江苏省1999—2005年霍乱弧菌的脉冲场凝胶电泳分析

目的了解江苏省1999-2005年间分离的霍乱弧菌核酸特征,及O139群与O1群霍乱弧菌在基因组水平上的关联。方法 NotⅠ酶切霍乱弧菌基因组,脉冲场凝胶电泳(PFGE)得到指纹图谱,并进行比较分析。结果 PFGE指纹图谱将全部136株霍乱弧菌分为6类,PFGE-A型由1株临床来源El Tor、120株临床来源和9株环境来源的O139组成,PFGE-B型由1株临床来源的El Tor组成,PFGE-C型由1株临床来源的El Tor和1株临床来源的O139组成,PFGE-D型由1株环境来源的El Tor组成,PFGE-E型由1株环境来源的O139组成,PFGE-F型由1株临床来源的O139组成。结论PFGE指纹图谱提示O139起源于不同克隆的El Tor霍乱弧菌,为了解O139的进化路线和流行规律提供了有效证据。作为高分辨率的基因分型工具,PFGE可用于追踪霍乱弧菌的暴发流行传染源,并在其分子流行病学研究中发挥重要作用。     

中国O1群El Tor霍乱弧菌产毒株表型多态性研究

目的 了解近50年中国不同地区分离的O1群El Tor型霍乱弧菌产毒株的生物表型特征变化。方法 采用多粘菌素B敏感试验、第Ⅳ组霍乱噬菌体裂解试验、VP试验和溶血实验进行表型特征分析。结果 生物型表型特征分析表明133株菌具有典型的El Tor生物型表型特征,其余251株菌呈现不典型的El Tor生物型表型特征;综合ctxB、rstR基因型和生物型表型特征分析,385 株检测菌株中 64 株为典型 El Tor 生物型菌株,21 株菌有典型的 El Tor 生物型表型特征但携带古典型ctxB基因,杂合型特征菌有280株,根据ctxB、rstR和表型特征的不同组合可再分为45个杂合型。结论 中国O1群El Tor霍乱弧菌菌株呈现明显的表型多态性,传统的表型分型特征不能有效区分古典生物型和El Tor生物型菌株。     

O1群霍乱弧菌新变异株的研究进展

O1群霍乱弧菌(Vibrio cholerae)是霍乱的病原体[1]。根据其"O"抗原的不同可分为200多个血清群[2]。根据其表型差异,O1群霍乱弧菌又可分为古典型(Classical biotype V.cholerae,CVC)和El Tor型(El Tor biotype V.cholerae,EVC)2个生物型。古典型与El Tor型不仅在表型和基因特性上不     

基于癌症基因组图谱的体细胞拷贝数变异与基因分布相关性分析

目的 探索拷贝数变异(CNA)区域中的非中性选择,分析肿瘤基因组中CNA与基因密度之间的关系。方法 从ArrayMap公共数据库中收集注释过的肿瘤基因组数据,对来自16 264个癌症样本,代表62个肿瘤类型的体细胞CNA进行分析,使用Spearman相关系数评估小片段拷贝数丢失和染色体断点与基因富集区的相关性。结果 从基因数量和编码序列占比方面来看,在基因密集的区域中,小片段的CNA显著富集,Spearman相关系数R=0.342,P<0.001。与CNA相关的DNA断裂位点也与富含基因的区域呈正相关,平均Spearman相关系数R=0.460,P<0.001。相反,染色体臂级CNA的频率与各个染色体臂上的总基因数呈负相关,Spearman相关系数R=-0.449,P=0.004,并且在各种肿瘤类型的数据中均观察到类似的结果。结论 通过大数据得到的肿瘤基因组图谱揭示了小片段CNA与基因密度之间存在正相关,而染色体臂级的CNA与其基因数则呈现负相关性。这些结果体现了CNA在肿瘤基因组进化过程中的非中性选择。     

福建省非典型埃尔托霍乱弧菌的遗传多样性研究

目的探索福建省非典型埃尔托霍乱弧菌(aEVC)的进化变迁规律,了解aEVC的变异株种类、基因特征,并分析不同类别变异株对霍乱流行趋势的影响。方法运用单基因片段分析技术,选择1962-2005年代表性O1群埃尔托霍乱菌株49株,对ctxB基因扩增产物进行序列测定和比对分析,确定ctxB基因型;同时对毒力因子tcpA和rstR基因分别进行古典型(Cl)和埃尔托型(El)特异的PCR扩增,确定其基因型别。结合ctxB、tcpA、rstR3种基因不同型别的组合形式,确定福建省aEVC菌株的多样性。结果1986年前菌株ctxB基因以埃尔托型B3型为主占92.00%(23/25),而1994年后菌株以古典型B1型为主占95.83%(23/24)。根据ctxB、rstR、tcpA3种基因不同型别的组合形式,1986年前菌株88.00%(22/25)表现为埃尔托型基因特征B3、El、El,1994年后菌株100.00%(24/24)表现为杂合型(Hybrid)变异株基因特征B1、El和(或)Cl、El。结论福建省霍乱菌株存在aEVC进化事件。ctxB、rstR、tcpA基因表现为B1、El和(或)Cl、El的杂合型变异株是福建省aEVC的主要特征型别,此类菌株于1994年后全面取代原来流行的埃尔托霍乱弧菌。不同血清型的aEVC变异株对菌株流行能力的影响不同,稻叶型埃尔托型菌株比杂合型流行时间要长,而小川型杂合型菌株比埃尔托型流行时间要持久。     

霍乱弧菌VPI毒力岛的tcpI-P和tcpH-A间隔区序列在不同菌株中的差异分析

目的：TcpA是霍乱弧菌主要定居因子毒素共调菌毛的主要亚单位，基因位于VPI毒力岛上。古典生物型和EI Tor生物型霍乱弧菌菌株的tcpA序列有较大差异，同时毒力岛上其他一些区域，主要包括部分基因的间隔区序列也存在差异。我们已在不同EI Tor流行株与非流行株以及非01／非O139菌株中又发现4种新的tcpA序列。笔拟探明这4种不同类型tcpA序列的菌株中，VPI毒力岛上其他几个差异片段(tcpI—P间隔区和tcpH—A间隔区)是否与相应tcpA序列有平行的变异关系、进而分析毒力岛和菌株分化的多样性。方法：在tcpI—P和tcpH—A间隔区外侧保守区中设计引物，扩增包含这两个间隔区的片段并进行测序，计算比较序列差异。结果：基因tcpH与tcpA之间的间隔区序列两两间比较的一致性在63．3％至95．3％之间；基因tcpI与tcpP之间的间隔区序列两两间比较的一致性在26．7％至100％之间。这些间隔区序列并没有像tcpA序列在这些菌株中的差异那样显示出类似的变异水平，甚至一些菌株的tcpA序列有较大差异时、某些区域反而高度一致。结论：在不同tcpA序列的菌株中tcpI—P间隔区和tcpH—A间隔区及tcpA这三个片段分化变异的速率不同，并不具有平行性。这些序列的多样性反映了霍乱弧菌VPI毒力岛以及菌株分化的多样性。这种分化也提示可能与这些区域具有结合一些调控因子、从而对环境信号产生不同方式或程度的应答的功能有关。     

辽宁省1997-2002年霍乱弧菌分离株核糖体分型和脉冲场凝胶电泳分型分析

为探讨辽宁省不同地区、不同来源霍乱病原体之间的遗传相关性，并对从鸭绿江水中分离出来的霍乱弧菌进行基因水平的分析，我们采用16SRNA核糖体基因分型(RT)及脉冲场凝胶电泳(PRGE)等分子生物学方法，对23株分离自1997—2002年辽宁省患者、外环境霍乱菌株进行研究。所用菌株经鉴定均为El tor生物型。染色体DNA提取、Southern杂交、PFGE参照文献[1]进行。     

珠江河口水体O1群和O139群霍乱弧菌监测

目的 分析珠江河口水体O1群和O139群霍乱弧菌菌型特征,探讨环境水体监测的方法和疫情监测中的作用.方法 2006年3月至2007年2月,在珠江河口选择24个水样采集点,每月采集一次,进行O1群和O139群霍乱弧菌的分离培养,并利用实时PCR监测样品增菌液中的O1群和O139群霍乱弧菌.采样同时测定气温、水温等气象资料.用脉冲场凝胶电泳(PFGE)对分离菌株进行分型分析.结果 监测期间共采样862份,霍乱弧菌分离阳性率7.77%,实时PCR阳性率为26.33%.按月的水样检测阳性率与水温变化趋势相似;城区监测点阳性率高于其他区域,在一家海产品批发市场排水口下游检测到产毒O139群菌株;菌株的菌型构成中,分离菌株主要为非产毒株;O1群E1 Tor小川和稻叶型以及O139群菌株的分离无季节变化趋势;PFGE分析75株分离株被分为49种带型,相似性为57.4%～100%,表现出明显的多样性.结论 霍乱弧菌在珠江河口水体中广泛存在,并呈现多样性.水体监测提供产毒菌株的指示,可作为环境危险评价的指标,且能在霍乱弧菌的监测和霍乱疫情预警中发挥作用.     

非霍乱弧菌在不同食品中的生存和生长

非霍乱弧菌在生化学性状上与霍乱弧菌两种生物型(古典生物型和El Tor生物型)有关,但与O 1抗血清不凝集。这种弧菌引起腹泻疾病的报告在亚洲和其他地方不断增加,并发现在水和海味食品中广泛存在。作者从腹泻病人中分离到三株非霍乱弧菌:1株不发酵蔗糖、血清群O 32(Heiberg V);1株发酵蔗糖、血清群O 24(HeibergⅡ);另1株亦发酵蔗糖、血清群O 37(HeibergⅠ),用此三菌株进行了在不同PH和温度条件下,在各种食品中的生长情况的研究。不同温度下的生长情况:置10 g样品(米     

北美洲人感染霍乱弧菌后血清抗毒素反应的持续时间

为研究人体对霍乱弧菌病原感染后血清产生霍乱抗毒素的时间及其维持多久,以便提供血清流行病学调查的参考数据和评价,作者对31例北美洲人志愿者作了人工霍乱弧菌口服试验。志愿者一次或多次摄入10~6个菌体,并以小苏打或食物冲淡胃酸。病原菌株包括古典稻叶型569B,古典小川型395,El Tor稻叶型P27459和N16961,El Tor小川型N15870,N18117和E7946。以微量法酶联免疫吸附试验(ELISA)检测血清IgG的抗毒素并根据标本比色的光密密(opticaldensity,O.D)为结果判断标准。结果:31例中7例(23%)粪便培养阳性,但未腹泻;18例出现中型腹泻(排便最平均为1.5升);6例为重型腹泻(排便量平     

Genetic characterization of Vibrio cholerae O1 strains isolated in Zambia during 1996-2004 possessing the unique VSP-II region of El Tor variant

New variants of Vibrio cholerae O1 have appeared in different time-frames in various endemic regions, especially in Asia and Africa. Sixty-nine strains of V. cholerae O1 isolated in Zambia between 1996 and 2004 were investigated by various genotypic techniques to determine the lineage of virulence signatures and clonality. All strains were positive for Vibrio seventh pandemic Islands (VSP)-I and VSP-II and repeat toxin (RTX) gene clusters attesting their El Tor lineage. Interestingly, strains isolated in recent times (2003-2004) were identified as an altered variant (El Tor biotype that harbours El Tor type rstR but produce classical ctxB) that replaced completely the progenitor El Tor strains prevalent in 1996-1997. Recent altered variant strains differed from prototype El Tor strains isolated earlier in that these strains lacked two ORFs, VC0493 and VC0498, in the VSP-II region. PFGE analysis revealed two major clonal lineages in the strains; cluster A represented the strains isolated before 2003 and cluster B the altered strains isolated in 2003-2004. Cluster A was closely related to prototype El Tor reference strain isolated in Bangladesh in 1971. Cluster B was found to be matched with Bangladeshi altered strains but was different from the hybrid strains isolated from Mozambique and Bangladesh. This report provides important information on the genesis of altered strains of V. cholerae O1 isolated in Zambia and emphasizes the need for further studies to follow the trends of evolutionary changes.     

Notes from the field: Identification of Vibrio cholerae serogroup O1, serotype Inaba, biotype El Tor strain - Haiti, March 2012

On October 20, 2010, an outbreak of cholera was confirmed in Haiti for the first time in more than a century. As of April 10, 2012, a total of 534,647 cases, 287,656 hospitalizations, and 7,091 deaths have been reported in Haiti as a result of the outbreak. The Vibrio cholerae strain that caused the Haiti epidemic has been characterized as toxigenic V. cholerae, serogroup O1, serotype Ogawa, biotype El Tor.     

霍乱疫情分离株流行菌型及耐药性分析

目的了解霍乱疫情分离株的流行菌型及其耐药性,为霍乱疫情的快速有效控制和临床治疗提供科学依据。方法对2008年6月海南省某地霍乱疫情采集的6株霍乱弧菌分离株进行血清学分型、噬菌体-生物分型、霍乱毒素基因检测、脉冲场凝胶电泳(PFGE)分子分型和药物敏感性试验。结果 6株菌均属埃尔托生物型霍乱弧菌,除1株为O1群小川型霍乱弧菌流行株(1c),其余5株均为O1群稻叶型霍乱弧菌流行株(1c);6株菌均具有霍乱毒素基因;PFGE分型为2个型,但聚类图谱分析相似性为100%;6株菌均对链霉素、复方新诺明、磺胺异噁唑、多粘菌素B耐药;对诺氟沙星、头孢噻肟、头孢噻吩等9种抗生素均敏感;结论该起霍乱疫情的流行菌型为O1群埃尔托霍乱弧菌稻叶型流行株(1c);诺氟沙星、头孢噻肟、头孢噻吩等可作为病例和带菌者治疗的指导用药,链霉素、复方新诺明、磺胺异噁唑、多粘菌素B则不宜使用。     

Comparison of amplified fragment length polymorphism and pulsed-field gel electrophoresis for subtyping of Vibrio cholerae serogroups O1 and O139

Molecular typing of Vibrio cholerae strains is a powerful tool for the surveillance of cholera. Amplified fragment length polymorphism (AFLP) is considered to be a powerful subtyping technique to distinguish bacterial strains at the genetic level. Optimization and standardization of AFLP protocol is required to allow data comparisons across different laboratories in a surveillance network. Here, we performed AFLP using different restriction enzymes and primer pairs for subtyping of V. cholerae serogroups O1 and O139 and compared the optimized AFLP protocol with pulsed-field gel electrophoresis (PFGE) to evaluate the applicability of AFLP for conducting epidemiological surveillance of cholera. The discriminatory index (D-value) of PFGE for serogroup O1 strains was similar when digested with NotI and SfiI, whereas that for O139 strains was higher for NotI digestion than for SfiI. EcoRI-G/MseI-T was the restriction enzyme and primer combination with highest discriminatory index used in the AFLP analysis. Capillary electrophoresis-based AFLP showed higher discriminatory power than that of polyacrylamide gel electrophoresis-based AFLP. When the two methods were compared using 72 epidemiologically unrelated serogroup O1 El Tor isolates, AFLP had a lower D-value than PFGE with NotI and SfiI digestions, respectively. For 54 epidemiologically unrelated serogroup O139 isolates, NotI PFGE had the highest discriminatory power, and SfiI PFGE and AFLP yielded almost the same but lower discriminatory power. We conclude that NotI and SfiI are both suitable for the PFGE of V. cholerae serogroup O1, whereas NotI should be defined as the primary enzyme for serogroup O139. The applicability of AFLP in V. cholerae subtyping and outbreak investigations is limited.     

多重PCR检测霍乱弧菌的毒素相关基因

目的 探索建立一种快速、敏感地检测霍乱弧菌O1群、O139群、非O1／非O139群的毒素相关基因的方法。方法 针对霍乱弧菌霍乱肠毒素A亚单位基因(ctxA)、小带联结毒素基因(zot)、辅助霍乱肠毒素基因(oce)、霍乱弧菌毒素共调菌毛A基因(tcpA)、毒素表达调控蛋白基因(toxR)分别设计引物，建立多基因PCR方法；通过一次扩增反应之产物经琼脂糖凝胶电泳，可检测出菌株的毒素相关基因的携带情况。结果 阳性对照菌株：MO45(霍乱弧菌O139群)检出5种毒素相关基因，结果符合设计要求；其他不同来源的霍乱弧菌(O1群、O139群、非O1／非O139群)检出1—5种不等的毒素相关基因；根据毒素相关基因携带情况，可将菌株分为5个基因型，并可区分为产毒株和非产毒株；多重PCR敏感度可达10^2cfu／ml。结论 该方法快速、特异、敏感，具有较大的应用价值。     

霍乱弧菌山梨醇发酵相关基因序列特征分析

目的 比较霍乱弧菌流行株与非流行株山梨醇发酵相关基因的差异，为采用分子生物学方法快速区分两类菌株提供理论依据。方法采用基因测序的方法，分别对42株霍乱弧菌(01群埃尔托型33株、0139群9株)流行株和非流行株的糖发酵激活蛋白、外周质麦芽糖结合蛋白、外周质磷酸盐结合蛋白和外周质氨基酸结合蛋白编码基因进行序列比较。结果糖发酵激活蛋白编码基因在霍乱弧菌流行株与非流行株共发现三个位置有单个碱基的差异，即第106、150和378位核苷酸(在流行株分别为A、A和T，而在非流行株分别为G、G和C)。第106位碱基的差异导致了氨基酸的不同(编码蛋白的第36个氨基酸在流行株和非流行株分别为苏氨酸和丙氨酸)。外周质麦芽糖结合蛋白和外周质磷酸盐结合蛋白编码基因各发现两个碱基的规律变化，外周质氨基酸结合蛋白编码基因未发现一致性碱基改变。结论在这些基因中存在着多个单核苷酸多态性，可能会成为快速区分两类菌株的重要依据。糖发酵激活蛋白第36位氨基酸的改变，可能会引起该蛋白活性的改变。     

Outbreak-associated Vibrio cholerae genotypes with identical pulsotypes, Malaysia, 2009

A cholera outbreak in Terengganu, Malaysia, in November 2009 was caused by 2 El Tor Vibrio cholerae variants resistant to typical antimicrobial drugs. Evidence of replacement of treatable V. cholerae infection in the region with antimicrobial-resistant strains calls for increased surveillance and prevention measures.     

Molecular analysis of vibrio cholerae strains isolated in Malaysia: public health implications

The genetic diversity or clonality among Vibrio cholerae O1, O139 and non-O1/ non-O139 of clinical and environmental origin using ribotyping and PFGE was performed in order to ascertain the public health implications of the different genotypes circulating within the Malaysian environment. Using an in-house typing scheme, of the 214 strains included, 202 strains were isolated locally between 1992 and 1998, seven were obtained from Bangladesh and five were reference strains. Amongst the 176 El Tor O1 strains, 152 clinical strains demonstrated five ribotypes--E1a, E1b, E2a, E3 and E1c. E1b was the most predominant ribotype demonstrated by 84% of the El Tor O1 strains and was present in all years demonstrating that this strain was intrinsic to Malaysia. PFGE analysis of these strains demonstrated minimal variation amongst the 15 PFGE profiles obtained. Ribotpye E2a amongst five clinical and two environmental O1 strains, were from one location and had previously been reported in Indonesia and the Philippines, thus demonstrating strong evidence that these strains may have been imported into Malaysia. Among Vibrio cholerae O139 strains, 91.7% were of ribotype A1a similar to the original O139, while two others were of ribotype A1b and one of A1e, corresponding to ribotypes 1, 2 and 3 of Dalsgaard and colleagues' scheme for O139 strains. PFGE analysis demonstrated that 89% of ribotype A1a could be differentiated into three PFGE genotypes which were very closely related. The eight non-O1/non-O139 serogroup strains were heterogeneous in both ribotype and PFGE patterns.     

Clonal origins of Vibrio cholerae O1 El Tor strains, Papua New Guinea, 2009-2011

We used multilocus sequence typing and variable number tandem repeat analysis to determine the clonal origins of Vibrio cholerae O1 El Tor strains from an outbreak of cholera that began in 2009 in Papua New Guinea. The epidemic is ongoing, and transmission risk is elevated within the Pacific region.     

Identification of Vibrio cholerae by enzyme electrophoresis.

Zymovar analysis of 260 strains of Vibrio cholerae plus 3 reference strains of V. mimicus, using 13 structural loci, led to the grouping of strains in 73 zymovars (strain or group of strains sharing the same alleles). Effective separation of strains, distinction of V. cholerae strains from closely related V. mimicus and the detection of 2 vibrio strains, including one with two O1 serovars, in supposedly pure collection cultures, illustrate the potential of zymovar analysis in the identification of V. cholerae isolates. Two El Tor strains from USA, one CT+ and the other CT-, shared the same zymovar 71, while 127 typical El Tor strains belonged to zymovar 14.