Animals and CDI Model

To induce the CDI model, 6–8-week-old male C57BL/6 mice were obtained from the Animal Experiment Center of Guangdong Province (China) and reared under specific pathogen-free conditions in the Southern Medical University animal facility. All animals had appropriate space, with free access to tap water and a standard rodent diet. The CDI mouse model was induced as previously described (Hutton et al., 2014; Yan et al., 2014) with some modifications. The detailed procedure is shown in Figure ​Figure1A.1A. Water containing antibiotics, comprising of kanamycin (0.8 mg/mL; Sigma), gentamicin (0.07 mg/mL; Sigma), colistin (0.1135 mg/mL; Sigma), MTZ (0.43 mg/mL; Sigma) and vancomycin (0.09 mg/mL; Sigma), was provided for 7 consecutive days starting on Day -9, and then replaced with autoclaved water from Day -2. A dose of clindamycin (10 mg/kg; Sigma) was given intraperitoneally at Day -1. The prepared toxigenic spores were administered to the model mice at a dose of 3 × 10⁸ colony forming units (cfu) by oral gavage from Day 0 for 3 consecutive days. The animals were observed every 4 h, mainly for the presence of diarrhea and death after the spore challenge. Body weight was measured daily from Day -9 to Day 2 (data after Day 3 not provided due to death of the mice). Cecal contents and cecum and colon tissue samples were collected, and C. difficile toxin was detected in supernatants of cecal contents, soon after the death of mice. The surviving mice were euthanized by CO₂ inhalation at Day 7. Tissue samples including cecum and colon tissues were collected for histopathologic and immunohistochemical analysis and cecal contents were collected for 16S rRNA analysis. Mean optical density of the immunohistochemical images were qualified using Image J 1.51 software.

Bacteroides fragilis ZY-312 prevents CDI-associated death. (A) Experimental design for B. fragilis ZY-312 prophylactic treatment of the CDI model mice. The first step of inducing the CDI mouse model was to use a large dosage of antibiotics to disrupt the normal gut microbiota. From D –9 to D –2, drinking water with antibiotics including kanamycin (0.8 mg/mL), gentamicin (0.07 mg/mL), colistin (0.1135 mg/mL), metronidazole (0.43 mg/mL), and vancomycin (0.09 mg/mL) was offered to male C57BL/6 mice (6–8 weeks old, n = 6–8). Clindamycin (10 mg/kg) was intraperitoneally injected the day (D –1) after the cessation of drinking water with antibiotics. Then the mice were orally challenge with 3 × 10⁸ cfu of Clostridium difficile VPI 10463 spores from D0 to D2. To explore the prophylactic effects of B. fragilis on preventing C. difficile-associated diseases, 1 × 10⁷ or 1 × 10⁸ cfu/day B. fragilis were given, respectively, to CDI model mice from D –9 to D2. Metronidazole (1 mg/day) was given orally from D0 to D5, as the positive control. (B) Survival rates of mice in the Blank, MTZ (Metronidazole treated), CDI model, and BFT (B. fragilis treatment) groups. (C) Visual observations of colon tissues from CDI model mice (top) and BFT (10⁸ cfu) mice (means ± SEM; n = 6–8 per group).