Research ArticleGenetics and Diet

The Effect of Diet on the Human Gut Microbiome: A Metagenomic Analysis in Humanized Gnotobiotic Mice

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Science Translational Medicine  11 Nov 2009:
Vol. 1, Issue 6, pp. 6ra14
DOI: 10.1126/scitranslmed.3000322
  • Fig. 1

    Design of human microbiota transplant experiments. (A) The initial (first-generation) humanization procedure, including the diet shift. Dark red arrows indicate fecal collection time points. (B) Reciprocal microbiota transplantations. Microbiota from first-generation humanized mice fed LF/PP or Western diets was transferred to LF/PP diet– or Western diet–fed germ-free recipients. (C) Colonization of germ-free mice starting with a frozen human fecal sample. (D) Characterization of the postnatal assembly and daily variation of the humanized mouse gut microbiota. (E) Sampling of the humanized mouse gut microbiota along the length of the gastrointestinal tract.

  • Fig. 2

    The effects of switching from the LF/PP diet to the Western diet on the humanized mouse gut microbiota. (A) 16S rRNA gene surveys (analyzed by unweighted UniFrac-based PCoA) from the human donor (green), first-generation humanized mice fed LF/PP (red) or Western (blue) diets, second-generation microbiota transplant recipients consuming the LF/PP (teal) or Western (purple) diets, and mice humanized with a frozen sample fed LF/PP (yellow) or Western (orange) diets (total of 340 samples with >800 sequences per sample). Weighted UniFrac resulted in a similar overall clustering pattern (data not shown). Principal coordinate 1 (PC1) and PC2 are the x axis and y axis, respectively, and have been scaled on the basis of percent variance. PC3 is depicted by the shading of each point. The percent variance explained by each coordinate is shown in parentheses. dpc, days after colonization with a human donor sample; dpd, days after diet switch. (B) Taxonomic distribution [Ribosomal Database Project (RDP) level 3 (class-level taxa) (27)] of two generations of humanized mice fed a LF/PP or Western diet. Values represent the average relative abundance across all samples within the indicated group. c, cecal samples (whereas all other samples are fecal); M, month. (C) 16S rRNA gene sequences (analyzed by unweighted UniFrac-based PCoA) from the mice in the experiment described in Fig. 1A. The x and y axes are scaled on the basis of the percent variance accounted for by each component (shown in parentheses). Each box corresponds to a single time point.

  • Fig. 3

    Postnatal assembly of the humanized gut microbiota. (A) Rarefaction curves measuring bacterial diversity in the fecal communities (species-level phylotypes defined by ≥97% identity). The curves are based on V2 16S rRNA gene sequences obtained from mice before weaning (P14) and after weaning (P28). Values are mean ± 95% confidence interval. (B) Taxonomic distribution [RDP level 3 (27)] of the gut microbiota sampled from mice from P14 to P85. Values represent the average relative abundance across all samples within a given group.

  • Fig. 4

    Clustering and taxonomic analysis of the gut microbiota of humanized mice consuming a LF/PP or Western diet. (A) 16S rRNA gene surveys (analyzed by unweighted UniFrac PCoA) of the humanized microbiota along the length of the gut (n = 148 samples with >500 sequences per sample). Weighted UniFrac results in a similar overall clustering pattern. PC1 and PC2 are the x axis and y axis, respectively. The percent variance explained by each coordinate is shown in parentheses. (B) Taxonomic distribution [RDP level 3 (27)] in communities distributed along the length of the gut. Values represent the average relative abundance across all samples within a given group. SI, small intestinal segment.

  • Fig. 5

    Clustering of the distal gut microbiome, the C. innocuum SB23 transcriptome, and the community meta transcriptome in the ceca of humanized mice. Microbiome or transcriptome profiles were normalized by z score, used to construct a correlation distance matrix, clustered with UPGMA (unweighted pair group method with arithmetic mean), and visualized (see Supplementary Material; Matlab version 7.7.0). (A) Clustering of fecal microbial gene content in the human donor’s microbiome and two groups of humanized mice starting at day 1 after colonization (n = 3 to 5 mice per group; fecal DNA was pooled before sequencing). All mice were maintained on the LF/PP diet (red) for 28 days, at which point group 2 was transferred to the Western diet (blue). (B) Clustering of C. innocuum SB23 gene expression in humanized mice fed the LF/PP (red) or Western (blue) diet. (C) Clustering of the gut microbiome’s meta transcriptome in humanized mice fed a LF/PP (red) or a Western (blue) diet. Black circles represent validated clusters (inconsistency threshold = 0.75, “cluster” function in Matlab version 7.7.0). (D) qRT-PCR validation of C. innocuum SB23 gene expression in humanized mice (n = 3 to 5 samples per group; see Supplementary Material). Mean values ± SEM are plotted (*P < 0.05, Student’s t test).

  • Fig. 6

    Transmissibility of adiposity from humanized mice to germ-free recipients. (A) The effects of Western and LF/PP diets on epididymal fat pad weight (expressed as a percentage of total body weight) in humanized gnotobiotic mice (n = 5 to 8 mice per group; n = 2 independent groups). (B) Percent increase in total body fat (measured by DEXA) after colonization of germ-free mice with a cecal microbiota harvested from humanized donors fed the Western or the LF/PP diet (n = 4 to 5 mice per group). Recipients were fed a LF/PP diet. Mean values ± SEM are plotted (*P < 0.05, Student’s t test).

  • Table 1

    Metabolic pathways altered by diet in the humanized mouse gut microbiome. Based on a bootstrap analysis of gut microbiome data sets from humanized mice fed the LF/PP diet [7 to 35 dpc (days after colonization with a human fecal sample)] or the Western diet (29 to 35 dpc; confidence interval = 0.95, 10,000 samples; inclusion criterion, pathways must be found at ≥0.6% relative abundance in at least two samples).

    KEGG pathways enriched on LF/PP dietCyanoamino acid metabolism
    Glycosaminoglycan degradation
    Glycosphingolipid biosynthesis—ganglioseries
    N-glycan degradation
    Nucleotide sugar metabolism
    Other ion-coupled transporters
    Pentose and glucuronate interconversions
    Phenylpropanoid biosynthesis
    Sphingolipid metabolism
    Starch and sucrose metabolism
    KEGG pathways enriched on Western dietABC transporters
    PTS
    Transporters

Additional Files

  • Supplementary Material for:

    The Effect of Diet on the Human Gut Microbiome: A Metagenomic Analysis in Humanized Gnotobiotic Mice

    Peter J. Turnbaugh, Vanessa K. Ridaura, Jeremiah J. Faith, Federico E. Rey, Rob Knight, Jeffrey I. Gordon*

    *To whom correspondence should be addressed. E-mail: jgordon{at}wustl.edu

    Published 11 November 2009, Sci. Transl. Med. 1, 6ra14 (2009)
    DOI: 10.1126/scitranslmed.3000322

    This PDF file includes:

    • Materials and Methods
    • References
    • Fig. S1. Phylogeny of the Erysipelotrichi.
    • Fig. S2. Rarefaction analysis of the gut microbiota of humanized mice.
    • Fig. S3. Unweighted UniFrac-based clustering of V2 16S rRNA gene surveys.
    • Fig. S4. Assembly of the human gut microbiota in suckling, weaning, and young adult C57BL/6J mice.
    • Fig. S5. Analysis of the in vivo Erysipelotrichi population, anchored to the C. innocuum SB23 draft genome.
    • Table S1–16 legends. [See below for instructions on downloading tables.]

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:
    (available at www.sciencetranslationalmedicine.org/cgi/content/full/1/6/6ra14/DC1)

    Tables S1 – S16. (In Microsoft Excel format).

    Files are packaged as a compressed archive, in *.zip format; users should download the compressed file to their machine and decompress the file on their local hard drive, using the instructions below.

    3000322_tables_s1_s16.zip (222 KB)

    • Table S1. V2 16S rRNA gene sequencing statistics from human donor and two generations of recipient humanized mice.
    • Table S2. Full-length 16S rRNA gene sequencing statistics.
    • Table S3. Abundance of class-level bacterial taxa in the gut microbiota.
    • Table S4. Abundance of genus-level bacterial taxa in the gut microbiota.
    • Table S5. V2 16S rRNA gene sequencing statistics from transplantation of a frozen human fecal sample.
    • Table S6. V2 16S rRNA gene sequencing statistics from the assembly of the humanized mouse gut microbiota.
    • Table S7. V2 16S rRNA gene sequencing statistics from humanized mouse biogeography analysis.
    • Table S8. Microbiome sequencing statistics (fecal samples).
    • Table S9. Genomes in the human gut microbe database.
    • Table S10. Relative abundance of KEGG orthologous groups (KOs) in Western diet–associated pathways (% of KO assignments).
    • Table S11. C. innocuum strain SB23 genome sequencing statistics.
    • Table S12. Number of genes assigned to glycoside hydrolase family 1 in gut Firmicutes and Bacteroidetes.
    • Table S13. cDNA sequencing statistics.
    • Table S14. C. innocuum strain SB23 genes up-regulated in the ceca of humanized mice fed the Western diet relative to the LF/PP diet.
    • Table S15. Primers used for qRT-PCR analysis.
    • Table S16. Community gene clusters differentially expressed in the ceca of humanized mice fed the Western diet relative to the LF/PP diet.

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