The idea of transplanting human gut communities into germ-free animals is longstanding (17
). However, we have now used newly available metagenomic methods to demonstrate that (i) a human distal gut microbiota can be transferred to germ-free mice with remarkable preservation of structure and diversity, even if the starting material is frozen feces, (ii) the community can subsequently be reliably transmitted from mothers to their offspring, (iii) the microbiota in these humanized mice has characteristic and reproducible variations in its composition along the length of the gut, and (iv) the configuration of the microbiota, its microbiome and meta-transcriptome change in a rapid, dramatic and reproducible fashion after switching from a plant polysaccharide-rich/low fat diet to a high-fat/high-sugar Western diet. We also show that a host phenotype, Western diet-induced increased adiposity, can be transmitted for a period of time to recipient gnotobiotic mice via transplantation of their gut microbiota. Together, these findings establish the justification for a translational medicine pipeline for conducting proof-of-principle and proof-of-mechanism clinical metagenomic studies where the impact of representative human microbiomes on host biology can be initially characterized in mice consuming diets resembling those of the humans being studied, and where the effects of various environmental exposures (e.g., foods, xenobiotics, etc.), and host genotypes on the dynamic operations of their gut microbial communities can be modeled under controlled conditions.
The phylogenetic tree of bacterial life in the gut consists primarily of shallow twigs that represent species- or genus-level diversity: these twigs stem back to a few deep splits near the base of the tree that represent the dominant phyla present in this body habitat (Firmicutes, Bacteroidetes, and Actinobacteria). This phylogenetic structure contrasts with other ecosystems such as the soil, where there are many deep branches of the tree represented (23
), and suggests that the gut selects for diversification in the few deep evolutionary lineages that flourish there. Microbiota transplants into germ-free mice living in gnotobiotic isolators where inadvertent contact with microorganisms living in the world outside of the isolator is avoided allow us to consider the selective forces that operate to assemble a gut community. We had previously attempted to transplant a foreign gut microbiota into adult germ-free mouse recipients, where the donors were conventionally raised zebrafish. Members of the donor gut community that most closely resembled the phylogenetic lineages normally present in the mouse gut microbiota survived in the mouse intestine, as judged by 16S rRNA surveys of the input (donor) community and selected recipient communities (24
). The factors that operated to select these phylotypes were not defined. The results from reciprocal microbiota transplants performed in the current study, in which various combinations of donor and host diets were tested, demonstrate that although the initial environmental exposure (legacy effect) affects early community composition, diet can supersede legacy effects and shape the composition of the microbiota and microbiome as well as its expressed gene repertoire. The ability of a fecal human community to establish and sustain itself in the intestines of adult germ-free mice with a unanticipated level of preservation of microbial diversity likely reflects the confluence of a number of factors, ranging from the initial openness of the germ-free mouse’s gut ecosystem, diet (including endogenous nutrient substrates available in the gut habitat such as mucus glycans which are readily foraged by members of the Bacteroidetes and shared with other components of the community) (25
), the relative immaturity of the innate and adaptive immune systems at the time of initial colonization, the fact that mice are coprophagic which allows repeated microbial inoculation, and the implicit notion that there must be a large number of shared features of the biochemical milieus of the mouse and human gut that have yet to be defined.
Our metagenomic analyses of humanized gnotobiotic mice also disclosed that in adult mice, community composition is dramatically altered over a time scale of hours when animals are switched from mouse chow to a Western diet. This bloom in members of the Erysipelotrichi and Bacilli classes of the Firmicutes occurred along the length of the gut. These findings have several implications. First, they indicate that it is possible to identify diets having large effects on the gut microbiota and microbiome over short time intervals using humanized mice, and that subsequent testing of the effects of these diets on humans could involve study designs where diet exposures do not have to be prolonged. Second, the ability to apply a diet selection to identify organisms that bloom in the microbiota of humanized mice, and then to recover these organisms and characterize their attributes in silico, in vitro and in vivo (in gnotobiotic mice) represents an attractive pipeline for the discovery of new classes of probiotics that affect nutrient harvest in a given diet context.
A previous study demonstrated that the ability to produce equol from a soy-isoflavone containing diet could be transmitted to germ-free rats upon colonization with a fecal sample from a high equol-producing human subject, but not with a sample from a low equol-producing individual (22
). Our experiments show the feasibility of applying metagenomic methods, including microbial community mRNA profiling (meta-transcriptomics) to microbiota transplantation experiments in order to begin to understand the microbial organismal and microbial genetic basis of how a human metabolic phenotype (metabotype) can be transferred via the gut microbiota. A key finding from our study is that similar microbial communities can be formed in gnotobiotic recipients using fresh and frozen aliquots of a human donor’s fecal sample. The fact that diversity can be captured after collected samples are quickly frozen and then stored at −80°C for almost a year not only reflects the capacity of strict anaerobes to survive freeze-thaw cycles but also speaks to the ability of the gut to select and support the rapid expansion of adapted organisms that may be present in only small numbers in a sample subjected to prolonged storage at this temperature. The ability to use frozen fecal samples has broad implications for human microbiome projects focusing on the gut ecosystem, since it means that humans populations with various physiologic or pathophysiologic states can be sampled, their biospecimens archived, and the functional attributes of their microbial communities compared and contrasted both in silico
based on metagenomic datasets, and experimentally in humanized gnotobiotic mice. In cases where the focus is on the interrelationships between diet, gut microbial ecology and nutrient/energy harvest in obese or underfed populations living in various cultural contexts, recreating both the microbiotas and diets of the studied populations in humanized gnotobiotic mice should yield more relevant and personalized animal models. Microbial-based biomarkers can be discovered and validated, and therapeutic tests performed in these animals before they are translated to human studies, or in more sublime way, these mice can become part of a clinical study. Finally, the successful transfer of a human gut microbiota across generations of mice without a significant drop in diversity creates a model for addressing a very intriguing question in genetics: does intergenerational transfer of a microbiome, like methylation, RNAi, and other short-term heritable influences, explain effects ‘induced by the environment’ that last a small number of generations?