Culture-independent analysis of the gut microbiota in colorectal cancer and polyposis

A role for the intestinal microbiota is routinely cited as a potential aetiological factor in colorectal cancer initiation and progression. As the majority of bacteria in the gut are refractory to culture we investigated this ecosystem in subjects with colorectal cancer and with adenomatous polyposis who are at high risk of developing colorectal cancer, using culture-independent methods. Twenty colorectal cancer and 20 polypectomized volunteers were chosen for this analysis. An exploration of the diversity and temporal stability of the dominant bacteria and several bacterial subgroups was undertaken using 16S rRNA gene denaturing gradient gel electrophoresis and ribosomal intergenic spacer analysis (RISA). Metabonomic analysis of the distal gut microbiota's environment was also undertaken. A significantly reduced temporal stability and increased diversity for the microbiota of subjects with colorectal cancer and polyposis was evident. A significantly increased diversity of the Clostridium leptum and C. coccoides subgroups was also noted for both disease groups. A clear division in the metabonome was observed for the colorectal cancer and polypectomized subjects compared with control volunteers. The intestinal microbiota and their metabolites are significantly altered in both colorectal cancer and polypectomized subjects compared with controls.     

Culture-independent analysis of gut bacteria: the pig gastrointestinal tract microbiota revisited

The phylogenetic diversity of the intestinal bacterial community in pigs was studied by comparative 16S ribosomal DNA (rDNA) sequence analysis. Samples were collected from a total of 24 pigs representing a variety of diets, ages, and herd health status. A library comprising 4,270 cloned 16S rDNA sequences obtained directly by PCR from 52 samples of either the ileum, the cecum, or the colon was constructed. In total, 375 phylotypes were identified using a 97% similarity criterion. Three hundred nine of the phylotypes (83%) had a <97% sequence similarity to any sequences in the database and may represent yet-uncharacterized bacterial genera or species. The phylotypes were affiliated with 13 major phylogenetic lineages. Three hundred four phylotypes (81%) belonged to the low-G+C gram-positive division, and 42 phylotypes (11.2%) were affiliated with the Bacteroides and Prevotella group. Four clusters of phylotypes branching off deeply within the low-G+C gram-positive bacteria and one in the Mycoplasma without any cultured representatives were found. The coverage of all the samples was 97.2%. The relative abundance of the clones approximated a lognormal distribution; however, the phylotypes detected and their abundance varied between two libraries from the same sample. The results document that the intestinal microbial community is very complex and that the majority of the bacterial species colonizing the gastrointestinal tract in pigs have not been characterized.     

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.     

二甲双胍调控肠道菌群并抑制结直肠癌的生长

目的 探究二甲双胍抑制肠癌的生长及调控肠道菌群的作用。 方法 20只C57BL/6J小鼠通过AOM DSS诱导结直肠癌发生,分成2组每天分别灌胃生理盐水及二甲双胍溶液,12周后处死小鼠,收集结直肠组织及粪便,比较2组小鼠肠道肿瘤个数及大小并进行H&E染色分析,运用16S rRNA基因测序技术检测小鼠肠道菌群组成,使用QIIME软件分析菌群物种分类、物种多样性指数及组间显著性差异。 结果 2组小鼠体质量随周龄增加均呈现增长趋势,且实验结束时体质量差异无统计学意义(t=0.743 1,P=0.469 7)。与对照组相比,接受二甲双胍灌胃的实验组小鼠结直肠肿瘤数量减少(t=2.260 0,P=0.040 3)且尺寸偏小(t=2.570 0,P=0.014 4)。粪便菌群测序结果显示实验组小鼠肠道菌群丰富度增加(P0.05)。在属水平上,实验组Akkermansia、Ruminococcus及Clostridium Ⅹa和Ⅳ等细菌的丰富度增加。 结论 二甲双胍能够调控肠道菌群的组成,增加肠道内产SCFAs细菌的定植,并抑制肠癌的发生发展。     

Culture-independent analysis of indomethacin-induced alterations in the rat gastrointestinal microbiota

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly prescribed for a variety of inflammatory conditions; however, the benefits of this class of drugs are accompanied by deleterious side effects, most commonly gastric irritation and ulceration. NSAID-induced ulceration is thought to be exacerbated by intestinal microbiota, but previous studies have not identified specific microbes that contribute to these adverse effects. In this study, we conducted a culture-independent analysis of approximately 1,400 bacterial small-subunit rRNA genes associated with the small intestines and mesenteric lymph nodes of rats treated with the NSAID indomethacin. This is the first molecular analysis of the microbiota of the rat small intestine. A comparison of clone libraries and species-specific quantitative PCR results from rats treated with indomethacin and untreated rats revealed that organisms closely related to Enterococcus faecalis were heavily enriched in the small intestine and mesenteric lymph nodes of the treated rats. These data suggest that treatment of NSAID-induced ulceration may be facilitated by addressing the microbiological imbalances.     

Structural segregation of gut microbiota between colorectal cancer patients and healthy volunteers

Despite a long-suspected role in the development of human colorectal cancer (CRC), the composition of gut microbiota in CRC patients has not been adequately described. In this study, fecal bacterial diversity in CRC patients (n=46) and healthy volunteers (n=56) were profiled by 454 pyrosequencing of the V3 region of the 16S ribosomal RNA gene. Both principal component analysis and UniFrac analysis showed structural segregation between the two populations. Forty-eight operational taxonomic units (OTUs) were identified by redundancy analysis as key variables significantly associated with the structural difference. One OTU closely related to Bacteroides fragilis was enriched in the gut microbiota of CRC patients, whereas three OTUs related to Bacteroides vulgatus and Bacteroides uniformis were enriched in that of healthy volunteers. A total of 11 OTUs belonging to the genera Enterococcus, Escherichia/Shigella, Klebsiella, Streptococcus and Peptostreptococcus were significantly more abundant in the gut microbiota of CRC patients, and 5 OTUs belonging to the genus Roseburia and other butyrate-producing bacteria of the family Lachnospiraceae were less abundant. Real-time quantitative PCR further validated the significant reduction of butyrate-producing bacteria in the gut microbiota of CRC patients by measuring the copy numbers of butyryl-coenzyme A CoA transferase genes (Mann–Whitney test, P<0.01). Reduction of butyrate producers and increase of opportunistic pathogens may constitute a major structural imbalance of gut microbiota in CRC patients.     

Therapeutic potential of melatonin in colorectal cancer: Focus on lipid metabolism and gut microbiota

Colorectal cancer (CRC) is one of the most common gastrointestinal malignancies. The occurrence and development of CRC are complicated processes. Obesity and dysbacteriosis have been increasingly regarded as the main risk factors for CRC. Understanding the etiology of CRC from multiple perspectives is conducive to screening for some potential drugs or new treatment strategies to limit the serious side effects of conventional treatment and prolong the survival of CRC patients. Melatonin, a natural indoleamine, is mainly produced by the pineal gland, but it is also abundant in other tissues, including the gastrointestinal tract, retina, testes, lymphocytes, and Harder's glands. Melatonin could participate in lipid metabolism by regulating adipogenesis and lipolysis. Additionally, many studies have focused on the potential beneficial effects of melatonin in CRC, such as promotion of apoptosis; inhibition of cell proliferation, migration, and invasion; antioxidant activity; and immune regulation. Meaningfully, gut microbiota is the main determinant of all aspects of health and disease (including obesity and tumorigenesis). The gut microbiota is of great significance for understanding the relationship between obesity and increased risk of CRC. Although the current understanding of how the melatonin-mediated gut microbiota coordinates a variety of physiological and pathological activities is fairly comprehensive, there are still many unknown topics to be explored in the face of a complex nutritional status and a changeable microbiota. This review summarizes the potential links among melatonin, lipid metabolism, gut microbiota, and CRC to promote the development of melatonin as a preventive and therapeutic agent for CRC.     

Culture-independent analysis of desulfovibrios in the human distal colon of healthy, colorectal cancer and polypectomized individuals

The production of hydrogen sulphide, an end product of metabolism by the sulphate-reducing bacteria (SRB) has been cited as a potential aetiological agent in gastrointestinal disease. Quantitative PCR (Q-PCR) assays to enumerate desulfovibrios from two gastrointestinal disease groups: colorectal cancer (CRC) n =27 and polypectomized individuals (PP) n =27, and two healthy control groups, elderly (H1) n =8 and young adults (H2) n =30 was performed. Analysis of Desulfovibrio sp. diversity using the dissimilarity sulphite reductase ( dsrAB ) gene as a molecular marker was also undertaken. Q-PCR detected Desulfovibrio sp. in all samples and no significant difference was observed for PP, H1, H2 with gene copy numbers of Desulfovibrio sp. averaging at 10⁶ g⁻¹ of faeces. Significantly reduced numbers of Desulfovibrio sp. were observed for CRC (10⁵ g⁻¹) compared with both PP and H2 groups ( P <0.05). Diversity analysis indicated that a low Desulfovibrio sp. diversity and the predominance of Desulfovibrio piger was a feature of both healthy and disease groups. In addition, a dsrAB gene sequence distantly related to a Gram-positive SRB was also recovered, highlighting the importance of cultivation-independent techniques for furthering our understanding of the diversity of the human gastrointestinal ecosystem.     

How the interplay among the tumor microenvironment and the gut microbiota influences the stemness of colorectal cancer cells

Colorectal cancer (CRC) remains the third most prevalent cancer disease and involves a multi-step process in which intestinal cells acquire malignant characteristics. It is well established that the appearance of distal metastasis in CRC patients is the cause of a poor prognosis and treatment failure. Nevertheless, in the last decades, CRC aggressiveness and progression have been attributed to a specific cell population called CRC stem cells (CCSC) with features like tumor initiation capacity, self-renewal capacity, and acquired multidrug resistance. Emerging data highlight the concept of this cell subtype as a plastic entity that has a dynamic status and can be originated from different types of cells through genetic and epigenetic changes. These alterations are modulated by complex and dynamic crosstalk with environmental factors by paracrine signaling. It is known that in the tumor niche, different cell types, structures, and biomolecules coexist and interact with cancer cells favoring cancer growth and development. Together, these components constitute the tumor microenvironment (TME). Most recently, researchers have also deepened the influence of the complex variety of microorganisms that inhabit the intestinal mucosa, collectively known as gut microbiota, on CRC. Both TME and microorganisms participate in inflammatory processes that can drive the initiation and evolution of CRC. Since in the last decade, crucial advances have been made concerning to the synergistic interaction among the TME and gut microorganisms that condition the identity of CCSC, the data exposed in this review could provide valuable insights into the biology of CRC and the development of new targeted therapies.     

老年结直肠癌患者肠道菌群变化与机体免疫炎症营养相关性

目的探讨老年结直肠癌患者肠道菌群变化与机体免疫、炎症、营养指标的相关性,为结直肠癌的防治提供参考。方法纳入2016年1月至2018年6月在我院接受治疗的老年结直肠癌患者30例和同期健康查体的老年人群30例,收集粪便标本,采用FloraCheck~(TM)高通量测序技术对粪便样本中所有细菌的16S rRNA V3-V4区进行DNA测序,序列通过MiSeq 16S宏基因组APP及QIIME软件,分析两组人群间属水平上显著差异性菌群。比较两组人群炎性指标(TNF-α、IL-6、IL-1、IL-12)、营养指标(总蛋白、白蛋白、前白蛋白、转铁蛋白)和免疫指标(CD3~+、CD4~+、CD8~+、NK)的差异。分析老年结直肠癌患者肠道菌群变化与营养、炎症、免疫的内在联系。结果老年结直肠癌组和老年健康组人群免疫指标CD3~+、CD4~+、CD8~+、NK和CD4~+/CD8~+比较差异无统计学意义;炎性指标老年结直肠癌组TNF-α含量较对照组明显增高(t=3.769,P=0.001),两组人群IL-1、IL-6、IL-12比较差异无统计学意义;营养指标老年结直肠癌组白蛋白、前白蛋白较对照组明显下降(t=-4.107,P=0.001;t=-4.366,P=0.001),两组人群总蛋白、转铁蛋白比较差异无统计学意义。老年结直肠癌组肠道差异性菌属变化,包括Eubacterium、Ruminococcaceae_UCG-002、Peptostreptococcus、Porphyromonas和部分炎症指标、营养指标、免疫指标有明显相关性。结论老年结直肠癌患者肠道菌群结构和功能异常,可能是导致患者机体免疫损伤、炎症反应、营养不佳的主要原因之一。有针对性地对肠道菌群结构进行优化,改善肠道菌群对宿主的影响,是一种帮助老年结直肠癌患者促进健康的策略。     

Culture-independent analysis of fecal microbiota in infants, with special reference to Bifidobacterium species

Fecal microbiota of 31 breast-fed, 26 mix-fed, and 11 bottle-fed infants were analyzed by using terminal restriction fragment length polymorphism (T-RFLP), and culture method. We first determined the total and cultivated bacterial counts in infant fecal microbiota. Only approximately 30% of bacteria present in fecal microbiota were cultivable while the remainder was yet-to-be cultured bacteria. Sixty-eight fecal samples were divided into two clusters (I and II) by T-RFLP analysis, and then subdivided into five subclusters (Ia, Ib, IIa, IIb and IIc). There was no clear relationship between clusters and feeding method. A proportion of bifidobacteria was detected in the fecal material by PCR method using species-specific primers. The predominant Bifidobacterium spp. was Bifidobacterium longum longum type (43 samples (63.2%)), followed by B. longum infantis type (23 samples (33.8%)) and B. breve (16 samples (23.5%)). The distribution of Bifidobacterium spp. was similar in the three feeding groups. In contrast, the high incidence of B. breve in cluster I, especially subcluster Ia and B. longum longum type in cluster II, especially subcluster IIa and IIc were characterized by T-RFLP method. Our results showed that the colonization of Bifidobacterium spp. in infant feces correlated with the T-RFLP clusters.     

结直肠癌与肠道微生物群研究进展

肠道微生物群是人体内环境的重要组成部分,与宿主共进化、共代谢、共发育,并与宿主之间相互调控,影响宿主健康。近年研究显示,肠道微生物群参与了结直肠癌的发生和发展。了解肠道微生物群的特征性变化及其诱发结直肠癌的机制对于结直肠癌的防治有着重要意义。目前以肠道微生物群为靶点的干预性基础研究也取得了一些突破性的研究进展。本文主要对结直肠癌患者肠道微生物群的变化、其可能的致病机制及临床相关研究进展等进行综述。     

The association of diet,gut microbiota and colorectal cancer: what we eat may imply what we get

Despite the success of colonoscopy screening and recent advances in cancer treatment, colorectal cancer (CRC) still remains one of the most commonly diagnosed and deadly cancers, with a significantly increased incidence in developing countries where people are adapting to Western lifestyle. Diet has an important impact on risk of CRC. Multiple epidemiological studies have suggested that excessive animal protein and fat intake, especially red meat and processed meat, could increase the risk of developing CRC while fiber could protect against colorectal tumorigenesis. Mechanisms have been investigated by animal studies.Diet could re-shape the community structure of gut microbiota and influence its function by modulating the production of metabolites. Butyrate, one of the short-chain fatty acids (SCFAs), which act as a favorable source for colonocytes, could protect colonic epithelial cells from tumorigenesis via anti-inflammatory and antineoplastic properties through cell metabolism, microbiota homeostasis, antiproliferative, immunomodulatory and genetic/epigenetic regulation ways. In contrast, protein fermentation and bile acid deconjugation, which cause damage to colonic cells through proinflammatory and proneoplastic ways, lead to increasedriskofdevelopingCRC.In conclusion, abalanced diet with an increased abundance of fiber should be adopted to reduce the risk and prevent CRC.     

Molecular analysis of gut microbiota in obesity among Indian individuals

Obesity is a consequence of a complex interplay between the host genome and the prevalent obesogenic factors among the modern communities. The role of gut microbiota in the pathogenesis of the disorder was recently discovered; however, 16S-rRNA-based surveys revealed compelling but community-specific data. Considering this, despite unique diets, dietary habits and an uprising trend in obesity, the Indian counterparts are poorly studied. Here, we report a comparative analysis and quantification of dominant gut microbiota of lean, normal, obese and surgically treated obese individuals of Indian origin. Representative gut microbial diversity was assessed by sequencing fecal 16S rRNA libraries for each group (n=5) with a total of over 3000 sequences. We detected no evident trend in the distribution of the predominant bacterial phyla, Bacteroidetes and Firmicutes. At the genus level, the bacteria of genus Bacteroides were prominent among the obese individuals, which was further confirmed by qPCR (P less than 0.05). In addition, a remarkably high archaeal density with elevated fecal SCFA levels was also noted in the obese group. On the contrary, the treated-obese individuals exhibited comparatively reduced Bacteroides and archaeal counts along with reduced fecal SCFAs. In conclusion, the study successfully identified a representative microbial diversity in the Indian subjects and demonstrated the prominence of certain bacterial groups in obese individuals; nevertheless, further studies are essential to understand their role in obesity.     

Comparative analysis of human gut microbiota by barcoded pyrosequencing

Humans host complex microbial communities believed to contribute to health maintenance and, when in imbalance, to the development of diseases. Determining the microbial composition in patients and healthy controls may thus provide novel therapeutic targets. For this purpose, high-throughput, cost-effective methods for microbiota characterization are needed. We have employed 454-pyrosequencing of a hyper-variable region of the 16S rRNA gene in combination with sample-specific barcode sequences which enables parallel in-depth analysis of hundreds of samples with limited sample processing. In silico modeling demonstrated that the method correctly describes microbial communities down to phylotypes below the genus level. Here we applied the technique to analyze microbial communities in throat, stomach and fecal samples. Our results demonstrate the applicability of barcoded pyrosequencing as a high-throughput method for comparative microbial ecology.     

Beneficial modulation of the gut microbiota

The human gut microbiota comprises approximately 100 trillion microbial cells and has a significant effect on many aspects of human physiology including metabolism, nutrient absorption and immune function. Disruption of this population has been implicated in many conditions and diseases, including examples such as obesity, inflammatory bowel disease and colorectal cancer that are highlighted in this review. A logical extension of these observations suggests that the manipulation of the gut microbiota can be employed to prevent or treat these conditions. Thus, here we highlight a variety of options, including the use of changes in diet (including the use of prebiotics), antimicrobial-based intervention, probiotics and faecal microbiota transplantation, and discuss their relative merits with respect to modulating the intestinal community in a beneficial way.     

三种跳虫肠道菌群的多样性分析及功能预测

【目的】跳虫在土壤生态系统中发挥着重要的作用。本研究旨在调查Sinella(Coecobrya)oligoseta,Proisotoma minuta和Tomocerus missus 3种跳虫肠道菌群的结构和多样性以及潜在功能。【方法】采用16S rDNA扩增子测序法对以上3种跳虫成虫肠道内容物中的菌群进行分析和比较;应用Tax4Fun法对其肠道菌群基因进行功能预测。【结果】3种跳虫中成虫肠道菌群多样性最高的是T.missus,最低的是S.(C.)oligoseta。在门水平上3种跳虫成虫肠道中最主要的菌群均为变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes),放线菌门(Actinobacteria)也具有较高的丰度;在属水平上S.(C.)oligoseta肠道中假单胞菌属Pseudomonas的丰度(16.21%)明显高于P.minuta和T.missus肠道中的丰度(分别为0.87%和1.37%);P.minuta肠道中弧菌属Vibrio的丰度(25.81%)明显高于S.(C.)oligoseta和T.missus肠...     

结直肠癌患者肠道黏膜菌群多样性变化研究

目的应用PCR-DGGE技术对结直肠癌患者肠道黏膜局部菌群多样性进行研究,为结直肠病变的防治提供微生态调节思路。方法收集正常对照组、结直肠息肉组及结直肠癌组患者各30例,采集结直肠黏膜局部肛拭子,提取细菌基因组DNA,采用PCR-DGGE对肠道黏膜局部菌群进行指纹图谱分析。结果正常对照组、结直肠息肉组和结直肠癌组患者PCR-DGGE指纹图谱分析显示3组肠道黏膜局部菌群多样性发生了显著的变化,3组肠道黏膜局部菌群发生了显著的菌群变迁。结论 PCR-DGGE分析结直肠癌患者肠道黏膜局部菌群多样性变化对监测肠道局部微生态变化在结直肠癌的发生过程中的作用具有重要价值。