灭活的双歧杆菌对小鼠血清中细胞因子水平的影响

目的：观察灭活的双歧杆菌对小鼠血清中I-—1β、IL-6和IFN-γ水平的影响。方法：小鼠腹腔注射环磷酰胺造成免疫低下动物模型,分别以新鲜BS肉汤培养基、耗尽培养上清(SCS)以及灭活的和活的双歧杆菌菌液进行灌胃。采用ELISA法检测血清中IL-1β、IL-6和IFN-γ的含量。结果：灭活的双歧杆菌与双歧杆菌活菌均可提高免疫低下小鼠血清中IL-1β、IL-6和IFN-γ的含量,二者的作用效果差异无显著性(P＞0．05),SCS也具有一定的免疫促进作用,但与双歧杆菌活菌相比差异有显著性(P＜0．05)。结论：灭活的双歧杆菌具有与双歧杆菌活菌相同或相近的免疫学活性,两者均可提高小鼠血清中细胞因子水平。     

灭活双歧杆菌调整小鼠抗生素相关性菌群失调

目的：观察灭活的双歧杆菌及其耗尽培养上清液（SCS）对小鼠肠道生理菌群的影响。方法：应用腹腔注射青霉素造成肠菌群失调动物模型,分别以灭活的双歧杆菌菌液,耗尽培养上清液以及活菌菌液对菌群失调小鼠进行灌胃治疗。结果：活菌组、死菌组及SCS组同自然恢复组的肠道生理菌群相比差异均有显著性,死菌组与SCS组相比,差异也有显著性。结论：灭活的双歧杆菌及其SCS对小鼠肠道菌群失调的恢复具有调整作用,尤其对双歧杆菌和乳酸杆菌有更明显的扶持作用。     

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。     

低pH处理对两歧双歧杆菌KLDS2.0603黏附能力的影响

【目的】确定低pH处理对两歧双歧杆菌KLDS2.0603黏附能力及其表面物理化学性质的影响。【方法】将两歧双歧杆菌KLDS2.0603菌体在不同低pH的PBS溶液中处理一定时间后,采用平板菌落计数法和直接镜检法,测定其经历不同pH的酸性环境后的黏附能力,及其表面疏水性和自动聚集能力。【结果】不同pH的PBS溶液处理后的双歧杆菌菌体,其黏附能力均不同程度下降,除pH 5.0的处理组外,其余处理组均显著低于空白组。此外,经不同pH的PBS溶液处理后,仅pH 3.0和3.5的两处理组,双歧杆菌表面疏水性显著提高。除pH 1.0、1.5和5.0的处理组外,其余处理组的自动聚集能力均显著下降。【结论】低pH的酸性环境会降低两歧双歧杆菌KLDS2.0603的黏附能力,并且双歧杆菌的自动聚集能力和表面疏水性也发生相应变化。除pH 3.0和3.5的处理组外,三者之间呈现一定的正相关性。     

双歧杆菌对EPEC和ETEC粘附的竞争抑制作用

观察双歧杆菌与肠上皮细胞系Lovo 细胞粘附后对肠致病性大肠杆菌(EPEC)及产毒性大肠杆菌(ETEC)粘附的竞争性抑制作用。发现双歧杆菌能完全抑制EPEC与ETEC的粘附,这种作用可能是由于双歧杆菌的占位性保护机制,在空间上阻止了病原菌与Lovo细胞的进一步接近     

嗜酸乳杆菌黏附鸡胚肠黏膜上皮细胞能力的研究

目的建立肠黏膜上皮细胞模型和测定嗜酸乳杆菌的黏附能力。方法将嗜酸乳杆菌用胃蛋白酶和HCl-H2O低pH以及反复冻融、灭活等方法处理后测定其黏附能力。结果成功地建立了鸡胚肠黏膜上皮细胞模型;经胃蛋白酶和HCl-H2O低pH以及灭活处理后,嗜酸乳杆菌的黏附能力和对照组差异有显著性。反复冻融后的嗜酸乳杆菌黏附能力与对照组差异无显著性。结论胃蛋白酶和HCl-H2O pH2.0会使嗜酸乳杆菌黏附肠黏膜上皮细胞能力下降,热灭活可使嗜酸乳杆菌的黏附能力提高,反复冻融对嗜酸乳杆菌的黏附能力无明显影响。     

灭活的双歧杆菌对肠上皮细胞粘附及其影响因素的研究

目的 观察灭活的青春双歧杆菌对人大肠癌细胞系ＣＣＬ－２２９的粘附以及影响粘附的因素。方法 通过与双歧杆菌活菌比较,灭活的双歧杆菌同样能粘附于肠上皮细胞,并且耗尽培养上清有利于双歧杆菌粘附。结果 粘附具有显著的浓度效应;粘附效果与孵育环境的ｐＨ值有关;高温处理耗尽培养上清对粘附无明显影响。结论 灭活的双歧杆菌可能具有与活菌相同的生态效应。     

两种生物状态肠道益生菌的粘附和粘附拮抗效应的对比研究

目的:研究活菌和灭活菌两种生物状态的肠道主要益生菌--德氏乳杆菌、双歧杆菌和肠球菌对肠黏膜上皮细胞粘附性及其对肠道几种常见病原菌的粘附拮抗效应.方法:用光镜和电镜技术分析了两种生物状态的三种益生菌对肠黏膜上皮细胞的粘附指数,通过排除实验、竞争实验和替代实验研究了两种生物状态益生菌对侵袭性大肠埃希菌、产毒性大肠埃希菌和痢疾志贺菌的粘附拮抗效应,应用平板扩散法观察了三种益生菌的代谢乏液对上述肠道病原菌的抑制能力.结果:德氏乳杆菌和肠球菌的灭活状态较活菌状态对肠黏膜上皮细胞的粘附性显著增高,双歧杆菌经灭活后对细胞的粘附性与活菌相比差异无显著性,两种生物状态的三种益生菌对肠道致病菌均具有粘附拮抗作用.滤过后的德氏乳杆菌、双歧杆菌和肠球菌的代谢乏液对侵袭性大肠埃希菌、产毒性大肠埃希菌和痢疾志贺菌均具有较明显的抑制作用,经42℃、65℃和100℃加热不影响德氏乳杆菌和双歧杆菌代谢乏液的抑菌作用.结论:灭活状态的德氏乳杆菌、双歧杆菌和肠球菌是具有潜在开发价值的微生态制剂.     

双歧杆菌四联活菌片体外生物拮抗作用的研究

目的实验通过双歧杆菌四联活菌片(思连康)对肠道致病菌体外生物拮抗作用的研究,揭示此药治疗腹泻等疾病的作用机制,为临床应用提供科学依据。方法对组成药物的4株益生菌发酵培养,定时取样,检测活菌数与pH值。先分别单独培养大肠埃希菌、沙门氏菌、志贺氏菌、思连康活菌片,调整菌液浓度,然后将思连康与每一个致病菌的菌液共同接种于GAM肉汤,并将每种菌液单独接种作为对照组,厌氧培养,定时检测致病菌与双歧杆菌的活菌数并分析。结果思连康4株菌株发酵终点活菌数均在109 CFU/mL以上,发酵过程中3株原籍菌的pH值逐渐下降,蜡样芽孢杆菌pH值先下降后升高。思连康与致病菌共培养6h,思连康显著影响大肠埃希菌生长(P0.05),而对沙门氏菌和志贺氏菌的生长无影响(P0.05);共培养12h,思连康对3株致病菌均产生明显抑菌作用(P0.05);共培养24h,未检测到致病菌的存在(P0.01)。结论双歧杆菌四联活菌片对致病菌抑制作用强。     

对双歧杆菌DM9227株的实验研究 Ⅲ.双歧杆菌DM9227株与蜡样芽胞杆菌DM423株共生关系的初步研究

本文对厌氧的双歧杆菌DM9227株与需氧的蜡样芽胞杆菌DM423株的共生关系进行了初步研究。将DM9227株与DM423株分别单独及按一定比例等量混合接种于BS肉汤内进行培养,间隔一定时间检测两菌在培养基内菌数的消长情况。结果表明DM423株与DM9227株之间呈偏生关系,即DM423株对DM9227株有促进作用,且在两菌比例适宜(1:100)情况下促进作用更明显,而DM9227株对DM423株有抑制作用。进一步将接种DM423株的Nb琼脂平板与接种DM9227株的BS琼脂平板在普通环境下一起密闭培养,可见厌氧的DM9227株生长良好,提示DM423株可能是通过消耗氧气,降低培养基的Eh,从而促进DM9227株生长的。     

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     

Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibition of enteropathogen-cell interactions.

Thirteen human bifidobacterial strains were tested for their abilities to adhere to human enterocyte-like Caco-2 cells in culture. The adhering strains were also tested for binding to the mucus produced by the human mucus-secreting HT29-MTX cell line in culture. A high level of calcium-independent adherence was observed for Bifidobacterium breve 4, for Bifidobacterium infantis 1, and for three fresh human isolates from adults. As observed by scanning electron microscopy, adhesion occurs to the apical brush border of the enterocytic Caco-2 cells and to the mucus secreted by the HT29-MTX mucus-secreting cells. The bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage. The adhesion to Caco-2 cells of bifidobacteria did not require calcium and was mediated by a proteinaceous adhesion-promoting factor which was present both in the bacterial whole cells and in the spent supernatant of bifidobacterium culture. This adhesion-promoting factor appeared species specific, as are the adhesion-promoting factors of lactobacilli. We investigated the inhibitory effect of adhering human bifidobacterial strains against intestinal cell monolayer colonization by a variety of diarrheagenic bacteria. B. breve 4, B. infantis 1, and fresh human isolates were shown to inhibit cell association of enterotoxigenic, enteropathogenic, diffusely adhering Escherichia coli and Salmonella typhimurium strains to enterocytic Caco-2 cells in a concentration-dependent manner. Moreover, B. breve 4 and B. infantis 1 strains inhibited, dose dependently, Caco-2 cell invasion by enteropathogenic E. coli, Yersinia pseudotuberculosis, and S. typhimurium strains.     

New knowledge on pathogenesis of bacterial enteric infections as applied to vaccine development.

This review attempts to synthesize the new knowledge of pathogenesis of bacterial enteric infections and relate this information to vaccine development. Discussion focuses on human infections and to those in which significant strides have been made. As a general theme in the pathogenesis of bacterial enteric infections, pathogens can be characterized into 5 groups on the basis of their degree of ultimate invasiveness after ingestion by a susceptible hose: mucosal adherence and enterotoxin production; mucosal adherence and brush border dissolution -- enteropathogenic E. coli (EPEC) of "classical" serotypes; mucosal invasion and intraepithelial cell proliferation; mucosal translocation followed by bacterial proliferation in the lamina propria and mesenteric lymph nodes; and mucosal translocation followed by generalized infection. The review covers cholera (motility and chemotaxis, mucosal adhesion, flagellar sheath protein, hemagglutinins, outer membrane proteins, enterotoxin production, quality and duration of infection derived immunity, immune response in humans, LPS, flagellar sheath protein, cholera lectin, other cholera hemagglutinins, outer membrane protein, previous cholera vaccines, killer whole cell vaccines, toxoids, combination vaccines, attenuated versus cholerae vaccines): enterotoxigenic Escherichia coli (ETEC) (entertoxins, O:H serotypes and enterotoxin phenotypes, colonization factors, immune response in humans, vaccines against ETEC, and toxiods); EPEC (vaccines against EPEC); Shigella (smooth LPS O antigen, epithelial cell invasiveness, Shigella toxin, and Shigella vaccines); and typhoid fever (caccines against typhoid fever). The major attraction of a nonliving oral cholera vaccine is its safety. A review of available information leads to the conclusion that an oral vaccine consisting of a combination of antigens, intending to stimulate both antibacterial and antitoxic immunity, would be most likely to succeed. Current approaches to immunoprophylaxis of ETEC infection involve vaccines that stimulate antitoxic or antiadhesion immunity or both by means of killed antigens or attenuated strains. It is likely that the most effective vaccines will contain appropriate antigens intended to simultaneously stimulate both antibacterial and antitoxic immunity, thereby leading to a synergistic protective effect. Now that the speical enteroadhesive properties of EPEC have been characterized and shown to be associated with a plasmid, it should be possible to identify the phenotypic gene products responsible for this phenomenon. It is likely that fimbriae or outer membrane proteins will prove to be the organelle of adhesion. When such information becomes available, it should be possible to prepare oral vaccines consisting of the purified antigen. Efficacy has been shown for attenuated Shigella strains utilized as oral vaccines. The major thrust in the development of new immunization agensts against typhoid fever is to identify immunizing agents at least equal in efficacy to the parenteral acetone killed vaccine but which cause no adverse reactions.     

A plasmid-encoded type IV fimbrial gene of enteropathogenic Escherichia coli associated with localized adherence

Enteropathogenic Escherichia coli (EPEC) form adherent microcolonies on the surface of tissue culture cells in a pattern termed localized adherence. Localized adherence requires the presence of a large EPEC adherence factor (EAF) plasmid. Recently a bundle-forming pilus has been described in EPEC possessing the EAF plasmid. An analysis of 22 non-invasive EPEC TnphoA mutants revealed that seven have insertions in the EAF plasmid and are incapable of localized adherence. We report here the mapping of the TnphoA insertions in these mutants. The nucleotide sequence of the gene interrupted in these TnphoA mutants (bfpA) was determined and found to correspond to the N-terminal amino acid sequence of the major structural protein of the bundle-forming pilus. The bfpA gene bears sequence similarities to members of the type IV fimbrial gene family and encodes a potential site for processing by a prepilin peptidase. A plasmid containing bfpA as the only open reading frame directs the synthesis of a protein recognized by antiserum raised against the bundle-forming pilus. TnphoA mutants at this locus are unable to synthesize BfpA, but synthesis is restored by introduction of a plasmid containing the cloned gene. The minimum fragment of DNA required to restore localized adherence is considerably greater than that required to restore BfpA synthesis. BfpA expression, as assessed by alkaline phosphatase activity in bfpA::TnphoA mutants, is affected by temperature and growth medium. These studies describe an EPEC plasmid-encoded fimbrial gene, a candidate for the elusive EPEC adherence factor responsible for localized adherence.     

Lactobacillus acidophilus LAP5 able to inhibit the Salmonella choleraesuis invasion to the human Caco-2 epithelial cell

The mechanisms for lactic acid bacteria (LAB) to inhibit Salmonella invasion appear to be multifactorial and include the adhesion of LAB to host intestine epithelium, the production of organic acids, or bacteriocin by LAB cells. Previously, we found a strain of Lactobacillus acidophilus isolated from swine, i.e. strain LAP5, was with antagonistic effect against Salmonella typhimurium. This strain LAP5 was also found to meet the requirements for probiotic use. In this study, we evaluate the potential of LAP5 strain to protect the human or swine from infection by Salmonella choleraesuis. We present evidence that the culture of LAP5 was able to inhibit the invasion of S. choleraesuis to human Caco-2 cell line. The LAP5 cell culture showed a higher inhibitory effect on the invasion of S. choleraesuis to Caco-2 cells than the spent culture supernatant (SCS) of LAP5 did. Also, the pH, organic acids or the bacteriocin, which act at low pH conditions, may play the role of antagonistic effect. The addition, adhesion of LAP5 cells to Caco-2 cell line may also play roles to reduce the invasion of S. choleraesuis.