The Story Beyond the Stool Sample

If you’re puzzled by the plethora of information and advertisements about the microbiome, good bacteria, bad bacteria, probiotics and prebiotics, you aren’t alone. This talk answered some common questions about the human microbiome, including: “What even is the human microbiome anyway? Spoiler Alert: it’s more than just bacteria in your poop! How do you measure the microbiome? Are my microbes unique to me? How many microbes are living in and on me and how did they get there? What are these microbes doing for me and why does it matter?

Noel Britton, a postdoctoral researcher at Johns Hopkins University, presented her dissertation work on the human bacterial environment. Throughout the talk, she often referred to the idea of balance. She highlighted that bacteria are not always good or bad and that bacterial species keep one another in check.

The rapid increase in microbiome publications and general research in this field is due to technological advancements. These include DNA sequencing, next-generation sequencing, and pyrosequencing. The avalanche of scientific publications has even made its way into the general person’s daily life through microbiome-focused advertising and products. However, there is still much we have to learn. A microbiome means a collection of microbes and genes that share a common environment. In this presentation, we focused on the human body and its microbiome. The metagenome is the genetic material, while the microbiota refers to the organisms in this environment. Examples of microbiota include bacterial, fungal, and viral species. Microbiota research mainly focuses on bacterial bias rather than fungal or viral organisms.

You may be asking yourself how many microbes are in a person’s microbiome. There are likely more microbes in the human body than there are stars in the Milky Way. The estimated amount of microbes in a single person is one hundred trillion cells and this makes up 57% of the cells in the human body. Various types of microbial quantification methods center around the central dogma. The central dogma is a scientific theory that genetic information flows from DNA to RNA to proteins. As a result, different processes, ranging from microscopy to metabolomics, take advantage of the central dogma to quantify the number of cells in a microbiome. There are different body sample sites used for microbiology sampling like stool, swabs, biopsies, saliva, and urine. For each sample site, there are similar processing steps overall. These steps include DNA extraction, library prep for sequencing, sequencing, and DNA analysis.

In order to extract the DNA from a sample, you must create a solution via vortexing. Next, the solution is centrifuged. This allows for the removal and separation of noncellular debris at the bottom of the centrifuged mix. Researchers continue on working with the supernatant, which is the fluid on top that houses the microbes. The supernatant undergoes high heat exposure and physical beading to break microbe cell walls and extract DNA. Once the DNA is obtained, it is purified and washed for further experimentation.

In preparation for sequencing, adaptation and amplification occur, to ensure there is adequate data for sequencing. During the sequencing process, fragments of the DNA are put together to assemble the entire structure for later data processing. The adapters are removed and the taxonomy of the sample is classified. Similar data are grouped together in an OTU, or operational taxonomic unit. Now that this data is obtained, how can it be analyzed and what questions can it answer? Looking at the data researchers will focus on alpha versus beta diversity. Alpha diversity is the variation within a single sample/group while beta diversity is the dissimilarity between multiple samples/groups. Relative abundance, or the taxonomic composition of each group, is also an important factor for researchers. Researchers also rely on network and correlational analysis to piece together the relationships between the clinical symptoms of their patients along with the samples collected in order to develop a story for their data. In previous research, it has been determined that the phyla makeup mainly remains the same between patients, but the relative abundance and ratios of these groups vary between patients. Even in the case of the McFarlane twins, who are monozygotic twins and probiotic entrepreneurs, they share 100% of their DNA, but only 30% of their gut microbiota. The gut microbiome is a unique identifier between people and there is a possibility of harnessing it as a forensic tool.

Taking a step back, the majority of these studies focus on stool science for gut investigation. Nevertheless, researchers could really sample from anywhere on the human body since microbial communities cover the entire body. Studies depict that there is a similarity between what we commonly think of as good versus bad bacteria. It is truly the amount and ratio of these species and how they interact with one another which leads to beneficial or detrimental effects. Notably, different body sites have their own normal ratios of disease-causing bacteria. For example in the urinary tract, Candida species are normally considered healthy; however, excess amounts can lead to yeast infection. All this information begs the question of how we get our microbes in the first place. They can really come from anywhere. Even a fetus gets microbes from its parent, which go on to influence their immunity early in life. Babies receive additional microbes from the birth canal, breast milk, and their environment in the early stages of life. Microbes are even present on the body after death; though they do decline as the host begins to erode.

Stay tuned for the In Sickness and In Health seminar in which Noel returns to discuss what our microbes do for us and how we can best help them!