The cleavage of Rabs results in Rab1B dispersal within cells and loss of Rab1B density in the intestinal tissue of infected mice. Collectively, our work describes an extracellular bacterial mechanism whereby MCF is activated by ARFs and subsequently induces the degradation of another small host guanosine triphosphatase (GTPase), Rabs, to drive organelle damage, cell death, and promote pathogenesis of these rapidly fatal infections.

Isolation of Vibrio cholerae and Vibrio vulnificus from Estuarine Waters, and Genotyping of V. vulnificus Isolates Using Loop-Mediated Isothermal Amplification.

Bacteria in the genus Vibrio are ubiquitous in estuarine and coastal waters. Some species (including Vibrio cholerae and Vibrio vulnificus) are known human pathogens causing ailments like cholera, diarrhea, or septicemia. Notably, V. vulnificus can also cause a severe systemic infection (known as vibriosis) in eels raised in aquaculture facilities. Water samples were periodically collected from the estuary of the Asahi River, located in the southern part of Okayama City, Japan. These samples were directly plated onto CHROMagar Vibrio plates, and colonies displaying turquoise-blue coloration were selected. Thereafter, polymerase chain reaction was used to identify V. cholerae and V. vulnificus. A total of 30 V. cholerae strains and 194 V. vulnificus strains were isolated during the warm season when the water temperature (WT) was higher than 20 °C. Concurrently, an increase in coliforms was observed during this period. Notably, V. vulnificus has two genotypes, designated as genotype 1 and genotype 2. Genotype 1 is pathogenic to humans, while genotype 2 is pathogenic to both humans and eels. The loop-mediated isothermal amplification method was developed to rapidly determine genotypes at a low cost. Of the 194 strains isolated, 80 (41.2%) were identified as genotype 1 strains. Among the 41 strains isolated when the WTs were higher than 28 °C, 25 strains (61.0%) belonged to genotype 1. In contrast, of the 32 strains isolated when the WTs were lower than 24 °C, 27 strains (84.4%) belonged to genotype 2. These results suggest that the distribution of the two genotypes was influenced by WT.

Characterization of Outer Membrane Vesicles Produced by Vibrio vulnificus.

Vibrio vulnificus (V. vulnificus) is a halophilic gram-negative bacterium that inhabits coastal warm water and induce severe diseases such as primary septicemia. To investigate the mechanisms of rapid bacterial translocation on intestinal infection, we focused on outer membrane vesicles (OMVs), which are extracellular vesicles produced by Gram-negative bacteria and deliver virulence factors. However, there are very few studies on the pathogenicity or contents of V. vulnificus OMVs (Vv-OMVs). In this study, we investigated the effects of Vv-OMVs on host cells. Epithelial cells INT407 were stimulated with purified OMVs and morphological alterations and levels of lactate dehydrogenase (LDH) release were observed. In cells treated with OMVs, cell detachment without LDH release was observed, which exhibited different characteristics from cytotoxic cell detachment observed in V. vulnificus infection. Interestingly, OMVs from a Vibrio Vulnificus Hemolysin (VVH) and Multifunctional-autoprocessing repeats-in -toxin (MARTX) double-deletion mutant strain also caused cell detachment without LDH release. Our results suggested that the proteolytic function of a serine protease contained in Vv-OMVs may contribute to pathogenicity of V. vulnificus by assisting bacterial translocation. This study reveals a new pathogenic mechanism during V. vulnificus infections. J. Med. Invest. 71 : 102-112, February, 2024.

Wildfire Ashes from the Wildland-Urban Interface Alter Vibrio vulnificus Growth and Gene Expression.

Climate change-induced stressors are contributing to the emergence of infectious diseases, including those caused by marine bacterial pathogens such as Vibrio spp. These stressors alter Vibrio temporal and geographical distribution, resulting in increased spread, exposure, and infection rates, thus facilitating greater Vibrio-human interactions. Concurrently, wildfires are increasing in size, severity, frequency, and spread in the built environment due to climate change, resulting in the emission of contaminants of emerging concern. This study aimed to understand the potential effects of urban interface wildfire ashes on Vibrio vulnificus (V. vulnificus) growth and gene expression using transcriptomic approaches. V. vulnificus was exposed to structural and vegetation ashes and analyzed to identify differentially expressed genes using the HTSeq-DESeq2 strategy. Exposure to wildfire ash altered V. vulnificus growth and gene expression, depending on the trace metal composition of the ash. The high Fe content of the vegetation ash enhanced bacterial growth, while the high Cu, As, and Cr content of the structural ash suppressed growth. Additionally, the overall pattern of upregulated genes and pathways suggests increased virulence potential due to the selection of metal- and antibiotic-resistant strains. Therefore, mixed fire ashes transported and deposited into coastal zones may lead to the selection of environmental reservoirs of Vibrio strains with enhanced antibiotic resistance profiles, increasing public health risk.

Sensitive colorimetric detection of Vibrio vulnificus based on target-induced shielding against the peroxidase-mimicking activity of CeO2@PtRu nanozyme.

Vibrio vulnificus infection caused by contaminated aquatic products and seawater can lead to severe disease and high mortality. The development of a rapid and sensitive detection method for Vibrio vulnificus is vital to effectively prevent infection in advance. In this study, CeO2@PtRu with high peroxidase activity was used to construct a colorimetric immunoassay for Vibrio vulnificus detection by conjugating polyclonal antibodies via the biotin-streptavidin system. The developed colorimetric biosensor for Vibrio vulnificus demonstrated rapid operability and good sensitivity with a detection range from 104 CFU/mL to 109 CFU/mL, and the limit of detection (LOD) is 193 CFU/mL. Moreover, the colorimetric biosensor showed excellent specificity and good recoveries from 98.70% to 102.10% with RSD < 7.45% for spiked real samples. This novel CeO2@PtRu-based colorimetric biosensor has great application potential for the sensitive detection of Vibrio vulnificus in seafood.

Mobile-CRISPRi as a powerful tool for modulating Vibrio gene expression.

CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) is a gene knockdown method that uses a deactivated Cas9 protein (dCas9) that binds a specific gene target locus dictated by an encoded guide RNA (sgRNA) to block transcription. Mobile-CRISPRi is a suite of modular vectors that enable CRISPRi knockdowns in diverse bacteria by integrating IPTG-inducible dcas9 and sgRNA genes into the genome using Tn7 transposition. Here, we show that the Mobile-CRISPRi system functions robustly and specifically in multiple Vibrio species: Vibrio cholerae, Vibrio fischeri, Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio campbellii. We demonstrate efficacy by targeting both essential and non-essential genes that function to produce defined, measurable phenotypes: bioluminescence, quorum sensing, cell division, and growth arrest. We anticipate that Mobile-CRISPRi will be used in Vibrio species to systematically probe gene function and essentiality in various behaviors and native environments.IMPORTANCEThe genetic manipulation of bacterial genomes is an invaluable tool in experimental microbiology. The development of CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) tools has revolutionized genetics in many organisms, including bacteria. Here, we optimized the use of Mobile-CRISPRi in five Vibrio species, each of which has significant impacts on marine environments and organisms that include squid, shrimp, shellfish, finfish, corals, and multiple of which pose direct threats to human health. The Mobile-CRISPRi technology is easily adaptable, moveable from strain to strain, and enables researchers to selectively turn off gene expression. Our experiments demonstrate Mobile-CRISPRi is effective and robust at repressing gene expression of both essential and non-essential genes in Vibrio species.

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.

Tracking Vibrio: population dynamics and ecology of Vibrio parahaemolyticus and V. vulnificus in an Alabama estuary.

Vibrio is a genus of halophilic, gram-negative bacteria found in estuaries around the globe. Integral parts of coastal cultures often involve contact with vectors of pathogenic Vibrio spp. (e.g., consuming raw shellfish). High rates of mortality from certain Vibrio spp. infections demonstrate the need for an improved understanding of Vibrio spp. dynamics in estuarine regions. Our study assessed meteorological, hydrographic, and biological correlates of Vibrio parahaemolyticus and V. vulnificus at 10 sites in the Eastern Mississippi Sound System (EMSS) from April to October 2019. During the sampling period, median abundances of V. parahaemolyticus and V. vulnificus were 2.31 log MPN/L and 2.90 log MPN/L, respectively. Vibrio spp. dynamics were largely driven by site-based variation, with sites closest to freshwater inputs having the highest abundances. The E-W wind scalar, which affects Ekman transport, was a novel Vibrio spp. correlate observed. A potential salinity effect on bacterial-particle associations was identified, where V. vulnificus was associated with larger particles in conditions outside of their optimal salinity. Additionally, V. vulnificus abundances were correlated to those of harmful algal species that did not dominate community chlorophyll. Correlates from this study may be used to inform the next iteration of regionally predictive Vibrio models and may lend additional insight to Vibrio spp. ecology in similar systems. IMPORTANCE: Vibrio spp. are bacteria found in estuaries worldwide; some species can cause illness and infections in humans. Relationships between Vibrio spp. abundance, salinity, and temperature are well documented, but correlations to other environmental parameters are less understood. This study identifies unique correlates (e.g., E-W wind scalar and harmful algal species) that could potentially inform the next iteration of predictive Vibrio models for the EMSS region. Additionally, these correlates may allow existing environmental monitoring efforts to be leveraged in providing data inputs for future Vibrio risk models. An observed correlation between salinity and V. vulnificus/particle-size associations suggests that predicted environmental changes may affect the abundance of Vibrio spp. in certain reservoirs, which may alter which vectors present the greatest vibrio risk.

Bactericidal effect of water-washing methods on Vibrio vulnificus contaminated in a raw fish Konosirus punctatus: water type, temperature, and pH.

This study aimed to evaluate a method for effectively reducing Vibrio vulnificus contamination in fish based on the type of washing water and method. Texture profiles and sensory evaluations were performed to determine the effect of the developed method on the quality and preference of the samples. The selected fish sample was Konosirus punctatus, which is mainly consumed in Asian countries. Various factors that could affect the survival rate of V. vulnificus were reviewed, including water type, temperature, exposure time, organic acids, pH, and washing methods. As a result, immersion and washing with filtered water with pH adjusted to 4.0 using acetic acid showed a high bactericidal effect of 2.5 log MPN/100 g. Furthermore, this method showed no statistically significant effect on the texture and sensory characteristics of fish. The results of the present study suggest a simple and effective method for preventing V. vulnificus infection in raw fish.

Finger Necrotizing Fasciitis and Septicemia Caused by Vibrio vulnificus.

Background: Vibrio vulnificus infections develop rapidly and have high mortality and disability rates. Vibrio vulnificus can cause local wound infection, gastroenteritis, or septicemia. Case Presentation: In this case, an 86-year-old male was accidentally stabbed in the middle of his right thumb while cleaning whitewater fish and came to the emergency department with high fever and painful swelling of the right hand. Physical examination revealed hemorrhagic bullae in the right hand. Emergency surgery and bacterial culture were performed. Because of timely antibiotic use and surgical treatment, the patient eventually recovered and was discharged from the hospital. Conclusions: This case suggests that the possibility of Vibrio vulnificus should be considered in cases of severe infection of the extremities, even without a history of seafood consumption or seawater exposure. Early recognition, rational choice of antibiotic agents, and timely wound debridement can substantially improve the prognosis of patients and reduce mortality.

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.

A One-Step RPA-CRISPR Assay Using crRNA Based on Suboptimal Protospacer Adjacent Motif for Vibrio vulnificus Detection.

Vibrio vulnificus is a hazardous foodborne pathogen responsible for approximately 95% of seafood-related deaths. This highlights the urgent requirement for specialized detection tools to be developed and used by food enterprises and food safety authorities. The DETECTR (DNA endonuclease targeted CRISPR trans reporter) system that combines CRISPR/Cas and recombinase polymerase amplification (RPA) has been utilized to develop a molecular detection assay for V. vulnificus. However, because the incompatibility between RPA and Cas12a cleavage has not been addressed, it is a two-step assay that lacks convenience and presents contamination risk. Here, we developed a one-step RPA-CRISPR assay for V. vulnificus using a special crRNA targeting a sequence with a suboptimal protospacer adjacent motif (PAM). The entire assay, conducted at 37°C, takes only 40-60 min, yields results visualized under blue light, and exhibits exceptional specificity and sensitivity (detecting 4 pathogen genome copies per reaction). This study offers a valuable tool for detecting V. vulnificus, aiding in foodborne infection prevention, and exemplifies one-step RPA-CRISPR assays managing Cas-cleavage activity through PAM adjustments.

Combined TCBS and CHROMagar Analyses Allow for Basic Identification of Vibrio vulnificus within a 48 h Incubation Period in the Coastal Baltic Sea.

With rising infection rates in recent years, Vibrio vulnificus poses an increasing threat to public safety in the coastal brackish Baltic Sea. It is therefore important to monitor this organism and assess the V. vulnificus infection risk on a more regular basis. However, as the coastline of the Baltic Sea is 8000 km long and shared by nine nations, a convenient, fast, inexpensive, yet efficient V. vulnificus identification method is essential. We evaluated the effectiveness of a two-step agar-based approach consisting of successive Vibrio isolation and cultivation on thiosulphate-citrate-bile salt sucrose (TCBS) agar and CHROMagar™ Vibrio for V. vulnificus in comparison with V. cholerae, V. parahaemolyticus, and V. alginolyticus. Our study contains isolates from water and sediment across a broad expanse of the Baltic Sea including 13 locations and two different summers, the time of year during which Vibrio infections are usually much more frequent. Confirmation of isolate species identity was carried out using molecular analyses. The two-step agar plating method performed well across different locations and timeframes in correctly identifying V. vulnificus by more than 80%, but the sensitivity in other Vibrio species varied. Thus, our approach yielded promising results as a potential tool for early V. vulnificus detection across a broad timeframe and transect of the Baltic Sea and potentially other brackish environments.