酶联免疫吸附试验检测产不耐热肠毒素大肠杆菌的研究

本文报道了ELISA法检测产不耐热肠毒素大肠杆菌(ETEC/LT⁺)的研究。用酶标抗-CT建立的ELISA,检测杭州市医院病人分离的70株和动物分离的56株大肠杆菌,以及来源于腹泻患者的10株沙门氏菌、亲水气单胞菌、类志贺氏邻单胞菌和耶尔森氏菌,结果仅从病人菌株中检出3株ETEC/LT₊,阳性率为4.3%;未发现与其它4种肠道病原菌有交叉反应。这是一种特异、敏感、快速检测ET EC的方法,在感染性腹泻的病原研究中有推广应用价值。     

上海市徐汇区2011年感染性腹泻病原学及流行病学特征分析

[目的]了解上海市徐汇区感染性腹泻的病原学及流行病学特征,为感染性腹泻的有效控制提供依据。[方法]对徐汇区6家医院肠道门诊的腹泻病例进行流行病学分析,并采用PCR方法检测其常见致病菌,对肠产毒性大肠埃希菌(ETEC)阳性菌株进行耐热肠毒素(ST)和不耐热肠毒素(LT)基因检测和药敏试验。[结果]共检出常见致病菌54株,检出率为9.31%,其中ETEC占阳性菌株的57.41%,20～39岁年龄段人群致病菌检出率最高(14.15%),ETEC肠毒素类型以ST为主(ST>LT>ST/LT),ETEC对头孢他啶全部敏感。[结论]ETEC是徐汇区感染性腹泻的主要致病菌,应对ETEC引起的感染性腹泻给予足够的重视。     

1995～2010年滕州市腹泻病人致泻性大肠杆菌检测结果分析

[目的]探讨滕州市致泻性大肠杆菌病原学特征,为制定相关措施提供依据。[方法]对1995~2010年滕州市级索、西岗镇腹泻病监测点及市财贸医院、市中心人民医院腹泻病门诊腹泻病人资料进行分析。[结果]共检测腹泻病人粪便标本2 756份,检出致泻性大肠杆菌482株,检出率为17.49%。其中ETEC占61.20%,EPEC占22.20%,EIEC占16.60%。病原菌检出率,男性为55.37%,女性为44.63%(P>0.05);0~4岁为26.64%,5~19岁为20.32%,ETEC肠毒素类型呈以ST为主(LT>ST>ST/LT)。[结论]致泻性大肠杆菌在引起婴幼儿腹泻病中占有一定重要地位。     

从急性腹泻病人粪便中检测肠毒素性大肠杆菌

本文报告从海南地区111例急性腹泻患者粪便中,检测肠毒素性大肠杆菌(ETEC)的结果,用乳鼠灌胃法检出耐热性肠毒素(ST)9例11株,病例阳性率8.11％;用DNA基因探针技术检测不耐热性肠毒素(LT),阳性24例30株,病例阳性率为21.62％,ST和LT均阳性的1例,ETEC病例阳性率(ST和LT阳性合计)为28.8％,证实了本地区存在ETEC引起急性腹泻。同时对检出的ETEC菌株做了药敏试验。     

广东省2013年副溶血弧菌暴发与散发菌株的病原学特征

目的 了解2013年广东省副溶血弧菌暴发与散发分离株的血清型别、抗菌药物耐药性、毒力基因携带情况以及分子分型特征.方法 对36株暴发分离株和43株散发分离株进行血清分型、药敏试验以及耐热直接溶血毒素基因(tdh)、耐热相关溶血毒素基因(trh)、GS-PCR和orf8基因的PCR检测,并进行脉冲场凝胶电泳(PFGE)分型.结果 36株暴发分离株全部为O3:K6血清型,43株散发分离株的优势血清型为O3:K6型(23株,53.49%).药敏检测结果显示,对氨苄西林(96.20%)和头孢噻吩(40.50%)的耐药率较高;对复方新诺明和氯霉素则高度敏感,敏感性均为100%.多重耐药分析显示,83.33%(30/36)的暴发分离株同时耐受≥3种抗菌药,37.21%(16/43)的散发分离株同时耐受≥3种抗菌药物.毒力基因PCR检测显示,36株暴发分离株均为tdh⁺tdh^-型菌株.86.05%(37/43)的散发分离株为tdh⁺tdh^-型菌株,11.63%(5/43)为tdh^-tdh⁺型菌株,仅1株为tdh⁺tdh⁺型菌株.暴发分离株全部携带GS-PCR和/或orf8基因,51.16%(22/43)的散发分离株携带GS-PCR和/或orf8基因.PFGE显示,79株副溶血弧菌经NotⅠ酶切后的PFGE图谱可分为3个聚类,32种PFGE型别,相似值为59.8%～100.0%.暴发菌株聚集在同一个聚类中,散发菌株散布在各个聚类中.结论 2013年广东省副溶血弧菌优势血清型为O3:K6型,菌株对多数抗菌药物仍然比较敏感,但存在多重耐药现象,多数菌株携带tdh基因,大部分O3:K6型菌株携带GS-PCR和/或orf8基因;PFGE结果提示广东省副溶血弧菌存在遗传多样性.     

4种致泻性大肠埃希菌分子诊断方法的评估及其在监测中的应用

目的评估和建立用于常规监测4种致泻性大肠埃希菌(DEC)的分子诊断方法,并应用于上海市腹泻人群DEC的监测.方法使用丹麦SSI分子诊断试剂盒对DEC参考菌株进行验证试验及制定DEC-PCR诊断、分离的操作规程(DEC-PCR-SOP),并检测2012年6-9月上海市3家临床医院腹泻病例粪便标本.结果经26株DEC参比菌株验证,SSI分子诊断试剂盒的特异性为100%; 1887份腹泻病例标本共分离得到218株DEC(乳糖阳性181株,乳糖阴性37株),其中致病性大肠埃希菌(EPEC) 118株、产毒性大肠埃希菌(ETEC)90株、侵袭性大肠埃希菌(EIEC)9株、产志贺毒素大肠埃希菌(STEC)1株、志贺菌18株,总阳性率为l1.6%;监测地区DEC腹泻病例中以EPEC占优势,而EPEC腹泻病例中又以2岁以下婴幼儿为主;外籍DEC病例以ETEC占优势,新生儿ETEC病例占5岁以下低年龄组腹泻病例的1/3.结论经评估DEC-PCR-SOP用于4种DEC常规监测的数据结果可信.国内食源性监测网络实验室应不断完善4种DEC诊断和参比能力.     

上海市1962-2011年霍乱弧菌表型特征及分子分型研究

目的 分析1962-2011年上海市霍乱弧菌的表型及分子分型特征.方法 采用WHO推荐的改良K-B纸片法,对222株霍乱弧菌进行11种抗菌药物(头孢曲松、强力霉素、诺氟沙星、环丙沙星、复方新诺明、丁胺卡那霉素、四环素、氯霉素、萘啶酸、氨苄西林、庆大霉素)敏感试验.以PCR检测霍乱毒素基因(ctxA)、小带联结毒素基因(zot)、辅助霍乱肠毒素基因(ace)、溶血素基因(hlyA)、毒素协调菌毛基因(tcpA)、外膜蛋白基因(ompU)和调控蛋白基因(toxR).采用脉冲场凝胶电泳(PFGE)方法对菌株进行分子分型,用BioNumerics软件分析电泳图谱.结果 222株霍乱弧菌经药物敏感试验分析显示,1962-1996年的菌株对多种药物敏感,2005-2011年的菌株对多种药物耐药.O139群耐药率明显高于O1群,O139群产毒株的耐药率比非产毒株高.毒力基因分析显示,1962-1996年霍乱患者来源菌株多为O1群产毒株,2005-2011年患者来源菌株多为O139群产毒株,水体来源菌株未检出ctxA基因,O1群水产来源菌株以hlyA⁺ toxR⁺ ompU⁺为主,占25.6％(11/43),O139群水产来源菌株以hlyA⁺ toxR⁺ompU⁺ctxA ⁺ace ⁺zot⁺ tcpA⁺为主,占76.1％(16/21).PFGE分析将222株菌分为121个PFGE型,O139群分为3个聚类,O1群分为5个聚类.结论 上海市霍乱弧菌随着时间推移表型及分子特征均发生了很大变化,耐药情况加重.     

2010年福建南平及永安监测点致泻大肠杆菌的监测报告

目的:为了解福建省腹泻病人中致泻大肠杆菌的感染情况。方法:应用多重PCR或单重PCR方法检测致泻大肠杆菌的EPEC/EHEC eaeA基因、EHEC stx基因、EAEC aggR基因、EIEC ipaH、EIEC virA、ETEC ST、ETEC LT、EPEC bfp基因。API 20E生化鉴定条进行生化试验。血清学鉴定。结果:2010年度共分离得19株致泻大肠杆菌,检出率为10.9%。1株EAEC;1株tEPEC;4株aEPEC;13株ETEC。19株分离的致泻大肠杆菌经API 20E鉴定均为大肠埃希菌。1株tEPEC与EPEC三种多价诊断血清型均不凝集,而1株aEPEC血清型为O111:K58(B4)。13株ETEC菌株血清型:多数为O6:K15,1株O25:K19(L),3株未能分型。结论:监测结果对于我们了解福建省致泻大肠杆菌感染情况,预警潜在发生疫情的可能性有其重要性。这些菌种的获得可以为下一步研究其毒力特征、分子分型及耐药性积累原始材料。     

延平区2010—2012年致泻性大肠杆菌检测分析

目的了解延平区感染性腹泻患者致泻性大肠杆菌(DEC)检出情况及菌株特征。方法用多重和单重PCR方法检测DEC的EPEC/EHECeaeA基因、EHECstx基因、EAEC aggR基因、EIECipaH和virA基因、ETEC ST和LT基因,对阳性菌株进行API20E系统生化鉴定、血清凝集试验和PFGE试验。结果 2010—2012年DEC监测点共检测腹泻病人粪便标本504份,检出DEC 39株(7.7%),其中EPEC 23株(59.0%,以aEPEC为主,18株),ETEC 14株(35.9%),EAEC 2株(5.1%)。ETEC中ST基因阳性9株,LT基因阳性3株,均阳性2株。不同类型的DEC生化反应特性不同;对18株aEPEC进行PFGE,DNA片段得到较好分离。DEC检出率各年龄组差异无统计学意义。结论 DEC是该监测点腹泻患者重要病原菌,以EPEC为主,建立健全基层疾控机构对DEC的检测很有必要。     

HIV/AIDS手术切口愈合与CD4+T淋巴细胞计数的关系

目的 探讨HIV/AIDS手术预后及切口愈合与CD₄⁺T淋巴细胞计数的关系.方法 以北京地坛医院2008年1月至2012年12月手术治疗的234例HIV/AIDS住院患者为研究对象,采用回顾性分析的方法,对患者的年龄、性别、发现抗HIV(+)时间、手术时CD₄⁺T淋巴细胞计数、是否为急诊手术、手术部位、切口分类、术后切口愈合级别、切口感染情况、术后并发症及预后进行分析.统计学采用Wilcoxon 秩和检验、χ²检验、Kruskal-Wallis H检验和Spearman相关分析,比较不同的切口愈合级别的患者CD₄⁺T淋巴细胞计数水平的差异、不同CD₄⁺T淋巴细胞计数水平的甲级愈合率和HIV/AIDS相关因素与手术切口愈合率的关系.结果 (1)共有234例患者,男性125例,女性109例,性别比为1.15:1,平均年龄(36.17±11.56)岁.发现抗HIV(+)时间为0～204个月.CD₄⁺T淋巴细胞计数M为388.5 cell/μl.其中23.93%患者CD₄⁺T 淋巴细胞计数<200 cell/μl.(2)急诊手术占7.26%.发病部位涉及23个器官,48种疾病.Ⅰ类切口占21.37%,Ⅱ类切口49.57%,Ⅲ类切口29.06%.86.32%切口为甲级愈合,11.97%为乙级愈合,1.71%为丙级愈合.4.27%患者出现术后并发症.术后出现并发症与未出现并发症患者的CD₄⁺T淋巴细胞计数差异无统计学意义(P>0.05),两组患者感染HIV的时间差异无统计学意义(P>0.05).甲级愈合与乙、丙级愈合的CD₄⁺T淋巴细胞计数水平差异无统计学意义(P>0.05).不同CD₄⁺T淋巴细胞计数水平的甲级愈合率差异无统计学意义(P>0.05).手术切口愈合情况与CD₄⁺T淋巴细胞计数水平、抗病毒治疗时间长短、HIV感染时间无明显相关性(P>0.05).结论 严格把握手术适应症和禁忌症,对需要手术的HIV/AIDS进行手术治疗,总体预后良好.低CD₄⁺T淋巴细胞计数并不是手术的绝对禁忌.切口愈合情况与CD₄⁺T淋巴细胞计数、抗病毒治疗时间长短及HIV感染时间无明显相关性.     

Enterotoxigenic Escherichia coli associated diarrhoea among infants aged less than six months in Calcutta, India

The role of enterotoxigenic Escherichia coli (ETEC) as etiologic agents of diarrhoea in infants aged less than six months was assessed in a hospital based study in Calcutta, India. Of the 218 cases examined, ETEC strains were isolated from 26 (11.9%) cases. Among these, in 17 cases ETEC was the sole infecting pathogen (p = 0.0085). Of the 26 isolates (each isolate representing a case), 24 were distributed among seven different O:K:H serotypes and two different colonization factor antigens (CFAs) I and II. Two of the remaining isolation were untypable, non-haemagglutinating, and were nonhydrophobic as measured by the salt aggregation test (SAT). Of the 26 ETEC strains detected, 15 (57.7%) produced heat-labile toxin (LT) only, 8 (30.8%) liberated heat-stable toxin (ST) only, and the remaining 3 (11.5%) produced both LT and ST. No ETEC strain was isolated from the 102 age-matched controls included in this study. All the ETEC isolates were multiple drug resistant. The study showed that the diarrhoea due to ETEC was of brief duration, mostly within the range of 3 to 7 days.     

Demonstration of enterotoxigenic Escherichia coli in diarrheic broiler chicks

An investigation was made to survey the possible presence of enterotoxigenic Escherichia coli (ETEC) in the stools of diarrheal chicks.We analyzed two outbreaks of diarrhea in broiler chicks at two independent farms in the Philippines, from which no pathogens other than Escherichia coli were found. In one outbreak at Farm # 1, all 42 isolates produced heat-labile enterotoxin (LT), with 3 of these isolates also producing heat-stable enterotoxin (ST). The 0 serotypes of 15 strains tested randomly could not be identified as any known serotype (0-antigen; 1–170). In another outbreak at Farm #2, 7 out of 52 isolates produced only LT, their subtypes being identified as O-149 or O-8, common serotypes in pig ETEC. Strains from Farm #1 did not produce any pili usually found in human ETEC. We believe this to be the first isolation of ETEC from diarrheal chicks.Corresponding author.     

三对引物同时PCR分型检测产毒素大肠杆菌的方法及在分子流行病学研究中的应用

在产毒素大肠杆菌（ＥＴＥＣ）肠毒素基因内设计合成三对引物，建立了三对引物同时ＰＣＲ检测ＥＴＥＣ的方法，一次ＰＣＲ即可扩增出６２７ｂｐ（ＬＴｈ）、２４０ｂｐ（ＳＴＩａ）和１６９ｂｐ（ＳＴＩｂ）三种肠毒素基因片段，可同时分型检测出ＬＴｈ、ＳＴＩａ、ＳＴＩｂ、ＬＴｈ－ＳＴＩａ、ＬＴｈ－ＳＴＩｂ五种基因型的ＥＴＥＣ，与非ＥＴＥＣ对照菌无交叉反应，最小检出量为１０ｃｆｕ，显示了很高的特异性和敏感性。将建立的方法用于山东省六县市６２３例大肠杆菌致泻标本的检测，阳性率为４０．３％，并能鉴别ＥＴＥＣ的基因型，为ＥＴＥＣ腹泻的分子流行病学研究提供了有效的检测手段     

三对引物同时PCR分型检测产毒素大肠杆菌的方法及在分子流行病学研究中的应用

在产毒素大肠杆菌肠毒素基因内设计合成三对引物，建立了三对引物同时ＰＣＲ检测ＥＴＥＣ的方法，一次ＰＣＲ即可扩增出６２７ｂｐ（ＬＴｈ）、２４０ｂｐ（ＳＴ Ｉａ）和１６９ｂｐ（ＳＴ Ｉｂ）三种肠毒素基因片段，可同时分型检测出ＬＴｈ，ＳＴ Ｉａ、ＳＴ Ｉｂ、ＬＴｈ－ＳＴ Ｉａ、ＬＴｈ－ＳＴ Ｉｂ五种基因型的ＥＴＥＣ，与非ＥＴＥＣ对照菌无交叉反应，最小检出量为１０ｃｆｕ，显示了很高的特异性和敏感性。将建立的     

Enterotoxigenic Escherichia coli and Vibrio cholerae diarrhea, Bangladesh, 2004

Flooding in Dhaka in July 2004 caused epidemics of diarrhea. Enterotoxigenic Escherichia coli (ETEC) was almost as prevalent as Vibrio cholerae O1 in diarrheal stools. ETEC that produced heat-stable enterotoxin alone was most prevalent, and 78% of strains had colonization factors. Like V. cholerae O1, ETEC can cause epidemic diarrhea.     

Immunogenicity of multivalent Shigella-ETEC candidate vaccine strains in a guinea pig model

Shigella and enterotoxigenic Escherichia coli continue to be significant causes of diarrheal disease in infants and young children in developing countries as well as prevalent agents of traveler's diarrhea. A vaccine which provides protection against disease caused by both pathogens would serve common at-risk populations. Such a vaccine would require inclusion of multiple Shigella strains as well as multiple ETEC antigens. The use of attenuated strains of Shigella as live vectors for the expression of ETEC antigens is one strategy for the development of such a multivalent vaccine. Live attenuated strains of S. flexneri 2a, S. sonnei and S. dysenteriae 1 containing deletions in guaBA biosynthetic pathway genes as well as in genes encoding enterotoxins, were constructed. Each strain was subsequently used as a live vector for the expression of one or two critical ETEC antigens. The resulting three Shigella derivative strains were tested for immunogenicty and protective capacity alone or as mixtures in the guinea pig model. S. flexneri strain CVD 1208(pCFA/I-CS3), S. sonnei strain CVD 1233(pCS4-LThK63) and S. dysenteriae 1 strain CVD 1252(pCS2) were able to elicit serum and mucosal antibody responses against the live vector as well as the guest ETEC antigens. Vaccination with combinations of two or three of these strains was able to elicit specific immune responses against each live vector as well as each ETEC antigen represented in the mixture. These studies demonstrate the potential of the use of mixtures of live Shigella derivatives expressing ETEC antigens to serve as an immunogenic multivalent vaccine.     

Surface water of a perennial river exhibits multi-antimicrobial resistant shiga toxin and enterotoxin producing Escherichia coli

The high incidences of waterborne diseases are frequently associated with shiga toxin (STEC) and enterotoxin producing Escherichia coli (ETEC). Therefore, in the present study, surface water samples collected from the river Saryu were analyzed for the presence of multi-antimicrobial resistant ETEC and STEC. Forty-two E. coli isolates were screened for virulence determinants of STEC and ETEC. Eighteen E. coli isolates exhibit both stx1 and stx2 genes (66.6%) or only stx1 (33.3%) gene. eaeA, hlyA, and chuA genes were present in 94.5%, 83.3%, and 55.6% of STEC, respectively. Further, it was observed that 12 isolates exhibit only ST1 gene (25%) or both LT1 and ST1 genes (75%). The resistance to multi-antimicrobials was observed in 100% and 27.7% of ETEC and STEC isolates, respectively. The presence of multi-antimicrobial resistant diarrheagenic E. coli in surface waters of south Asia is an important health concern due to risk of developing waterborne outbreaks.     

Isolation of enterotoxigenic Escherichia coli from British troops in Saudi Arabia.

Specimens from 181 patients with diarrhoea were examined by a Military General Hospital in a 3-month period during deployment of troops to Saudi Arabia in 1990/1. DNA probes for heat labile (LT) and heat stable (ST) enterotoxin genes identified enterotoxigenic Escherichia coli (ETEC) in 47 of the specimens (26%) and 49 ETEC strains were isolated. The majority (55%) belonged to a novel ETEC serotype having the O-antigen 159 and a flagellar antigen designated as a provisional new type. They produced ST and the coli surface associated antigen (CS)6. Strains of serotype O6:H16 represented 22% of the ETEC examined. They produced ST, LT and CS3 together with either CS1 or CS2. The remaining ETEC belonged to seven O:H serotypes. Overall, ST was the only enterotoxin gene identified in 73% of the ETEC and 67% of the strains expressed CS6 in the absence of other colonization antigens. Resistance to three or more antibiotics was observed in 53% of the ETEC, including most of the O159 strains.     

产毒性和致病性大肠埃希菌中小肠结肠炎耶尔森菌强毒力岛分布的研究

目的：了解小肠结肠炎耶尔森菌强毒力岛在产毒性（ETEC）和致病性大肠埃希菌（EPEC）中的分布。方法：用聚合酶链反应扩增和原位杂交方法。结果：在检测的93株ETEC和10株EPEC的毒力岛的检出率分别为32.25%(32/93)和30.00%(3/10),1株肠聚集性大肠埃希菌（EAggEC)也检出了毒力岛,而且这些阳性菌株中的岛大部分连接到天门冬氨酸tRNA（asntRNA)位点。结论：ETEC、EPEC以及EAggEC是致病性较强的病原菌,而小肠结肠炎耶尔森菌强毒力岛在ETEC和EPEC中具有阳性率较高的分布,对于进一步研究大肠埃希菌毒力变化和毒力的调控以及细菌毒力的进化具有重要意义。     

Outbreaks of cholera-like diarrhoea caused by enterotoxigenic Escherichia coli in the Brazilian Amazon Rainforest

The relationship between enteropathogens and severe diarrhoea in the Brazilian Amazon is poorly understood. In 1998, outbreaks of acute diarrhoea clinically diagnosed as cholera occurred in two small villages localized far from the main cholera route in the Brazilian rainforest. PCR was performed on some enteropathogens and heat-labile (LT) and/or heat-stable (STh) toxin genes, the virulence determinants of enterotoxigenic Escherichia coli (ETEC), were detected. Further characterization of ETEC isolates revealed the presence of two clones, one from each outbreak. One presenting serotype O167:H5 harboured LT-I and STh toxin genes and expressed the CS5CS6 colonization factor. The other, a non-typeable serotype, was positive for the LT-I gene and expressed the CS7 colonization factor. The current study demonstrates the importance of molecular diagnosis in regions such as the Amazon basin, where the enormous distances and local support conditions make standard laboratory diagnosis difficult. Here we also show that the mis-identified cholera cases were in fact associated with ETEC strains. This is the first report of ETEC, molecularly characterized as the aetiological agent of severe diarrhoea in children and adults in the Brazilian Amazon Rainforest.