产肠毒素大肠杆菌诱导肠上皮细胞凋亡的研究进展

产肠毒素大肠杆菌(enterotoxigenic Escherichia coli, ETEC)是引起人和动物腹泻的重要病原菌之一,其中黏附素和肠毒素是其感染引起腹泻的主要毒力因子。首先,黏附素介导ETEC与宿主小肠上皮细胞的黏附和定殖。随后,定殖的细菌产生肠毒素,导致水、电解质代谢紊乱,最终引起水样腹泻。传统的观点认为ETEC属于非侵袭性大肠杆菌,并不会引起肠上皮细胞凋亡和破坏肠道的屏障结构。但是越来越多的研究证据表明,在体外和体内ETEC感染均可诱导肠上皮细胞凋亡,破坏宿主肠黏膜屏障的完整性,促进疾病发展。本文将就ETEC不同毒力因子诱导细胞凋亡的具体机制、细胞凋亡与疾病发展的相关性以及在临床如何利用抗凋亡治疗预防ETEC感染等方面进行综述,旨为进一步深入阐明ETEC的分子致病机制提供参考,为防治ETEC引起的腹泻提供新策略。     

肠出血性大肠杆菌Ⅲ型分泌系统效应因子与宿主细胞相互作用研究进展

肠出血性大肠杆菌(Enterohemorrhagic Escherichia coli,EHEC)通过其Ⅲ型分泌系统将效应因子注入到宿主细胞内,破坏宿主细胞内的多种信号通路从而有利于细菌的感染及定植。近年来对于EHEC Ⅲ型分泌系统效应因子与宿主细胞相互作用研究成为EHEC致病机制研究新的热点,研究表明,除了经典的效应因子外,一些新发现的效应因子在细菌的致病过程中也发挥着重要作用,有些效应因子能够抑制宿主细胞内正常的信号通路,有些效应因子还具有抑制细胞凋亡,干扰炎症信号通路和抑制吞噬的作用。这些发现揭示了EHEC效应因子具有多种功能,它们通过与宿主细胞间的相互作用,在细菌的感染过程中发挥着重要作用。     

053肠聚集性大肠埃希菌的聚集黏附性菌毛引起肠上皮细胞的炎性反应

肠聚集性大肠埃希菌（大肠杆菌）是导致腹泻的病原菌，在发展中国家儿童中常引起水样腹泻。最近，有学者认为肠聚集性大肠杆菌是引起旅行者腹泻的最主要原因。感染肠聚集性大肠杆菌后，最典型的症状是黏性水样便，同时伴有发热、恶心、呕吐等。肠聚集性大肠杆菌的发病机制包括3步：①黏附到肠黏膜；②诱导黏液生物膜的产生与沉积；③炎症反应和细胞因子的释放。肠聚集性大肠杆菌黏附在人的肠黏膜上需要聚集黏附性菌毛（AAf），     

病毒感染引起的炎症反应：宿主对抗病毒的“双刃剑”

先天性免疫系统作为宿主抵抗外来病原入侵的第一道防线,也是最迅速的防御系统。宿主先天性免疫系统中的模式识别受体识别入侵信号并激活炎症信号通路,诱导产生大量促炎性细胞因子,引起炎症反应。病毒感染是激活炎症反应的条件之一,诱导机体产生强烈的免疫应答,强大的炎症反应调控网络在宿主抗病毒过程中发挥关键作用,以维持机体的平衡。本文综述了病毒感染引起的炎症反应,重点介绍了宿主对炎症反应的调控网络,以及DNA和RNA病毒对炎症反应的调节机制,为病毒感染引起的免疫性疾病的治疗提供参考。     

肠小体在猪肠道冠状病毒感染中的研究进展

猪肠道冠状病毒是引起仔猪腹泻的重要致病因素,主要感染小肠绒毛上皮细胞,给养猪业造成了巨大的经济损失。由于缺乏能模拟胃肠道高度复杂生理特性的体外研究模型,猪肠道冠状病毒感染与宿主肠上皮之间相互作用的研究也受到了极大的限制。随着干细胞技术的快速发展,一种能模拟肠道复杂的细胞类型及空间结构的体外模型——肠小体引起了人们的广泛关注。与传统的细胞系相比,肠小体不仅能模拟肠的结构和功能,同时还保留宿主的遗传特性,有望成为研究宿主-肠道病原相互作用的一种理想模型。本文就猪肠道冠状病毒以及肠小体在肠道病原研究中的应用进行综述,以期为猪肠道冠状病毒的基础研究提供新的思路与见解。     

新腹泻原性大肠杆菌的致病机理

<正> 以往的研究将肠道病原性大肠杆菌分成三类。一类是主要引起婴幼儿流行性腹泻的致病性大肠杆菌(BPEC);另一类是引起旅游者腹泻和发展中国家婴幼儿腹泻的产毒性大肠杆菌(ETEC);再一类是导致痢疾等疾病的侵袭性大肠杆菌(EIEC)。关于ETEC和EIEC的致病机理研究的比较清楚,ETEC中要产生LT和ST肠毒素,EIEC象志贺氏菌一样能侵入肠上皮细胞并繁殖。EPEC的致病性可能与某种粘附因子和细胞毒素产生有关。近来人们又提出两类新的腹泻性大肠杆菌,即肠出血性大肠杆菌(En-     

肠道病原菌III型分泌系统效应蛋白调控宿主细胞NF-κB和MAPK信号通路的研究进展

病原细菌感染对人类健康构成了严重的威胁,一类具有III型分泌系统(Type III secretion system,T3SS)的肠道致病细菌可以通过T3SS将效应蛋白“注射”到宿主细胞中,模拟和操纵宿主细胞的多种信号转导通路,包括细胞凋亡、细胞自噬和炎症反应等,从而有效地逃逸宿主的防御,增强感染性和致病性。本文综述了肠道病原菌T3SS效应蛋白在调控宿主炎症反应中NF-κB和MAPK通路的最新研究进展。     

应用斑点ELISA检测产肠毒素性大肠杆菌定居因子抗原

产肠毒素性大肠杆菌(ETEC)在发展中国家是引起婴幼儿和旅游者腹泻的最主要病原菌。据WHO预测,全世界每年因ETEC引起腹泻而致死的5岁以下婴幼儿多达80万。该菌的致病机制是,它能粘附并定居到宿主小肠表皮细胞,繁殖产生耐热肠毒素或不耐热肠毒素,前者主要由定居因子抗原(CFA)负责,     

结核分枝杆菌感染实验模型

结核分枝杆菌是引起人结核病的主要病原,全世界约有1/3人口感染结核分枝杆菌。尽管该病原可感染并引起许多动物疾病,但人类是其中心宿主。为研究结核分枝杆菌的致病机理及宿主对本病原的保护性和免疫病理学反应,选择合适的动物模型非常必要。本文阐述了结核病研究中常用的实验模型及各种模型的优缺点。实验模型的合理应用将促进我们对结核病的认识,从中获取的资料将有助于我们发现更好的预防和治疗方案。     

致犊牛脑膜炎大肠杆菌新疆分离株ibeB基因的特性分析北大核心CSCD

【目的】分析致犊牛脑膜炎大肠杆菌分离株ibeB基因的分子生物学信息。【方法】以自脑炎死亡犊牛脑组织、肝组织分离鉴定的O161-K99-STa致病性大肠杆菌牛-EN株和牛-EG分离株为材料。根据GenBank中公布的脑膜炎大肠杆菌K1株RS218 ibeB基因序列设计1对引物,采用PCR方法,从分离株中成功克隆ibe B基因,比较分离株ibeB基因与不同来源大肠杆菌ibeB基因的部分生物信息学特性。【结果】分离株ibeB基因序列全长1500 bp,包含1371 bp开放阅读框,共编码457个氨基酸;生物信息学分析显示,牛-EN株与致人脑膜炎大肠杆菌K1 RS218的核苷酸和氨基酸同源性分别为90.5%和96.9%,牛-EG株与大肠杆菌K12的核苷酸和氨基酸同源性分别为99.4%和100.0%;ibeB蛋白为亲水性蛋白,分子质量为50.26 kDa,理论等电点为6.05;该蛋白无跨膜区,但具有信号肽序列;亚细胞定位显示,分泌信号通路位点(SP)占比例为0.939,说明该蛋白属于分泌型蛋白。【结论】从致脑膜炎大肠杆菌分离株中成功克隆ibeB基因,该基因与致人脑膜炎大肠杆菌K1 RS218 ibeB基因有较高的同源性,均有相似的生物学特性,属肠外致病性大肠杆菌。     

Biosafety evaluation of bacteriophages for treatment of diarrhea due to intestinal pathogen Escherichia coli 3-2 infection of chickens

Intestinal Escherichia coli caused diarrhea in chicken makes serious damage directly to the chicken culture industry. Bacteriophage therapy is able to control the diarrhea in chickens effectively. In this study, the biosafety of bacteriophages was evaluated for treating intestinal pathogenic E. coli, which induced diarrhea in chickens. Ten bacteriophages were isolated from feces of chickens with diarrhea using the ill-chicken intestinal pathogenic E. coli 3-2 as target organism. Three bacteriophages propagated on E. coli 3-2 with relative big and clear plaques were selected and used together for toxicity experiment and evaluating the effect of therapy on chicken weight gain. In 3 weeks of trial, no mice given with or without mixed bacteriophages died, and the weight of mice of the experimental group did not show significant difference to the control group after 3 weeks infection. Besides remarkable decreasing the death rate of chickens with diarrhea, treatment of mixed bacteriophages also promoted the weight gain and saved the diet consumption as the utilize rate of diet increased 11% compared with the control group. These observations indicated that a mixture of three bacteriophages would be biosafe for rapid and effective preventing pathogenic E. coli infections.     

First isolation and further characterization of enteropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> (EPEC) O157:H45 strains from cattle

Background  

Enteropathogenic Escherichia coli (EPEC), mainly causing infantile diarrhoea, represents one of at least six different categories of diarrheagenic E. coli with corresponding distinct pathogenic schemes. The mechanism of EPEC pathogenesis is based on the ability to introduce the attaching-and-effacing (A/E) lesions and intimate adherence of bacteria to the intestinal epithelium. The role and the epidemiology of non-traditional enteropathogenic E. coli serogroup strains are not well established. E. coli O157:H45 EPEC strains, however, are described in association with enterocolitis and sporadic diarrhea in human. Moreover, a large outbreak associated with E. coli O157:H45 EPEC was reported in Japan in 1998. During a previous study on the prevalence of E. coli O157 in healthy cattle in Switzerland, E. coli O157:H45 strains originating from 6 fattening cattle and 5 cows were isolated. In this study, phenotypic and genotypic characteristics of these strains are described. Various virulence factors (stx, eae, ehxA, astA, EAF plasmid, bfp) of different categories of pathogenic E. coli were screened by different PCR systems. Moreover, the capability of the strains to adhere to cells was tested on tissue culture cells.     

Inactivation and injury of Escherichia coli O157:H7 and Staphylococcus aureus by pulsed electric fields

Two pathogenic microorganisms Escherichia coli O157:H7 and Staphylococcus aureus, suspended in peptone solution (0.1% w/v) were treated with 12, 14, 16 and 20 kV/cm electric field strengths with different pulse numbers up to 60 pulses. Pulsed electric field (PEF) treatment at 20 kV/cm with 60 pulses provided nearly 2 log reduction in viable cell counts of E. coli O157:H7 and S. aureus. S. aureus cells were slightly more resistant than E.coli O157:H7 cells. The results related to the effect of initial cell concentration of E. coli O157:H7 on the PEF inactivation showed that more inactivation was obtained by decreasing initial cell concentration. Any possible injury by PEF was also investigated after applying 20 kV/cm electric field to the microorganisms. As a result, it was determined that there was 35.92 to 43.36% injury in E. coli O157:H7 cells, and 17.26 to 30.86% injury in S. aureus cells depending on pulse number. The inactivation results were also described by a kinetic model.     

通辽地区犊牛腹泻大肠杆菌耐药性检测及一株多重耐药菌全基因组测序分析

【背景】大肠杆菌(Escherichia coli)是引起犊牛腹泻的最主要病原菌,其耐药性菌株的不断出现引起广泛关注。【目的】了解内蒙古自治区通辽市犊牛腹泻大肠杆菌耐药性及耐药基因流行情况。【方法】从通辽市多个旗县采集犊牛腹泻样品40份,经细菌分离纯化及16S rRNA基因测序,最终鉴定出20株大肠杆菌。采用药敏试验和PCR方法对分离菌进行耐药性及耐药基因检测分析,并对其中1株多重耐药菌株进行全基因组测序。【结果】20株分离菌均具有多重耐药性,对链霉素、环丙沙星、恩诺沙星和复方新诺明的耐药率达80%以上。所检耐药基因中,aphA1、strB、TEM-1和qnrS检出率达100%。通过对代表性菌株TL-13全基因组测序发现,其基因组大小为4897185bp,GC含量为50.68%,同时携带2个质粒,大小分别为108288bp(pTL13-1)和64018bp(pTL13-2)。质粒中共携带18个可移动耐药基因。【结论】通辽地区犊牛腹泻大肠杆菌多重耐药性普遍存在,4种常见耐药基因普遍流行。     

Generation of antibacterial oligosaccharides derived from chitin using heterologous endochitinase synthesized in Escherichia coli

Aims: To synthesize two heterologous endochitinases in Escherichia coli and demonstrate their potential for applied use in generating antibacterial chitin-derived oligosaccharides (OGS). Methods and Results: Heterologous endochitinase genes, chiA Nima and chiA74, were expressed in E. coli. Endochitinases were secreted by the E. coli export machinery and by ∼20 h maximal chitinolytic activity was observed. The highest chitinolytic activity was observed with ChiA Nima, which produced antibacterial OGS with activities against Enterobacter cloacae, Escherichia coli, Staphylococcus aureus and S. xylosus. Conclusions: It was shown that the export machinery of E. coli is well suited for the secretion of bioactive ChiA74 and ChiA Nima endochitinases, and that the latter can generate antibacterial OGS. Significance and Impact of the Study: Our study suggests that it is feasible to synthesize endochitinases ChiA Nima and ChiA74 codified by E. coli and mass-produce these enzymes in culture supernatants. As signal peptides in native ChiA Nima and ChiA74 were recognized by the protein export molecular apparatus in E. coli, these short peptides could be included as signal sequences for transport in E. coli of other proteins with applied value. This is the first report suggesting that ChiA Nima can be used to produce OGS to control food-borne pathogenic bacteria.     

Phylogenetic group distribution and prevalence of virulence genes in <Emphasis Type="Italic">Escherichia coli</Emphasis> isolates from food samples in South Korea

We analyzed the distribution of phylogenetic groups of foodborne Escherichia coli isolates. We also investigated the prevalence of virulence-associated genes of diarrheagenic E. coli. In total, 162 E. coli isolated from foods (raw meat, fish, and processed foods) were collected in Korea. Approximately 90% of the foodborne isolates belonged to phylogenetic groups A and B1, whereas 1.2% were allocated to group B2, and 9.3% to D. Multiplex polymerase chain reaction (PCR) assays were used to detect the following: stx ₁ and stx ₂ to identify Shiga toxin-producing E. coli (STEC), eae and bfpA to identify enteropathogenic E. coli (EPEC), ipaH for enteroinvasive E. coli, CVD432 for enteroaggregative E. coli, and lt and st for enterotoxigenic E. coli (ETEC). The presence of daaD in diffusely adherent E. coli was examined by singleplex PCR. Of the 162 foodborne E. coli isolates, three (1.9%) were confirmed to be pathogenic E. coli: STEC, ETEC, and atypical EPEC based on their possession of stx ₁, st, and eae, and the pathogenic strains were isolated in beef, rockfish, and pork, respectively. Molecular typing was conducted by multilocus sequence typing to investigate the genetic relationships among the pathogenic strains. All isolates positive for virulence genes had different mulilocus sequence typing profiles representing different sequence types (ST) of ST101, ST1815, and ST1820. These results indicate that some food samples were contaminated with pathogenic E. coli.     

Analysis of the tellurite resistance determinant on the pNT3B derivative of the pTE53 plasmid from uropathogenic Escherichia coli

We have found and sequenced a significant part of the previously described tellurite resistance determinant on mini-Mu derivative pPR46, named pNT3B, originally cloned from a large conjugative plasmid pTE53, found in Escherichia coli. This plasmid contains genes essential for tellurite resistance, together with the protective region bearing genes terX, Y, W, and the conserved spacing region bearing several ORFs of unknown function. Computer analysis of obtained sequence revealed a close similarity to the formerly described ter operons found on the Serratia marcescens plasmid R478 and the chromosome of Escherichia coli O157:H7. This finding confirms the presence of a whole region on the large conjugative plasmid that pTE53 originated from a uropathogenic E. coli strain, and suggests its possible role in horizontal gene transfer, resulting in the development of new pathogenic E. coli strains.     

Induction of cytokine formation by human intestinal bacteria in gut epithelial cell lines

Aims: To investigate the effects of human gut micro‐organisms on cytokine production by human intestinal cell lines. Methods and Results: Quantitative real‐time PCR assays were developed to measure the production of pro‐inflammatory (IL‐1α, IL‐6, IL‐18 and TNFα) and anti‐inflammatory (TGF‐β1, TGF‐β2, TGF‐β3, IL‐4 and IL‐10) cytokines in HT‐29 and Caco‐2 cell lines. They were co‐cultured with a range of mucosal bacteria isolated from ulcerative colitis patients, together with lactobacilli and bifidobacteria obtained from healthy people. HT‐29 cells were also co‐cultured with Campylobacter jejuni, enterotoxigenic Escherichia coli (ETEC), enteropathogenic E. coli and Salmonella typhimurium. The majority of commensal bacteria tested suppressed the expression of anti‐inflammatory cytokine mRNA, increased IL‐18, reduced IL‐1α, and with the exception of nonpathogenic E. coli, reduced TNF‐α. All overtly pathogenic species increased both pro‐inflammatory and anti‐inflammatory cytokine mRNA. Conclusion: Commensal and pathogenic species induced fundamentally different cytokine responses in human intestinal epithelial cell lines. Significance and Impact of the Study: Interactions between commensal bacteria tested in this study and the innate immune system were shown to be anti‐inflammatory in nature, in contrast to the pathogenic organisms investigated. These data contribute towards our understanding of how potential probiotic species can be used to suppress the pro‐inflammatory response in inflammatory bowel disease.     

牦牛源产细菌素屎肠球菌的分离鉴定和益生特性

【背景】抗生素的滥用导致牦牛肠道常见病原菌耐药性增加,益生菌作为对抗耐药性细菌的新型武器,应用前景广阔。【目的】获取益生特性优良的牦牛源益生菌。【方法】将20份牦牛粪便样本在含0.5%CaCO3的MRS培养基上分离纯化,以大肠杆菌和金黄色葡萄球菌为指示菌,用牛津杯法筛选有抑菌活性的菌株;排除酸和过氧化氢后,经耐酸耐热试验和蛋白酶敏感试验筛选产细菌素菌株,用形态学和16S rRNA基因序列分析鉴定;通过对大肠杆菌、沙门氏菌等腹泻病原菌体外抑菌试验、耐模拟胃肠液、测定自聚集能力和疏水性及抗生素敏感试验分析益生特性。【结果】从20份牦牛粪便样本中共分离出11株产生溶钙圈的菌株,其中6株对大肠杆菌和金黄色葡萄球菌抑菌效果显著,经复筛得到2株产细菌素的乳酸菌SC6和SC9,经鉴定均为屎肠球菌(Enterococcus faecium)。其中SC9对腹泻病原菌抑菌效果明显,有良好的耐受性和肠道黏附能力,对5种常用抗生素均敏感。【结论】屎肠球菌SC9有一定的抗逆性和潜在的益生能力,具备作为益生菌的潜力。