四种致泻性大肠埃希氏菌实验室检测研究进展

肠致病性大肠埃希氏菌（EPEC）、肠产肠毒素大肠埃希氏菌（ETEC）、肠侵袭性大肠埃希氏菌（EIEC）、肠出血性大肠埃希氏菌（EHEC）四种致泻性大肠埃希氏菌是引起人体以腹泻症状为主的全球性疾病的常见病原菌,也是我国绝大多数地区引起腹泻的主要病原菌,因此快速准确地检测这四种病原菌对预防和控制由其引起腹泻有着十分重要的作用。本文介绍了这四种致泻性大肠埃希氏菌实验室检测技术的研究进展,主要包括细菌分离培养、免疫学检测、多重PCR、实时荧光PCR等分子生物学方法。     

大肠埃希菌质粒型AmpC酶基因的检测分析

目的 调查大肠埃希菌质粒型Ampc酶基因的存在状况。方法 采用MH药敏平板对40株临床分离的大肠埃希菌进行抗菌药物敏感试验和ESBLs纸片法确诊,PcR方法检测DHA和ACT-1型Ampc酶基因。结果 40株大肠埃希菌呈多重耐药,20株ESBLs阳性菌AmpCM基因阳性率90％,20株ESBLs性菌Ampc酶基因阳性率45％,40株大肠埃希菌ACT-1和DHA基因阳性率分别为57．5％,40．0％,有12株大肠埃希菌同时携带ACT-1和DHA基因。结论 临床分离的大肠埃希菌多重耐药严重,质粒型AmpC酶基因携带率高。     

肠致病性大肠埃希菌病原学检验探析

目的:本文将对腹泻患儿进行肠致病性大肠埃希菌病原学检验,从而探讨肠致病性大肠埃希菌的病原学特点。方法:对所有腹泻患者进行粪便采集样本,之后应用病原学检验方法对样本中肠致病性大肠埃希菌进行分离,给予病原学检查,包括形态特征分析、培养特征分析以及生化反应特征分析等,得出结论。结果:231例腹泻患儿粪便样本中均检测出肠道致病性大肠埃希菌,检出率为100.00%。肠道致病性大肠埃希菌菌落呈现出灰色,可略带白色,菌落具有整齐的边缘以及光泽表面;对肠致病性大肠埃希菌进行革兰氏染色可知,其结果为阴性,无芽孢出现,但出现鞭毛,属于短小杆菌,两端呈现出圆顿形。结论:研究肠致病性大肠埃希菌的病原学特点能够有效提高肠道致病性大肠埃希菌引发腹泻等疾病的确诊率,使患者及时得到具有针对性的治疗措施,提高患者治疗效果。     

Identification of Shiga- like toxin Escherichia coli isolated from children wi th diarrhea by polymerase chain reaction

目的：探讨产志贺样毒素大肠埃希菌（SLTEC）在小儿腹泻中的病原学地位。方法：根据肠出血性大肠埃希菌（EHEC）0157：H7SLT1、SLT2、eaeA基因片段设计了3对引物,采用PCR法检测大肠埃希菌中SLT1、SLT2、eaeA毒力基因。结果：29株标准致泻大肠埃希菌（EHEC、EPEC、ETEC）和10株其它肠道病原菌中,只有1株EHEC检出SLT1、SLT2、eaeA毒力基因,其它均为阴性。从1032例腹泻患儿粪便中分离的474株未定型大肠埃希菌中,检出SLT1 20株（4.2％）,SLT2 7株（1.5％）;74株产志贺样毒素侵袭性大肠埃希菌（ESIEC）中检出SLT115株（20.3％）,SLT2 5株（6.8％）。结论：SLTEC是太原地区引起小儿腹泻的一种重要的致泻病原菌。     

多重荧光PCR法与细菌培养法在食品中致泻性大肠埃希氏菌检测中的应用

目的通过多重荧光PCR法与细菌培养法在食品中致泻性大肠埃希氏菌检测中的应用,比较两种方法的敏感性、特异性、时限性、重复性。方法采集10类食品200份样品进行细菌培养法和荧光PCR法检测致泻性大肠埃希氏菌。结果在10类食品200份样品中细菌培养法检出普通大肠埃希氏菌30株,致泻性大肠埃希氏菌未检出。多重荧光PCR法检出EHEC阳性4份。其中生禽畜肉检出2份、水产品2份。EPEC、ETEC、EAEC、EIEC均为阴性。结论与传统的细菌培养法相比,多重荧光PCR法可以为细菌分离培养提供初步信息,提示实验室在进行分离培养时应有针对地重点考虑,减轻分离培养繁杂过程,有利于实验室诊断。     

泌尿系感染患者大肠埃希菌fimH基因检测及尿便来源菌株同源性分析

目的：检测尿路致病性大肠埃希菌I型菌毛相关基因fimH携带率,调查相同泌尿系感染患者尿便大肠埃希菌的同源性,对相关基因fimH进行序列分析。方法：89株来自反复发作性尿路感染和急性单纯性膀胱炎患者清洁中段尿分离的大肠埃希菌。PCR方法检测I型菌毛基因fimH,采用Fisher确切概率法比较两组携带率是否有差异。取9名患者尿便同时培养的大肠埃希菌进行药敏初筛分型,同源性分析采用脉冲场凝胶电泳（PFGE）技术,对其fimH基因PCR扩增产物进行序列分析。结果：89株检出81株fimH阳性,两组检出率分别为（64／70）91．4％和（17／19）89．4％,P〉0．05。9名患者中5名患者尿便同时分离大肠埃希菌各自存在相同的PFGE型,同一患者尿便同型株中fimH阳性者序列比对无差异,不同型株fimH阳性者其序列均有6～7处变异。结论：fimH基因存在于绝大多数尿路致病性大肠埃希菌中。引起泌尿系感染的大肠埃希菌部分来源于肠道。     

腹泻症候群致泻性大肠埃希氏菌血清型及毒力基因检测

目的:通过对腹泻症候群中致泻大肠埃希菌的血清型和多重PCR检测,了解本地区致泻大肠埃希菌的血清群、型分布规律,所携带毒力基因及相互之间的相关性,为腹泻症候群中致泻大肠埃希菌的检测、鉴定提供科学依据,以提高阳性株的检出率。方法:对本地区2012-2013年腹泻症候群监测医院门诊未使用过抗生素腹泻患者的稀便、水样便、黏脓便或脓血便标本通过増菌、各种选择性培养基筛选出病原菌,后经生化试验、多重PCR试验和血清型分型试验进行鉴定。结果:本次检测共检出65株血清凝集致泻大肠埃希菌,分属EPEC、EIEC、ETEC,以EPEC为主,占83.08%,共8个血清型,其中血清型O55:H59、O128:H67占58.47%;共检出4种相关毒力基因,其中escV 49株(75.38%)。未分血清型菌株27株,检出相关毒力基因共5种,分属EPEC、EIEC、ETEC、EAEC,其中escV 15株(55.56%)。结论:本地区腹泻症候群中血清学阳性致泻大肠埃希菌以EPEC为主,常见血清型为O55:H59、O128:H67,毒力基因为escV、escV+bfpB、invE、elt。未分血清型致泻大肠埃希菌毒力基因为escV、escV+bfpB、invE、elt、astA,与已分型菌株携带基因基本一致。腹泻患者的大肠埃希菌检测中,在传统的细菌分离培养、生化试验、血清学鉴定的基础上,有必要进行大肠埃希菌的毒力基因检测,以提高阳性检出率,避免漏检,提高致泻性大肠埃希菌的诊断。     

血流感染大肠埃希菌对多黏菌素耐药机制研究

目的探讨血流感染大肠埃希菌对多黏菌素的耐药机制及流行特点,为其治疗提供依据。方法收集2018年1月至2019年12月浙江省人民医院和杭州市余杭区第二人民医院门诊及住院患者中分离的血流感染大肠埃希菌241株,通过VITEK-2Compact系统检测大肠埃希菌药物敏感性;应用WHONET5.6软件统计药敏结果;通过PCR法检测多黏菌素耐药相关基因;应用多克隆序列位点（MLST）测定菌株克隆型别;应用接合试验验证多黏菌素耐药基因转移。结果241株血流感染大肠埃希菌中,分离出6株多黏菌素耐药大肠埃希菌,分离率为2.5%。PCR检测显示6株多黏菌素耐药大肠埃希菌均携带mcr-1耐药基因,MLST分析结果显示这些菌株属于不同克隆型别。此外,6株多黏菌素耐药大肠埃希菌mcr-1耐药基因均可以成功传递至受体菌大肠埃希菌J53菌株中。结论血流感染大肠埃希菌对多黏菌素的耐药机制主要是携带mcr-1基因,该基因定位于质粒进行水平播散,需密切监测携带mcr-1基因的菌株,防止其进一步传播。     

214株大肠埃希菌耐药情况分析

目的：了解本院临床分离大肠埃希菌分不情况及耐药性情况。方法：利用法国梅里埃药敏分析系统对2004—2006年分离的大肠埃希菌进行药敏试验。结果：214株大肠埃希氏菌主要来自尿液（33．64％）和痰液（26．64％）样本,大肠埃希菌对亚胺培南敏感性为100％,对阿莫西林、环丙沙星、庆大霉素、替考西林等抗生素总体耐药率在40％以上,大肠埃希菌耐药率总体呈升高趋势。结论：临床分离大肠埃希菌耐药率呈现逐年上升趋势,抗生素的应用规范应该给予足够重视和监控。     

实时荧光多重聚合酶链反应-高分辨率溶解曲线分析鉴别6种致腹泻性大肠埃希菌的研究

目的建立一种鉴别6种致腹泻性大肠埃希菌的实时荧光多重聚合酶链反应-高分辨率溶解曲线分析方法。方法根据6种致腹泻大肠埃希菌的8种特异性毒力基因设计8对引物,采用实时荧光多重聚合酶链反应后溶解曲线分析,根据曲线的峰值、高度、宽度、面积不同鉴别不同的致病菌。结果成功设计出8对引物,扩增出针对6种致腹泻大肠埃希菌的8种不同毒力基因,获得了特异性的溶解曲线。优化反应体系后在同一反应管中成功获得针对8种特异基因的溶解曲线,除肠侵袭性大肠杆菌、细胞致死膨胀性大肠杆菌具有相同的两种基因不能鉴别外,其中5种致腹泻性大肠埃希菌均可以明确鉴别。结论多重聚合酶链反应-高分辨率溶解曲线分析鉴别致腹泻性大肠埃希菌是一种简单、特异、高效的方法,在腹泻病与食源性疾病的暴发调查和日常监测中具有广泛的应用价值。     

携带毒力岛大肠杆菌性小儿腹泻

目的：探讨携带毒力岛大肠杆菌（HPIEC）在小儿腹泻中的病原学地位。方法：采用PCR扩增和菌落原位杂交检测HPIEC－irp2毒力岛基因,并用血清学分型和聚合酶链反应（PCR）法检测各类致泻大肠杆菌。结果：从1032例腹泻患者粪便中分离出652株大肠杆菌,经血清学和PCR毒力基因分型,检出各类致泻大肠杆菌225株,其中肠致病性大肠杆菌（EPEC）20株,ETEC81株,产志贺样毒素大肠杆菌（SLTEC）47株,产志贺样毒素侵袭性大肠杆菌（ESIEC）74株,产毒素大肠杆菌（EIEC）3株。所有致泻大肠杆菌和未能分型的普通大肠杆菌株,再用HPIEC－irp2探针作菌落原位杂交,共检出携带毒力岛大肠杆菌（HPIEC－irp2)112株,总阳性率为17．2％。其中41例是从致泻大肠杆菌ESIEC（24／74）和SLTEC（17／47）中检出。携带毒力岛大肠杆菌性小儿腹泻的主要临床表现为食欲不振（87．5％）,腹痛（58．0％）,腹泻（＞6次／d,75．9）,发热（50．9％）,以粘液便为主（69．6％）。结论：携带毒力岛大肠杆菌是引起小儿腹泻的重要病原菌之一。     

Sporadic occurrence of hemorrhagic colitis associated with Escherichia coli O157:H7 in Newfoundland.

During a 9-month period in 1984, 113 fecal samples obtained from 92 patients with diarrheal illness were cultured for Escherichia coli serotype O157:H7 to determine the occurrence of this agent in diarrheal illness in Newfoundland. E. coli O157:H7 was isolated in almost pure culture from 12 stool specimens obtained from 7 (15%) of the 47 patients who had grossly bloody diarrhea; none of the 12 yielded any of the usual enteric pathogens. The agent was not isolated from the stool specimens obtained from the remaining 45 patients, who did not have bloody diarrhea. All seven patients whose specimens were positive for E. coli O157:H7 had clinical manifestations typical of hemorrhagic colitis, but the syndrome was clinically suspected and a specific test requested in only two cases. The seven cases were not clustered geographically or temporally, and no common exposure was identified. To determine whether hamburger meat was the source of E. coli O157:H7, 66 samples obtained from various retail outlets were tested; none were found to be positive. Hemorrhagic colitis may be a common disease, and E. coli O157:H7 should be considered as an agent in bloody diarrheal illness.     

婴儿腹泻患者分离出大肠杆菌的药敏及R质粒检测

作者对从婴儿腹泻患者粪便分离的84株大肠杆菌,做了药敏性和接合性R质粒的检测。共检出对链霉素、四环素、氯霉素、卡那霉素、庆大霉素及氨苄、青霉素6种抗生素的耐药菌80株,占95.2％。对检出的80株耐药菌进行接合试验,R质粒的平均检出率为92.5％。讨论部分还将本文结果与正常人群大肠杆菌的药敏及R质粒检出结果,做了比较分析。     

江苏省徐州市分离的大肠杆菌O157:H7菌株携带志贺毒素2型别的变化

目的 研究我国分离的大肠杆菌O157：H7菌株的志贺毒素型别的变化。方法使用针对志贺毒素2及其变种的引物对进行PCR检测、细菌染色体DNA的PstⅠ酶切后的Southern印迹实验和PCR产物的HaeⅢ、RasⅠ酶切分析,以及：PCR产物克隆后的序列分析方法以及致病性大质粒的PstⅠ酶切分析。结果1999年分离自徐州市的人的5株菌均携带slt1、slt2,而2000年从江苏省徐州市患者分离的10株和蜣螂标本分离的4株共计14株大肠杆菌O157：H7菌株仅携带slt2vha毒素基因,其致病性质粒的限制性酶切图谱与1999年的菌株相比也发生了改变。结论鉴于江苏省徐州市1999年的分离的5菌株全部携带slt1和slt2基因,结果提示该地的优势菌型发生了改变,可能当地大肠杆菌O157：H7感染的流行病学特点有关。针对slt2变异基因而设计的引物及其以此为基础进行的限制性片段长度多态性分析的方法对于研究O157：H7菌株的志贺毒素型别的变化能够满足特异性及灵敏性的要求。     

Serological identification of Escherichia coli isolated from cats and dogs

In a group of 112 cats, examined during an outbreak of diarrhea, 104 (92.8%) yielded Escherichia coli with identifiable O groups as compared to 12 (21.8%) of 55 control cats apparently free of intestinal ailments. E. coli group O6 occurred in 49.1% of the cats with diarrhea, and persisted in this group of cats at about the same rate when examined for a period of ten months. E. coli group O6 was not isolated from the control group.Specimens were also collected from dogs with various clinical signs. E. coli O4 and O6 strains appear to be important potential pathogens for canine species and were found to spread from one dog to another when in close contact. E. coli group O4 was identified in ten and O6 in three of 14 dogs examined. All isolates in which the O group could be identified were hemolytic.     

南京地区儿童肠道感染致病性大肠埃希菌血清型的分布及耐药性

目的:了解南京地区儿童细菌性腹泻中致病性大肠埃希菌(Escherichia coli,E.coli)的主要血清型和对抗生素的敏感性.方法:对5 409例粪便标本进行分离培养并用血清学方法鉴定E.coli血清型,采用纸片扩散法测定E.coli对常用抗生素的敏感性.结果:5 409例粪便标本中分离出633株E.coli,检...     

Polymicrobial bacteremia caused by Escherichia coli, Edwardsiella tarda, and Shewanella putrefaciens

Edwardsiella tarda, a member of Enterobacteriaceae, is found in freshwater and marine environments and in animals living in these environments. This bacterium is primarily associated with gastrointestinal diseases, and has been isolated from stool specimens obtained from persons with or without clinical infectious diseases. Shewanella putrefaciens, a saprophytic gram-negative rod, is rarely responsible for clinical syndromes in humans. Debilitated status and exposure to aquatic environments are the major predisposing factors for E. tarda or S. putrefaciens infection. A 61-year-old woman was febrile with diarrhea 8 hours after ingesting shark meat, and two sets of blood cultures grew Escherichia coli, E. tarda and S. putrefaciens at the same time. She was successfully treated with antibiotics. We present this rare case of polymicrobial bacteremia caused by E. coli, E. tarda and S. putrefaciens without underlying disease, which is the first found in Taiwan. This rare case of febrile diarrhea with consequent polymicrobial bacteremia emphasizes that attention should always be extended to these unusual pathogens.     

Response of gnotobiotic pigs to Escherichia coli

In a study of the response of gnotobiotic pigs to coliform infections, 45 one-week-old germfree pigs were divided into five groups and each group was inoculated orally with a different strain of Escherichia coli. Three of these were enteropathogenic swine strains, P307[08:K87(B), K88 a,b (L):H19]; P570 [0138:K81]; P568[0141:K85a,b(B), K88a,b(L):H4], one was a virulent human strain, H224, [026:K60(B6)], and one was a non-enteropathogenic swine strain, P581[OX13:K68]. It was attempted to protect a portion of the pigs with orally administered specific antisera and sera from non-immunized specific pathogenfree (SPF) pigs. Observations were made on the clinical response, bacterial counts of feces and intestinal contents, gross pathological changes, distribution of the organisms in organs and serum hemagglutinin titers.Infection with E. coli P307 resulted in diarrhea, dehydration and death, unless the pig was protected with specific antiserum. The pigs infected with E. coli P570 had a transient diarrhea but retained their appetites and recovered. Those infected with the other three strains remained healthy throughout. No circulating hemagglutinating antibody against the test strains of E. coli could be detected in any of the pigs seven days or earlier post-inoculation.Relationship could not be established between the numbers of viable E. coli in the feces and the presence of clinical colibacillosis. Orally administered specific antiserum afforded protection against strain P307, but did not reduce the number of E. coli in the gut or alter their distribution in the internal organs. This suggested that the protective effect of specific antibody in the intestine was due to its action on a metabolite (enterotoxin) produced by E. coli P307 rather than the organism itself.     

Studies of escherichia coli in gnotobiotic pigs. VI. Effects of feeding bacteria-free filtrates of broth cultures

Nine gnotobiotic pigs derived from one gilt were fed bacteria-free filtrates prepared from: 1) cultures of an enteropathogenic strain of Escherichia coli 09:K.:NM (Strain 340), 2) cultures of a nonenteropathogenic strain of E. coli 08.K..H16 (Strain CDC-1466-56), and 3) uninoculated culture medium.Diarrhea was observed initially two to four hours after feeding the filtrate prepared from the enteropathogenic E. coli. The duration of diarrhea was five to ten hours. No diarrhea was observed after feeding filtrate prepared from uninoculated medium or cultures of nonenteropathogenic E. coli.The pH values of the feces increased with the onset of diarrhea and decreased to normal after diarrhea stopped.No histopathological lesions were found.     

Molecular detection and antimicrobial resistance of diarrheagenic Escherichia coli strains isolated from diarrheal cases

OBJECTIVE: To identify and classify Iranian isolates of diarrheagenic Escherichia coli (E. coli) on the basis of presence of virulence genes and to determine antibiotic susceptibility of isolated strains. METHODS: The current cross-sectional study was conducted in 2005 at the Pasteur Institute, Tehran, Iran. One hundred and ninety-three diarrheagenic E. coli isolated from diarrheal patients in different regions of Iran were included in current study. Virulence factor genes for diarrheagenic E. coli were detected by polymerase chain reaction. RESULTS: Of the 193 diarrheagenic E. coli detected by PCR, 86 (44.5%) were Shiga toxin-producing E. coli STEC, 74 (38.4%) enteropathogenic E. coli EPEC, 19 (9.8%) enteroaggregative E. coli, and 14 (7.3%) enterotoxigenic E. coli isolates. Susceptibility to 12 clinically important antimicrobial agents was determined for 193 strains of diarrheagenic E. coli. A high incidence of resistance to tetracycline (63%), ampicillin (62%), streptomycin (56%), amoxicillin/clavulanic acid (44.5%), trimethoprim/sulfamethoxazole (39.5%), and cephalothin (37%) was observed. CONCLUSION: The STEC and EPEC strains with high resistance to tetracycline and ampicillin, but highly susceptible to quinolones are among the most important causative agent of diarrhea in Iran. This study suggests that antimicrobial resistance is widespread among E. coli strains colonizing Iranian patients. Guidelines for appropriate use of antibiotics in developing countries require updating.