分型检测致泻性大肠埃希氏菌PCR技术研究进展

致泻性大肠埃希氏菌是一类能够引起人类和动物腹泻的食源性致病菌,迅速确定致泻性大肠埃希氏菌的污染来源可有效缩小疫情影响范围,建立简便高效的致泻性大肠埃希氏菌检测与分型技术是保障食品安全和控制疫情的关键。为适应对时间敏感度较高要求的现场或在线检测,基于PCR技术的检测分型方法不断地被标准化和规范化。对近年来国内外的致泻性大肠埃希氏菌分子检测与分型的PCR技术研究进展进行了综述,并详细地介绍了多重聚合酶链反应、荧光实时定量聚合酶链反应和核酸等温扩增技术的原理及其优缺点。为致病菌溯源方法的选择提供参考,对防御并控制致病菌引起流行病传播具有重要的意义。     

大肠埃希菌的分型研究

目的分析上海某医院各科室分离大肠埃希菌的药敏状况和致病性,了解大肠埃希菌在该院流行情况。方法采用K-B琼脂法进行药敏试验,多重PCR技术进行基因分型。结果药敏结果显示该菌对多种常用抗生素具有耐药性,仅对阿米卡星等药物敏感。85株菌分为4个基因型,其中B2型25株,致病性最强;D型37株,致病性次之。菌株间亲缘关系表明可能存在院内流行。结论实验获得菌株具有较强耐药性和致病性,应当采取相应的措施预防院内感染的流行。     

四株大肠埃希氏菌国际参考菌株动力和鞭毛抗原检定的研究

Authors identified four strains of Escherichia coli from Collaborative Centre for reference and research on Escherichia coli-Klebsiella (WHO) in Denmark. Our results were different from original record and reference. It was shown that strain H311b and H5 were motile strains, and its H antigen respectively is H11 and H27. The strain W27 is nonmotile. H antigen of the strain H511 is H40, not H8. Antigen formula of four strains respectively is 26:60:11 (strain H311b), 81:97:27 (strain H5), 115:-:- (strain W27), 102:-:40 (strain H511).     

一种快速提取禽源性大肠埃希氏菌外膜蛋白的方法

介绍一种快速提取禽源性大肠埃杀氏外膜蛋白的方法,该法是对Ｋａｐｕｒ等发表的方法的改进,全过程只需超速离心一次,比Ｋａｐｕｒ等的方法缩短了４ｈ,所得样品可直接用于禽源性大肠埃希氏菌外膜蛋白模式的测定。     

泌尿系感染产ESBLs大肠埃希菌基因分型

目的 了解深圳市人民医院临床泌尿系感染标本中产ESBLs大肠埃希菌的基因型特点.方法 收集近几年深圳市人民医院临床尿液标本中非重复的产ESBLs大肠埃希菌43株,PCR分别扩增菌株的TEM、SHV、CTX-M基因,阳性株进行DNA测序分型.结果 43株产ESBLs大肠埃希菌中40株CTX-M基因阳性,分别为CTX-M-14型36株,CTX-M-9型2株,CTX-M-15型2株,其中17株CTX-M-14型菌株检出TEM-1基因;所有菌株均未检出SHV基因.结论 本地区致泌尿系感染产ESBLs大肠埃希菌中,CTX-M-14型为主.     

协同凝集法检测大肠埃希氏菌LT毒素的研究

产毒素大肠埃希氏菌（ＥＴＥＣ）是致急性腹泻、旅游者腹泻的重要病原。该菌可产生不耐热（ＬＴ）和耐热（ＳＴ）二类肠毒素。ＳＴ因分子量小,难以制备出单（多）克隆抗体,故一般免疫学检查手段难以检出。ＬＴ分子量较大,且与霍乱毒素（ＣＴ）具有共同的抗原性,故可用...     

抗大肠埃希氏菌K88ab,K88ac和K88ad特异单克隆抗体

A panel of twelve hybridoma cell lines, secreting specific antibodies to K88 adhesin antigens of enterotoxigenic Escherichia coli (ETEC) were established from eight separate fusions between mouse myeloma cell line Sp 2/0-Ag-14 and spleen cells from mice immunized with purified K88 antigens. Among the 12 monoclonal antibodies (MCA), K-A, K-35, K-11, and K-15 were K88a specific and reacted with all K88 adhesin bearing Escherichia coli strains tested, whatever K88ab, K88ac or K88ad they might be, as shown either in enzyme-linked immunosorbent assay (ELISA) or in direct agglutination test, whereas K32, K-4, and K-3 were specific for G88ab, K88ac, and K88ad respectively. The antigen patterns of 33 K88 bearing Escherichia coli strains covering 3 serotypes of K88ab, K88ac, and K88ad were analyzed by the use of these MCAs. The preliminary results showed that all Escherichia strains with the same serotype of K88 antigen shared at least one common type-specific antigenic determinant, that K88ad and K88ac strains enjoyed one common antigenic determinant that did not exist on K88ab strains, and that there were a few K88 antigenic determinants that appeared only on limited Escherichia coli strains of the same K88 serotype.     

大肠埃希菌异质性耐药的研究进展

大肠埃希菌(Escherichia coli)是人类和动物中常见细菌病原体,E.coli已对多种抗菌药物产生耐药,且由于抗菌药物选择压力越来越大,致使菌株的适应性和致病性更强,耐药的传播更快、定植率更高,给全球流行病学和临床治疗带来极大的挑战。近年来,E.coli的异质性耐药(heteroresistance,HR)相关研究相继被报道,异质性耐药是指细菌中的同源亚群对某种抗菌药物表现出不同的敏感性,是敏感菌进化为耐药菌的中间阶段,不同于完全耐药,异质性耐药在临床上不易及时检出,常导致临床抗菌药物治疗失败。因此,为有效防控E.coli感染及耐药的演变,深入剖析E.coli异质性耐药的流行特征及可能的机制尤为重要,本文就E.coli的异质性耐药研究进行了梳理,并对异质性耐药从表型特征、检测方法和可能的机制等方面进行了概述。旨在加强对E.coli异质性耐药的认识和研究,以期为全面了解E.coli耐药过程及发生机制、优化抗菌药物使用策略、减缓完全耐药菌种的出现提供参考。     

产ESBLs大肠埃希菌、肺炎克雷伯菌的耐药特征和基因分型研究

目的了解广东省中医院产超广谱β-内酰胺酶（ESBLs）的大肠埃希菌、肺炎克雷伯菌的流行、耐药特点和基因型分布。方法对该院2001年7月至2003年8月间临床分离保存的208株大肠埃希菌和肺炎克雷伯菌,用VITEK-32细菌鉴定仪进行细菌鉴定,用K—B法进行药敏试验,用双纸片法进行ESBLs初筛,用NCCLS1999年推荐的确证方法进行ESBLs确证。并采用PCR扩增和PCR产物测序方法对产ESBLs菌株进行基因分型。结果分离到产ESBLs细菌76株,总检出率为36．5％,其中肺炎克雷伯菌阳性率为41．9％（39／93）、大肠埃希菌阳性率为32．2％（37／115）,产酶株的耐药率明显高于非产酶株,产ESBLs细菌对青霉素类、环丙沙星及头孢菌素类耐药率较高,加酶抑制剂克拉维酸或他唑巴坦后耐药率有明显下降;PCR初步分型结果表明：TEM型42株（55．3％）,均为TEM-1型,CTX-M型27株（35．5％）,SHV型33株（43．4％）。结论产ESBLs细菌具有多重耐药的特点;CTX-M型和SHV型是该院产ESBLs大肠埃希菌和肺炎克雷伯菌中流行的基因型。     

大肠埃希菌耐药性及其基因同源性分析

目的 研究临床分离的大肠埃希菌对常用抗生索的耐药性及其基因分型,了解其耐药性趋势与传播流行情况,为临床合理治疗大肠埃希菌引起的感染提供参考依据。方法 采用常规鉴定技术鉴定细菌;采用K—B纸片扩散法测定77株大肠埃希菌对19种药物的耐药性;K—B法鉴定产超广谱β-内酰胺酶（ESBLs）;通过脉冲场凝胶电泳（PFGE）法对其进行基因分型以确定菌株之间的亲缘关系;FINGERPRINT Ⅱ软件进行细菌基因指纹图谱分析。结果 大肠埃希菌对青霉素类、喹诺酮类药物和氨曲南的耐药性明显增高,亚胺培南和美罗培南是大肠埃希菌感染患者的首选药物;经ESBLs确证试验,ESBLs阳性率为28．60％（22／77）;产ESBLs大肠埃希菌经PFGE指纹图谱分析,除第62株和第70株相似性系数为78．27％外,其余相似度均低于70．0％;ESBLs大肠埃希菌阴性株中除少数几对菌株相似性系数较高外,其余呈散在分布,且电泳带存有6条以上的不同条带,为流行病学无关的不同克隆。结论 大肠埃希菌对常用抗生索耐药性明显增高,且呈多重耐药趋势;该研究尚不能证明存在大肠埃希菌爆发性流行感染,提示可能存在院内感染大肠埃希菌的优势克隆;PFGE基因分型方法是耐药性与流行状况分析的有效手段。     

Comparison of five rep-PCR genomic fingerprinting methods for differentiation of fecal Escherichia coli from humans, poultry and wild birds

The development of a methodology to identify the origin of fecal pollution is important both for assessing the degree of risk posed to public health and for developing strategies to mitigate the environmental loading of pathogens associated with waterborne disease transmission. Five rep-PCR genomic fingerprinting methods, such as rep-PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR, ERIC2-PCR, BOX-PCR and (GTG)(5)-PCR, were assessed for their potential in differentiation of 232 fecal Escherichia coli isolates obtained from humans, poultry (chicken, duck and turkey) and wild birds (Canada goose and gull). Based on the results of cluster analysis and discriminant function analysis, (GTG)(5)-PCR was found to be the most suitable method for molecular typing of fecal E. coli, followed by BOX-PCR, REP-PCR, ERIC-PCR and ERIC2-PCR. A discriminant function analysis of (GTG)(5)-PCR fingerprints showed that 94.1%, 79.8%, 80.5%, 74.4%, 86.7% and 88.6% of turkey, chicken, duck, Canada goose, gull and human E. coli isolates were classified into the correct host group, respectively. Subsequently, (GTG)(5)-PCR was tested for its ability to track the origin of 113 environmental E. coli isolated from natural pond water. In conclusion, the (GTG)(5)-PCR genomic fingerprinting method can be considered as a promising genotypic tool for epidemiological surveillance of fecal pollution in aquatic environments.     

大肠杆菌生产异戊二烯的代谢工程研究进展

异戊二烯作为一种重要的化工原料,主要用于合成橡胶。此外,还广泛应用于医药或化工中间体、食品、粘合剂及航空燃料等领域。利用微生物法生产异戊二烯因具有环境友好、利用廉价的可再生原料、可持续发展等优势而成为当今研究的热点。这里介绍了大肠杆菌生产异戊二烯的代谢途径及关键酶,从代谢工程的角度出发综述了目前为提高大肠杆菌异戊二烯产量所应用到的方法和策略,并对今后的发展方向进行了展望。     

Biofilm formation by avian Escherichia coli in relation to media, source and phylogeny

AIMS: To assess the abilities of 105 avian pathogenic Escherichia coli (APEC) and 103 avian faecal commensal E. coli (AFEC) to form biofilms on a plastic surface and to investigate the possible association of biofilm formation with the phylotype of these isolates. METHODS AND RESULTS: Biofilm production was assessed in 96-well microtitre plates using three different media, namely, M63 minimal medium supplemented with glucose and casamino acids, brain-heart infusion broth, and diluted tryptic soy broth. Avian E. coli are highly variable in their ability to form biofilms. In fact, no strain produced a strong biofilm in all three types of media; however, most (75.7% AFEC and 55.2% APEC) were able to form a moderate or strong biofilm in at least one medium. Biofilm formation in APEC seems to be mostly limited to nutrient deplete media; whereas, AFEC are able to form biofilms in both nutrient deplete and replete media. Also, biofilm formation in E. coli from phylogenetic groups B2, D and B1 was induced by nutrient deplete conditions; whereas, biofilm formation by members of phylogenetic group A was strongest in a rich medium. CONCLUSIONS: Biofilm formation by APEC and phylotypes B2, D and B1 is induced by nutrient deplete conditions, while AFEC are able to form biofilms in both nutrient rich and deplete media. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to investigate biofilm formation by a large sample of avian E. coli isolates, and it provides insight into the conditions that induce biofilm formation in relation to the source (APEC or AFEC) and phylogenetic group (A, B1, B2 and D) of an isolate.     

Atypical enteropathogenic Escherichia coli genomic background allows the acquisition of non-EPEC virulence factors

Atypical enteropathogenic Escherichia coli (aEPEC) has been associated with infantile diarrhea in many countries. The clonal structure of aEPEC is the object of active investigation but few works have dealt with its genetic relationship with other diarrheagenic E. coli (DEC). This study aimed to evaluate the genetic relationship of aEPEC with other DEC pathotypes. The phylogenetic relationships of DEC strains were evaluated by multilocus sequence typing. Genetic diversity was assessed by pulsed-field gel electrophoresis (PFGE). The phylogram showed that aEPEC strains were distributed in four major phylogenetic groups (A, B1, B2 and D). Cluster I (group B1) contains the majority of the strains and other pathotypes [enteroaggregative, enterotoxigenic and enterohemorrhagic E. coli (EHEC)]; cluster II (group A) also contains enteroaggregative and diffusely adherent E. coli ; cluster III (group B2) has atypical and typical EPEC possessing H6 or H34 antigen; and cluster IV (group D) contains aEPEC O55:H7 strains and EHEC O157:H7 strains. PFGE analysis confirmed that these strains encompass a great genetic diversity. These results indicate that aEPEC clonal groups have a particular genomic background – especially the strains of phylogenetic group B1 – that probably made possible the acquisition and expression of virulence factors derived from non-EPEC pathotypes.     

Development of a 5'-nuclease PCR assay for the identification of Escherichia coli strains expressing the flagellar antigen H21 and their detection in food after enrichment

Aims: To develop a real‐time PCR assay targeting the Escherichia coli flagellar antigen H21 for identification and surveillance of clinically important Shiga toxin‐producing E. coli (STEC) serotypes classified in seropathotype C. Methods and Results: The fliC allele of STEC O91:H21 strain B2F1 was amplified and sequenced. The nucleotide sequence obtained was compared with fliC genes of E. coli O157:H21, O8:H21 and O113:H21 strains. A pair of oligonucleotide primers and a TaqMan^® minor groove binder probe specific for fliC‐H21 were designed and used in a 5′‐nuclease PCR assay. This method was evaluated using a panel of 138 diverse bacterial strains and was shown to be 100% specific for H21. PCR amplification of fliC‐H21 from one cell per reaction mixture was possible, and an initial inoculum of 10 STEC H21 colony‐forming units per 25 g of ground beef was detected after overnight enrichment. Conclusions: The PCR assay developed was found to be highly sensitive and specific for the identification and detection of E. coli H21 strains in ground beef. Significance and Impact of the Study: The real‐time PCR assay targeting the H21 flagellar antigen described here offers a valuable method for the rapid detection and molecular typing of pathogenic STEC H21 strains in food.     

多杀性巴氏杆菌分子分型方法简述

多杀性巴氏杆菌是一种能感染多种动物甚至是人的重要革兰氏阴性病原菌。目前临床上用于多杀性巴氏杆菌诊断的分型方法主要包括血清学分型方法和分子分型方法。其中血清学分型方法主要基于免疫学实验技术建立,操作过程繁琐,技术要求高,工作量大,不适用于临床上大规模快速开展多杀性巴氏杆菌流行病学调查的需要;而基于分子生物学手段建立的分子分型方法相对于传统的血清学分型方法而言具有快速、简单、灵敏、灵活等特点,特别是某些分子分型方法与传统的分型方法形成了较为精确的对应关系,因而在临床上得到了广泛的应用。目前适用于临床上开展多杀性巴氏杆菌分离鉴定的分子分型方法主要包括多重PCR方法及多位点序列分型法(MLST),其中多重PCR方法又包括基于荚膜编码区及脂多糖外核编码簇建立的PCR方法。本文将重点就这3种常用的多杀性巴氏杆菌分子分型方法进行综述,介绍其建立原理、实现手段以及各自的优缺点,为临床上开展多杀性巴氏杆菌的流行病学调查特别是分子流行病学调查提供参考。     

Identification of Escherichia coli O172 O-antigen gene cluster and development of a serogroup-specific PCR assay

AIM: To characterize the locus for O-antigen biosynthesis from Escherichia coli O172 type strain and to develop a rapid, specific and sensitive PCR-based method for identification and detection of E. coli O172. METHODS AND RESULTS: DNA of O-antigen gene cluster of E. coli O172 was amplified by long-range PCR method using primers based on housekeeping genes galF and gnd Shot gun bank was constructed and high quality sequencing was performed. The putative genes for synthesis of UDP-FucNAc, O-unit flippase, O-antigen polymerase and glycosyltransferases were assigned by the homology search. The evolutionary relationship between O-antigen gene clusters of E. coli O172 and E. coli O26 is shown by sequence comparison. Genes specific to E. coli O172 strains were identified by PCR assays using primers based on genes for O-unit flippase, O-antigen polymerase and glycosyltransferases. The specificity of PCR assays was tested using all E. coli and Shigella O-antigen type strains, as well as 24 clinical E. coli isolates. The sensitivity of PCR assays was determined, and the detection limits were 1 pg microl(-1) chromosomal DNA, 0.2 CFU g(-1) pork and 0.2 CFU ml(-1) water. The total time required from beginning to end of the procedure was within 16 h. CONCLUSION: The O-antigen gene cluster of E. coli O172 was identified and PCR assays based on O-antigen specific genes showed high specificity and sensitivity. SIGNIFICANCE AND IMPACT OF THE STUDY: An O-antigen gene cluster was identified by sequencing. The specific genes were determined for E. coli O172. The sensitivity of O-antigen specific PCR assay was tested. Although Shiga toxin-producing O172 strains were not yet isolated from clinical specimens, they may emerge as pathogens.