Mouse Models for Fecal Microbiota Transplantation Studies

The role of the microbiome in health remains one of the most rapidly growing areas of research today, with newly discovered links to many diverse disease states. Many studies have demonstrated that it is possible to transfer phenotypes via microbiota transplantation in mice, reinforcing rodent models as an important research tool that may aid in determining causality.

Models available to researchers today include germ-free (axenic) and antibiotic-treated mice to serve as recipients of transplants. Although germ-free animals remain the ideal in serving as a “blank slate” to evaluate the properties of transplanted microbial communities, the use of antibiotic-treated mice can be used as an alternative model. Both models have their caveats, and it is key that researchers understand the benefits and limitations of each model before making a decision on which one to proceed with for their studies. This review compares and contrasts both germ-free and antibiotic-treated mouse models, and discusses how the choice of model may affect experimental results.

Advantages

Germ-free mice are defined as being devoid of any detectable microorganisms: bacteria, fungi, parasites, and viruses, with the exception of endogenous viral elements. The advantage of germ-free mice as recipients of fecal microbiota transplantation (FMT) can be condensed into two main points.

First, the lack of competition and interference by existing microorganisms ensures optimal conditions for the transplanted microbes to colonize the new host. Second, the unambiguous definition of germ-free mice represents an experimentally controlled and highly reproducible situation. Repeated experiments in germ-free mice of the same strain and under the same experimental and environmental conditions are likely to have consistent outcomes.

Studies of germ-free mice and comparisons to mice harboring microorganisms have taught us about the importance of microorganisms for normal physiology, immunology, and even anatomy of the host. Germ-free mice have altered metabolism and thus special nutritional requirements, altered intestinal motility and structure, and a less developed mucosal immune system in the gut.¹ The abnormalities of germ-free mice expand beyond the gastrointestinal tract, as altered behavioral profiles and altered brain physiology and structure also have been reported.² The realization that microorganisms, and bacteria in particular, are essential for normal development of the host constitutes the one major disadvantage of germ-free mice in biomedical research. Many, but not all, of these abnormal traits can be normalized by microbiota transplantation later in life. Nonetheless, the developmental influence of a germ-free early life is still considered a potential and significant confounding factor.

The way to circumvent the effect of an early-life germ-free period is to breed germ-free mice transplanted with the microorganism(s) of interest and use their offspring for experiments, with these subsequent generations having the microbiota transmitted in a natural way from birth. This approach is useful for studies aiming to evaluate the effects of microbial candidates for health modulation or for introducing dysbiotic fecal microbiota from mouse disease models or human patients.

For studies aiming to evaluate the impact of dysbiosis occurring later in life, antibiotic treatment of mice otherwise harboring a complex microbiota is likely to be a more relevant model than germ-free mice. Microbiota disruption by antibiotic treatment allows for studying the effect during different life stages of the host, and further enables the generation of hypotheses about which classes of bacteria are responsible for changes by administration of targeted antibiotics. However, when using this approach, it must be recognized that antibiotics may affect the host via pathways other than the microbiota. Usually, an effect of antibiotic treatment on disease pathogenesis is hypothesized to involve alteration of the microbiome. While this is very likely, direct effects of antibiotics on disease progression and cellular pathways have been described in germ-free mice^3,4 and should be considered when evaluating the results.

Pseudo-germ-free state

Limited access to different germ-free mouse strains, financial considerations, and practical limitations to the housing and handling of germ-free mice often prompt researchers to pursue a pseudo-germ-free state for FMT experiments by depleting the entire microbiota by antibiotics. There are, however, several and often neglected caveats to this approach.

• It is not possible to obtain a completely germ-free state. Even after long-term administration of broad-spectrum antibiotics, it is possible to detect residual bacteria in the gut that can affect the microbial profile after FMT with the new microbiota.^5–7 Some of these surviving species may be hiding in the mucus layer of the intestinal lining and are as such potentially in close interaction with the host and important immunoregulators. Others may migrate from the fur and skin and recolonize the gut. The unpredictable nature of which species are present, and their abundance, after antibiotic treatment is likely to reduce the reproducibility of the model.
• The success of FMT is dependent on characteristics of the microbiota before antibiotic depletion. This was shown in mice with high and low richness microbiota profiles that had their microbiota equally depleted by antibiotics. Though the antibiotic treatment had a similar effect in the two groups, mice with different richness profiles before antibiotic treatment displayed variable colonization success after FMT.⁸ Such observations add to the reproducibility concerns of the antibiosis approach.
• Antibiotics are usually administered to mice by an oral route. Systemic absorption from the gastrointestinal tract and bioavailability in different tissues varies significantly between different classes of antibiotics, and the level of microbial depletion outside the gut is therefore very much dependent on the drug pharmacokinetics. To our knowledge, the effect on the skin microbiota of the most commonly used antibiotics for depletion of the gut microbiota has not been investigated, though it may play a role in reintroduction of species to the gut.
• The most commonly used antibiotic cocktails for gut microbiota depletion are bacteriostatic or bactericidal of nature.⁹ Fungicides and anti-parasitic agents may be included in the mix, whereas bacteriophages and eukaryotic viruses are never targeted. This inconsistency may influence colonization after FMT and again reduce reproducibility.

One additional consideration is that some journal editors—for reasons listed here—are increasingly requiring FMT experiments performed in antibiotic-treated mice to be replicated in germ-free mice.

This review described advantages and limitations of both germ-free and antibiotic-treated mice that will hopefully help researchers in making informed decisions when planning microbiome studies in mice. Reproducibility of the chosen model—between different experiments in the same lab as well as between labs—is a virtue in the field of laboratory animal science that should not be overlooked.

References

1. Al-Asmakh, M.; Zadjali, F. Use of Germ-Free Animal Models in Microbiota-Related Research. J. Microbiol. Biotechnol 2015, 25 (2510), 1583–1588 DOI: 10.4014/jmb.1501.01039.

2. Luczynski, P.; McVey Neufeld, K.-A.; Oriach, C. S.; Clarke, G.; Dinan, T. G.; Cryan, J. F. Growing up in a Bubble: Using Germ-Free Animals to Assess the Influence of the Gut Microbiota on Brain and Behavior. Int. J. Neuropsychopharmacol. 2016, 19 (8), pyw020 DOI: 10.1093/ijnp/pyw020.

3. Han, D.; Walsh, M. C.; Kim, K. S.; Hong, S.-W.; Lee, J.; Yi, J.; Rivas, G.; Surh, C. D.; Choi, Y. Microbiota-independent ameliorative effects of antibiotics on spontaneous th2-associated pathology of the small intestine. PLoS One 2015, 10 (2), e0118795 DOI: 10.1371/journal.pone.0118795.

4. Morgun, A.; Dzutsev, A.; Dong, X.; Greer, R. L.; Sexton, D. J.; Ravel, J.; Schuster, M.; Hsiao, W.; Matzinger, P.; Shulzhenko, N. Uncovering effects of antibiotics on the host and microbiota using transkingdom gene networks. Gut 2015, 64 (11), 1732–1743 DOI: 10.1136/gutjnl-2014-308820.

5. Ge, X.; Ding, C.; Zhao, W.; Xu, L.; Tian, H.; Gong, J.; Zhu, M.; Li, J.; Li, N. Antibiotics-induced depletion of mice microbiota induces changes in host serotonin biosynthesis and intestinal motility. J. Transl. Med. 2017, 15 (1), 13 DOI: 10.1186/s12967-016-1105-4.

6. Ellekilde, M.; Selfjord, E.; Larsen, C. S.; Jakesevic, M.; Rune, I.; Tranberg, B.; Vogensen, F. K.; Nielsen, D. S.; Bahl, M. I.; Licht, T. R.; et al. Transfer of gut microbiota from lean and obese mice to antibiotic-treated mice. Sci. Rep. 2014, 4, 5922 DOI: 10.1038/srep05922.

7. Hansen, A. K. Antibiotic treatment of nude rats and its impact on the aerobic bacterial flora. Lab. Anim. 1995, 29 (1), 37–44.

8. Ericsson, A. C.; Personett, A. R.; Turner, G.; Dorfmeyer, R. A.; Franklin, C. L. Variable Colonization after Reciprocal Fecal Microbiota Transfer between Mice with Low and High Richness Microbiota. Front. Microbiol. 2017, 8, 196 DOI: 10.3389/FMICB.2017.00196.

9. Hansen, A. K.; Krych, Ł.; Nielsen, D. S.; Hansen, C. H. F. A Review of Applied Aspects of Dealing with Gut Microbiota Impact on Rodent Models. ILAR J. 2015, 56 (2), 250–264 DOI: 10.1093/ilar/ilv010.