Scatter plots of scores and loadings from PCA on total microbiota.

<p><b>(A)</b> Scores from PC1 and PC2. <b>(B)</b> Loadings from PC1 and PC2. <b>(C)</b> Scores from PC3 and PC4. <b>(D)</b> Loadings from PC3 and PC4. The samples in the score plots (A and C) are colored and marked according to fiber type and dose. The size of the loading plot bubbles (B and D) correspond to abundance of the OTUs, and the colored bubbles are those that were found to be significantly related to tumor load in the PLS regression: red = positive and blue = negative, and the color saturation corresponds to the magnitude of the correlation with tumor load. The largest bubble (blue) represent OTU 181719 (<i>Bacteroides</i>).</p

Number of mice fed the different experimental diets (CE, BSG or IN) at a dose level of 5% or 15%.

<p>Number of mice fed the different experimental diets (CE, BSG or IN) at a dose level of 5% or 15%.</p

Effect of fiber type and fiber dose on the alpha diversity.

<p>Box plot of alpha diversity (observed species after 50,000 rarefactions). Grouping is according to the Turkey method (a single-step multiple comparison procedure and statistical test used to find means that are significantly different from each other). The different letters A, B and C are used to illustrate whether the mean difference between any pair of groups is statistically significant. Groups that do not share a letter are significantly different. The * represent outliers (less than 3/2 times of the lower quartile).</p

Effect of Dietary Fibers on Cecal Microbiota and Intestinal Tumorigenesis in Azoxymethane Treated A/J Min/+ Mice

<div><p>Foods naturally high in dietary fiber are generally considered to protect against development of colorectal cancer (CRC). However, the intrinsic effect of dietary fiber on intestinal carcinogenesis is unclear. We used azoxymethane (AOM) treated A/J Min/+ mice, which developed a significantly higher tumor load in the colon than in the small intestine, to compare the effects of dietary inulin (IN), cellulose (CE) or brewers spent grain (BSG) on intestinal tumorigenesis and cecal microbiota. Each fiber was tested at two dose levels, 5% and 15% (w/w) content of the AIN-93M diet. The microbiota was investigated by next-generation sequencing of the 16S rRNA gene (V4). We found that mice fed IN had approximately 50% lower colonic tumor load than mice fed CE or BSG (p<0.001). Surprisingly, all three types of fiber caused a dose dependent increase of colonic tumor load (p<0.001). The small intestinal tumor load was not affected by the dietary fiber interventions. Mice fed IN had a lower bacterial diversity than mice fed CE or BSG. The <i>Bacteroidetes/Firmicutes</i> ratio was significantly (p = 0.003) different between the three fiber diets with a higher mean value in IN fed mice compared with BSG and CE. We also found a relation between microbiota and the colonic tumor load, where many of the operational taxonomic units (OTUs) related to low tumor load were significantly enriched in mice fed IN. Among the OTUs related to low tumor load were bacteria affiliated with the <i>Bacteroides</i> genus. These results suggest that type of dietary fiber may play a role in the development of CRC, and that the suppressive effect of IN on colonic tumorigenesis is associated with profound changes in the cecal microbiota profile.</p></div

Overview of the responses caused by the different experimental factors (ANOVA tables for all biological parameters).

<p>The responses are shown as explained variance (%).</p

Venn diagram showing the overlap between OTUs identified by 50–50 MANOVA and PLS regression.

<p>The different statistical analysis was performed on 507 OTUs that passed the initial filtration. The 50–50 MANOVA identified 434 OTUs of which 118 OTUs overlapped with the 144 OTUs identified by the PLS regression. Twenty six OTUs of the OTUs identified from the PLS regression were not significant according to the dietary intervention.</p

The different fibers’ effect on the composition of the cecal microbiota.

<p>The relative abundances of the cecal microbiota from mice in the different fiber groups were averaged. All families with average above 1% in at least one fiber group are represented. The remaining taxonomy is represented by “Other Taxonomy”. Phylum = p and family = f. The <i>Bacteroidetes</i> are represented by the red/orange colors while the <i>Firmicutes</i> are represented by the blue/turquoise colors.</p

The joint analysis of microbiota and colon tumor load on male min mice, with overall results from Two-way ANOVA, 50–50 MANOVA and PLS regression.

<p>Note that the numbers for tumor load are different from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155402#pone.0155402.t001" target="_blank">Table 1</a>, since this joint analysis was performed on male mice only. The numbers are % of explained variance and number of significant OTUs for each experimental factor and analysis method.</p

Overview (family level) of the OTUs (144) that were found to have significant relation to tumor load in the PLS regression.

<p>The negatively correlated OTUs are the 39 blue bubbles in the loading plots of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155402#pone.0155402.g006" target="_blank">Fig 6</a>, while the 105 positively correlated OTUs are the red bubbles. Phylum = p; class = c; order = o and family = f.</p