A Western Diet Ecological Module Identified from the ‘Humanized’ Mouse Microbiota Predicts Diet in Adults and Formula Feeding in Children

<div><p>The interplay between diet and the microbiota has been implicated in the growing frequency of chronic diseases associated with the Western lifestyle. However, the complexity and variability of microbial ecology in humans and preclinical models has hampered identification of the molecular mechanisms underlying the association of the microbiota in this context. We sought to address two key questions. Can the microbial ecology of preclinical models predict human populations? And can we identify underlying principles that surpass the plasticity of microbial ecology in humans? To do this, we focused our study on diet; perhaps the most influential factor determining the composition of the gut microbiota. Beginning with a study in ‘humanized’ mice we identified an interactive module of 9 genera allied with Western diet intake. This module was applied to a controlled dietary study in humans. The abundance of the Western ecological module correctly predicted the dietary intake of 19/21 top and 21/21 of the bottom quartile samples inclusive of all 5 Western and ‘low-fat’ diet subjects, respectively. In 98 volunteers the abundance of the Western module correlated appropriately with dietary intake of saturated fatty acids, fat-soluble vitamins and fiber. Furthermore, it correlated with the geographical location and dietary habits of healthy adults from the Western, developing and third world. The module was also coupled to dietary intake in children (and piglets) correlating with formula (vs breast) feeding and associated with a precipitous development of the ecological module in young children. Our study provides a conceptual platform to translate microbial ecology from preclinical models to humans and identifies an ecological network module underlying the association of the gut microbiota with Western dietary habits.</p></div

Temporal and diet-regulated dynamics of ecological bacterial modules.

<p><b>A</b>. Bacteroides module. Each stacked bar represents the Z score normalized abundance of the component genera indicated in the legend. The diet and time points are indicated on the x-axis and can generally be followed in a temporal manner from left to right. Diets shown are human (undefined starting material), LF/PP (low fat/plant polysaccharide rich chow), WESTERN (Harlan-Teklad TD96132), FASTING (no food). I; initial fecal donor sample, N/A; initial sample prior to inoculation, pg; day following gavage into mouse, pw; day following shift to Western diet, pb: day following return to LF/PP diet, 1pf: day 1 fasting, pf: day after fasting. <b>B</b>. Temporal and diet-regulated dynamics of the <i>Pseudoflavonifractor</i> module. Each stacked bar represents the Z score normalized abundance of the component genera indicated in the legend. X-axis as for A. <b>C</b>. The abundance of the <i>Bacteroides</i> and <i>Pseuodoflavonifractor</i> modules associate with Western-diet. The average Z score for each fecal sample was calculated for both the <i>Bacteroides</i> and <i>Pseudoflavonifractor</i>-module component genera and arranged in order of abundance from left to right. Each bar is coloured by the sample diet.</p

The Bacteroides module distinguishes geographical dietary intake and formula feeding in children and piglets.

<p><b>A</b>. The <i>Bacteroides</i> module is endemic to all ages of the developed and developing world. The average Z score per fecal sample was calculated and combined to compare the three populations. The number of samples included with the given criteria (Count), average of the calculated Z scores/sample (Avg), the standard deviation (StdDev), and the lower 95% and upper 95% confidence limits (L95 and U95, respectively) are displayed in the Tables. The top panel includes all available samples in the study, regardless of age annotation. The middle panel includes only adults with an annotated age 18 years old or more. The bottom panel includes children with an annotated age 3 years old or less. In all cases, the Malawi samples have significantly lower abundance of the <i>Bacteroides</i> module than those from the USA and Venezuela. <b>B</b>. The Western diet-associated <i>Bacteroides</i> module also associates with formula feeding in children. Average Z scores per sample were calculated as in A. The p-values for <i>Bacteroides</i> module Z score association with diet by ANOVA are indicated. Top panel: all samples from the USA population that were annotated for formula or breast feeding were included in the analysis. Middle panel: Distribution of ages of annotated breast vs formula fed samples. Age matched samples all ≤0.8 years old (as indicated by top box plot) were analyzed. Only USA samples were annotated as formula fed as indicated. Bottom panel: The association with formula feeding remained significant in age matched samples across populations. <b>C</b>. The <i>Bacteroides</i> module associates with formula feeding in piglets as well as humans. Piglets suckled for 7 days (right) were switched to formula for 14 days (left), or left to suckle an additional 14 days (middle). The average Z score for the <i>Bacteroides</i> module from each sample was plotted.</p

Bacterial abundance in humanized mice is a poor predictor of dietary intake in humans.

<p><b>A</b>. Dynamic association of control ‘abundance module’ profile with dietary intake in the mouse. The top panel displays the Z score abundance of each of the genera indicated across time and dietary intake in the humanized mouse. The bottom panel shows the average Z scores for each fecal sample placed in order and coloured according to dietary intake of the mouse. <b>B</b>. The control ‘abundance module’ derived from humanized mice does not associate with diet in a controlled human study. Annotation is the same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083689#pone-0083689-g003" target="_blank">Figure 3A</a>.</p

Detection of Diet-associated Ecological Communities.

<p><b>A</b>. Hierarchical clustering of fecal 16S rRNA sequences in samples derived from humanized gnotobiotic mice. On the y-axis the human donor (1 or 2), diet (LF/PP; low-fat plant polysaccharide-rich mouse chow, Western; Harlan-Teklad TD96132, Fasting; no food, N/A; human sample prior to inoculation) and time point (indicated by day following dietary change and condition; N/A; human sample cultured prior to inoculation, initial; human fecal sample prior to inoculation, pw; post-Western diet change, pf; post fasting, pg; post-gavage, pb; post-return to mouse chow). On the x-axis the composite bacterial genera are indicated. Heat map; red (high abundance), blue (low abundance), grey (none detected). Hierarchical clustering for both rows and columns was performed using UPGMA clustering method with Euclidean distance measure, ordering weight by average value, normalization on a scale between 0 and 1 with empty value replacement by constant value given as 0. <b>B</b>. Identification of diet-related genera/genera interactive pairs. Pairwise genera p-values were calculated by co-occurrence analysis using Spearman correlation. Each point on the graph represents the relationship between two bacterial genera plotted across the p-value observed in the Western diet and the LF/PP (low fat, plant polysaccharide rich)-associated diets. The resulting Western diet-specific (green box) and LF/PP diet-specific (red box) pairs were selected for further analysis. <b>C</b>. Network map of Western diet-specific bacterial interactions. The Western diet-specific pairwise associations identified in B (green box) were assembled to visualize the diet-associated ecologic module. Purple edges indicate positive Spearman correlations, blue indicate negative correlations.</p

Diet Intake Correlation with Ecological Module: RECALL and FFQ Questionnaires [3].

<p>Significant (p<0.01) Spearman correlations of ‘<i>Bacteroides</i>’ module abundance (Average Z Score) with dietary intake as reported in COMBO. No significant correlations were found for either ‘High Abundance’ or ‘<i>Pseudoflavonifractor</i>’ modules. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083689#pone.0083689.s006" target="_blank">Table S3</a> for complete dataset.</p

The <i>Bacteroides</i> module is associated with Western diet in human patients.

<p>On the left is plotted the average Z score for each sample and placed in order of decreasing abundance from left to right. The quartile distribution of high fat diet samples/total is shown for each module. On the right the same average Z scores are plotted with annotation from each patient. The temporal order for each patient is from left to right. Only samples on a controlled Western or low-fat diet (as indicated) were included in the analysis – starting samples prior to commencing the controlled diet were excluded.</p

Community Structure Comparison: Libshuff and Parsimony Statistical Analysis of Colon-Associated Bacterial Populations.

<p>Community Structure Comparison: Libshuff and Parsimony Statistical Analysis of Colon-Associated Bacterial Populations.</p

Comparison of physical and histological parameters and bacterial load of WT and Nod2 KO littermates following DSS damage.

<p><b>A.</b> Timelines and histology assessment for individual mice. No significant difference was observed between the two genotypes for physical parameters (body weight loss, colon length: not shown) nor histological scores between the two groups (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030273#pone.0030273.s004" target="_blank">Figure S4</a> for data from 2 additional independent experiments). <b>B.</b> Colon, mesenteric lymph node and spleen tissue-associated bacterial loads assessed by FACS 42 days following DSS damage. ** = p≤0.01 by Anova with Bonferroni's multiple comparison test. <b>C.</b> Residence of commensal bacteria in the muscle layer in Nod2 KO mice. Examples of bacterial staining by EUB338 FISH probes are indicated by closed arrows in these representative images.</p

Richness, diversity and taxonomic analysis of the WT and Nod2 KO colon tissue-associated bacterial communities.

<p>Top panel: Incidence of phyla from treatment groups of WT and Nod2 KO littermates. Mean +/− SEM (n = 4–6) for each group is shown. * p≤0.05, ** p<0.01 by 2 way ANOVA with Bonferroni's multiple comparison test. Bottom panels: the Chao and Shannon estimates for richness and diversity were calculated from individual mice from each group as indicated (n = 4–6). The mean +/− SEM are shown. No differences were statistically significant by ANOVA.</p