Less than four years since the groundbreaking study linking gut microbiota to obesity (Ridaura VK et al, Science 2013), the race to develop microbiome therapies is in full sprint. Microbiome-focused biotech companies and pharmaceutical divisions are popping up in an effort to discover treatments for diseases ranging from autism, neurological disease, metabolic disorder, C. difficile, inflammatory bowel disease, asthma, cancer, diabetes, and more. Several companies already have leads in clinical trials, but most are in the preclinical discovery phase. One key decision for teams early in the drug discovery process is creating the right preclinical model. Germ-free mice, which are demonstrably free of microbes throughout their lifetime, are uniquely valuable to study the interaction between the host and its microbiota. Thus, drug discovery pipelines will rely on the best preclinical germ-free models and practices to advance microbiome therapies.
Interest in our gut microbes is more than a recent trend. Since the late 1950s, researchers have used gnotobiotic animals (those with only known strains of bacteria) to study the symbiotic relationship between an animal and their microbiome. It has been clear for over half a century that our microbiome is important (germ-free animals typically have weaker immune systems, poor muscle development, etc.) but only recently have researchers begun to understand how these complex interactions work.
Unfortunately, many bacteria in the gut present challenges to traditional culture. The gut is a very complex environment and many bacteria species require extremely low levels of oxygen, or specific nutrients provided by another microorganism. There are anaerobic chambers to limit oxygen and co-culture techniques when species are known to be interdependent. However, the complex microenvironment of the gut is best modeled in a whole animal, particularly when we don’t fully understand all the interactions.
To get to a known set of bacteria in an animal, one must start with a clean slate. Embryo transfer re-derivation—literally surgically transferring embryos to a surrogate mother—is a common technique used to purge a research colony of pathogens. (Similar techniques are used to create germ-free mice, who can then be inoculated with specific bacteria, or mixtures of bacteria for study. Gnotobiotic animals (those with only specific known bacteria) have been available for decades, and recently common strains have become commercially available that are germ-free. A major challenge is maintaining the biosecurity of the animal’s microenvironment to keep the animals from encountering any bacteria and prevent them from no longer being germ-free. Of course, what can be called germ-free is limited by the ability to detect microorganisms. Recent advances in detection and analysis technologies, such as next generation sequencing (NGS) have shed light on what organisms are present, and how those populations are altered in disease.
NGS has enabled research into our microbiota at an astounding rate. With sequencing, scientists can take snapshots of relative prevalence of different bacteria and see how that changes during a treatment or during disease. Before NGS added sensitivity and computational power, a thorough survey of the diversity of our gut bacteria was an insurmountable task. By focusing on a ribosomal subunit that is common to all bacteria, but modified slightly by each species, NGS can rapidly determine relative populations of bacteria in a sample. Profiling can be frequent, as non-invasive fecal samples are often used to examine the gut.
A good preclinical model is essential to predicting effectiveness of new treatments (pharmaceuticals, altered diets, etc.). Bringing together germ-free animals and profiling with NGS, means that preclinical models can now be established. Germ-free mice can be inoculated with microbiomes that are seen in different healthy and disease states. They are a better system to test new treatments by asking how altering diet changes the microbiome, or if different antibiotics (or formulations) are able to clear pathogens while preserving important probiotic species, etc. For example, in humans, C. difficile is often present at low levels in the gut without causing problems. However, when patients are on antibiotics, the normal profile of the gut can change dramatically. If C. difficile overgrows it can lead to severe colitis. An animal model with a microbiome showing C. difficile overgrowth can model colitis, and be used to test new treatments. Conversely, a model with a more normal profile (including some C. difficile) can be used to ask which antibiotics (or enteric coatings, etc.) have the lowest risk of causing overgrowth and colitis.
Interest in the microbiome and research into potential treatments has leaped forward because finally all the tools are in place for good clinical models. Rederivation and biosecurity techniques have been honed to generate and maintain animals in a delicate germ-free status. More thorough and sensitive testing allows better characterization of the microbiome. NGS can profile the complex gut with simple fecal samples, enabling understanding of subtle changes. The treatment potential of our microbiome is largely untapped, and the best preclinical germ-free models and practices will drive microbiome-based therapies through our drug discovery pipelines.
