RESEARCH ARTICLE SUMMARY
`
`Gut Microbiota from Twins Discordant
`for Obesity Modulate Metabolism
`in Mice
`
`Vanessa K. Ridaura, Jeremiah J. Faith, Federico E. Rey, Jiye Cheng, Alexis E. Duncan, Andrew
`L. Kau, Nicholas W. Griffi n, Vincent Lombard, Bernard Henrissat, James R. Bain, Michael J.
`Muehlbauer, Olga Ilkayeva, Clay F. Semenkovich, Katsuhiko Funai, David K. Hayashi, Barbara
`J. Lyle, Margaret C. Martini, Luke K. Ursell, Jose C. Clemente, William Van Treuren, William A.
`Walters, Rob Knight, Christopher B. Newgard, Andrew C. Heath, Jeffrey I. Gordon*
`
`Introduction: Establishing whether specifi c structural and functional confi gurations of a human gut
`microbiota are causally related to a given physiologic or disease phenotype is challenging. Twins dis-
`cordant for obesity provide an opportunity to examine interrelations between obesity and its associ-
`ated metabolic disorders, diet, and the gut microbiota. Transplanting the intact uncultured or cultured
`human fecal microbiota from each member of a discordant twin pair into separate groups of recipient
`germ-free mice permits the donors’ communities to be replicated, differences between their properties
`to be identifi ed, the impact of these differences on body composition and metabolic phenotypes to be
`discerned, and the effects of diet-by-microbiota interactions to be analyzed. In addition, cohousing
`coprophagic mice harboring transplanted microbiota from discordant pairs provides an opportunity to
`determine which bacterial taxa invade the gut communities of cage mates, how invasion correlates with
`host phenotypes, and how invasion and microbial niche are affected by human diets.
`
`Methods: Separate groups of germ-free mice were colonized with uncultured fecal microbiota from
`each member of four twin pairs discordant for obesity, or with culture collections from an obese (Ob) or
`lean (Ln) co-twin. Animals were fed a mouse chow low in fat and rich in plant polysaccharides (LF-HPP)
`or one of two diets refl ecting the upper or lower (Hi or Lo) tertiles of consumption of saturated fats (SF)
`and fruits and vegetables (FV) based on the U.S. National Health and Nutrition Examination Survey
`(NHANES). Ln or Ob mice were cohoused 5 days after colonization. Body composition changes were
`defi ned by quantitative magnetic resonance. Microbiota or microbiome structure, gene expression, and
`metabolism were assayed by 16S ribosomal RNA profi ling, whole-community shotgun sequencing, RNA-
`sequencing, and mass spectrometry. Host gene expression and metabolism were also characterized.
`
`Results and Discussion: The intact uncultured and culturable bacterial component of Ob co-twins’
`fecal microbiota conveyed signifi cantly greater increases in body mass and adiposity than those of
`Ln communities. Differences in body composition were correlated with differences in fermentation of
`short-chain fatty acids (increased in Ln), metabolism of branched-chain amino acids (increased in Ob),
`and microbial transformation of bile acid species (increased in Ln and correlated with down-regulation
`of host farnesoid X receptor signaling). Cohousing Ln and Ob mice prevented development of increased
`adiposity and body mass in Ob cage mates and transformed their microbiota’s metabolic profi le to a
`leanlike state. Transformation correlated with invasion of members of Bacteroidales from Ln into Ob
`microbiota. Invasion and phenotypic rescue were diet-dependent and occurred with the diet represent-
`ing the lower tertile of U.S. consumption of saturated fats and upper tertile of fruits and vegetables but
`not with the diet representing the upper tertile of saturated fats and lower tertile of fruit and vegetable
`consumption. These results reveal that transmissible and modifi able interactions between diet and
`microbiota infl uence host biology.
`
`READ THE FULL ARTICLE ONLINE
`http://dx.doi.org/10.1126/science.1241214
`
`Cite this article as V. K. Ridaura et al.,
`Science 341, 1241214 (2013).
`DOI: 10.1126/science.1241214
`
`FIGURES IN THE FULL ARTICLE
`
`Fig. 1. Reliable replication of human donor
`microbiota in gnotobiotic mice.
`
`Fig. 2. Cohousing Obch and Lnch mice
`transforms the adiposity phenotype of cage
`mates harboring the obese co-twin’s culture
`collection to a leanlike state.
`
`Fig. 3. Effect of cohousing on metabolic
`profi les in mice consuming the LF-HPP diet.
`
`Fig. 4. Effects of NHANES-based LoSF-HiFV
`and HiSF-LoFV diets on bacterial invasion,
`body mass, and metabolic phenotypes.
`
`Fig. 5. Invasion analysis of species-level taxa
`in Obch or Lnch mice fed the NHANES-based
`LoSF-HiFV diet.
`
`Fig. 6. Acylcarnitine profi le in the skeletal
`muscle of mice colonized with the Ob or Ln
`culture collections from dizygotic twin pair 1
`and fed the LoSF-HiFV diet.
`
`SUPPLEMENTARY MATERIALS
`
`Materials and Methods
`Supplementary Text
`Figs. S1 to S17
`Tables S1 to S17
`References and Notes
`
`RELATED ITEMS IN SCIENCE
`
`A. W. Walker, J. Parkhill, Fighting obesity
`with bacteria. Science 341, 1069–1070 (2013).
`DOI: 10.1126/science.1243787
`
`Cohousing Ln and Ob
`mice prevents adiposity
`phenotype in Ob cage
`mates (Obch). (A) The adi-
`posity change after 10
`days of cohousing. *P <
`0.05 versus Ob controls
`(Student’s t test). (B) Bac-
`teroidales from Lnch micro-
`biota invade the Obch
`microbiota. Columns show
`individual mice.
`
`The list of author affi liations is available in the full article online.
`*Corresponding author. E-mail: jgordon@wustl.edu
`
`www.sciencemag.org SCIENCE VOL 341 6 SEPTEMBER 2013
`Published by AAAS
`
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`
`Genome Ex. 1045
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`RESEARCH ARTICLE
`
`Gut Microbiota from Twins
`Discordant for Obesity Modulate
`Metabolism in Mice
`
`Vanessa K. Ridaura,1 Jeremiah J. Faith,1 Federico E. Rey,1 Jiye Cheng,1 Alexis E. Duncan,2,3
`Andrew L. Kau,1 Nicholas W. Griffin,1 Vincent Lombard,4 Bernard Henrissat,4,5
`James R. Bain,6,7,8 Michael J. Muehlbauer,6 Olga Ilkayeva,6 Clay F. Semenkovich,9
`Katsuhiko Funai,9 David K. Hayashi,10 Barbara J. Lyle,11 Margaret C. Martini,11
`Luke K. Ursell,12 Jose C. Clemente,12 William Van Treuren,12 William A. Walters,13
`Rob Knight,12,14,15 Christopher B. Newgard,6,7,8 Andrew C. Heath,2 Jeffrey I. Gordon1*
`
`The role of specific gut microbes in shaping body composition remains unclear. We transplanted
`fecal microbiota from adult female twin pairs discordant for obesity into germ-free mice fed
`low-fat mouse chow, as well as diets representing different levels of saturated fat and fruit and
`vegetable consumption typical of the U.S. diet. Increased total body and fat mass, as well as
`obesity-associated metabolic phenotypes, were transmissible with uncultured fecal communities
`and with their corresponding fecal bacterial culture collections. Cohousing mice harboring an
`obese twin’s microbiota (Ob) with mice containing the lean co-twin’s microbiota (Ln) prevented the
`development of increased body mass and obesity-associated metabolic phenotypes in Ob cage
`mates. Rescue correlated with invasion of specific members of Bacteroidetes from the Ln
`microbiota into Ob microbiota and was diet-dependent. These findings reveal transmissible,
`rapid, and modifiable effects of diet-by-microbiota interactions.
`
`Microbial community configurations
`
`vary substantially between unrelated in-
`dividuals (1–9), which creates a chal-
`lenge in designing surveys of sufficient power to
`determine whether observed differences between
`disease-associated and healthy communities dif-
`fer significantly from normal interpersonal var-
`iation. This challenge is especially great if, for
`a given disease state, there are many associated
`states of the microbial species (microbiota) or mi-
`crobial gene repertoire (microbiome), each shared
`by relatively few individuals. Microbiota config-
`
`1Center for Genome Sciences and Systems Biology, Washing-
`ton University School of Medicine, St. Louis, MO 63108, USA.
`2Department of Psychiatry, Washington University School of
`Medicine, St. Louis, MO 63110, USA. 3George Warren Brown
`School of Social Work, Washington University, St. Louis, MO
`63130, USA. 4Architecture et Fonction des Macromolécules
`Biologiques, CNRS and Aix Marseille Université, CNRS UMR
`7257, 13288 Marseille, France. 5Department of Cellular and
`Molecular Medicine, Faculty of Health and Medical Sciences,
`University of Copenhagen, DK-2200, Copenhagen, Denmark.
`6Sarah W. Stedman Nutrition and Metabolism Center, Duke
`University Medical Center, Durham, NC 27710, USA. 7Depart-
`ment of Medicine, Duke University Medical Center, Durham,
`NC 27710, USA. 8Department of Pharmacology and Cancer
`Biology, Duke University Medical Center, Durham, NC 27710,
`USA. 9Department of Medicine, Washington University School
`of Medicine, St. Louis, MO 63110, USA. 10Mondelez Interna-
`tional, Glenview, IL60025, USA. 11Kraft Foods Group, Glenview,
`IL 60025, USA. 12Department of Chemistry and Biochemistry,
`University of Colorado, Boulder, CO 80309, USA. 13Molecular,
`Cellular and Developmental Biology, University of Colorado,
`Boulder, CO 80309, USA. 14Biofrontiers Institute, University of
`Colorado, Boulder, CO 80309, USA. 15Howard Hughes Medical
`Institute, University of Colorado, Boulder, CO 80309, USA.
`*To whom correspondence should be sent. E-mail: jgordon@
`wustl.edu
`
`urations are influenced by early environmental
`exposures and are generally more similar among
`family members (2, 7, 10, 11).
`There have been conflicting reports about
`the relation between interpersonal differences
`in the structure of the gut microbiota and host
`body mass index (BMI). Taxonomic profiles
`for obese and lean individuals may have dis-
`tinct patterns between human populations, but
`technical issues related to how gut samples are
`processed and community members are identified
`by 16S ribosomal RNA (rRNA) gene sequencing
`may also play a role in observed differences.
`The relative contributions of the microbiota and
`dietary components to obesity and obesity-related
`metabolic phenotypes are unclear and likely multi-
`faceted (2, 12–17). Transplants of fecal microbiota
`from healthy donors to recipients with metabolic
`syndrome have provided evidence that the micro-
`biota can ameliorate insulin-resistance, although
`the underlying mechanisms remain unclear (18).
`Monozygotic (MZ) or dizygotic (DZ) twins
`discordant for obesity (19, 20) provide an attract-
`ive model for studying the interrelations between
`obesity, its associated dietary and lifestyle risk fac-
`tors, and the gut microbiota/microbiome. In the
`case of same-sex twins discordant for a disease
`phenotype, the healthy co-twin provides a valuable
`reference control to contrast with the co-twin’s disease-
`associated gut community. However, this comparison
`is fundamentally descriptive and cannot establish
`causality. Transplanting a fecal sample obtained
`from each twin in a discordant pair into separate
`groups of recipient germ-free mice provides an
`opportunity to (i) identify structural and functional
`
`differences between their gut communities; (ii)
`generate and test hypotheses about the impact of
`these differences on host biology, including body
`composition and metabolism; and (iii) determine
`the effects of diet-by-microbiota interactions
`through manipulation of the diets fed to these
`“humanized” animals and/or the representation of
`microbial taxa in their gut communities.
`
`Reproducibility of Microbiota Transplants
`from Discordant Twins
`We surveyed data collected from 21- to 32-year-old
`female twin pairs (n = 1539) enrolled in the Mis-
`souri Adolescent Female Twin Study [MOAFTS;
`(21, 22); for further details, see ref. (23)]. We
`recruited four twin pairs, discordant for obesity
`(obese twin BMI > 30 kg/m2) with a sustained
`multiyear BMI difference of ≥5.5 kg/m2 (n =
`1 MZ and 3 DZ pairs) (Fig. 1A). Fecal samples
`were collected from each twin, frozen immedi-
`ately after they were produced, and stored at
`–80°C. Each fecal sample was introduced, via a
`single oral gavage, into a group of 8- to 9-week-
`old adult male germ-free C57BL/6J mice (one
`gnotobiotic isolator per microbiota sample; each
`recipient mouse was individually caged within
`the isolator; n = 3 to 4 mice per donor microbiota
`sample per experiment; n = 1 to 5 independent
`experiments per microbiota). All recipient mice
`were fed, ad libitum, a commercial, sterilized mouse
`chow that was low in fat (4% by weight) and high
`in plant polysaccharides (LF-HPP) (23). Fecal
`pellets were obtained from each mouse 1, 3, 7,
`10, and 15 days post colonization (dpc) and, for
`more prolonged experiments, on days 17, 22, 24,
`29, and 35.
`Unweighted UniFrac-based comparisons of
`bacterial 16S rRNA data sets generated from the
`input human donor microbiota, from fecal sam-
`ples collected from gnotobiotic mice and from
`different locations along the length of the mouse
`gut at the time they were killed (table S1A), plus
`comparisons of the representation of genes with
`assignable enzyme commission numbers (ECs)
`in human fecal and mouse cecal microbiomes
`(defined by shotgun sequencing), disclosed that
`transplant recipients efficiently and reproducibly
`captured the taxonomic features of their human
`donor’s microbiota and the functions encoded by
`the donor’s microbiome (see Fig. 1B; fig. S1, A
`to E; fig. S2; table S1B; and table S2, A to D)
`(23). The 16S rRNA data sets allowed us to iden-
`tify bacterial taxa that differentiate gnotobiotic
`mice harboring gut communities transplanted
`from all lean versus all obese co-twins [analysis of
`variance (ANOVA) using Benjamini-Hochberg
`correction for multiple hypotheses] [table S3; see
`(23) for details].
`
`Reproducible Transmission of Donor Body
`Composition Phenotypes
`Quantitative magnetic resonance (QMR) anal-
`ysis was used to assess the body composition
`of transplant recipients 1 day, 15 days, and, in
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`RESEARCH ARTICLE
`
`the case of longer experiments, 8, 22, 29, and
`35 days after transplantation. The increased adi-
`posity phenotype of each obese twin in a discor-
`dant twin pair was transmissible: The change in
`adipose mass of mice that received an obese co-
`twin’s fecal microbiota was significantly greater
`than the change in animals receiving her lean
`twin’s gut community within a given experiment
`and was reproducible across experiments (P ≤
`0.001, one-tailed unpaired Student’s t test; n = 103
`mice phenotyped) (Fig. 1, C to E). Epididymal
`fat pad weights (normalized to total body weight)
`were also significantly higher in mice colonized
`with gut communities from obese twins (P ≤ 0.05,
`one-tailed unpaired Student’s t test). These dif-
`ferences in adiposity were not associated with
`statistically significant differences in daily chow
`consumption (measured on days 1, 8, and 15 after
`gavage and weekly thereafter for longer exper-
`iments) or with appreciably greater inflamma-
`tory responses in recipients of obese compared
`with lean co-twin fecal microbiota as judged by
`fluorescence-activated cell sorting (FACS) anal-
`ysis of the CD4+ and CD8+ T cell compartments
`in spleen, mesenteric lymph nodes, small intes-
`tine, or colon [see (23) for details].
`
`Functional Differences Between Transplanted
`Microbial Communities
`Fecal samples collected from gnotobiotic mice
`were used to prepare RNA for microbial RNA se-
`quencing (RNA-Seq) of the transplanted mi-
`crobial communities’ meta-transcriptomes (table
`S1C). Transcripts were mapped to a database of
`sequenced human gut bacterial genomes and as-
`signed to Kyoto Encyclopedia of Genes and Ge-
`nomes (KEGG) Enzyme Commission numbers
`(EC numbers) [see ref. (23)]. Significant differences
`and distinguishing characteristics were defined
`using ShotgunFunctionalizeR, which is based on a
`Poisson model (24) (see table S4 and table S5 for
`ECs and KEGG level 2 pathways, respectively).
`Transcripts encoding 305 KEGG ECs were dif-
`ferentially expressed between mice harboring mi-
`crobiomes transplanted from lean or obese donors
`[ShotgunFunctionalizeR, Akaike's information
`−30].
`criterion (AIC) < 5000; P ≤ 10
`Mice harboring the transplanted microbiomes
`from the obese twins exhibited higher expression
`of microbial genes involved in detoxification and
`stress responses; in biosynthesis of cobalamin;
`metabolism of essential amino acids (phenyl-
`alanine, lysine, valine, leucine, and isoleucine)
`
`and nonessential amino acids (arginine, cysteine,
`and tyrosine); and in the pentose phosphate path-
`way (fig. S3, A and B; table S4, B to G; and
`table S5). Follow-up targeted tandem mass spec-
`trometry (MS/MS)–based analysis of amino acids
`in sera obtained at the time mice were killed dem-
`onstrated significant increases in branched-chain
`amino acids (BCAA: Val and Leu/Ile), as well as
`other amino acids (Met, Ser, and Gly), plus trends
`to increase (Phe, Tyr, and Ala), in recipients of
`microbiota from obese compared with lean co-
`twins in discordant twin pairs DZ1 and MZ4
`(tables S1D and S6A). These specific amino
`acids, as well as the magnitude of their differ-
`ences, are remarkably similar to elevations in
`BCAA and related amino acids reported in obese
`and insulin-resistant versus lean and insulin-
`sensitive humans (25). This finding suggested
`that the gut microbiota from obese subjects
`could influence metabolites that characterize
`the obese state.
`In contrast, the transplanted microbiomes from
`lean co-twins exhibited higher expression of genes
`involved in (i) digestion of plant-derived poly-
`saccharides [e.g., a-glucuronidase (EC 3.2.1.139),
`a-L-arabinofuranosidase (EC 3.2.1.55)]; (ii) fermen-
`
`***
`
`15
`
`10
`
`05
`
`-5
`
`D
`
`% Change in fat mass
`
`Fat mass
`Lean body mass
`
`All lean
`
`co-twin donors
`
`All obese
`co-twin donors
`
`All lean
`co-twin donors
`
`All obese
`co-twin donors
`
`Twin pair 1
`Obese co-twin
`Lean co-twin
`
`0246
`
`-2
`
`C
`
`% Change in body composition
`
`E
`
`30
`
`20
`
`10
`
`0
`
`% Change in fat mass
`
`Twin Pair
`
`1 (DZ)
`
`2 (DZ)
`
`3 (DZ)
`
`4 (MZ)
`
`BMI (kg/m2)
`
`23
`
`32
`
`25.5
`
`31
`
`19.5 30.7
`
`24
`
`33
`
`Twin pair 1
`
`Lean donor
`
`Obese donor
`
`A
`
`B
`
`PC1 (23%)
`
`dpc
`
`1
`
`3
`
`7
`
`10
`
`15
`
`8dpc
`
`15dpc
`
`22dpc
`
`29dpc
`
`35dpc
`
`Input samples
`Experiment 1
`Experiment 2
`Experiment 3
`Fig. 1. Reliable replication of human donor microbiota in gnotobiotic
`mice. (A) Features of the four discordant twin pairs. (B) Assembly of bacterial
`communities in mice that had received intact and uncultured fecal microbiota
`transplants from the obese and lean co-twins in DZ pair 1. Principal coordinates
`analysis plot of principal coordinate 1 (PC1) based on an unweighted UniFrac
`distance matrix and 97%ID OTUs present in sampled fecal communities. Circles
`correspond to a single fecal sample obtained at a given time point from a given
`mouse and are colored according to the experiment (n = 3 independent ex-
`periments). Note that assembly is reproducible within members of a group of
`mice that have received a given microbiota, as well as between experiments. (C)
`Body composition, defined by QMR, was performed 1 day and 15 dpc of each
`mouse in each recipient group. Mean values (T SEM) are plotted for the percent
`increase in fat mass and lean body mass at 15 dpc for all recipient mice of each
`of the four obese co-twins’ or lean co-twins’ fecal microbiota, normalized to the
`
`-10
`initial body mass of each recipient mouse. A two-way ANOVA indicated that
`there was a significant donor effect (P ≤ 0.05), driven by a significant difference
`in adiposity and total body mass between mice colonized with a lean or obese
`co-twin donor’s fecal microbiota (adjusted P ≤ 0.05; Siˇdák’s multiple comparison
`test). (D) Mean values (T SEM) are plotted for the percent change in fat mass at
`15 dpc for all recipient mice of each of the four obese co-twins’ or lean co-twins’
`fecal microbiota. Data are normalized to initial fat mass (n = 3 to 12 animals
`per donor microbiota; 51 to 52 mice per BMI bin; total of 103 mice). ***P ≤
`0.001, as judged by a one-tailed unpaired Student’s t test. (E) More prolonged
`time course study for recipients of fecal microbiota from co-twins in discordant
`DZ pair 1 (mean values T SEM plotted; n = 4 mice per donor microbiota).
`The difference between the gain in adiposity calculated relative to initial fat
`mass (1 dpc) between the two recipient groups of mice is statistically signif-
`icant (P ≤ 0.001, two-way ANOVA).
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`
`biota of Obch mice were reconfigured so that they
`came to resemble the microbiota of Lnch cage
`mates. In contrast, the microbiota of the Lnch cage
`mates remained stable (fig. S9, A to C). We per-
`formed a follow-up analysis to identify species-
`level taxa that had infiltrated into and/or had been
`displaced from the guts of mice harboring the Ln
`andOb culture collections. We did so by character-
`izingthedirectionandsuccessofinvasion.Microbial
`SourceTracker estimates, for every species-level
`taxon or 97%ID OTU, the Bayesian probability
`(P) of its being derived from each of a set of source
`communities (30). The fecal microbiota of Ln or
`Ob controls sampled 5 days after colonization
`were used as source communities to determine the
`direction of invasion. The fecal communities
`belonging to each Lnch and Obch mouse were then
`traced to these sources. We defined the direction of
`invasion for these bacterial taxa, by calculating the
`log odds ratio of the probability of a Ln origin
`(PLn) or an Ob origin (POb) for each species-level
`taxon or 97%ID OTU, i, as follows:
`
`
`log2 PLni=PObi
`
`
`
`
`
`.
`
`
`
`Aij
`
`tation to butyrate [acetyl-CoA C-acetyltransferase
`(EC 2.3.1.9), 3-hydroxybutyryl-CoA dehydrogenase
`(EC 1.1.1.157), 3-hydroxybutyryl-CoA dehydratase
`(EC 4.2.1.55), butyryl-CoA dehydrogenase (EC
`1.3.8.1)] (fig. S3, C and D); and (iii) fermentation to
`propionate [succinate dehydrogenase (EC 1.3.99.1),
`phosphoenolpyruvate carboxykinase (EC 4.1.1.32),
`methylmalonyl-CoA mutase (EC 5.4.99.2)] (table
`S4A). Follow-up gas chromatography–mass spec-
`trometry (GC-MS) of cecal contents confirmed that
`levels of butyrate and propionate were significantly
`increased and that levels of several mono- and
`disaccharides significantly decreased in animals
`colonized with lean compared with obese co-twin
`gut communities (P ≤ 0.05, unpaired Student’s t
`test) (fig. S4, A and B, and table S6B). Procrustes
`analysis, using a Hellinger distance matrix (26), re-
`vealed significant correlations between taxonomic
`structure [97% identity (97%ID) threshold for de-
`fining distinct operational taxonomic units (OTU)
`in fecal samples], transcriptional profiles (enzyme
`representation in fecal mRNA populations), and
`metabolic profiles (GC-MS of cecal samples), with
`separation of groups based on donor microbiota
`and BMI (Mantel test, P ≤ 0.001) (fig. S5).
`These results suggest that, in this diet context,
`transplanted microbiota from lean co-twins had a
`greater capacity to breakdown and ferment poly-
`saccharides than the microbiota of their obese co-
`twins. Previous reports have shown that increased
`microbial fermentation of nondigestible starches
`is associated with decreased body weight and de-
`creased adiposity in conventionally raised mice that
`harbor a mouse microbiota [e.g., refs. (27–29)].
`
`Phenotypes Produced by Bacterial
`Culture Collections
`We followed up these studies of transplanted, in-
`tact, and uncultured donor communities with a set of
`experiments involving culture collections produced
`from the fecal microbiota of one of the discordant
`twin pairs. Our goal was to determine whether cul-
`tured bacterial members of the co-twins’ microbiota
`could transmit the discordant adiposity phenotypes
`and distinctive microbiota-associated metabolic pro-
`files when transplanted into gnotobiotic mouse re-
`cipients that received the LF-HPP chow diet.
`Collections of cultured anaerobic bacteria were
`generated from each co-twin in DZ pair 1 and
`subsequently introduced into separate groups of
`8-week-old germ-free male C57BL/6J mice (n =
`5 independent experiments; n = 4 to 6 recipient
`mice per culture collection per experiment). The
`culture collections stabilized in the guts of re-
`cipient mice within 3 days after their introduc-
`tion [see (23); fig. S6, A to E; and table S7 for
`documentation of the efficient and reproducible
`capture of cultured taxa and their encoded gene
`functions between groups of recipient mice].
`As in the case of uncultured communities, we
`observed a significantly greater increase in adi-
`posity in recipients of the obese twin’s culture col-
`lection compared with the lean co-twin’s culture
`collection (P ≤ 0.02, one-tailed unpaired Student’s t
`test) (Fig. 2, A and B). Nontargeted GC-MS showed
`
`that the metabolic profiles generated by the trans-
`planted culture collections clustered with the
`profiles produced by the corresponding intact
`uncultured communities (fig. S6E). In addition,
`the fecal biomass of recipients of the culture col-
`lection from the lean twin was significantly greater
`than the fecal biomass of mice receiving the cul-
`ture collection from her obese sibling; these differ-
`ences were manifest within 7 days (P ≤ 0.0001,
`two-way ANOVA) (fig. S7A).
`
`Cohousing Ob and Ln Animals Prevents
`an Increased Adiposity Phenotype
`Because mice are coprophagic, the potential for
`transfer of gut microbiota through the fecal-oral
`route is high. Therefore, we used cohousing to
`determine whether exposure of a mouse harboring
`a culture collection from the lean twin could
`prevent development of the increased adiposity
`phenotype and microbiome-associated metabolic
`profile of a cage mate colonized with the culture
`collection from her obese co-twin or vice versa.
`Five days after gavage, when each of the inoculated
`microbial consortia had stabilized in the guts of
`recipient animals, a mouse with the lean co-twin’s
`culture collection was cohoused with a mouse with
`the obese co-twin’s culture collection (abbreviated
`Lnch and Obch, respectively). Control groups con-
`sisted of cages of dually housed recipients of the
`lean twin culture collection and dually housed
`recipients of the obese co-twin’s culture collection
`(n = 3 to 5 cages per housing configuration per
`experiment; n = 4 independent experiments; each
`housing configuration in each experiment was
`placed in a separate gnotobiotic isolator) (Fig.
`2A). All mice were 8-week-old C57BL/6J males.
`All were fed the same LF-HPP chow adlibitum that
`was used for transplants involving the correspond-
`ingunculturedcommunities.Beddingwaschanged
`before initiation of cohousing. Fecal samples were
`collectedfrom allrecipients 1,2,3, 5, 6,7,8, 10,and
`15 days after gavage. Body composition was mea-
`sured by QMR 1 and 5 days after gavage, and after
`10 days of cohousing.
`Obch mice exhibited a significantly lower in-
`crease in adiposity compared with control Ob ani-
`mals that had never been exposed to mice harboring
`the lean co-twin’s culture collection (P ≤ 0.05, one-
`tailed unpaired Student’s t test). Moreover, the adi-
`posity of these Obch animals was not significantly
`different from Ln controls (P > 0.05, one-tailed un-
`paired Student’s t test) (Fig. 2B). In addition, expo-
`sure to Obch animals did not produce a significant
`effect on the adiposity of Lnch mice: Their adiposity
`phenotypes and fecal biomass were indistinguish-
`able from dually housed Ln controls (Fig. 2B; and
`fig. S7, B and C). Cohousing caused the cecal meta-
`bolic profile of Obch mice to assume features of
`Lnch and control Ln animals, including higher lev-
`els (compared with dually housed Ob controls) of
`propionate and butyrate and lower levels of cecal
`mono- and disaccharides, as well as BCAA and
`aromatic amino acids (Fig. 2, C and D, and fig. S8).
`Principal coordinates analysis of unweighted
`UniFrac distances revealed that the fecal micro-
`
`A positive log odds ratio indicated that a
`species or 97%ID OTU was derived from a Ln
`source; a negative log odds ratio indicated an Ob
`source. An invasion score was calculated to
`quantify the success of invasion of each species
`or 97%ID OTU, i, into each cohousing group, j, as
`follows:
`Invasion Scoreij ¼ log2
`
`Bij
`where Aij is the average relative abundance of
`taxoniinallfecalsamplescollectedfromgroupjafter
`cohousing, and Bij is its relative abundance in all
`samples taken from that group before cohousing.
`The observed mean of the distribution of
`invasion scores for Obch animals was significantly
`higher than that for dually housed Ob-Ob controls
`(P ≤ 0.0005, Welch’s two-sample t test) (fig.
`S10A). This was not the case for Lnch animals
`when compared with dually housed Ln-Ln
`controls (P > 0.05), which suggested that there
`was significant invasion of components of the
`Lnch microbiota into the microbiota of Obch cage
`mates, but not vice versa. To quantify invasion
`further, we used the mean and standard deviation
`of the null distribution of invasion scores (defined
`as the scores from recipients of the Ln or Ob
`microbiota that had never been cohoused with
`each other) to calculate a z value and a Benjamini-
`Hochberg adjusted P value for the invasion score
`of each species in Lnch and Obch mice. We con-
`servatively defined a taxon as a successful invader
`if it (i) had a Benjamini-Hochberg adjusted P ≤
`0.05, (ii) was represented in ≥75% of Obch or Lnch
`mice when sampled 7 and 10 days after initiation
`of cohousing, and (iii) had a relative abundance of
`≤0.05% before cohousing and ≥0.5% in the fecal
`microbiota at the time mice were killed. We de-
`fined a taxon that was displaced from an animal’s
`microbiota upon cohousing as having a relative
`
`www.sciencemag.org SCIENCE VOL 341
`
`6 SEPTEMBER 2013
`
`1241214-3
`
`Genome Ex. 1045
`Page 4 of 12
`
`

`

`RESEARCH ARTICLE
`
`abundance ≥1% in Lnch or Obch mice before they
`were cohoused and a relative abundance <0.5%
`after cohousing.
`
`The direction and success of invasion are
`shown in Fig. 2E and table S8A. The most suc-
`cessful Lnch invaders of the Obch microbiota were
`
`members of the Bacteroidetes (rank order of
`their invasion scores: Bacteroides cellulosilyticus,
`B. uniformis, B. vulgatus, B. thetaiotaomicron,
`
`***
`
`***
`
`***
`
`***
`
`***
`
`***
`
`Ob
`Obch
`
`Ln
`Lnch
`
`GFch
`
`D
`
`15
`
`Log2(spectral abundance)
`
`10
`
`5
`
`***
`
`**
`
`*
`
`*
`
`** *** ** ***
`
`C
`
`10
`
`8
`
`6
`
`4
`
`2
`
`µmol/mg wet cecal contents
`
`**
`
`*
`
`*
`
`*
`
`15
`
`10
`
`5
`
`0
`
`-5
`
`B
`
`% Change in fat mass
`
`Controls
`
`A
`
`Isolator
`
`Cage
`
`Ln
`
`Ob
`
`Ob
`
`Ob
`
`Ln
`Ln
`
`Ob
`Ob
`
`Obch
`Lnch
`
`5 dpc
`
`5 dpc
`
`5 dpc
`
`5 dpc
`
`Ln
`
`Ln
`
`Obch
`2xGFch
`Lnch
`
`-10
`
`Ob-Ob Ln-Ln Obch
`
`Lnch GF
`
`ch
`
`0
`
`Propionate
`
`Butyrate
`
`Cellobiose
`
`0
`
`Maltose
`or similar
`disaccharide
`
`GFch
`co-housing
`8
`10
`
`RA
`
`12 15
`
`Lnch
`
`Obch
`
`prior to co-housing
`1
`2
`3
`5
`
`co-housing
`8
`10
`
`7
`
`12
`
`15
`
`RA
`
`prior to co-housing
`1
`2
`3
`
`5
`
`6
`
`co-housing
`10
`7
`8
`
`12 15
`
`RA
`
`6
`
`6
`
`7
`
`E
`
`dpc
`
`*
`
` Clostridium clostridioforme
` Butyricimonas virosa
` Clostridium asparagiforme
` Turicibacter sanguinis
` Ruminococcus sp. 5_1_39BFAA
` Clostridium glycolicum
` Alistipes putredinis
` Clostridium tertium
` Bacteroides finegoldii
` Clostridium sp. MLG480
` Clostridium symbiosum
` Firmicutes Other
` Enterococcus faecium
` Roseburia faecis
` Ruminococcus sp. CCUG 37327 A
` Clostridium disporicum
` Odoribacter splanchnicus
` Alistipes shahii
` Bifidobacterium longum
` Roseburia spp.
` Anaerotruncus colihominis
` Bacteroides thetaiotaomicron
` Bacteroides cellulosilyticus
` Bacteroides vulgatus
` Bacteroides uniformis
` Bacteroides caccae
` Ruminococcus sp. DJF VR70k1
` Parabacteroides merdae
` Akkermansia muciniphila
` Clostridium sp. NML 04A032
` Clostridium ramosum
` Eubacterium limosum
` Oscillibacter spp.
` Enterobacteriaceae Other
` Alistipes indistinctus
` Holdemania filiformis
` Betaproteobacteria Other
` Clostridium spp.
` Bacteroides spp.
` Ruminococcus torques
` Blautia producta
` Bacteroides massiliensis
` Clostridium hathewayi
` Blautia hansenii
` Alistipes finegoldii
`
`**
`
`**
`*
`
`*
`
` -20
`
` 0
`
` 20
`
`Ob source
`
`Ln source
`
`Direction of invasion
`
`Not detected
`Species present in
` both Ob and Ln
`
`0
`
`5
`
`10 15
`
`Relative abundance
`(RA) (%) b (before),
`a (after) co-housing
`
`−6 −2
`
`2
`
`6
`
`Fold change (fc)
`(Log(RAach/RAbch))
`
`Fig. 2. Cohousing Obch and Lnch mice transforms the adiposity pheno-
`type of cage mates harboring the obese co-twin’s culture collection to a
`lean-like state. (A) Design of cohousing experiment: 8-week-old, male, germ-
`free C57BL/6J mice received culture collections from the lean (Ln) twin or the
`obese (Ob) co-twin in DZ twin pair 1. Five days after colonization, mice were
`cohoused in one of three configurations: Control groups consisted of dually
`housed Ob-Ob or Ln-Ln cage mates; the experimental group consisted of dually
`housed Obch-Lnch cage mates (data shown from five cages per experiment; two
`independent experiments) or Obch-Lnch- GFch-GFch cage mates (n = 3 cages per
`experiment). All mice were fed a LF-HPP diet. (B) Effects of cohousing on fat
`mass. Changes from the first day after cohousing to 10 days after cohousing
`were defined using whole-b