大肠杆菌肠毒素基因多重PCR检测方法的建立

目的 建立一种快速检测大肠杆菌耐热肠毒素(heat-stable enterotoxin, STa, STb)和不耐热肠毒素(heat-labile enterotoxin, LT-Ⅰ, LT-Ⅱ)基因的多重PCR方法。方法 参照文献合成四对可扩增产肠毒素大肠杆菌(Enterotoxigenic Escherichia coli, ETEC)耐热肠毒素基因(estA、estB)和不耐热肠毒素基因(elt-Ⅰ、elt-Ⅱ)的特异性引物,通过反应条件的优化,敏感性、特异性试验和临床样品检测,建立检测大肠杆菌肠毒素的多重PCR方法。结果 用所建立的多重PCR方法可特异性扩增出estA(229 bp)、estB(480 bp)、elt-Ⅰ(605 bp)和elt-Ⅱ(300 bp)基因片段,最低检出量分别为2.55×10¹ CFU/μL、2×10¹ CFU/μL、2×10¹ CFU/μL和2.47×10³ CFU/μL。从22株大肠杆菌分离株中检测到estA基因(2/22),elt-Ⅱ基因(3/22),未检测到estB和elt-Ⅰ基因,检测结果与常规PCR检测结果一致。结论 建立了检测大肠杆菌肠毒素基因(estA、estB、elt-Ⅰ和elt-Ⅱ)的多重PCR方法,该方法具有良好的特异性和敏感性,能够满足对细菌培养物的检测要求。     

大肠杆菌耐热性肠毒素（ST）的检测

产肠毒素性大肠杆菌(ETEC)是通过产生肠毒素而致婴幼儿、旅游者及幼畜腹泻的,临床检测表明,到发展中国家旅游者中的急性腹泻,至少有1/3～1/2是ETEC引起的。ETEC能产生两种肠毒素:热敏性肠毒素(Heat-Labile Enterotoxin,LT),为大分子具有免疫原性毒素,其作用机理、理化住质以及免疫学特性和检测方法均与霍乱毒素(CT)相似;耐热性肠毒素(Heat-Stable Enterotoxin,ST),根据其是否溶于甲醇而分为     

辽宁地区腹泻患者大肠杆菌分离株Ⅱ型不耐热肠毒素的检测及测序分析

目的对辽宁地区人源性腹泻样本中致病性大肠杆菌(Pathogenic Escherichia coli)的毒力因子进行检测,掌握产Ⅱ型不耐热肠毒素(Type Ⅱ heat labile enterotoxin,LT-Ⅱ)大肠杆菌在我国北方地区的流行现状并阐明LT-Ⅱ与其他致病因子间的关系。方法于2014年3月至2015年3月收集辽宁地区人源性腹泻粪便样品,用麦康凯培养基和生化试验的方法分离出大肠杆菌,并用多重PCR方法对毒力基因(elt-Ⅱ、elt-I、sta、stb、K88、K99等)进行检测,对分离到的LT-Ⅱ阳性大肠杆菌中elt-Ⅱ基因进行测序及分型。结果 354份腹泻样品中共分离到携带有毒力因子的大肠杆菌139株,检出率为39.2%。毒力因子阳性大肠杆菌中,有12株携带elt-Ⅱ(8.6%)基因。在12株携带elt-Ⅱ的大肠杆菌中,2株单独携带elt-Ⅱ,6株携带F1,1株携带K88,1株携带astA,2株携带irp2/astA;测序结果表明12株elt-Ⅱ阳性大肠杆菌中有10株携带elt-Ⅱc1亚型,2株携带elt-Ⅱc4亚型。结论在引起人源性腹泻的毒力因子中有大肠杆菌Ⅱ型不耐热肠毒的存在,且主要以LT-Ⅱc1为主要流行型。     

旅行者腹泻

发达国家的人群去其他国家(首先是发展中国家)旅游时,易产生一种称之为旅行者腹泻的疾病.该病可由多种病原引起,但50～75%病例系由产肠毒素大肠杆菌(ETEC)所致.目前已知ETEC可产生一种或两种肠毒素:1.不耐热肠毒素(LT),与霍乱肠毒素有交叉免疫反应;2.耐热肠毒素(ST),为一种小分子量肠毒素,无抗原性.因而存在着LT、ST、及LT/ST菌株.这些菌株有明显的地理差异,例如墨西哥和摩洛哥以LT/ST菌株为优势株;肯尼亚以LT株为主;在孟加拉国,ST株较LT/ST株稍多见,而明显多于LT株.但无论何地均可发现所有这三型菌株.此外,从25%病例检出志贺氏痢疾杆菌和沙门氏菌属,从1～5%病例     

重组超抗原金黄色葡萄球菌肠毒素B基因的克隆和序列分析

目的　采用基因工程技术制备金黄色葡萄球菌B型肠毒素 (SEB)。方法　根据SEB已知序列 ,设计一对引物 ,用PCR方法从金黄色葡萄球菌染色体DNA上扩增出基因片段 ,克隆到原核表达质粒 pET32a上 ,转化大肠埃希菌JM10 9感受态细胞 ,经酶切和PCR鉴定 ,然后进行测序。结果　PCR扩增产物大小为 74 0bp ,重组质粒经双酶切PCR鉴定表明已正确重组 ,测序结果与已知序列基本吻合。结论　成功地克隆了金黄色葡萄球菌B型肠毒素 ,为下一步研究发病机制奠定基础。     

大肠杆菌表面CS3菌毛展示随机肽库的构建

目的:在肠毒素性大肠杆菌CS3菌毛表面构建 10肽随机肽库．方法:首先将原有的单酶切CS3菌毛呈现载体改造为双酶切载体,并证实改造后的载体能正确形成CS3菌毛．同时设计合成2条寡核苷酸序列,链1含有1个10肽随机编码序列(NNS)10, 链2可与链1的3′端互补．两条链经过退火、延伸、酶切和回收与经过同样酶切的呈现载体连接,连接产物纯化后分多次电击转化,获得随机肽库．随机挑选10个克隆进行测序并对测序结果进行分析．结果:获得1个库容量为1．8×106大小的随机肽库．测序结果显示,所构建肽库的基本框架与预期设计相符,4个寡核苷酸出现的频率也与理论值相接近．结论:在肠毒素性大肠杆菌CS3菌毛表面成功构建库容量为1．8×106大小的10肽随机肽库．为下一步利用肽库进行筛选奠定了基础．     

大肠杆菌耐热肠毒素的纯化及部分特性分析

本文介绍一种简单、快速的提取和纯化大肠杆菌耐热肠毒素(ST)的方法,纯化过程包括:硫酸铵沉淀,Sephadex G-25 Bio-Gel P-4凝胶过滤。纯化后的纯毒素比活性为5×10~5鼠单位/mg蛋白。肠毒素被纯化167倍,回收率为33.7％。     

大肠杆菌不耐热肠毒素无毒突变体mLT63毒性检测及佐剂效果研究

目的对已构建的大肠杆菌不耐热肠毒素无毒突变体mLT63进行表达、纯化、毒性检测,并对其佐剂活性进行研究。方法采用已知的最佳诱导表达条件进行诱导表达,诱导表达产物经亲和层析、纯化浓缩后,进行毒性检测。将纯化后的mLT63联合幽门螺旋杆菌(Hp)亚单位疫苗UreB、Omp11经口途径免疫BALB/c小鼠,免疫后取血清、胃组织提取液、粪便提取液进行ELISA试验,检测其中的特异性抗体水平,将结果进行统计分析。结果经家兔回肠袢毒性试验验证本室构建大肠杆菌不耐热肠毒素无毒突变体mLT63没有毒性,mLT63联合Hp亚单位疫苗UreB、Omp11免疫小鼠实验证实mLT63具有佐剂活性。结论本室构建、表达的大肠杆菌不耐热肠毒素无毒突变体mLT63无毒,并具有免疫佐剂的活性。     

肠毒素性大肠杆菌的粘附素

<正> 肠毒素性大肠杆菌(ETEC)引起腹泻的直接致病机制归因于两种不同的肠毒素:不耐热毒素(LT)和耐热毒素(ST)。近十年来人们对这类细菌在宿主小肠上的定居能力日益重视。细菌的定居能力是由细菌表面上的菌毛为媒介的,人们称它为定居因子(Colonization factor antige CFA)或粘附素(Adhesins)。本文主要就ETEC粘附素的物理生化特性、遗传学及免疫前景作简要综述。 一、粘附素的宿主特异性及与O血清群关系 在引起不同宿主动物腹泻的ETEC中,已经发现了几种重要的粘附素,每种粘附素都有宿主特异性(表1)。发现较早的粘附素是猪源ETEC中的K88和987P,以及牛、羊源ETEC的K99和F41。后来,在引起人腹泻的ETEC中失后发现了CFAⅠ、CFAⅡCFAⅢ和E8775等不同类型的定居因子。近来又在幼     

直接从粪便中检测致犊牛腹泻产肠毒素大肠杆菌的双重PCR方法的建立与应用

目的产肠毒素大肠杆菌(Enterototxigenic,Escherichia Coli,ETEC)是造成人和动物腹泻的主要病原之一,也是造成新生乳用犊牛腹泻的主要原因,一株ETEC可能产生一种或者多种肠毒素。根据产肠毒素大肠杆菌产生的两种主要毒素的基因序列,设计了两对引物,建立多重PCR方法。该方法仅用4.5小时即可检测出导致犊牛腹泻的产肠毒素大肠杆菌菌株,具有敏感度高、特异性强和检测速度快等特点。本试验的目的是用所建立的多重PCR方法来确定ETEC在导致犊牛腹泻中的作用。采用该方法对乳用犊牛的203个腹泻样品进行了检测,结果,用传统的分离培养方法得到的203株典型大肠杆菌中有135株为ETEC;其中LT阳性为105株,ST阳性为126株,LT+ST阳性的为96株,阳性率为66.5%。而采用直接从粪样中提取PCR模板的方法进行PCR,检测到146个犊牛粪样为ETEC阳性,其中LT阳性为112株,ST阳性为137株,LT+ST阳性为103株,阳性率为71.9%,明显高于传统的检测方法;并且所检测到的146个ETEC阳性的犊牛粪样完全包含用传统的分离培养方法得到的135个阳性粪样,且基因分型结果相同。     

中国肠产毒性大肠杆菌中耶尔森菌强毒力岛的检测

目的　了解耶尔森菌强毒力岛在中国肠产毒性大肠杆菌中的分布及插入位点。方法　使用PCR扩增、DNA打点杂交和DNA测序及分析方法。结果　在 94株分离自中国的肠产毒性大肠杆菌中 ,14株耶尔森菌强毒力岛核心区的 8个基因PCR扩增阳性 ,除 3株菌的整合酶基因外 ,PCR扩增产物长度均与预期一致 ,但天冬酰胺转运RNA(asnTtRNA)位点扩增阴性 ;上述 14株菌进行全菌DNA打点杂交试验 ,均与irp2和 fyuA探针杂交 ;选择上述 3株菌中的 1株 ,对其整合酶基因的PCR产物测序 ,并与鼠疫耶尔森菌强毒力岛的整合酶基因序列比较 ,发现该整合酶基因于 5’端缺失了 347bp的片段。 结论　 14 %肠产毒性大肠杆菌携带的耶尔森菌强毒力岛 ,且均插入在天冬酰胺转运RNA位点处 ;部分菌株所携带的耶尔森菌强毒力岛的整合酶基因发生了缺失。     

Toxins and colonization factor antigens of enterotoxigenic Escherichia coli among residents of Jakarta, Indonesia

Infection caused by enterotoxigenic Escherichia coli (ETEC) poses a serious health problem among children and adults in developing countries. Colonization of the small intestinal mucosa by ETEC strains is mediated by antigenically specific fimbriae, also known as colonization factor antigens (CFA). The significance of this study arises from reports that active and passive immunization with ETEC strains harboring CFAs has previously been shown to induce protective immunity against diarrhea in animal models. The aim of this study was to determine toxin-associated CFAs of ETEC isolated from a diarrheal disease case-control study in Jakarta, Indonesia. Thirteen hundred and twenty-three diarrheic and control patients with lactose-fermenting colonies were screened by ganglioside GM1-enzyme-linked immunosorbent assay (GM1-ELISA) for heat-labile (LT) and heat-stable (ST) toxins. Two hundred and forty-six (19%) ETEC isolates identified by GM1-ELISA for the LT/ST toxins were screened for CFAs by Dot blot assay using monoclonal antibodies against CFA/I, II, and IV and against the putative colonization antigens (PCF) PCFO159, PCFO166, CS7, and CS17. Of the 246 ETEC isolates, 177 (72%) elaborated ST, 56 (23%) produced LT, while 13 (5%) elicited both the ST and LT toxins. CFA testing of the 246 ETEC isolates showed that 21 (8%) expressed CFA/I, 3 (1%) exhibited CFA/II, 14 (6%) elaborated CFA/IV, while 7 (3%) expressed PCFO159 and PCFO159 plus CS5. No CFAs or PCFs could be associated with 201 (82%) of the ETEC strains. This report documents the types of CFAs associated with ETEC strains in Jakarta, Indonesia. These data may help current research efforts on the development of CFA-based vaccines for humans against ETEC and provide additional information for future ETEC vaccine trials in Southeast Asia.     

Improved detection of enterotoxigenic Escherichia coli among patients with travelers' diarrhea, by use of the polymerase chain reaction technique

This study sought to determine whether a specific polymerase chain reaction (PCR) for enterotoxigenic Escherichia coli (ETEC) toxins after chaotropic extraction of DNA from stool would increase the detection of ETEC over that of conventional oligonucleotide probe hybridization of 5 E. coli colonies per stool sample (a standard method). By DNA hybridization, 29 (21%) of 140 patients were positive for ETEC, and 59 (42%) of 140 were positive for ETEC when PCR was used. Sensitivity of the PCR assay was confirmed through spiked stool experiments to be approximately 100-1000 ETEC colonies per sample. Specificity of the assay was determined by showing an absence of ETEC by the PCR technique in a subgroup of 48 subjects and by confirming the presence of ETEC DNA of positive samples by dot blot procedure. PCR technique detected significantly more ETEC infections in these subjects than did the hybridization method (P<.0001).     

Human antibody response to longus type IV pilus and study of its prevalence among enterotoxigenic Escherichia coli in Bangladesh by using monoclonal antibodies

Mouse monoclonal antibodies (MAbs) were derived against longus (CS20), a type IV pilus expressed by human enterotoxigenic Escherichia coli (ETEC). One MAb (ICA39) detected longus in 56 (8.5%) of 662 ETEC isolates obtained from a routine surveillance of diarrheal stools from children and adults. Five patients with diarrhea from whom longus-positive ETEC were isolated were also recruited. Of these 61 isolates, 50 were positive for other colonization factors (CFs; 61% for CFA/II and 21% for CFA/I), and 11 were negative for any of the other 8 CFs that were tested. They were either positive for the heat-stable enterotoxin (ST; n=29) or for the heat-labile enterotoxin (LT) and ST (n=32). All longus-positive ETEC were confirmed by polymerase chain reaction to harbor lngA, the longus structural pilin gene. Sera and/or fecal extracts from the patients reacted with the 22-kDa pilin polypeptide in immunoblots and ELISA. These studies show that longus is prevalent among ETEC in Bangladesh and that longus gives rise to IgA antibody responses in patients.     

Disease due to enterotoxigenic Escherichia coli in Bangladeshi adults: clinical aspects and a controlled trial of tetracycline

The clinical characteristics of disease due to enterotoxigenic Escherichia coli (ETEC) were determined in 88 adult males admitted to a hospital in Dacca, Bangladesh, with moderate to severe dehydration. Persons infected with ETEC strains producing both heat-labile toxin (LT) and heat-stable (ST) toxin had more dehydration and acidosis, longer duration of illness, and greater stool volume than persons infected with strains producing only ST. Tetracycline therapy, evaluated in 63 cases, resulted in slightly earlier termination of illness in patients with LT-ST strains but had no effect on illness in the patients with ST strains. In both groups of patients tetracycline shortened the duration of excretion of organisms. Because of its limited effectiveness and the generally excellent response of ETEC diarrhea to rehydration therapy alone, tetracycline is not warranted for use in treatment of ETEC diarrhea in adults in this population.     

多重聚合酶链反应检测产毒性大肠杆菌三种毒力基因

目的提高普通的单基因聚合酶链反应（ＰＣＲ）检测产毒性大肠杆菌（ＥＴＥＣ）的时效。方法以ＥＴＥＣ４４８１３（ＳＴｐ＋）、ＥＴＥＣ１９４４９（ＳＴｈ＋）和ＥＴＥＣ４４８１５（ＬＴ＋）三个标准株为模板,建立了检测产毒性大肠杆菌多重ＰＣＲ扩增系统。结果杂交工程株Ｈ１０９０７（ＳＴｐ＋）,ＰＳＬＭ００４（ＳＴｈ＋）和ＰＭＭ０３０（ＬＴ＋）,杂交的结果都是阳性,５４株菌株经ＰＣＲ检测,其中ＬＴ＋８株、ＳＴｐ＋２株、ＳＴｈ＋７株、ＬＴ＋＋ＳＴｐ＋１０株、ＬＴ＋＋ＳＴｈ＋１０株、ＳＴｐ＋＋ＳＴｈ＋４株和ＬＴ＋＋ＳＴｐ＋＋ＳＴｈ＋１３株。结论用一次ＰＣＲ扩增可检测和鉴别ＥＴＥＣ,比单基因ＰＣＲ更加快速、经济。     

Phosphodiesterase 5 (PDE5) restricts intracellular cGMP accumulation during enterotoxigenic Escherichia coli infection

ABSTRACT

Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) has a continuing impact on residents and travelers in underdeveloped countries. Both heat-labile (LT) and heat-stable (ST) enterotoxins contribute to pathophysiology via induction of cyclic nucleotide synthesis, and previous investigations focused on intracellular signal transduction rather than possible intercellular second messenger signaling. We modeled ETEC infection in human jejunal enteroid/organoid monolayers (HEM) and evaluated cyclic nucleotide pools, finding that intracellular cAMP was significantly increased but also underwent apical export, whereas cGMP was minimally retained intracellularly and predominantly effluxed into the basolateral space. LT and virulence factors including EatA, EtpA, and CfaE promoted ST release and enhanced ST-stimulated cGMP production. Intracellular cGMP was regulated by MK-571-sensitive export in addition to degradation by phosphodiesterase 5. HEMs had limited ST-induced intracellular cGMP accumulation compared to T84 or Caco-2 models. Cyclic nucleotide export/degradation demonstrates additional complexity in the mechanism of ETEC infection and may redirect understanding of diarrheal onset.     

Phenotypic diversity of enterotoxigenic Escherichia coli (ETEC) isolated from cases of travelers' diarrhea in Kenya.

BACKGROUND: The aim of this study was to characterize phenotypically enterotoxins, colonization factors (CFs) and the antibiotic susceptibility of enterotoxigenic Escherichia coli (ETEC) strains isolated from cases of acute diarrhea that occurred in Europeans traveling to resorts in Mombasa, Kenya; this information is critical for the development of vaccines and empirical treatment. METHODS: Over a 1-year period from 1996 to 1997, five E. coli-like colonies were obtained from each of 463 cases with acute diarrhea. These strains were characterized for enterotoxins using GM-1 ELISA, for CFs using a dot-blot assay, and for antibiotic susceptibility using antibiotic disks. RESULTS: Of 164 strains characterized for ETEC phenotype, 30 (18%) expressed heat-labile toxin (LT) only, 83 (51%) heat-stable toxin (ST) only, and 51 (31%) both LT and ST. Analysis for CF expression demonstrated that 107 (65%) of the strains were positive for CFs, including CFA/IV (46%), CFA/II (35%), and CFA/I (5%), while less than 4% expressed less common CFs. All ETEC strains tested were resistant to erythromycin and sensitive to ceftriaxone. Over one-third of the strains were resistant to sulfamethoxazole-trimethoprim or tetracycline. Six strains were resistant to nalidixic acid; none of these were resistant to ciprofloxacin. CONCLUSIONS: Cumulatively, our findings indicate that ETEC in this region comprises a highly diverse group of bacterial enteropathogens, and that the development of prophylactic agents against ETEC faces major challenges because of this diversity.