脉冲场凝胶电泳方法在霍乱暴发溯源中的应用

目的应用脉冲场凝胶电泳(PFGE)方法对不同类型的霍乱暴发事件进行分析,了解霍乱菌株的PFGE特征,追溯菌株的来源及其演变过程,探讨霍乱暴发流行的特点和规律。方法采用限制性内叨酶NotⅠ,对2003—2005年广州地区霍乱暴发疫情中41株霍乱弧菌进行PFGE分子分型,用BioNumerlcs Version 4.0软件(复选Dice相关系数和UPGMA方法)进行聚类分析,并与噬菌体-生物分型及致病相关基因分型方法作比较。结果在霍乱暴发事件中,PFGE能有效地区分流行病学上密切相关菌株与无关菌株,与噬菌体-生物分型和致病相关基因分型方法相比具有更高的分辨能力。结论PFGE分型可揭示人和环境分离的霍乱菌株之间的流行病学联系,为霍乱疫情溯源提供分子流行病学证据和支持。     

广州地区小川型霍乱弧菌的脉冲场凝胶电泳分析

目的分析小川型霍乱弧菌的致病相关基因型,探讨广州地区小川型霍乱流行趋势和规律。方法采用多重PCR方法检测小川型菌株的4种致病相关基因,应用脉冲场凝胶电泳技术（PFGE）对菌株进行分子分型,对PFGE图谱采用分子分型软件BioNumerics Version 4.0进行聚类分析。结果广州地区小川型菌株中存在3种致病相关基因型,即A型、B型和C型,20%感染者分离株为致病相关基因A型,80%环境分离株为致病相关基因C型。25株霍乱弧菌分为14个不同的PFGE型,归为3个聚类群（Ⅰ、Ⅱ、Ⅲ群）。每一次小川型暴发中分离的菌株PFGE克隆型相同或相近,而散发病例分离株与暴发株的PFGE型有些相同,有的差异较大。环境小川型菌株中存在PFGE克隆型的多样性,部分环境株与感染者分离株具有相近的PFGE型。结论应建立广州地区霍乱菌株的PFGE分子分型数据库,加强霍乱的预防、控制和预警。     

脉冲场电泳技术用于霍乱暴发的流行病学研究

[目的]探讨大连市2004年霍乱暴发中不同来源的霍乱弧菌之间的遗传相关性,进行同源性分析,并与表型分形、PCR检测CT毒素技术进行比较。[方法]2007年,选择2004年大连市霍乱暴发、散发疫情分离的34株稻叶型霍乱代表菌株,经噬菌体-生物分型和PCR技术检测CT毒素基因后,进行脉冲场电泳(PFGE)分型。[结果]分离自霍乱患者的22株霍乱菌株均为稻叶1b型,CT阳性。分离自暴发地区自备水的6株霍乱菌株为稻叶1b型,CT阳性。分离自市内外环境的5株霍乱菌株为非流行株,CT阴性。检测的34株霍乱弧菌PFGE分型为2大亚类4种带型,其中从金州区散发、开发区暴发疫点获得的人源菌株在PFGE图谱上带型相同,为2型;其他分离自散发病例的菌株在PFGE图谱上同优势2型的相似性分别高达97.50%、97.10%;分离自暴发地区自备水的霍乱菌株PFGE带型同样为2型,来自市内外环境的霍乱菌株PFGE带型都为4型,同优势2带型有很大差异。[结论]霍乱流行期间分离于患者和疫点自备水的霍乱弧菌PFGE图谱基本一致,与外环境分离的非流行株关系较远。     

一起霍乱疫情分子流行病学溯源分析

目的分析大连市2004年外环境中和1起霍乱疫情中不同来源的霍乱弧菌之间的遗传相关性。方法选择2004年暴发,散发的22株稻叶型霍乱代表菌株,先做噬菌体-生物分型和PCR技术检测CT毒素基因后,再进行PFGE分型研究。结果分离患者的霍乱菌株都为稻叶1b型,CT阳性。分离自暴发地区外环境的霍乱菌株也为稻叶1b型,CT阳性。但市内外环境的霍乱菌株都为非流行株,CT检测阴性。所试22株霍乱菌株PFGE分型共2大亚类3种带型。其中从开发区疫点所获得的人源菌株在PFGE图谱上带型相同,为2型;分离自散发病例的菌株在PFGE图谱上同优势2型之间相互略有差异,相似度为97.1%;分离自暴发地区自备水的霍乱菌株PFGE带型同样为2型,而来自市内外环境的霍乱菌株PFGE带型都为3型,同优势2带型有很大差异。结论霍乱流行期间分离于患者和疫点自备水的霍乱弧菌PFGE图谱基本一致,与外环境分离的非流行株关系较远。     

脉冲场凝胶电泳分型方法追溯霍乱传染源

目的　了解四川省 2 0 0 4年霍乱疫情分离菌株与常规监测的外环境和水产品中霍乱弧菌分离株之间的遗传相关性 ,追溯传染源 ,为预测疫情和制定防治措施提供依据。方法　O139群霍乱弧菌编码脂多糖 (LPS)特异性基因引物聚合酶链反应 (PCR)复核菌株 ,霍乱毒素 (CT)基因引物PCR检测霍乱毒力基因 ,脉冲场凝胶电泳 (PFGE)进行菌株的分子分型。结果　 2 5株受试菌株LPS基因 2对引物PCR结果均为阳性。所有 6株河水中分离的菌株均为CT基因阴性 ,其余均具有CT基因。 2 4株菌PFGE可分为 13个型。结论　LPS基因PCR检测结果从分子水平证实受试菌株均为O139群霍乱弧菌。河水中分离的 6株菌是非产毒株 ,其余菌株均具有致病性。环境水分离的O139群非产毒株之间以及产毒株与非产毒株之间遗传相关性较远。产毒株之间具有较高的同源性。四川省从甲鱼分离的O139群霍乱弧菌与同期流行的O139群霍乱弧菌分型的带型一致 ,在国内首次以分子流行病学分析提示甲鱼可能是四川省近年霍乱疫情主要传染来源之一。     

脉冲场凝胶电泳分型追溯江西省霍乱暴发传染源

目的分析2006年5月江西省聚餐暴发霍乱疫情的分离菌株与常规监测在外环境及水产品中分离的霍乱弧菌之间分子分型特征和遗传相关性。方法利用聚合酶链反应检测霍乱毒素基因，用脉冲场凝胶电泳（PFGE）对菌株进行分子分型，所得结果以BioNumerics V4．0软件UPGMA方法进行聚类分析。结果30株与暴发相关分离菌株中，无论来自病例、带菌者、疫区污水、水产品的菌株均为产毒株。30株O139群霍乱弧菌以NotⅠ酶切后PFGE可分为4个型别。从甲鱼、牛蛙分离的O139群霍乱弧菌的型别与此次暴发的优势PFGE型别一致，并且也为产毒株。两起暴发包括多起聚餐，从中分离的菌株优势型别相同，传播媒介为甲鱼、牛蛙，均购于同一水产批发部，说明为同一传染源。结论携带O139群霍乱毒素基因的甲鱼、牛蛙是引起本次多起聚餐暴发霍乱的主要传染源。     

脉冲场凝胶电泳分型技术在追溯O139霍乱传染来源中的应用

目的分析四川省2004年7起因聚餐暴发霍乱疫情的分离菌株之间,及其与常规监测中从外环境、水产品中分离的霍乱弧菌之间的分子分型特征和遗传相关性,查找霍乱传染来源.方法利用聚合酶链反应检测霍乱毒力基因（ctxAB）,用脉冲场凝胶电泳（PFGE）对菌株进行分子分型,所得结果以BioNumerics V4.0软件UPGMA方法进行聚类分析.结果所试72株O139群霍乱弧菌中所有4株从河水分离的菌株ctxAB阴性,其余均具有ctxAB,为产毒株.对其中的67株菌以Not I酶切后PFGE可分为16个型别.从甲鱼分离的O139群霍乱弧菌与同期流行的O139群霍乱弧菌优势PFGE型别一致,并且也为产毒株.7起暴发中分离的菌株型别各不相同.结论2004年四川省O139霍乱暴发感染来源复杂,提示并非因为菌株在该地持续存在而引起,但甲鱼可能是2004年度霍乱聚餐暴发的主要传染来源之一.     

四川省2005年霍乱分子流行病学特征

目的：分析四川省2005年霍乱弧菌分子特征,以及霍乱暴发疫情分离的菌株与菌株之间,疫情分离的菌株与海、水产品监测分离的霍乱弧菌之间的遗传相关性,查找霍乱传染来源,为预测疫情和制定防治措施提供依据.方法：利用聚合酶链反应（PCR）检测O139群霍乱弧菌特异性编码脂多糖基因（LPS）和霍乱毒力基因（ctxAB）;脉冲场凝胶电泳对菌株进行分子分型,所得结果以BioNumerics V4.0软件UPGMA方法进行聚类分析.结果：所试16株霍乱弧菌14株LPS阳性,为O139群霍乱弧菌;另2株为O1群稻叶型霍乱弧菌.2株稻叶型霍乱弧菌和1株O139群霍乱弧菌ctxAB阴性,其余13株菌均具有ctxAB,为产毒株.对16株菌以NotⅠ酶切后PFGE可分为5个型别.O139群霍乱弧菌优势流行株与从甲鱼分离的O139群霍乱弧菌PFGE型别一致,且同为产毒株.结论：甲鱼等海、水产品被O139群霍乱弧菌污染情况严重,甲鱼中分离的O139群霍乱弧菌与霍乱疫情分离菌株之间高度同源,被污染的甲鱼可能是本年度食源性霍乱暴发的主要传染来源之一,海、水产品的监测是近期霍乱防控的重点.     

湖南省2005年-2010年霍乱疫情分离株的毒力基因PCR及PFGE分型分析

目的:了解2005年-2010年湖南省霍乱疫情分离到的O139群霍乱弧菌菌株的病原学特征,研究疫情分离株之间的克隆相关性。方法:采用K-B法进行药敏试验;聚合酶链反应(PCR)检测ctxAB毒力基因;脉冲场凝胶电泳对疫情分离代表株进行PFGE分型分析。结果:33株霍乱弧菌对强力霉素、复方新诺明的耐药率较高,分别为39.39%和75.76%,对环丙沙星、诺氟沙星以及丁胺卡那100%敏感;毒力基因的PCR结果显示为所有疫情分离的O139霍乱弧菌均为产毒株,即霍乱肠毒素基因ctxAB阳性;24株分离自2005年和2010年的7起疫情里的O139霍乱弧菌进行PFGE分型及聚类分析后,共分为3个PFGE带型,所有菌株的带型相似率在83%～100%之间。结论:湖南省2005年-2010年霍乱疫情以O139群为主,引起疫情的全部为产毒株,不同年份不同地区之间霍乱疫情的分离菌株之间存在着紧密相关的流行克隆群,对分离菌株进行耐药性监测和进一步的分子分型分析,有助于霍乱的主动监测和传染来源的追踪。     

江西省霍乱外环境监测菌株分子分型研究

目的分析江西省1999-2006年外环境监测分离的O139群霍乱弧菌之间的分子分型特征和遗传相关性,为霍乱防制提供科学依据。方法利用聚合酶链反应检测霍乱毒素基因,用脉冲场凝胶电泳对菌株进行分子分型,所得结果以BioNumerics V5.01软件UPGMA方法进行聚类分析。结果 43株O139群霍乱弧菌中15株为产毒株,28株为非产毒株;43株O139群霍乱弧菌以NotⅠ酶切后脉冲场凝胶电泳可分为22个型别。结论江西省外环境分离菌株PFGE型别呈多态性。要加强对环境水体霍乱弧菌污染状况的监测工作,结合流行病学调查分析,为霍乱暴发或流行提供预测预警。     

湖南省2012年霍乱弧菌病原学特征分析

目的了解2012年湖南省不同地区不同时间各疫情菌株的病原学特征,分析比较各疫情分离株之间以及与常规监测分离株之间的遗传相关性,为追溯传染源提供依据。方法利用生化鉴定系统进行菌株鉴定,血清学方法生物分型,PCR方法检测毒力基因和脉冲场凝胶电泳技术(PFGE)进行分子分型。结果 2012年从湖南省疫情和监测样品中分离的17株O139群霍乱弧菌均带毒力基因,均为产毒株。在进行PFGE分子分型的17株菌中,有2起疫情酶切图谱完全相同,而该2起疫情与其他的3起疫情以及这3起疫情之间的酶切图谱不完全相同。结论湖南省2012年从甲鱼中分离的O139群霍乱弧菌毒力基因携带率高,是疫情频发的一个重要原因。从病人和食品中分离的菌株具有高度同源,进一步证实该疫情为食源性传播。分子分型图谱相似率100%的2起疫情传染来源一致,而其他各起疫情之间关联性很小或者没有,传染来源均不同。     

Comparison of pulsed-field gel electrophoresis and ribotyping for subtyping of Vibrio cholerae O139 isolated in Thailand.

Pulsed-field gel electrophoresis (PFGE) of Cpo I-digested genomic DNA and ribotyping (Bgl I) were applied to 60 Vibrio cholerae strains including 48 V. cholerae O139 from Thailand to compare their value in differentiating strains of the present V. cholerae O139 epidemic. PFGE patterns were divided into groups A and B representing five and four subtypes, respectively, while ribotyping showed four different patterns. PFGE group B subtypes were only presented among O139 isolates from Thailand, whereas four O139 strains from Bangladesh and India showed identical PFGE group A subtypes observed in O139 isolates from Thailand. Two nontoxigenic O139 isolates from Thailand showed different and unique PFGE types as did five V. cholerae non-O139 isolates containing a gene virulence complex found in V. cholerae O139. These results indicate that PFGE (Cpo I) can resolve recent evolutionary divergence within V. cholerae O139 and offers a useful supplementary tool for following the progressing V. cholerae O139 epidemic.     

安徽省2010年霍乱弧菌病原学特征分析

目的对2010年度安徽省霍乱疫情的流行菌株进行实验检测,从病原学角度分析本年度霍乱的特征。方法应用常规细菌学方法、聚合酶链式反应(PCR)方法和脉冲场凝胶电泳技术(PFGE)对46株霍乱弧菌的基本特性、致病毒力基因及分子分型特征进行实验检测。结果 46株菌株O1群小川型44株,O139群2株,实验菌株对15种抗生素表现出程度不同的耐药性;所有菌株均携带有肠毒素(CT)和毒素共调菌毛(TCP)毒力基因;PFGE图谱聚类可分作5个型别,93.2%的O1群菌株(41/44)具有相同的分子分型。结论 2010年度安徽省流行的霍乱以O1群小川血清型为主要病原菌,流行菌型的更迭明显,6个市发生的疫情检获的O1群菌株93.2%具有相同的分子分型,表现出极高的病原同源性。     

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。     

Molecular typing of Vibrio cholerae O1 isolates from Thailand by pulsed-field gel electrophoresis

The aim of the present study was to genotypically characterize Vibrio cholerae strains isolated from cholera patients in various provinces of Thailand. Two hundred and forty V. cholerae O1 strains, isolated from patients with cholera during two outbreaks, i.e. March 1999-April 2000 and December 2001-February 2002, in Thailand, were genotypically characterized by NotI digestion and pulsed-field gel electrophoresis (PFGE). In total, 17 PFGE banding patterns were found and grouped into four Dice-coefficient clusters (PF-I to PF-IV). The patterns of V. cholerae O1, El Tor reference strains from Australia, Peru, Romania, and the United States were different from the patterns of reference isolates from Asian countries, such as Bangladesh, India, and Thailand, indicating a close genetic relationship or clonal origin of the isolates in the same geographical region. The Asian reference strains, regardless of their biotypes and serogroups (classical O1, El Tor O1, O139, or O151), showed a genetic resemblance, but had different patterns from the strains collected during the two outbreaks in Thailand. Of 200 Ogawa strains collected during the first outbreak in Thailand, two patterns (clones)--PF-I and PF-II--predominated, while other isolates caused sporadic cases and were grouped together as pattern PF-III. PF-II also predominated during the second outbreak, but none of the 40 isolates (39 Inaba and 1 Ogawa) of the second outbreak had the pattern PF-I; a minority showed a new pattern--PF-IV, and others caused single cases, but were not groupable. In summary, this study documented the sustained appearance of the pathogenic V. cholerae O1 clone PF-II, the disappearance of clones PF-I and PF-III, and the emergence of new pathogenic clones during the two outbreaks of cholera. Data of the study on molecular characteristics of indigenous V. cholerae clinical isolates have public-health implications, not only for epidemic tracing of existing strains but also for the recognition of strains with new genotypes that may emerge in the future.     

Preferential association of the heat-stable enterotoxin gene (stn) with environmental strains of Vibrio cholerae belonging to the O14 serogroup

Toxigenic Vibrio cholerae O1 and O139 serogroups have the capacity of causing epidemic and pandemic cholera but are infrequently found in the environment. The other serogroups are abundant in aquatic environments but do not possess the virulence genes necessary for causing the disease. Of the 559 environmental strains of V. cholerae, collected during different periods from environmental samples in Calcutta, 9 (1.6%) harboured the heat-stable enterotoxin gene (stn). Six of the 9 strains belonged to the O14 serogroup. Thus, V. cholerae strains carrying the stn gene revealed preferential association with the O14 serogroup. Three of the six strains harboured the tcpA gene of the E1 Tor type, which is an unusual feature among environmental V. cholerae strains. A strain that possessed the E1 Tor type tcpA also had the CTX prophage. Pulsed field gel electrophoresis (PFGE) revealed that the stn gene positive O14 strains of V. cholerae were not clonal.     

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.     

Fluoroquinolone-resistant Vibrio cholerae isolated during a cholera outbreak in India

During the cholera epidemic of 2002 in and around Hubli, south India, Vibrio cholerae strains resistant to fluoroquinolones were isolated. Among the isolates of V. cholerae non-O1, non-O139 serogroups, 55.9% and 47.1% were resistant to norfloxacin and ciprofloxacin, respectively. However, only 12.5% of the O1 serogroup strains were resistant to both norfloxacin and ciprofloxacin. Though the O139 serogroup strains were susceptible to these antibiotics, they exhibited multidrug resistance. Emergence of fluoroquinolone-resistant V. cholerae that also exhibited multidrug resistance is of great significance in the epidemiology and control of cholera.