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Category: Environmental Microbiology; Applied and Industrial Microbiology
Stable Isotope Probing and the Human Gut, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555816896/9781555815370_Chap11-1.gif /docserver/preview/fulltext/10.1128/9781555816896/9781555815370_Chap11-2.gifAbstract:
This chapter describes the recent developments that have started to shed some light on the fermentative capacity of the colonic microbiota and its contribution to health and disease. The combined use of stable isotope probing (SIP) with analytical, gastroenterological, nanotechnological, and phylogenetic microarray techniques is highlighted in the chapter. The human gastrointestinal (GI) tract comprises a series of organs ranging from the stomach to the distal colon, with complex and dynamically changing conditions, that harbors immense microbial assemblages that are known to be critical for human health and disease. Now, many tools and techniques are available to comprehensively characterize the microbial diversity in the human gut. The study of the composition of the (intestinal) microbiota has recently gained enormous momentum through the development of DNA microarray methods to study the presence of hundreds of species at the same time. Endogenous proteins either are of secretory origin or enter the gut lumen as desquamated epithelial cells. Most microorganisms in the colon prefer to ferment carbohydrates and switch to protein fermentation when fermentable carbohydrates have been used up. The flux model currently describes the collective intestinal microbiota as a single entity. Since the bacteria that primarily used the glucose are known from the SIP results, this model could be deconvoluted into the individual microorganisms contributing to the production of the labeled metabolites. Galacto-oligosaccharides (GOS) are nondigestible carbohydrates that are resistant to GI digestive enzymes but can be fermented by specific colonic bacteria.
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Principle of nucleic acid (DNA and RNA) SIP. (Adapted from Kovatcheva-Datchary et al., 2009b .) A 13C-labeled substrate is incubated with a complex microbiota in vitro (a) or in vivo (b). Over time, samples are taken and nucleic acids are isolated. Using density gradient centrifugation (with cesium chloride for DNA or cesium trifluoroacetate for RNA), unlabeled (12C; indicated in gray) and labeled (13C; indicated in black) nucleic acids are separated. After fractionation, several molecular profiling techniques can be used to analyze the microbial composition of the heavy (13C) and light (12C) fractions. Differences in composition reveal that members of the microbiota were involved in fermentation of the labeled substrate.
Similarity of microbiota samples in TIM-2 to fecal samples from human volunteers. (A) Similarity of the total microbiota and major phylogenetic groups between TIM-2 profile and human fecal profiles. Samples were analyzed using the Human Intestinal Tract Chip (HIT-Chip) (Rajilic-Stojanovic et al., 2009 ). (Adapted from Kovatcheva-Datchary et al., 2009b.) (B) Clone libraries were generated from 16S rRNA template from TIM-2 samples. From these, 23 RFLP groups were defined and 96 representative clones were selected for sequencing. The phylogenetic analyses showed that the clone library consisted of phylotypes belonging to the three major phyla found in the intestinal microbiota, namely Bacteroidetes, Firmicutes (Clostridium clusters IV and XIVa), and Actinobacteria (Bifidobacterium).
Distribution of RNA over several fractions after density gradient centrifugation. (A) Fractions collected after density gradient centrifugation were subjected to RT-PCR using primers against 16S rRNA. Due to differences in G+C content of the RNA of the different members of the microbiota, the 16S rRNA already distributes over several fractions (with the high-G+C RNA present in the lower numbered fractions) before addition of a labeled substrate, as also indicated in the rightmost tube in panel B. Four hours after addition of a 13C-labeled substrate, the distribution of the RNA shifts to more dense fractions. (B) When a single microorganism is probed with the substrate, more clear-cut separation is achieved. [13C]DNA (or RNA) segregates to a denser fraction (ii) than 12C (i), even when both are present in the same culture (iii). When a complex mixture of bacteria is present, the RNA segregates over almost all fractions (iv).
TRFLP profiles of the [13C]glucose experiment. TRFLP profiles were created from various fractions and at different time points. Representative profiles are shown. The densest fractions are shown on top. These densest fractions (fraction 4 after 1 h; fraction 3 after 2 h) contain peaks that have been attributed to Streptococcus bovis and Clostridium perfringens. S, Streptococcus bovis; C, Clostridium perfringens; E, Enterococcus faecium/faecalis. Pseudo-TRFs are indicated with an asterisk. (Adapted from Egert et al., 2007.)
Gut microbial metabolic network used for 13C-isotopomer-based pathway analysis upon [13C]glucose fermentation, with the main experimentally observed isotopomer labeling patterns indicated. (A) VXXX represents the metabolic fluxes that have been quantitatively determined (displayed in panel B), with xxx denoting reaction steps as follows: EMP, Embden-Meyerhof-Parnas pathway (glycolysis) of pyruvate synthesis from labeled glucose; PYR, synthesis of pyruvate from unlabeled protein sources; ACCOA, synthesis of acetyl-coenzyme A from unlabeled protein and fatty acid sources; PDH, pyruvate-dehydrogenase complex; LDH, lactate dehydrogenase; PFCX, pyruvate-formate carboxyl group transfer; PFL, pyruvate-formate lyase; FHL, formate-hydrogen lyase; WLP, Wood-Ljungdahl pathway; ACK, acetate kinase; BUK, butyrate kinase. VLACt, VFORt, VAct, and VBUt represent transmembrane transport of intra- and extracellular indicated metabolites, whereas VLACex, VFORex, VACex and VBUex represent rates of disappearance of the indicated metabolites from TIM-2 due to dialysis.
Several profiling techniques to evaluate which members of the microbiota ferment inulin. (A) TRFLP profile of heavy (13C-labeled) and light (12C-containing) fractions. A peak of 623 bp (circled) is specifically increased in the heavy fraction. (B) HIT-Chip profiling of the major groups/families present in the microbiota. Especially the Clostridium cluster XIVa is increased in the labeled fraction compared to the unlabeled fraction. (C) When zooming in on this Clostridium cluster XIVa, it is clear that several phylotypes (e.g., Ruminococcus obeum, Eubacterium rectale, E. hallii, Clostridium colinum, and Bryantella formatexigens) are increased in the labeled fraction, whereas others decrease (e.g., Coprococcus eutactus and Clostridium nexile).
The two pathways through which propionate can be produced. The acrylate pathway (dark gray) leads to fully labeled propionate, with fully labeled pyruvate and lactate (and acrylate) as precursors. The succinate decarboxylating pathway (light gray) also has pyruvate as a precursor, but propionate is formed through oxaloacetate, malate, fumarate, and succinate. This results in equal portions of the M+1, M+2, and fully labeled propionate-isotopomers, due to mixing of the label in the precursors of this pathway. When concentrations of the individual isotopomers are known, one can calculate fluxes through the two pathways using the formulae that are plotted in the figure.
Example of information that becomes available using the combination of (one- or two-dimensional) NMR and LC-MS. On the left the NMR information of the labeling around carbon atom 2 (top) and carbon atom 3 (bottom) is given. For carbon atom 3 the signals are the sum of two different isotopomers (as shown). On the top right, the information gathered with LC-MS is displayed. On the bottom right the combined measurements provide information of all the individual isotopomers, measured with either NMR, LC-MS, or both. This information is then used to calculate fluxes through the two pathways of propionate production as indicated in the legend of Fig. 7 .
Cross-feeding within the intestinal microbiota. (A) Cumulative production of butyrate (and acetate [inset]). The different mass-isotopomers are shown. Butyrate M+1, butyrate with one of the four carbon atoms labeled by 13C; M+2, two carbon atoms are 13C; M+3, three C atoms are 13C; M+4, all four C atoms are labeled. Similarly: acetate M+1, acetate with one of the two C atoms labeled by 13C; M+2, both C atoms labeled. Combining the LC-MS and NMR data (latter not shown), it was clear that the vast majority (>;99%) of the M+2 butyrate was formed by coupling of an unlabeled (12C) and a labeled (13C) acetate. (B) Within the first approximately 20 min, this is likely to have been produced by Ruminococcus bromii. After that, a lag time in the production is observed (between approx. 20 and 150 min). The subsequent accumulation of the M+2 isotopomer is hypothesized to be produced by E. rectale. For this the M+2 acetate (produced by R. bromii and/or the Prevotella species) first had to diffuse out of the cells of the primary fermenters and subsequently be taken up by E. rectale. The fact that there is very little fully labeled (M+4) butyrate argues against the involvement of R. bromii in the production of M+2 butyrate in this second stage of production (from 150 min onward).
Example of SIP on GOS. The three-dimensional plot shows the labeling of 16S-rRNA of Bifidobacterium longum in time after feeding [U-13C] GOS. Tzero is the sample taken before addition of the labeled substrate. Most B. longum 16S-rRNA fractionates into fraction 4. At 1 h after addition of GOS, this has already shifted to fraction 3. Two hours after addition, RNA can even be observed in the heaviest fraction (fraction 1). Of note is that after 8 h, the distribution shifts back to the less dense fractions. This means that the RNA becomes diluted with 12C again from other substrates that are fermented after GOS has been used to completion. SIP was combined here with the I-Chip microarray platform ( Rose et al., 2010 ; Maathuis et al., 2009 ). This means that besides B. longum, information has been gathered for all other microbes for which a signal was present after hybridization to the microarray. This gives powerful information about the members of the microbiota involved in fermentation of the substrate.
SIP combined with HIT-Chip (Rajilic-Stojanovic et al., 2009 ) to study fermentation of lactose. (A) HIT-Chip profiling of the major groups/families present in the microbiota. Especially the Actinobacteria family is increased in the labeled fraction compared to the unlabeled fraction. (B) When zooming in on this family, it is clear that it is composed primarily of bifidobacterial species. Of these, particularly B. adolescentis was increased by lactose, as indicated by TRFLP profiling (not shown).
Use of the nasal catheter for substrate delivery and sample taking. (A) A 4.5-m catheter is inserted through the nose or throat and past the pyloric valve. Then, a balloon attached to the tip is inflated and the catheter is pulled into the GI tract through regular peristaltic movements. When the catheter has reached the terminal ileum or proximal colon, it is fixed, and the 13C-substrate is delivered through the lumen of the catheter. Subsequently, over time, samples are taken for SIP and/or metabolite analyses through the sample ports. (B) X-ray photograph of the actual catheter positioned with the tip in the terminal ileum. The tip of the catheter can be recognized as the white cylinder in the middle-left of the photo. This catheter can be in place for several hours, up to a day, before it has to be removed. Successful pilot experiments have been performed with this catheter (Vanhoutvin et al., in preparation; He et al., 2008 ; see Fig. 13 ). (Disclaimer: This photograph does not contain any confidential patient information.)
Breath hydrogen and 13CO2 and plasma [13C]glucose concentrations after influsion of [13C]lactose through the nasal catheter in two individuals. (A) Individual 1: breath hydrogen is low, while plasma is enriched for [13C]glucose. This is indicative of the fact that the catheter had not reached the proximal colon yet, but resided in the terminal ileum, where part of the lactose was digested by lactase, allowing uptake of [13C]glucose (and [13C]galactose; not measured). Breath 13CO2 increases due to oxidative use of the [13C]glucose by the host. (B) Individual 2: breath hydrogen increases approximately 30 min after influsion of the substrate, indicative of bacterial fermentation of the substrate. Breath 13CO2 starts to increase after approximately 45 min. Plasma does not become enriched for [13C]glucose, indicating that all glucose that is released from lactose is fermented by the intestinal microbiota.
Chemical memory Chip (CMC). (A) CMC with a main channel through which GI fluids are sampled and side chambers that store the “chemical memory.” A large quenching chamber is partly shown, which contains a liquid that stops continuing chemical reaction from occurring. (B) Drawing and electron microscopic photograph of the restriction at the beginning of the main channel that defines the chip’s filling time. (C) Incorporation of the silicon wafer on a 000-sized capsule to facilitate swallowing of the CMC. The configuration shown here is a long channel (up to 25 m!) that stores the intestinal fluid.