粪便中大肠埃希菌的分离鉴定

2004年4月采集大连金州湾沿岸畜禽养殖场猪、牛、鸡和某中学人粪便样品。经改良的生化鉴定方法鉴定,得大肠埃希菌(Escherichia coli)猪源75株、牛源78株、鸡源69株、人源68株,占粪大肠菌比率分别为85.23%、92.86%、80.23%、80.00%。为验证改良的生化鉴定方法准确性,从中随机选取8株E.coli,扩增16S rRNA序列并与Genebank中E.coli16S序列比对,确定这8株细菌与E.coli同源相似率达99%以上,进而验证改良的生化鉴定方法的可行性。     

泌尿系统感染高龄患者大肠埃希菌耐药性分析

目的:探究高龄患者泌尿系统感染大肠埃希菌对常用药物的耐药性,指导临床医生合理用药。方法:按照标准操作规程采集我院泌尿系统感染高龄患者的尿液,作常规尿标本培养和分离,应用微生物分析仪进行细菌鉴定,采用纸片扩散法进行药物敏感试验,采用纸片扩散法和双纸片确证法完成产超广谱β-内酰胺酶菌株的检测。结果:129株大肠埃希菌中,检测出产超广谱β-内酰胺酶菌株有62株,占48.1%;在17种常用抗生素敏感试验中,亚胺培南敏感性最高(100%),无耐药株出现,对第一、二、三和四代头孢类抗生素均出现了不同程度的耐药性,对氨苄西林、四环素和哌拉西林高度耐药(均超过了95%)。结论:泌尿系统感染高龄患者大肠埃希菌对临床常用抗生素耐药性逐年升高,且产超广谱β-内酰胺酶菌株也逐渐增多,这就要求临床医生严格合理应用抗生素,尽量避免耐药菌株的出现。     

非致病性大肠埃希菌能增多大肠埃希菌O157:H7志贺毒素的产生

大肠埃希菌(简称大肠杆菌)O157：H7毒力因子为志贺毒素2(stx2),其基因由温和噬菌体编码,由晚期基因启动子调控表达。stx2的合成与释放需要诱导噬菌体溶菌周期,而且正常肠道大肠杆菌感染了毒素编码的噬菌体就能制造毒素和噬菌体,使毒素水平远远超过病原性菌株本身的产量,作者在体外以及鼠肠道验证了这一假设。     

高赖氨酸蛋白基因在大肠埃希菌中的表达

从四棱豆中克隆高赖氨酸蛋白基因wblys,通过PCR扩增wblys片段,转入原核表达载体pGEX-4T-1,构建pGEX-4T-1/wblys大肠埃希菌工程菌,表达重组蛋白,IPTG诱导后,发现细菌全蛋白在44 ku(含GST标签)处多出1条明显条带。HPLC检测赖氨酸含量。诱导后菌体总赖氨酸含量比正常菌体提高15.84 mg/g。在大肠埃希菌中高效表达植物源高赖氨酸蛋白基因,为该基因在益生菌中表达提供研究工作基础。     

儿童泌尿系感染大肠埃希菌的耐药性分析

目的 了解大肠埃希菌在儿童中的感染情况及其耐药性.方法 对中段尿标本进行分离培养及鉴定,药敏试验用K-B法,检测ESBLs用表型确证试验,药敏结果分析用WHONET 5.5软件,统计分析采用x2检验.结果 2009年1月至2011年12月从儿科门诊和病房送检的1755份中段尿标本中共检出85株大肠埃希菌,检出率为4.8％,男女比为6:11; ＜3岁的婴幼儿组57例,占67.1％;3～6岁育龄前儿童17例,占20.0％;＞6岁儿童11例,占12.9％;产ESBLs的大肠埃希菌占60.0％;85株大肠埃希菌对亚胺培南和美罗培南全部敏感,对头孢哌酮/舒巴坦、哌拉西林/他唑巴坦和阿米卡星的敏感率均为90％以上,产ESBLs的大肠埃希菌对氨苄西林、头孢唑啉、头孢他啶、头孢曲松、头孢吡肟、头孢西丁、头孢呋新酯、左旋氧氟沙星和罗红霉素的耐药率较非产ESBLs菌株的耐药率高,P值均＜0.05,差异有统计学意义.结论 儿童泌尿系感染大肠埃希菌,可以选用的抗菌素已极为有限,临床要谨慎用药,并及时根据药敏结果修正药物种类.     

大肠埃希菌α溶血素的研究进展

大肠埃希菌α溶血素是RTX毒素家族的典型代表,也是大肠埃希菌肠外感染的重要毒力因子之一。详细介绍了大肠埃希菌α溶血素的成熟、分泌、溶血机理及其生物学效应,这将对大肠埃希菌肠外感染的研究具有重要意义,同时对原核细胞与真核细胞之间的相互作用以及其他细菌溶血素致病机制研究有借鉴作用。     

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。     

大肠埃希菌蛋白指纹图谱的建立

目的建立大肠埃希菌（Escherichia coli,E．coli）蛋白指纹图谱,为Ecoli感染快速诊断奠定基础。方法收集临床分离E.coli88株,提取细菌DNA,PCR检测Ecoli 16S rRNA。蛋白提取液提取细菌蛋白,干化学法测蛋白浓度,应用表面增强激光解析电离飞行时间质谱技术（SELDI-TOF-MS）检测Ecoli蛋白,采用Ciphergen Pro-teinchip软件自动采集数据。重复测定20次Ecoli混合标本,评价SELDI检测Ecoli蛋白分子量的重复性。结果E．coil标准菌株ATCC 25922和临床分离株均可检出16S rRNA。AU芯片能捕获近30个E．coli蛋白峰,其中19个蛋白峰构成E．coli特征性蛋白指纹图谱,各蛋白峰在临床分离E．coli间分子量变异系数≤0．2％。SELDI重复检测20次E．coli混合标本显示同一蛋白峰的分子量变异系数≤0．05％。结论E．coli在分子量3～20kD范围内具有特征性蛋白指纹图谱,为快速诊断E.coli感染提供了新思路。     

肠出血性大肠埃希菌产生的Vero毒素

本文简要述及Ｖｅｒｏ毒素的分子结构,生物学活性,作用机理,毒素分子中氨基酸置换对毒素活性的影响和Ｖｅｒｏ毒素及其基因的检测。     

Systematic high-yield production of human secreted proteins in Escherichia coli

Human secreted proteins play a very important role in signal transduction. In order to study all potential secreted proteins identified from the human genome sequence, systematic production of large amounts of biologically active secreted proteins is a prerequisite. We selected 25 novel genes as a trial case for establishing a reliable expression system to produce active human secreted proteins in Escherichia coli. Expression of proteins with or without signal peptides was examined and compared in E. coli strains. The results indicated that deletion of signal peptides, to a certain extent, can improve the expression of these proteins and their solubilities. More importantly, under expression conditions such as induction temperature, N-terminus fusion peptides need to be optimized in order to express adequate amounts of soluble proteins. These recombinant proteins were characterized as well-folded proteins. This system enables us to rapidly obtain soluble and highly purified human secreted proteins for further functional studies.     

嗜酸氧化亚铁硫杆菌基因组分泌蛋白的初步分析

利用信号肽预测软件SignalP v3.0、跨膜螺旋结构预测软件TMHMM v2.0和非经典分泌蛋白预测软件SecretomeP对嗜酸氧化亚铁硫杆菌全基因组的3 218个氨基酸序列进行预测分析.结果表明在嗜酸氧化亚铁硫杆菌中有507个蛋白为分泌蛋白,其中分泌型信号肽120个(其中有9个为RR-motif亚组型信号肽),脂蛋白信号肽3个,Prepilin-like信号肽4个,非经典分泌蛋白380个.并对分泌型信号肽的长度分布、氨基酸使用频率和酶切位点的氨基酸使用频率作了统计.得分最高的100个非经典分泌蛋白中,有36个具有功能分类,主要是参与细胞壁、能量代谢及转运和结合的蛋白质.嗜酸氧化亚铁硫杆菌的这507个分泌蛋白所参与的生化过程可能发生在膜外的周质空间或是菌体外的场所,为该物种与矿物相互作用,以及对环境做出响应服务.     

抗菌肽GK1在大肠杆菌中的融合表达

为高效表达抗菌肽GK1并避免GK1的高抗菌活性对大肠杆菌宿主菌的致命影响, 将经改造后的人胰岛素原(mhPI)与GK1的融合基因(mhPI-GK1)克隆到表达载体pET28a中, 构建出表达质粒pET28a-mhPI-GK1, 转化至大肠杆菌BL21(DE3)中进行表达。融合蛋白在大肠杆菌中以包涵体形式表达, 表达量占菌体总蛋白的20%。经CNBr裂解、阳离子交换层析和RP-HPLC纯化后, 每升发酵液可获得5.7 mg纯度大于97%的重组GK1。质谱检测显示重组GK1的分子量为2794.0 D, 抑菌活性实验表明纯化后的重组GK1和化学合成GK1具有相同的抗菌活性。为利用基因工程方法大规模生产GK1奠定了基础。     

Genome-wide identification of the subcellular localization of the Escherichia coli B proteome using experimental and computational methods

Escherichia coli K-12 and B strains have most widely been employed for scientific studies as well as industrial applications. Recently, the complete genome sequences of two representative descendants of E. coli B strains, REL606 and BL21(DE3), have been determined. Here, we report the subproteome reference maps of E. coli B REL606 by analyzing cytoplasmic, periplasmic, inner and outer membrane, and extracellular proteomes based on the genome information using experimental and computational approaches. Among the total of 3487 spots, 651 proteins including 410 non-redundant proteins were identified and characterized by 2-DE and LC-MS/MS; they include 440 cytoplasmic, 45 periplasmic, 50 inner membrane, 61 outer membrane, and 55 extracellular proteins. In addition, subcellular localizations of all 4205 ORFs of E. coli B were predicted by combined computational prediction methods. The subcellular localizations of 1812 (43.09%) proteins of currently unknown function were newly assigned. The results of computational prediction were also compared with the experimental results, showing that overall precision and recall were 92.16 and 92.16%, respectively. This work represents the most comprehensive analyses of the subproteomes of E. coli B, and will be useful as a reference for proteome profiling studies under various conditions. The complete proteome data are available online (http://ecolib.kaist.ac.kr).     

羊布鲁氏菌的分泌蛋白质组分析

分泌蛋白是指那些分泌到细胞外的蛋白质。布鲁氏菌的分泌蛋白可能介导了病原与宿主之间的相互作用, 在布鲁氏菌的毒力方面发挥一定的作用, 但是研究方法的局限性限制了分泌蛋白的研究。本文报道了利用蛋白质组的方法来寻找羊布鲁氏菌的分泌蛋白。首先用TCA-丙酮法提取布鲁氏菌培养上清中的分泌蛋白, 双向电泳进行分离, 然后用质谱来鉴定这些蛋白, 最终鉴定到40种蛋白。通过生物信息学分析, 发现这些蛋白主要是ABC转运系统的底物结合蛋白、外膜蛋白和热休克蛋白。这些蛋白的识别不仅有助于对布鲁氏菌致病机制的理解, 而且也可为     

禾谷镰刀菌基因组中含寄主靶向模体分泌蛋白功能的初步分析

基于生物信息学的方法,以SignalP v3.0、TargetP v1.01、Big-PI Predictor和TMHMM v2.0四个分析软件对禾谷镰刀菌(Fusarium graminearum Schw.)全基因组的11 640个蛋白编码基因的N-端氨基酸序列进行信号肽分析,预测出606个潜在的分泌蛋白编码基因.通过对这些潜在的分泌蛋白进行MEME软件分析,发现其中有157个分泌蛋白的剪切点下游120氨基酸残基范围内具有一个保守的RXLX模体,其中有79个分泌蛋白具有可预测的功能性描述,包括FG00023.1具有与草酸盐氧化酶1有关的功能,FG01588.1具有与1,4-α-葡糖苷酶葡聚糖有关的功能,这些基因可作为禾谷镰刀菌致病相关的候选基因.其中FG04097.1编码的具有与丝氨酸型蛋白酶有关的功能在致病疫霉和疟原虫真核寄生物中具有相似保守的寄主靶标模体的效应蛋白中也观察到,但FG09127.1,FG05287.1编码的蛋白尚未被证明参与致病过程的研究报道.深入研究分泌蛋白将有助于明确植物与病原微生物互作的分子机制.利用禾谷镰刀菌基因组学研究成果,结合计算机技术和生物信息学的方法,分析其分泌蛋白组学,将有助于全面掌握其致病因子的结构与功能.对于这些蛋白功能的比较研究,将有利于对禾谷镰刀菌致病性的分子机制的探索,并为设计新的防治措施提供理论依据.     

Prediction of protein solubility in Escherichia coli using logistic regression

In this article we present a new and more accurate model for the prediction of the solubility of proteins overexpressed in the bacterium Escherichia coli. The model uses the statistical technique of logistic regression. To build this model, 32 parameters that could potentially correlate well with solubility were used. In addition, the protein database was expanded compared to those used previously. We tested several different implementations of logistic regression with varied results. The best implementation, which is the one we report, exhibits excellent overall prediction accuracies: 94% for the model and 87% by cross‐validation. For comparison, we also tested discriminant analysis using the same parameters, and we obtained a less accurate prediction (69% cross‐validation accuracy for the stepwise forward plus interactions model). Biotechnol. Bioeng. 2010; 105: 374–383. © 2009 Wiley Periodicals, Inc.     

Fimbriation, surface hydrophobicity and serum resistance in uropathogenic strains of Escherichia coli

Abstract A total of 80 Escherichia coli strains were examined for expression of P-fimbriae, mannose-sensitive haemagglutination (MSHA) and mannose-resistant haemagglutination (MRHA) of human group A erythrocytes and guinea pig erthrocytes, cell surface hydrophobicity and resistance to serum bactericidal activity. Isolates were obtained from urine of children and adults, either with acute pyelonephritis ( n = 15 and n = 12) or lower urinary tract infection (UTI) ( n = 30 and n = 23, respectively). Results obtained showed that, in E. coli strains isolated both from children and adults with lower UTI, significant differences were not found concerning the incidence of P-fimbriae, cell surface hydrophobicity and serum resistance. In pyelonephritogenic E. coli isolated from children and adults, the incidence of P-fimbriae and cell surface hydrophobicity was associated more frequently with the former (87% vs. 42% and 100% vs. 67%, P < 0.05), while serum resistance was associated with the latter (47% vs. 67%, P < 0.05).     

Penicillin-binding proteins of pathogenic Escherichia coli strains

The penicillin-binding proteins of 11 pathogenic Escherichia coli strains, including enteropathogenic, enterotoxigenic, enteroinvasive, enteroaggregative, and enterohemorrhagic E. coli, were detected in gels following the labeling of isolated cell envelopes with [³H]benzylpenicillin. The electrophoretic profiles, sensitivities to and morphological changes induced by β-lactam antibiotics showed that the penicillin-binding proteins of most pathogenic E. coli possess structural and physiological functions similar to those of E. coli K12.     

Shiga toxin-producing Escherichia coli and enteropathogenic Escherichia coli in healthy goats in India: occurrence and virulence properties

AIMS: To describe the occurrence and virulence gene pattern of shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) in healthy goats of Jammu and Kashmir, India. METHODS AND RESULTS: A total of 220 E. coli strains belonging to 60 different 'O' serogroups was isolated from 206 local (nonmigratory) and 69 migratory goats. All the 220 strains were screened for the presence of stx(1), stx(2), eaeA and hlyA genes. Twenty-eight E. coli (75.6%) strains from local and nine (24.3%) strains from migratory goats belonging to 18 different serogroups showed at least presence of one virulence gene studied. Twenty-eight strains (16.47%) (belonging to 13 different serogroups) from local goats carried stx(1) gene alone or in combination with stx(2) gene, while as only one strain (2%) from migratory goats possessed stx(2) gene alone. Interestingly in the present study none of the STEC strains carried eaeA gene. Similarly, none of the strains from local goats possessed eaeA and none of the migratory goats possessed stx(1) gene. Eight strains (16%) (belonging to four different serogroups) from migratory goats carried eaeA gene. Twenty-five (14.7%) and seven (14%) strains from local and migratory goats harboured hlyA gene respectively. CONCLUSIONS: Healthy goats of Jammu and Kashmir state serve as a reservoir of STEC and EPEC. Further studies in this direction are needed to work out whether or not they are transmitted to humans in this part of world. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report of isolation of STEC and EPEC strains from healthy goats in Jammu and Kashmir State of India, which could be a source of infection to humans.