Dietary fat source modulates gut microbiota profiles

As described above, one main difference between GF and SPF mice is caecum weight. Therefore, the caecum to body weight ratio was calculated. BA, P- and L-HFD-BA feeding of GF reduced the caecum weight by over 1.5-fold (Figure 34A). As in the literature described, we also observed reduced Figure 33:Bile acid composition was modulated by colonization status and diet.

(A) PCA analysis of the systemic bile acid composition within the colonization and feeding groups. (B) Measurement of CA, TCA, DCA, TDCA and T-β-MCA in systemic plasma. CD; BA; P-HFD-BA; L-HFD-BA; For detailed description of the statistical analysis see section 3.17; Number of mice in each group are indicated below the x-axis (n = number of mice measured).

caecum weight in SPF mice fed HFD. Measurement of viable bacteria in caecal content of SPF mice by anaerobic cultivation revealed no differences between diets (Figure 34B). No viable bacteria were observed in GF mice, which was also confirmed by gram staining (data not shown).

High-throughput sequencing of 16S rRNA gene amplicon libraries was performed to identify diet-induced shifts in caecal microbiota profiles from SPF mice. Beta-diversity analysis revealed a significant clustering of samples according to diet. (Figure 34C). A clear separation induced by the diet was also observed in the 16 weeks feeding experiment (Suppl. Fig. S14A). Analysis of alpha-diversity, assessed by Shannon effective counts, was 1.2-fold lower in BA- than in CD-fed mice (Figure 34D). However, dietary intervention did not change microbial richness. In contrast, no differences in alpha-diversity were observed in the long-term feeding experiment (Suppl. Fig. S14B).

Diet-induced shifts in caecal microbiota composition were already visible at the phylum level (Figure 34D). The relative abundance of Firmicutes was 1.2-fold higher in CD- and P-HFD-BA- than in BA-fed mice. BA feeding increased the relative abundance of Deferribacteres by 32 % and of Proteobacteria by 41 % compared to CD feeding. In the first feeding experiment, the abundance of Firmicutes was also 1.2-fold lower and the abundance of Proteobacteria 40 % higher in BA than CD mice (Suppl. Fig.

S14C). The relative abundance of Coriobacteriaceae was reduced in BA-fed groups but this difference did not reach significance (Figure 34F).

A deeper look at the level of single molecular species showed that the four dietary interventions were characterized by the presence of specific OTUs (Figure 34G). Most of the OTUs characteristic for CD mice belonged to the families Ruminococcaceae and Rikenellaceae, whereas BA feeding increased the relative abundance of OTUs belonging to Oscillospiraceae spp. and Alistipes spp.

Interestingly, P-HFD-BA as well as the long-term P-HFD feeding increased the relative abundance of an OTU with closest match to Acetatifactor muris. L-HFD-BA feeding was characterized by the OTUs with was closest to Clostridium lactatifermentans.

As the duration of dietary intervention is also important for modulating the gut microbiota, we looked at changes in the gut microbiota induced by CD and BA diet fed 8 or 16 weeks (Figure 35). A clear separation of the feeding groups and duration was found when looking at beta-diversity (Figure 35A). Of note, different DNA isolation protocols were used for these two studies (see section 3.7 for technical details). Therefore, the results have to be handled with caution.

Alpha diversity analysis revealed a moderate but significant reduction in Shannon effective score induced by 8 weeks BA feeding (Figure 35B). No differences were observed in richness of molecular species. Interestingly, the relative abundance of Proteobacteria and the corresponding family

and D). Moreover, BA diet increased the abundance of Desulfovibrionaceae independent of duration.

In the CD-fed groups, long-term feeding resulted in a reduced relative abundance of Deferribacteraceae whereas Rikenellaceae were increased in their relative abundance. In contrast, long-term BA feeding decreased the relative abundance of Lachnospiraceae and increased the one of Bacteroidaceae. With respect to Coriobacteriaceae, BA feeding reduced their relative abundance compared to CD regardless of the feeding duration (Figure 35E). However, this difference did not reach significance. Furthermore, each diet as well as feeding duration was characterized by specific OTUs (Figure 35F). Interestingly, short-term CD feeding was characterized by one OTU with a 100 % sequence similarity to Faecalibaculum rodentium.

Summarizing all aforementioned results, the dietary fat source had important effects on host metabolism like glucose tolerance and hepatic lipid metabolism which then seems to modulate WAT mass. Furthermore, it changed caecal microbiota composition which may consequently affect the host. Further investigation is needed to dissect these differences in more detail.

C

E D

(12/12) (12/12) (12/12) (12/12) 110

(12/12) (12/12) (12/12) (12/12) 30

effective number of species

* *

CD BA P-HFD-BA L-HFD-BA

(12/12) (12/12) (12/12) (12/12) 0

(12/12) (12/12) (12/12) (12/12) 0

(12/12) (12/12) (12/12) (12/12) 0

(12/12) (12/12) (12/12) (12/12) 0

(12/12) (12/12) (12/12) (12/12) 0

(12/12) (12/12) (12/12) (12/12) 0

(12/12) (12/12) (12/12) (12/12) 0

(12/12) (12/12) (12/12) (12/12) 0

(12/12) (12/12) (12/12) (12/12) 0

(12/12) (12/12) (12/12) (12/12) 0.0

caecum to body weight ratio

[mg/g]

cfu/ g caecal content

Figure 34:Diet modulated caecal microbiota profile of SPF mice.

(A) Measurement of caecum to body weight ratio showed colonization status and diet induced changes. (B) Viable bacterial cell counts were assessed by anaerobic cultivation of caecal content. (C) Analysis of β-diversity revealed significant differences in bacterial composition induced by the diet. (D) Shannon effective species counts and richness. (E) At phylum level, significant differences between the four feeding groups could be observed. (F) The relative abundance of the family Coriobactericeae. (G) Relative abundance heat map of OTUs characteristic for the different diets (classification was done with EzTaxon). CD; BA; P-HFD-BA; L-HFD-BA;

*p<0.05; **p<0.01; ***p<0.001 (serial comparison was performed with the Rhea R package [232]). NGS was performed on the 16S ribosomal RNA gene amplicons of the V3/V4 region (450bp) of caecal content using MiSeq platform. IMNGS and Rhea were used for sequence analysis. Number of mice in each group are

A B

effective number of species

CD BA

Figure 35: Caecal microbiome is modulated by diet.

(A) Analysis of β-diversity via MDS plot. (B) Shannon effective and richness species counts. At phylum (C) and family level (D), changes were observed between the two diets as well as the two feeding periods. (E) The relative abundance of Coriobacteriaceae. (F) Relative abundance heat maps of OTUs characteristic for the different groups, diets and feeding times (classification was done with EZBioCloud). CD; BA; w., weeks;

*p<0.05; **p<0.01; ***p<0.001; NGS was performed on the 16S ribosomal RNA gene amplicons of the V3/V4 region (450bp) of caecal content using the MiSeq platform. Sequence analysis was done using IMNGS and Rhea. Number of mice in each group are indicated below the x-axis (n = positive mice per total number of mice).

5 Discussion

Coriobacteriaceae are prevalent and dominant members of the human gut microbiota and are able to metabolize bile acids, steroid hormones, and lipids. Furthermore, several studies associated their occurrence with increased cholesterol and lipid metabolism as well as T2D and obesity. However, no mechanistic study about the role of Coriobacteriaceae on host metabolism is currently available.