胆固醇结石患者胆囊细菌的T-RFLP研究

目的应用T-RFLP分析胆固醇结石中微生物群落结构。方法应用末端标记限制性片段长度多态性（Terminal Restriction Fragment Length Polymorphism,T-RFLP）和克隆文库分析,以微生物群落16S rRNA基因（16S rDNA）为目标,对8例常规细菌培养阴性的纯胆固醇患者胆囊结石和胆汁中的微生物群落结构进行解析和比较。结果发现8例纯胆固醇结石患者胆汁中未找到细菌存在的证据,结石中细菌16S rDNA阳性率为37.5%（3/8）,细菌16S rDNA片段测序表明细菌群落与肠杆菌科和微球菌科的微生物有较高的相似性,同时细菌群落中检出了大量的未培养微生物（Uncultured bacterium clone）。结论运用T-RFLP方法分析16S rDNA克隆片段能够有效评估纯胆固醇结石中的细菌群落结构的多样性。     

Comparison of bacterioplankton communities in three heavily polluted streams in China

Objective To compare the bacterioplankton communities in streams exposed to pollution of different types. Methods The bacterioplankton communities in three selected heavily polluted streams were investigated by using terminal‐restriction fragment length polymorphism (T‐RFLP) analysis in combination with 16S rRNA gene clone library analysis. Results Both T‐RFLP and 16S rRNA gene clone library revealed a great difference in bacterioplankton community composition in the different streams. Conclusion This work ...     

Diet- and colonization-dependent intestinal dysfunction predisposes to necrotizing enterocolitis in preterm pigs

BACKGROUND & AIMS: Preterm birth and formula feeding are key risk factors associated with necrotizing enterocolitis (NEC) in infants, but little is known about intestinal conditions that predispose to disease. Thus, structural, functional, and microbiologic indices were used to investigate the etiology of spontaneous NEC development in preterm pigs. METHODS: Piglets were delivered by cesarean section at 92% gestation, reared in infant incubators, and fed infant formula or colostrum every 3 hours (n = 120) until tissue collection at 1-2 days of age. RESULTS: Clinical and histopathologic signs of NEC were observed in 57% of pigs fed FORMULA (26/46) and in 5% of pigs fed COLOSTRUM (2/38) (P < .05). Relative to COLOSTRUM, both healthy and sick FORMULA pigs had reduced intestinal villous heights, enzyme activities, nutrient absorption, and antioxidant levels and higher inducible nitric oxide synthetase activity (P < .05). In healthy pigs, mucosal microbial diversity remained low and diet independent. NEC pigs showed bacterial overgrowth, and a high mucosal density of Clostridium perfringens was detected in some but not all pigs. Germ-free conditions and antiserum against Clostridium perfringens toxin prevented intestinal dysfunction and NEC in formula-fed pigs, whereas the gut trophic factors, epidermal growth factor, and glucagon-like peptide 2 had limited effects. CONCLUSIONS: A subclinical, formula-induced mucosal atrophy and dysfunction predispose to NEC and bacterial overgrowth. The adverse feeding effects are colonization dependent and may be reduced by factors in colostrum that include antibodies against aggressive toxins such as those of Clostridium perfringens.     

Comparative evaluation of establishing a human gut microbial community within rodent models

The structure of the human gut microbial community is determined by host genetics and environmental factors, where alterations in its structure have been associated with the onset of different diseases. Establishing a defined human gut microbial community within inbred rodent models provides a means to study microbial-related pathologies, however, an in-depth comparison of the established human gut microbiota in the different models is lacking. We compared the efficiency of establishing the bacterial component of a defined human microbial community within germ-free (GF) rats, GF mice, and antibiotic-treated specific pathogen-free mice. Remarkable differences were observed between the different rodent models. While the majority of abundant human-donor bacterial phylotypes were established in the GF rats, only a subset was present in the GF mice. Despite the fact that members of the phylum Bacteriodetes were well established in all rodent models, mice enriched for phylotypes related to species of Bacteroides. In contrary to the efficiency of Clostridiales to populate the GF rat in relative proportions to that of the human-donor, members of Clostridia cluster IV only poorly colonize the mouse gut. Thus, the genetic background of the different recipient rodent systems (that is, rats and mice) strongly influences the nature of the populating human gut microbiota, determining each model's biological suitability.     

Use of culture-independent analysis to reveal alteration of intestinal microflora by heat-killed Lactobacillus pentosus in a mouse model of endogenous sepsis

In this study we evaluated alteration of intestinal microflora by terminal-restriction fragment length polymorphism (T-RFLP) analysis and quantitative PCR (qPCR) for specific microbes. The effects of orally administered heat-killed Lactobacillus pentosus strain b240 (HK-b240) in immunosuppressed mice with endogenous Pseudomonas aeruginosa sepsis was estimated. By T-RFLP analysis, 5 dominant operational taxonomic units (OTUs) including Bacteroides spp. (OTU460) and Lactobacillus spp. (OTU657) were consistently observed, irrespective of treatment, at all time points. A significantly higher population of segmented filamentous bacteria (SFB) was observed by qPCR after 3 weeks of HK-b240 administration; thereafter, the difference was not sustained during immunosuppression and progression of sepsis. Although not significant, Lactobacillus spp. accounted for a larger population in the HK-b240-treated group. In conclusion, this study demonstrated successful application of culture-independent assays for evaluating biological agents by detecting changes in microflora even if the protection was not sufficient to result in significant survival change.     

结香与未结香白木香内生细菌群落结构及变化

研究采用不依赖培养方法T-RFLP,比较结香和非结香的白木香内生细菌的群落结构及变化,研究发现结香后内生细菌的T-RFs、香农多样性指数有增加的趋势,各样品间差异的T-RFs片段数占总T-RFs片段数60%以上,说明结香前后白木香内生细菌的种类发生显著变化。在非结香样品中,内生细菌的优势菌群是Anoxybacillus, Clostridium, Candidatus endobugula, Lysinibacillus,结香样品中内生细菌的优势菌群为Clostridium,Lysinibacillus,Luteimonas,phytoplasma。另外,在结香和非结香样品中都出现一些特异的T-RFs片段,这些明显的变化使得样品聚类分析时,结香样品内生细菌群落结构相似性比较高聚在一起,非结香样品聚在一起,说明结香前后白木香内生细菌发生显著的、有规律的变化。研究旨在利用不依赖培养的方法首次探讨白木香内生细菌的群落结构及结香中的变化,为合理利用白木香内生细菌资源提供科学依据。     

Dietary cholesterol lowers plasma and cecal equol concentrations in mice

This study examined the effects of cholesterol on mouse intestinal microflora and on isoflavonoids in the cecum and plasma. Dietary cholesterol affects bile acid metabolism and bile acids can influence the intestinal microorganisms. Intestinal microflora appear to play an important role in isoflavone metabolism. We hypothesized that dietary cholesterol changes the metabolism of isoflavonoids and intestinal microorganisms in mice. Male mice were randomly divided into two groups, which were fed a cholesterol-daidzein (CDA) or daidzein (DA) diet (control diet) for 60d. Plasma equol and cecal equol concentrations were significantly higher in the DA group (control group) than in the CDA group. However, plasma cholesterol concentrations were significantly higher in the CDA group compared to the DA group. The composition of cecal microorganisms differed between the two dietary groups. The occupation ratios of Clostridium cluster XI, Clostridium subcluster XIVa, and Lactobacillales were significantly higher in the CDA group. The occupation ratio of Bifidobacterium was significantly lower in the CDA group. This study suggests that dietary cholesterol has the potential to affect the metabolism of equol from daidzein by altering the metabolic activity of the intestinal microorganisms and gut physiological function.     

小鼠粪便及肠道各部位内容物细菌群落结构差异分析

【摘要】 目的 分析小鼠粪便及肠道各部位内容物细菌群落结构差异,探讨粪便取样研究肠道微生物与疾病关系的科学性和客观性,为相关实验设计提供参考。 方法 利用末端限制性片段长度多态性、变性梯度凝胶电泳和荧光定量PCR技术对BALB/c小鼠肠道不同部位（十二指肠、空肠、回肠、盲肠、结肠和直肠）内容物及粪便中细菌群落组成和丰度进行比较,分析细菌群落在小鼠粪便及肠道各部位的分布差异。 结果 粪便与直肠、结肠中优势片段均为244 bp、255 bp和449 bp,而小肠内容物（十二指肠、空肠及回肠）的优势片段则为60 bp、73 bp、261 bp、268 bp和272 bp,且小肠各部位之间细菌群落结构差异较大;十二指肠和空肠内容物中的细菌丰度较低,分别为6.9 log(copies)/g和8.3 log(copies)/g,而粪便中细菌丰度高达11.8 log(copies)/g,约2倍于十二指肠细菌丰度,显著高于十二指肠和空肠P ＜ 0.05）,与大肠各部位及回肠内容物细菌丰度相当,差异不显著（P ＞ 0.05）。结论 粪便和大肠各部位内容物的细菌群落结构个体差异较小肠小,适合进行肠道微生态与某些相关疾病的研究;粪便与大肠特别是结直肠部位细菌群落组成相似,可通过粪便取样进行该部位的微生物生态学相关分析。     

拭子提取黏膜部细菌DNA方法的优化

目的:探索一种适合于黏膜部微生物组学分析的DNA抽提方法.方法:评价玻璃珠机械破碎法+溶菌酶裂解法结合DNA提取试剂盒法(Beads+ Lysozyme+ QIA法)和溶菌酶裂解法结合DNA提取试剂盒法(Lysozyme+ QIA法)对9例鼻咽拭子和5株常见细菌的DNA提取效果.利用NanoDrop 2000测定DNA的浓度;以末端限制性片段长度多样性(T-RFLP)分析DNA的生态结构多态性.结果:两种方法对鼻咽拭子总DNA提取量无明显差别(P=0.288);T-RFLP结果显示,Beads+Lysozyme+ QIA法可得到92种末段限制性片段(T-RF),高于Lysozyme+ QIA法所得的43种,Beads+ Lysozyme+ QIA法SDI为(2.34±0.43)明显高于Lysozyme+ QIA法(1.61±1.09)(P=0.03),Beads+ Lysozyme+ QIA法可以获得更丰富的T-RF;Beads+ Lysozyme+ QIA法在表皮葡萄球菌、金黄色葡萄球菌、大肠埃希菌中可获得更高的DNA提取量(均P＜0.05),在甲型溶血性链球菌和枯草芽孢杆菌中两种方法无明显差异(P＞0.05).结论:优化的拭子提取细菌DNA的方法可以更大程度地反映样本中微生物的多样性,减少偏倚;有利于黏膜部微生物组学的研究.     

Vertical distribution of bacteria in a lake sediment from Antarctica by culture-independent and culture-dependent approaches

Bacterial diversity of the subsurface (18-22 cm), middle (60-64 cm) and bottom (100-104 cm) of a 136-cm-long sediment core sampled from a freshwater lake in Antarctica was determined by the culturable approach, T-RFLP and 16S rRNA gene clone libraries. Using the culturable approach, 41 strains were isolated and, based on phylogenetic analysis, they could be categorized into 14 groups. Representatives of the 14 groups varied in their growth temperature range (4-30 °C), in their tolerance to NaCl (0-2 M NaCl) and in the growth pH range (5-11). Eleven of fourteen representative strains exhibited either amylase, lipase, protease and (or) urease activities at 4 °C. Bacterial diversity at the phyla level using T-RFLP and 16S rRNA clone libraries was similar and clones were affiliated with Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes. TRFs affiliated with Spirochaetes were detected only by the T-RFLP approach and clones affiliated with Caldiserica only in the clone libraries. Stratification of bacteria along the depth of the sediment was observed both with the T-RFLP and the 16S rRNA gene clone library methods, and results indicated that stratification was dependent on the nature of the organism, aerobic or anaerobic. For instance, aerobic Janthinobacterium and Polaromonas were confined to the surface of the sediment, whereas anaerobic Caldisericum was present only in the bottom portion of the core. It may be concluded that the bacterial diversity of an Antarctic lake sediment core sample varies throughout the length of the core depending on the oxic-anoxic conditions of the sediment. Furthermore, these psychrophilic bacteria, due to their ability to produce extracellular cold active enzymes, might play a key role in the transformation of complex organic compounds.