Our gut microbiota is rich with biological insights, which can help clinical investigators better understand factors that influence both sickness and health. But despite its huge importance, this area of research is plagued by challenges. Stool samples from a diverse patient set are needed for researchers to gather valuable insights. Yet, collection, storage, and shipping issues constrain the quantity and quality of testable material. A new approach is needed to help researchers more fully explore the clues that live in the intestinal microbiota.

Why is studying the gut microbiome so important?

Dr. Freinkman: The gut microbiome is composed of the microbes, primarily bacteria, that live in our digestive tract. These tiny organisms not only help break down our food, but also play an important role in other areas of our health, especially the development and regulation of the immune system. Therefore, it’s no surprise that gut dysbiosis contributes to metabolic disorders, including obesity, diabetes, as well as to diseases that involve unhealthy immune responses centered on the gut, such as Crohn’s disease and inflammatory bowel disease. Perhaps less obvious is the emerging association between the gut microbiome and neurological disorders, including autism, epilepsy and Parkinson’s disease, through the gut-brain axis. The list of diseases and conditions linked to specific gut flora continues to expand, with new scientific discoveries emerging nearly every week. But in most cases, we are missing key information about how gut microbiota exert their effects on the host to promote either health or disease. Direct analysis of gut microbiota composition in the fecal metabolome is critical in order to decipher this functional information, and that’s where microbiome research presents some special challenges.

To learn more about the importance of the gut microbiome, check out our recent interview with Dr. Sean Gibbons from the Institute for Systems Biology.

What are some of the challenges with feces sample collection?

Mr. Rabuka: The biggest challenge most researchers face when collecting fecal samples is the cold-chain requirement associated with the transport and storage of the samples. They must get the samples from the donor to the lab while maintaining temperature-controlled shipping conditions. This forces researchers to request that donors collect their samples on-site in the lab or in a clinical setting. Alternatively, home collection requires expensive packaging, ice-packs, and costly, temperature-controlled courier services in addition to the inconvenience of donors having to store collected sample(s) in their home freezers. What’s worse, from our years of experience in the gut microbiome space, we know that these cold-chain transport methods are often still inadequate at providing the temperature stability required to ensure the integrity of the sample between the time of collection and the time of sample processing in the lab. A lack of temperature stability means that the microbial profile analysis from these samples will include inherent biases, negatively affecting the accuracy of the metabolomic signature.

Traditionally, fecal samples are collected either in plastic specimen containers using a wooden stick, in a tube including an integrated spoon attached to the lid, or through a very basic plastic ice-cream type container. None of these offer any homogenization of the sample at the time of collection and make the subsequent sample extraction step laborious. The lack of homogenization also introduces biases through improper distribution and lack of uniform surface contact between the sample and the stabilizer, if present. This can lead to inadequate stabilization action of the chemistry, leaving portions of the fecal sample prone to microbiome profile changes associated with post-collection microbial growth and/or decay during transport and storage.

We also have to think about the individuals asked to provide the samples. It is extremely difficult, if not impossible, to collect these samples on demand. Most donors are incapable of producing a sample if asked to do so within a short amount of time. Fecal sample collection does not compare to any other human sample type: blood, saliva, or urine. Because of this, allowing donors to collect samples from the privacy of their own homes is crucial for successful studies.

Although snap-freezing (e.g., use of dry ice or liquid nitrogen to freeze the sample) is the “gold standard” for fecal metabolomics analysis, this method may limit the cohort enrollment to participants who will be able to collect and bring samples to the lab or clinic within a very restricted timeframe (usually a few hours) or alternatively will have to come on-site to collect and provide their samples. This restricts the number of potential participants, especially when trying to collect from individuals who have health conditions or mobility impairments.

What can we learn from the intestinal microbiome through metabolomics?

Dr. Freinkman: Metabolomics is the systematic measurement of hundreds to thousands of small molecules, or metabolites, in biological samples. In every cell and organism, metabolites serve as the currency of energy harvesting and storage, as well as the building blocks of DNA and protein, among many critical functions. But, within the context of the gut microbiome, another key property of metabolites is their ability to work as chemical signals, especially among microbial cells and between microbes and their hosts. From a gut microbe’s point of view, metabolites are great signaling molecules because, compared to protein and nucleic acids, small molecules are less energetically costly to produce, easier to export out of the cell, typically more stable in the extracellular environment, and less likely to trigger a host immune response. That explains why so many of the gut microbiome’s known effects on our health are mediated by metabolites. Therefore, from the researcher’s standpoint, metabolomics can provide the key causal link between the composition of the gut microbiome – usually derived from metagenomic sequencing – and the phenotype of interest. This mechanistic understanding can form the basis for rationally designed treatments to support a healthy gut, such as supplementing a patient’s diet with a beneficial small molecule or inhibiting a microbial enzyme to prevent the formation of harmful metabolites. Knowing which metabolites are behind the microbiome’s role in a particular disorder can also provide a way to monitor the effectiveness of any treatment. For all these reasons, metabolomics provides a much more complete and actionable understanding of the gut microbiome than metagenomic analysis alone.

Mr. Rabuka: Many investigators studying the human gut microbiome have generated fantastic amounts of data on the microorganisms that may be present in people’s gastrointestinal tract and the impact that these microbial communities can have on their health status. Nonetheless, knowing which microorganisms are present is often insufficient in allowing us to truly understand what biological and biochemical pathways are active and what chemical product they are releasing into their environment. If we want to regulate or modify effects caused by or modulated by the gut microbiota, we need additional information regarding these said pathways.

For example, many studies in the fecal microbiota transplant space have shown that inactivated gut bacteria present in a fecal sample, when transplanted into another individual via fecal material transfer (FMT), can cause a significant and clinically relevant change in their state of health. This clearly suggests that not only the microorganisms but also the accompanying compounds, substrates, by-products, etc. influence a body’s response to the transplant. Unless we can understand how each component present in the fecal matter is produced and how they can directly interact with the host’s biological processes, we can only see one part of the equation. Metabolomics provides us with an additional layer of insights crucial to a broader, more accurate interpretation and understanding of the microbiome.

Ready to see what new insights metabolomics can help your research reveal? Contact us today at hello@metabolon.com to learn more.