Evolution of mammals and their gut microbes

Mammals are metagenomic in that they are composed of not only their own gene complements but also those of all of their associated microbes. To understand the coevolution of the mammals and their indigenous microbial communities, we conducted a network-based analysis of bacterial 16S ribosomal RNA gene sequences from the fecal microbiota of humans and 59 other mammalian species living in two zoos and in the wild. The results indicate that host diet and phylogeny both influence bacterial diversity, which increases from carnivory to omnivory to herbivory; that bacterial communities codiversified with their hosts; and that the gut microbiota of humans living a modern life-style is typical of omnivorous primates.     

Microfluidic digital PCR enables multigene analysis of individual environmental bacteria

Gene inventory and metagenomic techniques have allowed rapid exploration of bacterial diversity and the potential physiologies present within microbial communities. However, it remains nontrivial to discover the identities of environmental bacteria carrying two or more genes of interest. We have used microfluidic digital polymerase chain reaction (PCR) to amplify and analyze multiple, different genes obtained from single bacterial cells harvested from nature. A gene encoding a key enzyme involved in the mutualistic symbiosis occurring between termites and their gut microbiota was used as an experimental hook to discover the previously unknown ribosomal RNA-based species identity of several symbionts. The ability to systematically identify bacteria carrying a particular gene and to link any two or more genes of interest to single species residing in complex ecosystems opens up new opportunities for research on the environment.     

Innate immune homeostasis by the homeobox gene caudal and commensal-gut mutualism in Drosophila

Although commensalism with gut microbiota exists in all metazoans, the host factors that maintain this homeostatic relationship remain largely unknown. We show that the intestinal homeobox gene Caudal regulates the commensal-gut mutualism by repressing nuclear factor kappa B-dependent antimicrobial peptide genes. Inhibition of Caudal expression in flies via RNA interference led to overexpression of antimicrobial peptides, which in turn altered the commensal population within the intestine. In particular, the dominance of one gut microbe, Gluconobacter sp. strain EW707, eventually led to gut cell apoptosis and host mortality. However, restoration of a healthy microbiota community and normal host survival in the Caudal-RNAi flies was achieved by reintroduction of the Caudal gene. These results reveal that a specific genetic deficiency within a host can profoundly influence the gut commensal microbial community and host physiology.     

肠道微生物宏基因组文库构建及酯解酶基因筛选

利用宏基因组学方法,以人肠道微生物样品为原材料,构建了1个约30 000个克隆的fosmid文库.以三丁酸甘油酯为底物,通过功能筛选,获得1个酯解酶阳性克隆.对该阳性克隆构建亚克隆,挑选具有酯解酶活性的阳性亚克隆进行测序分析,最终获得1个肠道微生物来源的酯解酶基因(GenBank登录号:JQ972699).结果表明,文库克隆的平均插入片段约为40 kb,没有重复插入片段克隆.获得的酯解酶基因推演蛋白与Pyramidobacter piscolens W5455的patatin样磷脂酶同源性最高,氨基酸一致性为95％.生物信息学分析结果表明该基因可能通过Vd型分泌方式进行分泌并发挥功能.本研究是通过构建人肠道微生物宏基因组大片段文库并结合重组子功能筛选获得酯解酶的首次报道,可为食品工业提供新的酯解酶来源和筛选方法.     

Germinated brown rice relieves hyperlipidemia by alleviating gut microbiota dysbiosis

Hyperlipidemia is a frequent metabolic disorder that is closely associated with diet.  It is believed that brown rice, containing the outer bran layer and germ, is beneficial for the remission of hyperlipidemia.  This study established a rat model of hyperlipidemia by feeding a high-fat diet.  The hypolipidemic potential of germinated brown rice (Gbrown) and germinated black rice (a germinated black-pigmented brown rice, Gblack) were explored in the model rats, mainly in the aspects of blood lipids, lipases, apolipoproteins, and inflammation.  The gut microbiota in hyperlipidemic rats receiving diverse dietary interventions was determined by 16S rDNA sequencing.  The results showed that the intervention of Gbrown/Gblack alleviated the hyperlipidemia in rats, evidenced by decreased TC, TG, LDL-C, and apolipoprotein B, and increased HDL-C, HL, LPL, LCAT, and apolipoprotein A1.  Gbrown/Gblack also weakened the inflammation in hyperlipidemia rats, evidenced by decreased TNF-α, IL-6, and ET-1.  In addition, 16S rDNA sequencing revealed that the diet of Gbrown/Gblack elevated the abundance and diversity of gut microbiota in hyperlipidemia rats.  At the phylum level, Gbrown/Gblack decreased Firmicutes, increased Bacteroidetes, and decreased the F/B ratio in hyperlipidemia rats.  At the genus level, Gbrown/Gblack decreased Streptococcus and increased Ruminococcus and Allobaculum in hyperlipidemia rats.  Some differential microbial genera relating to lipid metabolism were also determined, such as the Lachnospira and Ruminococcus in the Gblack group, and the Phascolarctobacterium, Dorea, Turicibacter, and Escherichia-Shigella in the Gbrown group.  Notably, the beneficial effect of Gblack was stronger than Gbrown.  To sum up, the dietary interventions of Gbrown/Gblack contributed to the remission of hyperlipidemia by alleviating the dysbiosis of gut microbiota.     

Environmental genome shotgun sequencing of the Sargasso Sea

We have applied "whole-genome shotgun sequencing" to microbial populations collected en masse on tangential flow and impact filters from seawater samples collected from the Sargasso Sea near Bermuda. A total of 1.045 billion base pairs of nonredundant sequence was generated, annotated, and analyzed to elucidate the gene content, diversity, and relative abundance of the organisms within these environmental samples. These data are estimated to derive from at least 1800 genomic species based on sequence relatedness, including 148 previously unknown bacterial phylotypes. We have identified over 1.2 million previously unknown genes represented in these samples, including more than 782 new rhodopsin-like photoreceptors. Variation in species present and stoichiometry suggests substantial oceanic microbial diversity.     

Insights into the effects of pulsed antimicrobials on the chicken resistome and microbiota from fecal metagenomes

Antimicrobial resistance has become a global problem that poses great threats to human health. Antimicrobials are widely used in broiler chicken production and consequently affect their gut microbiota and resistome. To better understand how continuous antimicrobial use in farm animals alters their microbial ecology, we used a metagenomic approach to investigate the effects of pulsed antimicrobial administration on the bacterial community, antibiotic resistance genes (ARGs) and ARG bacterial hosts in the feces of broiler chickens. Chickens received three 5-day courses of individual or combined antimicrobials, including amoxicillin, chlortetracycline and florfenicol. The florfenicol administration significantly increased the abundance of mcr-1 gene accompanied by floR gene, while amoxicillin significantly increased the abundance of genes encoding the AcrAB-tolC multidrug efflux pump (marA, soxS, sdiA, rob, evgS and phoP). These three antimicrobials all led to an increase in Proteobacteria. The increase in ARG host, Escherichia, was mainly attributed to the β-lactam, chloramphenicol and tetracycline resistance genes harbored by Escherichia under the pulsed antimicrobial treatments. These results indicated that pulsed antimicrobial administration with amoxicillin, chlortetracycline, florfenicol or their combinations significantly increased the abundance of Proteobacteria and enhanced the abundance of particular ARGs. The ARG types were occupied by the multidrug resistance genes and had significant correlations with the total ARGs in the antimicrobial-treated groups. The results of this study provide comprehensive insight into pulsed antimicrobial-mediated alteration of chicken fecal microbiota and resistome.     

单一益生菌(屎肠球菌)介导的断奶仔猪肠道菌群变化规律

通过细菌16SrRNA基因的高通量测序,对断奶仔猪的肠道菌群结构和饲喂两株单一益生菌(屎肠球菌)后肠道菌群的变化情况和规律进行分析。结果显示,断奶仔猪肠道微生态中拟杆菌门Bacteroidetes和厚壁菌门Firmicutes的细菌所占比例分别为50.5%和42.2%,为绝对优势菌群。饲喂屎肠球菌后,肠道菌群的多样性和细菌丰度均增加;厚壁菌门Firmicutes,变形菌门Proteobacteria、无壁菌门Tenericutes、酸杆菌门Actidobacteria、芽单胞菌门Gemmatimonadetes、放线菌门Actinobacteria细菌的丰度明显上升,而拟杆菌门Bacteroidetes的细菌丰度则发生了明显下降。屎肠球菌所在的肠球菌属Enterococcus的比例虽由0.07%(对照组)上升至0.12%(SF1组)和0.14%(SF2组),但在肠道菌群中所占比例仍然很小,说明屎肠球菌对仔猪肠道微生态的作用主要是影响其他细菌的丰度和多样性进而改变菌群结构。与对照组相比,SF1组的上述菌群变化幅度高于SF2组,通过与3组仔猪生长性能数据的对比,上述菌群变化的幅度与仔猪生长性能提高的程度呈现正相关。     

利用Illumina MiSeq测序平台分析肉鸡盲肠微生物多样性

为对肉鸡盲肠微生物的多样性进行探索,选取15只饲养在相同条件下的科宝-500肉鸡,利用Illumina MiSeq测序平台与Qiime等16srRNA序列分析工具在22日龄时采集盲肠内容物进行研究。结果显示:1)测序共获得359 158条有效序列与3 210个OTU,并且由稀释曲线可以证明此次测序结果比较全面的覆盖了肉鸡盲肠微生物群落。2)各样品菌群之间存在差异,得到序列的数量以及菌群多样性差别较大。3)通过核心菌群分析可知,4个共享菌群在门水平上分别为:拟杆菌门Bacteroidetes(70.0%±12.1%)、厚壁菌门Firmicutes(25.4%±11.2%)、变形菌门Proteobacteria(4.3%±3.7%)和蓝菌门Cyanobacteria(0.3%±0.5%)。4)有益菌群如乳酸杆菌属Lactobacillus、双歧杆菌属Bifidobacterium虽然存在于盲肠微生物中,但相对丰度不高。5)PICRUSt分析盲肠菌群对应的基因功能发现:功能转运、嘌呤代谢、DNA修复重组蛋白、氧化磷酸化和甲烷代谢等基因功能丰度最高。由此可见,个体差异虽然对肉鸡盲肠微生物结构影响较大,但是仍然存在不少共性。     

耐硼赖氨酸芽孢杆菌的保护剂筛选及其对罗非鱼肠道微生物群落的影响

本研究目的是为了探究耐硼赖氨酸芽孢杆菌（Lysinibacillus boronitolerans,YS11）的保存条件及其对罗非鱼肠道微生物多样性和丰度的影响,为微生物饲料添加剂的研发提供科学依据。选择海藻糖、乳糖、蔗糖和脱脂奶粉4种保护剂,并通过浓度梯度优化筛选最佳保护剂,结果表明,YS11在10%海藻糖中,干燥后保持较高的存活率。罗非鱼幼苗饲喂菌体发酵液,喂养70 d后,通过高通量测序观察YS11对罗非鱼肠道微生物群落的影响,结果表明,添加YS11的处理改变了罗非鱼肠道微生物群及其多样性,提高罗非鱼肠道中有益菌的丰度,降低某些潜在致病菌的丰度。本研究结果证实,10%海藻糖为最佳冷冻干燥保护剂;耐硼赖氨酸芽孢杆菌能够调节肠道微生物群,促进益生菌生长,抑制病原菌生长。     

Alleviating cancer drug toxicity by inhibiting a bacterial enzyme

The dose-limiting side effect of the common colon cancer chemotherapeutic CPT-11 is severe diarrhea caused by symbiotic bacterial β-glucuronidases that reactivate the drug in the gut. We sought to target these enzymes without killing the commensal bacteria essential for human health. Potent bacterial β-glucuronidase inhibitors were identified by high-throughput screening and shown to have no effect on the orthologous mammalian enzyme. Crystal structures established that selectivity was based on a loop unique to bacterial β-glucuronidases. Inhibitors were highly effective against the enzyme target in living aerobic and anaerobic bacteria, but did not kill the bacteria or harm mammalian cells. Finally, oral administration of an inhibitor protected mice from CPT-11-induced toxicity. Thus, drugs may be designed to inhibit undesirable enzyme activities in essential microbial symbiotes to enhance chemotherapeutic efficacy.     

PCR-DGGE 技术分析不同染料对厌氧污泥中微生物群落结构的影响

为了揭示在生物法处理印染废水过程中废水所含染料种类的变化对厌氧脱色菌群群落结构的影响,采用8种不同的染料进行浓度梯度驯化从厌氧污泥中富集得到的脱色菌群,测定了驯化后菌群对其驯化染料的脱色率,并将获得的各个菌群基因组DNA以细菌16S rDNA V3区通用引物进行PCR扩增,将扩增产物进行变性梯度凝胶电泳(DGGE),获得菌群的基因组DNA特征指纹图谱.脱色试验结果显示,经偶氮染料驯化后的菌群对其驯化染料的脱色率要高于经蒽醌染料驯化的菌群.DGGE图谱表明经不同染料驯化后,菌群结构发生了显著变化,其中菌群经活性黑5和酸性蓝127驯化后菌种数减少明显.另外,对菌群的12条优势菌条带进行克隆测序,通过Blast比对,它们分别属于乳杆菌属(Lactobacillus)、肠球菌属(Enterococcus)、乳球菌属(Lactococcus)和双歧杆菌属(Bifidobacterium),在菌群对染料的脱色过程中发挥了重要作用.     

Comparative metagenomics of microbial communities

The species complexity of microbial communities and challenges in culturing representative isolates make it difficult to obtain assembled genomes. Here we characterize and compare the metabolic capabilities of terrestrial and marine microbial communities using largely unassembled sequence data obtained by shotgun sequencing DNA isolated from the various environments. Quantitative gene content analysis reveals habitat-specific fingerprints that reflect known characteristics of the sampled environments. The identification of environment-specific genes through a gene-centric comparative analysis presents new opportunities for interpreting and diagnosing environments.     

蜜蜂肠道菌群的调节作用及影响因素

近年来, 蜜蜂数量的大量减少导致全球养蜂业面临重要的经济损失,并对农业各个方面造成了严重后果。最新的研究表明肠道菌群与宿主的健康息息相关。综述了肠道菌群的种类和特性,阐述了肠道菌群在蜜蜂等昆虫健康中的调节作用及影响肠道菌群的因素,并对今后的研究方向进行了展望。     

Predicting a human gut microbiota's response to diet in gnotobiotic mice

The interrelationships between our diets and the structure and operations of our gut microbial communities are poorly understood. A model community of 10 sequenced human gut bacteria was introduced into gnotobiotic mice, and changes in species abundance and microbial gene expression were measured in response to randomized perturbations of four defined ingredients in the host diet. From the responses, we developed a statistical model that predicted over 60% of the variation in species abundance evoked by diet perturbations, and we were able to identify which factors in the diet best explained changes seen for each community member. The approach is generally applicable, as shown by a follow-up study involving diets containing various mixtures of pureed human baby foods.     

奥利司他干预对高脂饮食大鼠肠道微生物的影响

将大鼠随机分为正常组(10只)与造模组(高脂饮食组,20只),连续喂养10周后造模成功;再将造模组随机分为模型组(10只)和奥利司他组(10只),正常组、模型组和奥利司他组分别给予正常饮食、高脂饮食、高脂饮食+奥利司他灌胃,再持续饲喂10周后采集样品进行血清生化指标检测、组织病理学观察和肠道菌群分析,以探究奥利司他对高脂饮食诱导肥胖大鼠的影响。结果表明：奥利司他干预可显著降低高脂饮食大鼠的体质量;降低长期高脂饮食所引起的肝脏脂质堆积,减少肝损伤及体内脂肪的堆积;降低高脂饮食大鼠的血清总胆固醇和低密度脂蛋白胆固醇水平,升高高密度脂蛋白胆固醇水平,改善肥胖大鼠的血脂代谢异常问题;升高肥胖大鼠血清中脂联素水平,同时降低血清瘦素以及游离脂肪酸水平;奥利司他干预会造成大鼠小肠和盲肠菌群丰度和多样性的减少,增加大鼠结肠内微生物群落的物种多样性;奥利司他干预降低了高脂饮食大鼠结肠中厚壁菌门与拟杆菌门相对丰度的比值,在小肠和盲肠中则提高了该比值;高脂饮食后大鼠盲肠中螺旋体门和疣微菌门的相对丰度显著高于正常组的,但奥利司他治疗后螺旋体门的相对丰度较模型组有所降低;奥利司他促进高脂饮食大鼠小肠中的乳杆菌属(Lactobacillus)和罗姆布茨菌属(Romboutsia),盲肠中的阿克曼菌属(Akkermansia)和布劳特氏菌属(Blautia),结肠中的布劳特氏菌属、毛形杆菌属(Lachnobacterium)和罗斯伯里氏菌属(Roseburia)等肠道有益菌的生长,并抑制小肠中链球菌属(Streptococcus)等有害菌的繁殖。可见,奥利司他干预可有效地改善因高脂饮食引起的肥胖及其代谢紊乱,并可改善因高脂饮食造成的肠道菌群结构失调。     

PCR-DGGE 技术分析不同染料对厌氧污泥中微生物群落结构的影响

为了揭示在生物法处理印染废水过程中废水所含染料种类的变化对厌氧脱色菌群群落结构的影响,采用8 种不同的染料进行浓度梯度驯化从厌氧污泥中富集得到的脱色菌群,测定了驯化后菌群对其驯化染料的脱色率,并将获得的各个菌群基因组DNA 以细菌16S rDNA V3 区通用引物进行PCR 扩增,将扩增产物进行变性梯度凝胶电泳（DGGE）,获得菌群的基因组DNA 特征指纹图谱。脱色试验结果显示,经偶氮染料驯化后的菌群对其驯化染 料的脱色率要高于经蒽醌染料驯化的菌群。DGGE 图谱表明经不同染料驯化后,菌群结构发生了显著变化,其中菌群经活性黑5 和酸性蓝127 驯化后菌种数减少明显。另外,对菌群的12 条优势菌条带进行克隆测序,通过Blast 比对,它们分别属于乳杆菌属（Lactobacillus）、肠球菌属（Enterococcus）、乳球菌属（Lactococcus）和双歧杆菌属（Bifidobacterium）,在菌群对染料的脱色过程中发挥了重要作用。     

The inhibitory receptor PD-1 regulates IgA selection and bacterial composition in the gut

Immunoglobulin A (IgA) is essential to maintain the symbiotic balance between gut bacterial communities and the host immune system. Here we provide evidence that the inhibitory co-receptor programmed cell death-1 (PD-1) regulates the gut microbiota through appropriate selection of IgA plasma cell repertoires. PD-1 deficiency generates an excess number of T follicular helper (T(FH)) cells with altered phenotypes, which results in dysregulated selection of IgA precursor cells in the germinal center of Peyer's patches. Consequently, the IgAs produced in PD-1-deficient mice have reduced bacteria-binding capacity, which causes alterations of microbial communities in the gut. Thus, PD-1 plays a critical role in regulation of antibody diversification required for the maintenance of intact mucosal barrier.     

斜带石斑鱼幼鱼消化道与养殖水体中可培养菌群的研究

利用海水琼脂、TCBS和MRS培养基对斜带石斑鱼Epinephelus coioides幼鱼消化道和养殖水体中的细菌进行分离纯化,采用生理生化鉴定结合16SrRNA基因测序进行细菌鉴定,探讨了幼鱼消化道中的菌群与养殖水体中菌群的关系。结果表明：幼鱼消化道中可培养细菌总数（9．0×106cfu／g）高于养殖水体中可培养细菌总数（5．4×105cfu／mL）,幼鱼消化道中细菌种类也明显多于养殖水体;在幼鱼消化道和养殖水体中均检测到副溶血弧菌、哈维氏弧菌、短小芽孢杆菌、克劳氏芽孢杆菌、鲍氏不动杆菌、嗜冷杆菌和洋葱伯克霍尔德菌等7种细菌。细菌系统进化分析结果表明,这些细菌隶属于β-变形杆菌纲、γ-变形杆菌纲和芽孢杆菌纲。幼鱼消化道和养殖水体的优势菌中均有芽孢杆菌和嗜冷杆菌,提示芽孢杆菌和嗜冷杆菌能通过养殖水体很好地进入消化道。3种乳酸菌（乳酸乳球菌、干酪乳杆菌和屎肠球菌）仅见于幼鱼消化道,且仅占消化道菌群数量的0．70％。弧菌在消化道和养殖水体中所占比例均很小,分别仅占细菌总数的1．07％和0．10％。     

Selective silencing of foreign DNA with low GC content by the H-NS protein in Salmonella

Horizontal gene transfer plays a major role in microbial evolution. However, newly acquired sequences can decrease fitness unless integrated into preexisting regulatory networks. We found that the histone-like nucleoid structuring protein (H-NS) selectively silences horizontally acquired genes by targeting sequences with GC content lower than the resident genome. Mutations in hns are lethal in Salmonella unless accompanied by compensatory mutations in other regulatory loci. Thus, H-NS provides a previously unrecognized mechanism of bacterial defense against foreign DNA, enabling the acquisition of DNA from exogenous sources while avoiding detrimental consequences from unregulated expression of newly acquired genes. Characteristic GC/AT ratios of bacterial genomes may facilitate discrimination between a cell's own DNA and foreign DNA.