双歧杆菌及肠致病性大肠杆菌粘附的细胞膜通透性研究

采用乳酸脱氢酶释放法比较研究双歧杆菌１０２７株及肠致生大肠杆菌（ＥＰＥＣ）对体外肠上皮细胞Ｌｏｖｏ细胞株粘附的细胞膜通透性,探讨它们对肠上皮细胞的不同生物学交谈２。结果表明,双歧杆菌粘附Ｌｏｖｏ细胞后,宿主细胞释放ＬＤＨ远较ＥＰＥＣ粘附的效果低,提示双歧杆菌粘附对主细胞膜通生影响不大,而ＥＥＣ则可损伤宿主细胞膜而增加其通性。因此,双歧杆菌作为生理性细菌可与肠上皮细胞和谐共生,这与ＥＰＥＣ的粘附损伤     

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-     

灭活的双歧杆菌对EPEC的黏附抑制作用

目的：研究灭活的青春双歧杆菌DMS8504对肠致病灶大肠埃希菌（EPEC）黏附抑制作用。方法：通过与活菌比较,观察灭活的双歧杆菌粘附于人大肠癌CCL－229细胞后对EPEC的黏附抑制作用。结果：用SCS或pH5.0新鲜BS肉汤悬浮的双歧杆菌能够安全抑制EPEC的黏附,而仅用SCS或pH5.0新鲜BS肉汤均不能抑制其黏附。     

Caspase信号通路在双歧杆菌脂磷壁酸诱导结肠癌细胞凋亡中的作用

目的探讨Caspase信号通路在双歧杆菌脂磷壁酸（LTA）诱导结肠癌细胞凋亡中的作用。方法RT-PCR检测经双歧杆菌LTA处理后,结肠癌Lovo细胞中MyD88和FADD mRNA的表达变化;AnnexinV检测经Caspase通用抑制剂（Z-Val-Ala-Asp-FMK）预先处理后,双歧杆菌LTA诱导结肠癌Lovo细胞凋亡率的变化;荧光法检测经双歧杆菌LTA处理后,Lovo细胞中Caspase-8活性的变化。结果经双歧杆菌LTA处理后,Lovo细胞中MyD88的mRNA表达明显升高（P〈0．05）,而FADD信号分子的mRNA表达无明显变化;双歧杆菌LTA能够增强Lovo细胞中Caspase-8的活性（P〈0．05）,且其诱导Lovo细胞凋亡的作用能够被Caspase抑制剂所抑制（P〈0．05）。结论MyD88信号分子在双歧杆菌LTA诱导Lovo细胞凋亡中可能起着承接上游分子TLRs与下游信号分子FADD的作用;而Caspase信号通路可能是双歧杆菌LTA诱导结肠癌Lovo细胞凋亡的主要信号传导途径。     

Toll样受体在双歧杆菌脂磷壁酸诱导结肠癌细胞凋亡中的作用

目的探讨双歧杆菌脂磷壁酸（LTA）对Toll样受体（TLRs）表达的影响及其与诱导结肠癌细胞凋亡之间的关系。方法用AnnexinV检测在双歧杆菌LTA处理前后结肠癌Lovo细胞凋亡的变化;流式细胞术检测Lovo细胞表面TLRs的表达,并用相应的TLRs封闭抗体作用后,AnnexinV检测经双歧杆菌LTA诱导的Lovo细胞凋亡的变化。结果经双歧杆菌LTA处理后,结肠癌Lovo细胞发生了明显的凋亡,并有一定的时间和剂量依赖关系;结肠癌Lovo细胞有TLR受体的基础表达,经双歧杆菌LTA处理后,TLR2和TLR4在Lovo细胞上的表达增加,其中尤以TLR2增加更为明显;用相应的TLRs抗体封闭作用后,双歧杆菌LTA诱导Lovo细胞凋亡的能力下降。结论双歧杆菌LTA能诱导肿瘤细胞凋亡,并且TLRs特别是TLR2在LTA诱导肿瘤细胞凋亡中可能发挥着主要作用,TLR4可能仅起着协同作用。     

实验性痴呆动物的肠道菌群和粘附性研究

用ＡＦ６４Ａ复制实验性痴呆动物模型,分析该动物的肠道菌群,并以双歧杆菌和大肠杆菌作为肠道菌的代表,初步探讨它们对实验性痴呆动物肠道粘膜上皮细胞表面的粘附特性。结果表明,实验性痴呆动物的肠道菌群是紊乱的,二种试验菌均能粘附到正常小鼠肠上皮细胞上,双歧杆菌的粘附率明显高于大肠杆菌,而双歧杆菌对实验性痴呆小鼠肠上皮细胞的粘附率明显低于对照组小鼠,大肠杆菌则相反。     

细菌对肉鸡肠粘液的粘附作用

研究两歧双歧杆菌、嗜酸乳杆菌、禽大肠杆菌O78、大肠杆菌 ATCC 25922、鸡白痢沙门氏菌和鼠伤寒沙门氏菌与肉鸡不同部位肠粘液糖蛋白的粘附性能,探讨两歧双歧杆菌和嗜酸乳杆菌对所试病原菌的抗粘附作用。结果表明：在不同的肠道部位,两歧双歧杆菌、嗜酸乳杆菌、鸡白痢沙门氏菌和鼠伤寒沙门氏菌与肠粘液糖蛋白均有不同的粘附作用,而禽大肠杆菌O78、大肠杆菌 ATCC 25922在各肠段粘液上的粘附性能则相近;在相同的肠道部位,所试益生菌的粘附能力大于病原菌;两歧双歧杆菌和嗜酸乳杆菌对所试病原菌的粘附有不同的阻断作用,同时二者有时还存在互补抗粘附作用。     

大肠杆菌-双歧杆菌穿梭表达载体的构建及内皮抑素基因的表达

目的:构建大肠杆菌-长双歧杆菌穿梭表达载体,并通过此载体使人内皮抑素基因在大肠杆菌和长双歧杆菌中得到表达。方法:以质粒pDG7、pBCSK( )、pET-9C为基础,构建大肠杆菌-长双歧杆菌穿梭表达载体pET-1128,并将人内皮抑素基因插入到新构建的表达载体中,分别转化大肠杆菌BL21(DE3)和长双歧杆菌NQ-1501。诱导表达,表达产物经SDS-PAGE和WesternBlot鉴定。结果:成功构建了大肠杆菌-双歧杆菌穿梭载体,人内皮抑素基因在大肠杆菌和长双歧杆菌中均可表达。结论:构建的穿梭载体为今后用双歧杆菌作为生理菌载体进行肿瘤的基因治疗奠定了基础。     

双歧杆菌的粘附特性及其对肠道致病菌的体外拮抗作用

本文采用体外细胞培养法, 从实验室现有的20株不同生境来源的双歧杆菌中筛选具有较强粘附能力的菌株, 并通过混合培养和牛津杯方法, 研究了具有粘附特性的双歧杆菌对肠道致病菌的体外拮抗作用。结果显示, 长寿老人源菌株A03和I06的粘附能力最强, 其菌液对金黄色葡萄球菌及大肠杆菌均具有显著的抑制性, 但是菌体及中和后的发酵液均没有抑菌性, 说明受试菌主要通过其代谢产物中的有机酸来发挥其抑菌性能; 此外, 通过分析比较受试菌和肠道致病菌分别与Caco-2细胞粘附后释放的乳酸脱氢酶量, 证实双歧杆菌与致病菌对细胞的作用具有本质上的区别, 双歧杆菌的粘附能减缓致病菌对细胞所造成的损害。     

灭活的青春双歧杆菌对人大肠癌细胞的粘附

针对灭活的青春双歧杆菌DM850 4与人大肠癌CCL 2 2 9细胞之间的粘附现象及粘附机制进行研究。结果发现灭活的双歧杆菌具有与活菌相同的粘附定植能力 ,两者粘附于体外培养的肠上皮细胞均依赖于耗尽培养上清 (SCS)的存在。青春双歧杆菌粘附素有可能是存在于细胞壁中及分泌至SCS中的脂磷壁酸 (LTA)。LTA与细菌细胞壁耐热蛋白相互粘连 ,并且伸出胞壁之外。此外 ,肠上皮细胞表面的粘附素受体可能为糖类或糖蛋白。     

Single multiplex assay to identify simultaneously enteropathogenic, enteroaggregative, enterotoxigenic, enteroinvasive and Shiga toxin-producing Escherichia coli strains in Brazilian children

A multiplex PCR to differentiate typical and atypical enteropathogenic Escherichia coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic (ETEC), enteroinvasive E. coli (EIEC) and Shiga toxin-producing E. coli (STEC) strains was developed and evaluated. The targets selected for each group were eae and bfpA for EPEC, aggR for EAEC, elt and est for ETEC, ipaH for EIEC and stx for STEC isolates. This PCR was specific and sensitive for rapid detection of target isolates in stools. Among 79 children with acute diarrhea, this technique identified 13 (16.4%) with atypical EPEC, four (5%) with EAEC, three (3.8%) with typical EPEC, one (1.3%) with ETEC and one (1.3%) with EIEC.     

双歧杆菌粘附体外肠上皮细胞的钙信号传递的研究

本文采用钙荧光探剂 Ｆｌｕｏ—３／ＡＭ染色法,定量研究了双歧杆菌１０２７株、肠致病性大肠杆菌（ＥＰＥＣ）对体外肠上皮细胞Ｌｏｖｏ细胞株粘附的钙信号传递机制。结果表明,双歧杆菌１０２７株粘附可引起Ｌｏｖｏ细胞内Ｃａ~＋２随时间延长而梯度升高,但双歧杆菌１０２７株的作用远不如ＥＰＥＣ明显。同时发现双歧杆菌粘附引起Ｌｏｖｏ。细胞内Ｃａ~２＋升高主要源于细胞外Ｃａ~２＋内流所致,这与ＥＰＥＣ粘附引起宿主细胞内Ｃａ~２＋升高主要源于细胞内Ｃａ~２＋储池的Ｃａ~２＋释放不同。ＥＰＥＣ粘附引起宿主细胞内Ｃａ~２＋大幅度升高是其致病的重要信号传递基础;而双歧杆菌粘附仅引起宿主细胞内Ｃａ~２＋轻度升高,可能是其作为生理性细菌与肠上皮细胞和谐共生的信号传递基础。     

Bacteriological studies on Iraqi milk products.

Samples from different types of domestic milk products including cheese, kishfa and gaymer were assessed for bacteriological quality over a 4-month period. A total of 400 samples were randomly selected across Mosul city and tested for faecal coliform counts, enteropathogenic Escherichia coli (EPEC) and enterotoxigenic E. coli (ETEC). Faecal coliforms were present at levels greater than 10(2) cfu/g in 72.5% and less than 10(2) cfu/g in 27.5% of samples. Of the 430 E. coli strains isolated from the 400 samples of milk products, 138 were serotypes of EPEC. These were found in 81 (40.5%) samples of cheese, 35 (23.8%) of kishfa and 22 (29.7%) of gaymer. During this period, 26 strains of ETEC were also isolated, all of which demonstrated heat-labile or heat-stable toxins. The high proportion of strains of three groups of E. coli showing resistance to antibiotics is discussed in relation to widespread use of antibiotics and the possible public health implication.     

Vero cytotoxin production and HeLa cell adherence of enteropathogenic Escherichia coli of infantile diarrhoea in Iran

A total of 70 enteropathogenic Escherichia coli (EPEC) strains belonging to 11 serogroups, isolated from infantile diarrhoea in Tehran, Iran, were tested for the production of verocytotoxin (VT), enterotoxin, and also for their adherence to HeLa cells. In total 55 (78.5%) strains were either VT (32 strains) or enterotoxin (23 strains) producers, and of these 8 strains produced both VT and enterotoxins. 57 (81.4%) strains showed either Localized (LA) or Diffuse adherence (DA) or both types of adhesion (LA/DA) on HeLa cells, with strains showing LA/DA in the same preparations being dominant (32 strains), followed by those showing LA (14 strains) and DA (11 strains). Among adherent EPEC, 26 (37.1%) strains belonging to the serogroups 020, 086, 0119, 0125, 0126, 0127 and 0128 also produced VT. These findings suggest that production of VT and enterotoxin is an important factor in the pathogenesis of EPEC diarrhoea in Iran and that the combination of adherence and production of toxins is a common feature of EPEC strains which cause diarrhoea in this country.     

Prevalence of diarrheagenic Escherichia coli in children with diarrhea in Salvador, Bahia, Brazil

We report the frequency of the different diarrheagenic Escherichia coli (DEC) categories isolated from children with acute endemic diarrhea in Salvador, Bahia. The E. coli isolates were investigated by colony blot hybridization with the following genes probes: eae, EAF, bfpA, Stx1, Stx2, ST-Ih, ST-Ip, LT-I, LT-II, INV, and EAEC, as virulence markers to distinguish typical and atypical EPEC, EHEC/STEC, ETEC, EIEC, and EAEC. Seven of the eight categories of DEC were detected. The most frequently isolated was atypical EPEC (10.1%) followed by ETEC (7.5%), and EAEC (4.2%). EHEC, STEC, EIEC, and typical EPEC were each detected once. The strains of ETEC, EAEC, and atypical EPEC belonged to a wide variety of serotypes. The serotypes of the others categories were O26:H11 (EHEC), O21:H21 (STEC), O142:H34 (typical EPEC), and O:H55 (EIEC). We also present the clinical manifestations and other pathogenic species observed in children with DEC. This is the first report of EHEC and STEC in Salvador, and one of the first in Brazil.     

Multiplex PCR-DNA probe assay for the detection of pathogenic Escherichia coli

A multiplex PCR-DNA probing assay was developed to detect four major Escherichia coli virotypes. Six highly specific polymerase chain reaction (PCR) primer sets and DIG-labeled chemiluminescent probes were designed to target the Shiga-like toxin I and II genes (stxI and stxII) of verotoxigenic E. coli (VTEC), heat-stable and heat-labile toxin genes of enterotoxigenic E. coli (ETEC), adherence factor (EAF) of enteropathogenic E. coli (EPEC) and a fragment of the invasiveness plasmid (IAL) of enteroinvasive E. coli (EIEC). The primer pairs generate products of 350, 262, 170, 322, 293 and 390 bp in length, respectively. The multiplex primers and probes were tested for specificity against 31 pathogenic E. coli strains, nine nonpathogenic E. coli and non-E.coli enteric and environmental bacterial strains. The results showed a high degree of specificity of the primers and probes for strains from corresponding virotypes and no reaction with the nontarget bacterial strains. The proposed multiplex PCR-DNA probing assay provides rapid and specific detection of four major virotypes of E. coli.