Faecalibacterium prausnitzii and a Prebiotic Protect Intestinal Health in a Mouse Model of Antibiotic and Clostridium difficile Exposure

Abstract

Background: Clostridium difficile (CD) infection (CDI) increases patient morbidity, mortality and healthcare costs. Antibiotic treatment induces gut dysbiosis and is both a major risk factor for CD colonization and treatment of CDI. Probiotics have been trialed to support commensal gut microbiota and reduce CDI. This study investigated commensal microbe Faecalibacterium prausnitzii (FP) and a prebiotic, both known to yield butyrate and be anti-inflammatory and immunomodulatory, on CD colonization and gut integrity in mice.

Methods: Mice were randomly grouped and supplemented daily with FP, prebiotic, FP + prebiotic, FP/prebiotic supernatant, or saline throughout the entire study. Following treatment with clindamycin for 3 days, mice were exposed to CD. Feces were collected at baseline, the day after antibiotic, and 1, 3, and 5 days after CD exposure and cultured for bacterial overgrowth and CD colonization. On days 1 and 5 after CD exposure, mice were randomly euthanized, and proximal colon was dissected for histological analysis and preparation of RNA for analysis of proinflammatory and anti-inflammatory cytokines.

Results: Although all mice exhibited bacterial overgrowth and CD colonization, bacterial burden resolved quicker in the FP + prebiotic group. This was associated with induction and resolution of innate immune responses, anion exchanger, and tight junction protein preservation in proximal colon. CD toxin virulence potential was questionable as expression of CD toxin B receptor was depleted in the FP + prebiotic group.

Conclusion: Supplementation with anti-inflammatory butyrate-supporting commensal bacteria and prebiotic may support innate immune responses and minimize bacterial burden and negative effects during antibiotic and CD exposure.

Keywords: antibiotics; butyrate, Clostridium difficile, innate immunity, intestine, microbiome, prebiotic, probiotics.

Figure 1

Effect of antibiotic treatment on bacterial overgrowth. Mice were randomized into groups and supplemented daily with Faecalibacterium prausnitzii (FP), potato starch (PS), FP+PS, supernatant, or saline. Mice were treated with daily subcutaneous injections of clindamycin (1.4 mg/d) for 3 days. Following transfer to clean cages, fresh feces were collected, and concentration of enterococcus and gram-negative bacteria was measured by plating serially diluted samples on selective agar at (A) baseline before antibiotics and (B) 1 day after last dose of antibiotic. Data are presented as mean log10 CFU/g ± SEM and percentage of animals in each group with range of log₁₀ CFU/g set as none, 1–3, 4–5, 6–7, 8–9, and >9 log₁₀ CFU/g. n = 12–13 animals/group.

Figure 2

Bacterial colonization after Clostridium difficile (CD) exposure. Mice were randomized into groups and treated with clindamycin as described in Figure 1. Three days after the last dose of clindamycin, mice were orally exposed to VA17 (4 log₁₀ CFU/mL). Mice received the randomized supplements daily. Concentration of enterococcus, gram-negative bacteria, and CD was measured by plating serially diluted fresh stool samples on selective agar on (A) day 1, (B) day 3, and (C) day 5 after CD challenge. Data are presented as mean log₁₀ CFU/g ± SEM, and percentage of animals in each group with range of log₁₀ CFU/g set as none, 1–3, 4–5, 6–7, 8–9, and >9 log₁₀ CFU/g. n = 10 animals/group. FP, Faecalibacterium prausnitzii; PS, potato starch.

Figure 3

Effect of Faecalibacterium prausnitzii (FP) and potato starch (PS) on bacterial colonization recovery. Mice received supplementation with FP+PS or saline and received clindamycin and Clostridium difficile (CD) as described in Figures 1 and 2. Fresh stool was cultured on selective agar for concentration of enterococcus, gram-negative bacteria, and CD measured by plating serially diluted samples on (A) day 1, (B) day 3, and (C) day 5 after CD challenge. Data are presented as mean log₁₀ CFU/g ± SEM. *P < .05.

Figure 4

Effects of Clostridium difficile (CD) on tight junction protein and an anion exchanger expression in proximal colon. Mice were treated as described in Figures 1 and 2. Proximal colon was collected and embedded in optimal cutting temperature medium (OCT) for histology on days 1 and 5 after CD exposure. (A) Occludin (red), zona occludin-1 (ZO-1; green), (B) claudin-3 (green), and (C) Na⁺/H⁺ exchanger isoform 3 (NHE₃; green) were visualized by immunohistochemistry in sections of proximal colon frozen in OCT. A selected area was cropped and enlarged. All images were acquired using a 40 × objective. Images are representative of at least replicate images captured per mouse in 3–6 mice per treatment group. FP, Faecalibacterium prausnitzii; PS, potato starch.

Figure 5

Comparison of Faecalibacterium prausnitzii (FP) plus potato starch (PS) with saline on butyrate transporter and pathogen receptors. Mice were treated as described in Figures 1 and 2. Proximal colon was collected and used to prepare RNA or embedded in optimal cutting temperature medium (OCT) for histology on days 1 and 5 after Clostridium difficile (CD) exposure. (A) Expression of TLR2 messenger RNA was detected in proximal colon of mice using quantitative real-time reverse transcription polymerase chain reaction. Data are the mean fold-change ± SEM. *P < .05. (B) SLC5A8 (green) and (C) FZD7 (green) were visualized by immunohistochemistry in sections of proximal colon frozen in OCT. All images were acquired using a 40 × objective. Images are representative of at least replicate images captured per mouse in 6 mice per treatment group at time point 5 days after CD exposure.

Figure 6

Effect of Faecalibacterium prausnitzii (FP) plus potato starch (PS) on chemokine and cytokines following Clostridium difficile (CD) exposure. Mice were treated as described in Figures 1 and 2. Proximal colon was collected and used to prepare RNA. (A–F) Expression of interleukin (IL)-1β, monochemoattractant protein-1 (MCP1), IL-8, neutrophil elastase (ELANE), IL-10, and inducible nitric oxide synthase (iNOS) messenger RNA were detected in proximal colon of mice using quantitative real-time reverse transcription polymerase chain reaction. Data are the mean fold-change ± SEM. *P < .05.