Protocol for fecal microbiota transplantation: A microaerophilic approach for mice housed in a specific pathogen-free facility

Abstract

Recently, studies have emerged exploring the potential application of fecal microbiota transplantation (FMT) in pre-clinical settings. Here, we present a protocol for FMT for mice housed in a specific pathogen-free (SPF) facility. We describe steps for sample collection, microaerophilic processing of freshly collected fecal pellets, and administration through oral gavage. We then detail procedures for the engraftment of the bacterial community. This protocol focuses on age- and gender-matched, healthy donor mice using a mobile and cost-effective alternative to an anoxic cabinet.

Keywords: health sciences; microbiology; model organisms; sequence analysis; systems biology.

Graphical abstract

Figure 1

Preparation of sterile anoxic saline Deoxygenated saline is transferred to clean glass septum bottles inside the anoxic glove box isolator. Clean septa and aluminum crimp closure caps are used to seal the bottles. The filled bottles are autoclaved and ready to be used. A witness solution, saline solution containing resazurin, is used as indicator to confirm anoxia.

Figure 2

Custom-made stainless-steel trolley enabling microaerophilic processing of FMT donor material—Top view The trolley is used to secure the argon gas bottles, enables the use of a microaerophilic glove bag for processing FMT donor material inside an animal facility.

Figure 3

Collection of fresh feces (Left) Prepare the changing station of the mice to collect fresh fecal pellets. Additionally, a scale to weigh the mice and a device to provide the mice with ear tags was present in the changing station on the day the picture was taken. These are not required for fecal sampling. (Right) Gently immobilize the mouse to collect the fecal pellet directly from the rectum.

Figure 4

FMT product preparation (Left) Vortexed and centrifuged FMT product sample displaying a visually clear separation between the turbid supernatant and clumpy pellet. (Right) The turbid FMT supernatant and transparent saline (placebo) are administered using gavage needles.

Figure 5

Fecal processing has a minimal impact on the bacterial ecology of the fecal donor samples Donor fecal samples and FMT product was freshly collected and processed at day 1 of FMT administration. (A) MDS plot of Bray-Curtis diversity – each dot represents an individual fecal donor sample or the processed FMT product (B) Observed bacterial richness measured as absolute OTU counts. Boxplot representation: the middle line is the median, the box represents the interquartile range (IQR), the whiskers display 1.5 × IQR, and the points indicate outliers. (C) Taxonomic stacked bar plot representing the quantitative genus abundances of the identified OTUs. One-Sample Wilcoxon Signed Rank Test (n = 6 in donor group). FMT, fecal microbiota transplantation; MDS, multidimensional scaling; OTU, operational taxonomic unit.