脉冲场电凝胶技术在肠道致病菌分子流行病学调查中的应用研究

目的：探讨建立快速敏感的肠道病原菌的诊断分型方法，有效地追踪溯源和控制传染源。方法：用脉冲场电泳胶与噬菌体分型实验方法对29株S．sonnei分离株与12株EHEC O157：H7菌株进行分型。结果：4起S．sonnei暴发事件的PFGE的试验结果显示，同起事件的DNA条带几乎一致；2起EHEC O157：H7暴发事件中，同起事件的PFGE条纹几乎一致，而3株散发菌株的PFGE条纹各异；另外对12株EHEC O157：H7进行噬菌体分型试验，也显示了比较好的分型力。结论：PFGE具分型力强、重复性高等特点，能直观地判断肠道致病菌的亲缘关系，及时查明暴发流行的传染源，从而有效地控制疫情的蔓延。     

肠出血性大肠埃希菌O157:H7不同分型方法比较

目的探讨建立快速敏感的EHEC O157：H7诊断分型方法，有效地追踪溯源和控制传染源。方法分别用反向被动乳胶凝集试验(RPLA)、噬菌体分型、vt毒力基因PCR试验、脉冲场电泳胶(PFGE)4种实验方法对EHEC O157：m菌株进行分型。结果对同起事件菌株，4种方法均有较好的分型能力；PFGE对散发菌株有很强的分型能力，而RPLA与vt毒力基因PCR试验尚有不足，但其操作简便，实验要求相对不高，可以作为EHEC O157：H7致病毒素的初筛方法。结论几种分型技术联合使用比任一种技术单独使用能提供更为可靠的分型结果。     

我国部分产生志贺毒素肠出血性大肠埃希菌O157：H7脉冲电场凝胶电泳分型

目的 探讨我国部分地区产生志贺毒素的肠出血性大肠埃希菌（EHEC）O157：H7菌株的分布，流行以及分型情况。方法 用脉冲电场凝胶电泳（PFGE）方法进行分型，辅以药物敏感性试验对51株产生志贺毒素的EHECO157：H7菌株进行研究。结果 根据细菌染色体DNA的XbaI酶切图谱，可将从江苏省徐州市等地分离的51株产生志贺毒素的EHECO157：H7菌株分成8个PFGE型别（PFGE1-PFGE8），宁夏菌株为PFGE1型和2型，徐州菌株有6个PFGE型，1986-1988年的分离菌株属于PFGE7型，1999、2000年病人分离株的优势型别分别是PFGE5型与3型，1999年爆发性流行期间从患者分离的5株菌属于PFGE5型，从家畜家禽的粪便，食品，蔬菜等标本分离的19株菌，可以分为PFGE3-6等4个型别，其中，PFGE5型占优势（10株菌）。结论 爆发性流行的发生与携带病原菌的家畜家禽粪便污染的食品，蔬菜等有关，从我国部分地区分离的EHECO157：H7菌株的PFGE型别有地区性差异。     

上海地区分离的大肠埃希菌O157的分子分型研究

目的对2006年-2014年在上海地区分离的大肠埃希菌O157进行分子分型研究。方法通过脉冲场凝胶电泳(PFGE)和多位点序列分型(MLST)技术的联合应用,对来自中国上海地区的16株大肠埃希菌O157分离株和4株标准株进行分子分型研究。结果 PFGE方法可将20株菌株分为12种型别,分辨力为0.921 1。其中14株O157:H7菌株处于同一聚类,其与6株O157:H?菌株为不相关菌株。MLST方法可将20株菌株分为6种ST型,分辨力为0.726 3。7个管家基因的核苷酸多态性范围为0.000 784~0.009 449。其中14株O157:H7菌株分为ST2966型和ST11型,属于同一克隆群。6株O157:H?分离株分为4种ST型,包括3种新的ST型,且与本研究中的O157:H7菌株间的管家基因型别相差至少6个。结论上海地区从动物和患者粪便中分离的大肠埃希菌O157:H7分离株间的遗传谱系接近,与本实验研究中的大肠埃希菌O157:H?菌株间亲缘关系较远。需加强对上海地区大肠埃希菌O157的病原学监测,并进一步比较2种分型技术对大肠埃希菌O157的分型效果。     

浙江省首株产志贺毒素大肠埃希菌0157：H7的分子生物学特性研究

目的：了解自浙江省杭州市腹泻婴儿中分离的1株大肠埃希菌O157：H7（HZI-11株）的分子生物学特性。方法：应用ATB1525细菌半动化生化鉴定系统鉴定菌种。应用0157特异性抗血清玻片凝集试验、H7特异性抗血清试管凝集试验、以及PCR检测O抗原特异性rfbE基因和H7特异性fliC基因，进行菌株血清型的鉴定。应用多重Real-time PCR和常规PCR检测stx1、stx2、hly和eae毒力基因。对菌株进行脉冲场凝胶电泳（PFGE）分型，并与国内代表菌株进行比较。ATB1525药敏检测仪和纸片法检测菌株的抗药性。结果：细菌生化鉴定为大肠埃希菌，山梨醇阴性。血清型为O157：H7。毒力基因stx2、hly和eae均阳性，stx1阴性。PFGE谱带同江苏分离O157：H7菌株几乎完全相同，带型的相似度为97％。结论：该菌株为浙江省首株产志贺毒素大肠埃希菌（STEC）O157：H7。与国内近年在江苏等地流行的STEC O157：H7菌株密切相关。STEC O157：H7已开始对浙江地区的人民健康构成了威胁。     

湖北省首次分离出肠出血性大肠埃希菌O157：H7

目的：为了解肠出血性大肠埃希菌O157:H7（EHEC O157:H7）在我省的动物宿主种类和菌型特征，制定相应流行病防治对策。方法：采用传统方法，在6～10月份肠道传染病高发季节，对武汉市3大养殖场、屠宰场的肉猪、奶牛及武汉7大城区的农贸商场的水产品进行调查采样，共采集811份样品，进行EHEC O157:H7检测。结果：341份猪粪样品中检出1株EHEC O157:H7，阳性率为0.29%；255份奶牛粪便样品中检出7株EHEC O157:H7，阳性率为2.75%；其他类标本中未检出。分离的这8株阳性菌株与传统的EHEC O157:H7存在许多不同之处，他们均发酵山梨醇，大部分菌株不分解棉子糖，也存在不分解卫茅醇的菌株，而β-葡萄糖醛酸酶试验为阳性，在致病因子上，检测现有的几种毒力基因均为阴性。结论：在我省首次检出8株EHEC O157:H7，说明其普遍存在于动物体内，对环境、食品构成较大威胁。     

对流行地区的E.coli O157：H7病原学分析及研究

目的：对流行地区O157：H7进行病原学分析。方法：PCR方法对O157：H7菌株毒力基因谱检测比较，同时用PFGE和RAPD方法对O157：H7菌株的同源性分析比较。结果：流行地区分离的O157：H7菌株，100％携带Hly,eaeA基因，95．35％携带SLT2基因，11．63％携带SLT1基因，PFGE图谱表明流行地区分离的O157：H7菌株与日本分离的O157：H7菌株有明显差异，为不相关菌株；与国内标准菌株882364为近似型（相似，但不相同），流行地区病人分离菌株与外环境家畜家禽类便与昆虫肠道分离菌的PFGE图谱完全相同，结论：携带O157：H7菌株的家畜家禽可能是导致疫情发生的传染源。PFGE用于O157：H7病原学分析，对流行病学研究有重要意义，但不适宜基层，RAPD方法用于O157：H7病原学分析，技术简便，省时。     

肠出血性大肠埃希菌遗传基因分型方法比较

目的 对肠出血性大肠埃希菌(EHEC)O157:H7的遗传基因型别进行不同分子分型方法的对比研究,以对EHEC的分子流行病学追踪提供更为行之有效的方法.方法 采用核酸脉冲场凝胶电泳(PFGE)与多位点串联重复序列(VNTR)分析(MLVA)两种方法对16株EHEC O157:H7进行分析.结果 在PFGE的研究中,依据经XbaI及确认BlnI酶切后,具有相同电泳图谱的菌株被定义为PFGE同一群落的菌株,将16株菌株分成6个型别.MLVA通过对9个位点的串联重复序列的比较研究,16株菌株在3个位点上没有显示出区别,在另外6个位点上等位基因的数目为2～7种,16株细菌被分为14个MLVA的型别.流行病学资料表明,这些菌株为散发病例分离株,分离地域上基本没有关联,因此,MLVA的结果与流行病学信息高度相关.结论 MLVA能够区分无法区分的PFGE型别,且可以区分来自于不同地理区域的散发菌株,具有区分肠出血性大肠埃希菌O157:H7的潜力.     

四川省大肠杆菌O157:H7分子流行病学、耐药性及对消毒剂抗性研究

目的研究从四川省分离的大肠杆菌O157：H7菌株流行病学特征。方法用多重PCR测定67株大肠杆菌O157：H7的sit、eaeA、hly毒力基因；对不同来源的大肠杆菌O157：H7进行质粒和PFGE分型；测定67株大肠杆菌O157：H7的耐药性和对消毒剂的抗力。结果62．7％的株菌（42／67）携带有毒力基因，毒力图谱类型主要为slt1＋slt2＋eaeA＋hly。67株菌共有6种质粒谱型和7种PFGE谱型。64．2％（43／67）的菌株分别对7种不同的抗生素耐药，其中有23、35、34株菌分别对氨苄青霉素、四环素和SMZ耐药。不同来源的大肠杆菌O157：H7抗性谱不同，80．6％（54／67）的菌株对酒精和季铵盐产生了抗性，所有菌株对洗必泰敏感。结论四川省不同来源的大肠杆菌O157：H7带毒率相似，但毒力基因谱型不完全相同。细菌有较高的耐药性。菌株对常用消毒剂有很高的抗性，在消毒灭菌时需要用较大剂量的消毒剂才能将其杀灭。不同来源的大肠杆菌O157：H7耐药性、质粒和PFGE谱型有一定的相似性，提示菌株可能在人、动物、食品以及外环境之间相互传播并存在流行的可能。     

2007年浙江省衢州地区动物中产志贺毒素大肠埃希菌O157:H7感染状况

目的:了解2007年浙江省衢州地区产志贺毒素大肠埃希菌O157:H7在动物中的分布情况及其耐药性、PFGE分型及毒力基因携带状况。方法:按全国O157:H7监测方案于5~10月份肠道传染病高发季节,在衢州地区采集各种动物粪便/肛拭,用免疫磁珠富集后进行O157:H7分离培养、鉴定,可疑菌株以PCR法检测O、H抗原及志贺样毒素(SLT1和SLT2)、粘附抹平因子(eaeA)及溶血素(hly)4种毒力基因。用脉冲场凝胶电泳(pulse field gel electrophoresis,PFGE)方法进行同源性分析,同时选择14种抗生素进行药敏试验,分析分离所得菌株的耐药状况。结果:共监测动物粪便标本300份,分离得产志贺毒素大肠埃希菌O157:H7菌株16株,分离率为5.33%。16株O157:H7菌株,毒力基因Hly、eaeA、SLT2均阳性,SLT1均阴性。脉冲场凝胶电泳分型显示,16株O157:H7菌株可分2个PFGE基因型,型间差异较小。耐药性分析显示这些菌株对红霉素、利福平的耐药率最高,达100.0%,对其他受试抗生素均敏感。结论:该地区动物中产志贺毒素大肠埃希菌O157:H7带菌率较高,所分离菌株主要携带SLT2基因,因此推测该地区存在发生产志贺毒素大肠埃希菌O157:H7感染暴发或流行的潜在危险,需增加对动物源性O157:H7的监测力度。     

Molecular typing methods to investigate transmission of Escherichia coli O157:H7 from cattle to humans.

The utility of phage typing, pulsed-field gel electrophoresis (PFGE), and plasmid profile analysis was compared, to differentiate between Canadian Escherichia coli O157:H7 strains of human (n = 27) and cattle (n = 24) origin. The diversity indices for phage typing, plasmid analysis and PFGE were 0.85, 0.69 and 0.93, respectively. PFGE and phage typing were also applied to study the role of direct transmission of E. coli O157:H7 from cattle to humans on isolates collected from two separate farm outbreaks. PFGE showed that more than one E. coli O157:H7 strain with varying PFGE DNA subtype profiles, may be responsible for an outbreak, and that more than one E. coli O157:H7 subtype may be circulating on a particular farm at any one time. To our knowledge, this is one of the first reports where PFGE typing was used to verify the direct transmission of E. coli O157:H7 from cattle to humans.     

Characterization and epidemiologic subtyping of Shiga toxin-producing Escherichia coli strains isolated from hemolytic uremic syndrome and diarrhea cases in Argentina

Argentina has a high incidence of hemolytic uremic syndrome (HUS); 12.2 cases per 100,000 children younger than 5 years old were reported in 2002. Shiga toxin (Stx)-producing Escherichia coli (STEC) is the primary etiologic agent of HUS, and STEC O157 is the predominant serogroup isolated. The main objective of the present work was to establish the phenotypic and genotypic characteristics of the STEC strains in general isolated from Argentine children during a prospective study and the clonal relatedness of STEC O157:H7 strains using subtyping techniques. One hundred and three STEC strains isolated from 99 children were included. The phenotypic and genotypic features were established, and a polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) was performed to determine stx2 variants. The clonal relatedness of E. coli O157 isolates was established by phage typing and pulsed-field gel electrophoresis (PFGE). The 103 STEC strains belonged to 18 different serotypes, and 59% were of serotype O157:H7. Stx2 was identified in 90.3%, and stx1 in 9.7%. Among the 61 STEC O157 strains, 93.4% harbored the stx2/stx2vh-a genes; PT4 (39.3%) and PT2 (29.5%) were the predominant phage types. Using PFGE with the enzyme XbaI, a total of 41 patterns with at least 80% similarity were identified, and seven clusters with identical profiles were established. Some of the clusters were further split by PFGE using BlnI as the second enzyme. Isolates with indistinguishable PFGE patterns were with one exception also indistinguishable by phage typing and stx genotyping. These findings confirmed that some isolates were genetically related. However, no epidemiological linkages were identified. STEC strains with different genotypes and belonging to diverse serotypes were isolated in Argentina. Some STEC O157 strains could not be distinguished by applying subtyping techniques such as PFGE and phage typing.     

Clonal diversity of Shiga toxin-producing Escherichia coli O103:H2/H(-) in Germany.

Shiga toxin producing Escherichia coli O103:H2/H(-) belong to the third most frequently isolated EHEC serotypes in Germany following isolates of O157:H7/H(-) and O26:H11/H(-). A total of 145 respective E. coli 103 isolates from single cases of diarrhoea and haemolytic uremic syndrome (HUS) in 1997-2000 were characterised by a range of molecular subtyping methods (PFGE, P-gene profiling, ribotyping, electrotyping) and phage typing in order to analyse their genetic relatedness and the practicability for new epidemiological tracing back. All isolates cluster into a distinct EHEC subgroup and reveal a high clonal diversity together with a considerable stability. Since strains of this serotype rank up to the third most frequently isolated EHEC in Germany a large population of this serotype, and therefore, a great supply of such strains may exist in this country.     

中国部分地区大肠埃希菌O157的分子分型及变异研究

目的了解出血性大肠埃希菌（EHEC）O157的分子流行特征和遗传变异关系并完善EHECO157：H7感染性腹泻监测制度。方法采用聚合酶链反应分析毒力基因的分布情况；用脉冲场凝胶电泳技术对1988-2005年部分地区分离到的249株EHECO157：H7（其中产志贺毒素的245株，不产志贺毒素的4株）和51株O157非H7进行分子流行病学分析。结果stx2基因在我国的EHECO157：H7菌株中有着很高的分布率，部分菌株携带stx2基因变种。300株O157共分为161种带型，其中51株O157非H7菌株共有42种带型，4株O157：H7非产毒株分别为4种带型，245株EHECO157：H7共有115种带型。结论EHECO157间的基因变异较大；产stx2原毒素的菌株和5衄2毒素发生变异的菌株带型相差较大。部分菌株之间存在着一定的交叉，说明虽然这两大类菌株有其特有的流行克隆系，但是它们的克隆系之间亲缘关系较近。     

脉冲场凝胶电泳技术对成都市两起细菌性痢疾暴发的分型研究

目的 运用脉冲场凝胶电泳技术(PFGE)和聚合酶链反应(PCR)技术对成都市两起细菌性痢疾暴发分离株进行分析.方法 利用PCR检测志贺菌分离株ipaH基因和ial基因.用PFGE对菌株进行分型,所得结果用BioNumeries V 4.0软件UPGMA方法进行聚类分析.结果 所试54株分离菌ipaH基因均为阳性,48株为ial阳性.以Xba Ⅰ酶切后PFGE分型,崇州可分为4个带型,有26株型别一致,占实验菌株(36株)的72%,为优势菌株.自凉拌白肉中分离的菌株带型与优势菌株相同.大邑县可分为8个带型,有10株型别一致,占实验菌株(18株)的56%.崇州和大邑县的优势菌株带型差异较大.结论 利用PFGE能够有效地鉴别细菌性痢疾的暴发和传播来源.     

Escherichia coli O157:H7: animal reservoir and sources of human infection

This review surveys the literature on carriage and transmission of enterohemorrhagic Escherichia coli (EHEC) O157:H7 in the context of virulence factors and sampling/culture technique. EHEC of the O157:H7 serotype are worldwide zoonotic pathogens responsible for the majority of severe cases of human EHEC disease. EHEC O157:H7 strains are carried primarily by healthy cattle and other ruminants, but most of the bovine strains are not transmitted to people, and do not exhibit virulence factors associated with human disease. Prevalence of EHEC O157:H7 is probably underestimated. Carriage of EHEC O157:H7 by individual animals is typically short-lived, but pen and farm prevalence of specific isolates may extend for months or years and some carriers, designated as supershedders, may harbor high intestinal numbers of the pathogen for extended periods. The prevalence of EHEC O157:H7 in cattle peaks in the summer and is higher in postweaned calves and heifers than in younger and older animals. Virulent strains of EHEC O157:H7 are rarely harbored by pigs or chickens, but are found in turkeys. The bacteria rarely occur in wildlife with the exception of deer and are only sporadically carried by domestic animals and synanthropic rodents and birds. EHEC O157:H7 occur in amphibian, fish, and invertebrate carriers, and can colonize plant surfaces and tissues via attachment mechanisms different from those mediating intestinal attachment. Strains of EHEC O157:H7 exhibit high genetic variability but typically a small number of genetic types predominate in groups of cattle and a farm environment. Transmission to people occurs primarily via ingestion of inadequately processed contaminated food or water and less frequently through contact with manure, animals, or infected people.     

Phage typing and drug resistance of Shigella sonnei isolated in England and Wales.

Phage typing of Shigella sonnei has been used to examine isolates from the 1991-2 sonnei dysentery outbreak in England and Wales and compare them with strains isolated during and following a widespread foodborne outbreak in 1994 which was associated with consumption of imported lettuce. The distribution of phage types was different in the three periods studied with PT 3 predominating during 1991-2, PT 2 during the ''lettuce'' outbreak in the summer months and PT 6 during the subsequent months. PT 6 was frequently associated with travel outside the UK. Variation was also seen in the distribution of drug resistance patterns.     

Optimal settings of fingerprint-type analysing computer software for the analysis of enterohaemorrhagic Escherichia coli pulsed-field gel electrophoresis patterns

Settings of fingerprint-type analysing computer software were optimized for analysis of enterohaemorrhagic Escherichia coli (EHEC) pulsed-field gel electrophoresis (PFGE) patterns. Under the lowest values of parameters, maximum value of similarities calculated using the Dice coefficient were obtained between PFGE patterns from one EHEC strain on the same gel when reference lanes for calibration of distortions during electrophoresis were set to every fourth lane. PFGE patterns of 15 EHEC strains on different gels were investigated. Similarity values calculated using the Pearson product-moment correlation coefficient (Pearson correlation) were significantly higher than those using the Dice coefficient with optimal values of parameters determined by the program (P < 0.01). When PFGE patterns of 45 EHEC strains were analysed by the computer program, EHEC strains from one mass outbreak and three intra-family outbreaks were each clustered and the similarity values within the clusters were > 90% using Pearson correlation.