In-vitro anti-<Emphasis Type="Italic">Vibrio</Emphasis> spp. activity and chemical composition of some Tunisian aromatic plants

The chemical composition of five aromatic plants (Mentha longifolia, M. pulegium, Eugenia caryophyllata, Thymus vulgaris and Rosmarinus officinalis) frequently used in food preparation in Tunisia was analysed by GC-MS. The antimicrobial effect of the essential oils obtained from these plants was tested against Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio fluvialis strains. Thyme oil exhibited a high level of antimicrobial activities against Vibrio spp. strains. The diameter of the zones of growth inhibition for V. parahaemolyticus species was interestingly high (ranging from 14.66 to 28 mm). The MIC and MBC values were interestingly low for thyme oil (MIC 0.078–0.156 mg/ml) and (MBC >0.31–1.25 mg/ml). These results showed that these plants especially thyme and clove, can be to be used for seafood preparation to protect against contamination by Vibrio spp. strains. An erratum to this article can be found at     

基于高通量共聚焦激光扫描显微镜方法定量分析副溶血性弧菌生物被膜结构

【目的】副溶血性弧菌是水产品中常见的食源性致病菌,生物被膜的形成对副溶血性弧菌的环境生存和传播至关重要。这项工作的目的是评估临床和环境中分离出的44株副溶血性弧菌菌株形成的生物被膜的结构多样性。【方法】该研究基于共聚焦激光扫描显微镜的高通量方法,使用与高分辨率成像兼容的96孔微量滴定板,结合结构分析软件ISA-2来研究生物被膜形成和结构,分析22株食品与22株临床来源的副溶血性弧菌菌株形成的生物被膜结构参数(生物体积、平均厚度、粗糙系数)。【结果】CLSM图像显示,44株副溶血性弧菌菌株在培养48h后能够形成3D结构,进一步比较分析了临床来源菌株与环境来源菌株形成的生物被膜结构异同,发现临床菌株生物被膜的变异系数比环境菌株生物被膜的变异系数小,且同时携带tdh和trh两种毒力因子的菌株生物被膜变异性最小。凝聚层次聚类分析结果显示,副溶血性弧菌生物被膜可以分为致密且表面光滑(39%)、斑驳且表面粗糙(27%)、疏松且表面坑洼(34%),临床菌株易形成致密且表面光滑和斑驳且表面粗糙的生物被膜,而环境菌株易形成致密且表面光滑和疏松且表面坑洼的生物被膜。【结论】该研究深入了解了副溶血性弧菌生物...     

Demonstration and Partial Characterization of a Bacterial Growth Enhancer in sera

During our research into the pathogenesis of Vibrio parahaemolyticus, we noticed that the concentration of serum added to the tissue culture medium (Dulbecco’s modified Eagle’s medium: DMEM) greatly affected its growth. Using gel filtration column chromatography, we clearly demonstrated that serum contains not only a bacterial growth inhibitor (BGI) but also a bacterial growth enhancer (BGE) for Vibrio parahaemolyticus. Our data indicate that the BGI is transferrin, whereas the BGE seems to be an undescribed small molecule (molecular weight of 1,000–3,000 Da) and is associated with magnesium and molybdenum ions. BGE activity was not decreased by heat treatment (at 60 or 100°C for 30 min) and affected the growth rate of a wide range of Gram-negative and Gram-positive bacteria. The addition of EDTA into DMEM lowered the growth rate, whereas the addition of BGE restored the growth activity. This study suggests that sera contain a previously undescribed small BGE molecule.     

信号分子AI-2合成关键基因luxS对副溶血性弧菌生物学特性的影响

[背景]副溶血性弧菌是全球范围重要的食源性病原菌,能引起急性肠胃炎。群体感应系统LuxS/AI-2影响细菌的生物学特性,为研究副溶血性弧菌的传播机制和控制技术提供了新的途径。[目的]探讨群体感应信号分子AI-2合成关键基因luxS对海产品中分离的副溶血性弧菌Vp2009027生物学特性的影响。[方法]利用自杀质粒同源重组技术敲除信号分子AI-2合成关键基因luxS,构建副溶血性弧菌Vp2009027的luxS基因缺失株,通过比较野生株与luxS基因缺失株的生长曲线、AI-2活性、运动能力、生物膜形成能力和耐药性,分析LuxS/AI-2系统对副溶血性弧菌生物学特性的影响。[结果]构建了副溶血性弧菌Vp2009027的luxS基因缺失株,野生株和luxS基因缺失株的生长无明显差异,luxS基因的缺失导致AI-2合成受阻、运动能力和生物膜形成能力增强、四环素耐药性降低。[结论]luxS基因对副溶血性弧菌的生物学特性具有重要的调控作用,为进一步研究副溶血性弧菌的传播机制和研发控制技术提供基础。     

Isolation and characterization of a toxic intracellular protein from Vibrio parahaemolyticus

A toxic factor released from disrupted cells of Vibrio parahaemolyticus was partially purified by gel filtration after precipitation with (NH₄)₂SO₄ at 40% saturation. The factor, which was a thermostable protein of 63 kDa, lysed human erythrocytes at a concentration of 0.15 g ml^-1. Its LD₅₀ by intravenous injection into mice was 6.4 g. Fluid accumulated in suckling mice force-fed with the toxic material (1 to 25 g). Haemolytic activity, which occurred maximall at 37°C and pH 7.0 was enhanced by Ca²⁺, Cu²⁺ and Zn²⁺, each at 1 mm. Anti-toxic-factor serum agglutinated V. parahaemolyticus cells. The factor may play a role in the pathogenesis of V. parahaemolyticus infections and in the host's defence mechanisms against infection by the microorganism.     

Inhibitory activity of probiotics <Emphasis Type="Italic">Streptococcus phocae</Emphasis> PI80 and <Emphasis Type="Italic">Enterococcus faecium</Emphasis> MC13 against Vibriosis in shrimp <Emphasis Type="Italic">Penaeus monodon</Emphasis>

Occurrence of widespread epizootics among larval and cultured shrimp has put on viable preventive approaches such as application of probiotics on a high priority in aquaculture. In the present study, four probiotics bacteria were isolated from marine fish and shrimp intestine based on the antagonistic activity and nonpathogenic to the host. The isolates of probiotics strains Streptococcus phocae PI80, Enterococcus faecium MC13, Lactococcus garvieae LC149, B49 and one commercial probiotics (ECOFORCE) were fed to post larvae of Penaeus monodon obtained from two different hatcheries to analyze the growth and protection against Vibrio harveyi and V. parahaemolyticus. Growth of P. monodon post larvae fed with probiotic strain S. phocae PI80 was significantly (P < 0.001) higher when compared with control and other three strains in both experiments. The treatment of post larvae with B49 reduced the growth as well as Specific growth rate. Among the three probiotic strains S. phocae PI80 and E. faecium MC13 have effectively inhibited the pathogens. In experiment I high survival (92%) were observed in S. phocae PI80 treated post larvae when challenged with Vibrio harveyi followed by E. faecium MC13 (84%), B49 (76%) and ECOFORCE (68%) but PI80 did not protect the post larvae in the same experiment when they were exposed to V. parahaemolyticus. The probiotic isolate of MC13 has protected the post larvae against V. parahaemolyticus when compared to other probiotics and control. Similarly in the second experiment feeding of S. phocae enhanced the survival of larvae when challenged with V. harveyi. The laboratory studies proved that bacterial probionts S. phocae and E. faecium isolated from shrimp and brackishwater fish has potential applications for controlling pathogenic vibriosis in shrimp culture.     

Lethal Effect of Fresh Sea Water on Vibrio parahaemolyticus and Isolation of Bdellovibrio Parasitic against the Organism

Halophilic Bdellovibrio, which is parasitic and lytic to Vibrio parahaemolyticus, was isolated from fresh sea water in the winter. It had a lethal effect on V. parahaemolyticus. The optimum temperature for multiplication ranged from 25 C to 30 C and growth was not observed at 35 C. Plaque numbers of the isolate reached a maximum in 17 hr under conditions of shaking at 25 C in autoclaved sea water supplemented with V. parahaemolyticus cells, and were as high as ten times the number of host cells. With respect to the host-suspended medium, the isolate multiplied in natural sea water ten times more than in Herbst's artificial sea water but did not grow in saline. V. parahaemolyticus, Vibrio alginolyticus and several species in the Vibrio genus were susceptible to the parasite on the basis of plaque formation but Escherichia coli and Staphylococcus aureus were not.     

副溶血性弧菌脂蛋白定位系统转运蛋白结构与功能的生物信息学分析

【目的】副溶血性弧菌是一种重要的人畜共患病原菌,脂蛋白定位系统(Localization of lipoprotein system,Lol)负责该菌脂蛋白的转运与定位,与其致病力及耐药性密切相关,对Lol系统转运蛋白进行系统的生物信息学分析,有助于推动副溶血性弧菌致病与耐药机理的进一步研究。【方法】本文通过生物信息学分析技术,结合ExPASy在线工具、SignalP 4.0 Server、TMHMM-2.0、STRING、SWISS-MODEL等软件,分析了副溶血性弧菌Lol系统转运蛋白LolA-E及LolCD_2E的基本性质、蛋白互作关系及三级结构。【结果】LolA和LolB为酸性亲水蛋白,含信号肽位点,无跨膜区域。LolC和LolE为碱性疏水膜蛋白,LolCD_2E为中性疏水膜蛋白,LolC-E及LolCD_2E均无显著的信号肽位点。蛋白相互作用网络显示,LolA–E五个蛋白的编码基因均共表达,负责脂蛋白的合成与转运,并与BamA、Pal、MacB、CmeC等外膜蛋白具有密切的互作关系。三级结构同源建模发现,副溶血性弧菌与大肠杆菌拥有相似的LolA和LolB结构,LolC-E含有MacB蛋白的同源结构,赋予了该系统消耗ATP运输脂蛋白的重要功能。此外,本研究还首次发现了副溶血性弧菌LolC和LolE中存在一段保守的Hook结构,是LolCD_2E复合物与LolA结合并转运脂蛋白的关键区域。【结论】本研究为副溶血性弧菌Lol系统转运蛋白的表达纯化、结构与功能的研究提供了重要的数据基础,为后续抗菌药物的研发提供了新型作用靶点。     

Production of a recombinant chitin oligosaccharide deacetylase from Vibrio parahaemolyticus in the culture medium of Escherichia coli cells

An open reading frame (ORF) encoding chitin oligosaccharide deacetylase (Pa-COD) gene and its signal sequence was cloned from the Vibrio parahaemolyticus KN1699 genome and its sequence was analyzed. The ORF encoded a 427 amino acid protein, including the 22 amino acid signal sequence. The deduced amino acid sequence was highly similar to several bacterial chitin oligosaccharide deacetylases in carbohydrate esterase family 4. An expression plasmid containing the gene was constructed and inserted into Escherichia coli cells and the recombinant enzyme was secreted into the culture medium with the aid of the signal peptide. The concentration of the recombinant enzyme in the E. coli culture medium was 150 times larger than that of wild-type enzyme produced in the culture medium by V. parahaemolyticus KN1699. The recombinant enzyme was purified to homogeneity from culture supernatant in an overall yield of 16%. Substrate specificities of the wild-type and the recombinant enzymes were comparable.     

红海榄内生细菌多样性及其抑制鱼类致病菌活性研究

红海榄为红树科红海属植物,广泛分布于热带海岸的红树林中,为真红树植物的典型代表。该研究以广西山口红树林国家级自然保护区红海榄为对象,应用稀释涂布法和三线法从红海榄各组织中分离出17株内生细菌,通过各菌株形态特征观察及利用16S rRNA序列基因和韦恩图分析其内生细菌多样性。结果表明:17株内生细菌分属3个门5科7属8种,Micromonospora和Mangrovibacter属为其优势类群。进一步分析发现红海榄根茎叶等组织的内生细菌类群差别较大,仅有1个相同菌属,17株菌中有3株菌与已有细菌物种典型菌株的全长16S rRNA基因相似性低于97%,代表着潜在的新属或新种。运用纸片法研究8株内生细菌发酵液的乙酸乙酯提取物对鱼类致病菌副溶血弧菌活性抑制效果,发现3株内生细菌(H003、H013、H009,浓度5 mg·m L-1)的代谢产物对副溶血弧菌具有较强的抑菌活性,其抑菌圈直径分别达到(8.4±0.07)、(8.2±0.07)、(8.3±0.14)mm。该研究结果表明红海榄中具有较好的内生细菌多样性和抑菌活性,为今后研究其内生细菌的化学多样性及其应用提供了重要的物质基础。     

水产动物副溶血性弧菌的拮抗菌研究进展

细菌性疾病的爆发常造成水产养殖业的巨大经济损失,其中副溶血性弧菌(Vibrio parahaemolyticus)引起的细菌性疾病更是引起了人们的关注.拮抗菌在代谢活动中通过分泌抗菌物质直接对病原菌产生抑制或竞争作用来抑制或杀死病原菌,在病害防治中发挥着重大作用.对副溶血性弧菌拮抗菌的种类、产生拮抗物质的种类、筛选拮抗...     

副溶血性弧菌耐热性直接溶血素(TDH)的研究进展

副溶血性弧菌(Vibrio parahaemolyticus)是海产品中一种常见的食源性致病菌,常导致水产养殖动物患病或者引起食物中毒。耐热性直接溶血素(thermotolerant direct hemolysin,TDH)是副溶血性弧菌最为重要的致病因子之一。本文围绕tdh基因在弧菌属中的广泛分布与传播、tdh基因的多样性及其表达调控、TDH的蛋白结构及其生物活性进行了综述,并对未来TDH的研究方向进行了展望。旨在进一步了解由副溶血性弧菌感染所引起的病症,为预防副溶血性弧菌的感染和临床治疗提供理论支撑。     

Occurrence and distribution of virulent strains of Vibrio parahaemolyticus in seafoods marketed from Cochin (India)

This study was aimed for the detection of Vibrio parahaemolyticus by biochemical and molecular methods in seafood samples collected from the markets of Cochin located at the southwest coast of India. A total of seventy-two V. parahaemolyticus cultures were isolated by selecting sucrose and cellobiose non-fermenting colonies. All the biochemically confirmed strains were found to have 368-bp toxR gene fragment, while an additional 24% of the samples were confirmed as V. parahaemolyticus by toxR based polymerase chain reaction (PCR) from enrichment broths. PCR based methods are used to detect tdh, trh, and orf8 genes for the identification of pathogenic and pandemic V. parahaemolyticus. Only one out of two urease positive isolates amplified the trh (500bp) gene. About 10% of the isolates showed weak haemolysis and none were found to amplify tdh (269 bp) and orf8 (746 bp) genes, thus indicating the meager incidence of pandemic strains from this area. The incidence of trh positive isolates from market samples signals towards the adoption of stringent seafood safety measures for the products meant for human consumption.     

Heterodisaccharide 4-O-(N-acetyl-β-D-glucosaminyl)-D-glucosamine is an effective chemotactic attractant for Vibrio bacteria that produce chitin oligosaccharide deacetylase

Aims: To investigate the attractant effect of 4‐O‐(N‐acetyl‐β‐d ‐glucosaminyl)‐d ‐glucosamine (GlcNAc‐GlcN) in the chemotaxis of Vibrio bacteria that produce carbohydrate esterase (CE) family 4 chitin oligosaccharide deacetylase (COD), an enzyme that catalyzes the production of GlcNAc‐GlcN from N,N′‐diacetylchitobiose (GlcNAc)₂. Methods and Results: The chemotactic effect of disaccharides from chitin on several strains of Vibrio bacteria was investigated using an agar gel lane‐migration method. The results demonstrated that GlcNAc‐GlcN functions as an effective chemoattractant in the CE family 4 COD‐producing vibrios, Vibrio parahaemolyticus and Vibrio alginolyticus. In contrast, this phenomenon was not observed in Vibrio nereis or Vibrio furnissii, which lack genes encoding this enzyme. From transmission electron microscope observation of V. parahaemolyticus cells following the chemotaxis assay, GlcNAc‐GlcN appears to stimulate polar flagellum rotation. Conclusions: GlcNAc‐GlcN is a specific chemoattractant for the CE family 4 COD‐producing vibrios, V. parahaemolyticus and V. alginolyticus. Significance and Impact of the Study: It was clarified for the first time that GlcNAc‐GlcN functions as a signalling molecule in the chemotaxis of Vibrio bacteria that have an ability to produce CE family 4 COD, which generate GlcNAc‐GlcN from (GlcNAc)₂.     

The unique role of siderophore in marine-derived Aureobasidium pullulans HN6.2

The l-ornithine-N ⁵-monooxygenase structural gene (SidA gene, accession number: FJ769160) was isolated from both the genomic DNA and cDNA of the marine yeast Aureobasidium pullulans HN6.2 by inverse PCR and RT-PCR. An open reading frame of 1,461 bp encoding a 486 amino acid protein (isoelectric point: 7.79) with calculated molecular weight of 55.4 kDa was characterized. The promoter of the gene (intronless) was located from −1 to −824 and had three HGATAR boxes which were putative binding motifs for the respective DNA-binding motifs and one CATA box. The SidA gene in A. pullulans HN6.2 was disrupted by integrating the hygromycin B phosphotransferase (HPT) gene into Open Reading Frame of the SidA gene using homologous recombination. Of all the disruptants obtained, one strain S6 (∆sidA) did not synthesize both intracellular and extracellular fusigen so that it could not inhibit growth of the pathogenic bacteria Vibrio anguillarum and Vibrio parahaemolyticus. The disruptant S6 did not grow in the iron-deplete medium and seawater medium because cell budding was stopped, but could grow in the iron-replete medium with 10 μM Fe³⁺ and Fe²⁺. H₂O₂ in the medium was more toxic to the disruptant S6 than to its wild type HN6.2. Thus, we infer that the fusigen produced by the marine-derived A. pullulans HN6.2 can play a unique role in chelating, uptake and concentration of iron to maintain certain proper physiological functions within the cells and secretion of siderophore may represent an efficient tool to eliminate competitors to compete for limiting nutritional resources in marine environments.     

Activities of Alkyl Hydroperoxide Reductase Subunits C1 and C2 of Vibrio parahaemolyticus against Different Peroxides

Alkyl hydroperoxide reductase subunit C gene (ahpC) functions were characterized in Vibrio parahaemolyticus, a commonly occurring marine food-borne enteropathogenic bacterium. Two ahpC genes, ahpC1 (VPA1683) and ahpC2 (VP0580), encoded putative two-cysteine peroxiredoxins, which are highly similar to the homologous proteins of Vibrio vulnificus. The responses of deletion mutants of ahpC genes to various peroxides were compared with and without gene complementation and at different incubation temperatures. The growth of the ahpC1 mutant and ahpC1 ahpC2 double mutant in liquid medium was significantly inhibited by organic peroxides, cumene hydroperoxide and tert-butyl hydroperoxide. However, inhibition was higher at 12°C and 22°C than at 37°C. Inhibiting effects were prevented by the complementary ahpC1 gene. Inconsistent detoxification of H₂O₂ by ahpC genes was demonstrated in an agar medium but not in a liquid medium. Complementation with an ahpC2 gene partially restored the peroxidase effect in the double ahpC1 ahpC2 mutant at 22°C. This investigation reveals that ahpC1 is the chief peroxidase gene that acts against organic peroxides in V. parahaemolyticus and that the function of the ahpC genes is influenced by incubation temperature.     

Genetic analysis of the capsule polysaccharide (K antigen) and exopolysaccharide genes in pandemic <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> O3:K6

Background  

Pandemic Vibrio parahaemolyticus has undergone rapid changes in both K- and O-antigens, making detection of outbreaks more difficult. In order to understand these rapid changes, the genetic regions encoding these antigens must be examined. In Vibrio cholerae and Vibrio vulnificus, both O-antigen and capsular polysaccharides are encoded in a single region on the large chromosome; a similar arrangement in pandemic V. parahaemolyticus would help explain the rapid serotype changes. However, previous reports on "capsule" genes are controversial. Therefore, we set out to clarify and characterize these regions in pandemic V. parahaemolyticus O3:K6 by gene deletion using a chitin based transformation strategy.     

Development of a loop-mediated Isothermal amplification assay for sensitive and rapid detection of <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis>

Background  

Vibrio parahaemolyticus is a marine seafood-borne pathogen causing gastrointestinal disorders in humans. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are known as major virulence determinants of V. parahaemolyticus. Most V. parahaemolyticus isolates from the environment do not produce TDH or TRH. Total V. parahaemolyticus has been used as an indicator for control of seafood contamination toward prevention of infection. Detection of total V. parahaemolyticus using conventional culture- and biochemical-based assays is time-consuming and laborious, requiring more than three days. Thus, we developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the sensitive and rapid detection of Vibrio parahaemolyticus.     

Molecular characterization of Vibrio parahaemolyticus of similar serovars isolated from sewage and clinical cases of diarrhoea in Calcutta,India

Vibrio parahaemolyticus is a seafood-borne halophilic pathogen that causes acute gastroenteritis in humans. During the course of an investigation on the incidence of V. parahaemolyticus in sewage water samples of Calcutta, India, we isolated eight (26.7%) strains of V. parahaemolyticus from 30 samples. Among these strains, five (62.5%) carried the thermostable direct hemolysin (tdh) gene, a major virulence marker of V. parahaemolyticus. Two strains belonged to serovar O5:K3 and the remaining three to O5:KUT, which is common among clinical strains of V. parahaemolyticus isolated from hospitalized patients of Calcutta with acute diarrhoea. The tdh positive sewage strains of V. parahaemolyticus were compared by randomly amplified polymorphic DNA (RAPD)-PCR and pulsed-field gel electrophoresis (PFGE) with strains of similar serovars selected from our culture collection to determine the genetic relatedness. Our results showed that except for sharing the similar serovar, sewage and clinical strains of V. parahaemolyticus were genetically different. In addition, toxRS-targeted group-specific (GS) PCR and open reading frame 8 (ORF-8) PCR showed that the sewage strains did not belong to the pandemic genotype. Since the sewage in Calcutta is directly used for cultivation of vegetables and for pisciculture, the presence of tdh positive V. parahaemolyticus in the sewage highlights the need for constant monitoring of the environment.