Refining a Protocol for Faecal Microbiota Engraftment in Animal Models After Successful Antibiotic-Induced Gut Decontamination

Abstract

Background: There is mounting evidence for the therapeutic use of faecal microbiota transplant (FMT) in numerous chronic inflammatory diseases. Germ free mice are not always accessible for FMT research and hence alternative approaches using antibiotic depletion prior to FMT in animal studies are often used. Hence, there is a need for standardising gut microbiota depletion and FMT methodologies in animal studies. The aim of this study was to refine gut decontamination protocols prior to FMT engraftment and determine efficiency and stability of FMT engraftment over time.

Methods: Male C57BL/6J mice received an antibiotic cocktail consisting of ampicillin, vancomycin, neomycin, and metronidazole in drinking water for 21 days ad libitum. After antibiotic treatment, animals received either FMT or saline by weekly oral gavage for 3 weeks (FMT group or Sham group, respectively), and followed up for a further 5 weeks. At multiple timepoints throughout the model, stool samples were collected and subjected to bacterial culture, qPCR of bacterial DNA, and fluorescent in-situ hybridisation (FISH) to determine bacterial presence and load. Additionally, 16S rRNA sequencing of stool was used to confirm gut decontamination and subsequent FMT engraftment.

Results: Antibiotic treatment for 7 days was most effective in gut decontamination, as evidenced by absence of bacteria observed in culture, and reduced bacterial concentration, as determined by FISH as well as qPCR. Continued antibiotic administration had no further efficacy on gut decontamination from days 7 to 21. Following gut decontamination, 3 weekly doses of FMT was sufficient for the successful engraftment of donor microbiota in animals. The recolonised animal gut microbiota was similar in composition to the donor sample, and significantly different from the Sham controls as assessed by 16S rRNA sequencing. Importantly, this similarity in composition to the donor sample persisted for 5 weeks following the final FMT dose.

Conclusions: Our results showed that 7 days of broad-spectrum antibiotics in drinking water followed by 3 weekly doses of FMT provides a simple, reliable, and cost-effective methodology for FMT in animal research.

Keywords: antibiotics; faecal microbiota transplant; gut decontamination; gut engraftment; microbiome.

Figure 1

Study design and mouse body weight. (A) Study design. From five weeks of age, mice were given antibiotics in drinking water for 21 days. FMT of human donor stool sample (FMT group) or Saline (Sham group) was then orally administered weekly for 3 weeks, followed by a 5 week follow up period. Stool was collected every 3–4 days from day 0 until day 21, and then collected weekly until the completion of the experiment. F0–F3, FMT (Faecal Microbiota Transplant) weeks 0–3; E1–E5, Engraftment weeks 1–5. (B) Mouse body weight over the course of 12 weeks, with different treatments. Values shown are average ± SD (n =10 mice/group).

Figure 2

Antibiotic treatment reduced bacterial content of mouse gut after three days. (A) Bacterial culture of stool samples on 10% horse blood agar plates; (B) DNA concentration (ng/mg of stool) of stool samples during the 21-day course of antibiotic treatment with ampicillin, vancomycin, neomycin and metronidazole (****p < 0.0001, One-way ANOVA); (C) Variation in Ct value of 16S rRNA gene amplification during antibiotic treatment (****p < 0.0001, One-way ANOVA). Positive and negative controls for 16S are represented in the graph; (D) Representative pictures of fluorescent in-situ hybridisation at day 0 (D0; left panel), day 3 (D3; middle panel) and day 7 (D7; right panel) of antibiotic treatment. Scale bar 2 μm.

Figure 3

Microbiome composition shift and engraftment after FMT. (A) PCoA plot showing beta-diversity of different treatment groups. (B) Bray Curtis dissimilarity index comparing distances to donor microbiota. (C,D) Alpha-diversity indices during and after FMT. (E) taxonomy data (Phylum level) showing successful engraftment of donor microbiota. F0–F3: FMT weeks 0–3; E1–E5, Engraftment weeks 1–5; OTUs, operational taxonomic units.