中药91—4对抗生素相关性腹泻小鼠肠道膜菌群及腔菌群的影响

本实验以肠道菌检测与电镜观察法,初步研究了中药９１－４对抗生素相关性腹泻小鼠肠道菌群的影响。结果表明,盐酸林可霉素对小鼠肠道膜菌群、腔菌群干扰显著、乳酸杆菌、双歧杆菌及肠杆菌等比正常对照组明显减少,致小鼠腹泻或肠炎;中药９１－４能够促进肠道乳酸杆菌、双歧杆菌等生长繁殖,对肠道膜菌群、腔菌群均具有调整作用,加速肠粘膜病变的愈合及修复,可有效地控制ＡＡＤ模型小鼠的腹泻症状。     

四君子汤对实验性脾虚小鼠肠道菌群的影响

本文报道了四君子汤对脾虚小鼠肠道内与人类关系密切的主要菌群的影响。用大量大黄水煎液灌胃给予小鼠,造成实验性脾虚模型,引起小鼠肠道内菌群紊乱,其中双歧杆菌、乳杆菌菌量均下降,与对照组小鼠相比具有显著性差异。当小鼠服用四君子汤后,双歧杆菌,乳杆菌均上升至正常值水平。四君子汤对拟杆菌数量也有一定的影响。本研究表明,四君子汤对小鼠肠道菌群的失调具有调节功能,对脾虚小鼠的康复具有一定的促进作用。     

玉米紫色植株花色苷色素对小鼠肠道菌群的影响

目的了解玉米紫色植株花色苷色素对小鼠肠道菌群的影响。方法按中华人民共和国卫生部《保健食品检验与评价技术规范》中的动物实验调节肠道菌群功能的检验方法,观察玉米紫色植株色素对小鼠肠道中肠杆菌、乳杆菌、双歧杆菌数量的影响。结果灌服后小鼠肠道乳杆菌、双歧杆菌数量变化差异均无显著性,低剂量组肠扦菌数量明显低于灌服前（P〈0．05）,其他组肠杆菌数量变化差异无显著性。结论玉米紫色植株色素对小鼠肠道菌群可能有影响。     

中药91－4对抗生素相关性腹泻小鼠肠道膜菌群及腔菌群的影响

本实验以肠道菌群检测与电镜观察法,初步研究了中药９１－４对抗生素相关性腹泻（ＡＡＤ）小鼠肠道菌群的影响。结果表明,盐酸林可霉素对小鼠肠道膜菌群、腔菌群干扰显著,乳酸杆菌、双歧杆菌及肠杆菌等比正常对照组明显减少,致小鼠腹泻或肠炎;中药９１－４能够促进肠道乳酸杆菌、双歧杆菌等生长繁殖,对肠道膜菌群、腔菌群均具有调整作用,加速肠粘膜病变的愈合及修复,可有效地控制ＡＡＤ模型小鼠的腹泻症状。     

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     

酪酸梭菌-婴儿型双歧杆菌二联活菌制剂的研究

对酪酸梭菌－婴儿型双歧杆菌二联活菌制剂的特性及功效进行研究和分析。方法：通过实验鼠进行刺激生长试验、活菌数测定、毒理试验、调节肠道菌群试验、免疫调节作用来观察制剂的作用。结果：小鼠肠道中双歧杆菌、乳杆菌、酪酸梭菌数量明显增加,高剂量能增加小鼠抗体生成细胞数。     

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。     

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     

扶正固本丸对小鼠肠道菌群的影响

本文报告应用扶正固本丸对小鼠肠道内与人类关系密切的主要菌群的影响。用大黄水煎液灌胃予小鼠,造成实验性脾虚模型,引起小鼠肠道内菌群紊乱,其中双歧杆菌,乳杆菌量均下降,与对照组相比有显著性差异。当服用扶正固本丸后,双歧杆菌、乳杆菌均上升至正常水平。本研究表明,扶正固本丸对小鼠肠道菌群失调具有一定的调整作用。     

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。     

悉生小鼠体内大肠杆菌和青春型双歧杆菌对艰难梭菌的拮抗作用

本文应用悉生小鼠做模型,研究了大肠杆菌(E.coli)和青春型双歧杆菌(Bifidobacterium adolescentis)对艰难梭菌(Clostridium diffi-cile)的拮抗作用。E.coli和B.adolescentis预先接种无菌SSB小鼠,再用C.difficile攻击。结果表明,E.coli和E.coli B.adolescentis对小鼠均有保护作用,保护平分别为87.5%(7/8)和100%(8/8)。B.adolescentis定值后数量达10~(10.28)CFU/g,且对E.coli数量和小鼠本身无影响。E.coli和B.adolescentis联合比E.coli单独抑制C.difficile在肠道中繁殖的作用更强(0.02>P>0.01),但对其毒素产生和粘附力的作用无明显差异。C.difficile攻击后的1～14天,小鼠粪便中C.difficile菌数在10~4至10~8CFU/g内变化,细胞毒素为10~3CFU/g,A毒素滴度为10~2/g,B.adolescentis也一度下降10~2CFU/g。接种C.difficile后,小鼠虽无明显的腹泻症状,但组织学仍可观察到肠粘膜有充血和分泌增加等轻度损害。扫描电镜和普通光镜均发现E.coli单独或与B.adolescentis共同吸附在肠粘膜微绒毛表面,未见有C.difficile吸附。     

双歧杆菌对免疫抑制小鼠白色念珠菌感染的保护作用

为探讨双歧杆菌对白色念珠菌感染的保护作用,作者采用环磷酰胺腹腔注射方法复制了免疫抑制小鼠模型,研究白色念珠菌感染前后两歧双歧杆菌灌饲的保护作用。结果表明：白色念珠菌感染前灌饲两歧双歧杆菌能有效地抑制白色念珠菌在小鼠肠道中的定植,并且保护肠粘膜的完整性。对其机理的研究认为,两歧双歧杆菌可能是通过调整肠道菌群平衡、增加肠道中生理菌群的数量、降低肠道ｐＨ值、以及产生抗菌物质等途径发挥保护作用     

Effect of antibiotics on the human intestinal flora in mice

Antibiotics used during selective decontamination were studied for their effect on the human intestinal flora in mice. Polymyxin B and neomycin were found to eliminate Escherichia coli from the gastrointestinal tract but did not alter total numbers of obligate anaerobes. Neomycin induced an increase of the percentage of gram-negative obligate anaerobes. Cephradine did not affect the numbers of obligate and facultative anaerobes but increased the percentage of gram-negative obligately anaerobic rods in the flora. The selective effect of polymyxin B and neomycin on the flora is accounted for by a relative insusceptibility of the anaerobic flora as compared with E. coli. Low concentrations of polymyxin B and neomycin were detected in caecal supernatants. This was found to be due to strong binding of both antibiotics to the solid fraction of intestinal contents.     

Chemostat modeling of Escherichia coli persistence in conventionalized mono-associated and streptomycin-treated mice

We have previously shown that Escherichia coli BJ4 has similar doubling time in mice that are mono-associated (having only the inoculated E. coli BJ4) or streptomycin-treated (having mainly gram-positive bacteria plus the inoculated E. coli BJ4). We also showed that when the mice were conventionalized (fed cecum homogenate from conventional mice or ones with a complete microbial flora), the introduction of complete flora in both cases increased the in vivo doubling time, while decreasing the colony counts in fecal samples. To determine whether the increase in doubling time could explain the decrease in colony counts, we analyzed our previous results by a chemostat model. The analysis shows that the increasing doubling time alone is sufficient to explain the decrease in colony counts in mono-associated mice, but not in the streptomycin-treated mice. The observed decreasing rate in colony counts in streptomycin-treated mice is slower than predicted. Furthermore, whereas the model predicted a decrease to extinction in both mice, the E. coli persist at a frequency 10-80 times higher in streptomycin-treated mice than in mono-associated mice. Thus, while a chemostat model is able to explain some of the population dynamics of intestinal bacteria in mice, additional factors not included in the model are stabilizing the system. Because we find that E. coli declines more slowly and to a higher stabilization frequency in streptomycin-treated mice, which have a more diverse flora before conventionalization, we take these results to suggest that the persistence of E. coli populations is promoted by species diversity. We propose that a mechanism for the persistence may be the presence of new E. coli niches created by keystone species in the more diverse flora.     

The intestinal microflora regulates cytokine production positively in spleen-derived macrophages but negatively in bone marrow-derived macrophages.

Besides its role as a barrier against potential pathogens, intestinal flora is presumed to protect the host by priming the immunological defense mechanisms. In this respect, the influence of intestinal flora on macrophage precursors was examined, and its modulating effect was compared on LPS-induced cytokine production by macrophages derived from bone marrow and spleen precursors (BMDM and SDM respectively). The regulation of IL-1, IL-6, TNF-alpha and IL-12 production in macrophages from germ-free and from three groups of flora-associated mice, conventional, conventionalized and E. coli-mono-associated mice, was investigated. The whole flora inhibited IL-1, TNF-alpha and IL-12 secretion by BMDM, whereas it had a stimulatory effect on IL-12 secretion by SDM. Implantation of E. coli alone enhanced cytokine secretion by BMDM but had a more limited effect than whole flora on SDM, enhancing only TNF-alpha and IL-12 secretion. Study of expression of mRNA showed a correlation with protein secretion for IL-6 but not for TNF-alpha and IL-1. IL-12 enhancement in BMDM seemed to be dependent on regulation of p35 mRNA expression while it was correlated to increased p40 mRNA expression in SDM. The results demonstrated that intestinal flora modulated bone marrow and spleen macrophage cytokine production in a differential manner and suggested a role for bacteria other than E. coli among the whole flora. The contrasting effects exerted by the intestinal flora on bone marrow and spleen precursors are an interesting observation in view of the different functions of these organs in immunity. The finding that intestinal flora enhanced IL-12 production in spleen is also potentially important since this cytokine is implicated in the determination of the relative levels of Th1 and Th2 responses and plays a pivotal role in host defense against intracellular microorganisms.     

Gram-negative bacteria aggravate murine small intestinal Th1-type immunopathology following oral infection with Toxoplasma gondii

Oral infection of susceptible mice with Toxoplasma gondii results in Th1-type immunopathology in the ileum. We investigated gut flora changes during ileitis and determined contributions of gut bacteria to intestinal inflammation. Analysis of the intestinal microflora revealed that ileitis was accompanied by increasing bacterial load, decreasing species diversity, and bacterial translocation. Gram-negative bacteria identified as Escherichia coli and Bacteroides/Prevotella spp. accumulated in inflamed ileum at high concentrations. Prophylactic or therapeutic administration of ciprofloxacin and/or metronidazole ameliorated ileal immunopathology and reduced intestinal NO and IFN-gamma levels. Most strikingly, gnotobiotic mice in which cultivable gut bacteria were removed by quintuple antibiotic treatment did not develop ileitis after Toxoplasma gondii infection. A reduction in total numbers of lymphocytes was observed in the lamina propria of specific pathogen-free (SPF), but not gnotobiotic, mice upon development of ileitis. Relative numbers of CD4(+) T cells did not differ in naive vs infected gnotobiotic or SPF mice, but infected SPF mice showed a significant increase in the frequencies of activated CD4(+) T cells compared with gnotobiotic mice. Furthermore, recolonization with total gut flora, E. coli, or Bacteroides/Prevotella spp., but not Lactobacillus johnsonii, induced immunopathology in gnotobiotic mice. Animals recolonized with E. coli and/or total gut flora, but not L. johnsonii, showed elevated ileal NO and/or IFN-gamma levels. In conclusion, Gram-negative bacteria, i.e., E. coli, aggravate pathogen-induced intestinal Th1-type immunopathology. Thus, pathogen-induced acute ileitis may prove useful to study bacteria-host interactions in small intestinal inflammation and to test novel therapies based on modulation of gut flora.     

低出生体重儿肠道微生态构建影响因素的 动态监测

目的动态监测低出生体重儿肠道菌群,分析不同体重、不同喂养方式及疾病状态等因素对患儿肠道微生态的影响,为规范临床低出生体重儿宫外营养支持措施及治疗手段提供依据。方法应用16SrRNA荧光定量PCR技术检测正常新生儿和低出生体重儿生后第1、3、7天粪便中大肠埃希菌、肠球菌、乳杆菌及双歧杆菌的含量。结果 (1)在生后7d内,无论正常新生儿还是低出生体重儿,其粪便中大肠埃希菌、肠球菌、乳杆菌和双歧杆菌的含量均随日龄的增加而增加,且生后7d内正常新生儿的粪便中大肠埃希菌、肠球菌、乳杆菌和双歧杆菌的含量均显著高于低出生体重儿(P0.05),正常新生儿生后7d内粪便中各细菌的增长率均高于低出生体重儿。(2)体重2 000~2 500g的低出生体重儿粪便中大肠埃希菌和肠球菌在各日龄中的含量明显高于体重2 000g的新生儿(P0.05);同时其粪便中双歧杆菌和乳杆菌含量在3日龄和7日龄阶段明显高于体重2 000g的新生儿(P0.05)。(3)3日龄和7日龄母乳喂养组的低出生体重儿粪便中双歧杆菌和乳杆菌含量明显高于乳制品喂养组(P0.05);且母乳喂养组新生儿生后7日内粪便中大肠埃希菌、乳杆菌和双歧杆菌含量的增长率均高于乳制品喂养组,尤其是双歧杆菌的增长率(126.49%vs 54.81%)。(4)合并并发症的3日龄和7日龄的低出生体重儿,粪便中乳杆菌和双歧杆菌含量均明显低于无合并症的低出生体重儿(P0.05);且无并发症组的低出生体重儿其粪便中肠球菌、乳杆菌和双歧杆菌的增长率均高于有并发症组的低出生体重儿,大肠埃希菌增长率则低于有并发症组。结论低出生体重儿肠道菌群的定植时间晚且数量少,体重、喂养方式及有无并发症是影响新生儿肠道菌群丰度的重要因素。母乳喂养可促进低出生体重儿肠道中益生菌的定植。疾病因素会导致肠道菌群丰度的降低,使肠道菌群紊乱,其程度可能与病情的严重程度相关。     

Effects of the Human Intestinal Flora on Germ-free Mice

The effects of complete human intestinal flora and of intestinal anaerobes on germ-free mice were studied. The gross composition of the flora of mice was similar to that of the flora with which the animals had been contaminated and appeared to be stable provided that the animals were kept isolated. The complete flora and the anaerobes reduced caecal weight to normal values and induced an antagonistic effect against Escherichia coli. In contrast to the complete flora, the anaerobes were not invasive in immunosuppressed mice and induced colonization resistance and antagonism against Pseudomonas aeruginosa.     

Enterocyte TLR4 mediates phagocytosis and translocation of bacteria across the intestinal barrier

Translocation of bacteria across the intestinal barrier is important in the pathogenesis of systemic sepsis, although the mechanisms by which bacterial translocation occurs remain largely unknown. We hypothesized that bacterial translocation across the intact barrier occurs after internalization of the bacteria by enterocytes in a process resembling phagocytosis and that TLR4 is required for this process. We now show that FcgammaRIIa-transfected enterocytes can internalize IgG-opsonized erythrocytes into actin-rich cups, confirming that these enterocytes have the molecular machinery required for phagocytosis. We further show that enterocytes can internalize Escherichia coli into phagosomes, that the bacteria remain viable intracellularly, and that TLR4 is required for this process to occur. TLR4 signaling was found to be necessary and sufficient for phagocytosis by epithelial cells, because IEC-6 intestinal epithelial cells were able to internalize LPS-coated, but not uncoated, latex particles and because MD2/TLR4-transfected human endothelial kidney (HEK)-293 cells acquired the capacity to internalize E. coli, whereas nontransfected HEK-293 cells and HEK-293 cells transfected with dominant-negative TLR4 bearing a P712H mutation did not. LPS did not induce membrane ruffling or macropinocytosis in enterocytes, excluding their role in bacterial internalization. Strikingly, the internalization of Gram-negative bacteria into enterocytes in vivo and the translocation of bacteria across the intestinal epithelium to mesenteric lymph nodes were significantly greater in wild-type mice as compared with mice having mutations in TLR4. These data suggest a novel mechanism by which bacterial translocation occurs and suggest a critical role for TLR4 in the phagocytosis of bacteria by enterocytes in this process.     

The Escherichia coli biofilm-promoting protein Antigen 43 does not contribute to intestinal colonization

Abstract Escherichia coli is a versatile organism capable of causing a variety of intestinal and extraintestinal diseases, as well as existing as part of the commensal flora. A variety of factors permit specific attachment to host receptors including fimbrial adhesins and outer membrane proteins such as autotransporters. One of the better characterized autotransporters is Antigen 43 (Ag43), the major phase-variable surface protein of E. coli. Ag43 is associated with bacterial cell-cell aggregation and biofilm formation. Nevertheless, the precise biological significance and contribution to intestinal colonization remain to be elucidated. Here we investigated the contribution of Ag43 to E. coli adherence to intestinal epithelial cells and colonization of the mouse intestine. These investigations revealed that Ag43 increased in vitro adherence of E. coli to epithelial cells by promoting bacterial cell-cell aggregation but that Ag43 did not promote specific interactions with the mammalian cells. Furthermore, Ag43 did not contribute significantly to colonization of the mouse intestine and expression of Ag43 was lost a few days after colonization of the mouse was established. Unexpectedly, considering its similarity to other adhesins, our findings suggest that Ag43 does not act as a direct colonization factor by binding to mammalian cells.