INTRODUCTION
The human microbiome, consisting of trillions of microorganisms living on our body surfaces, has a profound impact on our health and well-being. Scientific research over the past two decades has uncovered the vital role of the gut microbiome in everything from digestion and immunity to mental health and disease prevention. This chapter presents an overview of our understanding of the gut microbiome and its importance to human health and disease revealed through studies of the gut microbiomes of humans and animals. It explores some of the latest research on the gut microbiome, including how probiotics are being used to prevent necrotizing enterocolitis (NEC) in premature infants, the success of using fecal microbiota transplantation (FMT) to treat Clostridioides difficile infection, the emerging field of next-generation probiotics as a promising therapeutic approach to a variety of health conditions, and the associations between a disrupted gut microbiome and several disease states such as Type 2 diabetes, various neurological pathologies, and the development of certain cancers.
Complex communities of microorganisms reside on every surface of the human body. These organisms, and the information encoded in their genomes, are collectively referred to as the human gut microbiome. Microbial colonization is generally thought to begin at birth, though there are studies that suggest this process may begin in utero. Different body ecosystems (e.g., the integument [skin], oral cavity, vaginal canal, the gut) each harbor a unique microbiome. Over the past 20 years, significant advancements in high-throughput genomic sequencing, transcriptomic, proteomic, and metabolomic technologies, combined with the development of powerful computational tools, have allowed scientists to explore and examine the structural intricacies of these microbial communities. Most microbiome research to date focuses on the gut, which in recent years has emerged as an attractive target for clinical interventions.
The neonatal human gut microbiota has low complex ity. The composition becomes more complex and developed between the ages of 3 and 5 and appears to be governed by interactions between the microbiota and the host immune system, as well as diet and other environmental exposures. Microbiota constituents include primarily bacteria, but also archaea, unicellular eukaryotes, and even viruses. Most bacte ria are obligate anaerobes, with the majority belonging to the Bacteroidota (formerly Bacteroidetes) and Bacillota (formerly Firmicutes) phyla, and also include members of the Pseudo monadota (formerly Proteobacteria), Verrucomicrobia, and Actinomycetota phyla. Both animal and human studies reveal remarkable diversity among gut bacteria in a mature microbiome in healthy individuals. Any given individual adult gut can harbor >1000 unique microbial species. These diverse microbial communities have an extensive metabolic repertoire that promotes synergistic interactions among the gut microbial constituents: one organism’s waste product serves as another one’s metabolic substrate. In this way, each person cultivates a unique profile of microbial residents. There is little evidence to support the idea of a “core” set of microbial species that defines a healthy gut microbiome. Instead, the functional capacities conferred by the metabolic repertoire of an individual’s microbiome are conserved due in part to genetic redundancy among the multitudes of microbial taxa that comprise the gut microbiota. Phylogenetically unrelated microbes will still utilize similar strategies to adapt and survive in a particular ecosystem, including the gut.
FACTORS THAT SHAPE THE GUT MICROBIOME
The gastrointestinal tract is composed of distinct compartments: the stomach, followed by the duodenum, jejunum, ileum, and the colon. The microenvironment of each segment (e.g., pH, levels of oxygen, types of mucins covering the epithelium, etc) exerts selective pressures on the microbiota. The microbiota itself can influence the abundance and complexity of organisms in each segment. Microbes produce diffusible molecules that impact inter-microbe (and microbe–host!) interactions. Together, the host anatomy and physiology, and microbial activity dynamically shape the assembly and succession of the microbiome along the gastrointestinal tract.
There are other factors that influence microbiome composition. Studies show that host genetics account for some of the observed variation, but the actual contribution is still under active investigation. Diet, household members (including pets), and medications contribute roughly 20% of the observed variation, but most person-to-person variability remains unexplained. This is a complex and challenging topic to explore because the microbiome composition within each person is also dynamic and variable over time, even in healthy individuals.
Antibiotics have an enormous influence on microbiome com position and structure. These molecules can eliminate sensitive or low-abundance taxa from the microbiome or, conversely, create opportunities for other group(s) of microbiota to expand. One well-characterized example is the antibiotic-associated expansion of Clostridioides difficile (formerly Clostridium difficile) in some patients treated with antibiotics. Healthy people can harbor this organism in their gut; however, after a course of antibiotics that do not affect this organism, they are at risk of developing C. difficile colitis due to overgrowth of the bacteria, which produces exotoxins that cause pseudomembranous colitis.
FUNCTIONS CARRIED OUT BY THE GUT MICROBIOME
Early studies using animal models, particularly gnotobiotic mice, were instrumental in uncovering important functions carried out by the microbiome. Gnotobiotic animals are derived by aseptic caesarian section and are reared using sterile technics such that the gut colonized by a microbiome is either fully defined or completely free of all detectable microorganisms (referred to as anexic, or germ-free). More recently, clinical studies in humans are contributing to our understanding of the myriad and complex influences that the microbiome has on human health and disease. We are still in the early days of elucidating the biology and functional traits of many of the individual microbial taxa that make up the microbiome.
Energy Generation from Undigestible Carbohydrates
Humans lack many enzymes necessary to catabolize several types of complex dietary carbohydrates. However, the microbiome synthesizes an extensive array of fermentative enzymes that convert carbohydrates into alcohols, gases, and short-chain fatty acids (SCFA). There are data suggesting that many SCFAs are beneficial to the host. SCFAs maintain intestinal homeostasis by enhancing gut barrier function, exerting anti-inflammatory and anti-carcinogenic effects, modulating immune functions, and fueling ATP generation by epithelial cells lining the colon and other organs, such as the liver. Different individual’s gut micro biomes have varying abilities to harvest energy. Consider the symbiotic relationship between host and microbiota in obesity. In a landmark study, researchers transplanted fecal microbiomes obtained from identical human twins, one who was lean and the other obese, into germ-free mice. After feeding these mice the same diet, they measured a significantly greater increase in total body fat in the mice harboring the “obese microbiota” compared to “lean microbiota.” Fed an identical diet, the “obese microbiota” had an increased capacity to harvest energy, which promoted obesity in these mice.
As fiber and other complex carbohydrates are replaced in industrialized countries by processed, simple carbohydrates and fat, the incidence of obesity, as well as colorectal cancer and inflammatory bowel diseases have also increased. Distinguishing whether the microbiome is simply correlated with these conditions or plays an active role in the development of these disease states will require careful study to identify the specific mechanisms by which changes in the microbiome impact these trends and risk for disease.
Vitamin Biosynthesis
The gut microbiome synthesizes several vitamins, including several quinones and the B vitamins thymine, riboflavin, niacin, biotin, pantothenic acid, and folate. B vitamin micronutrients are essential for organ and immune function, mucosal barrier integrity, energy metabolism, and cellular growth and differentiation. Indeed, certain Vitamin B deficiencies are associated with gut inflammatory diseases.
Immune Development & Function & Colonization Resistance
We now understand that relationships between the gut microbiome and the host immune system both shape microbiota structure and regulate immune system development and responses. The impact of the microbiome on immune system development has been greatly advanced through the use of gnotobiotic mouse model systems, including germ-free mice and mice harboring defined microbial communities. While these animal models imperfectly capitulate the complexity of the human intestinal environment and its microbiota, they have provided key insights to our understanding of host–microbiome interspecies communication in the context of disease and are crucial for evaluating new therapeutic and diagnostic tools that target the microbiome.
Early studies demonstrated that germ-free mice have com promised gut immune function, impaired secretion of antibodies and antimicrobial peptides, diminished gut-associated lymphoid tissue, and altered proportions of Th1, Th2, and Th17 CD4+ T cells that are critical for sensing and responding to pathogens in the gut. Remarkably, reconstituting gut microbiota harvested from healthy mice into these germ-free animals restored normal immune function. This is due in part to the production of SCFAs. Not only do these molecules serve as energy sources for human cells as described above, but they also act as signaling molecules that enhance epithelial barrier function, promote an anti-inflammatory environment, and boost the function of various immune cells throughout the body. In addition, SCFAs dampen inflammatory responses by intestinal macrophages. These initial studies demonstrated that early interactions between the gut microbiota and the nascent immune system are critical for promoting and maintaining immune homeostasis.
The microbiota also protects against colonization of exogenous microbial species (colonization resistance). Studies have indicated that a few mechanisms likely underly colonization resistance. Established microbiota limit the growth of invading organisms by competing for nutrients, and many members of the microbiota produce antimicrobials that inhibit microbes that are not yet established.
Microbiome & the Gut-Brain Axis
In addition to SCFAs, the gut microbiota synthesizes important molecules including neuroactive compounds that modulate the gut-brain axis in mice. A growing body of evidence suggests that gut microbes influence neurological pathways and mental health behaviors including depression and anxiety, Alzheimer’s disease, autism spectrum disorder, multiple sclerosis, and Parkinson’s disease. The complex mechanisms used to modulate mental health and neurological function by microbes are under active investigation, and likely involve endocrine, immune, and metabolic pathways.
Microbiome & Systemic Disease
A growing body of literature reports an association between a disrupted microbiome and the development of systemic dis eases such as diabetes, inflammatory bowel diseases, psoriatic arthritis, Celiac disease, certain liver diseases, and even some cancers. While some of this research is emergent, there is robust and compelling evidence to support using the microbiome as a diagnostic and/or prognostic marker for type 2 diabetes mellitus (T2D). Patients with T2D typically have imbalanced blood glucose levels, high blood pressure, and dyslipidemia. In addition to genetic risk factors, a high-fat diet and a sedentary lifestyle increase the risk for developing T2D. Work by several groups has correlated disruptions in the gut microbiome with progression of insulin resistance in T2D. The working hypothesis is that the disrupted microbiome negatively impacts intestinal barrier function and various signaling pathways that contribute to the development of insulin resistance, though exactly how the dynamics of host-microbiome interactions do this is yet to be elucidated. Dozens of human observational studies investigating the microbiota in patients with and without T2D report associations between specific taxa and disease. Organisms belonging to the Bifidobacterium, Bacteroides, Faecalibacterium, Akkermansia, and Roseburia genera were negatively associated with T2D, while members of the Ruminococcus, Fusobacterium, and Blautiagenera were positively associated with T2D. Several clinical trials are now underway to identify specific bacteria that are involved in the progression of T2D and to evaluate the effects and change of gut microbiota after berberine (diabetes medication) and/or probiotics administration in patients with newly diagnosed T2D.
There is increasing evidence for the role of members of the human gut microbiome in the development of certain types of cancers. The most well-known example of a bacterial carcinogen is Helicobacter pylori, which can cause MALT lymphoma and gastric adenocarcinoma. However other microbiome constituents are also implicated in cancer. For example, certain periodontal pathogens including Fusobacterium nucleatum and Porphyromonas gingivalis play an important role in the development of colorectal and pancreatic cancer, likely by their abilities to induce chronic inflammation and host cell proliferation, as well as producing various carcinogenic substances.
HOW CAN THE MICROBIOME TRANSFORM MEDICINE?
With the growing body of evidence linking the gut microbiome to the function of various organs, and numerous studies demonstrating that gut microbiome alterations factor in the pathogenesis of many systemic diseases, it is tempting to consider how modulating the gut microbiome might be used for prophylaxis or to treat disease. There are many obstacles and challenges to surmount before this becomes a mainstay of practice:
Poor study design and a lack of standardization in sample collection, processing, and analysis. Collecting data from a single time point, variations in sample collection or processing, differences in DNA extraction techniques, sequencing methods, and analysis are all factors that vary considerably between studies and make it challenging to interpret or validate results across studies.
Most microbiome research to date has relied heavily on animal models. These animal studies often rely on inbred mouse strains that do not reflect variation observed in the human population. It is critical to determine if these observations extend to humans.
We lack a comprehensive and mechanistic understanding of how microbiome diversity impacts its function. The inter-individual variation in microbiome composition observed among people and the changes in complexity and abundance along the intestinal tract, not to mention the external influences on microbiome variation like diet, environment, and medications, make it challenging to formulate universal principles that would guide how the microbiome could be manipulated to aid treatment. For example, we now understand that many members of the gut microbiota produce enzymes that can directly metabolize drugs (e.g., certain antibiotics, cardiovascular medications, and cancer immunotherapies among others), which can have profound effects on the efficacy of treatment. One specific example is Digoxin, which is a medication used to treat atrial fibrillation and congestive heart failure. Interestingly, some patients do not respond to this treatment. Using a combination of transcriptional profiling, comparative genomics, and culture-based assays, scientists discovered that certain, but not all, strains of the gut bacterium Eggerthella lenta reduce digoxin to a form that is not readily absorbed in the intestine. This correlated with changes to drug concentration in the serum and urine, providing a working hypothesis that patients who do not respond to digoxin may be colonized by bacterial strains that impact metabolism of the drug leading to variable responses in patients. These results also highlight that genome-level differences likely matter much more than simply detecting the presence of species in the gut. Further research examining the role of the gut microbiome in drug metabolism and how much it accounts for varying patient responses to pharmaceuticals may lead to new therapies or modifications in the way drugs are prescribed.
Despite these challenges, there have been clinical successes. NEC in very low birth weight infants causes significant morbidity and mortality and several studies implicate a role for the gut microbiota in the pathology. Clinical studies demonstrate that early oral administration of two bacteria (Lactobacillus acidophilus and Bifidobacterium infantis, prominent early colonizers of the healthy infant gut) as a probiotic reduce the incidence, severity of, and death due to NEC. Another example is probiotic intervention with B. infantis for infants with severe acute malnutrition. This intervention promoted weight gain that was associated with reduced intestinal inflammation markers.
In other studies looking at the role of the gut microbiome in cancer chemotherapy, many groups have demonstrated that the microbiome affects the response to chemotherapy and immunotherapy with checkpoint inhibitors (in both positive and negative ways). One striking study demonstrated a relationship between T-cell responses specific for Bacteroides thetaiotaomicron and Bacteroides fragilis and the efficacy of CTLA-4 blockade both in animal models and in patients with cancer. The study showed that tumors in antibiotic-treated or germ free mice did not respond to CTLA blockade. Importantly, the response was restored by either instilling B. fragilis into the guts of these animals, or immunizing them with B. fragilis polysaccharides, or transferring B. fragilis–specific T cells into the antibiotic-treated or germ-free animals. In a follow-up study, the authors instilled fecal contents from human patients that were treated for melanoma with antibodies against CTLA-4 into mice. These mice showed outgrowth of B. fragilis with anticancer properties, revealing a critical role for Bacteroides in the immunostimulatory effects of CTLA-4 blockade.
Perhaps the gold standard of modulating the microbiome to treat disease is FMT. This technique can be traced back to fourth-century Chinese medical literature, which describes treating severe diarrhea by instilling fecal material from a healthy person into the rectum of a person experiencing diarrhea. In 2013, the U.S. Food & Drug Administration approved FMT as a therapeutic intervention for C. difficile infection (CDI). During this process, a minimally manipulated microbial community from healthy donor stool is infused into the patient’s intestinal tract, which reconstitutes the patient’s gut microbiota with a community that closely resembles that of the donor. Importantly, resident C. difficile is also displaced. FMT therapy has emerged as the best option for patients with acute, severe, and complicated forms of CDI that fail to respond to other conventional treatments. Currently, there are clinical trials to identify a more defined set of microbes that can be used as FMT alternatives, with the idea that this will be a safer and more reproducible therapy. Based on its remarkable success, FMT is now being considered as a potential therapy for other GI dis eases, including inflammatory bowel disease. In April of 2023, the U.S. Food and Drug Administration approved the first orally administered fecal microbiota product to prevent recurrent CDI in adults (Vowst). The effectiveness of this product was evaluated in a randomized, placebo-controlled clinical study in which some participants received Vowst while others received placebo. Through 8 weeks after treatment, CDI recurrence in Vowst-treated participants was lower compared to placebo treated participants (12.4% compared to 39.8%).
The past two decades have seen remarkable advances in the field of gut microbiome research. Uncovering the underlying mechanisms of microbiota-host interactions may lead not only to the development of microbiome-based therapies, but also deeper insight into how our “microbial organ” supports human health. In fact, currently there are hundreds of clinical trials currently being carried out, to investigate how the gut microbiome influences a variety of clinical conditions. Discovering exactly how the microbiome and its interactions with host metabolism and development influences health will require painstaking, rigorous, and robust research, coupled with critical analysis of the resulting data.
