Antibiotic Cocktail: An Excellent Tool to Probe Physiology by Perturbing Gut Microbiota: A Mice Model

Abstract

Antibiotics have become an excellent tool for understanding the role of gut microbes. While the effect of antibiotic treatment on adults is studied, its impact during adolescence is unclear. In the current study, we treated C57BL/6 mice with an antibiotic cocktail composed of nine antibiotics from weaning (3 weeks old) until they became young adults (10 weeks old). We investigated the effect of antibiotic treatment (1) on gut microbiota composition, (2) on the brain by studying the behavior, Hypothalamic-Pituitary-Adrenal (HPA axis), neuroinflammation, neurotransmitters, neuronal health, and appetite regulators, and (3) on systemic circulation by recording changes in the stress hormones, insulin resistance, and metabolic profile. In the gut, we found that the antibiotic treatment significantly increased the Proteobacteria and Actinobacteria while decreasing the Bacteroidetes phylum. In the brain, we observed HPA axis activation, elevated proinflammatory response, decline in neurotrophins, neurotransmitter abundance, and appetite regulators expression, which could be linked with behavioral changes. We found increased insulin resistance and altered metabolite profiles in the peripheral system. Moreover, our association study highlights the role of Proteobacteria and Actinobacteria in altering the host behavior, brain function, and systemic circulation. Altogether this study demonstrates, how prolonged antibiotic exposure during adolescence disrupts gut microbiota and is associated with physiological and behavioral alterations through gut peripheral system and brain interactions.

Fig. 1

Alteration in the gut microbiota profile followed by the antibiotic treatment: Panels a–d show the Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria, respectively. Panels e–g show the significant genera under the Proteobacteria phylum. Panels h, i represent the relative abundance of Corynebacterium and Nocardioides under the Actinobacteria phylum. Panel j shows the effect of antibiotic treatment on the genus of Bacteroidetes phylum. The statistics shown here were calculated based on the two-way ANOVA. The significance level is indicated as *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001

Fig. 2

Effect of antibiotic treatment on behavior: the time spent at the center and the distance traveled by mice in the open field apparatus (a) and (b), respectively. The time spent at the open arm and distance traveled in the elevated maze apparatus are shown in c and d. e and f Shows the results of the forced swim test while g shows the marble burying test, respectively. We used two-way ANOVA followed by the Bonferroni test. * represents the P value ≤ 0.05, while **, ***, and **** represent the P value ≤ 0.01, 0.001, and 0.0001, respectively. The data is represented here as mean ± SEM. For the figures a, b, and d, we used n = 10, while for c, we used five mice. For the gene expression study, we used n = 3

Fig. 3

Antibiotic treatment affects the HPA axis, insulin resistance, and inflammation: a shows the fold change in the CRH level at the hypothalamus. Adrenocorticotropic hormone (ACTH) and corticosterone levels in the serum sample are shown in b and c, respectively. The fasting blood glucose (d) and serum insulin levels (e). f Represents the insulin resistance. g–i Represent the fold change value of cytokines in the antibiotic-treated group compared to their respective control group in the PFC, hippocampus, and hypothalamus. j–l The table shows the statistical significance in PFC, hippocampus, and hypothalamus. We presented the data as mean ± SEM. Here we have used three mice. We performed the two-way ANOVA followed by the Bonferroni test to determine the statistical significance. P ≤ 0.05 is indicated as *. P ≤ 0.01, P ≤ 0.001, and P ≤ 0.0001 are represented as **, ***, ****

Fig. 4

The neuronal health and the abundance of neurotransmitters in the brain are followed by the antibiotic treatment: the LC–MS measured the concentration of glutamate in the prefrontal cortex (a). b and c Show the concentration of GABA and norepinephrine in the prefrontal cortex. The fold change in the gene expression of cFOS and BDNF at the prefrontal cortex, hippocampus, and hypothalamus is shown in d, e, and f, respectively. Table (g–i) shows the statistical significance between the control and treatment groups for the PFC, hippocampus, and hypothalamus gene expression. We used two-way ANOVA and the post hoc Bonferroni test to determine the statistical significance. We used n = 3. P ≤ 0.05, P ≤ 0.01, P ≤ 0.001, and P ≤ 0.0001 represented by *, **, ***, ****, respectively

Fig. 5

The effect of antibiotic treatment on the feeding activity: a indicates the food intake by the mice, where n = 5. b and c Represent the gene expression NPY and AgRP, whereas d and e describe POMC and CART expression. For b–e, we used three mice. We used two two-way ANOVA and post hoc Bonferroni tests to determine the statistical significance. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001