Gut Microbiota Manipulation in Irritable Bowel Syndrome

Increased knowledge suggests that disturbed gut microbiota, termed dysbiosis, might promote the development of irritable bowel syndrome (IBS) symptoms. Accordingly, gut microbiota manipulation has evolved in the last decade as a novel treatment strategy in order to improve IBS symptoms. In using different approaches, dietary management stands first in line, including dietary fiber supplements, prebiotics, and probiotics that are shown to change the composition of gut microbiota, fecal short-chain fatty acids and enteroendocrine cells densities and improve IBS symptoms. However, the exact mixture of beneficial bacteria for each individual remains to be identified. Prescribing nonabsorbable antibiotics still needs confirmation, although using rifaximin has been approved for diarrhea-predominant IBS. Fecal microbiota transplantation (FMT) has recently gained a lot of attention, and five out of seven placebo-controlled trials investigating FMT in IBS obtain promising results regarding symptom reduction and gut microbiota manipulation. However, more data, including larger cohorts and studying long-term effects, are needed before FMT can be regarded as a treatment for IBS in clinical practice.


Introduction
Irritable bowel syndrome (IBS) has become the most common gastrointestinal disease for referral to the gastroenterologists due to the patients' complaints of abdominal pain, bloating and mixed bowel movements of diarrhea and/or constipation, which can range from mild to severe [1,2]. IBS can cause several extraintestinal symptoms such as headache, tiredness, fibromyalgia and poor social functioning and emotional well-being [3]. Though IBS does not cause increased mortality or cancer incidence [4], the severity of the symptoms reduces the quality of life of the patients, which leads them to skip work or school, reducing their daily productivity and causing a financial burden to society [5,6].
According to a systemic review and meta-analysis [7], the global prevalence of IBS using Rome IV criteria for the diagnosis of IBS and its subtypes [8] is 3.8% [7], but this prevalence was higher in the Western world population using the older criteria Rome III, reaching to 9.2% [7,9]. Diarrhea-predominant IBS is the most common subtype using Rome IV criteria, while mixed-type IBS was the most common subtype using Rome III criteria [7]. IBS is more common in women than in men [7]. The diagnosis of IBS is mainly a diagnosis of exclusion due to the common symptoms that can mask those of other organic diseases, most importantly, celiac disease, inflammatory bowel diseases and colorectal cancer [10].

Diet
The first-line treatment for IBS has concentrated on changing the patient's dietary habits [49]. Dietary management has focused on reducing the consumption of certain carbohydrates that are highly fermentable but poorly absorbable, mainly the fermentable oligo-, di-, mono-saccharides and polyols (FODMAPs) that aggravate IBS symptoms, Table 1. When FODMAPs are poorly absorbed in the small intestines, they reach the colon, where they become fermented by the bacteria there, thus producing gas that causes bloating and osmotic changes leading to altered bowel motions [50]. There are at least 10 randomized controlled trials or randomized comparative trials that show that following a low FODMAPs diet improves global IBS symptoms, bloating, flatulence and diarrhea in 50-80% of the patients [51]. Previous publications have shown that changing the type of the consumed diet can change the gut microbiota in these patients with a parallel improvement in their symptoms [44,[52][53][54], Table 2. Several studies showed that using a low FODMAPs diet can reduce the abundance of several bacteria such as Bifidobacterium, Clostridium and Faecalibacterium prausnitzii in feces and increase the richness of Actinobacteria, Firmicutes and Clostridiales [44,53,54], but reported conflicting results concerning other bacterial types and the levels of microbiota metabolites, summarized in Table 2. A study by Staudacher et al. following IBS patients using a low FODMAPs diet for 12 months showed no difference in Bifidobacteria abundance in stool microbiota analysis [55]. However, there were lower concentrations of total fecal short-chain fatty acids, acetate, propionate and butyrate, before and after dietary management [55]. The study concludes that after completing all three phases of the low FODMAPs diet (restriction, reintroduction and personalization), it is safe and effective to follow a low FODMAPs diet for long-term when patients are supervised by a dietician [55]. The responsiveness to a low FODMAPs diet may be predicted by fecal microbiota profiles; for example, Phascolarctobacterium are more abundant in responders, and Firmicutes (Bacilli and Clostridia), Streptococcus, Dorea, Coprobacillus and Ruminococcus gnavus are more abundant in non-responders [56]. However, coadministration of multi-strain probiotics preparation (VSL#3) containing Streptococcus thermophilus, Bifidobacterium breve, B. longum, B. infantis, Lactobacillus acidophilus, L. plantarum, L. paracasei, L. delbrueckii subsp. bulgaricus, to the low FODMAPs diet restores levels of Bifidobacterium species [52].
Dietary fibers play an important role as stool bulking agents as they cause water retention and may increase transit time [57]. They are divided into water-soluble and waterinsoluble fibers and consist of short-and long-chain carbohydrates and lignin, Table 1. In IBS, using water-soluble fibers (psyllium) improves IBS symptoms, while water-insoluble fibers exacerbate them [20]. In addition, supplementing one's diet with fructo-and galactooligosaccharides increase the abundance of Bifidobacterium due to their prebiotic activity, thus having a beneficial effect on the colon. Moreover, fermenting the dietary fibers by the colonic bacteria leads to the production of fecal short-chain fatty acids (propionate and butyrate) that reflect the activity of the gut microbiota [57][58][59][60].
In studies performed by our group, the aforementioned dietary modifications, using low FODMAPs and changing the dietary fiber intake, also showed significant changes in the densities of the enteroendocrine cells [20]. These cells are scattered throughout the whole gastrointestinal tract and are responsible for releasing the gut hormones that control the functions of the gastrointestinal tract after stimulating their microvilli with different types of nutrients [20]. In biopsies taken from different parts of the gastrointestinal tracts (stomach, small and large intestines) and dyed using special immunohistochemical staining, the densities of the different enteroendocrine cells in IBS patients at baseline were abnormal compared to healthy controls; however, they normalized toward the cells densities measured for healthy controls after dietary modifications [12,20,[61][62][63][64][65][66].

Prebiotics, Probiotics and Antibiotics
Prebiotics, such as trans-galactooligosaccharide, are fermentable poorly-absorbable food elements that provide essential nutrients to enhance the growth of beneficial bacteria, such as Bifidobacterium and Lactobacillus, and have an anti-inflammatory effect that deters harmful pathogens in the bowels, which may contribute to the improvement of the global symptoms of IBS [67,68]. Probiotics are living microorganisms that consist of bacteria (mainly Bifidobacterium and Lactobacillus) and yeast, which are friendly to the gut and confer health benefits to the host when given in adequate amounts, usually in tablet forms or consumed in yogurt [58]. Probiotics have been used to beneficially manipulate the dysbiotic gut in IBS patients by improving the function of the gut barrier, inhibiting the overgrowth of pathogenic bacteria and producing short-chain fatty acids and several neurotransmitters [69], normalizing IL-10 and IL-12 levels and suppressing pro-inflammatory cytokine expression [70], Figure 1. Fifty-three randomized-controlled trials involving 5545 patients showed that probiotics appeared to have beneficial effects on global IBS symptoms and abdominal pain [71]. When it comes to the type of probiotics to be used, those that contain multiple bacterial strains (such as a combination of Bifidobacterium longum, B. bifidum, B. lactis, Lactobacillus acidophilus, L. rhamnosus and Streptococcus thermophilus, known as LacClean Gold [72,73] or the combination of seven bacterial strains, namely; Lactobacillus acidophilus, L. plantarum, L. rhamnosus, Bifidobacterium breve, B. lactis, B. longum and Streptococcus thermophilus [74], are more beneficial than monostrain probiotics in alleviating IBS symptoms; however, the effects that the probiotics have on improving IBS symptoms are short-termed and do not last for a long time [58,75]. dysbiotic gut in IBS patients by improving the function of the gut barrier, inhibiting the overgrowth of pathogenic bacteria and producing short-chain fatty acids and several neurotransmitters [69], normalizing IL-10 and IL-12 levels and suppressing pro-inflammatory cytokine expression [70], Figure 1. Fifty-three randomized-controlled trials involving 5545 patients showed that probiotics appeared to have beneficial effects on global IBS symptoms and abdominal pain [71]. When it comes to the type of probiotics to be used, those that contain multiple bacterial strains (such as a combination of Bifidobacterium longum, B. bifidum, B. lactis, Lactobacillus acidophilus, L. rhamnosus and Streptococcus thermophilus , known as LacClean Gold [72,73] or the combination of seven bacterial strains, namely; Lactobacillus acidophilus, L. plantarum, L. rhamnosus, Bifidobacterium breve, B. lactis, B. longum and Streptococcus thermophilus [74], are more beneficial than monostrain probiotics in alleviating IBS symptoms; however, the effects that the probiotics have on improving IBS symptoms are short-termed and do not last for a long time [58,75]. On the other hand, the use of nonabsorbable antibiotics, such as rifixamin, has been investigated in several double-blinded and placebo-controlled randomized controlled trials [71,76] and has been approved by the US Food and Drug Administration for treating patients with diarrhea-predominant IBS [77]. It is suggested that the beneficial effect of using rifixamin in repeated courses occurs by reducing the total load of the gut microbiota On the other hand, the use of nonabsorbable antibiotics, such as rifixamin, has been investigated in several double-blinded and placebo-controlled randomized controlled trials [71,76] and has been approved by the US Food and Drug Administration for treating patients with diarrhea-predominant IBS [77]. It is suggested that the beneficial effect of using rifixamin in repeated courses occurs by reducing the total load of the gut microbiota and modulating intestinal permeability, thus improving bloating and diarrhea in these patients [78]. The combination of rifixamin and neomycin has improved constipation and bloating in patients with constipation-predominant IBS [79].

Fecal Microbiota Transplantation
Fecal microbiota transplantation (FMT) has recently become popular as a novel method for modulating gut microbiota in gastrointestinal disorders such as inflammatory bowel syndrome, IBS and recurrent C. difficile infection [80][81][82][83], and non-gastrointestinal diseases such as chronic fatigue syndrome, obesity and even some neuropsychiatric disorders [83,84]. During FMT, a suspension made from fecal material, which is collected from healthy individuals, is infused into the gut of the patient via naso-jejunal tube, gastroscope or colonoscope [83], Figure 2. FMT is currently only used in clinical research trials and is considered a safe procedure once one adheres to the current guidelines [85]. Before performing FMT, screening of the donors should include performing thorough physical and laboratory investigations by blood and stool analysis and culture to rule out organic disorders, infectious agents and contagious diseases, most importantly, HIV, viral hepatitis, syphilis, malaria, tuberculosis and trypanosomiasis, to avoid transmitting them to the recipient [86]. It is advisable that the donors of the feces have not recently used antibiotics, travelled to tropical areas, had high-risk sexual behavior or had a bout of gastroenteritis or diarrhea within 4 weeks of donation [85]. It is not yet clear what is the correct dose or the frequency of FMT that should be performed on patients with IBS; however, according to the consensus guidelines, at least 30 g of donor feces should be added to the saline solution in order to prepare the fecal suspension that should be either stored at −80 • C or infusion directly on the same day of preparation [85]. After performing FMT, the stool will be collected from the recipients and stored in a special freezer at −80 • C for further microbial analysis [80]. Table 3 describes the different randomized controlled FMT trials, the dosages and the frequency of fecal transplants. and modulating intestinal permeability, thus improving bloating and diarrhea in these patients [78]. The combination of rifixamin and neomycin has improved constipation and bloating in patients with constipation-predominant IBS [79].

Fecal Microbiota Transplantation
Fecal microbiota transplantation (FMT) has recently become popular as a novel method for modulating gut microbiota in gastrointestinal disorders such as inflammatory bowel syndrome, IBS and recurrent C. difficile infection [80][81][82][83], and non-gastrointestinal diseases such as chronic fatigue syndrome, obesity and even some neuropsychiatric disorders [83,84]. During FMT, a suspension made from fecal material, which is collected from healthy individuals, is infused into the gut of the patient via naso-jejunal tube, gastroscope or colonoscope [83], Figure 2. FMT is currently only used in clinical research trials and is considered a safe procedure once one adheres to the current guidelines [85]. Before performing FMT, screening of the donors should include performing thorough physical and laboratory investigations by blood and stool analysis and culture to rule out organic disorders, infectious agents and contagious diseases, most importantly, HIV, viral hepatitis, syphilis, malaria, tuberculosis and trypanosomiasis, to avoid transmitting them to the recipient [86]. It is advisable that the donors of the feces have not recently used antibiotics, travelled to tropical areas, had high-risk sexual behavior or had a bout of gastroenteritis or diarrhea within 4 weeks of donation [85]. It is not yet clear what is the correct dose or the frequency of FMT that should be performed on patients with IBS; however, according to the consensus guidelines, at least 30 g of donor feces should be added to the saline solution in order to prepare the fecal suspension that should be either stored at −80 °C or infusion directly on the same day of preparation [85]. After performing FMT, the stool will be collected from the recipients and stored in a special freezer at −80 °C for further microbial analysis [80]. Table 3 describes the different randomized controlled FMT trials, the dosages and the frequency of fecal transplants.  A recent meta-analysis, including seven placebo-controlled randomized controlled studies, has investigated the effect of this novel method involving 470 patients with IBS [87]. Five out of these trials used fresh/frozen fecal material [88][89][90][91][92], while the other two trials used frozen oral FMT capsules compared to placebo capsules [93,94]. These studies showed conflicting results. The meta-analysis [87] suggested that the form of transplantation used in each study had a significant effect on the study outcome, indicating that the fresh/frozen fecal material might be superior to frozen oral capsules in improving IBS global symptoms and having lasting alteration of gut microbiota, Table 3.
In several studies, butyrate-producing bacteria in fecal samples of the recipient IBS patients were not lower than that of the donors, for example, Eubacterium halli, Eubacterium rectale, Megasphera elsdenii, Faecalibacterium prausnitzii [90], Alistipes spp. [90,91], Eubacterium biforme and Lactobacillus spp. [91] were increased, while Bacteroides spp. was decreased in responders following FMT [91]. Moreover, Lactobacillus spp. was negatively correlated with the clinical outcome of IBS-symptom severity score [91]. In addition, our group and other publications also reported that IBS patients with low fecal Alistipes spp. were most likely to not respond to FMT [41,95] and that FMT also increased the total fecal short-chain fatty acids levels, namely; butyric acid [41,96], which was inversely correlated with IBS symptoms [96]. Donor selection seems to be important, but it remains to be investigated whether single or mixed donors is the preferred choice and at which time intervals should FMT be performed. Several trials showed contradictory results when using either single [91,93,97] or mixed donors [92,94], but most of them showed that the bacterial composition of FMT recipients shifted closer to that of the donors, Table 3. A study showed that increasing the dose of fecal transplant to 60 g and/or using repetitive FMT may increase the response rate in IBS patients to FMT [98].
In several open-labeled studies performed by our group, we investigated the effect of FMT from the healthy relatives of the patients on IBS symptoms, gut microbiota, short-chain fatty acids, stem cells and enteroendocrine cells in diarrhea-predominant IBS patients [41,66,80,99,100]. According to our studies, the IBS symptom severity score improved significantly following FMT [41,80]. The bacterial strains signals for Ruminococcus gnavus, Actinobacteria and Bifidobacteria and the fecal short-chain fatty acids were significantly different between IBS patients and their donors, which became insignificantly different starting at 3 weeks after FMT and lasting up to 6 months following FMT [41]. However, the beneficial effect of FMT on IBS symptoms tends to fade over time, as observed in other trials [41,80,89,91]. The gut microbiota profile for IBS patients became more or less similar to that of their donors following FMT [41,80], which is consistent with the findings of several randomized control trials mentioned in Table 3. Furthermore, our studies also showed that altering the gut microbiota following FMT was paralleled by changes in the densities of the duodenal stem cells progenitors and the densities of the enteroendocrine cells in the duodenum and colon toward the densities measured in healthy controls [66,99,100]. This suggests that manipulating the gut microbiota by FMT changes the so-called "gut microenvironment" in the gastrointestinal tract of IBS patients, which may be responsible for improving the global symptoms of IBS.

Conclusions
There is strong evidence that dysbiosis plays an important role in the pathophysiology of IBS. There are different non-pharmacological methods that can improve the symptoms of IBS, which affect the gut microenvironment. Manipulating the gut microenvironment not only changes the composition of gut microbiota but also affects the other components of the gut microenvironment, namely short-chain fatty acids that represent the gut microbiota function and the enteroendocrine cells densities with a total impact on the symptoms of IBS. More studies with larger cohorts and for longer terms are required to investigate this issue.
Funding: This research received no external funding.