健康老年人肠道双歧杆菌和乳杆菌种群多样性分析

从31个60岁以上的符合中华医学会老年医学分会健康老年人标准的健康老人中随机选取4例作为研究对象,使用分子生物学方法,对他们的肠道双歧杆菌和乳杆菌种群多样性进行分析.研究结果表明,健康老人肠道中双歧杆菌的优势种群为长双歧杆菌（Bifi∥dobacterium longum）和假小链双歧杆菌（Bifidobacterium pseudocatenulatum）,分别占双歧杆菌种群的55%和45%;而肠道乳杆菌则有唾液乳杆菌（Lactobacillus salivarius）50%、Lactobacillus mocosae 31.1%、口腔乳杆菌（Lactobacillus oris）6.3%、鼠李糖乳杆菌（Lactobacillus rhamnosus）6.3%和瑞士乳杆菌（Lactobacillus helveticus）6.3%,其中唾液乳杆菌、Lactobacillus mocosae为健康老人肠道优势乳杆菌种群.     

成都地区母乳双歧杆菌对婴儿肠道双歧杆菌构建的影响

目的 观察不同喂养方式婴儿肠道双歧杆菌的构建规律，初步探索母乳双歧杆菌与婴儿肠道双歧杆菌之间的相关性。方法 在四川大学华西第二医院纳入57对母婴志愿者，根据喂养方式分为母乳（BF）组（n=31）和混合喂养（MF）组（1月龄内添加奶粉时间>8 d,n=26），收集产妇产后0天、第30天的母乳，以及婴儿出生后0天、第7天、第30天的粪便。采用实时荧光定量PCR测定母乳和婴儿粪便中双歧杆菌属、角双歧杆菌（B. angulatum）、链双歧杆菌（B. catnulatum）、齿双歧杆菌（B. dentium）、短双歧杆菌（B.breve）、两歧双歧杆菌（B. bifidum）、长双歧杆菌长亚种（B. longum subsp. longum）、长双歧杆菌婴儿亚种（B.longum subsp. infantis）及青春双歧杆菌（B. adolescentis）的丰度。观察2组婴儿肠道双歧杆菌0―7―30天的构建规律；分析母乳双歧杆菌与婴儿肠道双歧杆菌之间的相关性。结果 0―7―30天2组婴儿肠道双歧杆菌构建过程存在差异。母乳双歧杆菌与婴儿肠道双歧杆菌存在相关：0 d时，BF组母乳B. a...     

婴儿肠道细菌及乳杆菌种群多样性研究

目的 对婴儿肠道细菌及乳杆菌组成进行分析。方法 以66例出生1～7d的婴儿为调查对象,用分子生物学的手段,借助肠道细菌DNA指纹图谱和16S rDNA PCR扩增克隆分类（RFLP）技术。结果 婴儿肠道菌群随出生天数增加而变化;乳杆菌特异性引物对PCR扩增结果表明,出生4h后乳杆菌已在婴儿肠道内定植,且定植时间与分娩方式无关;克隆多样性研究则显示婴儿肠道乳杆菌优势菌群为植物乳杆菌（74％）、嗜酸乳杆菌（14％）和约氏乳杆菌（12％）。结论 婴儿肠道中的优势乳杆菌主要为植物乳杆菌。     

双歧杆菌与肠道黏膜免疫关系的研究现状

双歧杆菌（Bifidobacterium）是人体肠道内重要的生理菌,它在机体内的多少是衡量健康状况的指标之一。众多研究表明,双歧杆菌具有维持肠道微生态平衡、生物拮抗、免疫、抗衰老、抗肿瘤和营养等功能,在人体健康和疾病方面起着重要作用。新生儿出生时,肠道是无菌的,但在出生后不久,外界的菌就逐渐进入体内,有的菌和宿主细胞有一定的亲和力,通过粘附和定植在肠道定居,成为肠道的正常菌群。     

酪酸梭菌与婴儿型双歧杆菌对产气荚膜梭菌试管内生物拮抗作用的研究

用酪酸权菌（Clostridium butyricum）和婴儿型双歧杆菌（Bifidobacterium Infantis）对产气荚膜梭菌（Clostriduim perfringens）进行试管内的生物拮抗试验。将酪酸梭菌、婴儿型双歧杆菌及酪酸梭菌和婴儿型双歧杆菌分别对产气荚膜酸菌以一定的比例等量混合接种于GAM液体培养基中进行厌氧培养。实验证明酪酸梭菌和婴儿型双歧能明显抑制产气荚膜梭菌的生长,并且比各自单独培养时显示了较强的生物拮抗作用。     

双歧杆菌的有关酶系

按照悉生生物学理论,肠内菌群对宿主一人的健康有重大的影响,双歧杆菌（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ）是典型的能在健康人肠道内定植的有益菌在母乳喂养的婴儿肠道中,该菌占菌群总数的９２％以上,两瓶养婴儿粪便中优势菌则为嗜乳酸杆菌（Ｌａｃｔｏｂｃｉｌｌｕｓａｃｉｄｏｐｈｉｌｌｕｍ）,现已确认双歧菌数量是衡量婴儿健康状况的重要指标之一［１］．在健康成年和长寿老人肠道中双歧苗也是优势菌,某些动物如猪、狗、老鼠、蜜蜂等,消化道中也存在大量双歧杆菌。双歧杆菌数量随年龄变化而呈动态变化趋势,并与机体的许多生理、…     

婴儿肠道双歧杆菌多样性的研究进展

双歧杆菌是婴儿肠道中最丰富的微生物,对婴儿肠道微生物的成熟和稳定有显著影响,与婴儿健康紧密相关。大量研究表明,婴儿肠道中最广泛存在的双歧杆菌有长双歧杆菌、短双歧杆菌和两歧双歧杆菌。母婴之间垂直传递可直接影响婴儿肠道中双歧杆菌各种属的早期定植和相对丰度变化,特别是分娩方式和喂养方式会显著影响婴儿肠道双歧杆菌。此外,胎龄和辅食等因素对婴儿肠道中双歧杆菌的组成与多样性也有一定影响。本文对婴儿肠道双歧杆菌的种属组成、相对丰度变化以及影响其多样性的因素进行了综述,为婴儿双歧杆菌群落的认识提供一定的理论依据。     

酪酸梭菌与婴双歧杆菌对艰难梭菌体外生物拮抗作用的作用

用酪酸梭菌（Clostridium butyricum)和婴双歧杆菌（Bifidobacterium infantis)单独或联合艰难菌（Clostridium difficile)进行试管内的生物拮抗作用,将酪酸梭菌、婴儿双歧杆菌单独或酪酸梭菌和婴儿双歧杆菌联合分别与艰难梭菌以一定的比例等量混合后,接种于GAM液体培养基中进行厌氧培养。证明酪酸梭菌和婴儿双歧杆菌能明显抑制艰难梭菌的生长,并且两菌联合比各自单独培养时显示出更强的生物拮抗作用。     

酷酸梭菌——婴儿型双歧杆菌二联活菌制剂对人体肠道菌群的调节试验

目的 观察酷酸梭菌－－婴儿型双歧杆菌二联活菌制剂对受试人群肠道菌群的影响。方法 检测受试者服用前后的肠道菌群并计数。结果 肠杆菌、肠球菌、拟杆菌的数量无明显变化,双歧杆菌、乳杆菌的数量明显增加,差异有非常显著性（Ｐ〈０．０１）,结论 酪酸梭菌－－婴儿型双歧杆菌二联活菌制剂具有一定的调节人体肠道菌群、增殖双歧杆菌和乳杆菌的作用。     

大豆多糖对双歧杆菌及人肠道菌群生长的影响

目的研究大豆多糖对双歧杆菌及肠道菌群生长的影响。方法替换Bs培养基中的碳源,分为不加糖、加葡萄糖2％、加大豆多糖2％、加大豆多糖5％、加低聚果糖2％五组,加3种双歧杆菌（长双歧、青春双歧、两歧双歧）菌液1％,测其24h后的活菌数,比较大豆多糖对双歧杆菌生长的影响;替换Bs培养基中的碳源,分为不加糖、加葡萄糖2％、加大豆多糖2％,加低聚果糖2％四组,加人体粪便菌液1％,模拟人体肠道环境厌氧培养24h后,用选择性培养基测其肠杆菌、肠球菌、双歧杆菌、乳酸杆菌的活菌数,观察大豆多糖对人体肠道菌群的影响。结果大豆多糖添加量为5％时对长双歧的促进作用明显优于不加糖组（P〈0．05）;大豆多糖对人体肠道各菌群的生长促进作用与低聚果糖差异无显著性（P〉0．05）。结论大豆多糖对长双歧杆菌的体外促进作用较明显;以粪菌群发酵糖试验表明,大豆多糖对乳杆菌和双歧杆菌均有促进作用,和低聚果糖作用效果相比差异无显著性（P〉0．05）,具有益生元的特性。     

Comparison of mucosal adhesion and species identification of bifidobacteria isolated from healthy and allergic infants

Fifty bifidobacteria strains were isolated from fecal samples of allergic and age matched healthy infants. Allergic infants were found to have an adult type Bifidobacterium flora with high levels of Bifidobacterium adolescentis. Healthy infants had a typical infant Bifidobacterium flora with high levels of Bifidobacterium bifidum. These isolates were tested for their adhesive properties to human intestinal mucus. The adhesion of the fecal bifidobacteria from healthy infants was significantly higher (P<0.0001) than for allergic infants. This suggests a correlation between allergic disease and the composition of the intestinal bifidobacteria flora which has reduced adhesive abilities to the intestinal mucus. Therefore, dietary supplementation of bifidobacteria typical for healthy infants, may be beneficial in the treatment of allergic disorders.     

儿童肠道双歧杆菌和乳杆菌种群结构分析

以21例2～5岁中国儿童的肠道菌群为研究对象,利用传统培养计数法和分子生物学技术,对此年龄段健康儿童的肠道菌群分布及其中关键益生菌的种群结构进行了定量研究。实验表明,儿童肠道厌氧菌的数量高达109CFUg(湿重),其肠道菌群的定植抗力(平均BE=2.38)较强;不同的个体之间所能检测到的关键益生菌的种类有所不同,一般能检测到其中的1～4种双歧杆菌和1～5种乳杆菌;长双歧杆菌和假小链双歧杆菌的平均数量多达107CFUg(湿重),检出率分别为90.48%和85.71%,为儿童肠道内双歧杆菌的优势菌种;L.mucosae和发酵乳杆菌的数量较多,平均为3.68log10CFUg(湿重)和3.97log10CFUg(湿重),检出率分别为71.43%和52.38%,为稳定定植于儿童肠道内的优势乳杆菌;不同种类的益生菌在不同样本之间的数量组成均存在有很大差异,双歧杆菌的样本差异为1.86～3.85,乳杆菌的为2.43～4.07。     

健康蒙古族人肠道中乳酸菌和双歧杆菌多样性

【背景】越来越多的研究发现人类的诸多疾病与肠道菌群失衡有关。乳酸菌和双歧杆菌属于肠道中的有益菌,在不同人群肠道中的多样性不尽相同。【目的】在种水平上分析健康蒙古族人群肠道菌群中乳酸菌和双歧杆菌的多样性。【方法】以27名健康蒙古族志愿者为研究对象,其中14名来自中国内蒙古,13名来自蒙古国。首次采用乳酸菌和双歧杆菌的特异性引物扩增与PacBioSMRT三代测序技术相结合,在种水平上探讨志愿者肠道中乳酸菌和双歧杆菌的丰度和生物多样性,并进一步分析性别、BMI(Bodymassindex)值和地域对上述两者可能的影响,以及优势菌种之间的相关性。【结果】在种的水平上,27名志愿者肠道样品中共鉴定到68个乳酸菌和11个双歧杆菌,其中平均相对含量在1%以上的乳酸菌有8个,包括唾液链球菌(Streptococcus salivarius,36.41%)、瘤胃乳酸杆菌(Lactobacillus ruminis,17.94%)、德氏乳杆菌(Lactobacillus delbrueckii,3.11%)、罗氏乳杆菌(Lactobacillus rogosae,2.23%)、轻型链球菌(Streptococcus mitis,2.18%)、阴道乳杆菌(Lactobacillus vaginalis,2.02%)、魏斯氏乳杆菌(Weissella confusa,1.54%)和鼠李糖乳杆菌(Lactobacillus rhamnosus,1.09%);双歧杆菌有5个,包括青春双歧杆菌(Bifidobacterium adolescentis,39.88%)、长双歧杆菌(Bifidobacterium longum,27.15%)、链状双歧杆菌(Bifidobacterium catenulatum,26.30%)、两歧双歧杆菌(B. bifidum,3.92%)和角双歧杆菌(Bifidobacterium angulatum,1.71%),聚类分析分为链状双歧杆菌和青春双歧杆菌2个主要的类群。分析结果显示:性别、BMI值和地域均未能显著影响志愿者肠道中乳酸菌和双歧杆菌的菌群结构(P0.05),但男性和女性之间、中国内蒙古地区和外蒙古国的志愿者之间的个别乳酸菌菌种相对含量存在显著差异(P0.05)。对样品中的优势乳酸菌和双歧杆菌进行Spearman相关性分析发现,乳酸菌和双歧杆菌彼此之间相关性较为密切,不同菌种间相关性不尽相同,与具体的菌种有关。【结论】首次采用PacBio SMRT测序技术在种的水平揭示了健康蒙古族人肠道中乳酸菌和双歧杆菌菌种多样性,为在种水平上解析肠道中乳酸菌和双歧杆菌多样性提供了新的研究思路和实施方案。     

PCR and real-time PCR primers developed for detection and identification of <Emphasis Type="Italic">Bifidobacterium thermophilum</Emphasis>in faeces

Background  

Culture-independent methods based on the 16S ribosomal RNA molecule are nowadays widely used for assessment of the composition of the intestinal microbiota, in relation to host health or probiotic efficacy. Because Bifidobacterium thermophilum was only recently isolated from human faeces until now, no specific real-time PCR (qPCR) assay has been developed for detection of this species as component of the bifidobacterial community of the human intestinal flora.     

Quantitative PCR with 16S rRNA-gene-targeted species-specific primers for analysis of human intestinal bifidobacteria

A highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR with Bifidobacterium genus- and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 10(6) to 10 cells per PCR assay. It was also found that the method was applicable to the detection of Bifidobacterium in feces when it was present at concentrations of >10(6) cells per g of feces. Concerning the distribution of Bifidobacterium species in intestinal flora, the Bifidobacterium adolescentis group, the Bifidobacterium catenulatum group, and Bifidobacterium longum were found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition of Bifidobacterium species in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period.     

婴幼儿腹泻患者肠道菌群分布特点研究

目的研究不同年龄段腹泻患儿肠道菌群分布特点,探讨不同年龄腹泻患儿肠道菌群与疾病的关联。方法选取9例符合临床诊断标准的0~1岁婴儿腹泻患者和8例符合临床诊断标准的1~3岁幼儿腹泻患者的粪便样本,同时于健康儿童中随机选取6例粪便样本作为对照,提取各组对象粪便总DNA,采用PCR-DGGE进行菌群多样性与差异性分析。结果 0~1岁腹泻患儿肠道菌群与健康对照组相比,肠道菌群构成差异显著,条件致病菌巴黎链球菌数量显著增加。1~3岁腹泻患儿肠道菌群与健康对照组相比,条件致病菌解没食子酸链球菌、屎肠球菌数量显著增加,长双歧杆菌数量下降。结论婴幼儿腹泻患者肠道菌群的构成与健康对照组相比差异显著,该特点可作为婴幼儿腹泻早期诊断的实验依据。     

Mother-to-Infant Transmission of Intestinal Bifidobacterial Strains Has an Impact on the Early Development of Vaginally Delivered Infant's Microbiota

Objectives

Bifidobacterium species are one of the major components of the infant''s intestine microbiota. Colonization with bifidobacteria in early infancy is suggested to be important for health in later life. However, information remains limited regarding the source of these microbes. Here, we investigated whether specific strains of bifidobacteria in the maternal intestinal flora are transmitted to their infant''s intestine.

Materials and Methods

Fecal samples were collected from healthy 17 mother and infant pairs (Vaginal delivery: 12; Cesarean section delivery: 5). Mother''s feces were collected twice before delivery. Infant''s feces were collected at 0 (meconium), 3, 7, 30, 90 days after birth. Bifidobacteria isolated from feces were genotyped by multilocus sequencing typing, and the transitions of bifidobacteria counts in infant''s feces were analyzed by quantitative real-time PCR.

Results

Stains belonging to Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium catenulatum, Bifidobacterium longum subsp. longum, and Bifidobacterium pseudocatenulatum, were identified to be monophyletic between mother''s and infant''s intestine. Eleven out of 12 vaginal delivered infants carried at least one monophyletic strain. The bifidobacterial counts of the species to which the monophyletic strains belong, increased predominantly in the infant''s intestine within 3 days after birth. Among infants delivered by C-section, monophyletic strains were not observed. Moreover, the bifidobacterial counts were significantly lower than the vaginal delivered infants until 7 days of age.

Conclusions

Among infants born vaginally, several Bifidobacterium strains transmit from the mother and colonize the infant''s intestine shortly after birth. Our data suggest that the mother''s intestine is an important source for the vaginal delivered infant''s intestinal microbiota.     

Quantitative PCR with 16S rRNA-Gene-Targeted Species-Specific Primers for Analysis of Human Intestinal Bifidobacteria

A highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR with Bifidobacterium genus- and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 10⁶ to 10 cells per PCR assay. It was also found that the method was applicable to the detection of Bifidobacterium in feces when it was present at concentrations of >10⁶ cells per g of feces. Concerning the distribution of Bifidobacterium species in intestinal flora, the Bifidobacterium adolescentis group, the Bifidobacterium catenulatum group, and Bifidobacterium longum were found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition of Bifidobacterium species in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period.     

Molecularly assessed shifts of Bifidobacterium ssp. and less diverse microbial communities are characteristic of 5-year-old allergic children

The composition of intestinal microbiota and the Bifidobacterium group community in 20 allergic and 20 nonallergic 5-year-old children was visualized by PCR-denaturing gradient gel electrophoresis (DGGE). The number of dominant bands in the DGGE profiles was smaller in allergic children than in nonallergic children (P<0.001). Allergic children mainly formed a single group upon cluster analysis, whereas nonallergic children were divided between four different groups. In allergic children the Bifidobacterium adolescentis species prevailed, and in nonallergic children the Bifidobacterium catenulatum/pseudocatenulatum prevailed (P=0.01 and P=0.01, respectively). The less diverse composition of intestinal microbiota and prevalence of particular species of Bifidobacterium were characteristic of allergic children even at the age of 5 years.