【作者】 郝小燕；

【导师】 曹虹；

【作者基本信息】 南方医科大学 ， 病原生物学， 2010， 硕士

【摘要】 一、研究背景和目的细菌性脑膜炎是新生儿时期中枢神经系统最常见最严重的感染。在过去三十年中,发达国家和发展中国家,细菌性脑膜炎发病率一直都没有明显变化,占新生儿疾病的1‰,但死亡率达17%-38%,未死亡者遗留神经系统后遗症者也高达58%。B族链球菌和大肠埃希菌是引起新生儿细菌性脑膜炎的最常见病原体,由于对B族链球菌携带孕妇的产时预防和对新生儿的抗生素选择性治疗,所以新生儿B族链球菌感染率明显减低,但同时也降低了新生儿对非B族链球菌感染的抵抗力,致使大肠埃希菌(E.coli)K1株成为目前新生儿细菌性脑膜炎的首要致病菌。大肠埃希菌(E. coli)K1株在40%败血症或80%脑膜炎患儿中都可分离到,它主要源于母亲肠道,孕妇因其特殊体质,肠道定殖的E. coli K1株容易易位于阴道,致使新生儿在通过产道时获得感染。研究表明新生儿20%肠道中有E. coli K1株定植,其中孕妇肠道有该菌定植的占77%。E. coli K1株一旦定植于新生儿肠道,0.5%可发生肠道侵袭性定位转移而入血,并穿过血脑屏障而引发脑膜炎。随着广谱抗生素的广泛应用,耐药性大肠埃希菌不断增多,致使新生儿尤其是低出生体重、极低出生体重儿的大肠埃希菌性脑膜炎病死率逐渐增高。抗生素耐药性已成为过去十年中一个严重的公共健康问题。补充和替代抗生素治疗的药物和方法研究也越来越受到关注。目前E. coli K1株如何通过肠屏障造成中枢神经系统感染,仍不清楚,但孕妇肠道易位于阴道的E.coli K1株,因发生了易位才由机会致病菌转变为致病菌,产生了致病性,所以调节肠道微生态平衡,抑制致病菌的肠道粘附侵袭,成为在孕妇阶段预防本病发生的关键步骤,另外,在新生儿脑膜炎的发生过程中,E. coli K1株首先定植于新生儿肠道,之后才会穿透肠屏障和血脑屏障,引起菌血症和脑膜炎,所以预防E. coli K1株穿透肠上皮入血,也成为有效预防其引发菌血症和脑膜炎的关键环节。研究证实益生菌可预防和治疗多种感染性疾病,其机制主要包括：直接与致病微生物相互作用,通过替代、排斥和竞争机制抑制致病菌生长和粘附定植；调节肠道微生态,促进内环境共生稳态；通过空间位点和营养竞争等阻断其对肠黏膜诱导破坏和血行转移,发挥其抗感染作用；诱导结肠Mucin基因表达的上调,肠道黏膜表面粘蛋白的分泌增多,从而拮抗致病菌的粘附侵袭和跨细胞易位转移。如将益生菌应用于早期新生儿脑膜炎的预防,可克服广谱抗生素的诸多缺点。最近已有研究首次证明乳酸菌LGG能显著抑制E. coli Kl株在新生大鼠肠道粘附与入侵从而显著降低菌血症与脑膜炎发生率。但是活菌片中益生菌如何粘附于肠黏膜,是否诱导上调粘液蛋白基因表达,和如何提高肠道微生态的稳定性,目前仍不清楚。本研究的主要目的：评价三联活菌片中的益生菌对大肠埃希菌K1株在肠上皮粘附的抑制作用和预防新生儿脑膜炎的保护作用。(1)应用SYBR Green Real-time PCR法,对小鼠肠道中的益生菌-双歧杆菌和保加利亚乳杆菌,以及致病菌-E. coli K1株,进行定量检测,观察小鼠肠道益生菌的定植和对致病菌粘附的拮抗作用。(2)对益生菌和对照组小鼠肠道菌群进行变性梯度凝胶电泳图谱(DGGE)分析,观察益生菌对肠道微生态的调节作用。(3)应用活菌计数法,对乳鼠肠道、血液和脑脊液中E. coli K1株进行定量检测,观察益生菌对E. coliK1株血行转移脑膜炎的拮抗作用。(4)采用竞争性排除方法,探讨益生菌抑制E. coli K1株粘附侵袭肠上皮Lovo细胞作用。通过检测细胞培养液中乳酸脱氢酶的释放量,观察益生菌对肠上皮细胞的保护作用和E.coli K1株对细胞膜通透性的损伤作用。(5)运用RT-PCR法验证益生菌对主要粘蛋白基因MUC2表达的调节作用。益生菌能否拮抗E.coli K1株诱导的粘蛋白基因表达下降,发挥其拮抗致病菌粘附侵袭和易位作用。通过以上研究,观察活菌片中益生菌能否调节肠道微生态,初步探讨益生菌抑制致病菌的粘附、侵袭和预防血行转移入脑是否与调节肠道粘液蛋白基因表达相关。二、研究方法1、小鼠模型观察益生菌对致病菌的粘附抑制作用提取活菌片中双歧杆菌、保加利亚乳杆菌、和E. coli K1株基因组DNA,双歧杆菌、保加利亚乳杆菌引物参照文献,E. coli K1株针对IbeA基因设计引物PCR,PCR产物克隆入pMD19-T Simple Vector载体中,转化至DH-5a宿主细胞,提取质粒倍比稀释后作标准品,将雌性BALB/c小鼠10只,用活菌片PBS稀释液灌胃,连续14天,每天取肠道标本连续观察益生菌定殖情况。根据益生菌肠道定殖时间,将雌性BALB/c小鼠40只,随机分成四组,①益生菌组(L)、②益生菌+致病菌组(L+P)、③致病菌组(P)、④对照组(N),留取各组不同时间粪便标本。抽提其细菌DNA,进行Real-time PCR反应,观察益生菌肠道的粘附及拮抗致病菌粘附侵袭作用。2、益生菌对肠道微生态的影响以益生菌组和对照组小鼠粪便标本总细菌为模板,以肠道总细菌16S rDNA可变区V3区为靶基因,引物序列V3-357f-GC;V3-R519,进行PCR扩增和DGGE分析。观察益生菌的肠道微生态的调节作用。3、益生菌抑制E. coli K1株粘附、侵袭、和损伤肠上皮细胞作用参照文献采用竞争性排斥,将益生菌、E. coli K1株与Lovo细胞共孵育,检测益生菌拮抗E.coli K1株粘附与侵袭Lovo细胞的效果。设细胞只单纯孵育E.coliK1株的为对照组,计数其粘附和侵袭的菌落数,并分别计算益生菌干预下E. coliK1株粘附与侵袭的菌落数和相对黏附率、相对侵袭率。公式为相对粘附或侵袭率=(益生菌组的菌落数／对照组的菌落数)×100%将益生菌、E. coli K1株同时和分别与Lovo细胞共孵育,通过检测细胞培养液中乳酸脱氢酶的释放量,以观察益生菌对细胞保护和细菌对细胞膜通透性的损伤作用。4、E.coliK1株肠道血源性转移的新生大鼠模型参照文献将Sprague Dawley乳鼠随机分成益生菌组和PBS组,每组15只,分别用益生菌和PBS处理后,灌胃E. coli K1株,取肠道、血液和脑脊液标本进行定量培养,涂布于利福平抗性平板,血标本同时涂布MRS平板过夜培养并计菌落数。观察益生菌对血行转移细菌性脑膜炎的预防作用。5、益生菌和E. coli K1株影响肠道MUC2基因表达把Sprague Dawley乳鼠随机分成益生菌组、益生菌+致病菌组、致病菌组和对照组,每组3只分别用益生菌和PBS处理3天后,给乳鼠灌胃用E. coli K1株,取肠道提取结肠组织总RNA,采用GAPDH基因做内参,用RT-PCR法观察益生菌和致病菌的诱导肠道MUC2基因表达的作用。三、研究结果1、小鼠模型观察益生菌对致病菌的粘附抑制作用的研究结果表明,灌胃益生菌组小鼠肠道中的双歧杆菌和保加利亚乳杆菌基因拷贝数在第3天时开始明显增加,在第7天时达到稳定,且随服药天数的增加两种益生菌基因拷贝数没有明显变化；灌胃益生菌和致病菌组小鼠肠道中E. coli K1株基因拷贝数明显低于单独灌胃致病菌组。2、变性梯度凝胶电泳图谱(DGGE)分析显示,正常BALB/c小鼠肠道细菌的多样性相似,说明用其作为动物模型对肠道菌群进行相关研究具备可行性。用非权重配对法(UPGMA)进行相似性聚类分析,提示服药后益生菌组组内个体相似性较对照组提高,说明服用益生菌可提高肠道菌群构成的一致性和肠道微生态的稳定性,可减少宿主及环境的很多因素对肠道微生态的影响。3、益生菌抑制E. coli K1株粘附、侵袭和损伤肠上皮细胞的研究显示,益生菌能显著抑制E. coli K1株粘附Lovo细胞(P<0.01),且呈剂量依赖性。益生菌对E. coli Kl株侵袭的抑制也呈剂量依赖性(P<0.01)。E. coli K1株对Lovo细胞的粘附导致较高水平的LDH释出,明显高于益生菌对Lovo细胞所造成的影响,益生菌与致病菌共同粘附于Lovo细胞后,LDH的释放虽高于益生菌组,却明显低于致病菌组粘附引起的LDH的释放。4、在E. coli K1株肠道血源性转移的新生大鼠模型中,将益生菌+致病菌组乳鼠用益生菌处理后再灌胃E.coli K1 (109CFU／只),同时设只灌胃E. coli Kl株的对照组,48 h后取肠道、血液、脑脊液标本检测。结果发现,益生菌+致病菌组乳鼠肠道定植的E.coli K1株数量显著少于对照组(P<0.05),未发生菌血症,脑脊液中也未检测到E. coli K1株。对照组灌胃给予E. coli K1株(109CFU／只)后,15只乳鼠中9只出现菌血症(>105 CFU/mL),2只脑脊液中出现E. coli K1株。5、用RT-PCR法对益生菌拮抗致病菌粘附侵袭的机制进行了研究。提取各组乳鼠肠道组织总RNA,采用GAPDH基因做内参,RT-PCR法观察益生菌和致病菌诱导结肠Mucin基因的表达。结果显示灌胃益生菌组乳鼠肠道MUC2基因明显上调,而灌服E. coli K1组的MUC2基因表达则显著降低,共同灌服组基因表达无明显变化。说明益生菌诱导的MUC2基因表达上调可能成为其拮抗致病菌易位的保护机制之一。四、结论1、活菌片中益生菌能粘附于肠黏膜,抑制大肠埃希菌K1株肠道粘附定植。2、活菌片中益生菌可提高肠道微生态的稳定性。3、活菌片中益生菌能显著抑制大肠埃希菌K1株对肠上皮的粘附、侵袭和损伤,以及血行转移,可能有预防细菌性脑膜炎发生的作用。4、活菌片中益生菌可上调肠道粘液蛋白基因表达,保护肠道免受致病菌的损伤,这可能成为拮抗致病菌粘附侵袭和易位的保护机制之一。更多还原

【Abstract】 Background and objectiveBacterial sepsis and meningitis in neonates are the most common serious infections in the world.Over the last 30 years, morbidity of bacterial sepsis and meningitis has not changed substantially despite advances in antimicrobial chemotherapy and supportive care.Neonatal infection with Escherichia coli (E. coli) K1 occurs in one in 1000 live births. But it is a potentially devastating illness that is associated with 17%-38% mortality and 58% long-term neurological sequelae. Currently, E. coli is a leading Gram-negative bacterium causing neonatal bacterial meningitis (NBM).Approximately 40% of septicemia isolates and 80% of meningitis isolates from neonates are K1-producing strains. Meningitis-causing strains of E. coli K1 is mainly from the mother intestinal, which is usually transferred from maternal to infant gastrointestinal tract at the time of parturition and there are approximately 20% of newborns colonized with E. coli Kl in their stool.The intestinal colonization of neonates with E. coli Kl occurs most frequently by perinatal acquisition from the mother:77% of infants had positive stool cultures for K1 by the second day of life when they born to Kl colonized mothers.Once E. coli K1 colonized, approximately one in 200 infants develops an invasive infection and NSM.E.coli K1 colonized in neonatal intestine is mainly from the mother intestinal, so, it is important to inhibit strongly pathogens adherence ability and improve the intestinal symbiotic homeostasis.Because the primary site of K1 colonization is in the gastrointestinal tract, K1 penetration of the gastrointestinal epithelium and subsequent translocation to the blood-stream would presumably have to occur for bacteremia to develop, which need to protect intestinal epithelial cell,inhibit strongly the meningitic pathogens and intestinal hematogenous metastasis.Studies demonstrated that probiotics have shown a protective effect for the prophylaxis of infectious diseases.Multiple mechanisms of probiotic therapy have been postulated, including the production of antimicrobial agents, competition for space or nutrients, immunomodulation and the decline in bacterial translocation (BT) by probiotics is mediated by up-regulation of epithelial MUC-2.Studies demonstrated that this particular Lactobacillus strain could prevent the onset of urogenital infections by interfering with the epithelial colonization by uropathogenic Staphylococcus. aureus.However, it is unknown whether probiotics could colonize in intestinal mucosal, inhibit strongly pathogens adherence ability and improve the intestinal symbiotic homeostasis and whether probiotics are effective in preventing NSM.In this work, our purpose is to evaluate the potential inhibition of the probiotics in the adherence of E. coli K1 to murine intestinal mucosa. SYBR Green Real-time PCR was used to detect quantitatively Bifidobacteria and Lactobacillus bulgaricus in probiotics as well as pathogens (E. coli K1)in mice of the above groups administered orally. The murine intestinal microbiota of probiotics group was examined by denaturing gradient gel electrophoresis.The viable bacteria counting was used to detect quantitatively E. coli K1 in the intestinal colonization and metastasis in rat pubs and to determine the number of CFU of E. coli K1 adhesion and invasion to Lovo cells that were incubated with probiotics in vitro in competitive exclusion assays, respectively. The RT-PCR was used to further investigate the impact of the bacterial as an environmental factor affecting mucin gene expression. We used rat pups associated with microbiota of different compositions.Gene modulation in rats fed by probiotics or E. coli K1 was observed.Methods1、Probiotics in live tablets could inhibit strongly pathogens adherence abilityAccording to the E. coli K1 gene IbeA, we designed a pair of primers.DNA primers of Bifidobacteria and Lactobacillus bulgaricus gene 16S rDNA were obtained according to the reference.These primers were used to amplify the 16S rDNA and IbeA from the extraction of probiotics in live tablets and E. coli K1 genomic DNA via PCR. The production were cloned into pMD19-T and transformatics in E. coli DH-5αto amplify. Extraction of plasmid as a standard product.Ten female BALB/c mice were administered orally with probiotics for 14 days. The intestinal colonization probiotics were determined at every day. According to the intestinal colonization time of probiotics, Female BALB/c mice randomly assigned to the following experimental groups;(Ⅰ)probiotics group,(Ⅱ)probiotics + pathogen group, (Ⅲ) pathogen group, and (Ⅳ) the non-treated group as control.Caecal samples in mice of the above groups administered orally were taken. DNA level of Bifidobacteria and Lactobacillus bulgaricus in probiotics were determined by quantitative PCR using SYBR Green dye.2、The impact of probiotics on the intestinal microfloraMicrobial DNA was extracted from feces in mice of the probiotics group and control group, and the pair of primers (V3-357f-GC;V3-R519) were used to amplify the V3 region of the 16S rDNA gene was amplified by PCR and analyzed by DGGE3、The inhibition of the probiotics in the adherence and invasion of E. coli K1 to murine intestinal mucosa of Lovo cellsDifferent doses of probiotics and the same doses of E. coli K1 strain were added to the wells coated with Lovo cells.The effect of probiotic on the adhesion and invasion of pathogens to Lovo cells was examined by competitive exclusion assays.The relative adhesion and invasion rate of E. coli K1 was calculated by [100%×(number of intracellular bacteria recovered in probiotics+E. coli K1 group)/(number of intracellular bacteria recovered in E. coli K1 group)].The release of LDH from Lovo cells which were differently treated with probiotics and E. coli K1 were determined by using commercially available Kit.4、Neonatal rat model of probiotics on prevention E. coli K1 MeningitisTo investigate the role of probiotics on prevention E. coli K1 Meningitis, the rat pups were randomly divided into the probiotics group and control group. Pups at 2 days of age were infected with probiotics in the probiotics group.Control rats received PBS only. All pups at 5 days of age were fed by E. coli K1.The stool,bloods and CSF specimen were cultured in a rifampin-resistant agar plates to determine the number of colony forming units (CFUs) of E. coli K1.The bloods Samples also were cultured in MRS plates to determine the number of CFUs of probiotics.5、Probiotics up-regulate MUC-2 mucin gene expressionThe rat pups were randomly divided into the probiotics group and control group. Pups at 2 days of age were infected with probiotics in the probiotics group. Control rats received PBS only. All pups at 5 days of age were administered orally by E. coli K1.After a 5-day probiotic and 1-day E. coli K1 treatment, total RNA was isolated from each of the treated Intestine samples Kit, Then, cDNA was amplified.The RT-PCR products were electrophoresed in 2% agarose gel and visualized via image analysis software.Result1、DNA level of Bifidobacteria and Lactobacillus bulgaricus in probiotics increased significantly in the third day and chieved stability in the seventh day. The level of DNA of E. coli K1 in the probiotics combined with pathogens group was decreased remarkably when compared to the pathogens group only.2、Denaturing gradient gel electrophoresis analysis demonstrated that no differences were detected in the composition of the overall fecal bacterial community. The UPGMA algorithm was used for construction of dendrograms, as implemented in the software and demonstrated the greater homogeneity of intestinal flora after administration with probiotics.3、The viable bacteria counting was applied to determine the number of E. coli K1 adhesion and invasion to Lovo cells that were incubated with probiotics in vitro in the competitive exclusion assays.The probiotics were found to significantly decrease the adhesion and invasion of pathogens to Lovo cells in a dose-dependent manner in vitro(P<0.01).The amounts of LDH released from Lovo cells suggested that the adherence of probiotics were essentially different with E. coli K1.The adhesion of probiotics could prevent the harmful effect of E. coli K1 on Lovo cells(P<0.01).4、The viable bacteria counting was used to detect quantitatively pathogens (E. coli K1)in the intestinal colonization and metastasis in rat pubs of the above groups administered orally. In the probiotics group, no E. coli K1 was founded in CSF specimens, no bacteremia occurred and the number of intestinal E. coli K1 in the probiotics combined with pathogens group was decreased remarkably when compared to the control group only (P<0.01).The cultures of probiotics were also done with the blood samples from the pups feeding probiotics. No probiotics was detected.5、The mRNA levels of MUC2 mucin were upregulated in the probiotics group, In rats fed with E. coli K1,MUC2 gene expression was lower than in control animals, Conversely, no changes in mucins were detected in rats fed intragastrically by probiotics and E. coli K1.Conclusion1、Probiotics in live tablets could colonize in intestinal mucosal, inhibit strongly pathogens adherence ability.2、Probiotics could improve the intestinal symbiotic homeostasis.3、Probiotics could protect intestinal epithelial cell,inhibit strongly the meningitic pathogens injury and intestinal hematogenous metastasis.4、These results indicated that the expression of MUC2 in rat colon could be selectively stimulated by probiotics and these rats may up-regulate Mucin synthesis as a defense mechanism.更多还原