Heterogeneity of the gut microbiome in mice: guidelines for optimizing experimental design

Abstract

Targeted manipulation of the gut flora is increasingly being recognized as a means to improve human health. Yet, the temporal dynamics and intra- and interindividual heterogeneity of the microbiome represent experimental limitations, especially in human cross-sectional studies. Therefore, rodent models represent an invaluable tool to study the host-microbiota interface. Progress in technical and computational tools to investigate the composition and function of the microbiome has opened a new era of research and we gradually begin to understand the parameters that influence variation of host-associated microbial communities. To isolate true effects from confounding factors, it is essential to include such parameters in model intervention studies. Also, explicit journal instructions to include essential information on animal experiments are mandatory. The purpose of this review is to summarize the factors that influence microbiota composition in mice and to provide guidelines to improve the reproducibility of animal experiments.

Keywords: animal facility; animal models; confounding factors; microbiome; microbiota.

Figure 1.

Main functions of bacteria in the gut. Bacteria benefit the host in many ways. Besides breaking down food compounds and synthesizing vitamins and other nutrients, they play an important role in the development and training of the immune system (Hill and Artis ; Renz, Brandtzaeg and Hornef ; Sonnenberg and Artis 2012). They provide colonization resistance (Kamada et al.; Lawley and Walker 2013), protect against epithelial injury (Rakoff-Nahoum et al.2004) and promote angiogenesis (Stappenbeck, Hooper and Gordon ; Reinhardt et al.2012) and fat storage (Bäckhed et al.2004). They are also able to modulate bone-mass density (Sjögren et al.2012), modify the nervous system (Hsiao et al.2013) and metabolize therapeutics into active compounds (Claus et al.2011).

Figure 2.

Experimental variables that influence microbiome analysis. During a typical experimental workflow (donor selection, sampling, DNA extraction, sequencing and analysis of the data), variation is systematically introduced and complicates inter-experimental comparisons.

Figure 3.

The development of the murine microbiota and the immune system over time. In utero, few, if any, bacteria are present in the mouse gut and the immune system is not yet matured. Upon birth, the neonate is inoculated with microorganisms by the mother and the environment, and rapidly develops an immune system that enables the pup to fight infections. Genetic background co-determines the composition of the microbiota, for example when the genotype increases intestinal inflammation. After weaning, diet changes induce a novel surge in microbiota development and maturation of the immune response. At this time point, the microbiota is fully established but still susceptible to changes in its composition by manipulation (e.g. diet) or natural influences. When the mouse reaches adulthood around eight weeks, the microbiota displays a stable homeostatic state. At each of these four stages, the microbiota can be studied in conventional animals using the different experimental approaches that are listed. ILCs: innate lymphoid cells; Treg: regulatory T cells.