L. fermentum CECT5716 treatment ameliorated the gut dysbiosis
The effects of L. fermentum on the intestinal microbiota were explored since gut dysbiosis plays a key role in metabolic disorders, including obesity. Several ecological features of the gut bacterial communities were evaluated in the three experimental groups by different parameters including Chao1 richness (diversity estimation), Phylogenetic diversity (PD) whole tree (consider the phylogeny to estimate diversity across a tree), Observed OTUs (count of unique OTUs in each sample) and Shannon diversity (a richness and evenness estimator). Microbial richness, evenness and diversity were significantly decreased in the HFD group compared to the control diet group, whereas L. fermentumadministration was able to restore all these ecological parameters to normal values, excepting Shannon index. Although it reached normal values, it did not differ from the control group (Figure 7A). Furthermore, the principal coordinates analysis (PCoA) showed evident differences between control diet- and HFD-fed groups, thus indicating two extremely different gut environments (Figure 7B). Of note, whenL. fermentum was administered to obese mice, marked differences could be appreciated in comparison with non-treated obese mice (Figure 7B). To further investigate this remarkable shift in the gut microbial environment after probiotic treatment, the bacteria composition at phylum level was examined (Figure 7C). The 16S rDNA analysis revealed that the most abundant phyla were Bacteriodetes (B) andFirmicutes (F) in all experimental groups, although HFD induced a dramatic shift in both phyla when compared to non-obese mice, significantly increasing Firmicutes (from 57.66% to 78.68%) and reducing Bacteroidetes (from 23.72% to 13.02%). This resulted in an increased F/B ratio, which was significantly restored by L. fermentum treatment, reaching similar values to control diet-fed mice (Figure 7). Similarly, Verrumicrobia phylum also showed a reduced proportion in HFD-fed mice, which was significantly increased withL. fermentum administration (Figure 7C). Interestingly, the relative abundance of Erysipelotrichi (class) was enriched in untreated obese mice in comparison with non-obese mice, being this situation ameliorated after probiotic treatment (Figure 7C).
At genus level, control obese mice revealed a reduced proportion in the sequences in two genera, Bacteroides and Akkermansia , belonging to Bacteriodetes and Verrumicrobia , respectively, and an increase in Clostridium , in comparison with control mice, which was significantly ameliorated after L. fermentum treatment (Figure 7C).
Correlation of major bacteria genera and main obesity-related markers using the non-parametric test of Spearman suggested that beneficial effects observed in L. fermentum -treated mice could be derived from microbiota modulation. In fact, a positive correlation betweenAkkermansia genus abundance and adipokines and intestinal barrier markers expression, as well as, genus derived fromErysipelotrichaceae and inflammatory markers expression were found (Figure S1). Thus, increased abundance of Akkermansia and reduced presence of Erysipelotrichi (class) observed afterL. fermentum treatment could be involved in its beneficial effects.
The PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) analysis of the KEGG orthologs associated with these bacterial signatures also showed different clusters among the three groups (Figure 8A). LEfSe bar graph indicated that non-obese mice group was characterized by a significantly higher (p<0.01) proportion of bacterial taxa associated with the energy metabolism, transport and catabolism, metabolism of cofactors and vitamins, replication and repair processes and other glycan degradation (Figure 8A). In contrast, HFD mice harbored a higher (p < 0.01) proportion of bacterial taxa associated with the cellular antigens, biosynthesis of lipopolysaccharides and fatty acids, membrane transporters and metabolism of amino sugar and nucleotide sugar (Figure 8A). When we evaluated the L. fermentum treatment in obese mice bacteria related with glycan biosynthesis and metabolism, arginine and proline metabolism, membrane and intracellular structural molecules, nucleotide metabolism or secondary metabolism biosynthesis were overrepresented (p < 0.01), which means that all of these pathways were less represented (p < 0.01) in the other two experimental groups of mice (Figure 8A).
Finally, we estimated the relationship between taxonomic and functional enrichments in each group using the non-parametric test of Spearman’s rank correlation. Results, as depicted in the form of hierarchical heat map, showed distinct clusters of positive and negative associations between bacterial taxa and metagenomic functions (Figure 8B). To further simplify these clusters, we extracted the significant correlation subsets (Spearman’s rho > 0.7 and p-value < 0.01) and built separate correlation networks for each experimental group (Figure 8C). Moreover, different co-occurrence networks arrays were observed between non-treated and treated obese mice, confirming previous results.