Regulatory Effect of Lactiplantibacillus plantarum 2-33 on Intestinal Microbiota of Mice With Antibiotic-Associated Diarrhea

Abstract

Diarrhea is one of the common adverse reactions in antibiotic treatment, which is usually caused by the imbalance of intestinal flora, and probiotics play an important role in the structure of intestinal flora. Therefore, this experiment studied the regulatory effect of Lactiplantibacillus plantarum 2-33 on antibiotic-associated diarrhea (AAD) mice. First, the AAD mice model was established by the mixed antibiotic solution of gentamicin sulfate and cefradine. Then, the physiological indexes and diarrhea of mice were observed and recorded by gastric perfusion of low dose (1.0 × 10⁷ CFU/ml), medium dose (1.0 × 10⁸CFU/ml), and high dose (1.0 × 10⁹ CFU/ml) strain 2-33. 16S rRNA gene V3-V4 regions were sequenced in colon contents of mice in control group, model group, self-healing group, and experimental group, respectively, and the diversity of intestinal flora and gene function prediction were analyzed. The results showed that the intestinal flora of AAD mice was not significantly regulated by gastric perfusion of strain 2-33 to 7 days, but the relative abundance and diversity of intestinal flora of AAD mice were significantly improved by gastric perfusion to 14 days (p < 0.05). In addition, at the genus level, the relative abundance of Lactobacillus increased significantly, and the relative abundance of Enterococcus and Bacillus decreased significantly (p < 0.05). In addition, the regulation of strain 2-33 on intestinal flora of AAD mice was time- and dose-dependent, short-term gastric perfusion, and low dose had no significant effect (p > 0.05). Strain 2-33 can significantly increase the levels of anti-inflammatory cytokines IL-4 and IL-10, significantly decrease the levels of proinflammatory cytokines TNF-α and IFN-γ (p < 0.05), and can also adjust carbohydrate metabolism, amino acid metabolism, and energy metabolism to normal levels, thus accelerating the recovery of intestinal flora structure of AAD mice. In summary, strain 2-33 can improve the structure and diversity of intestinal flora of AAD mice, balance the level of substance and energy metabolism, and play a positive role in relieving diarrhea, maintaining and improving the intestinal microecological balance.

Keywords: Lactiplantibacillus plantarum; antibiotic-associated diarrhea; inflammatory cytokines; intestinal flora; mice; regulating function.

FIGURE 1

Effect of strain 2-33 on physiological characteristics of AAD mice. Diarrhea index score (A); body weight (B); food intake (C); water intake (D). C, control group; A, model group; S, self-healing group; L, low-dose group; M, middle-dose group; H, high-dose group. The experimental results are expressed as the $\overline{\text{x}}$ ± SD, n = 6.

FIGURE 2

Effect of strain 2-33 on cytokines in serum of AAD mice. IL-4 cytokines (A); IL-10 cytokines (B); TNF-α cytokines (C); IFN-γ cytokines (D). There are significant differences among different letters (p < 0.05).

FIGURE 3

Rarefaction curves (A) and Shannon index curves (B).

FIGURE 4

Analysis of bacterial beta diversity of intestinal flora. PCoA figure (A); NMDS figure (B).

FIGURE 5

Strain 2-33 regulates the changes of AAD mice flora. The relative abundance of bacterial communities is at phylum level (A) and genus level (B); significant difference of the relative abundance of genus level in the top 5 (C). *(p < 0.05); **(p < 0.01).

FIGURE 6

Different analyses of intestinal flora in mice. (A) Bar chart of LDA discrimination result, in which LDA value is > 2.0, and the length of bar chart represents LDA value. (B) LEfSe hierarchical tree diagram of multi-level species, showing the classification of intestinal flora and its main bacterial structure. In the diagram, the circles radiating from inside to outside are phylum, class, order, family, genus, etc. At different classification levels, each small circle represents a classification at that level. The diameter of the small circle is proportional to the relative abundance. Species with significant differences are colored according to groups, whereas those without significant differences are yellow.

FIGURE 7

Prediction and analysis of genes function relative abundance based on PICRUSt2 analysis. (A) KEGG pathway level 1; (B) KEGG pathway level 2.