Comparison of co-housing and littermate methods for microbiota standardization in mouse models

The intestinal microbiota is emerging as a fundamental factor that broadly influences physiology. Thus, studies using transgenic animals should be designed to limit the confounding effects of microbiota variation between strains. Here, we report the impact on intestinal microbiota of co-housing versus F2-generation littermates, two commonly used techniques to standardize microbiota in animal models. Our results establish that while fecal microbiota was partially normalized by extended co-housing, mucosal communities associated with the proximal colon and terminal ileum remained stable and distinct. In contrast, strain intercrossing to generate F2 littermates allowed robust microbiota standardization in fecal, colon and ileum sampling locations. Using reciprocal intercrosses of P1 parents, we further identified dissymmetry in F2 community structures caused by maternal transmission, in particular of the Verrucomicrobiaceae. Thus, F2 littermate animals from a unidirectional P1 cross should be used as a standard method to minimize the influence of the microbiota in genotype/ phenotype studies.