思连康治疗儿童功能性消化不良临床分析

目的探讨思连康治疗儿童功能性消化不良的效果和临床应用安全性。方法采用随机、对照方法,使用思连康治疗3—14周岁患有儿童功能性消化不良症状的患儿各70例。所有参加临床验证的患儿均严格按照人选标准、排除标准选择病例,试验方法和给药方法完全统一。观察日服药物后的起效时间及第6、10天的症状缓解率。结果服药后上腹痛、嗳酸及上腹不适、烧心感症状的半小时缓解率,思连康治疗组优于多酶片对照组,分别为41．42％和11．42％、45．71％和17．14％、35．71％和15．71％（P均〈0．01）。思连康治疗组治疗后的第6天与第10天,临床症状总积分较治疗前显著下降（P〈0．01）,有效率分别为80％和75．71％,均高于多酶片对照组（38．57％、48．57％,P均〈0．01）,其中上腹痛、上腹不适与烧心症状的改善更为明显。整个疗程中,思连康治疗组未发现任何不良反应。结论思连康对儿童功能性消化不良症状具有显著疗效,本试验期间未观察到任何毒副作用,口味香甜,受到患儿欢迎。     

长双歧杆菌NCC2705株果糖结合蛋白BL0033基因的克隆及其在大肠杆菌中的表达

目的：克隆长双歧杆菌NCC2705株果糖结合蛋白BL0033的基因,利用大肠杆菌表达GST-BL0033融合蛋白并纯化。方法：以长双歧杆菌NCC2705株基因组为模板,PCR扩增BL0033基因,并将其插入pGEX-4T-1表达载体,转化至大肠杆菌DH5α;提取质粒,经PCR、质粒双酶切及测序鉴定后,转入大肠杆菌BL21,并对表达条件进行摸索;用谷胱甘肽-Sepharose4B树脂对可溶性GST-BL0033融合蛋白进行纯化。结果：PCR扩增的BL0033基因长度接近1000bp,与预期值一致;重组菌在IPTG浓度为0.05mmoL/L的条件下,于16℃诱导过夜后,SDS-PAGE分析可见可溶性表达条带,相对分子质量约60×103,与预期值一致;亲和纯化后,SDS-PAGE结果显示单一的表达条带。结论：克隆了BL0033蛋白的基因,并表达纯化了融合蛋白GST-BL0033,为进一步研究长双歧杆菌NCC2705株BL0033蛋白功能奠定了基础。     

青春双歧杆菌的波状层次生长特征观察

通过观察青春双歧杆菌在体外培养条件出现波状层次生长的情况,了解该过程中青春双歧杆菌形态的改变及其生物学意义。采用青春双歧杆菌低浓度接种于BS固体平板培养基中,延长培养时间至6 d,使其单个菌落长成较大的菌落后,置4℃冰箱中2~4周,在其培养结束时和放置冰箱后对菌落及菌体形态特征进行观察。在特定的培养时间及其后的低温处理后,青春双歧杆菌部分菌落出现了半透明环和不透明环交替的层次状结构,在半透明环区域和不透明环区域,菌体的形态以及对温度的敏感性存在差异,且在透明环区域,菌体发生了自溶现象。证明青春双歧杆菌在一定条件下形成了波状层次生长过程,且在该过程中,青春双歧杆菌的生长、生存方式以及增殖方式均发生了适应性变化。     

Identification and characterization of a sulfoglycosidase from Bifidobacterium bifidum implicated in mucin glycan utilization

Human gut symbiont bifidobacteria possess carbohydrate-degrading enzymes that act on the O-linked glycans of intestinal mucins to utilize those carbohydrates as carbon sources. However, our knowledge about mucin type O-glycan degradation by bifidobacteria remains fragmentary, especially regarding how they decompose sulfated glycans, which are abundantly found in mucin sugar-chains. Here, we examined the abilities of several Bifidobacterium strains to degrade a sulfated glycan substrate and identified a 6-sulfo-β-d-N-acetylglucosaminidase, also termed sulfoglycosidase, encoded by bbhII from Bifidobacterium bifidum JCM 7004. A recombinant BbhII protein showed a substrate preference toward 6-sulfated and 3,4-disulfated N-acetylglucosamines over non-sulfated and 3-sulfated N-acetylglucosamines. The purified BbhII directly released 6-sulfated N-acetylglucosamine from porcine gastric mucin and the expression of bbhII was moderately induced in the presence of mucin. This de-capping activity may promote utilization of sulfated glycans of mucin by other bacteria including bifidobacteria, thereby establishing the symbiotic relationship between human and gut microbes.     

Oral administration of Bifidobacterium longum prevents gut-derived Pseudomonas aeruginosa sepsis in mice

Aims: The aim of the study was to evaluate the efficacy of probiotics on gut‐derived sepsis caused by Pseudomonas aeruginosa in immunocompromised mice. Methods and Results: After oral inoculation of P. aeruginosa, mice were treated with cyclophosphamide to induce leucopenia and translocation of the intestinal P. aeruginosa into blood, thereby producing gut‐derived sepsis. In this model, administration of 1 × 10⁹ CFU of Bifidobacterium longum strain BB536 for 10 days significantly (P < 0·01) increased the survival rate compared with groups of mice administered either with Bifidobacterium breve strain ATCC 15700 or excipients contained in the probiotic bacterial powder. Administration of B. longum significantly decreased viable counts of P. aeruginosa in the liver and blood compared with other groups. Culture of intestinal contents revealed a significantly lower viable count of P. aeruginosa in the jejunum of B. longum‐treated mice compared with other groups of mice. Furthermore, in vitro data demonstrated that B. longum possessed apparently higher adherent activity to Caco‐2 cell monolayers and significantly suppressed the adherence of P. aeruginosa to the monolayers of cells compared with other groups. Conclusion: Oral administration of B. longum protects mice against gut‐derived sepsis caused by P. aeruginosa, and the effect may be due to interference of P. aeruginosa adherence to intestinal epithelial cells. Significance and Impact of this Study: This study demonstrated that oral administration of B. longum BB536 is effective to protect against opportunistic infection with drug‐resistant bacteria such as P. aeruginosa. The results suggest that probiotics may play an important role even in the immunocompromised patients.     

Interactions between salivary Bifidobacterium adolescentis and other oral bacteria: in vitro coaggregation and coadhesion assays

Coaggregation assays were performed to investigate interactions between oral Bifidobacterium adolescentis and other oral bacterial species. Bifidobacterium adolescentis OLB6410 isolated from the saliva of healthy humans did not coaggregate with Actinomyces naeslundii JCM8350, Streptococcus mitis OLS3293, Streptococcus sanguinis JCM5708, Veillonella parvula ATCC17745 or Porphyromonas gingivalis OB7124, but it did coaggregate with Fusobacterium nucleatum JCM8532. Subsequent examination of biofilm formation on saliva-coated hydroxyapatite discs using FISH revealed that B. adolescentis OLB6410 could not directly adhere to the coated discs. It did, however, adhere to biofilms of A. naeslundii, V. parvula, and F. nucleatum, although it did not coaggregate with A. naeslundii nor with V. parvula. These results suggest that the adhesion of B. adolescentis to tooth surfaces is mediated by other oral bacteria. Heat- or proteinase K-treated F. nucleatum could not coaggregate with B. adolescentis. Similarly, the coaggregation and coadhesion of proteinase K-treated B. adolescentis were strongly inhibited. It is therefore probable that proteinaceous factors on the cellular surface of B. adolescentis and F. nucleatum are involved in their interaction. The data presented in this study add to our understanding of bifidobacterial colonization in the human oral cavity.     

Genome comparison of Bifidobacterium longum strains NCC2705 and CRC-002 using suppression subtractive hybridization

Because probiotic effects are strain dependent, genomic explanations of these differences will contribute to understanding their mechanisms of action. The genomic sequence of the Bifidobacterium longum probiotic strain NCC2705 was determined, but little is known about the genetic diversity between strains of this species. Suppression subtractive hybridization (SSH) is a powerful method for generating a set of DNA fragments differing between two closely related bacterial strains. The purpose of this study was to identify genetic differences between genomes of B. longum strains NCC2705 and CRC-002 using PCR-based SSH. Strain CRC-002 produces exopolysaccharides whereas NCC2705 is not known for reliable exopolysaccharide production. Thirty-five and 30 different sequences were obtained from the SSH libraries of strains CRC-002 and NCC2705, respectively. Specific CRC-002 genes found were predicted to be involved in the biosynthesis of exopolysaccharides and metabolism of other carbohydrates, and these genes were not present in the genome of strain NCC2705. The identification of an endo-1,4-beta-xylanase gene in the CRC-002 SSH library is an important difference because xylanase genes have previously been proposed as a defining characteristic of the NCC2705 strain. The results demonstrate that the SSH technique was useful to highlight potential genes involved in complex sugar metabolism that differ between the two probiotic strains.     

Safety evaluation of Bifidobacterium pseudocatenulatum G4 as assessed in BALB/c mice

AIMS: To assess the safety of Bifidobacterium pseudocatenulatum G4 in BALB/c mice that involves examination of bacterial translocation, changes in the internal organs and histology of the intestinal lining. METHODS AND RESULTS: Forty male BALB/c mice were randomly assigned into five groups (n = 8). Three groups were orally fed with 50 microl of three different concentrations of B. pseudocatenulatum G4 (2 x 10(4), 1 x 10(8) and 1 x 10(11) CFU day(-1)) for 4 weeks. One group was orally administered with 50 microl of 1 x 10(8) CFU B. longum BB536 per day for 4 weeks and last group was used as a nonbifidobacterial treatment control, which received 50 microl of skim milk. The administered strains did not affect the general health of mice and incapable of carrying out translocation to blood or liver. There were no significant differences in the internal organ (liver, heart, kidney and spleen) indices, serum enzymes of liver (aspartate aminotransferase, alkaline phosphate, alanine aminotransferase) and kidney (urea and creatinine) and histology (villi height, crypts height, mucosa thickness and epithelial cell height) of caecum, ileum and colon. CONCLUSION: Administration of high dose of up to 1 x 10(11) CFU B. pseudocatenulatum G4 per day to mice did not show any health threatening symptoms. SIGNIFICANCE AND IMPACT OF THE STUDY: Bifidobacterium pseudocatenulatum G4 is none pathogenic to BALB/c mice and could be safe probiotic for human consumption.     

应用生态制剂对肝硬化患者血浆内毒素水平和自发性细菌性腹膜炎发生的影响

对肝硬化腹水患才应用双歧杆菌制剂预防原发性腹膜炎效果进行探索。68例患者随机分为培菲康组和对照组。入院时两组患者的年龄、诊断、并发症、肝肾功能和凝血酶原活动度比较无显著差异。68例患者共发性原发性腹膜炎8例,其中治疗组34例中,发生1例,发生率为2.9％,对照组34例发生7例,发生率为20.6％。8例腹膜炎患者中5例腹水分离出细菌,以革兰阴性菌为主,其中大肠杆菌2例,培菲康口服后血浆内毒素水平显著降低,结果表明,双歧杆菌制剂预防肝炎肝硬化腹水患者的自发性腹膜炎有一定效果。     

Multiplex PCR using 16S rRNA gene-targeted primers for the identification of bifidobacteria from human origin

Three multiplex polymerase chain reactions (PCRs) targeted on Bifidobacterium and related species were designed to identify human species. The selected primers yielded amplified products of various sizes, each specific for a species. Three to four pairs were gathered in one PCR reaction and their specificity under multiplex conditions was confirmed using DNA from 26 reference strains. Using this technique on unidentified faecal strains, B. bifidum, B. longum and B. breve species were commonly recovered in infants while B. adolescentis, B. catenulatum/B. pseudocatenulatum continuum and B. longum species were predominant in adults. Thus, a single PCR can provide the assignment of a strain to one these species, reducing the number of PCR reactions and hands-on time for the identification of human isolates of bifidobacteria. Moreover, this technique is also applicable for the in situ detection of bifidobacteria in DNA extracts from human stools.