多重实时PCR检测产毒素性霍乱弧菌和副溶血弧菌

设计引物和探针,优化多重实时PCR条件,以同时检测霍乱弧菌霍乱毒素基因ctxA、副溶血弧菌种特异性基因gyrB和耐热肠毒素基因tdh。该多重实时PCR方法检测产毒素性的O1群(3株)和O139群(44株)霍乱弧菌菌株、不产毒素的O1群(12株)和O139群(6株)及非O1非O139群(7株)霍乱弧菌菌株的ctxA,阳性和阴性结果与普通PCR检测结果100%符合;检测副溶血弧菌种特异性gyrB,116株副溶血弧菌均阳性,而9株其它细菌和72株霍乱弧菌均阴性;检测tdh的阳性和阴性结果也与普通PCR结果完全一致。另外还建立了检测副溶血弧菌菌株trh1和trh2的单重实时PCR方法。     

O1群和O139群霍乱弧菌主要抗原研究进展

霍乱弧菌是引起人和动物烈性肠道传染病霍乱的病原体。在霍乱弧菌的200多个血清群中,只有O1群和O139群霍乱弧菌能引起霍乱。快速准确检测O1群和O139群霍乱弧菌是霍乱防治的关键。表面抗原在O1群和O139群霍乱弧菌检测中发挥着重要作用。简要综述了O1群和O139群霍乱弧菌的脂多糖、霍乱肠毒素、外膜蛋白W、毒素共调菌毛和甘露糖敏感血凝素等5种主要抗原的研究进展。     

PCR检测霍乱弧菌RTX吸毒素基因

建立了一种扩增最近发现的霍乱弧菌RTX毒素基因的PCR方法。在世界各地引起流行和散发霍乱病例的166株临床和环境霍乱弧菌分离物中,用PCR和Hep—2细胞细胞毒性试验发现它们都是产毒的。而在相关基因簇中有缺失的古典生物型标准株用这两种方法结果都是阴性。这是第一个用于鉴别O1群霍乱弧菌古典生物型菌株和E1　Tor生物型菌株以及包括O139血清型的其它非O1群菌株的快速基因分型方法。建立的PCR方法也可特异性地检测霍乱弧菌中的RTX毒素基因,因为用此RTX毒素特异性PCR以及Hep—2细胞毒性试验时副溶血弧菌、致腹泻性大肠杆菌、气单胞菌和邻单胞菌的临床分离株都是阴性。这些结果使霍乱弧菌中RTx毒素的特性明显了。其在细菌致病中的作用需要进一步研究。     

与霍乱毒素一致的模拟弧菌Vibrio Mimicus肠毒素的提纯

<正>霍乱病原菌、古典型和EI Tor生物型霍乱弧菌与一些其它弧菌即非O1群霍乱弧菌和模拟弧菌(最近才按蔗糖阴性分类为非O1群霍乱弧菌)密切相关。这些菌群的大多数菌种是非肠道致病性的或弱致病性的、或者引起不同于霍乱因子所致的腹泻,但有些菌种能产生一种与古典型霍乱弧菌的霍乱毒素密切相关的肠毒素。     

霍乱O139血清群多糖结合疫苗的生物合成研究

目的:霍乱是由霍乱弧菌引起的一种烈性传染病,其防治已成为一个全球性的公共卫生问题。其中1992年出现的O139血清群是除O1外另一种病原体,发病数日益增多。因此,有必要寻找一种安全有效适用于各种人群的疫苗。方法:敲除霍乱弧菌O139血清群93-3株脂多糖合成途径中O抗原连接酶基因waa L,在周间质产生游离的多糖,之后转入包含来自脑膜炎奈瑟球菌的糖基转移酶和霍乱毒素B亚单位(CTB)编码序列的共表达载体,经IPTG诱导后制备全菌蛋白样品,利用抗His抗体检测糖蛋白的表达,利用Ni柱和离子交换柱对糖蛋白进行纯化,并对其进行糖定量和蛋白定量。结果:以未糖基化、相对分子质量约为14×103的底物蛋白CTB为对照,当共表达CTB和糖基转移酶Pgl L时,通过Western印迹可检测到相对分子质量约为20×103的糖基化蛋白,经Ni柱及阳离子交换柱纯化,得到纯度较高的O139群霍乱O抗原多糖结合蛋白,其纯度约为84.2%,并计算得其糖-蛋白比为0.103∶1。结论:通过生物法合成了一种霍乱O139血清群的多糖结合疫苗,为后续进行动物评价打下了基础。     

霍乱弧菌01群E1 Tor型溶血素的氨基末端区在古典型菌株中的表达及其细胞毒性

<正>产生霍乱毒素(CT)的01群霍乱弧菌引起人霍乱。某些非01群霍乱弧菌株能产生与霍乱毒素相似的肠毒素,而另一些来自临床腹泻病例的非01群菌却不产生这种毒素,这表明还有其它因素能引起腹泻反应。现已证明这些菌株能产生溶血素——一种可能的腹泻因素,尽管其致病机理尚不清楚。Yamamoto等人证实了非01群菌株和周群E1 Tor型菌株所产生的溶血素在生物学、理化住质和免疫学方面是难区分的。Southern杂交分析表明两者的基因基本上是相同的。     

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。     

rBS-WC(O139)霍乱疫苗的小鼠免疫试验

1992年起在印度和孟加拉国相继发生的O139霍乱,目前已迅速传播到其周边国家,对我国亦已构成严重威胁。当前全世界预防霍乱的工作更加复杂,除了要针对O1群霍乱弧菌研制保护期长、保护效率高的疫苗外,还要对这一新发现的时隐时现的烈性腹泻病原菌快速作出反应,及时分析其保护性抗原,构建有效疫苗,以控制疫情发展。 O139霍乱弧菌与O1群霍乱弧菌有很大不同,特别在菌体抗原方面存在显著差异。O1群菌感染的患者康复后对同群菌的冉感染有免疫保护作用,而对O139群菌的感染则无此保护作     

霍乱弧菌和副溶血弧菌分离株的gyrB基因系统发育分析

依据gyrB基因部分编码序列构建系统发育树以分类和鉴别霍乱弧菌和副溶血弧菌,并探讨其种系发生关系。扩增并测序13株霍乱弧菌、8株副溶血弧菌、2株嗜水气单胞菌及1株类志贺邻单胞菌的gyrB基因(编码DNA促旋酶B亚单位)序列,并采用距离法与最大似然法构建系统发育树。两种方法所构建的树结构完全一致,霍乱弧菌、副溶血弧菌、嗜水气单胞菌及类志贺邻单胞菌各自形成一个独立的簇。其中,霍乱肠毒素基因(ctxA)阳性的霍乱弧菌(8株O139群与2株O1群ElTor型)聚类成一分枝;3株副溶血弧菌临床株(1株2002年流行株,2株2004年分离株)与1日本菌株及2001年1株自环境分离的毒力株聚类。系统发育分析靶分子gyrB基因可以良好区分上述4种常见病原菌。产毒O139群霍乱弧菌与产毒O1群ElTor型霍乱弧菌关系密切。副溶血弧菌环境毒力株与本地区临床主要流行株在系统发育关系上较为接近,可能是潜在的致病菌。     

非O1群、非O139群霍乱弧菌致败血症1例报道

对襄阳市中心医院分离的2株疑似霍乱弧菌进行鉴定及药物敏感性试验。利用MicroScan WalkAway 40鉴定仪进行生化鉴定及药物敏感性试验,玻片凝集法确定血清型别,PCR扩增16SrRNA保守区基因并将产物进行测序分析。此2株疑似霍乱菌株O1群及O139群霍乱弧菌诊断血清均不凝集,16SrRNA扩增产物测序Blast比对分析与数据库中霍乱弧菌相似性达100％,药敏结果显示对氨苄西林、庆大霉素、环丙沙星、阿米卡星、氯霉素、复方新诺明（SXT）、四环素均敏感。该病例为非O1、非O139群霍乱弧菌导致的败血症,可能经胃肠道途径传播。     

Method of DNA extraction and application of multiplex polymerase chain reaction to detect toxigenic Vibrio cholerae O1 and O139 from aquatic ecosystems

Vibrio cholerae is a free-living bacterium found in water and in association with plankton. V. cholerae non-O1/non-O139 strains are frequently isolated from aquatic ecosystems worldwide. Less frequently isolated are V. cholerae O1 and V. cholerae O139, the aetiological agents of cholera. These strains have two main virulence-associated factors, cholera toxin (CT) and toxin co-regulated pilus (TCP). By extracting total DNA from aquatic samples, the presence of pathogenic strains can be determined quickly and used to improve a microbiological risk assessment for cholera in coastal areas. Some methods suggested for DNA extraction from water samples are not applicable to all water types. We describe here a method for DNA extraction from coastal water and a multiplex polymerase chain reaction (PCR) for O1 and O139 serogroups. DNA extraction was successfully accomplished from 117 sea water samples collected from coastal areas of Perú, Brazil and the USA. DNA concentration in all samples varied from 20 ng to 480 micro g micro l-1. The sensitivity of the DNA extraction method was 100 V. cholerae cells in 250 ml of water. The specificity of multiplex O1/O139 PCR was investigated by analysing 120 strains of V. cholerae, Vibrio and other Bacteria species. All V. cholerae O1 and O139 tested were positive. For cholera surveillance of aquatic environments and ballast water, total DNA extraction, followed by V. cholerae PCR, and O1/O139 serogroup and tcpA/ctxA genes by multiplex PCR offers an efficient system, permitting risk analysis for cholera in coastal areas.     

Incidence and toxigenicity of Vibrio cholerae in a freshwater lake during the epidemic of cholera caused by serogroup O139 Bengal in Calcutta, India

Abstract The extent of contamination of a freshwater lake with Vibrio cholerae 0139 Bengal and the toxigenicity of all the V. cholerae isolates recovered during the period of the study were examined during and after an explosive outbreak of 0139 cholera in Calcutta. Strains biochemically characterized as V. cholerae could be isolated throughout the period of study examined from the freshwater lake samples. Most probable number of V. cholerae belonging to the 0139 serogroup in surface waters was 3 to 4 per 100 ml during major part of the study but isolation of this serogroup from sediment and plankton samples was infrequent. Of the total of 150 strains recovered, 23 (15.3%) agglutinated with the 0139 antiserum while the remaining belonged to the non-O1 non-O139 serogroups. None of the strains agglutinated with the O1 antiserum. All the 23 strains of V. cholerae O139 produced cholera toxin while 7.9% of the 127 non-O1 non-O139 strains also produced cholera toxin. Resistance to ampilicillin, furazolidone and streptomycin was encountered among strains belonging to both V. cholerae O139 and V. cholerae non-O1 non-O139 strains, but the percentage of resistant strains in the former was much higher than in the latter. During this cholera epidemic, possibly due to the introduction of large numbers of toxigenic V. cholerae such as the O139 serogroup, there was an increase in the number of toxigenic vibrios among the innocuous aquatic residents. This presumably occured through genetic exchange and, if substantiated, could play an important role in the re-emergence of epidemics.     

Strains of Vibrio cholerae serogroups O1 and O139 that produce the basic protective antigens

To find out stable and effective producers of major protective antigens intended for use as components of cholera chemical vaccine against V. cholerae strains of serogroups O and O139, the comparative analysis of the production of cholera toxin, toxin-coregulated pili (TCP), antigens O1 and O139, polysaccharide capsule and outer membrane protein OmpU in different V. cholerae strains groups O1 and O139 has been made. V. cholerae strain KM68, serogroup O1, has been found capable of the production of antigen O1, serovar Ogawa, protein OmpU at a sufficiently high level and the hyperproduction of cholera toxin and TCP, and thus suitable for use in the manufacture of cholera bivalent vaccine as the source of these antigens. Specially selected alysogenic noncapsular strain KM137 of serogroup O139, characterized by a high and stable level of the biosynthesis of this somatic antigen when grown in both laboratory and production conditions, may serve as the produces of antigen O139.     

A new type of conjugative transposon encodes resistance to sulfamethoxazole, trimethoprim, and streptomycin in Vibrio cholerae O139.

Vibrio cholerae O139 is the first non-O1 serogroup of V. cholerae to give rise to epidemic cholera. Apparently, this new serogroup arose from an El Tor O1 strain of V cholerae, but V. cholerae O139 is distinguishable from V. cholerae El Tor O1 by virtue of its novel antigenic structure and also its characteristic pattern of resistances to the antibiotics sulfamethoxazole, trimethoprim, streptomycin, and furazolidone. We found that the first three of these antibiotic resistances are carried on an approximately 62-kb self-transmissible, chromosomally integrating genetic element which we have termed the SXT element. This novel conjugative transposon-like element could be conjugally transferred from V. cholerae O139 to V cholerae O1 and Escherichia coli strains, where it integrated into the recipient chromosomes in a site-specific manner independent of recA. To study the potential virulence properties of the SXT element as well as to improve upon the live attenuated O139 vaccine strain Bengal-2, a large internal deletion in the SXT element was crossed on to the Bengal-2 chromosome. The resulting strain, Bengal-2.SXT(s), is sensitive to sulfamethoxazole and trimethoprim and colonizes the intestines of suckling mice as well as wild-type strains do, suggesting that the SXT element does not encode a colonization factor. Derivatives of Bengal-2.SXT(s) are predicted to be safe, antibiotic-sensitive, live attenuated vaccines for cholera due to the O139 serogroup.     

Molecular analysis of Vibrio cholerae O1, O139, non-O1, and non-O139 strains: clonal relationships between clinical and environmental isolates

A total of 26 strains of Vibrio cholerae, including members of the O1, O139, and non-O1, non-O139 serogroups from both clinical and environmental sources, were examined for the presence of genes encoding cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), and outer membrane protein (ompU), for genomic organization, and for the presence of the regulatory protein genes tcpI and toxR in order to determine relationships between epidemic serotypes and sources of isolation. While 22 of the 26 strains were hemolytic on 5% sheep blood nutrient agar, all strains were PCR positive for hlyA, the hemolysin gene. When multiplex PCR was used, all serogroup O1 and O139 strains were positive for tcpA, ompU, and tcpI. All O1 and O139 strains except one O1 strain and one O139 strain were positive for the ctxA, zot, and ace genes. Also, O1 strain VO3 was negative for the zot gene. All of the non-O1, non-O139 strains were negative for the ctxA, zot, ace, tcpA, and tcpI genes, and all of the non-O1, non-O139 strains except strain VO26 were negative for ompU. All of the strains except non-O1, non-O139 strain VO22 were PCR positive for the gene encoding the central regulatory protein, toxR. All V. cholerae strains were negative for the NAG-specific st gene. Of the nine non-ctx-producing strains of V. cholerae, only one, non-O1, non-O139 strain VO24, caused fluid accumulation in the rabbit ileal loop assay. The other eight strains, including an O1 strain, an O139 strain, and six non-O1, non-O139 strains, regardless of the source of isolation, caused fluid accumulation after two to five serial passages through the rabbit gut. Culture filtrates of all non-cholera-toxigenic strains grown in AKI media also caused fluid accumulation, suggesting that a new toxin was produced in AKI medium by these strains. Studies of clonality performed by using enterobacterial repetitive intergenic consensus sequence PCR, Box element PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) collectively indicated that the V. cholerae O1 and O139 strains had a clonal origin, whereas the non-O1, non-O139 strains belonged to different clones. The clinical isolates closely resembled environmental isolates in their genomic patterns. Overall, there was an excellent correlation among the results of the PCR, AFLP, and PFGE analyses, and individual strains derived from clinical and environmental sources produced similar fingerprint patterns. From the results of this study, we concluded that the non-cholera-toxin-producing strains of V. cholerae, whether of clinical or environmental origin, possess the ability to produce a new secretogenic toxin that is entirely different from the toxin produced by toxigenic V. cholerae O1 and O139 strains. We also concluded that the aquatic environment is a reservoir for V. cholerae O1, O139, non-O1, and non-O139 serogroup strains.     

Structure and arrangement of the cholera toxin genes in Vibrio cholerae O139

Abstract The sequence of the ctxB gene encoding the B subunit of cholera toxin has been determined for a strain of Vibrio cholerae of the novel O139 serotype associated with recent outbreaks of severe cholera throughout South-East Asia and found to be identical to the ctxB gene in V. cholerae O1 of the E1 Tor biotype. Analyses by Southern hybridization and PCR showed that all strains of the O139 serotype V. cholerae tested carried cholera toxin genes and other gene associated with a virulence cassette DNA region at two loci identical or homologous to those identified in the Classical rather than the E1 Tor biotype of V. cholerae serotype O1 although these loci in O139 could reside on restriction fragments of variable size.     

Role of moderate bacteriophage 139 in change in production of cholera toxin in a classic strain of Vibrio cholerae

New data were obtained concerning cell sensitivity of pathogenic strains of cholera vibrions, which belong to the serogroup O1 of classical biovar, to the temperate bacteriophage K139, the native host of which is Vibrio cholerae O139. Molecular-genetic and biochemical studies showed that phage 139 integrated into the chromosome of strains V. cholerae O1 can change their toxigenic properties. A change in the production of cholera toxin (CT) in lysogens is associated both with an increase in the activity of the toxR regulatory gene and with a distortion of the structure of a chromosomal DNA region that contains a copy of the operon ctxAB encoding CT biosynthesis.     

Alteration of cholera toxin biosynthesis in Vibrio cholerae 01 as a result of temperate phage 139 integration into bacterial chromosome

Infection of V. cholerae 01 (classical and eltor biovars) cells with the temperate cholera phage 139 derived from V. cholerae serogroup 0139 followed by integration of the phage genome into the bacterial chromosome significantly increased the production of cholera toxin, the main virulence factor. The level of toxin biosynthesis in the lysogenic V. cholerae classical strain increased 3-fold and that in V. eltor thirty times in comparison with the parental strains. Increased production of cholera toxin was not associated with an increase in the number of copies of genes involved in its biosynthesis but seemed to be due to changes in toxinogenesis regulation.     

Peptides mimicking Vibrio cholerae O139 capsular polysaccharide elicit protective antibody response

Vibrio cholerae is the etiological agent of cholera. V. cholerae serogroup O1 had been, until 1992, the only serogroup responsible for large epidemics and pandemics of cholera. In 1992, a new serotype of V. cholerae emerged in South-East Asia that caused a massive outbreak of cholera in India and neighboring countries. The new serotype was named V. cholerae O139. The main differences between V. cholerae O139 and O1 are that the former possesses a capsular polysaccharide and different lipopolysaccharide. Capsular polysaccharides are, in general, T-independent antigens giving rise to poor immune responses lacking immunological memory. In order to overcome this, monoclonal antibodies against the capsular polysaccharide of V. cholerae O139 were used to screen different phage-displayed random peptide libraries. Eight different phage clones were selected and characterized using enzyme immunoassay with the monoclonal antibodies, and then tested for specificity by competition with V. cholerae O139 capsular polysaccharide. Selected peptides were sequenced, synthesized and conjugated to bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). The conjugated peptides were used to immunize mice. It is evident that the anti-peptide mouse antibodies bind to the V. cholerae O139 capsular polysaccharide. In addition, the anti-peptide antibodies are protective in a suckling mouse model. The protective efficacy is both specific and dose-dependent. A PCT (PCT/IT2003/000489) with the publication number WO 2004/056851 has been filed for the sequences of the eight peptides.