Sensitivities of Vibrio vulnificus to Chinese Herbs and Effect of Medicinal Bath on Vibrio vulnificus

Vibrio vulnificus can cause rotten-skin disease in marine fish aquaculture, resulting in a large number of deaths. The occurrence of rotten-skin disease is becoming severer and severer, leading to serious economic losses. In this study, the sensitivities of V. vulnificus to the 18 kinds of herbs, such as stone calamus and Chinese rhubarb, were determined using the agar diffusion method （susceptibility paper）. The results showed that Chinese gall, dark plum, sea buckthorn and eucalyptus leaves have bacteriostatic and bactericidal effects, while sappan wood, Chi- nese rhubarb, pomegranate bark, mentha and clove have strong bacteriostatic ef- fects. The Chinese medicinal herb bath showed good control effect on V. vulnificus.

Dynamic Expressions of Liver Tissue Apoptosis-related Genes of Vibrio Vulnificus Sepsis Rats and the Effects of Antibacterial Agents

Dynamic changes in mRNA expressions of liver tissue apoptosis-promoting genes Fas and Bax and apoptosis-inhibiting gene Bcl-2 of vibrio vulnificus sepsis rats were detected and the effects of antibacterial agents were examined. The rat model with Vibrio vulnificus sepsis （VV group） was established and some of the Vibrio vulnificus sepsis rats were treated with antibacterial agents （AA group）. The mRNA expressions of Fas, Bax and Bcl-2 were measured by reverse transcription polymerase chain reaction （RT-PCR）. As compared with normal control group （NC group）, the ex- pressions of Fas and Bax mRNA in liver tissue at all different time points in VV group were increased significantly （P〈0.05）, and the highest levels of Fas and Bax mRNA expressions were 6 and 12 h after the infection, respectively. At the same time, the expression of Bcl-2 mRNA in liver tissue at all different time points in VV group were decreased significantly （P〈0.05）, and the lowest level of Bcl-2 mRNA expression appeared 2 h after the infection. The mRNA expressions of Bcl-2 in liver tissue 9 and 12 h after the infection in AA group were increased significantly （P〈0.05） compared with NC group, while the expressions of Fas and Bax mRNA were not significantly different from those of NC group. Compared with VV group, the expression of Fas mRNA in AA group was decreased （P〈0.05） and Bax rnRNA was decreased significantly 12 and 16 h after the infection （P〈0.05）, while the expressions of Bcl-2 mRNA were increased significantly 9, 12 and 16 h after the infection （P〈0.05）. It is concluded that the mRNA expressions of liver tissue apoptosis-promoting genes Fas and Bax were increased remarkably in vibrio vulnificus sepsis rats, whereas the expression of apoptosis-inhibiting gene Bcl-2 mRNA was decreased obviously in sepsis rats in early stage. The treatment with cefoperazone sodium and levofloxacin lactate could inhibit the expression of Fas mRNA and Bax mRNA and enhance the expression ofBcl-2 rnRNA at the same time.

Resveratrol Prevents Vibrio vulnificus-Induced Sepsis by Attenuating Necroptosis

Objective This study investigated how the natural phytophenol and potent SIRT1 activator resveratrol(RSV)regulate necroptosis during Vibrio vulnificus(V.vulnificus)-induced sepsis and the potential mechanism.Methods The effect of RSV on V.vulnificus cytolysin(VVC)-induced necroptosis was analyzed in vitro using CCK-8 and Western blot assays.Enzyme-linked immunosorbent assays and quantitative real-time polymerase chain reaction,western blot,and immunohistochemistry and survival analyses were performed to elucidate the effect and mechanism of RSV on necroptosis in a V.vulnificus-induced sepsis mouse model.Results RSV relieved necroptosis induced by VVC in RAW264.7 and MLE12 cells.RSV also inhibited the inflammatory response,had a protective effect on histopathological changes,and reduced the expression level of the necroptosis indicator pMLKL in peritoneal macrophages,lung,spleen,and liver tissues of V.vulnificus-induced septic mice in vivo.Pretreatment with RSV downregulated the mRNA of the necroptosis indicator and protein expression in peritoneal macrophages and tissues of V.vulnificusinduced septic mice.RSV also improved the survival of V.vulnificus-induced septic mice.Conclusion Our findings collectively demonstrate that RSV prevented V.vulnificus-induced sepsis by attenuating necroptosis,highlighting its potency in the clinical management of V.vulnificus-induced sepsis.

Development of Quantitative Real-time Polymerase Chain Reaction for the Detection of Vibrio vulnificus Based on Hemolysin (vvhA) Coding System

Objective To establish a TaqMan real-time fluorescent quantitative PCR to detect Vibrio vulnificus based on the hemolysin gene （vvhA） coding cytolysin. Methods Primers and probes in the conserved region of the vvhA gene sequence were designed for the TaqMan real-time PCR to detect 100 bp amplicon from V. vulnificus DNA. Recombinant plasmid pMD19-vvhA100 was constructed and used as a positive control during the detection. Minimal amplification cycles （Ct value） and fluorescence intensity enhancement （ARn value） were used as observing indexes to optimize the reaction conditions of TaqMan real-time PCR. The TaqMan assay for the detection of Vbirio vulnificus was evaluated in pure culture, mice tissue which artificially contaminated Vibrio vulnificus and clinical samples. Results The established TaqMan real-time PCR showed positive results only for Vibrio vulnificus DNA and pMD19-vvhA100. The standard curve was plotted and the minimum level of the vvhA target from the recombinant plasmid DNA was 103 copies with a Ct value of 37.94±0.19, as the equivalent of 0.01 ng purified genomic DNA of Vibrio vulnificus. The results detected by TaqMan PCR were positive for the 16 clinical samples and all the specimens of peripheral blood and subcutaneous tissue of mice which were infected with Vibrio vulnificus. Conclusion TaqMan real-time PCR is a rapid, effective, and quantitative tool to detect Vibro vulnificus, and can be used in clinical laboratory diagnosis of septicemia and wound infection caused by Vibrio vulnificus.

Vibrio vulnificus Disease Prevalence

Vibrio vulnificus is a deadly disease that has been increasing in prevalence. In this study, we review both primary and secondary data to discuss the factors that are contributing to the increase of vibrio disease, causing a 41% increase between 1996 and 2005. It has also been shown that public health campaigns to limit vibrio infections have focused on raw seafood consumption. However, an estimated 42% of infections are now being caused by wound infection, rather than through contaminated seafood. This shows the disparities in addressing vibrio contamination through contaminated seawater and open wounds. This is particularly stressing as global warming is causing an increase in risk of contaminated seawater. Reasons for this increase are discussed, and also possible solutions are presented for public health interventions to help mitigate this rise in vibrio infections.

<i>Vibrio vulnificus</i>septicemia and necrotizing fasciitis in the patients with liver cirrhosis and diabetes mellitus

Vibrio vulnificus (V. vulnificus) infection is a rare disease in Japan but the leading cause of death related to raw seafood consumption. We hereby reported a successfully treated case of V. vulnificus septicemia, severe necrotizing fasciitis, disseminated intravascular coagulation and multiple organ failure after raw perch consumption with underlying alcoholic liver cirrhosis and diabetes mellitus. It is the first report of a case of V. vulnificusinfection caused by eating raw perch, whereas V. vulnificus infection should be suspected in all of middle-aged to elderly men with underlying immunosuppressive diseases, who have recent consumption of raw seafood or contact with seawater, especially in the summer. The levels of HbA1c and glycoalbumin were not high in the present case, however, obvious hyperglycemia was found even after the infection had completely healed. On reviewing 166 case of V. vulnificus infection in Japan including ours, the complication of diabetes mellitus, one of immunocompromised condition, was found only in 11%, although it had been reported that individuals strongly suspected of having diabetes were 17.2% among the Japanese male population aged from 40 to 74 years. Because diabetes mellitus might be underdiagnosed in the previous reports, intensive examinations are considered to be necessary in order to correctly diagnose diabetes mellitus in patients with severe V. vulnificus infection.

A case report of Vibrio vulnificus infection in the lower limbs by sea fish stabbing and literature review

Objective: To summarize and analyze the clinical treatment experience of Vibrio vulnificus sepsis and improve the success rate of treatment. Methods: The clinical data of a patient with vibrio vulnificus sepsis admitted to the Department of Critical Care Medicine, the First Affiliated Hospital of Hainan Medical College in October 2019 were analyzed. Summarize the experience of treatment and compare with other literatures to analyze the most reasonable clinical treatment of vibrio vulnificus sepsis. Results: The patient was acutely ill, the disease progressed rapidly, fever, soft tissue swelling and pain, necrosis, ulceration and pus, systemic multiple organ failure, family members finally gave up treatment, microbiology culture prompted: Vibrio parahaemolyticus. Conclusions: Early intervention, blood purification, debridement, removal of infection sources and inflammatory mediators can improve the survival rate of patients with Vibrio vulnificus infection. Due to the high mortality rate, clinicians should pay attention to it.

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-resistantV. Vulnificus and the protection of their vital organs.Methods:An increasing concentration gradient method was used to induce multidrug-resistantV. 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-resistantV. 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-resistantV. 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)-1β, IL-6, and tumor necrosis factor (TNF)-α expression in serum (IL-1β:p = 0.010, IL-6:p = 0.029, TNF-α:p = 0.025) and inhibited the protein expression of the inflammatory factors IL-1β, IL-6, TNF-α in liver (IL-1β:p = 0.010, IL-6:p = 0.011, TNF-α:p = 0.037) and kidney (IL-1β:p = 0.016, IL-6:p = 0.011, TNF-α:p = 0.008) tissues. Bergamottin did not affect the proliferation of multidrug-resistantV. 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-resistantV. Vulnificus infection. This study can serve as a reference and guide the development of novel clinical treatment strategies forV. Vulnificus.

Extracellular matrix(ECM)pathway involved in skin immune response of Cynoglossus semilaevis upon Vibrio vulnificus infection

Half-smooth tongue sole(Cynoglossus semilaevis)is regarded as a significant commercial marine fish species in China,and frequent outbreaks of vibriosis has led to substantial economic losses.In this study,we investigated the molecular mechanisms underlying the involvement of the extracellular matrix(ECM)signaling pathway in the skin immune response of C.semilaevis infected with Vibrio vulnificus.The results showed that most differentially expressed proteins(DEPs)were identified through iTRAQ proteome analysis,and ten ECM-related proteins were screened at 12 and 36 h post-infection(hpi).Notably,several DEPs associated with ECM including HSPG,FN,COCH,LAMB1,LAMC1,TSPN,COL6A1,AMBP,HX and ITGA6 were tested for their expression at the transcriptional level during V.vulnificus infection using qRT-PCR assay.The analysis of protein-protein interaction(PPI)showed that the integrin ITGA6 exhibited obvious interactions among ECM-related DEPs.Furthermore,the spatio-temporal expression of the Csitga6 gene was highest in the skin,gill,muscle,and spleen,but lower in the liver,kidney and intestine.To our knowledge,this is the first report on the involvement of ECM pathway in the skin immune response to V.vulnificus infection,and provides a reference for further study of the ECM mechanism in the mucosal immune response of marine fish.

Effect of antimicrobial agents on the toll-like receptors and inflammatory cytokines in liver tissue of the alcohol-induced liver disease in rats with Vibrio vulnificus sepsis

Background Septicemia and inflammation-mediated septic shock caused by Vibrio vulnificus （VV） is strongly associated with chronic liver disease. This study examined the effects of antimicrobial therapy on expression of hepatic toll-like receptors and inflammatory cytokines in rats with alcohol-induced liver disease complicated by VV sepsis. Methods Male Sprague-Dawley rats were assigned to the following treatment groups： normal control （N）, alcoholic liver disease control （A）, antimicrobial-treated alcoholic liver disease control （AA）, alcoholic liver disease with VV sepsis （AV）, and antimicrobial-treated alcoholic liver disease with VV sepsis （AVA）. Alcohol-induced liver disease was observed in all groups except N. Expression of mRNAs encoding hepatic toll-like receptors 2 and 4, myeloid differentiation protein-2, tumor necrosis factor-α （TNF-α）, interleukin （IL）-1β, IL-6 and IL-10 was determined by RT-PCR. Results mRNAs encoding toll-like receptors 2 and 4 and myeloid differentiation protein-2 were significantly up-regulated in group AV as compared to control groups at 2-24 hours of sepsis; peak expression occurred at 12 hours. These mRNAs were also up-regulated in group AVA but to lesser degrees than in group AV at comparable time post-infection, mRNAs encoding TNF-α, IL-1β and IL-6 were significantly elevated in group AV as a function of infection. In group AVA as compared to AV, expression of TNF-α and IL-1β mRNAs was lower at 12-24 hours post-infection and expression of IL-6 mRNA was lower at 24 hours post-infection. Compared with control groups, IL-10 mRNA expression in group AV was markedly higher at 12-24 hours of sepsis. Expression of IL-10 mRNA was lower in group AVA as compared to AV at 24 hours of sepsis. Conclusions Antimicrobial therapy reduces expression of toll-like receptors and cytokines in rats with alcohol-induced liver disease complicated by VV sepsis. Monitoring hepatic toll-like receptor and cytokine expression during antibiotic therapy may be valuable for determining the course of VV sepsis in subjects with liver disease.

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

Necrotizing fasciitis （NF） caused by vibrio vulnificus （VV） is a rare but fatal clinical emergency characterized by hemorrhagic bullous skin lesions,septic shock and multiple organ dysfunction syndrome （MODS）,commonly occurring in patients with chronic liver disease.1,2 In general,mortality of these patients is higher than 60%,which could approach 100% when complicated with septic shock.Therefore,early diagnosis and effectively surgical intervention in emergency room are the mainstays for better outcomes of these patients.However,many emergency physicians lack experiences in diagnosing and managing vibrio NF.Furthermore,due to the preexisting severe medical complications including hypotension,lactic acidosis,coagulation disorders and thrombocytopenia,the optimal therapeutic strategies for these critical patients should obviously be evaluated on a patient-to-patient basis,which remains largely undefined.

Localization of Vibrio vulnificus infection in dendritic cells and its effects on the cytoskeleton

Background Vibrio vulnificus （Vv） is an estuadne bacterium that can cause primary septicemia as well as serious wound infections. However, little is known about the mechanisms by which Vv infects dendritic cells （DCs） and its effects on cytoskeleton. In this study, we aimed to investigate the invasion, internalization, and the organelles damage of the cultured dendritic cells （a DC 2.4 strain） during Vv infection. Methods The study model was the cultured DCs infected by a Vv 1.758 strain. Electron microscopy was used to observe the localization of bacteria at the different time points of infection, cell morphology, and the process of organelles changes. The cytoskeleton structure including the microfilaments and the microtubules rearrangement was examined under a fluorescence microscope. Results The Vv were pinocytosised into the DC cells through double-sides, and localized at 1-2 μm of the inner side membrane. It took 1.3, 1.9, and 3.4 hours to reach the infection ratio of 25%, 50%, and 75%, respectively. Using electron microscopy, the DCs had been observed to have developed chromatin aggregation within 4.0 hours, and significant cytoskeleton structure disruption was noted within 6.0 hours.

Evaluation of the antibacterial mechanism and biofilm removal effect of eugenol on Vibrio vulnificus and its application in fresh oysters

Eugenol is a natural active substance with high antibacterial activity,but its antibacterial activity against Vibrio vulnificus has not been extensively studied.The purpose of this study was to investigate the antibacterial and biofilm-clearing abilities and potential mechanisms of eugenol against V.vulnificus,and to provide knowledge for the use of eugenol to prevent oyster contamination.It was found that eugenol had an encouraging antibacterial effect on V.vulnificus with a minimum inhibitory concentration(MIC)of 0.2 mg/mL.The accumulation of reactive oxygen species(ROS)and the increase of malondialdehyde(MDA)content suggest that oxidative stress is involved in the bactericidal mechanism.Moreover,cell membrane hyperpolarization,changes in cell membrane integrity and morphology suggest that eugenol can reduce the permeability and integrity of cell membranes in V.vulnificus.In addition,eugenol produced a significant biofilm clearance effect on V.vulnificus,as evidenced by the reduced amount of biofilm and the reduction of polysaccharides and viable cells in the biofilm.Finally,eugenol was able to effectively inhibit the activity of V.vulnificus in artificially contaminated oyster at 4°C and 25°C.But sensory analysis showed that 0.10%eugenol was most acceptable to trained panelists.All of these highlight the great promise of eugenol as a natural bacteriostatic agent for the food industry.

创伤弧菌对水产养殖的影响及致病机制

随着水产养殖向集约化和工厂化养殖发展,水产养殖动物病害频繁发生,在诸多病原菌中,创伤弧菌(Vibrio vulnificus)是水产养殖动物的主要病原体之一,给水产养殖业造成巨大损失。文章简要介绍了创伤弧菌对水产养殖动物的影响及致病机理,同时对创伤弧菌疾病预防和治疗提出更环保的治疗方式,以期加强对创伤弧菌的致病性和危害性的重视与研究,有助于合理地进行疾病诊断和采取防控措施,同时为进一步深入研究创伤弧菌的致病机理奠定重要基础。

矛尾复虾虎鱼溃疡病病原创伤弧菌的鉴定

2009年8月江苏连云港赣榆县多家虾蟹养殖塘混养的矛尾复虾虎鱼(Synechogobius hasta)大量死亡,从病鱼深层溃烂组织及肝脏中分离出大量优势生长的细菌,人工感染试验证明分离菌对虾虎鱼的半数致死量(LD50)为1.63×106 cfu/g。对分离菌进行了形态特征、理化特性和分子生物学等方面的分析。一方面,分离菌株16S rRNA和gyrB基因序列的BLAST搜索及最大简约法分析均表明其与创伤弧菌(Vibrio vulnificus)亲缘关系最近;另一方面,分离菌革兰氏阴性,氧化酶阳性,葡萄糖发酵型。因此,引起江苏连云港赣榆县矛尾复虾虎鱼溃疡病病原菌是弧菌属的创伤弧菌。

三株弧菌热休克蛋白70基因的克隆和序列分析

根据GenBank中同类蛋白序列设计特异PCR引物,从2株创伤弧菌Vibrio vulnificus和1株河弧菌Vibriofluvialis中扩增出热休克蛋白70(heat shock protein,hsp70)基因片段。对这3个片段进行克隆、测序和分析的结果表明,3个片段长均为1 911 bp,包含完整的hsp70 ORF,编码636个氨基酸。它们的氨基酸序列与GenBank中其它物种hsp70的氨基酸序列比较发现,2株创伤弧菌hsp70基因序列和同种其它菌株的同源性高,达98%以上;而河弧菌的hsp70序列属首次克隆;与多种原核和真核生物的hsp70氨基酸序列一起构建了系统进化树,结果支持传统的分类结果。

Expression of high mobility group protein B1 in the lungs of rats with sepsis

BACKGROUND： Vibrio vulnifi cus inside the body could activate the NF-!B signaling pathwayand initiate the inflammatory cascade. The lung is one of the earliest organs affected by sepsisassociated with acute lung injury. High mobility group protein B1 （HMGB1） is an important late-actingpro-infl ammatory cytokine involving in the pathophysiology of sepsis. It is also involved in the injuryprocess in the lung, liver and intestine. There has been no report on the involvement of HMGB1 inVibrio vulnifi cus sepsis-induced lung injury.METHODS： Sixty rats were randomly divided into a normal control group （group A, n=10） anda Vibrio vulnificus sepsis group （group B, n=50）. Sepsis was induced in the rats by subcutaneousinjection of Vibrio vulnificus （concentration 6×108 cfu/mL, volume 0.1 mL/100g）） into the left lowerlimbs. The rats in group B were sacrifi ced separately 1, 6, 12, 24, and 48 hours after the infection.Their lungs were stored as specimens, lung water content was measured, and lung pathology wasobserved under a light microscope. The expressions of the HMGB1 gene and protein in the lungswere detected by RT-PCR and Western blot. Data were analyzed with one-way analysis of variance（ANOVA） and the LSD method for pair-wise comparison between the two groups. P〈0.05 wasconsidered statistically signifi cant.RESULTS： Compared to group A （0.652±0.177）, HMGB1 mRNA expression in the lungs ofgroup B was signifi cantly higher at 0 hour （1.161±0.358, P=0.013）, 24 hours （1.679±0.235, P=0.000）,and 48 hours （1.258±0.274, P=0.004） （P〈0.05）, and peaked at 24 hours. Compared to group A（0.594±0.190）, HMGB1 protein expression at 6 hours （1.408±0.567, P=0.026） after infection wassignificantly increased （P〈0. 05）, and peaked at 24 hours （2.415±1.064, P=0.000） after infection.Compared to group A （0.699±0.054）, lung water content was significantly increased at 6 hours（0.759±0.030, P=0.001）,12 hours （0.767±0.023, P=0.000）, 24 hours （0.771±0.043, P=0.000） and 48hours （0.789±0.137, P=0.000） after infection （P〈0.05）. Compared to group A, pathological changesat 12 hours in group B indicate marked pulmonary vascular congestion, interstitial edema andinfl ammatory infi ltration. Alveolar cavity collapse and boundaries of the alveolar septum could not beclearly identifi ed.CONCLUSION： Vibrio vulnifi cus sepsis can lead to injury in rat lungs, and increased HMGB1expression in lung tissue may be one of the mechanisms for injury from Vibrio vulnifi cus sepsis.

Successful surgical treatment of polybacterial gas gangrene confirmed by metagenomic next-generation sequencing detection:A case report

BACKGROUND We report on a case of Vibrio vulnificus(V.vulnificus)detected by metagenomics next-generation sequencing(mNGS)in a 53-year-old male patient with polymicrobial gas gangrene and successful treatment by surgery.This report raises awareness among dermatologists that when a patient is clinically suspected of a special type of pathogenic infection,the mNGS method should be preferred to identify the patient’s pathogen infection as soon as possible and then take effective treatment in time to save patients’lives.CASE SUMMARY A 53-year-old male who worked in the aquatic market complained of redness and swelling of the lower limbs,blisters and ulcers with fever for 3 d.We used mNGS to test the pathogens in ulcer secretions.The results were returned in 24 h and indicated:V.vulnificus,Fusobacterium necrophorum,Staphylococcus haemolyticus,Staphylococcus aureus,Streptococcus dysgalactiae and Klebsiella aerogenes.This patient was diagnosed with V.vulnificus infection.The emergency operation was performed immediately under combined lumbar and epidural anesthesia:Left leg expansion and exploration(August 10,2021).After surgery,we continued to use piperacillin sodium tazobactam sodium 4.5 g every 8 h and levofloxacin 0.5 g for anti-infection treatment.The patient underwent further surgery under lumbar anesthesia on August 17,2021 and August 31,2021:Left leg deactivation and skin grafting,negative pressure closed drainage and right thigh skin removal.After treatment,the transplanted flap survived.CONCLUSION We could confirm the diagnosis of Vibrio vulnificus infection within 24 h through mNGS detection and then immediately performed emergency surgery.

DISEASE RESISTANCE AND HUMORAL IMMUNOMODULATORY EFFECTS OF VITAMIN C ON GROUPER, EPINEPHELUS AWOARA

The disease resistance and humoral immunomodulatory effects of vitamin C administered orally to grouper, Epinephelus awoara maintained on a frozen fish diet supplemented with vitamin C at 500, 1000, 1500 and 2000 mg/kg were investigated. After 20 weeks, the growth rates of the groups with high level of vitamin C apparently increased. The untreated fish had symptoms of vitamin C deficiency. The endogenous liver tissue vitamin C levels were found to reflect well the dietary treatments. After intraperitoneal injection or bath challenge with a virulent strain of Vibrio vulnificus , fish fed with high level vitamin C showed significantly higher survival rate compared with the normal control group. Vaccination with formalin inactivated V. vulnificus significantly enhanced the specific antibody production in fish treated with vitamin C, and completely protected from strong bacterial challenge the groups fed on fish with vitamin C 1500 and 2000 mg/kg diet.

A culture-free method for detection of Vibrio vulnif icus from coastal seawater based on loop-mediated isothermal amplification targeting vcgC gene

Vibrio vulnificus is an opportunistic pathogen widely distributed in estuarine and coastal seawaters. In this study, a culture-free method was developed to rapid detection of Vo vulnificus in all seasons, based on loop-mediated isothermal amplification （LAMP） targeting virulent-correlated gene （vcg）. The new assay method allows differentiation between the virulent and non-virulent strain of V. vulnificus accurately. This method also allows effective detection of the pathogeny in winter when the bacterium lives in the viable but non-culturable （VBNC） state. A total of 30 costal seawater samples collected in all seasons were used for the evaluation of this method. The results show that the method is sensitive, accurate and convenient.