Bacillus licheniformis Reshapes the Gut Microbiota to Alleviate the Subhealth

Abstract

Subhealth is a condition between health and disease that has become a common public health risk. Therefore, it is necessary to find more scientific therapies that can alleviate the symptoms of subhealth effectively. The gut microbiota is closely associated with subhealth. As a mature probiotic preparation, Bacillus licheniformis (B. licheniformis) can regulate gut microbiota balance, which indicates that B. licheniformis has the potential in regulating subhealth. This study produced the subhealthy rats by using chronic stress for 4 weeks to simulate psychological stress, with excessive antibiotics for 1 week to simulate bad living habits. Then, they were treated for 4 weeks with B. licheniformis. The results showed that B. licheniformis could recover the gut microbiota balance that had been destroyed by subhealth. The serum corticosterone and the proinflammatory cytokine tumor necrosis factor-α decreased after being treated by B. licheniformis. B. licheniformis also reduced glutamic acid and norepinephrine levels while increasing γ-aminobutyric acid and 5-hydroxytryptamine levels in the brain. In addition to the physiological changes, B. licheniformis decreased the anxiety-like behaviors of rats. Therefore B. licheniformis could alleviate the subhealth state, mainly by remodeling the gut microbiota, reducing inflammation, inhibiting the hypothalamic-pituitary-adrenal axis hyperactivity, regulating neurotransmitter levels, and easing a negative mood.

Keywords: Bacillus licheniformis; antibiotics; gut microbiota; subhealth.

Figure 1

Experimental procedures.

Figure 2

Effect of simulated subhealth on body weight, food intake, liver index, spleen index, and adrenal index (A) Body weight in the modeling period (B) Food intake in the modeling period (C) Weight gain/food intake ratio in the modeling period. Week 0: the end of adaptation; week 1–4: the end of each week in modeling period. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. (D) Body weight in the recovery period (E) Food intake in the recovery period (F) Weight gain/food intake ratio in the recovery period. Week 0: the end of modeling period; week 1–4: the end of each week in recovery period. con_2 vs. sub ^△ p < 0.05, ^△△ p < 0.01, ^△△△ p < 0.001; con_2 vs. sub+bl ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001. (G) Liver index: liver/body weight ratio at the end of the modeling period (H) Spleen index: spleen/body weight ratio at the end of the modeling period (I) Adrenal index: adrenal/body weight ratio at the end of the modeling period (J) Liver index: liver/body weight ratio at the end of the recovery period (K) Spleen index: spleen/body weight ratio at the end of the recovery period (L) Adrenal index: adrenal/body weight ratio at the end of the recovery period. * p < 0.05.

Figure 3

B. licheniformis reduced anxiety-like behaviors. (A,B) Immobility time and immobility count in FST at the end of the modeling period. (C,D) Time spent in the closed-arms and the open-arms at the end of the modeling period. (E,F) Immobility time and immobility count in FST at the end of the recovery period. (G,H) Time spent in the closed-arms and the open-arms at the end of the recovery period. ** p < 0.01.

Figure 4

Changes in cytokines and nervous system-related metabolites (A) IL-6 levels in serum at the end of the modeling period (B) 5-HT levels in the brain at the end of the modeling period (C–I) Acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and hexanoic acid levels in the colon at the end of the modeling period (J) IFN-γ levels in serum at the end of the recovery period (K) IL-6 levels in serum at the end of the recovery period (L) TNF-α levels in serum at the end of the recovery period (M) CORT levels in serum at the end of the recovery period (N) Propionic acid levels in colon contents at the end of the recovery period (O) Isobutyric acid levels in colon contents (P) Isovaleric acid levels in colon contents at the end of the recovery period (Q) Tyr levels in the brain at the end of the recovery period (R) Levels of NE in the brain at the end of the recovery period (S) Levels of E in the brain at the end of the recovery period (T) DA levels in the brain at the end of the recovery period (U) GABA levels in the brain at the end of the recovery period (V) Glu levels in the brain (W) Gln levels in the brain at the end of the recovery period (X) Trp levels in the brain at the end of the recovery period (Y) 5-HT levels in the brain at the end of the recovery period (Z) 5-HIAA levels in the brain at the end of the recovery period. * p < 0.05; ** p < 0.01.

Figure 5

Changes in the gut microbiota. (A) PCoA of the gut microbiota at the end of the modeling period (B) Composition of the gut microbiota at the phylum level at the end of the modeling period (C) Composition of gut microbiota at the family level at the end of the modeling period (D) PCoA of gut microbiota at the end of the recovery period (E) Composition of the gut microbiota at phylum taxonomic ranking at the end of the recovery period (F) Composition of gut microbiota at family taxonomic ranking at the end of the recovery period (G) LEfSe for comparisons of the subgroup to the sub+bl group: taxonomic tree and effect size histograms (H) Microbial network at the end of the recovery period: the genus with degree >10 are marked. Different colors of the nodes represent different phylum. The red curve shows a positive correlation, and the green curve shows a negative correlation.

Figure 6

Correlation among relative abundance of the gut microbiota, and physiological and behavioral indices. (A) Correlation among the genera whose relative abundance changed significantly and the physiological or behavioral indices. The red cell means positive correlation, the blue cell means negative correlation, the value in the cell is the p-value, and the blank cell indicates that the correlation coefficient was not significant. * p < 0.05; ** p < 0.01; *** p < 0.001 (B) Propionic acid and NE (C) NE and TNF-α (D) Hexanoic acid and TNF-α (E) TNF-α and CORT (F) NE and CORT.

Figure 7

Graphical summary of the main findings. Effect of B. licheniformis on inhibiting HPA axis hyperactivity (reducing serum CORT) and inflammation (reducing serum TNF-α) by remodeling the gut microbiota and reducing anxiety (reducing anxiety-like behaviors and changing brain neurotransmitter levels) to alleviate the subhealth. The green arrow indicates a decrease, the red arrow indicates an increase, the green “−” indicates a negative effect, and the red “+” indicates a positive effect. GLU glutamic acid, GABA gamma-amino butyric acid, NE norepinephrine, CORT corticosterone, SCFAs short chain fatty acids.