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      Faecal Metaproteomic Analysis Reveals a Personalized and Stable Functional Microbiome and Limited Effects of a Probiotic Intervention in Adults

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          Abstract

          Recent metagenomic studies have demonstrated that the overall functional potential of the intestinal microbiome is rather conserved between healthy individuals. Here we assessed the biological processes undertaken in-vivo by microbes and the host in the intestinal tract by conducting a metaproteome analysis from a total of 48 faecal samples of 16 healthy adults participating in a placebo-controlled probiotic intervention trial. Half of the subjects received placebo and the other half consumed Lactobacillus rhamnosus GG for three weeks (10 10 cfu per day). Faecal samples were collected just before and at the end of the consumption phase as well as after a three-week follow-up period, and were processed for microbial composition and metaproteome analysis. A common core of shared microbial protein functions could be identified in all subjects. Furthermore, we observed marked differences in expressed proteins between subjects that resulted in the definition of a stable and personalized microbiome both at the mass-spectrometry-based proteome level and the functional level based on the KEGG pathway analysis. No significant changes in the metaproteome were attributable to the probiotic intervention. A detailed taxonomic assignment of peptides and comparison to phylogenetic microarray data made it possible to evaluate the activity of the main phyla as well as key species, including Faecalibacterium prausnitzii. Several correlations were identified between human and bacterial proteins. Proteins of the human host accounted for approximately 14% of the identified metaproteome and displayed variations both between and within individuals. The individually different human intestinal proteomes point to personalized host-microbiota interactions. Our findings indicate that analysis of the intestinal metaproteome can complement gene-based analysis and contributes to a thorough understanding of the activities of the microbiome and the relevant pathways in health and disease.

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          Relating the metatranscriptome and metagenome of the human gut.

          Although the composition of the human microbiome is now well-studied, the microbiota's >8 million genes and their regulation remain largely uncharacterized. This knowledge gap is in part because of the difficulty of acquiring large numbers of samples amenable to functional studies of the microbiota. We conducted what is, to our knowledge, one of the first human microbiome studies in a well-phenotyped prospective cohort incorporating taxonomic, metagenomic, and metatranscriptomic profiling at multiple body sites using self-collected samples. Stool and saliva were provided by eight healthy subjects, with the former preserved by three different methods (freezing, ethanol, and RNAlater) to validate self-collection. Within-subject microbial species, gene, and transcript abundances were highly concordant across sampling methods, with only a small fraction of transcripts (<5%) displaying between-method variation. Next, we investigated relationships between the oral and gut microbial communities, identifying a subset of abundant oral microbes that routinely survive transit to the gut, but with minimal transcriptional activity there. Finally, systematic comparison of the gut metagenome and metatranscriptome revealed that a substantial fraction (41%) of microbial transcripts were not differentially regulated relative to their genomic abundances. Of the remainder, consistently underexpressed pathways included sporulation and amino acid biosynthesis, whereas up-regulated pathways included ribosome biogenesis and methanogenesis. Across subjects, metatranscriptional profiles were significantly more individualized than DNA-level functional profiles, but less variable than microbial composition, indicative of subject-specific whole-community regulation. The results thus detail relationships between community genomic potential and gene expression in the gut, and establish the feasibility of metatranscriptomic investigations in subject-collected and shipped samples.
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            Meta-analyses of studies of the human microbiota

            Our body habitat-associated microbial communities are of intense research interest because of their influence on human health. Because many studies of the microbiota are based on the same bacterial 16S ribosomal RNA (rRNA) gene target, they can, in principle, be compared to determine the relative importance of different disease/physiologic/developmental states. However, differences in experimental protocols used may produce variation that outweighs biological differences. By comparing 16S rRNA gene sequences generated from diverse studies of the human microbiota using the QIIME database, we found that variation in composition of the microbiota across different body sites was consistently larger than technical variability across studies. However, samples from different studies of the Western adult fecal microbiota generally clustered by study, and the 16S rRNA target region, DNA extraction technique, and sequencing platform produced systematic biases in observed diversity that could obscure biologically meaningful compositional differences. In contrast, systematic compositional differences in the fecal microbiota that occurred with age and between Western and more agrarian cultures were great enough to outweigh technical variation. Furthermore, individuals with ileal Crohn's disease and in their third trimester of pregnancy often resembled infants from different studies more than controls from the same study, indicating parallel compositional attributes of these distinct developmental/physiological/disease states. Together, these results show that cross-study comparisons of human microbiota are valuable when the studied parameter has a large effect size, but studies of more subtle effects on the human microbiota require carefully selected control populations and standardized protocols.
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              Gut microbiota disturbance during antibiotic therapy: a multi-omic approach

              Objective Antibiotic (AB) usage strongly affects microbial intestinal metabolism and thereby impacts human health. Understanding this process and the underlying mechanisms remains a major research goal. Accordingly, we conducted the first comparative omic investigation of gut microbial communities in faecal samples taken at multiple time points from an individual subjected to β-lactam therapy. Methods The total (16S rDNA) and active (16S rRNA) microbiota, metagenome, metatranscriptome (mRNAs), metametabolome (high-performance liquid chromatography coupled to electrospray ionisation and quadrupole time-of-flight mass spectrometry) and metaproteome (ultra high performing liquid chromatography coupled to an Orbitrap MS2 instrument [UPLC-LTQ Orbitrap-MS/MS]) of a patient undergoing AB therapy for 14 days were evaluated. Results Apparently oscillatory population dynamics were observed, with an early reduction in Gram-negative organisms (day 6) and an overall collapse in diversity and possible further colonisation by ‘presumptive’ naturally resistant bacteria (day 11), followed by the re-growth of Gram-positive species (day 14). During this process, the maximum imbalance in the active microbial fraction occurred later (day 14) than the greatest change in the total microbial fraction, which reached a minimum biodiversity and richness on day 11; additionally, major metabolic changes occurred at day 6. Gut bacteria respond to ABs early by activating systems to avoid the antimicrobial effects of the drugs, while ‘presumptively’ attenuating their overall energetic metabolic status and the capacity to transport and metabolise bile acid, cholesterol, hormones and vitamins; host–microbial interactions significantly improved after treatment cessation. Conclusions This proof-of-concept study provides an extensive description of gut microbiota responses to follow-up β-lactam therapy. The results demonstrate that ABs targeting specific pathogenic infections and diseases may alter gut microbial ecology and interactions with host metabolism at a much higher level than previously assumed.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                12 April 2016
                2016
                : 11
                : 4
                : e0153294
                Affiliations
                [1 ]Department of Veterinary Biosciences, University of Helsinki, Helsinki, Finland
                [2 ]Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands
                [3 ]KU Leuven, Department of Microbiology and Immunology, Rega Institute, Leuven, Belgium
                [4 ]VIB, Center for the Biology of Disease, Leuven, Belgium
                [5 ]Valio Oy, Helsinki, Finland
                [6 ]Translational Proteomics, Turku Center for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland
                [7 ]Department of Bacteriology and Immunology, Immunobiology Research Program, University of Helsinki, Helsinki, Finland
                [8 ]Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands
                University of Illinois, UNITED STATES
                Author notes

                Competing Interests: The work was partially supported by Valio Ltd, a manufacturer of probiotics; one of the authors (RAK) is employed by Valio Ltd. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials, and the content of this paper was neither influenced nor constrained by that fact.

                Conceived and designed the experiments: WdV CK AS GC. Performed the experiments: CK. Analyzed the data: CK JS J. Ritari WdV AS J. Raes GF SVS. Contributed reagents/materials/analysis tools: WdV GC RAK AP MdB. Wrote the paper: CK WdV MdB AS JS GC AP J. Ritari J. Raes SVS.

                [¤]

                Current address: Van’t Hoff Institute for Molecular Sciences (HIMS), University of Amsterdam, Amsterdam, The Netherlands

                Article
                PONE-D-15-05251
                10.1371/journal.pone.0153294
                4829149
                27070903
                6d393946-b2c2-41d1-9efe-75fbbfe79fba
                © 2016 Kolmeder et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 5 February 2015
                : 28 March 2016
                Page count
                Figures: 7, Tables: 0, Pages: 23
                Funding
                Funded by: Doctoral Programme in Food Chain and Health
                Award Recipient :
                Funded by: Suomalainen Tiedeakatemia
                Award ID: 141130
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100002342, Suomalainen Tiedeakatemia;
                Award ID: 137389
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100002342, Suomalainen Tiedeakatemia;
                Award ID: 141140
                Award Recipient :
                Funded by: European Research Council
                Award ID: 250172
                Award Recipient :
                Support was provided by: Doctoral Programme in Food Chain and Health (to CK); Academy of Finland [ http://www.aka.fi/en-GB/A/] (141130 to WdV, 137389 to WdV, 141140 to WdV); European Research Council [ http://erc.europa.eu/] (250172 to WdV); FP7 [ http://cordis.europa.eu/fp7/home_en.html]; Spinoza award of the Netherlands Organization for Scientific Research [ http://www.nwo.nl/en/research-and-results/programmes/spinoza+prize] (to WdV); METACARDIS HEALTH-F4-2012- 305312 (to J. Raes); IWT-SBO 100016 (to J. Raes); Marie Curie Actions FP7 People COFUND [ http://cordis.europa.eu/project/rcn/100377_en.html] (Proposal 267139 (acronym OMICS@VIB) to SVS); Research Foundation Flanders (FWO) [ http://www.fwo.be/en/] (to J. Raes); The Flemish agency for Innovation by Science and Technology (IWT) http://www.flagera.eu/?q=iwt (to J. Raes); KU Leuven [ http://www.kuleuven.be/english] (to J. Raes); The Rega Institute [ http://rega.kuleuven.be/] (to J. Raes); Biocenter Finland, Turku Proteomics Facility [ http://www.biocenter.fi] (to GC). One of the authors (RAK) is employed by a commercial company (Valio Ltd) and contributed to the design of the clinical trial.
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