Variation in epitopes of the B subunit of Vibrio cholerae non-O1 and Vibrio mimicus cholera toxins.

Monoclonal antibodies reacting with the B subunit of Vibrio cholerae O1 strain 569B cholera toxin (CT-B) were used to identify unique and common epitopes of V. cholerae non-O1 and Vibrio mimicus CT-B. Vibrio cholerae non-O1 strains produced CT-B showing three monoclonal antibody reaction patterns (epitypes), which corresponded with epitypes described previously for V. cholerae O1 classical biotype CT-B (CT1), El Tor biotype CT-B (CT2), and a unique V. cholerae non-O1 CT-B (CT3), which lacked an epitope located in or near the GM1 ganglioside binding site of 569B CT-B. Vibrio mimicus CT-B was immunologically indistinguishable from 569B CT-B. These and previous results define six epitopes on 569B CT-B, and a fourth epitope in or near the GM1 ganglioside binding site.     

The use of liposomes for detection of the surface lipopolysaccharide antigen, Vibrio cholerae cells, and antibodies against them

A test system for determination of Vibrio cholerae cells, surface O-antigen, and antibodies against them was developed on the basis of complement-dependent lysis of liposomes sensitized by the lipopolysaccharide-dependent antigen from Vibrio cholerae 569B. The factors that affect the function of the liposomal reagent were studied, and the conditions for detecting antibodies and antigenic material were optimized. This system is highly specific and sensitive to be used for the determination of anticholeraic antibodies (30-50 times as effective as agglutination tests), lipopolysaccharide antigen (100 ng/ml, which corresponded to 3.0 ng of lipopolysaccharide in the sample studied), and Vibrio cholerae cells (3.3 x 10(7) m.b./ml, which corresponded to 10(6) m.b. in sample). It takes 30-40 min to detect the lipopolysaccharide antigen and 90 min to detect V. cholerae cells.     

DNA环介导恒温扩增技术快速检测霍乱弧菌

霍乱弧菌是一种重要的食源性致病菌,主要引起急性肠道传染病,其快速检测具有重要意义。根据霍乱弧菌的mdh管家基因序列,设计2对特异性检测引物,利用DNA环介导恒温扩增技术(Loop-mediated isothermal amplification,LAMP),经反应体系优化,成功建立了霍乱弧菌的LAMP快速检测方法。该方法最佳反应温度为65℃,60min完成检测,对培养菌的检测限为25CFU/mL,污染食品中霍乱弧菌的检测限为32CFU/g。对33株同种或近源细菌进行LAMP检测,仅霍乱弧菌得到阳性扩增。LAMP方法实践应用结果表明,对1057份虾、蟹、牡蛎、肉类、人腹泻物等样本进行检测,共检出85份阳性,与国际标准(ISO TS21872-1-2007)检测结果的符合率为100%。结果表明,本研究建立的霍乱弧菌LAMP检测方法特异性强、灵敏度高、操作简便,有利于霍乱弧菌疫情的监测。     

O139霍乱弧菌LPS基因在大肠杆菌中的克隆和表达

利用粘粒载体pCOS5构建了国内分离的O139霍乱弧菌的基因组文库,并从文库中筛选获得可以表达O139霍乱弧菌脂多糖的重组克隆株E.coliJM109(pMG310)。重组粘粒pMG310经酶切分析,所克隆的外源DNA片段大小为37kb。实验证明：重组克隆株E.coliJM109(pMG310)所表达的脂多糖具有良好的免疫原性及反应原性。     

O1或O139霍乱疫苗对异群霍乱缺乏交叉保护

O1和O139霍乱弧菌是引起急性腹泻的病原微生物,用这两群菌的灭活全菌体与重组霍乱毒素B亚单位构建的亚单位／菌体型疫苗免疫队CA小鼠,对本群菌的攻击可提供良好免疫保护,而对异群霍乱菌则缺乏交叉保护作用。     

霍乱弧菌检测方法的研究进展

烈性肠道传染病霍乱能引起大范围乃至世界性大流行,在我国被列为甲类传染病.霍乱弧菌是导致感染者严重腹泻、引起霍乱的病原菌.霍乱弧菌的快速、准确检测是霍乱预防、控制的重要依据.目前,国内、外针对霍乱弧菌建立了许多有效的检测方法,尤其是分子生物学相关技术的应用,为霍乱弧菌的检测提供了新的手段.本文综述了近年来霍乱弧菌检测方法...     

O-antigenic lipopolysaccharides isolated from a marine Vibrio, bio-serogroup 1875, possessing an antigenic factor in common with O1 Vibrio cholerae

The chemical and serological characteristics of lipopolysaccharides (LPS) isolated from Vibrio bio-serogroup 1875 were compared with those of O1 Vibrio cholerae LPS. Vibrio bio-serogroup 1875 LPS contained all the component sugars which were found in O1 V. cholerae LPS, i.e. glucose, L-glycero-D-manno-heptose, fructose, glucosamine, perosamine and quinovosamine, though the amount of perosamine, a characteristic component of O1 V. cholerae LPS, was very low compared with that of O1 V. cholerae LPS. Their LPS additionally contained mannose and two unidentified neutral sugars which are not regular constituents of O1 V. cholerae LPS. Definite serological cross-reactivity in the passive haemolysis test between LPS from Vibrio bio-serogroup 1875 and LPS from O1 V. cholerae was demonstrated.     

Response of pathogenic Vibrio species to high hydrostatic pressure.

Vibrio parahaemolyticus ATCC 17802, Vibrio vulnificus ATCC 27562, Vibrio cholerae O:1 ATCC 14035, Vibrio cholerae non-O:1 ATCC 14547, Vibrio hollisae ATCC 33564, and Vibrio mimicus ATCC 33653 were treated with 200 to 300 MPa for 5 to 15 min at 25 degrees C. High hydrostatic pressure inactivated all strains of pathogenic Vibrio without triggering a viable but nonculturable (VBNC) state; however, cells already existing in a VBNC state appeared to possess greater pressure resistance.     

Sequence Analyses of Type IV Pili from Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus

Bacterial surface structures called pili have been studied extensively for their role as possible colonization factors. Most sequenced Vibrio genomes predict a variety of pili genes in these organisms, including several types of type IV pili. In particular, the mannose-sensitive hemagglutinin (MSHA) and the PilA pili, also known as the chitin-regulated pilus (ChiRP), are type IVa pili commonly found in Vibrio genomes and have been shown to play a role in the colonization of Vibrio species in the environment and/or host tissue. Here, we report sequence comparisons of two type IVa pilin subunit genes, mshA and pilA, and their corresponding amino acid sequences, for several strains from the three main human pathogenic Vibrio species, V. cholerae, V. parahaemolyticus, and V. vulnificus. We identified specific groupings of these two genes in V. cholerae, whereas V. parahaemolyticus and V. vulnificus strains had no apparent allelic clusters, and these genes were strikingly divergent. These results were compared with other genes from the MSHA and PilA operons as well as another Vibrio pili from the type IVb group, the toxin co-regulated pilus (TCP) from V. cholerae. Our data suggest that a selective pressure exists to cause these strains to vary their MSHA and PilA pilin subunits. Interestingly, V. cholerae strains possessing TCP have the same allele for both mshA and pilA. In contrast, V. cholerae isolates without TCP have polymorphisms in their mshA and pilA sequences similar to what was observed for both V. parahaemolyticus and V. vulnificus. This data suggests a possible linkage between host interactions and maintaining a highly conserved type IV pili sequence in V. cholerae. Although the mechanism underlying this intriguing diversity has yet to be elucidated, our analyses are an important first step towards gaining insights into the various aspects of Vibrio ecology.     

Growth response of Vibrio cholerae and other Vibrio spp. to cyanobacterial dissolved organic matter and temperature in brackish water

Environmental control of growth and persistence of vibrios in aquatic environments is poorly understood even though members of the genus Vibrio are globally important pathogens. To study how algal-derived organic matter and temperature influenced the abundance of different Vibrio spp., Baltic Sea microcosms inoculated with Vibrio cholerae, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio alginolyticus and native bacterioplankton, were exposed to different temperatures (12-25 degrees C) and amended with dissolved organic matter from Nodularia spumigena (0-4.2 mg C L(-1)). Vibrio abundance was monitored by culture-dependent and molecular methods. Results suggested that Vibrio populations entered a viable but nonculturable state during the incubations. Abundance of Vibrio spp. and total bacterioplankton were orders of magnitude higher in microcosms amended with organic matter compared with reference microcosms. Vibrio cholerae abundances ranged from 0.9 to 1.9 x 10(5) cells mL(-1) in treatments amended with 4.2 mg C L(-1). Vibrio cholerae abundance relative to total bacterioplankton and other Vibrio spp. also increased >10-fold. In addition, V. vulnificus abundance increased in mesocosms with the highest organic matter addition (0.9-1.8 x 10(4) cells mL(-1)). Temperature alone did not significantly affect abundances of total bacterioplankton, total Vibrio spp. or individual Vibrio populations. By contrast, cyanobacterial-derived organic matter represented an important factor regulating growth and abundance of V. cholerae and V. vulnificus in brackish waters.     

The occurrence of alpha (1----2) linked N-acetylperosamine--homopolymer in lipopolysaccharides of non-O1 Vibrio cholerae possessing an antigenic factor in common with O1 V. cholerae

Chemical analysis was carried out on lipopolysaccharides from Vibrio cholerae bio-serogroup Hakata 487-85. The O-specific chain of the phenol-soluble lipopolysaccharides was demonstrated by 13C-NMR spectroscopy and methylation analysis to contain a linear homopolymer of alpha(1----2) linked N-acetylperosamine (4-acetamido-4,6-dideoxy-D-mannopyranose), which was closely similar to but not identical to a linear alpha(1----2) linked N-3-deoxy-L-glycerotetronyl (S-2,4-dihydroxybutyryl) perosamine-homopolymer constituting that of O1 Vibrio cholerae lipopolysaccharides.     

High Prevalence of Vibrio cholerae Non-O1 Carrying Heat-Stable-Enterotoxin-Encoding Genes among Vibrio Isolates from a Temperate-Climate River Basin of Central Italy

Vibrio spp. of clinical interest from the Arno River basin (Tuscany, Italy) were investigated in this study. Vibrios were isolated from 70% of water samples. Vibrio cholerae non-O1 was the most prevalent species (82% of isolates), followed by Vibrio mimicus (10%) and Vibrio metschnikovii (8%). Recovery of vibrios was correlated with temperature, pH, and various indicators of municipal pollution. None of the 150 Vibrio isolates carried ctx-related genomic sequences, whereas 18 (14.6%) of the 123 V. cholerae non-O1 isolates and 1 (6.7%) of the 15 V. mimicus isolates carried sto alleles. These findings indicate that considerable circulation of sto-positive vibrios may occur in temperate-climate freshwater environments.     

Sensitivity of Vibrio cholerae to the lethal and mutagenic effects of UV-irradiation mediated by the presence of plasmids

The effect of UV-irradiation on Vibrio cholerae cells and its changes mediated by the plasmid R245 have been studied. Vibrio cholerae strains 569B and RV31 have been shown to be considerably more sensitive to lethal effect of UV-irradiation as compared with Escherichia coli and Salmonella typhimurium cells. Highly toxigenic strain 569B and practically atoxigenic strain RV31 have the same UV-sensitivity. Lethal effect of UV-irradiation on Vibrio cholerae cells is increased when the irradiated cells are plated on enriched media. UV-induction of mutations was not registered in plasmidless strains of Vibrio cholerae. Plasmid R245 increases UV-resistance of vibrio cells and makes them UV-mutable.     

Comparative characteristics of the preparations of hemolysin of ctx+ and ctx- Vibrio cholerae eltor and Vibrio cholerae O139 strains

Hemolysin of ctx+ Vibrio cholerae strains was obtained and studied. Ctx+ Vibrio cholerae O1 and O139 strains produced the hemolysin during cultivation in triptone medium without FeCl3. Mol.wt. and the spectrum of lytic activities of hemolysins of ctx+ Vibrio cholerae did not differ from hemolysins of ctx- strains.     

Involvement of the GspAB complex in assembly of the type II secretion system secretin of Aeromonas and Vibrio species

The type II secretion system (T2SS) functions as a transport mechanism to translocate proteins from the periplasm to the extracellular environment. The ExeA homologue in Aeromonas hydrophila, GspA(Ah), is an ATPase that interacts with peptidoglycan and forms an inner membrane complex with the ExeB homologue (GspB(Ah)). The complex may be required to generate space in the peptidoglycan mesh that is necessary for the transport and assembly of the megadalton-sized ExeD homologue (GspD(Ah)) secretin multimer in the outer membrane. In this study, the requirement for GspAB in the assembly of the T2SS secretin in Aeromonas and Vibrio species was investigated. We have demonstrated a requirement for GspAB in T2SS assembly in Aeromonas salmonicida, similar to that previously observed in A. hydrophila. In the Vibrionaceae species Vibrio cholerae, Vibrio vulnificus, and Vibrio parahaemolyticus, gspA mutations significantly decreased assembly of the secretin multimer but had minimal effects on the secretion of T2SS substrates. The lack of effect on secretion of the mutant of gspA of V. cholerae (gspA(Vc)) was explained by the finding that native secretin expression greatly exceeds the level needed for efficient secretion in V. cholerae. In cross-complementation experiments, secretin assembly and secretion in an A. hydrophila gspA mutant were partially restored by the expression of GspAB from V. cholerae in trans, further suggesting that GspAB(Vc) performs the same role in Vibrio species as GspAB(Ah) does in the aeromonads. These results indicate that the GspAB complex is functional in the assembly of the secretin in Vibrio species but that a redundancy of GspAB function may exist in this genus.     

DNA damage and cell killing by nitrofurantoin in relation to its carcinogenic potential

Nitrofurantoin inhibited growth and produced loss of viability of Vibrio cholerae cells in a dose-dependent manner, the 10% (D10) and 37% (D37) survival doses being 18.0 and 5.5 micrograms/ml x hr. respectively. The drug also caused filamentation of the cells in a very significant manner. Ultraviolet absorption data and thermal chromatography through hydroxyapatite column revealed that nitrofurantoin treatment of Vibrio cholerae cells produced a maximum amount of 55% of DNA reversibly bihelical due to the formation of inter-strand cross-links. Helix-coil transition studies carried out by viscometric and also, spectrophotometric methods revealed that the nitrofurantoin-induced cross-links in Vibrio cholerae DNA, imparted to this DNA greater thermal stability than that of native DNA. The quantitative aspect and also the mode of nitrofurantoin action on DNA of Vibrio cholerae and Escherichia coli cells vis-à-vis the carcinogenic potential of the drug were discussed.     

Preliminary structure determination of the capsular polysaccharide of Vibrio cholerae O139 Bengal Al1837.

Vibrio cholerae O139 Bengal has recently been identified as a cause of epidemic cholera in Asia. In contrast to V. cholerae O1, V. cholerae O139 Bengal has a polysaccharide capsule. As determined by high-performance anion-exchange chromatography and 1H nuclear magnetic resonance analysis, the capsular polysaccharide of V. cholerae O139 Bengal strain Al1837 has six residues in the repeating subunit; this includes one residue each of N-acetylglucosamine, N-acetylquinovosamine (QuiNAc), galacturonic acid (GalA), and galactose and two residues of 3,6-dideoxyxylohexose (Xylhex). The proposed structure is [formula: see text]     

Occurrence and potential pathogenesis of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus on the South Coast of Sweden

During the summer of 2006, several wound infections - of which three were fatal - caused by Vibrio cholerae were reported from patients who had been exposed to water from the Baltic Sea. Before these reports, we initiated a sampling project investigating the occurrence of potential human pathogenic V. cholerae, Vibrio vulnificus and Vibrio parahaemolyticus in The Sound between Sweden and Denmark. The Blue mussel (Mytilus edulis) was used as an indicator to follow the occurrence of vibrios over time. Molecular analyses showed high frequencies of the most potent human pathogenic Vibrio spp.; 53% of mussel samples were positive for V. cholerae (although none were positive for the cholera toxin gene), 63% for V. vulnificus and 79% for V. parahaemolyticus (of which 47% were tdh(+) and/or trh(+)). Viable vibrios were also isolated from the mussel meat and screened for virulence by PCR. The mortality of eukaryotic cells when exposed to bacteria was tested in vivo, with results showing that the Vibrio strains, independent of species and origin, were harmful to the cells. Despite severe infections and several deaths, no report on potential human pathogenic vibrios in this area had been published before this study.