Physical exercise and vegetarian diet affected gut microbiome differently; a physical exercise increased the populations of Firmicutes and Actinobacteria, whereas a vegetarian diet increased the populations of Bacteroidetes and Actinobacteria.
To investigate the effect of the physiological condition of host and diet composition on the diversity of gut microbiome, we first recruited 30 ~ 50 years old volunteers depending on a meat-containing diet. The 75 volunteers were divided into three groups; one group shifting their diet from meat diet to a vegetarian diet (the VT group), second group adopting a 30 min physical exercise of a guided aerobic exercise in fitness center three times per week without changing their original diets (the EX group), and control continuing their life style (the Ctrl group). The fecal samples from each group were collected for metagenome analyses by NGS (Next-generation sequencing) (the VT group, n=20; the EX group, n=21; the Ctrl group n=22).
The NGS analyses showed that the compositions of gut microbiome changed significantly in both groups after the diet shift or the physical exercise (Fig. 1). The phylogenetic compositions of the microbial communities of the gut microbiome in each group were compared by combining gene abundance data at taxonomic levels from phylum to species. As shown in Fig. 1a, Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria were present as main dominant phyla in all of the experimental groups, and the members of 2 major phyla, Bacteroidetes and Firmicutes, dominated the gut microbiome of each groups (>88% of all 16S rRNA sequences), which is consistent with previous human gut microbiome studies . Bacteroidetes were more abundant than Firmicutes at the starting point, and their compositions were not changed in the gut microbiome of the control group. However, in the EX group, the abundance of Bacteroidetes and Proteobacteria decreased while the abundance of Firmicutes and Actinobacteria increased; whereas, in the VT group, the abundance of Bacteroidetes and Actinobacteria increased while correspondingly the abundance of Firmicutes and Proteobacteria decreased. The Bacteroidetes/Firmicutes ratio in the VT group altered 3 times more than that of the EX group during the experimental period (Fig. 1b). Bacteroidetes constituted 66.3% of the VT gut microbiome and 47.9% of the EX gut microbiome at the end of experiment respectively (Fig. 1, Additional file 1: Fig. S1).
The physical exercise increased the population of Megamonas funiformis while decreased Bacteroides, whereas a vegetarian diet increased the populations of Prevotella while decreased Bacteroides.
The five most dominant families at starting point of the experiment were Prevotellaceae, Ruminococcaceae, Bacteroidaceae, Lanchonospiraceae, and Bifidobacteriaceae (Fig. 2a). Exercise increased the composition of Bacteroidaceae, Lachnospiraceae, and Enterobacteriaceae while decreased the composition of Prevotellaceae, Ruminococcaceae, and Bifidobacteriaceae. Meanwhile, a vegetarian diet increased the composition of Prevotellaceae, Ruminococcaceae, and Lachnospiraceae while decreased the composition of Bacteroidaceae, Bifidobacteriaceae, and Enterobacteriaceae. The five most abundant genera in the VT group at the end of the experiment were Prevotella, Bacteroides, Faecalibacterium, Bifidobacterium, and Lachnospira, which accounted for 77.7% of the bacterial population. Although these five genera were also the most abundant in the VT group at the end of the experiment, these genera constituted only 51.6% in the EX group. The relative proportion of Bacteroides was decreased while the relative proportion of Prevotella and Faecalibacterium were increased in the VT group at the end of the experiment (Fig. 2b, Fig. 3, Additional file 2: Fig. S2, Additional file 3: Fig. S3). Interestingly, exercise made Megamonas genus flourish to constitute up to 7.0 % in the gut microbiome meanwhile the abundance of Prevotella and Bacteroides decrease. Although most of the species detected in the VT group were also detected in the EX group, it is worth noting that the relative abundances of Prevotella spp. (Prevotella copri, FJ678796_s, FJ511060_s and EU462041_s), Bacteroides spp. (Bacteroides vulgatus, Bacteroides coprocola), Faecalibacterium spp. (BABG01000051_s, Faecalibacterium prausnitzii), and Bifidobacterium spp. (Bifidobacterium pseudocatenulatum, Bifidobacterium adolescentis, and Bifidobacterium stercoris) in the VT group were higher than in the EX group, but the relative abundance of Prevotella stercorea, Bacteroides plebeius, Bacteroides uniformis, Bacteroides stercoris and Faecalibacterium spp. (DQ793299_s) in the VT group were lower than in the EX group (Fig. 4). Especially, exercise increased the population of Megamonas funiformis up to 17 times, constituting 6.9% of the gut microbiome, while reducing the population of Bacteroides_uc spp. to tenth.
Diet shift decreased Escherichia coli 22 times. It has been reported that a vegetarian diet shifts gut microbiome to contain less potentially pathogenic intestinal microbes responsible of diarrhea . This work showed the same tendency in which Enterobacteriaceae, such as Shigella and Escherichia, was significantly underrepresented in the VT group (Additional file 4: Fig. S4). The genus Prevotella, one of the leading sources of the inter-individual gut microbiome variation , has been reported to be dominant in a vegetarian diet . In the same context, another previous study has also shown that an animal-based diet led to the domination of Bacteroides genus, while a vegetarian diet led to the domination of Prevotella genus in gut microbiome . It is interesting to note that Prevotella is the only notable genus, which was significantly increased in its abundance, and that Bacteroides is the only notable genus, which was notably decreased in its abundance by diet shift in this work, which confirms the previous report of the positive correlation between a vegetarian diet and domination of Prevotella.
Alpha-diversity analysis showed that physical exercise and vegetarian diet modified the gut microbiomes in a different way.
Since the gross microbiome analysis showed the transformative effect of physical exercise and diet shift on gut microbiome (Fig. 1 ~ 4), we analyzed further the effect of physical exercise and vegetarian diet on the compositional changes of gut microbiome by using a-diversity analyses. Maximum-likelihood phylogenetic tree comprising all of the taxa in each group showed that the diversity of microbial composition slightly decreased as well as confirming the increase of Firmicutes and Actinobacteria by exercise and increase of Bacteroidetes and Actinobacteria by vegetarian diet (Fig. 5a). The taxonomic α-diversity measurement by ACE richness, Shannon and Simpson showed that the diversities of gut microbiome were slightly decreased in both cases of physical exercise and vegetarian diet although the differences were not statistically significant (Fig. 5b). Interestingly, HCL heatmap at Genus level indicated that exercise affected the microbial community more significantly than diet shift (Fig. 5c). Based on the results of Fig. 4 and Fig. 5, the slight decreases in microbial diversity by physical exercise and vegetarian diet seems to have its roots in inhibition of proliferation of a harmful group of bacteria.
Beta-diversity analysis confirmed that physical exercise and vegetarian diet modified the gut microbiomes in a different way.
Different effects of physical exercise and vegetarian diet on gut microbiome became more evident by β analyses which quantitate the ratio between location and local species method (Fig. 6). The NMDS plot based on Bray-Curtis distances showed that the original composition of gut microbiome became diverged into a different direction by either physical exercise or vegetarian diet (Fig. 6a). Also, the NMDS heatmap with Bray-Cirtis distance showed that physical exercise affected the composition of gut microbiome more significantly than vegetarian diet (Fig. 6b).
The information on the relative relatedness of microbial members based on phylogenetic distances between the microbial organisms was evaluated by using Fast UniFrac analysis  to analyze the effect of physical exercise and vegetarian diet. As shown in Fig. 6c, both a physical exercise and a vegetarian diet dramatically changed the compositions of the gut microbiome. Interestingly enough, however, the Fast UniFrac analysis showed that the physical exercise affected the composition of gut microbiome much more than the vegetarian diet. Principal coordinate analysis (PCA) also confirmed a same tendency (Fig. 6d). The first principal component (PC1) was dominant with 75.6% variability, and the microbial compositions of the EX, VT, and Ctrl were different each other. In consistent with the Fast UniFrac analysis, exercise affected the composition of gut microbiome more dramatically than a vegetarian diet.