The gut microbiota is made up of trillion microorganisms comprising bacteria, archaea, and eukaryota living in an intimate relationship with the host. This is a highly diverse microbial community and is essentially an open ecosystem despite being deeply embedded in the human body. The gut microbiome is continually exposed to allochthonous bacteria that primarily originates from food intake. Comprising more than 1000 bacterial species, the gut microbiota endows so many different functions—so many that can be considered as an endocrine organ of its own. In this book chapter, we summarize the importance of gut microbiota in the development and maintenance of a healthy human body. We first describe how the gut microbiota is formed during the birth of a human baby and how a healthy microflora is established overtime. We also discuss how important it is to maintain the microbiota in its homeostatic condition. A discussion is also given on how alterations in the microbiota are characteristic of many diseased conditions. Recent investigations report that reestablishing a healthy microbiota in a diseased individual using fecal microbial transplant can be used as a therapeutic approach in curing many diseases. We conclude this chapter with a detailed discussion on fecal microbial transplants.
We, animals, live in a microbe-dominated planet. We are all covered, filled, and fueled by bacteria. All body surfaces like the skin, gastrointestinal tract, urogenital, and respiratory tract are in constant contact with the environment and are, therefore, colonized by bacteria. The realities of life associated with a microbe-dominated planet have led to the coevolution of animals with bacteria. This coevolution has led to close inhabitation of bacteria on different surfaces of the human body, especially the gut. Here, many bacteria and their phages, viruses, fungi, archaea, protists, and nematodes intermingle to form a microbial consortia collectively called “microbiome” or “microbiota”. The presence and abundance of each taxonomic group may vary within population based on their access to adequate health care and local sanitation condition or within individuals based on their metabolic, medical, diet, and various other factors.
Despite being embedded deeply in the human body, the gut is essentially an open ecosystem—with constant exposure to environmental factors. The closure of the NIH-funded human microbiome project has given advanced understanding about the composition and functional characteristics of the gut microbiota composition. The composition of the human microbiome varies significantly depending on the habitat . For example, the gut microbiota is mainly populated by bacteria , while the skin harbors mainly fungi . It was always considered that human microbiome outnumbered human nucleated cells by at least a factor of 10. However, reports suggest that the ratio is closer to 1 . Recently metagenomic analysis concluded that the gene set of the human microbiome is 150 times larger than the human gene complement raising the possibility that the large bacterial genetic repertoire aids the human component in performing the essential functions that are not encoded by the human genome. Since more than 99% of these genes are bacterial in origin, the number of bacterial species were calculated to be around 1000–1150 bacterial species. The figures collectively emphasize the biological importance of the microbiome, and the genetic complement can be rightly considered as the second genome . However, the taxonomic diversity of the gut microbiome notwithstanding, this chapter will deal only with the bacterial populations and all further references to gut microbiota means gut bacterial microbiota.
A typical human gut microbiota contains about 1014 bacteria and is made up of more than 1000 bacterial species [5, 6]. The human gut microbiota is composed of six bacterial phyla—
The functions bestowed by the bacterial gut flora are so enormous that it can be considered as an endocrine organ on its own . It is well understood that the gut microbiota and their metabolites play important roles in host homeostasis, such as providing important nutrients like secondary bile salts and B/K group vitamins [10, 11], help in fermenting the otherwise indigestible complex plant carbohydrates such as dietary fibers into short-chain fatty acids (SCFA) , contributing to an effective intestinal epithelial barrier and activation of both innate/adaptive immune responses of the host . In addition, the healthy gut microbiome drives intestinal development by promoting vascularization, villus thickening, mucosal surface widening, mucus production, cellular proliferation, and maintaining epithelial junctions [14, 15, 16]. The influence of the gut microbiota, either directly or indirectly, affects the physiology of most host organs even the brain [14, 17, 18, 19, 20].
The taxonomic composition of the microbiota is very subtle—subject to change with variations in the diet , feeding time changes , sleep wake cycles, and even jet lag . The unique taxonomic signature of the gut microbiota has to be strictly maintained for a healthy gut. Any disruption of the taxonomic composition of the gut could lead to conditions such as inflammatory bowel syndrome (IBS), asthma, obesity, metabolic syndrome, and cancer . In some cases, resuscitation of the gut microbiota using probiotic bacteria like
In this chapter, we will sequentially detail how the microbiota establishes itself in a human body, how minor or major variations in the gut microbiome introduces diseased conditions, and how the recent therapeutic approaches aimed at resuscitating the healthy microbiota can cure many dysbiotic microbiota-associated conditions.
2. Our internal garden—on how gut microbiota is planted and nurtured
2.1 The initial colonization
The most prevalent concept, and a very incorrect one, is that babies are borne sterile and after birth, the body becomes immediately colonized with microbes from the surrounding environment. Placental mammals such as humans are borne through a birth canal, which is colonized by microbes. A baby acquires its first inoculum from the birth canal. A healthy vaginal microbiota is composed of few bacterial species [25, 26] and is predominated by
Cesarean section babies, in contrast to vaginally borne babies, are dominated by skin-associated bacteria like
2.2 Development of the microbiota
The establishment, composition, and the density of the microbiome in the gastrointestinal (GI) tract depends on the biochemical factors like pH, oxygen gradient, antimicrobial peptides (AMPs), bile salts, etc. There is a pH gradient across the GI tract—lowest in the stomach and gradually increase to the terminal ileum, and a drop in caecum and increases toward the distal colon. Oxygen also exhibits a gradient across the length of the tract. The levels are highest in the upper GI tract, which decrease to anaerobic conditions in the distal colon. Radially across the tract too, there exists a oxygen gradient. Anoxic conditions exist in the lumen, while there is an increase in the oxygen tension near the mucosa, and this oxygen is rapidly consumed by the facultative anaerobes . Each area in the gut produces its own AMPs. Saliva contains lysozyme, which is antibacterial. Small intestine produces α-defensins, C-type lectins, lysozyme, and phospholipase A2. The large intestine produces β-defensins, C-type lectins, cathelicidins, galectins, and lipocalin. Mucus also plays an important role in the distribution of the gut microbiota. The mucus in the stomach and colon can be discriminated into two layers—outer loose layer, which is densely populated by bacteria, and the inner “solid” layer, where bacteria is sparce [41, 42]. Only mucin-degrading bacteria like
The initially colonized microbiome has relatively few bacterial species. But during the initial phase of life, bacterial diversity increases in the microbiota. This may be because of the constant exposure of the baby to the environment. The gradual increase in the length of the GI tract can also provide a new niche for the bacteria to colonize. The bacterial diversity also depends on the diet as the introduction of a more plant-based diet increases the proportion of the Firmicutes . Though lifestyle, illness, puberty, and other variable factors affect the microbiome, family members tend to have similar microbiomes with shared bacterial strains . Thus, during the first year of life, the microbiome proceeds through a very variable phase. A distinct composition resembling the “adult microbiome composition” is established once an adult diet is established after weaning . Once established, the gut microbiome composition seems to remain stable for a long time, possibly lifelong .
3. Maintain the flora!—on how any alterations could be disastrous
Biologically, the gut microbiome is very essential for the normal functioning of the human body. This complex ecosystem is responsible for many critical functions like (1) metabolism and energy regulation —up to 10% of our daily consumed calories are provided by the microbes who break down complex plant-derived carbohydrates into short-chain fatty acids (SCFA), the main energy source of the enterocytes. From this perspective, alterations in the gut microbiome can contribute to obesity [48, 49] and consequently type II diabetes ; (2) immune system activation [51, 52, 53]—the colonic mucosal immune system plays a dual role and in that it must tolerate the gut microbiome and at the same time react against pathogenic organisms. This homeostasis is achieved by the intricate interplay between the microbiome and the host; (3) colonization resistance—physiologically colonized body surfaces are intrinsically protected from pathogen colonization. It is the intricate interplay between the above mentioned three major functions of the gut microbiota that brings about the physiological healthy state of the host.
Colonization resistance is the native ability of the host to suppress the invasion by exogenous microorganisms . The concept of colonization resistance originated from the studies of Dubos in 1965 who demonstrated that indigenous gut microbiota neutralize colonization by a potential pathogen . Slightly earlier, it was noted that loss of obligate anaerobic bacterial population in the lower intestinal tract correlated with infection, suggesting that the commensal anaerobic organisms were providing colonization resistance [56, 57, 58, 59]. At the time, colonization resistance was thought to result from microbe-mediated inhibition. We now know that the multiple mechanisms like microbiota-mediated activation of host immune responses are also involved. Colonization resistance provides broad protection against bacteria, virus, and other categories of pathogens . On the other hand pathogenic microorganisms can out compete commensal microorganisms, subvert the immune response and invade the epithelia. For example, some pathogens can cause inflammation in the gut and utilize the consequent nutrient-rich inflammatory environment to outgrow other Proteobacteria. However, in a healthy intestine, the gut microbiota maintains the stiff colonization resistance by three mechanisms (1) directly inhibiting or killing the invading organism, (2) maintaining a protective musical barrier, and (3) stimulating a strong immune response that can neutralize a pathogen.
3.1 Direct inhibition
Bacteria produce many bioactive molecules, such as antimicrobial peptides, bacteriocins, etc., to selectively kill or inhibit the growth of competing bacteria . These bioactive molecules are the primary source of antibiotics in the pharmaceutical industry .
3.2 Barrier maintenance
Gut microbiota regulates the strength of the intestinal barrier and sequesters themselves within the intestine. For example, the mucus layer is an important deterrent for many pathogenic microorganisms to reach the underlying epithelial cells. Mucus production is enhanced when a germ-free mice epithelium is exposed to some bacterial products , which means that an intact microbiota is essential to maintain the required thickness of the mucosal layer to keep pathogenic microorganism at bay. Diet can also influence the thickness of the mucosal layer. An assessment of intestinal microbiota localization with immunofluorescence shows that the absence of microbiota-accessible carbohydrates in the diet resulted in a thinner mucosal layer, thus exposing the underlying epithelial cells to pathogenic organisms . The thinning of the mucosal layer made mouse susceptible to colitis.
3.3 Immune maturation and inflammation
A healthy microbiota is essential for a healthy immune system. Almost one and half decades ago, it was observed that microbial products secreted from the microbiota induced the colonic immune system by activating anti-inflammatory cells and cytokines. In 2005, a polysaccharide from
Microbes can also directly activate the colonic immune system. Segmented filamentous bacterium (SFB) from the microbiota was shown to adhere tightly to epithelial cells of the terminal ileum with Th17 cells, and this adherence correlated with the induction of inflammatory/antimicrobial defense genes . More recently, bacteria in human feces were subjected to selection based on their potential to induce anti-inflammatory T regulatory cells. It was found that bacteria belonging to cluster XIVa clostridial group induced anti-inflammatory T regulatory cells along with bacteroides species [67, 68]. Gut microbiota can also activate the expression of bacterial C-type lectins in intestinal epithelial cells. The lectin, RegIIIγ, is essential to create a 50-μm clearance zone between the gut microbiota and small intestinal epithelial cells. Abrogation of the RegIIIγ synthesis increased the proximity of gut microbiota to the epithelial cells . This shows that microbiota-activated lectin synthesis can directly act to suppress bacterial activity. In addition, gut microbiota can also enhance systemic antiviral activity . Therefore, it is very important to maintain a healthy microbiota to drive efficient pro-inflammatory and anti-inflammatory immune responses in the host.
Simple alterations to the gut microbiota can often lead to very unhealthy consequences. For example, in the esophagus, the composition of the microbiome is heavily dependent on the microbes originating from the oral cavity and is dominated by
The most important stomach associated bacterium is
Sampling the fecal material represents the colonic microbial population. However, sampling the small intestine is difficult because it is accessible only by invasive sampling. The small intestine is populated by distinct microbial communities that are less diverse and are dominated by
3.4 Antibiotic-associated colitis
Antibiotics considered as “wonder drugs” were implemented into therapy years before and have saved millions of lives. Even though antibiotics can reduce morbidity and mortality associated with bacterial illness, no antibiotic is pathogen selective. The application of antibiotics lead to collateral damage of accompanying microorganisms in a population, for example in a microbiota. Studies investigating the impact of antibiotics on microbiota confirm that antibiotic treatment increases the susceptibility of an individual to bacterial pathogens by compromising colonization resistance [90, 91, 92, 93].
The observation that antibiotic therapy reduced colonization resistance making the host susceptible to bacterial infections was observed very early in the literature [56, 57, 58, 59]. Gut microbial compositional analysis of an antibiotic-treated mice showed the expansion of γ-proteobacteria and enterococci, suggesting that gut microbiota somehow suppressed the expansion of oxygen-tolerant species [94, 95]. A study on healthy volunteers treated for a week or less with antibiotics reported persistent effects on their bacterial flora that included a loss of biodiversity on the gut flora, insurgence of antibiotic resistance strains, and upregulation of antibiotic resistance genes . Antibiotic treatment can also induce long-term defects in the microbiota. For example, a single dose of clindamycin induced long-term susceptibility to
Microbiota establishes itself very early in the life cycle of every human, and this development is very crucial for a healthy lifestyle . So, administration of antibiotics in the early stages of life predisposes the individual to diseases in late infancy or adulthood, particularly allergic or metabolic syndromes . Antibiotic exposed prenatal mice resulted in exacerbated asthma following intranasal challenge with ovalbumin . This case is true in children who are administered with antibiotics in the first year of life and may develop asthma during sixth or the seventh year . Early use of marcolids in Finnish children led to the development of asthma and increased BMI associated with a dysbiotic gut microbiota . Effects of antibiotic administration in early life are not limited to development of asthma alone but also to obesity. A low dose of penicillin delivered at birth transiently shifted the microbiota, and this transient shift induced sustained effects in body composition, leading to obesity . All these reports emphasize the detrimental microbiota-associated effects of antibiotics and their implications in health.
3.5 Inflammatory bowel disease (IBD)
IBD perhaps is the first diseased condition where alterations in microbiota are studied most extensively. IBD is a term mainly used to describe two disease conditions—Crohn’s disease and ulcerative colitis. Here, intestinal cells play an important role in integrating the interactions among intestinal microbiota, mucosal immune system, and environmental factors . It was observed very early that IBD conditions had a genetic component—there was a 10-fold increase in risk if related closely to the patient . Genome-wide association studies (GWAS) reported many genetic factors that are associated with IBD . As more GWAS based studies began identifying genetic factors associated with IBD, it was soon noted that some IBD genetic factors were also associated with other disease conditions like diabetes . Curiously, GWAS and meta-analysis identified considerable overlap between susceptibility loci for IBD and mycobacterial infections . The genetic associations notwithstanding, alterations in the gut microbiome of IBD patients have always been an interesting topic for microbiome researchers. The most significant alteration in the composition of the gut microbiome associated with IBD is the reduction in the abundance of the protective bacterium
3.6 Colorectal cancers (CRC)
CRC is the fourth leading causes of death causing cancer and is the third important cause of malignancy. The CRC incidence is growing fast worldwide in low and middle east countries and is expected to increase by 60% by 2030 worldwide . The transformation from a healthy epithelial cell to a malignant cell requires three steps: (1) induction of oncogenic mutations within Lgr5+ intestinal stem cells, (2) altered β-catenin/Wnt signaling, and (3) proinflammatory cascades such as TNFα-NFκB and IL16-STAT3 catalyzing CRC development (Garret 2015 EN). Initially, there was increasing evidence about the role of bacteria in CRC. Bacteria such as
4. The therapeutic potential of the gut microbiota
Humans have used live bacteria, particularly probiotic bacteria, for therapeutic purposes from time immemorial. We have seen some examples in earlier sections of this chapter. Perhaps, the best example of using live bacteria to cure infectious disease comes from antibiotic-associated CDI illness.
FMT is the procedure where fecal matter is collected from a tested donor, diluted in an isotonic solution, strained, and transplanted into the patient using colonoscopy, endoscopy, sigmoidoscopy, or enema. The history of using stool of healthy donors to treat human diseases dates back to the fourth century in China, during the Dong Jin dynasty (AD 300–400 years) . Fecal suspensions or “yellow soup” was used to treat serious disorders such as food poisoning, febrile disease, typhoid fever, etc., becoming the first record of the utilization of human feces to treat human diseases. There are striking similarities between the earlier yellow soup and modern day FMT technology—(1) the inoculum originated from human fecal matter, (2) administration route is digestive tract, and (3) the fecal matter re-establishes the microbiota thereby treating the disease. This long tradition might be the reason why FMT is so well accepted in China . In Europe, the first report of using fecal enema to treat pseudomembranous colitis came in 1958 . Currently, FMT stands in the threshold of becoming a great technology to cure many disorders considered incurable in the past.
4.1 In treating diseases associated with gut microbiota-associated dysbiosis
CDI perhaps was the first condition in which a treatment was attempted using FMT. In 1983, it was shown that by re-establishing the healthy gut microbiota using FMT, mitigation of CDI can be achieved (Schwan 1983 EN). Severe CDI cases can lead to intensive care admission, sepsis, toxic megacolon, and can prove fatal. Colectomy is the standard method of treatment, but the mortality rate is 50%. In a study involving 29 patients who underwent FMT plus vancomycin for severe CDI cases, 62% of the patients were cured in a single FMT, while 38% needed multiple FMTs. Taken together, FMT was highly efficient for CDI infections . The primary and secondary cure rates with FMT using fresh fecal sample to cure CDI is 91 and 98% . FMT from frozen fecal sample also gave similar efficacies in treating CDI [133, 134]. By 2013, FMT was made officially the treatment strategy for CDI [103, 135]. Many pharmaceutical firms are actively working to bring easily consumable CDI-targeted drugs based on FMT. A defined microbial ecosystem therapeutics (MET-1 or RePOOPulate) was developed to cure recurrent CDI [136, 137]. The closest enema-based drug that is awaiting clinical approval is RBX2660, which depends upon the microbial suspension provided from the donor and is formulated for therapeutic delivery. With positive results in phase 2, the drug is currently in phase 3.
Similar to CDI, IBD is also a dysbiotic-associated disease where FMT is a potential therapy. However, in IBD, the use of FMT is a bit complicated and less efficient than in CDI. Early studies using FMT to treat IBD showed very promising result with good microbiota remission reported over long-term follow up . With years, the outcomes started to differ depending upon sample size, treatment approaches, and study designs . Even within IBD patients, remission rates were different. Crohn’s disease had a higher remission of 61%, while ulcerative colitis patients had a remission rate of 22%. It is clear that the FMT treatment for IBD is complicated by numerous factors like differences in treatment regimens, stool preparation/formulation, and dosing frequencies. Varied levels of dysbiosis and difference in the microbiota composition between donor and patient also complicate FMT treatments. However, it was reported that FMT with intensive doses and multiple donors induced clinical remission and endoscopic improvement in ulcerative colitis patients, and this treatment had distinctive improvements in the microbiota composition . It was also shown that a second FMT 3 months past the first one greatly improved the efficacy and safety in treating IBD with FMT [141, 142]. Here, the patients received FMT repeatedly in 3 month intervals—in a procedure called step-up FMT. The efficacy of the procedure increased at each step and was best suggested for patients with refractory IBD and immune-related diseases [143, 144]. There are currently 27 ongoing clinical trials using FMT targeting IBD with two additional trials on children with IBD .
Cancers like colorectal cancers that are associated with a dysbiotic microbiome opening the possibility for a therapeutic intervention using FMT. It was shown that bacteria like
FMT is also showing great promise in patients undergoing hematopoietic stem cell transplantation surgery. Administration of a series of prophylactic antibiotics during surgery can result in the loss of microbial gut diversity and antibiotic resistant strains like
4.2 Collection, preparation, and delivery of FMT samples
Collection and preservation of the stool samples carry the primary importance in FMT. Freshly collected feces can either be immediately used, lyophilized, or cryopreserved. The efficacy of FMT in treating CDI using fresh or frozen feces varied but not significantly . The cure rate was 100% in patients receiving fresh feces, 83% for the lyophilized group, and 78% for patients receiving frozen feces. But the efficacy was more pronounced in treating IBD, and this was demonstrated to be caused due to loss of bacteria in frozen feces . The laboratory preparation methods of FMT is also critical for the success of FMT. Recent studies have reported that some preparation methods can stress the living microbial cells affecting the efficacy of FMT  emphasizing the need for extreme care. For example,
Several groups are developing stool products that can be packaged, transported, commercialized, and easily administered by physicians or consumed by patients. These products range from basic (frozen or freeze-dried stool) to more advanced products like capsules of synthetic stool grown in culture and assembled. The most basic products, from stool banks like OpenBiome and Advancing Bio, provide hospitals with screened frozen material ready for clinical use. More advanced are products like RBX2660, a cryopreserved filtered microbiota derived from stool of selected donors and administered via an enema system. The most advanced is a lyophilized powder that can be reconstituted by rectal infusions developed by CIPAC Therapeutics.
4.3 Precision microbiome reconstitution
The lack of regulatory protocol and stiff resistance from clinicians treating chronically ill patients has dampened efforts to introduce FMT as a viable therapy. This led to the development of the concept of “precision microbiome reconstitution,” where a single bacterium can be used to restore colonization resistance in
4.4 The importance of SAFE FMT
Safety of the patient should be of prime importance when an FMT procedure is considered, especially if the patient is having a poor immune status [166, 167]. Middle gut FMT procedures can cause vomiting and aspiration . The nasojejunal tube could put the patient at high risk of aspiration and should be conducted with anesthesia . There is enough evidence that the long-term safety of the patient should be considered as well. Generally, a tested donor fecal sample is used for FMT. However, this carries the disadvantage that unwanted or potentially pathogenic bacterial phenotypes maybe carried from donors to recipients. A particular case was reported where the patient developed new onset obesity after obtaining a stool sample from a heterologous donor . Using the patient’s own stool sample can avoid the problems associated with donor stool samples. Here, a fecal sample of the patient is banked in the hospital before any procedure that requires antibiotic treatment. The banked sample may provide the vital resource to avoid hospital-acquired infections and to replenish the patient’s own microbiota. Preservation of the patient’s own or donor feces pose a second challenge . There are reports that fecal matter from patients with colon cancer promoted tumorigenesis in germ-free and carcinogenic mice. Potential cardiometabolic, autoimmune, and neurological disease also have been discussed. All these points to the tough screening and regulations are needed before a donor is selected for fecal sample prior FMT.
However, recent reports suggest that FMT is gaining wide acceptance among patients. A survey showed that among patients of Crohn’s disease who received FMT, 56% showed satisfactory clinical efficacy, 74% showed willingness for a second FMT, and 89% expressed willingness to recommend FMT to other patients . Also, the cost efficacy of FMT has been demonstrated worldwide [172, 173, 174, 175, 176, 177]. FMT is still very far from being implemented into routine therapy. The technique needs to undergo rigorous process of standardization before the therapy becomes applied in daily practice. Nevertheless the importance of gut microbiota in maintenance of a healthy lifestyle is demonstrated without doubt. In future therapeutic approaches including antibiotic therapy should take into consideration the impact it has on the gut microbiota and the clinicians should be mindful of the impact of the devastating secondary effects of these therapeutic approaches on the patient.