Initially, after the administration of lincomycin hydrochloride, all mice except the control group showed mental depression, a red anus, and diarrhea. Compared with the control group, the diarrhea status scores and stool water content of the model group was significant, which suggested that the AAD mouse model was successfully established. As shown in Figure 1B, a significant decrease in diarrhea status scores was observed in the Bu, Bi + Ba, and Bu + Ba groups. Among them, the Bu + Ba group showed a better result than the Bu group. Furthermore, the Bi + Ba and Bu + Ba groups exhibited significantly reduced stool water content. Although the Bi or Bu groups also showed a significant decrease, the effect was not as great as the mixed bacteria group (Figure 1C).

As shown in Figure 2, the colons of mice in the control group exhibited normal histological features. The mucosal epithelium remained intact, and the intestinal glands were abundant and closely arranged. Compared with the control group, the colonic mucosa of AAD mice showed a small inflammatory cell infiltration and mild edema in the submucosa. There was a slight reduction in the number of cupped glandular cells. These pathological characteristics were also observed in most treatment groups. However, no significant histopathological lesions were observed in mice treated with Bu or Bu + Ba, thereby indicating that it may reduce intestinal inflammation.

To compare the immunomodulatory effects of different strains on AAD mice, cytokines were detected in colonic tissues by ELISA (Figure 3). The levels of IL-6 in the model group were significantly higher than those in the control group. Among the tested strains, the Bu + Ba group showed reduced levels of IL-6. Although the levels of IL-1β, IL-17, and TNF-α in the colonic tissues of mice were not significantly different from those in the control group, they showed a modest increase, whereas the Bu + Ba treatment decreased these proinflammatory inflammatory factors.

To analyze the regulation of different treatments on the intestinal barrier, serum LPS levels were determined. As shown in Figure 4, the level of LPS was significantly higher in the model group, whereas the Bi + Ba and Bi + Bb groups showed decreased LPS levels in the serum. Moreover, the Bi + Ba group showed a significant difference compared with the model group. Compared with the control group, the mRNA expression of occludin and Mucin-2 significantly decreased in the model group. However, the Bu + Ba treatment enhanced the expression of occludin in the colon to the level of the control group. In addition, Bi + Ba and Bu + Ba also increased the Mucin-2 expression. In the model group, diarrhea induced a significant decrease in the expression of NHE3 in the colonic tissues of the mice, whereas a supplementation with Bi, Bi + Ba, and Bu + Ba significantly increased this expression up to the level of the control group.

After treatment with lincomycin hydrochloride, the concentrations of acetic acid, propionic acid, isobutyric acid, and isovaleric acid in the caecal specimens of the model group mice were significantly reduced (Figure 5). Compared with the model group, the Bi and Bu groups had significantly increased acetic acid, propionic acid, and isobutyric acid concentrations. The Bi + Ba treatment significantly increased propionic acid, isobutyric acid, and isovaleric acid, but did not significantly increase the level of acetic acid. Significant increases in acetic acid, propionic acid, isobutyric acid, and isovaleric acid concentrations were also observed in the Bu + Ba group. The Bi + Bb group showed significantly increased levels of propionic acid, isobutyric acid, and isovaleric acid in the caecal specimens. These results also indicated that the gavage of Bacteroides and the mixture of probiotics most strongly benefitted the recovery of SCFA concentrations in AAD mice.

The evenness and Shannon indexes presented the evenness and diversity of the gut microbiota, respectively. The results showed that the evenness and diversity were significantly reduced following the lincomycin hydrochloride treatment (Figure 6A,B). Compared with the model group, the Bi + Ba, Bu + Ba, and Bi + Bb groups showed increased evenness index, and there was a significant difference in the increase in the Bu + Ba group. The highest Shannon index values were observed in the Bi + Ba and Bu + Ba groups. The Bi and Bi + Bb groups also exhibited a significant increase. The purpose of the β-diversity analysis was to determine the similarities among the groups, and the principal component analysis (PCoA) of community β-diversity and clustering of distance based on the Jaccard index analysis indicated that the clusters of the microbial compositions of the Bi, Bu, Bi + Ba, Bu + Ba, and Bi + Bb groups were closer to the control group than the model group; this indicated that all these treatment groups potently restored the intestinal microbiota disorder (Figure 6C,D). In conclusion, the mixture of Bacteroides and Bifidobacterium most positively impacted the recovery of flora diversity in AAD mice, with the Bu + Ba group facilitating the best recovery rate.

In this study, Spearman’s correlation coefficient was used to express the correlation between the two indicators. As shown in Figure 6E, the water content in the stool was negatively correlated with the Shannon index and the concentrations of isobutyric acid, and positively correlated with the levels of IL-6. In addition, the concentration of acetic acid was positively correlated with occludin. The evenness index was negatively correlated with the diarrhea status scores.