Analysis of human and animal fecal microbiota for microbial source tracking

Microbial compositions of human and animal feces from South Korea were analyzed and characterized. In total, 38 fecal samples (14 healthy adult humans, 6 chickens, 6 cows, 6 pigs and 6 geese) were analyzed by 454 pyrosequencing of the V2 region of the 16S rRNA gene. Four major phyla, Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes, were identified in the samples. Principal coordinate analysis suggested that microbiota from the same host species generally clustered, with the exception of those from humans, which exhibited sample-specific compositions. A network-based analysis revealed that several operational taxonomic units (OTUs), such as Lactobacillus sp., Clostridium sp. and Prevotella sp., were commonly identified in all fecal sources. Other OTUs were present only in fecal samples from a single organism. For example, Yania sp. and Bifidobacterium sp. were identified specifically in chicken and human fecal samples, respectively. These specific OTUs or their respective biological markers could be useful for identifying the sources of fecal contamination in water by microbial source tracking.     

Comparative study of bacterial groups within the human cecal and fecal microbiota

The composition of the human cecal microbiota is poorly known because of sampling difficulties. Samples of cecal fluid from eight subjects were collected via an intestinal tube. Feces were also collected. Total anaerobes, facultative anaerobes, bifidobacteria, and Bacteroides were enumerated by culture methods, and the predominant phylogenetic groups were quantified by molecular hybridization using a set of six rRNA-targeted probes. The numbers of strict anaerobes, bifidobacteria, Bacteroides, and members of the Clostridium coccoides group and Clostridium leptum subgroup were lower in the cecum. Facultative anaerobes represented 25% of total bacteria in the cecum versus 1% in the feces.     

Culture-independent analysis of fecal microbiota in cattle

The phylogenetic diversity of the fecal bacterial community in Holstein cattle was determined by 16S ribosomal RNA gene sequence analysis. The sequences were affiliated with the following phyla: Firmicutes (81.3%), Bacteroidetes (14.4%), Actinobacteria (2.5%), and Proteobacteria (1.4%). The Clostridium leptum subgroup was the most phylogenetically diverse group in cattle feces. In addition, a number of previously uncharacterized and unidentified bacteria were recognized in clone libraries.     

粪便菌群移植的研究与应用

粪便菌群移植早在半个世纪前就有报道应用于临床治疗,但一直没有受到人们的重视。过去的几年间,粪便菌群移植又在针对Clostridium difficile(艰难梭状芽胞杆菌)感染的治疗中逐渐兴起,并且有非常良好的治疗效果,对Clostridium difficile感染性肠炎的病症完全消失。因此,越来越多的患者开始尝试粪便菌群移植的治疗方法,以代替传统的药物治疗方法。肠道菌群的研究逐渐深入我们意识到,肠道菌群已经不仅仅是人体肠道共生微生物而已,而是可以视为人体一个独立且密不可分的人体器官,承担着免疫系统与代谢系统中重要的生理活动。这些关于肠道菌群的研究对粪便菌群移植有着强有力的推动。许多疾病的发病根源,尤其是与微生物紊乱有直接联系的代谢疾病,都可以从微生物与人体的互作模式中寻找答案。本课题综述了国内外近年来粪便菌群移植在人体疾病研究中的应用研究。     

Molecular typing of fecal eukaryotic microbiota of human infants and their respective mothers

The micro-eukaryotic diversity from the human gut was investigated using universal primers directed towards 18S rRNA gene, fecal samples being the source of DNA. The subjects in this study included two breast-fed and two formula-milk-fed infants and their mothers. The study revealed that the infants did not seem to harbour any microeukaryotes in their gut. In contrast, there were distinct eukaryotic microbiota present in the mothers. The investigation is the first of its kind in the comparative study of the human feces to reveal the presence of micro-eukaryotic diversity variance in infants and adults from the Indian subcontinent. The micro-eukaryotes encountered during the investigation include known gut colonizers like Blastocystis and some fungi species. Some of these micro-eukaryotes have been speculated to be involved in clinical manifestations of various diseases. The study is an attempt to highlight the importance of micro-eukaryotes in the human gut.     

粪微生态制品保存方法

目的 探索粪微生态制品体外保存方法,为粪微生态制品的保存及运输提供依据。方法 将新鲜粪微生态制品按照加入甘油、蔗糖保护剂的不同以及制备冻干粉的方法分为6组,用10倍稀释法稀释粪菌液,微量点样法将各浓度的粪菌液涂于Wilkins-Chalgren培养基,30℃恒温箱中培养并计数。在保存2周、1个月、2个月、3个月时复苏部分微生态制品,用同样的方法培养并计数。结果 粪微生态制品活菌数随时间递减,在2周内下降速度最快,递减105~107数量级。结论 甘油、蔗糖这两种保护剂以及冻干粉的制备对粪微生态制品的保存差异不大,新鲜粪微生态制品仍为最佳选择。     

粪菌移植的临床应用研究进展

人类的肠道菌群种类及数量众多,目前被认为是人体的一个特殊器官。肠道菌群在维持肠道的正常生理功能和机体免疫功能方面发挥了重要作用,肠道微生态失衡与炎症性肠病、代谢综合征、肝病、心血管疾病、精神疾病、关节炎等多种肠内外疾病密切相关,纠正肠道微生态失衡将有助于上述疾病的治疗。粪菌移植（fecal microbiota transplantation,FMT）是指将健康人粪便中的功能菌群移植到患者胃肠道内,重建具有正常功能的肠道菌群,以达到治疗肠道和肠道外疾病的目的。目前报道FMT已应用于艰难梭菌感染、炎症性肠病、肠易激综合征、代谢综合征等多种疾病的治疗中。本文就FMT的临床应用现状作一综述。     

粪菌移植的现状及未来

粪菌移植是一种直接改变受体肠道微生物群以使其正常化,从而获得治疗效益的方法。自2013年美国食品和药物管理局批准粪菌移植用于治疗复发性和难治性艰难梭菌感染以来,粪菌移植成为研究热点。从此,粪菌移植应用的范围迅速扩大,不仅用于胃肠道疾病,而且用于胃肠外疾病。虽然目前的证据认为粪菌移植是一种普遍安全、副作用少的治疗方法,但其扩大适应症及安全性问题尚未形成共识,需要更深入的研究。本文就目前粪菌移植与胃肠内外疾病的关系及临床安全性问题进行综述,为临床更好地开展粪菌移植提供参考。     

Gene-targeted metagenomic analysis of glucan-branching enzyme gene profiles among human and animal fecal microbiota

Glycoside hydrolases (GHs), the enzymes that breakdown complex carbohydrates, are a highly diversified class of key enzymes associated with the gut microbiota and its metabolic functions. To learn more about the diversity of GHs and their potential role in a variety of gut microbiomes, we used a combination of 16S, metagenomic and targeted amplicon sequencing data to study one of these enzyme families in detail. Specifically, we employed a functional gene-targeted metagenomic approach to the 1-4-α-glucan-branching enzyme (gBE) gene in the gut microbiomes of four host species (human, chicken, cow and pig). The characteristics of operational taxonomic units (OTUs) and operational glucan-branching units (OGBUs) were distinctive in each of hosts. Human and pig were most similar in OTUs profiles while maintaining distinct OGBU profiles. Interestingly, the phylogenetic profiles identified from 16S and gBE gene sequences differed, suggesting the presence of different gBE genes in the same OTU across different vertebrate hosts. Our data suggest that gene-targeted metagenomic analysis is useful for an in-depth understanding of the diversity of a particular gene of interest. Specific carbohydrate metabolic genes appear to be carried by distinct OTUs in different individual hosts and among different vertebrate species'' microbiomes, the characteristics of which differ according to host genetic background and/or diet.     

Microbial species involved in production of 1,2-sn-diacylglycerol and effects of phosphatidylcholine on human fecal microbiota

1,2-sn-Diacylglycerols (DAGs) are activators of protein kinase C (PKC), which is involved in the regulation of colonic mucosal proliferation. Extracellular DAG has been shown to stimulate the growth of cancer cell lines in vitro and may therefore play an important role in tumor promotion. DAG has been detected in human fecal extracts and is thought to be of microbial origin. Hitherto, no attempts have been made to identify the predominant fecal bacterial species involved in its production. We therefore used anaerobic batch culture systems to determine whether fecal bacteria could utilize phosphatidylcholine (0.5% [wt/vol]) to produce DAG. Production was found to be dependent upon the presence of the substrate and was enhanced in the presence of high concentrations of deoxycholate (5 and 10 mM) in the growth medium. Moreover, its production increased with the pH, and large inter- and intraindividual variations were observed between cultures seeded with inocula from different individuals. Clostridia and Escherichia coli multiplied in the fermentation systems, indicating their involvement in phosphatidylcholine metabolism. On the other hand, there was a significant decrease in the number of Bifidobacterium spp. in the presence of phosphatidylcholine. Pure-culture experiments showed that 10 of the 12 strains yielding the highest DAG levels (>50 nmol/ml) were isolated from batch culture enrichments run at pH 8.5. We found that the strains capable of producing large amounts of DAG were predominantly Clostridium bifermentans (8 of 12), followed by Escherichia coli (2 of 12). Interestingly, one DAG-producing strain was Bifidobacterium infantis, which is often considered a beneficial gut microorganism. Our results have provided further evidence that fecal bacteria can produce DAG and that specific bacterial groups are involved in this process. Future strategies to reduce DAG formation in the gut should target these species.     

In vitro fermentation of linear and alpha-1,2-branched dextrans by the human fecal microbiota

The role of structure and molecular weight in fermentation selectivity in linear α-1,6 dextrans and dextrans with α-1,2 branching was investigated. Fermentation by gut bacteria was determined in anaerobic, pH-controlled fecal batch cultures after 36 h. Inulin (1%, wt/vol), which is a known prebiotic, was used as a control. Samples were obtained at 0, 10, 24, and 36 h of fermentation for bacterial enumeration by fluorescent in situ hybridization and short-chain fatty acid analyses. The gas production of the substrate fermentation was investigated in non-pH-controlled, fecal batch culture tubes after 36 h. Linear and branched 1-kDa dextrans produced significant increases in Bifidobacterium populations. The degree of α-1,2 branching did not influence the Bifidobacterium populations; however, α-1,2 branching increased the dietary fiber content, implying a decrease in digestibility. Other measured bacteria were unaffected by the test substrates except for the Bacteroides-Prevotella group, the growth levels of which were increased on inulin and 6- and 70-kDa dextrans, and the Faecalibacterium prausnitzii group, the growth levels of which were decreased on inulin and 1-kDa dextrans. A considerable increase in short-chain fatty acid concentration was measured following the fermentation of all dextrans and inulin. Gas production rates were similar among all dextrans tested but were significantly slower than that for inulin. The linear 1-kDa dextran produced lower total gas and shorter time to attain maximal gas production compared to those of the 70-kDa dextran (branched) and inulin. These findings indicate that dextrans induce a selective effect on the gut flora, short-chain fatty acids, and gas production depending on their length.     

粪菌移植在临床治疗中的研究进展

粪菌移植的历史可以追溯到中国东晋时期。大量研究证实粪菌移植有可能成为许多疾病的有效治疗方法,现已逐渐应用于临床上并取得较好的疗效。粪菌移植可以通过增加短链脂肪酸,特别是丁酸盐的产量来减少肠道通透性,从而降低疾病的严重程度,这有助于维持上皮屏障的完整性。粪菌移植还可以通过抑制Th1分化、T细胞活性、白细胞粘附和炎症因子的产生来恢复免疫生态失调。目前对粪菌移植的研究主要集中在治疗艰难梭菌感染,但其他领域对粪菌移植研究的热情也日益高涨,如用于治疗炎症性肠病、代谢综合征和肠易激综合征等。本文对最新的粪菌移植在临床治疗中的研究进展作一综述。     

Changes in fecal microbiota of healthy dogs administered amoxicillin

The effect of oral amoxicillin treatment on fecal microbiota of seven healthy adult dogs was determined with a focus on the prevalence of bacterial antibiotic resistance and changes in predominant bacterial populations. After 4–7 days of exposure to amoxicillin, fecal Escherichia coli expressed resistance to multiple antibiotics when compared with the pre-exposure situation. Two weeks postexposure, the susceptibility pattern had returned to pre-exposure levels in most dogs. A shift in bacterial populations was confirmed by molecular fingerprinting of fecal bacterial populations using denaturing gradient gel electrophoresis (PCR-DGGE) of the 16S V3 rRNA gene region. Much of the variation in DGGE profiles could be attributed to dog-specific factors. However, permutation tests indicated that amoxicillin exposure significantly affected the DGGE profiles after controlling for the dog effect ( P =0.02), and pre-exposure samples were clearly separated from postexposure samples. Sequence analysis of DGGE bands and real-time PCR quantification indicated that amoxicillin exposure caused a shift in the intestinal ecological balance toward a Gram-negative microbiota including resistant species in the family Enterobacteriaceae .     

1 067例不同年龄及地域人群肠道菌群特征

目的 探索不同人群在不同年龄阶段的菌群结构特点,了解环境因素对肠道菌群组成及发展的影响。方法 收集65例中国健康儿童粪便样本并进行16S rRNA检测,同时从已发表文献中获取来自亚洲、非洲、欧洲、北美洲及南美洲国家的1 002例健康人群肠道菌群数据,分析来自不同年龄阶段健康人群肠道菌群的特征。结果 3岁以下全球各地儿童肠道菌群的种类较为一致：拟杆菌属和双歧杆菌属在儿童肠道菌群中占据主导地位,且它们在免疫调节上有重要作用。3岁以上健康人群的肠道菌群则展示出了地域性特征：亚洲健康人群的肠道代表菌属为考拉杆菌属和巨单胞菌属,非洲健康人群的肠道代表菌属为粪杆菌属,欧洲健康人群的肠道代表菌属也是粪杆菌属,北美洲健康人群的肠道代表菌属为拟杆菌属,南美洲健康人群的肠道代表菌属为普氏菌属。另外,经济较发达的欧洲及北美洲健康人群的肠道菌群多样性显著高于其他经济较落后地区。结论 本研究加深了对不同地域人群肠道菌群特点以及它们随年龄增长而发展变化的认识。     

Effect of apple intake on fecal microbiota and metabolites in humans

The effects of apple intake on the fecal flora, water content, pH, and metabolic activities in eight healthy volunteers and the utilization of apple pectin in vitro were investigated. Although several isolates of Bifidobacterium, Lactobacillus, Enterococcus, and the Bacteroides fragilis group utilized apple pectin, most isolates of Escherichia coli, Collinsela aerofaciense, Eubacterium limosum, and Clostridium perfringens could not. When fecal samples from healthy adults were incubated in liquid broth with apple pectin present or absent, the numbers of Bifidobacterium and Lactobacillus in the former were higher than those in the later. After the intake of apples (2 apples a day for 2 weeks) by eight healthy adult humans, the number of bifidobacteria in feces increased (p < 0.05 on day 7 and p < 0.01 on day 14 of the intake period), and the numbers of Lactobacillus and Streptococcus including Enterococcus tended to increase. However, lecithinase-positive clostridia, including C. perfringens, decreased (p < 0.05), and Enterobacteriaceae and Pseudomonas tended to decrease. Moreover, the concentrations of fecal acetic acid tended to increase on apple intake. The fecal ammonia concentration showed a tendency to reduce and fecal sulfide decreased (p < 0.05) on apple intake. These findings indicate that apple consumption is related to an improved intestinal environment, and apple pectin is one of the effective apple components improving the fecal environment.     

Changes in human fecal microbiota due to chemotherapy analyzed by TaqMan-PCR, 454 sequencing and PCR-DGGE fingerprinting

Background

We investigated whether chemotherapy with the presence or absence of antibiotics against different kinds of cancer changed the gastrointestinal microbiota.

Methodology/Principal Findings

Feces of 17 ambulant patients receiving chemotherapy with or without concomitant antibiotics were analyzed before and after the chemotherapy cycle at four time points in comparison to 17 gender-, age- and lifestyle-matched healthy controls. We targeted 16S rRNA genes of all bacteria, Bacteroides, bifidobacteria, Clostridium cluster IV and XIVa as well as C. difficile with TaqMan qPCR, denaturing gradient gel electrophoresis (DGGE) fingerprinting and high-throughput sequencing. After a significant drop in the abundance of microbiota (p = 0.037) following a single treatment the microbiota recovered within a few days. The chemotherapeutical treatment marginally affected the Bacteroides while the Clostridium cluster IV and XIVa were significantly more sensitive to chemotherapy and antibiotic treatment. DGGE fingerprinting showed decreased diversity of Clostridium cluster IV and XIVa in response to chemotherapy with cluster IV diversity being particularly affected by antibiotics. The occurrence of C. difficile in three out of seventeen subjects was accompanied by a decrease in the genera Bifidobacterium, Lactobacillus, Veillonella and Faecalibacterium prausnitzii. Enterococcus faecium increased following chemotherapy.

Conclusions/Significance

Despite high individual variations, these results suggest that the observed changes in the human gut microbiota may favor colonization with C.difficile and Enterococcus faecium. Perturbed microbiota may be a target for specific mitigation with safe pre- and probiotics.     

Effects of green tea consumption on human fecal microbiota with special reference to Bifidobacterium species

Green tea is one of the most popular beverages in the world. Its beneficial health effects and components have been extensively reviewed. However, little is known about the influence of green tea consumption on the human intestinal microbiota (HIM), which plays a crucial role in human health. Ten volunteers who did not usually consume green tea, drank it for 10 days and then stopped drinking it for 7 days. Their fecal samples were collected at three time points: before beginning the 10‐day green‐tea regime, at the conclusion of that 10 days, and 7 days after stopping the regime. Their fecal samples were analyzed by terminal restriction fragment length polymorphism with specific primer‐restriction enzyme systems for HIM and by using a real‐time PCR method for the Bifidobacterium species. Although the HIM of each subject was relatively stable, the proportion of Bifidobacterium species played an important role in the classification of their fecal microbiota. Although there were inter‐individual differences in the Bifidobacterium species, an overall tendency for the proportion of bifidobacteria to increase because of green tea consumption was noted. However, little change was observed in the composition of Bifidobacterium species in each sample. This suggests that the change in proportion was induced, not by an inter‐species transition, but by an intra‐species increase and/or decrease. In conclusion, green tea consumption might act as a prebiotic and improve the colon environment by increasing the proportion of the Bifidobacterium species.     

Characteristics of fecal microbiota in non-alcoholic fatty liver disease patients

This study was designed to investigate the gut microbiota of patients with non-alcoholic fatty liver disease. The inclusive and exclusive criteria for NAFLD patients and healthy subjects were formulated, and detailed clinical data were collected. The genomic DNA of stool samples were extracted for 16S rDNA sequencing, and the amplified V4-region was sequenced on the Illumina Miseq platform. Metastats analysis was performed to identify the differential taxa between the groups. Redundancy analysis was used to evaluate the association between gut microbial structure and clinical variables. Thirty NAFLD patients and 37 healthy controls were involved. The 16S rDNA sequencing showed that there was a dramatic variability of the fecal microbiota among all the individuals. Metastats analysis identified eight families and 12 genera with significant differences between the two groups. When some clinical parameters, such as waist-to-hip ratio (WHR) and homeostasis model assessment of insulin resistance (HOMA-IR), were enrolled in Redundancy analysis, the distribution of the two group of samples was obviously changed. The compositional shifts in fecal bacterial communities of NAFLD patients from the healthy controls were mainly at family or genus levels. According to our Redundancy analysis, insulin resistance and obesity might be closely related to both NAFLD phenotype and intestinal microecology.