2000-2005年广州地区稻叶型霍乱弧菌的分子分型

目的：分析20002005年广州地区霍乱暴发点、散发事件和环境监测分离的稻叶型霍乱弧菌的致病相关基因型和脉冲场凝胶电泳技术（PFGE）分型，探讨该稻叶型霍乱的流行特征和趋势。方法：对分离株进行噬菌体生物分型，采用多重PCR方法检测菌株的4种致病相关基因，应用PFGE进行分子分型，并采用软件BioNumerics Version4．0进行聚类分析。结果：广州地区稻叶型霍乱菌株中存在3种致病相关基因型：A、B和C型。98％感染者分离株为致病相关基因A型，45％环境分离株为C型。82株菌分为39个不同的PFGE型。各年份稻叶型暴发与散发事件中均存在具有PFGE分型特征一致的菌株，而环境中菌株则存在多种不同的PFGE型，来源不同地区分离株也均有相似的分型特征。结论：噬菌体一生物分型、致病相关基因分型和PFGE分型存在一定的关联规律性，分子分型可为分析霍乱疫情和预警提供技术平台。     

湖北省2012年流行株O139霍乱弧菌脉冲场凝胶电泳分型分析

目的分析3起霍乱疫情病原O139霍乱弧菌分子分型特征和遗传相关性,探讨O139霍乱疫情流行特征。方法对2012年湖北省3起霍乱疫情分离鉴定的35株O139霍乱弧菌菌株用水煮法提取DNA,利用聚合酶链反应检测霍乱肠毒素CT基因;取新鲜培养的菌株,制备约4.3个麦氏单位的细菌悬浮液,经裂解、洗涤及限制性内切酶NotI和SfiI酶切后进行脉冲场凝胶电泳(PFGE)分子分型,用凝胶成像仪获取电泳图像,分析DNA片段并用BioNumerics V4.6软件UPGMA方法(复选Dice相关系数)进行聚类分析。结果 35株O139霍乱弧菌ctxA基因PCR扩增产物约为308bp,分离自聚餐食用的凉菜、病人、带菌者及厕所标本(对应病人家)O139霍乱弧菌均为产毒株,经NotI酶切分为9种PFGE带型,SfiI酶切分为6种PFGE带型;A市2株病人分离菌和4株带菌者分离菌PFGE带型为KZGN11O139.CN0077+KZGS12O139.CN0054,B市1株病人分离菌和1株厕所分离菌PFGE带型为KZGN11O139.CN0302+KZGS12O139.CN0058,C市和D市分离菌株优势型为KZGN11O139.CN0276+KZGS12O139.CN0007,包括3株食品分离菌、2株厕所分离菌及16株病人和带菌者分离菌,另有6株PFGE带型呈现多样性。结论 2012年湖北省霍乱疫情特点为散发以及聚餐导致的局限暴发,3起疫情的分离菌株带型各不相同,呈现多样性,其中2起为单一菌型感染,1起为混合菌型感染,传染来源复杂且不明确,提示应加强霍乱的病原学监测。     

O139群霍乱弧菌地方分离株的分子特征研究

目的研究本地分离出的O139群霍乱弧菌的分子特征,建立霍乱暴发和散发的快速检测及流行病学溯源的有效手段。方法采用PCR方法对霍乱弧菌进行4种毒力基因的检测,用随机扩增多态性分析(RAPD)及SPSS软件对以上菌株进行多态性分析,对霍乱弧菌毒力进行快速测定和分子分型。结果5株O139群霍乱弧菌均可检出四种毒力基因。所有霍乱弧菌的RAPD结果经聚类分析可分为3个聚类群,较好地反应了不同群的霍乱弧菌之间的亲缘关系。结论可将PCR和RAPD方法结合分析用于霍乱疫情的快速检测和流行病学溯源。     

河南省某一淡水养殖环节中非O1/O139群霍乱弧菌毒力基因及分子分型分析

目的 了解河南省淡水养殖环节中非O1/O139群霍乱弧菌毒力基因分布及分子分型情况。方法 对河南省淡水养殖环节中50株非O1/O139群霍乱弧菌和3株病人来源菌株进行全基因测序,利用PubMLST-Vc数据库分析其序列分型(ST),利用最小生成树关系图分析进化关系,通过VFDB数据库获得其毒力基因分布。结果 来源于淡水养殖环节和来源于病人的非O1/O139群霍乱弧菌53株菌株均具有黏附、趋化运动、抗吞噬、毒素及酶类等功能的8类毒力相关因子基因,缺失辅助定植、毒素共调、分泌等功能的毒力相关因子基因和副霍乱肠毒素等4个毒素基因。部分毒力相关因子或部分菌株的毒力相关因子毒力基因不全。与3株来源于病人的菌株相比,二者毒力因子基因携带情况相近,除MSHA Ⅳ型菌毛毒力因子mshA基因、荚膜多糖wbuB基因、wbfY基因、rmlB基因、血红素受体hutA基因及Ⅲ型分泌系统vscC2和vcrD2基因外,部分菌株二者携带相同的毒力因子基因。53株非O1/O139群霍乱弧菌分属19个ST型,ST4和ST5是优势ST型,养殖环节来源的菌株与病人来源的菌株分属不同的ST型。19个ST型等位基因位点变异差异数在1~7个,其中分离自淡水养殖环节的非O1/O139群霍乱弧菌菌株17个ST型等位基因位点变异差异数在1~7个,病人来源的非O1/O139群霍乱弧菌菌株2个ST型与分离自淡水养殖环节的非O1/O139群霍乱弧菌菌株ST1、ST2、ST6和ST10属同一簇,与ST1有6个等位基因位点存在差异。结论 河南省淡水养殖环节非O1/O139群霍乱弧菌菌株MLST分型多样化,携带多种毒力相关因子,不同来源菌株携带的毒力基因相同,虽然分属不同的ST型,但还是存在食品安全风险,提醒有关部门采取措施进行防控。     

云南省非O1/O139群霍乱弧菌分子特征分析

目的非O1/O139群霍乱弧菌能够引起人类急性腹泻性疾病,但与O1群和O139群相比,人们往往容易忽视其危害性。因此,我们对分离于云南省的31株非O1/O139群霍乱弧菌开展了分子特征分析。方法采用纸片法(K-B)开展抗生素敏感试验;多聚酶链反应(PCR)扩增检测毒力基因;同时,我们对菌株进行了脉冲场凝胶电泳(PFGE)和多位点序列分析(MLST)分子分型研究。结果药敏实验结果显示,67.74%的菌株对利福平耐药,对萘啶酸和复方新诺明的耐药率为29.03%,所有的菌株对庆大霉素和环丙沙星敏感;PCR结果显示,所有菌株的ompW基因为阳性,87.10%的菌株hly基因为阳性,25.81%的菌株rstREl tor为阳性,16.13%的菌株rstRClassical和tcpAEl tor为阳性,而CT、rfbO1和rfbO139基因全为阴性;PFGE结果显示,31株非O1/O139群霍乱弧菌呈现出高度的离散趋势,几乎没有相同带型的菌株出现;在MLST结果分析中,发现了1个gyrB新的等位基因,mdh基因发现了4个新的等位基因,metE基因发现了6个新的等位基因,pntA中发现了2个新的等位基因,purM中发现了3个新等位基因,pyrC中发现了4个新等位基因。经过排列组合后,共发现了17个新的霍乱弧菌ST型别(ST273-ST289)。结论非O1/O139群霍乱弧菌呈现出高度的多样性特征,同时,云南省的非O1/O139群霍乱弧菌具有一定的地域特点,丰富了现有的霍乱弧菌分子分型数据库。     

O1群霍乱弧菌PFGE分子分型和菌株流行能力关系研究

目的 调查福建省1962-2005年不同流行时期、不同流行血清群的O1群埃尔托型霍乱弧菌(EVC)的菌株间的遗传关联度和基因组的多态性;探讨不同PFGE型(簇)和霍乱菌株流行能力的关系。方法 运用脉冲场凝胶电泳分子分型(PFGE)技术,对福建省1962-2005年代表性霍乱菌株进行分型,结合流行病学背景资料探讨不同PFGE型(簇)和相关霍乱菌株的流行能力。结果 77株霍乱菌株按100%的相似度分为61个PFGE型,按照90%的相似度可得到5个主要的PFGE簇,命名为G1-G5;52株小川型霍乱弧菌,按照100%的相似度分为47个PFGE型,按照90%的相似度可得到4个主要的PFGE簇[G1(o)-G4(o)];25株稻叶型霍乱弧菌按照100%的相似度分为15个PFGE型,按照90%的相似度,可得到2个主要的PFGE簇[G1(i)-G2(i)]。各个流行时期的菌株均有优势的PFGE簇,散发的菌株呈现分散的PFGE型,没有集中趋势。结论 福建省3次流行的霍乱菌株有遗传关联性,在流行过程中产生基因变异出现新的优势PFGF簇;各地区间的霍乱流行有传播上的相关性。小川型G2(o)PFGE簇和稻叶型G1(i)PFGE簇的菌株有引起霍乱持续流行的能力,后者更有引发大规模病例数的能力。     

我国O139群霍乱弧菌CTX遗传单元基因分布特征

到目前为止 ,已发生的 7次霍乱世界大流行 ,都是由O1群霍乱弧菌引起的。 1992年在印度、孟加拉等国引起暴发流行的O139群霍乱弧菌 ,是一种新出现的由非O1群霍乱弧菌引起的霍乱流行的病原体 ,能产生与O1群霍乱弧菌相同的霍乱毒素 (CT) ,其所引起的霍乱在临床和流行病学上也与O1群所致的霍乱基本相同。近年来研究表明 ,与O1群霍乱弧菌相比 ,O139群霍乱弧菌具有独特的表型及分子特征。在霍乱弧菌中 ,CT是最主要的毒力因素 ,其结构基因位于染色体上大小为 7～ 9.7kb的CTX遗传单元上[1] ,它的核心区域 (4 .5kb)至少有 5个基因 ,依次排列是c…     

15株非O1群霍乱弧菌的致病性和毒力实验观察

近五年来国内外报道了非 O1群霍乱弧菌及 O_(139)血清群的霍乱暴发流行情况。我们于1996年5月至9月从本院肠道门诊腹泻患者粪便中分离出15株非O1群霍乱弧菌和1株 O1群 El-tor 小川型霍乱弧菌,并将其对小白鼠的致病性和肠毒素毒力、小肠病理变化进行了实验观察。现将结果报告如下。材料与方法一、菌株来源及鉴定15株非 O1群霍乱弧菌和1株 O1群 El-tor 小川型霍乱弧菌,均从本院肠道门诊患者粪便中分离所得。并将其分别编为1号至15号、El-tor 弧菌编为16     

霍乱弧菌的分子遗传学特征及其检测研究进展

目前霍乱弧菌至少被分为 1 90多个血清群 ,霍乱弧菌新菌株不断产生、并被发现 ,使流行情况变得复杂。此外 ,针对霍乱弧菌的不同生存状态 ,采用过去的免疫学和生物学方法不能很好检测、鉴别。目前 ,分子生物学和核酸技术的发展为我们快速检测、鉴别霍乱弧菌提供了可能。现就霍乱弧菌的分子遗传学特征及其基因检测方面的研究进展作一综述。一、分子遗传学特征目前 ,引起霍乱暴发流行的主要菌株为Ｏ1群的ＣＶＣ、ＥＶＣ流行株ＥＳＥＶＣ和Ｏ1 3 9群 ,其他非Ｏ1群、非Ｏ1 3 9群一般不致病或仅引起散发腹泻或肠道外感染。能造成霍乱流行的菌株是…     

杭州市健康人群粪便中检出O1和O139群霍乱弧菌无毒力株

目的　对杭州市饮食、服务行业人员粪便标本进行霍乱弧菌监测。方法　 2 0 0 0年 5月～ 2 0 0 1年 7月采集 2 6 2 5 2名饮食、服务行业从业人员健康体检者粪便标本 ,分别接种于碱性蛋白胨水和 4号琼脂中。通过培养特性、菌落形态、革兰染色、动力和生化试验等检查 ,对所分离的疑似霍乱弧菌菌株进行初步鉴定。采用霍乱弧菌O1群及O139群单克隆抗体的玻片凝集试验、噬菌体分型、霍乱弧菌ctxA和tcpA基因的PCR进一步鉴定各疑似霍乱菌株。 结果　上述标本中检出O1血清群和O139血清群霍乱弧菌各 2株。 2株O1血清群霍乱弧菌的噬菌体分型分别为 19k和 5k ,为埃尔托生物型非流行菌株 ;2株O139血清群霍乱弧菌的噬菌体分型分别为 2 0k和 31k ;但ctxA和tcpA基因PCR检测结果均为阴性。结论　尽管所检出的 4株霍乱弧菌均为无毒力的非流行菌株 ,但因携带者从事职业的特殊性 ,仍应引起高度重视。     

Plasmid-borne multiple drug resistance in Vibrio cholerae serogroup O1, biotype El Tor: evidence for a point-source outbreak in Bangladesh

In 1979, an outbreak of plasmid-borne, multiply drug-resistant Vibrio cholerae serogroup O1 biotype El Tor (V. cholerae O1) occurred in the Matlab area of Bangladesh. The outbreak could have resulted from the introduction into the area of a single resistant strain or from multiple conjugations of drug-sensitive V. cholerae O1 with C plasmids in other environmental flora. Resistant strains were phage typed to determine their relatedness, and plasmid studies were conducted to determine the frequency of C plasmids in nonvibrio flora of family contacts of cholera patients. Forty-one (85%) of 48 resistant strains of V. cholerae O1 examined belonged to two closely related phage types new to the area, whereas 59 drug-sensitive strains from the same period were primarily of two different phage types. Group C plasmids were in nonvibrio strains from five of 36 family contacts of patients with drug-resistant cholera but none of 82 family contacts of patients with sensitive cholera. This outbreak most likely began from the introduction into the area of a single, multiply drug-resistant strain of V. cholerae O1. C plasmids detected in the nonvibrio flora of family contacts probably came from the resistant strain of V. cholerae O1.     

Genomic diversity of 2010 Haitian cholera outbreak strains

The millions of deaths from cholera during the past 200 y, coupled with the morbidity and mortality of cholera in Haiti since October 2010, are grim reminders that Vibrio cholerae, the etiologic agent of cholera, remains a scourge. We report the isolation of both V. cholerae O1 and non-O1/O139 early in the Haiti cholera epidemic from samples collected from victims in 18 towns across eight Arrondissements of Haiti. The results showed two distinct populations of V. cholerae coexisted in Haiti early in the epidemic. As non-O1/O139 V. cholerae was the sole pathogen isolated from 21% of the clinical specimens, its role in this epidemic, either alone or in concert with V. cholerae O1, cannot be dismissed. A genomic approach was used to examine similarities and differences among the Haitian V. cholerae O1 and V. cholerae non-O1/O139 strains. A total of 47 V. cholerae O1 and 29 V. cholerae non-O1/O139 isolates from patients and the environment were sequenced. Comparative genome analyses of the 76 genomes and eight reference strains of V. cholerae isolated in concurrent epidemics outside Haiti and 27 V. cholerae genomes available in the public database demonstrated substantial diversity of V. cholerae and ongoing flux within its genome.     

Emergence and evolution of Vibrio cholerae O139

The emergence of Vibrio cholerae O139 Bengal during 1992-1993 was associated with large epidemics of cholera in India and Bangladesh and, initially, with a total displacement of the existing V. cholerae O1 strains. However, the O1 strains reemerged in 1994 and initiated a series of disappearance and reemergence of either of the two serogroups that was associated with temporal genetic and phenotypic changes sustained by the strains. Since the initial emergence of the O139 vibrios, new variants of the pathogen derived from multiple progenitors have been isolated and characterized. The clinical and epidemiological characteristics of these strains have been studied. Rapid genetic reassortment in O139 strains appears to be a response to the changing epidemiology of V. cholerae O1 and also a strategy for persistence in competition with strains of the O1 serogroup. The emergence of V. cholerae O139 has provided a unique opportunity to witness genetic changes in V. cholerae that may be associated with displacement of an existing serogroup by a newly emerging one and, thus, provide new insights into the epidemiology of cholera. The genetic changes and natural selection involving both environmental and host factors are likely to influence profoundly the genetics, epidemiology, and evolution of toxigenic V. cholerae, not only in the Ganges Delta region of India and Bangladesh, but also in other areas of endemic and epidemic cholera.     

Molecular tracking of the lineage of strains of Vibrio cholerae O1 biotype El Tor associated with a cholera outbreak in Andaman and Nicobar Islands, India

A large outbreak of acute watery diarrhoea involving all age groups of mongoloid tribal aborigines occurred during October-November, 2002 in the Nancowry group of Andaman and Nicobar Islands in the Indian Ocean. Twenty-one of the 67 stool samples from 67 patients were positive for toxigenic Vibrio cholerae O1, serotype Ogawa biotype El Tor, which showed striking similarity in its antibiogram with some of the strains of V. cholerae O1 Serotype Ogawa biotype El Tor isolated in Kolkata. The Nancowry and Kolkata isolates were compared with molecular tools involving random amplified polymorphic DNA (RAPD) fingerprinting, ribotyping and pulsed-field gel electrophoresis (PFGE). RAPD fingerprinting and ribotyping techniques revealed that all the V. cholerae strains associated with the outbreak in these islands were clonal in nature and identical to a population of isolates obtained from Kolkata since 1993. PFGE could discriminate within these Kolkata isolates further and established that a particular subtype of this population reached the remote Nancowry islands and was responsible for the outbreak.     

The Vibrio cholerae O139 serogroup antigen includes an O-antigen capsule and lipopolysaccharide virulence determinants.

Vibrio cholerae serogroup O139 emerged on the Indian subcontinent in October 1992 to become the first non-O1 V. cholerae serogroup documented to cause epidemic cholera. Although related to V. cholerae El Tor O1 strains, O139 strains have unique surface structures that include a capsular surface layer and lipopolysaccharide (LPS). Immunoblot analysis of either whole-cell lysates or LPS preparations revealed three electrophoretic forms of the O139 antigen: two slowly migrating forms and one rapidly migrating form that appeared identical to O139 LPS. All three forms of the antigen shared an epitope defined by an O139-specific monoclonal antibody. A serum-sensitive nonencapsulated mutant was isolated that lacks only the slow migrating forms. The slow migrating forms did not stain with silver whereas the rapidly migrating form did, suggesting that the former might constitute highly polymerized O-antigen side-chain molecules that were not covalently bound to core polysaccharide and lipid A (an "O-antigen capsule"). A single transposon insertion resulted in the loss of immunoreactivity of both the LPS and the O-antigen capsule, implying that there are genes common to the biosynthesis of both these macromolecules. The O139 LPS and O-antigen capsule were both important for colonization of the small intestine of the newborn mouse and for serum resistance, demonstrating that both of these forms of the O139 serogroup antigen are virulence factors.     

The O139 serogroup of Vibrio cholerae comprises diverse clones of epidemic and nonepidemic strains derived from multiple V. cholerae O1 or non-O1 progenitors

Sixty-four representative strains of Vibrio cholerae O139 were analyzed, to re-examine the origin of this serogroup. Ribotyping differentiated the strains into 3 HindIII and 7 BglI ribotypes. One HindIII and 5 BglI ribotypes were shared by all toxigenic O139 strains. Of 6 nontoxigenic O139 strains, 3 shared ribotypes with the toxigenic strains, carried genes encoding toxin coregulated pilus, and were susceptible to the cholera toxin-converting bacteriophage CTXPhi. The remaining 3 strains belonged to 2 different ribotypes distinct from toxigenic O139 strains and were resistant to CTXPhi and JA-1, an O139-specific lytic bacteriophage. Polymerase chain reaction amplicons corresponding to the gmhD gene carried by these 3 strains also differed from those of the toxigenic O139 strains but were identical to those of 15 environmental non-O1-non-O139 strains. Thus, the O139 antigen is present in different lineages, and this serogroup appears to comprise epidemic and nonepidemic strains derived separately from different progenitors.     

Toxigenic Vibrio cholerae serogroup O141-associated cholera-like diarrhea and bloodstream infection in the United States

Toxigenic Vibrio cholerae serogroup O141 has been associated with sporadic cholera-like diarrhea and bloodstream infection in the United States. Consumption of seafood and proximity to the coast may increase the risk of infection. All V. cholerae isolates recovered from stool samples of patients with diarrhea or from a normally sterile site should be serogrouped and assessed for cholera toxin production. Improved surveillance and case-control studies are needed to further characterize illness and risk factors for V. cholerae O141 infection.