**3. The role of the microbiota in specific diseases and conditions**

### **3.1 Inflammatory bowel disease**

Inflammatory Bowel Disease (IBD) defines a group of chronic disorders that includes Crohn's disease (CD) and Ulcerative colitis (UC). Though they are two different diseases, they both affect the intestinal tract and are characterized by intestinal inflammation with periods of remission and relapse [30]. The incidence of IBD is consistently growing in the recent few decades, having a peak onset age between 15 and 35 years that was initially described in the western populations, and now is also more frequent in other countries, as processed food and animal-based diets are overtaking the plant-based diet [31].

The etiology of IBD is an important subject of discussion as it is not fully understood. The key ways proposed as mechanisms for developing inflammation in IBD are the genetic susceptibility and environmental factors that interact with the immune system. Thus, the host gives an inappropriate immune response to changes of the gut microbiome and modulates inflammation and disease involvement and activity [32, 33].

The interaction between the host and different environmental factors, such as infections, smoking, dietary habits, psychological stress, medications, and alcohol consumption leads to alterations in the balance between gut microbiota and the genetically predisposed host. This imbalance changes the complex interactions of the immune system and products of the commensal microbiota that trigger immune responses using inflammatory mediators and signaling pathways. Hence, prolonged imbalance of the gut microbiota (including the microbiome, mycobiome, virome, and protozoa) with changes of the composition with a decrease of the commensal phyla and increase of potential pathological microorganisms, defined as dysbiosis, induce the alterations and dysregulations of mucosal barrier [34–36].

The dysfunction of the mucosal immune barrier has been shown in mouse studies that can regulate the development of T regulatory (T reg) cells and T helper 17 (Th17) cells with important differentiation in healthy and sick subjects. The activation of Th17 cells is important in bacterial and fungal infections, releasing pro-inflammatory interleukine (IL) 17 cytokines, important in the pathogenesis of colitis. T reg cells play an important role in the suppression of inflammation through transforming-growth factor B (TGF-B), interleukine (IL) 35, and IL10. The deficiency of T reg cells leads to inflammation and IBD [33, 37–39]. Their role is important against *Citrobacter rodentium* and *Salmonella enterica* and was shown to be decreased in *Bacteroides* increased microbiome. Also, *Clostridium* clusters showed the ability to act on the differentiation of T reg cells [34, 37, 40, 41].

The dysbiosis occurring in IBD affecting bacterial microbiota is the most studied section of the gut microbiota. The most frequent phyla that are seen in healthy subjects are *Bacteroides, Bifidobacterium spp, Fecalibacterium spp, Firmicutes spp, Roseburia spp, Actinobacteria, and Verrucomicrobia* are regarded as over 90% of the gut microbial families [30, 32, 34]. Patients affected by IBD, in general show a decreased presence of mentioned phyla and an increase in *Proteobacteria spp, Escherichia coli spp*, *Fusobacterium spp, Ruminococcus spp, Pasteurellaceae spp, Veillonellaceae, Campylobacter spp,* and *Clostridioides spp*. There have been shown differences in composition and diversity regarding UC and CD, regarding also the extension of disease, aggressivity, and activity, thus being able to use the microbiome changes as a biomarker for disease activity and response to treatment [30, 34].

Regarding composition and diversity, there is a common agreement that in CD patients is a greater degree of dysbiosis compared to UC. Studies using 16 s rRNA sequencing characterized the gut microbiome in IBDs, showing a decrease of *Anaerostipes, Methanobrevibacter, Fecalibacterium* (especially *F.prausnitzii*), *Peptostreptococcaceae, Collinsella, Bifidobacteria* (especially *Bifidobacterium adolescentis*), *Dialister invisus, Clostridioides* cluster XIVa, *Bacteroides fragilis, Roseburia, Firmicutes* and *Erysipelotrichales* in CD and an increase of *Proteobacteria (Campylobacter), Yersinia enterocolitica, Bacteroides (vulgatus, fragilis), Helicobacterhepaticus, Mycobacteria spp, Enterobacteriaceae* (pathogenic *E.coli, Shigella*), *Ruminococcus gnavus, Veillonellaceae, Fusobacteriaceae,* and *Pasteurellaceae*, in human and animal models [30, 34].

These bacterial taxa are different from those expressed in UC, where a decrease of *Roseburia, Eubacterium, Faecalibacterium, Akkermansia, Bifidobacterium* and an increase *Helicobacteraceae, Mucispirillum, Desulfovibrio, Clostridioides ramnosum,* and *Porphyromonas* differentiate from common alterations of the microbiome seen in both CD and UC [34, 35, 42, 43].

Regarding disease phenotype, there have been a few studies about a range of specific gut bacteria changes associated with different patterns in CD. Li et al. [44] showed that individuals with ileal CD showed an increase in *Actinobacteria spp* and *Firmicutes/Bacillus* and a decrease in *Ruminococcus spp* [44]. Also, this phenotype was associated with an absence of *Roseburia* and *F. prausnitzii*, and an increase of *E. coli* [45]. In addition, decreased presence of *F. prausnitzii* in patients with ileal resection in CD, showed an increase in recurrence [46].

The regulation of gut mucosal immunity and host immune response is made through bacterial physiology and interaction on cell growth and interaction with metabolites produced by the microbiome. The stability of mucosal inflammation is disrupted in IBDs with the alteration of immunomodulatory metabolites such as SCFAs (acetate, propionate, and butyrate), bile acids, and tryptophan metabolites. SCFAs are mostly represented by acetate and are produced by *Bacteroidetes* and *Firmicutes*, and there has been demonstrated an important reduction in IBDs while associated also with reduced SCFA-producing bacteria such as *F. prausnitzii, R.intestinalis*. Another study also demonstrated decreased specific taxa for CD as *Phascolarctobacterium* and *Roseburia* and for UC *Leuconostocaceae spp* [32, 38, 47].

Given the alterations of gut microbiota and metabolites in IBD, there have been developed and proposed several management strategies for controlling the microbiome. Probably the most studied approach is using probiotics, which are bacterial species that may promote the maintenance of the immunological balance [48]. The effectiveness of probiotics in improving IBD evolution has been exhibited using different strains of *Lactobacillus, Bifidobacterium, Streptococcus,* and *Saccharomyces*. Their efficacy was seen in maintaining remission in UC patients by reducing pro-inflammatory cytokines and restoring normal gut microbiota. Nevertheless, the use of probiotics in CD showed little or no implication [31, 48, 49]. Often administered with oral probiotics, are the substrates, such as fructooligosaccharides, pectins, starch, and fibers, targeting microbiome composition by aiding the development of normal gut microbiota [50].

The use of antibiotics for their role in the modulation of microbiota is controversial. They function by decreasing the concentrations of different bacteria in the gut and reducing tissue invasion and translocation, acting also on metabolism with a decrease of pro-inflammatory metabolites and an increase of SCFAs. However, the non or very little selectivity character of antibiotics alter also the composition of some beneficial bacterial strains and their use is kept for septic and infectious complications, such as *Clostridioides difficile* infection [32, 48, 51, 52].

An important method of influencing the microbiome is Fecal Microbiota Transplantation (FMT), a very attractive method with significant rates of success, that is known from as early as fourth century [53]. As well as probiotics, FMT was better studied and showed important results in UC, and less in CD [34, 54, 55]. In UC, in mild-tomoderate cases, usage is still modest as it managed to induce response and remission in 20–55% of cases being comparable with active treatment as reflected in decreasing Mayo score and reducing symptoms [54, 56]. An important use of FMT is also recommended in recent guidelines for recurrent infection [57]. It remains a subject of future studies' better selection of FMT donors as currently being no possibility of predicting the success of a given donor to an IBD patient, thus defining an "ideal" donor [53].

The changes in lifestyle and diet represent the most common intervention on the microbiome, and of paramount interest being the first recommendation and the easiest to accept the measure. Diets rich in vegetables, fermented foods probioticrich (kimchi, kefir, yogurt, and pickled vegetables), fibers, and prebiotics have a positive impact on intestinal barrier health and microbiome balance [35, 50]. Currently, there are some diet recommendations for IBD and the most studied diets are Low Fermentable Oligosaccharides, Disaccharides, Monosaccharides and Polyols (FODMAP), Crohn's disease exclusion diet, and Mediterranean diet (MD). A low FODMAP diet was found to have a good improvement in disease clinical scores in mild cases of IBD that are associated with IBS (Irritable Bowel Syndrome). MD characterized by low saturated fat, high monounsaturated fat, fiber, high vitamin B, C, E, and moderate ethanol intake showed in a few studies on CD patients' improvements of the quality of life and mild reducing fecal calprotectin an serum CRP [35, 58–61]. Another diet studied is a plant-based diet that exerts anti-inflammatory effects, composed of whole grains, cereals, fruits, vegetables, and nuts showed good improvements regarding symptoms, lowering serum CRP, overall WBC, but with the price of requiring supplementation of micronutrients [31, 62].

### **3.2 Acute and chronic pancreatitis**

Acute pancreatitis (AP) is defined as an inflammatory condition of the pancreas following the injury of the pancreatic serous acini, leading to premature activation

### *Intestinal Microbiomics in Physiological and Pathological Conditions DOI: http://dx.doi.org/10.5772/intechopen.110642*

of digestive enzymes (trypsin, chymotrypsin, lipase, and elastase) [63]. The clinical severity of AP cases depends on their complications, which can be localized (sterile or infected peri/pancreatic necrosis) or systemic (transient or persistent organ failure) into mild, moderate, severe, and critical AP [64]. The evolution of AP can be summarized in three stages: (1) local inflammation of the pancreas; (2) systemic inflammatory response syndrome; and (3) multiple organ dysfunction syndrome [65–67].

The revised Atlanta classification identifies two main stages of AP: (a) interstitial edematous pancreatitis and (b) necrotizing pancreatitis (NP) [68].

Although often overlooked, the gut microbial community and the gut barrier integrity disruption were described as aggravating factors responsible for the amplification of the initial inflammatory process accompanying AP [69]. Apparently, according to Liu et al. 2008 in AP patients, with mild and severe forms, there is an early gut mucosal dysfunction, leading to the development of multiple organ dysfunction [70]. The mucus layer integrity in the gut lining is lost after the onset of AP as shown by Fishman et al. 2014, leading to the failure of the gut barrier, apparently due to mechanisms independent of the activity of the pancreatic proteases in the intestinal lumen [71]. Pancreatic necrosis is accompanied by a lot of inflammatory cytokines and determines multiple changes in the gut such as a decrease in intestinal motility, favoring bacterial overgrowth and malnutrition and followed by gut barrier failure and increased permeability [72]. The intestinal permeability is highly increased in severe forms of AP and favors a poor prognosis.

The gut mucosal secretions also contain important quantities of secretory IgA, a key immunoglobulin that prevents the adhesion of pathogens and is responsible for the maintenance of immune homeostasis [73]. Usually, the amount of sIgA found in the small intestine is directly correlated with bacterial eubiosis and diversity. A decrease in sIgA is often correlated with low bacterial diversity in the small intestine and increased permeability and bacterial translocation leading to severe AP and infection [74].

The study by Yu et al. 2020 performed the 16S rRNA sequencing of gut microbiota species from fecal samples obtained through rectal swabs from 80 patients and described a correlation between gut microbiota and the severity of AP [75].

The microbiota profile was different, depending on the severity grade. In mild AP the main two phyla *Bacteroidetes* and *Firmicutes* were identified. *Bacteroides, Escherichia-Shigella,* and *Enterococcus* species were dominant while *Blautia* was highly decreased*. Finegoldia, Eubacterium hallii*, and *Lachnospiraceae* were considered to be potential diagnostic biomarkers for this stage of AP. In moderately severe AP, *Anaerococcus* was the most significantly increased and *E. hallii* the most decreased species, while in severe AP, *Enterococcus* was the most significantly increased and *E. hallii* the most decreased species. *Proteobacteria* phylum was the most increased in both, moderately severe and severe AP [75]. This study is impaired by several limitations such as possible contamination due to rectal swab samples and secondly by the impossibility to determine if microbiota dysbiosis is due to the presence of AP or is the main factor determining the AP severity. These findings are in correlation to those of the multihospital prospective clinical study performed by Tan et al. 2015 who describe dramatic alterations of the microbiota, determined by real-time quantitative polymerase chain reaction, in mild and severe forms of AP [76]. *Enterobacteriaceae* and *Enterococcus* were found to be increased by 3.2 and 9.3%, respectively, while the beneficial strains like *Bifidobacterium* were decreased by 9.2% in the severe forms of AP compared to mild forms [76]. The drawbacks of this study consist in the small sample size of patients with AP included and the lack of modern techniques like

high-throughput sequencing. Another study performed by Zhu et al. 2019 describes the reduction of other beneficial strains like *Blautia* in patients with severe AP [77].

The gut mucosal lining is affected by dysbiosis mainly through the metabolites produced by certain bacterial species. *Firmicutes* and *Bacteroidetes* are mainly responsible for the production of short-chain fatty acids (SCFAs), mainly acetate, propionate, and butyrate, the main energy source of enterocytes, colonocytes, and hepatocytes [78]. SCFAs are very important for the maintenance of tight junctions between the intestinal epithelial cells and also for the mucosal immune barrier [79]. In AP patients, there is a decrease in SCFAs promoted by dysbiosis and moreover, because of the decreased pH, it creates the condition for potential pathogenic and pathogenic bacteria, such as *E. coli* and *Shigella,* to grow and aggravate the evolution [80].

Experimental studies performed on mice suggested that microbiota regulation by fecal transplantation might reduce the damage at the intestinal barrier level and create a more stable evolution, preventing severe forms [80, 81]. Ding et al. 2021 showed in a randomized, controlled study registered at https://clinicaltrials.gov (NCT02318134) that the fecal microbiota transplantation had no beneficial effects in the evolution of severe forms of AP and moreover, the intestinal permeability might have been adversely affected [82].

Chronic pancreatitis (CP) is defined as a progressive and irreversible inflammation of the pancreas that leads to pancreatic exocrine insufficiency (PEI) and diabetes mellitus [83]. A normal pancreatic function provides antimicrobial peptides, bicarbonate, and digestive enzymes that are necessary for digestive function but also for the maintenance of healthy microbiota [84, 85].

The evidence accumulated in recent years regarding pancreatic exocrine deficiency advocates for small intestinal bacterial overgrowth (SIBO) and gut dysbiosisreduced diversity, and increased abundance of opportunistic pathogens [86, 87]. Capurso et al. 2016 also demonstrated in a meta-analysis that one-third of patients with CP have SIBO [88]. A study by Ní Chonchubhair et al. 2018 evaluated the relationship between SIBO and clinical symptoms in CP and found that SIBO was present in 15% of chronic pancreatitis patients [89]. Frost et al. 2020 recently determined the intestinal microbiota composition by bacterial 16S ribosomal RNA gene sequencing and found reduced alfa and beta microbial diversity index and an increased abundance of opportunistic pathogens in patients with CP. They found in CP cases an increase in abundance of *Enterococcus* and *Bacteroides* and an absolute reduction of *Faecalibacterium* and *Prevotella* [86]. Talukdar et al. 2017 also described in their study a reduction of *Fecalibacterium prausnitzii* and *R. bromii* in CP without and with diabetes. Apparently, the gut barrier integrity is disrupted due to low *Fecalibacterium* levels and this favors the passage of bacterial endotoxins in circulation followed by subsequent alterations in the functionality of beta pancreatic cells [90].

As the studies indicated, there are some significant alterations in the composition and function of the gut microbiota in patients with AP and CP, leading to severe forms of disease and in correlation with a poor prognosis. The disturbance of the gut microflora equilibrium needs to be further explored in close correlation with the gut mucosal integrity and systemic inflammatory status.

### **3.3 Colorectal cancer**

Colorectal cancer (CRC) is the third most frequent cancer worldwide with more than 1.9 million new cases and 930.000 deaths reported in 2020. It is predicted that

### *Intestinal Microbiomics in Physiological and Pathological Conditions DOI: http://dx.doi.org/10.5772/intechopen.110642*

by 2040, the burden of the disease will be increased to 3.2 million cases per year and 1.6 million deaths per year [91]. Approximately 90% of CRC cases are sporadic [92], and various environmental and genetic factors contribute to CRC tumorgenesis [93]. Studies show that only a small percentage of CRC cases are genetically predisposed [93, 94], underlining the importance of environmental factors in the development of CRC. Diets rich in red and grilled meat, tobacco, high alcohol intake, disruption of circadian rhythm, and preexisting conditions, such as obesity, inflammatory bowel disease, and diabetes, have been associated with CRC [95]. In addition, the intestinal microbiota is getting more and more recognition among environmental factors implicated in the development of CRC, evidence dating as early as the 1960s. One study published in the late 1960s demonstrated that glucoside cycasin failed to produce its carcinogenic effect in germ-free mice and was only able to induce cancer in conventional rats [96]. In 1975 Reddy et al. showed that a large dose of 1,2-dimethylhydrazine induced multiple colonic tumors in 93% of the conventional rats included in the study, whereas 1,2-dimethylhydrazine-induced colonic tumors were observed in only 20% of the germ-free mice [97]. Moreover, subcutaneous administration of azoxymethane led to an increased incidence of colonic tumors in germ-free rats, indicating that intestinal bacterial populations can alter the carcinogenic effects of certain compounds in the colon [98].

Studies on humans, that have analyzed both mucosal and fecal samples, demonstrate that the gut microbiota of CRC patients differs significantly from that of healthy subjects, CRC patients presenting diminished richness and bacterial diversity [99–101]. Also, Chen et al. 2012 observed that the microbial composition in cancerous tissue is significantly different from that found in the intestinal lumen [102]. Numerous bacteria have been correlated with CRC in spite of variations in intestinal microbiota [99, 100].

*B. fragilis*, a bacteria that colonizes most humans [103] *F. nucleatum, Prevotella intermedia, Parvimonas micra, Porphyromonas asaccharolytica, Alistipes finegoldii,* and *Thermanaerovibrio* are bacteria identified by one meta-analysis to be enriched in CRC [104]. In 2019 two more meta-analyses investigating the fecal metagenome in CRC have been published, expanding the list of CRC-enriched bacteria [105, 106].

Not only has an increase in the population of *F. nucleatum* been associated with CRC, but also it is thought to promote disease progression [107]) and its presence in CRC tissues might be indicative of a worsen prognosis [108, 109]. A recent study found increased levels of *P. intermedia* and *F. nucleatum* in adenocarcinomas compared with paired adenomatous polyps. The presence of this bacteria was shown to exert an additive effect on the migration and invasion of CRC cells and was also associated with lymph node involvement and distant metastasis [110].

Increased levels of *Enterococcus faecalis, E. coli,* and *Peptostreptococcus anaerobiusi* in CRC patients in comparison with healthy controls was also reported by several authors, but the exact mechanisms by which these bacteria promote cancer development is still to be determined [100].

The enriched bacteria are also associated with reduced levels of benefic bacteria, such as *Clostridium butyicum and Streptococcus thermophilus*, [104] bacteria belonging to the genus *Roseburia* and other butyrate-producing bacteria [111]. *Wang et al. 2012* highlight that the decrease in butyrate-producing bacteria and the opportunistic pathogen multiplication might be responsible for the structural imbalance of gut microbiota in patients suffering from CRC [111]. Short-chain fatty acids (SCFAs) are fermentation end products produced by bacteria, with butyrate being the most intensively studied SCFA. Apart from being considered the energy source for

colonocytes, they also promote the apoptosis of cancer cells [112]. The amount of SCFAs produced by the microbiota is however insufficient to inhibit CRC development and probiotic supplementation might result in increased SCFAs. One *in vitro* study showed that *Lactobacillus fermentum* NCIMB 5221 was able to increase SCFAs production, thus exerting antiproliferative effects against Caco-2 cancer cells and promoting normal epithelial cell growth [113]. Resistant starch (RS) is part of starch that is fermented into SCFAs in the cecum and this process leads to pH decrease. Prebiotic supplementation with RS has been demonstrated to reduce the proliferation of epithelial cells in the colon and rectum [114, 115]. Moreover, the administration of synbiotics, meaning the combinations of prebiotics and probiotics has also been investigated. In one RCT patients with a history of CCR received a synbiotic preparation composed of oligofructose-enriched inulin and two probiotics *Lactobacillus rhamnosus* GG and *Bifidobacterium lactis* Bb12. The synbiotic intervention resulted in significantly reduced colorectal proliferation, an increase in the number of beneficial bacteria, cytokine production modulation (decreased interleukin (IL) 2 and increased IFN-gamma production), and a decreased genotoxins exposure, which translates into a reduction in DNA alterations [116].

The role of the intestinal microbiota in CRC tumor progression is also supported by the differences in bacterial composition between patients with early-stage adenomas and those in advanced stages with definitive CRC [92].

Nevertheless, the CRC microbiome is also characterized by an imbalance in the composition of the viral and fungal species [92, 99]. A higher viral load has been observed in tumors compared to normal tissue of CRC patients [92]. Although some studies have identified cytomegalovirus, John Cunnningham virus, and human papilloma virus in CRC tumor samples, the data are however inconsistent [99]. Shotgun metagenomic analyses of viromes of fecal samples identified 22 viral taxa that differentiate the CRC virome from one of healthy controls [117]. Trans kingdom crosstalk between bacteria and viruses may play an important role in CRC tumorigenesis, as some studies indicate [118]. Although less studied, differences in terms of fungal composition were also observed [119, 120].

Existing studies suggest that several carcinogenesis mechanisms involved in the development of CRC are intimately linked to the gut microbiota. Among studies, authors have insisted on the mechanisms of inflammation, oxidative stress, pathogenic bacteria, genotoxins, and biofilm [100]. Studies have demonstrated that some bacterial species, such as *F. nucleatum* [121] and *P. anaerobius* [122], can induce a pro-inflammatory immune microenvironment, which leads to the progression of colorectal neoplasia. The immunomodulatory capacity of probiotics has led scientists to investigate probiotics in the management of CRC. Oral administration of a mixture of six viable strains of *Lactobacillus* and *Bifidobacterium* in patients with CRC 4 weeks after surgery resulted in a significant pro-inflammatory cytokine reduction compared to placebo administration. The levels of tumor necrosis factor (TNF-α), IL-6, IL-10, IL-12, IL-17A, IL-17C, and IL-22 were significantly reduced, and no severe adverse reactions were reported [123]. After comparing the intestinal microbiota of CRC patients with that of healthy patients, one study analyzed the possibility of preventing colorectal carcinogenesis by modulating the composition of the intestinal bacterial population using *L. gasseri*. Probiotic administration resulted in an increase in the *Lactobacillus* population and a decrease in the amount of *Clostridium perfringens* as well as a shift in fecal pH toward acidosis along with an increase in IL-1 and natural killer (NK) cell activity values starting with week 4 [124].

### *Intestinal Microbiomics in Physiological and Pathological Conditions DOI: http://dx.doi.org/10.5772/intechopen.110642*

Additionally, through their adhesion capacities, pathogens and their virulence factors adhere to the intestinal epithelial cells (IECs) and promote tumor formation [122, 125– 127]. Also, the gut microbiota can modulate the immune system response by stimulating the production of chemokine in tumoral cells with the purpose of recruiting T lymphocytes [128]. Moreover, bacterially produced genotoxins, exert DNA damage in IECs, which can further initiate carcinogenesis. For example, *E. coli* produces the genotoxin colibactin [129, 130] which is reported to induce transient DNA damage in epithelial cells [130]. Similarly, *Salmonella* damages the DNA in IECs by producing typhoid toxin [131]. Inflammation can lead to increased levels of ROS (reactive oxygen species) and RNS (reactive nitrogen species), its negative impact translating into DNA damage and the development of mutations. *E. faecalis* [132], *P. anaerobius* [133], *E. coli,* and enterotoxigenic *B. fragilis* [134, 135] promote ROS production by colonic cells. Enterotoxigenic *B. fragilis,* through its metalloprotease toxin and its effect on IL-17 pathway, is believed to promote carcinogenesis in colonic cell population [136, 137]. Microbiota, also found as a biofilm at the surface of the colon mucosa, can promote colonic tumor cell proliferation through modulating interleukin 6 and STAT3 signaling pathways [138, 139].
