Raman–deuterium isotope probing to study metabolic activities of single bacterial cells in human intestinal microbiota

Human intestinal microbiota is important to host health and is associated with various diseases. It is a challenge to identify the functions and metabolic activity of microorganisms at the single-cell level in gut microbial community. In this study, we applied Raman microspectroscopy and deuterium isotope probing (Raman–DIP) to quantitatively measure the metabolic activities of intestinal bacteria from two individuals and analysed lipids and phenylalanine metabolic pathways of functional microorganisms in situ. After anaerobically incubating the human faeces with heavy water (D₂O), D₂O with specific substrates (glucose, tyrosine, tryptophan and oleic acid) and deuterated glucose, the C–D band in single-cell Raman spectra appeared in some bacteria in faeces, due to the Raman shift from the C–H band. Such Raman shift was used to indicate the general metabolic activity and the activities in response to the specific substrates. In the two individuals' intestinal microbiota, the structures of the microbial communities were different and the general metabolic activities were 76 ± 1.0% and 30 ± 2.0%. We found that glucose, but not tyrosine, tryptophan and oleic acid, significantly stimulated metabolic activity of the intestinal bacteria. We also demonstrated that the bacteria within microbiota preferably used glucose to synthesize fatty acids in faeces environment, whilst they used glucose to synthesize phenylalanine in laboratory growth environment (e.g. LB medium). Our work provides a useful approach for investigating the metabolic activity in situ and revealing different pathways of human intestinal microbiota at the single-cell level.     

定量稳定性同位素探针技术及其在微生物生态学研究中的应用

定量稳定性同位素探针技术(qSIP)是将生态系统中微生物分类性状与代谢功能联系起来的有效工具,能够定量测定特定环境中单个微生物类群暴露于同位素示踪剂后微生物代谢活动或生长速率。qSIP技术采用定量PCR与高通量测序技术并结合稳定同位素探针技术(SIP),通过向环境样品添加标记底物进行培养,提取微生物生物标记物,利用超高速等密度梯度离心将被同位素标记的重链核酸与未被标记的轻链核酸进行分离,并对所有组分微生物类群进行绝对定量和测序分析,基于GC含量和未标记处理DNA密度曲线量化参与吸收转化的DNA同位素丰度。本文重点阐述qSIP的技术原理、数据分析流程及其在微生物生态学研究中的应用进展,并对该技术存在的问题进行了分析和展望。     

Catching a glimpse of the bacterial gut community of companion animals: a canine and feline perspective

Dogs and cats have gained a special position in human society by becoming our principal companion animals. In this context, efforts to ensure their health and welfare have increased exponentially, with in recent times a growing interest in assessing the impact of the gut microbiota on canine and feline health. Recent technological advances have generated new tools to not only examine the intestinal microbial composition of dogs and cats, but also to scrutinize the genetic repertoire and associated metabolic functions of this microbial community. The application of high-throughput sequencing techniques to canine and feline faecal samples revealed similarities in their bacterial composition, with Fusobacteria, Firmicutes and Bacteroidetes as the most prevalent and abundant phyla, followed by Proteobacteria and Actinobacteria. Although key bacterial members were consistently present in their gut microbiota, the taxonomic composition and the metabolic repertoire of the intestinal microbial population may be influenced by several factors, including diet, age and anthropogenic aspects, as well as intestinal dysbiosis. The current review aims to provide a comprehensive overview of the multitude of factors which play a role in the modulation of the canine and feline gut microbiota and that of their human owners with whom they share the same environment.     

In Vivo Pyro-SIP Assessing Active Gut Microbiota of the Cotton Leafworm,Spodoptera littoralis

The gut microbiota is of crucial importance for the host with considerable metabolic activity. Although great efforts have been made toward characterizing microbial diversity, measuring components'' metabolic activity surprisingly hasn''t kept pace. Here we combined pyrosequencing of amplified 16S rRNA genes with in vivo stable isotope probing (Pyro-SIP) to unmask metabolically active bacteria in the gut of cotton leafworm (Spodoptera littoralis), a polyphagous insect herbivore that consumes large amounts of plant material in a short time, liberating abundant glucose in the alimentary canal as a most important carbon and energy source for both host and active gut bacteria. With ¹³C glucose as the trophic link, Pyro-SIP revealed that a relatively simple but distinctive gut microbiota co-developed with the host, both metabolic activity and composition shifting throughout larval stages. Pantoea, Citrobacter and Clostridium were particularly active in early-instar, likely the core functional populations linked to nutritional upgrading. Enterococcus was the single predominant genus in the community, and it was essentially stable and metabolically active in the larval lifespan. Based on that Enterococci formed biofilm-like layers on the gut epithelium and that the isolated strains showed antimicrobial properties, Enterococcus may be able to establish a colonization resistance effect in the gut against potentially harmful microbes from outside. Not only does this establish the first in-depth inventory of the gut microbiota of a model organism from the mostly phytophagous Lepidoptera, but this pilot study shows that Pyro-SIP can rapidly gain insight into the gut microbiota''s metabolic activity with high resolution and high precision.     

肠道微生物多样性与能量代谢调控及肥胖关系的研究进展

肠道微生物与宿主之间相互选择,构成了一个相对稳定的超有机体。宿主基因型和遗传关系影响肠道微生物的生态学特征,而肠道微生物发酵肠道内多糖,为宿主提供可吸收利用的养分,增强肠道对养分吸收的能力。同时,肠道微生物还影响一些转录因子的活性,调控宿主基因的表达,增强宿主甘油三酯的合成和脂肪沉积,减少脂肪酸氧化分解,调控宿主能量代谢。     

Differential Effects of Antibiotic Therapy on the Structure and Function of Human Gut Microbiota

The human intestinal microbiota performs many essential functions for the host. Antimicrobial agents, such as antibiotics (AB), are also known to disturb microbial community equilibrium, thereby having an impact on human physiology. While an increasing number of studies investigate the effects of AB usage on changes in human gut microbiota biodiversity, its functional effects are still poorly understood. We performed a follow-up study to explore the effect of ABs with different modes of action on human gut microbiota composition and function. Four individuals were treated with different antibiotics and samples were taken before, during and after the AB course for all of them. Changes in the total and in the active (growing) microbiota as well as the functional changes were addressed by 16S rRNA gene and metagenomic 454-based pyrosequencing approaches. We have found that the class of antibiotic, particularly its antimicrobial effect and mode of action, played an important role in modulating the gut microbiota composition and function. Furthermore, analysis of the resistome suggested that oscillatory dynamics are not only due to antibiotic-target resistance, but also to fluctuations in the surviving bacterial community. Our results indicated that the effect of AB on the human gut microbiota relates to the interaction of several factors, principally the properties of the antimicrobial agent, and the structure, functions and resistance genes of the microbial community.     

Metagenomic dissection of the canine gut microbiota: insights into taxonomic,metabolic and nutritional features

Domestication of dogs from wolves is the oldest known example of ongoing animal selection, responsible for generating more than 300 dog breeds worldwide. In order to investigate the taxonomic and functional evolution of the canine gut microbiota, a multi-omics approach was applied to six wild wolves and 169 dog faecal samples, the latter encompassing 51 breeds, which fully covers currently known canine genetic biodiversity. Specifically, 16S rRNA gene and bifidobacterial Internally Transcribed Spacer (ITS) profiling were employed to reconstruct and then compare the canine core gut microbiota to those of wolves and humans, revealing that artificial selection and subsequent cohabitation of dogs with their owners influenced the microbial population of canine gut through loss and acquisition of specific bacterial taxa. Moreover, comparative analysis of the intestinal bacterial population of dogs fed on Bones and Raw Food (BARF) or commercial food (CF) diet, coupled with shotgun metagenomics, highlighted that both bacterial composition and metabolic repertoire of the canine gut microbiota have evolved to adapt to high-protein or high-carbohydrates intake. Altogether, these data indicate that artificial selection and domestication not only affected the canine genome, but also shaped extensively the bacterial population harboured by the canine gut.     

The interplay between host cellular and gut microbial metabolism in NAFLD development and prevention

Metabolism regulation centred on insulin resistance is increasingly important in nonalcoholic fatty liver disease (NAFLD). This review focuses on the interactions between the host cellular and gut microbial metabolism during the development of NAFLD. The cellular metabolism of essential nutrients, such as glucose, lipids and amino acids, is reconstructed with inflammation, immune mechanisms and oxidative stress, and these alterations modify the intestinal, hepatic and systemic environments, and regulate the composition and activity of gut microbes. Microbial metabolites, such as short-chain fatty acids, secondary bile acids, protein fermentation products, choline and ethanol and bacterial toxicants, such as lipopolysaccharides, peptidoglycans and bacterial DNA, play vital roles in NAFLD. The microbe–metabolite relationship is crucial for the modulation of intestinal microbial composition and metabolic activity. The intestinal microbiota and their metabolites participate in epithelial cell metabolism via a series of cell receptors and signalling pathways and remodel the metabolism of various cells in the liver via the gut–liver axis. Microbial metabolic manipulation is a promising strategy for NAFLD prevention, but larger-sampled clinical trials are required for future application.     

Bacteria,phages and pigs: the effects of in-feed antibiotics on the microbiome at different gut locations

Disturbance of the beneficial gut microbial community is a potential collateral effect of antibiotics, which have many uses in animal agriculture (disease treatment or prevention and feed efficiency improvement). Understanding antibiotic effects on bacterial communities at different intestinal locations is essential to realize the full benefits and consequences of in-feed antibiotics. In this study, we defined the lumenal and mucosal bacterial communities from the small intestine (ileum) and large intestine (cecum and colon) plus feces, and characterized the effects of in-feed antibiotics (chlortetracycline, sulfamethazine and penicillin (ASP250)) on these communities. 16S rRNA gene sequence and metagenomic analyses of bacterial membership and functions revealed dramatic differences between small and large intestinal locations, including enrichment of Firmicutes and phage-encoding genes in the ileum. The large intestinal microbiota encoded numerous genes to degrade plant cell wall components, and these genes were lacking in the ileum. The mucosa-associated ileal microbiota harbored greater bacterial diversity than the lumen but similar membership to the mucosa of the large intestine, suggesting that most gut microbes can associate with the mucosa and might serve as an inoculum for the lumen. The collateral effects on the microbiota of antibiotic-fed animals caused divergence from that of control animals, with notable changes being increases in Escherichia coli populations in the ileum, Lachnobacterium spp. in all gut locations, and resistance genes to antibiotics not administered. Characterizing the differential metabolic capacities and response to perturbation at distinct intestinal locations will inform strategies to improve gut health and food safety.     

Shuttling of information between the mucosal and luminal environment drives intestinal homeostasis

The gastrointestinal tract is a passageway for dietary nutrients, microorganisms and xenobiotics. The gut is home to diverse bacterial communities forming the microbiota. While bacteria and their metabolites maintain gut homeostasis, the host uses innate and adaptive immune mechanisms to cope with the microbiota and luminal environment. In recent years, multiple bi-directional instructive mechanisms between microbiota, luminal content and mucosal immune systems have been uncovered. Indeed, epithelial and immune cell-derived mucosal signals shape microbiota composition, while microbiota and their by-products shape the mucosal immune system. Genetic and environmental perturbations alter gut mucosal responses which impact on microbial ecology structures. On the other hand, changes in microbiota alter intestinal mucosal responses. In this review, we discuss how intestinal epithelial Paneth and goblet cells interact with the microbiota, how environmental and genetic disorders are sensed by endoplasmic reticulum stress and autophagy responses, how specific bacteria, bacterial- and diet-derived products determine the function and activation of the mucosal immune system. We will also discuss the critical role of HDAC activity as a regulator of immune and epithelial cell homeostatic responses.     

肠道菌群与胆汁酸的互作在 相关疾病中的作用

胆汁酸在人体的胆固醇代谢、脂质消化、宿主-微生物相互作用及通路调控等方面具有重要作用。大多数胆汁酸(95%)通过肝肠循环重回收,还有约5%作为结肠内细菌生物转化的基质。胆汁酸微生物转化中涉及的各种酶可通过肠道细菌培养而被验证,证明其有种属特异性。最近,生物信息学方法揭示了这些酶有多种亚型。因此,在胆汁酸转化中肠道菌群发挥重要的作用,微生物群落结构和功能对次级胆汁酸在胆汁酸池中的分布有深刻影响。研究认为胆汁酸和胆汁酸池的组成与几种疾病有关,包括炎症性肠病、代谢综合征和结直肠癌。最近,人们的重点放在肠道菌群如何改变胆汁酸进而导致或减轻某些疾病。本文总结了肠道菌群、胆汁酸生物转化和疾病状态之间的相互作用的研究进展。     

Metagenomic analysis reveals a functional signature for biomass degradation by cecal microbiota in the leaf-eating flying squirrel (Petaurista alborufus lena)

ABSTRACT: BACKGROUND: Animals co-evolve with their gut microbiota; the latter can perform complex metabolic reactions that cannot be done independently by the host. Although the importance of gut microbiota has been well demonstrated, there is a paucity of research regarding its role in foliage-foraging mammals with a specialized digestive system. RESULTS: In this study, a 16S rRNA gene survey and metagenomic sequencing were used to characterize genetic diversity and functional capability of cecal microbiota of the folivorous flying squirrel (Petaurista alborufus lena). Phylogenetic compositions of the cecal microbiota derived from 3 flying squirrels were dominated by Firmicutes. Based on end-sequences of fosmid clones from 1 flying squirrel, we inferred that microbial metabolism greatly contributed to intestinal functions, including degradation of carbohydrates, metabolism of proteins, and synthesis of vitamins. Moreover, 33 polysaccharide-degrading enzymes and 2 large genomic fragments containing a series of carbohydrate-associated genes were identified. CONCLUSIONS: Cecal microbiota of the leaf-eating flying squirrel have great metabolic potential for converting diverse plant materials into absorbable nutrients. The present study should serve as the basis for future investigations, using metagenomic approaches to elucidate the intricate mechanisms and interactions between host and gut microbiota of the flying squirrel digestive system, as well as other mammals with similar adaptations.     

Aging alters the metabolic flux signature of the ER‐unfolded protein response in vivo in mice

Age is a risk factor for numerous diseases, including neurodegenerative diseases, cancers, and diabetes. Loss of protein homeostasis is a central hallmark of aging. Activation of the endoplasmic reticulum unfolded protein response (UPR^ER) includes changes in protein translation and membrane lipid synthesis. Using stable isotope labeling, a flux “signature” of the UPR^ER in vivo in mouse liver was developed by inducing ER stress with tunicamycin and measuring rates of both proteome‐wide translation and de novo lipogenesis. Several changes in protein synthesis across ontologies were noted with age, including a more dramatic suppression of translation under ER stress in aged mice as compared with young mice. Binding immunoglobulin protein (BiP) synthesis rates and mRNA levels were increased more in aged than young mice. De novo lipogenesis rates decreased under ER stress conditions in aged mice, including both triglyceride and phospholipid fractions. In young mice, a significant reduction was seen only in the triglyceride fraction. These data indicate that aged mice have an exaggerated metabolic flux response to ER stress, which may indicate that aging renders the UPR^ER less effective in resolving proteotoxic stress.     

Raman-activated sorting of antibiotic-resistant bacteria in human gut microbiota

The antibiotic-resistant bacteria (ARB) and antibiotic-resistant genes (ARGs) in human gut microbiota have significant impact on human health. While high throughput metagenomic sequencing reveals genotypes of microbial communities, the functionality, phenotype and heterogeneity of human gut microbiota are still elusive. In this study, we applied Raman microscopy and deuterium isotope probing (Raman–DIP) to detect metabolic active ARB (MA-ARB) in situ at the single-cell level in human gut microbiota from two healthy adults. We analysed the relative abundances of MA-ARB under different concentrations of amoxicillin, cephalexin, tetracycline, florfenicol and vancomycin. To establish the link between phenotypes and genotypes of the MA-ARB, Raman-activated cell sorting (RACS) was used to sort MA-ARB from human gut microbiota, and mini-metagenomic DNA of the sorted bacteria was amplified, sequenced and analysed. The sorted MA-ARB and their associated ARGs were identified. Our results suggest a strong relation between ARB in human gut microbiota and personal medical history. This study demonstrates that the toolkit of Raman–DIP, RACS and DNA sequencing can be useful to unravel both phenotypes and genotypes of ARB in human gut microbiota at the single-cell level.     

肠道微生物培养的研究进展及应用

肠道中栖居着组成复杂、功能多样的微生物群,这些微生物群在宿主免疫、营养吸收、代谢调节等方面发挥着重要作用。随着测序技术的快速发展,肠道微生物研究通过16S rRNA基因测序和宏基因组测序产生了大量的数据,其中许多未组装的序列成为微生物“暗物质”。近年来,不少研究利用多种不同微生物分离培养方法,结合高通量鉴定技术,从人、小鼠、猪肠道中分离了大量的微生物,丰富了菌株资源,为解析微生物“暗物质”以及后续肠道微生物功能和应用研究提供了基础和保障。尽管微生物的可培养性受到多种因素的影响,大部分微生物尚处于“未培养”的状态,但无论是病因研究还是生理和遗传特征的解析都离不开微生物实体资源的获取。肠道微生物的分离培养对微生物研究从关联分析向菌群功能验证、因果机制解析和功能菌株开发的深入研究具有重要意义。本文旨在探讨和综述影响微生物可培养性的因素,总结回顾肠道微生物的培养方法并阐述肠道微生物培养研究的进展,以期为肠道微生物培养研究提供新的视角。     

Novel Polyfermentor Intestinal Model (PolyFermS) for Controlled Ecological Studies: Validation and Effect of pH

In vitro gut fermentation modeling offers a useful platform for ecological studies of the intestinal microbiota. In this study we describe a novel Polyfermentor Intestinal Model (PolyFermS) designed to compare the effects of different treatments on the same complex gut microbiota. The model operated in conditions of the proximal colon is composed of a first reactor containing fecal microbiota immobilized in gel beads, and used to continuously inoculate a set of parallel second-stage reactors. The PolyFermS model was validated with three independent intestinal fermentations conducted for 38 days with immobilized human fecal microbiota obtained from three child donors. The microbial diversity of reactor effluents was compared to donor feces using the HITChip, a high-density phylogenetic microarray targeting small subunit rRNA sequences of over 1100 phylotypes of the human gastrointestinal tract. Furthermore, the metabolic response to a decrease of pH from 5.7 to 5.5, applied to balance the high fermentative activity in inoculum reactors, was studied. We observed a reproducible development of stable intestinal communities representing major taxonomic bacterial groups at ratios similar to these in feces of healthy donors, a high similarity of microbiota composition produced in second-stage reactors within a model, and a high time stability of microbiota composition and metabolic activity over 38 day culture. For all tested models, the pH-drop of 0.2 units in inoculum reactors enhanced butyrate production at the expense of acetate, but was accompanied by a donor-specific reorganization of the reactor community, suggesting a concerted metabolic adaptation and trigger of community-specific lactate or acetate cross-feeding pathways in response to varying pH. Our data showed that the PolyFermS model allows the stable cultivation of complex intestinal microbiota akin to the fecal donor and can be developed for the direct comparison of different experimental conditions in parallel reactors continuously inoculated with the exact same microbiota.     

Outside the liver box: The gut microbiota as pivotal modulator of liver diseases

The gut microbiota affects host physiology and has evolved as an important contributor to health and disease. Gut and liver are closely connected and communicate via the portal vein and the biliary system so the liver is constantly exposed to gut-derived bacterial products and metabolites. The intestinal barrier is important for maintaining physical and functional separation between microbes in the gut and the interior of the host and disruption of the barrier function can lead to bacterial translocation and increased leakage of bacterial metabolites. Liver diseases have been associated with dysbiotic changes in the gut microbiota and impaired gut barrier integrity, thus a future strategy to treat liver disease may be to target the gut microbiota and thereby restore the gut barrier function. This review will summarize and discuss studies that have shown a link between the gut microbiota and liver disease with the main focus on non-alcoholic fatty liver disease and alcoholic liver disease.     

Human intestinal lumen and mucosa-associated microbiota in patients with colorectal cancer

Recent reports have suggested the involvement of gut microbiota in the progression of colorectal cancer (CRC). We utilized pyrosequencing based analysis of 16S rRNA genes to determine the overall structure of microbiota in patients with colorectal cancer and healthy controls; we investigated microbiota of the intestinal lumen, the cancerous tissue and matched noncancerous normal tissue. Moreover, we investigated the mucosa-adherent microbial composition using rectal swab samples because the structure of the tissue-adherent bacterial community is potentially altered following bowel cleansing. Our findings indicated that the microbial structure of the intestinal lumen and cancerous tissue differed significantly. Phylotypes that enhance energy harvest from diets or perform metabolic exchange with the host were more abundant in the lumen. There were more abundant Firmicutes and less abundant Bacteroidetes and Proteobacteria in lumen. The overall microbial structures of cancerous tissue and noncancerous tissue were similar; however the tumor microbiota exhibited lower diversity. The structures of the intestinal lumen microbiota and mucosa-adherent microbiota were different in CRC patients compared to matched microbiota in healthy individuals. Lactobacillales was enriched in cancerous tissue, whereas Faecalibacterium was reduced. In the mucosa-adherent microbiota, Bifidobacterium, Faecalibacterium, and Blautia were reduced in CRC patients, whereas Fusobacterium, Porphyromonas, Peptostreptococcus, and Mogibacterium were enriched. In the lumen, predominant phylotypes related to metabolic disorders or metabolic exchange with the host, Erysipelotrichaceae, Prevotellaceae, and Coriobacteriaceae were increased in cancer patients. Coupled with previous reports, these results suggest that the intestinal microbiota is associated with CRC risk and that intestinal lumen microflora potentially influence CRC risk via cometabolism or metabolic exchange with the host. However, mucosa-associated microbiota potentially affects CRC risk primarily through direct interaction with the host.     

Evidence for a core gut microbiota in the zebrafish

Experimental analysis of gut microbial communities and their interactions with vertebrate hosts is conducted predominantly in domesticated animals that have been maintained in laboratory facilities for many generations. These animal models are useful for studying coevolved relationships between host and microbiota only if the microbial communities that occur in animals in lab facilities are representative of those that occur in nature. We performed 16S rRNA gene sequence-based comparisons of gut bacterial communities in zebrafish collected recently from their natural habitat and those reared for generations in lab facilities in different geographic locations. Patterns of gut microbiota structure in domesticated zebrafish varied across different lab facilities in correlation with historical connections between those facilities. However, gut microbiota membership in domesticated and recently caught zebrafish was strikingly similar, with a shared core gut microbiota. The zebrafish intestinal habitat therefore selects for specific bacterial taxa despite radical differences in host provenance and domestication status.     

In Vitro Continuous Fermentation Model (PolyFermS) of the Swine Proximal Colon for Simultaneous Testing on the Same Gut Microbiota

In vitro gut modeling provides a useful platform for a fast and reproducible assessment of treatment-related changes. Currently, pig intestinal fermentation models are mainly batch models with important inherent limitations. In this study we developed a novel in vitro continuous fermentation model, mimicking the porcine proximal colon, which we validated during 54 days of fermentation. This model, based on our recent PolyFermS design, allows comparing different treatment effects on the same microbiota. It is composed of a first-stage inoculum reactor seeded with immobilized fecal swine microbiota and used to constantly inoculate (10% v/v) five second-stage reactors, with all reactors fed with fresh nutritive chyme medium and set to mimic the swine proximal colon. Reactor effluents were analyzed for metabolite concentrations and bacterial composition by HPLC and quantitative PCR, and microbial diversity was assessed by 454 pyrosequencing. The novel PolyFermS featured stable microbial composition, diversity and metabolite production, consistent with bacterial activity reported for swine proximal colon in vivo. The constant inoculation provided by the inoculum reactor generated reproducible microbial ecosystems in all second-stage reactors, allowing the simultaneous investigation and direct comparison of different treatments on the same porcine gut microbiota. Our data demonstrate the unique features of this novel PolyFermS design for the swine proximal colon. The model provides a tool for efficient, reproducible and cost-effective screening of environmental factors, such as dietary additives, on pig colonic fermentation.