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Refining a Protocol for Faecal Microbiota Engraftment in Animal Models After Successful Antibiotic-Induced Gut
Nadia Amorim1, Emily McGovern1, Anita Raposo1
1Microbiome Research Centre, St. George and Sutherland Clinical School, UNSW Sydney, Sydney, NSW, Australia.
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.
Insights
A 7-day antibiotic treatment effectively decontaminates the gut for faecal microbiota transplant (FMT) research. Three weekly FMT doses ensure successful donor microbiota engraftment and stability in mice.
Area of Science:
- Microbiology
- Immunology
- Gastroenterology
Background:
- Faecal microbiota transplant (FMT) shows therapeutic potential for chronic inflammatory diseases.
- Standardized methods for gut microbiota depletion and FMT are needed for animal research.
- Germ-free mice are not always accessible, necessitating alternative approaches.
Purpose of the Study:
- To refine gut decontamination protocols before FMT in animal models.
- To assess the efficiency and stability of FMT engraftment over time.
Main Methods:
- Mice received a 21-day antibiotic cocktail (ampicillin, vancomycin, neomycin, metronidazole).
- Following decontamination, mice received weekly FMT or saline (Sham) for 3 weeks.
- Bacterial presence, load, and microbiota composition were analyzed via culture, qPCR, FISH, and 16S rRNA sequencing.
Main Results:
- 7 days of antibiotics proved most effective for gut decontamination.
- Three weekly FMT doses led to successful donor microbiota engraftment.
- Engrafted microbiota remained stable and similar to the donor for 5 weeks post-treatment.
Conclusions:
- A 7-day broad-spectrum antibiotic regimen followed by 3 weekly FMT doses is a reliable method.
- This protocol offers a simple, cost-effective approach for FMT in animal studies.
