Bacteroides fragilis Protects Against Antibiotic-Associated Diarrhea in Rats by Modulating Intestinal Defenses

Abstract

Antibiotic-associated diarrhea (AAD) is iatrogenic diarrhea characterized by disruption of the gut microbiota. Probiotics are routinely used to treat AAD in clinical practice; however, the effectiveness and mechanisms by which probiotics alleviate symptoms remain poorly understood. We previously isolated a non-toxic Bacteroides fragilis strain ZY-312, which has been verified to be beneficial in certain infection disorders. However, the precise role of this commensal bacterium in AAD is unknown. In this study, we successfully established an AAD rat model by exposing rats to appropriate antibiotics. These rats developed diarrhea symptoms and showed alterations in their intestinal microbiota, including overgrowth of some pathogenic bacteria. In addition, gastrointestinal barrier defects, indicated by compromised aquaporin expression, aberrant tight junction proteins, and decreased abundance of mucus-filled goblet cells, were also detected in ADD rats compared with control animals. Of note, oral treatment with B. fragilis strain ZY-312 ameliorated AAD-related diarrhea symptoms by increasing the abundance of specific commensal microbiota. Interestingly, we demonstrated that these changes were coincident with the restoration of intestinal barrier function and enterocyte regeneration in AAD rats. In summary, we identified a potential probiotic therapeutic strategy for AAD and identified the vital roles of B. fragilis strain ZY-312 in modulating the colonic bacterial community and participating in microbiota-mediated epithelial cell proliferation and differentiation.

Keywords: Bacteroides fragilis; antibiotic-associated diarrhea; enterocyte regeneration; gut dysbiosis; intestinal barrier function.

Figure 1

An appropriate mixture of antibiotics induces diarrhea in SD rats. (A) A schematic of antibiotic-associated diarrhea model. Rats were gavaged daily with antibiotics mixture for 7 days. (B) Fecal consistency was assessed and classified by three visual grading scale (upper panels), and fecal consistency scores were shown in all different groups (lower panel). (C,D) Fecal water content and 120-min stool weight were measured in rats treated with normal saline or different doses of antibiotic cocktails (low, medium, and high), from day 4 to day 14, n = 8/group. Repeated measures ANOVA, *P < 0.05 and **P < 0.01 versus normal group.

Figure 2

Disruption of SD rats microbiota by a mixture of antibiotics. (A,B) Bacterial culture for microbial community composition analysis of normal control rats and antibiotic-associated diarrhea rats induced by the medium dose of antibiotic cocktail. Abbreviations: BD, Bacteroides (in BBE agar); BL, Bifidobacterium (in TPY medium); LC, Lactobacillus (in Lactobacillus selective agar); EB, Enterobacteriaceae (in eosin-methylene blue medium); EC, Enterococcus (in CATC agar); SB, Saccharomycete (in DRBC agar); SP, Staphylococcus aureus (in Mannitol salt agar); CD, Clostridium (in reinforced Clostridium agar); PS, Peptococcus (in CDC anaerobic agar). t-Test, *P < 0.05 and **P < 0.01 versus day 1.

Figure 3

Bacteroides fragilis ZY-312 improves antibiotic-associated diarrhea (AAD)-related gastrointestinal symptom. (A) Experimental design schematic of different treatments in AAD rats. Rats were pretreated with antibiotic mixtures for 7 days, followed by bacterial treatment. During the treatment period (day 8–14), rats were gavaged with normal saline, ZY-312 [10⁷, 10⁸, and 10⁹ colony-forming units (CFU)/day], Bifico (70 mg/day), or Bifico (70 mg/day) combined with ZY-312 (10⁸ CFU). (B–D) Fecal consistency scores, fecal water content, and 120-min stool weight of rats in each group from day 8 to day 17 were shown. n = 16/group. Repeated measures ANOVA, *P < 0.05 and **P < 0.01 versus AAD group.

Figure 4

Bacteroides fragilis ZY-312 restores specific microbiota changes in antibiotic-associated diarrhea (AAD) rats. (A) Microbial richness based on the Chao1 index, and unweighted UniFrac principal coordinate analyses based on all operational taxonomic units (OTUs) of normal rats (group A) and AAD rats (group B) on days 11 and 17, respectively. (B) Relative abundance of OTUs of the gut microbiota in all groups, including normal control (group A) and AAD groups gavaged with normal saline (group B), different doses of ZY-312 (10⁷, 10⁸, and 10⁹ CFU) (group C, D, and E), Bifico (70 mg/day) (group F), or Bifico (70 mg/day) combined with ZY-312 (10⁸ CFU) (group G) by gavage, at the rank of phylum (left panel) and genus (right panel). (C) Linear discriminant analysis effect size identifies differential abundance of bacteria between normal rats (group A) and AAD rats (group B) on days 11 and17, or between AAD rats (group B) and ZY-312 (10⁹ CFU) rats (group E) (LDA > 3.5), respectively. (D) Relative abundance of OTUs of the genus Akkermansia and Escherichia that are significantly altered by ZY-312 treatment (group E).

Figure 5

Bacteroides fragilis ZY-312 improves gut barrier integrity in antibiotic-associated diarrhea (AAD) rats. (A) Alcian blue staining for detecting goblet cells numbers were shown in normal rats, AAD rats treated with normal saline, ZY-312 [10⁹ colony-forming units (CFU)], Bifico (70 mg/day), or Bifico (70 mg/day) combined with ZY-312 (10⁸ CFU) on days 11 and 17. n = 8/group. (B) Immunohistochemistry of Muc2 protein located in colonic tissues was detected in all groups as mentioned above. (C) Immunofluorescence staining for ZO-1 and ZO-1 (TJP1) mRNA level in all the groups was shown as mentioned above. The magnifications of the above figures are 10× and 40×. (D) Colon protein level and mRNA expression of occludin in all groups on days 11 and 17 were shown. (E) qPCR analysis of AQP1, AQP3, and AQP8 gene in colonic tissues were detected in all groups as indicated in Figure 3, with mRNA fold changes normalized to those of normal control group. One-way ANOVA, *P < 0.05 and **P < 0.01 versus AAD group.

Figure 6

Bacteroides fragilis ZY-312 activates Erk signaling and promotes proliferation in antibiotic-associated diarrhea (AAD) rats. (A,B) Cell proliferation marker Ki67 and p-ERK1/2 were detected by immunohistochemistry in colon sections on days 11 and 17 in all groups as mentioned in Figure 5A. All images were taken at the same magnification at 40×. (C) Phosphorylation of extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 was also detected by western blot in colon tissues on day 11 in all groups as mentioned in Figure 5A.

Figure 7

Schematic of the therapeutic role of Bacteroides fragilis ZY-312 in AAD symptom. Antibiotic-associated diarrhea (AAD) rats exhibit dysbiosis of the gut microbiota, with overgrowth of some pathogenic bacteria and then resulting in defective gastrointestinal integrity and improper epithelial organization with low aquaporins, aberrant tight junction proteins, and a decreased number of mucus-filled goblet cells. Treatment of AAD rats with B. fragilis ZY-312 alters the composition of the gut microbiota, with promotion of commensal A. muciniphila growth, and subsequently remodeling of the colonic epithelium by improving enterocyte monolayer integrity, increasing mucus layer thickness, and water transport in rats.