**5. Microbiota**

The large intestine (colon) is populated by a large number of bacterial, archaeal, protozoan, viral and fungal species that are collectively known as the gut microbiota. The number of species ranges from hundreds to tens of thousands that outnumber the human genome by at least 100:1 and are known as the second human genome. The microbiota, especially the bacterial species, forms a community of organisms that metabolize nondigestible carbohydrates, plant cell wall material, and other oligosaccharides that make up dietary fiber, and they produce a wide variety of metabolites including short-chain fatty acids (SCFAs) [45]. Other substances, especially the polyphenols in the diet, serve as substrates for the microbiota. Other secondary molecules include vitamins, cholesterol, and their derivatives, along with the other remaining cell wall components such as lipopolysaccharides, which can also be found

as metabolites formed by the microbiota [46]. A high fat, low fiber diet reduces SCFAs producing increasing the numbers *Firmicutes* and decreasing the numbers of the *Bacteroidetes* phyla, and also decreasing the level of SCFAs (since there is less fiber) and the synthesis of other essential metabolites [47]. SCFAs such as acetate, propionate, and butyrate are synthesized as byproducts of resistance starch metabolism by the microbiota. On the other hand, these bacteria can also metabolize histidine into imidazole propionate which actually can modulate host inflammation, metabolism and cause insulin resistance [48].

To a certain extent, antibiotics, certain diseases (e.g., inflammatory bowel diseases; IBD), and the genetics of the host may contribute to dysbiosis; however, an individual's diet has a major impact on the growth and maturation of the microbiota [49]. SCFAs have distinct effects on colon epithelia, specific transport and signaling mechanisms, and profound effects on a human's health including diabetes.

## **5.1 The chemistry and metabolism of SCFAs**

SCFAs are mostly aliphatic carboxylic acids that have a carbon chain of six or less. A few SCFAs are branched and synthesized from various amino acids but contribute only about 5% of the overall SCFAs produced.

The microbiota consists mainly of *Bacteroidetes* and *Firmicutes* (combined are approximately 90% of the gut microbiota), while the remaining bacteria consist of Actinobacteria, Proteobacteria, and *Verrucomicrobiota*. Amazingly, even within the first month of life, the gastrointestinal (GI) tracts of children are colonized by different species of *Bacteroidetes* and *Firmicutes* at a much lower level. A child's GI tract begins germ-free but accumulates additional numbers of species until the child achieves adulthood. Diet, geographic location, and the genetics of the individual are all contributors to the microbiota colonization of the adult GI tract. In addition, it is now hypothesized that microbiota present in the birth canal during childbirth contributes to gastrointestinal colonization and those children born through the Cesarian section have more health problems than those who do not. Breastfeeding rather than simple formula (not exactly the same diet) also modulates the microbial species that colonize the GI tracts since breastmilk has bacteria from the skin microbiota. Therefore, it is conceivable that the diet can have a profound influence on the child's microbiota population and health. It is even more conceivable that an increase in childhood obesity could be influenced by changes and/or deficits in the developing microbiota [49–51].

Nondigested carbohydrates that are used as nutrients by the microbiota of the GI tract are referred to as prebiotics [52]. Enteric nutrition is given to certain patients as a supplement during their hospital stay to prevent malnutrition. Such treatments frequently result in diarrhea. Once prebiotics are added to the enteric nutrition, the symptoms of diarrhea disappear [53]. Thus, a diet with a proper prebiotic level is critical for maintaining a proper *Firmicutes* to *Bacteroidetes* ratio. Conversely, a high fat, low fiber (i.e., low prebiotic) diet will increase the *Firmicutes* to *Bacteroidetes* ratio [49]. It is well known that SCFAs are produced by the microbiota and when they are absorbed, the SCFAs stimulate Na+/water absorption [54]. Therefore, prebiotics are essential to maintain a proper ratio for any diet. Prebiotics are those foods that contain different types of nondigestible fiber as a fuel source for microbiota. The prebiotics include mostly vegetables such as artichokes, leeks, garlic, onions, asparagus, wheat bran/flour, bananas, and chicory root. It is important to note that when vegetables are cooked they lose at least a third of their fiber content [52].

### *Mitigating Diabetic Foot Ulcers: The Effect of Diet and Microbiome DOI: http://dx.doi.org/10.5772/intechopen.106629*

Once SCFAs are produced, most are transported into colonocytes and are metabolized by the colonocytes as a nutrient for cell growth and metabolism. Very little of the SCFAs are transported into the systemic circulation. Acetate levels are likely high enough to have effects on the other organs, but very little if any, butyrate or propionate is thought to leave the portal blood and liver [55]. A limited amount is produced and absorbed in the small intestine, but the colon is certainly the major source [56]. Receptors for SCFAs exist in most of the major organs including the gastrointestinal tract. In the intestine and colon, SCFAs are linked to motility and maintenance of the epithelial barrier. SCFAs stimulate water absorption, while at the same time increasing motility, maintaining the epithelial barrier and therefore preventing constipation and diarrhea [57]. When entering into the systemic circulation, SCFAs also have profound effects on whole body health and metabolism. SCFAs are the source of fuel for the heart and the cardiovascular system, they control body weight and insulin release, and are precursors for lipid and glucose production in the liver (gluconeogenesis). Commensal bacteria have a number of health benefits which include the development of the gastrointestinal tract and other tissues such as the central nervous system, maintaining the immune system, and increasing metabolism [58]. Therefore, an imbalance or dysregulation of microbiota populations could disrupt normal metabolism and water balance and result in obesity, diabetes, and its symptoms and complications such as foot ulcers.

The major SCFAs produced are acetate (C2), propionate (C3), and butyrate (C4) which contain two, three, and four carbons, respectively. These SCFAs are mainly synthesized by the Firmicutes species *Lactobacillus* and also the *Actinomycetota* species *Bifidobacteria*. *Bacteroidetes* bacteria can also produce SCFAs but at lower levels. The SCFAs act as the primary source of energy for colonocytes. Different species of bacteria with different metabolic steps are responsible for the formation of each of the SCFAs. None of the bacteria have all of the necessary enzymes to produce all three of the SCFAs directly from dietary fiber. Nondigestible carbohydrates enter the citric acid cycle of bacteria at different points in different bacteria. Propionate is formed from succinyl-CoA, an intermediate of the citric acid cycle, as well as lactate and propanediol. Acetate and butyrate are formed from acetyl-CoA [59, 60]. In addition to entering the citric acid cycle and being used as an energy source, SCFAs also have profound epigenetic effects to increase the rate of gene transcription by inhibiting histone deacetylase (HDAC) activity [61]. The SCFAs affect many genes that regulate transcription and colonocyte homeostasis [62, 63]. These accumulated epigenetic events are thought to increase the incidence of colorectal cancer [64, 65]. Butyrate is synthesized from acetate, while propionate is synthesized by butyrate-producing bacterial species such as *Faecalibacterium, Eubacterium, and Anaerostipes* [66, 67]. Additionally, to absorption (i.e., about 95% of SCFAs produced), some of the SCFAs are excreted in the feces [60]. The amount of SCFAs produced can vary greatly by the content and type of dietary fiber, and the number and species of bacteria. Other factors such as antibiotics, bypass surgery, and stress can also alter the microbiota species composition and SCFAs production. Local sanitation and the introduction of microbes into the diet determine whether the proper microbiota is developed in the GI tract. Amazingly, people in countries with the best sanitation introduce fewer microbes into the microbiota, which consequently leads to dysbiosis and diseases such as obesity and diabetes [49].

SCFAs can exert physiological function either during transport across the colonocytes or by binding to their receptors located on the gastrointestinal mucosa. A number of orphan G-protein-linked receptors were discovered and initially had no known ligands [68]. However, later studies demonstrated that SCFAs activated several of these G-protein-linked receptors, and several of them are identified to present in the mucosa of intestine and colon [69]. They include the G-protein coupled receptor (GPR) GPR43 (acetate and propionate) and GPR41 (propionate and butyrate), which have about 40% homology across species [70]. GPR43 and GPR41 are also known as free fatty acid receptors, (FFAR) FFAR2, and FFAR3, respectively. Acetate and propionate are ligands for GPR43 (FFAR2), while propionate and butyrate are ligands for GPR42 (FFAR3) [45]. Butyrate also binds to GPR109a, which is distributed along the colon, T-cells, and the microglia. GPR43 is highly expressed in immune cells, adipose tissue (stimulates fat deposition), distal ileum (increases motility, and stimulates peptide-YY (PYY) and glucagon-like peptide-1 (GLP-1) secretion), skeletal muscle, and the heart [71] that are tissues all involved in diabetes and obesity. Therefore, during dysbiosis and diabetes when there is a lack of SCFAs this could contribute to a decrease in immune responses and cause cardiovascular disorders. The main function of GPR43 is to maintain energy homeostasis within the body, GPR43 increases energy released by improving glucose tolerance and increasing energy utilization within the body [72]. GPR43 is also present in pancreatic β-cells and stimulates insulin secretion, which in turn increases glucose absorption into the tissues to aid increased energy utilization. These aspects were demonstrated using GPR43 knockout mice [73, 74]. Therefore, when SCFAs are not synthesized j in sufficient quantities then that would result in difficulties in maintaining normal energy homeostasis and glucose utilization. Although most of the SCFAs are utilized in the colon, more dietary fiber consumption, resulting in greater amounts of SCFAs, gets absorbed into the systemic circulation and thus interacts with other tissue receptors. Although further studies are required, it is likely that SCFAs increase body metabolism, as high fiber diets result in weight loss and reduce blood lipid levels. Additionally, increases in HDL levels are observed, which lowers the chances of atherosclerosis, but an increase in blood cholesterol levels has been shown in some studies [47, 75–77]. However, this reported evidence indicates that SCFAs activate receptors that release GLP-1 and increase insulin secretion to regulate blood glucose levels [70]. In the pulmonary system, SCFAs protect against inflammation by activating the GPR41 and reducing hematopoiesis and infiltration of immune cells [78]. Loss of these functions would result in weight gain, cardiovascular disorders, and insulin resistance.

Altered microbiota populations have been detected in several diseases including obesity and conditions resulting from the overuse of antibiotics. The overuse of antibiotics has been shown to decrease Bacteroidetes and increase Firmicutes [14]. Metabolic activity of Bacteroidetes and Firmicutes taxa determined by 16s RNA analyses revealed that the Firmicutes tend to be more active than that of Bacteroidetes taxa. Even with the cessation of antibiotic treatment, some species, mainly Firmicutes, never return to normal levels [76, 79].

When microbiota utilize fats and metabolize less fiber in the diet, the microbiota synthesize fewer SCFAs but also produce metabolites that contribute to obesity. When the ratio of *Firmicutes* to *Bacteroidetes* increases that contributes to an increased risk of diabetes and obesity [80]. However, a change in the ratio does not always lead to diabetes [81, 82] because there can be regional differences in the expression of the microbiota in lean and obese individuals which likely depends on the diet at that locale [83, 84]. However, dietary requirements for modulation of diabetes may not have enough impact but it is certainly important since increases in fiber in the diet reduced the symptoms of diabetes in patients [80, 85]. Many of these food items rich in fiber also contain large amounts of sugars and carbohydrates. It is interesting that

## *Mitigating Diabetic Foot Ulcers: The Effect of Diet and Microbiome DOI: http://dx.doi.org/10.5772/intechopen.106629*

with weight loss comes changes in microbiota, but the research design many times do not reflect enough which came first, the change in weight or the change in microbiota making interpretation of results difficult.

There are two major phyla of bacteria that are present in the gut microbiome. The ratio of these bacteria is balanced in healthy lean individuals. Firmicutes are considered to be the "bad" bacteria but they are the major phyla that produce the short chain fatty acids needed as a fuel source for the colon [86, 87]. Without the SCFA when the Bacteroidetes become the dominant phyla with fewer *Firmicutes* colon will become more permeable to bacterial infections and diseases such as Inflammatory Bowel Disease although some report even *Bacteroidetes* are also reduced [88]. Other phyla include Actinobacteria as they increase significantly decrease the risk of developing diabetes [89], *Proteobacteria* increases have been linked to dysbiosis and may not cause diabetes but those with diabetes have increased amounts, particularly in TTDM [90], and *Verrucomicrobia* is also increased in diabetic patients [91].

High fat low fiber diets tend to diminish and cause dysbiosis of the colon microbiota. Fermented foods are considered to be among the best foods that can replace or restore the normal balance of microbiota in humans [14]. Yogurt is considered to be among the best, but only recently have there been any definitive studies. *Bifidobacterium animalis* and *Lactobacillus* species are said to be the most beneficial and are found in yogurt and other fermented milk products [14, 92]; however, immune deficient patients should avoid yogurts and other fermented milk products since they could be susceptible to a fatal form of septicemia [93]. These short-chain fatty acids (SCFAs) are not just linked as a fuel source for colonocytes in the large intestine but also to proper immune responses and proper intestinal permeability to prevent bacterial infections [88]. Parasitic infections which can result from increased permeability such as toxoplasmosis, hydatidosis, and cysticercosis infect a large population worldwide. *Toxoplasma gondii* in the pancreas could damage the pancreatic cells. Hence, insulin secretion would be affected which leads to an increased risk of diabetes [94].
