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

Nadia Amorim; Emily McGovern; Anita Raposo; Saroj Khatiwada; Sj Shen; Sabrina Koentgen; Georgina Hold; Jason Behary; Emad El-Omar; Amany Zekry

doi:10.3389/fmed.2022.770017

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

Front Med (Lausanne). 2022 Feb 9:9:770017. doi: 10.3389/fmed.2022.770017. eCollection 2022.

Authors

Nadia Amorim¹, Emily McGovern¹, Anita Raposo¹, Saroj Khatiwada¹, Sj Shen¹, Sabrina Koentgen¹, Georgina Hold¹, Jason Behary^{1

2}, Emad El-Omar^{1

2}, Amany Zekry^{1

2}

Affiliations

¹ Microbiome Research Centre, St. George and Sutherland Clinical School, UNSW Sydney, Sydney, NSW, Australia.
² Department of Gastroenterology and Hepatology, St. George Hospital, Sydney, NSW, Australia.

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.