Alteration of the intestinal microbiota as a cause of ...

Wageningen Academic P u b l i s h e r s

Beneficial Microbes, 2014; 5(3): 247-261

Alteration of the intestinal microbiota as a cause of and a potential therapeutic option

http://www.wageningenacademic.com/doi/pdf/10.3920/BM2013.0033 - Sunday, September 13, 2015 10:27:05 PM - IP Address:52.2.249.46

in irritable bowel syndrome J. König and R.J. Brummer School of Health and Medical Sciences, Faculty of Medicine and Health, Örebro University, 701 82 Örebro, Sweden; [email protected] Received: 16 July 2013 / Accepted: 16 December 2013 © 2014 Wageningen Academic Publishers

REVIEW ARTICLE Abstract The intestinal microbiota forms a complex ecosystem that is in close contact with its host and has an important impact on health. An increasing number of disorders are associated with disturbances in this ecosystem. Also patients suffering from irritable bowel syndrome (IBS) show an altered composition of their gut microbiota. IBS is a multifactorial chronic disorder characterised by various abdominal complaints and a worldwide prevalence of 10-20%. Even though its aetiology and pathophysiology are complex and not well understood, it is widely accepted that aberrations along the microbe-gut-brain axis are involved. In this review, it will be discussed how exogenous factors, e.g. antibiotics, can cause disbalance in the intestinal microbiota and thereby contribute to the development of IBS. In addition, several new IBS treatment options that aim at re-establishing a healthy, beneficial ecosystem will be described. These include antibiotics, probiotics, prebiotics and faecal transplantation. Keywords: gut microbiota, antibiotics, probiotics, prebiotics, faecal transplantation

1. Introduction Irritable bowel syndrome (IBS) has a worldwide prevalence of 10-20%. Even though it is not life-threatening or associated with higher mortality, it strongly affects the patients’ quality of life and causes substantial economic costs due to the need for medical consultation and absenteeism in the workplace (El-Serag et al., 2002; Sandler et al., 2002). Symptoms vary between patients and include abdominal pain and cramps, constipation and/or diarrhoea, bloating, flatulence, faecal urgency, a sense of incomplete evacuation and relief of pain or discomfort upon defaecation (Longstreth et al., 2006).

function of the immune system in the pathogenesis of IBS. Mild immune activation has been found both locally in the gut and systemically (Barbara et al., 2011), and mucosal biopsies from IBS patients are characterised by an increased quantity of various immune cells, such as mast cells (Bischoff, 2009; Chadwick et al., 2002; Cremon et al., 2009). Own preliminary data applying immune fingerprinting of intraepithelial and lamina propia lymphocytes isolated from mucosal biopsies show that IBS patients display an altered composition of immune cells. Furthermore, psychological and environmental factors, such as anxiety, depression and significant negative life events, are believed to contribute to IBS development (Fichna and Storr, 2012).

IBS is a multifactorial disease, and its aetiology and pathophysiology are complex and not well understood. It is, however, accepted that a dysregulation of the microbe-gutbrain axis plays a profound role. Associated pathophysiologic aberrations include visceral hypersensitivity, abnormal gut motility, and autonomic nervous system dysfunction (Karantanos et al., 2010). In addition, there is a growing amount of data revealing a contributing role of an aberrant

A considerable body of evidence points to the presence of a disturbed intestinal microbiota composition in IBS. A specific subtype, the so-called post-infectious IBS, develops after an episode of acute infectious gastroenteritis and is causally linked to aberrations in the gut ecosystem (Spiller and Lam, 2012). Several studies have demonstrated that the composition of the microbiota in IBS patients is distinctly different from that of healthy controls (Simren et al., 2013),

ISSN 1876-2833 print, ISSN 1876-2891 online, DOI 10.3920/BM2013.0033247

J. König and R.J. Brummer


and IBS symptoms can be improved by treatments targeting the microbiota, such as antibiotics, probiotics and prebiotics (Kajander et al., 2008; Sachdev and Pimentel, 2012; Spiller, 2008). In this review, we will describe the aberrant gut ecosystem in IBS, discuss how exogenous factors might cause disbalance of this ecosystem, and assess how a healthy ecosystem could be re-established by known as well as novel treatments.

2. The intestinal microbiota The intestinal microbiota in healthy adults forms a complex ecosystem that is composed of up to 1000 microbial species (Zoetendal et al., 2008). These organisms normally live in a well-balanced symbiotic state with their host and have an important impact on our health. They form a crucial barrier against pathogens and are involved in the development and maturation of the immune system. In addition, they play a vital role in the metabolism of non-digestible compounds and in the supply of essential vitamins and short chain fatty acids. Each adult has its individual subject-specific intestinal microbiota composition, however, we all share a common microbial core (Qin et al., 2010). It was recently suggested that all humans can be classified into one of three so-called enterotypes, which are characterised by relatively high levels of one of the three genera Bacteroides, Prevotella or Ruminococcus (Arumugam et al., 2011). In healthy adults, the composition remains remarkably stable even during a 10-year period (Rajilic-Stojanovic et al., 2012). However, with increasing age, the core microbiota changes and shows a higher interindividual variability that is strongly influenced by diet and living situation (Claesson et al., 2011, 2012). Before birth, our intestinal tract is basically sterile. Colonisation with microbiota starts during the process of delivery by exposure to the extra-uterine environment, such as maternal vaginal, faecal and skin microbiota. During the first months of life, the composition becomes more and more complex, and varies widely between individuals. It takes about one year until this rather coincidental, chaotic intestinal microbiota is transformed into a more adult-like, stable community (Palmer et al., 2007). Different factors, such as delivery mode (vaginal or by caesarean section), infant diet (breast or formula feeding), and the use of antibiotics, are likely to influence the early microbiota composition and maturation of the immune system, which in turn might have long-term effects on adult microbiota and health later in life (Scholtens et al., 2012). The altered microbiota in children delivered by caesarean section might for instance contribute to their higher risk of developing allergic diseases (Bager et al., 2008). It is estimated that the bacterial cells in the human body outnumber the mammalian cells by a factor of 10, but the true extent and diversity of the human microbiota is still 248

unknown. Culture-independent techniques, which enable an unbiased detection of all bacteria present in the human gut based on 16S rRNA (small subunit ribosomal RNA) sequences, have only recently been developed. Assays, such as phylogenetic microarrays (Rajilic-Stojanovic et al., 2009) or barcoded pyrosequencing (Andersson et al., 2008), have revealed that the diversity of intestinal bacteria is much greater than previously thought, and new bacterial species and strains are being discovered continually.

Faecal compared to intestinal mucosa-adherent microbiota Most commonly, faecal samples are used to determine the composition of the intestinal microbiota. They can easily be collected non-invasively, facilitating repeated analysis. The microorganisms in human faeces account for about 50% of the dry matter, which otherwise consists mostly of fibres and soluble material, as analysed in healthy subjects on a Britishtype diet (Stephen and Cummings, 1980). Half of the faecal microbial cells are viable, while about one third are dead and one fifth damaged (Ben-Amor et al., 2005). However, even dead cells might still have functions in the intestine, as also non-viable probiotic bacteria possess a strong bioactivity in the small bowel (Van Baarlen et al., 2009). The amount of bacteria in the gastrointestinal tract increases gradually from very low numbers in the stomach to concentrations of approximately 1012 bacterial cells per g of luminal content in the colon, with an especially steep gradient at the ileocaecal valve (Sekirov et al., 2010). Slow transit as well as alternating anterograde and retrograde propulsive movements in the large intestine enable microbes to colonise on food particles, with which they proliferate during the passage through the colon. Bacteria at the interface of the substrate are found both as isolated dispersed cells as well as in biofilm populations (Macfarlane and Macfarlane, 2006). They depend strongly on the substrate provided and are susceptible to exogenous factors, such as changes in the diet and transit times. Probably only a small portion of the bacteria in the faeces is able to adhere to the mucosa and directly interacts with intestinal epithelial or immune cells. Thus the major effects of the luminal microbiota on the host are likely to be indirect, via bacterial metabolites, such as short-chain fatty acids, or through the secretion of antimicrobial substances (socalled bacteriocins) and immunomodulins. Studies have shown that the mucosa-adherent microbiota differ significantly from the faecal one (Carroll et al., 2011; Eckburg et al., 2005; Zoetendal et al., 2002). They probably consist of a subset of bacteria with specific properties that enable them to get attached to or enter the mucus layer covering the mucosal epithelium, e.g. pili. They are in close contact with the underlying epithelial, immune and endocrine cells, and are therefore able to Beneficial Microbes 5(3)


Alteration of the gut microbiota as a cause and a therapeutic option in IBS

interact both directly and indirectly with the host. Even though the mucosal microbiota composition might vary along the colon, e.g. due to variances in the availability of substrate and mucus, studies so far have not shown significant location-dependent differences (Eckburg et al., 2005; Momozawa et al., 2011; Zoetendal et al., 2002). However, only a few studies exist, and even fewer have used high-throughput methods for analysis. The effect of bowel cleansing on the intestinal microbiota composition will be discussed later in this review. It is likely that the luminal and the mucosal bacteria interact and influence each other. In addition, when taking samples, faecal samples might contain some mucus-associated bacteria due to mucosal shedding, while mucosal biopsies could be contaminated with faeces.

3. Altered intestinal microbiota in irritable bowel syndrome In recent years, the importance of a healthy intestinal microbiota has been more and more substantiated. Many disorders, such as inflammatory bowel diseases and obesity, have been associated with a disturbed composition of the gut microbiota (Frank et al., 2007; Ley et al., 2006). It is now widely accepted that a disbalanced gut ecosystem also plays an important role in the pathophysiology of IBS (Salonen et al., 2010). Numerous bacterial species have been found to be differentially abundant in IBS. However, the results are rather inconsistent and no species could be specifically linked to IBS so far. On the one hand, this is probably due to the heterogeneous character of the IBS pathophysiology, which shows a large inter-individual variation of aberrations along the microbe-gut-brain axis. On the other hand, studies apply various different techniques and use different classifications of patients according to symptoms. It is quite likely that the microbiota composition differs amongst the different IBS subgroups, such as constipation- or diarrhoeapredominant IBS. However, most reports do not distinguish between the different subtypes, or just focus on one specific subtype and/or include small patient groups. In addition, it is difficult to account for exogenous factors, and especially diet has a strong influence on the microbiota composition (Wu et al., 2011). Earlier studies investigating faecal microbiota in IBS patients using culture-based analyses detected decreased amounts of bifidobacteria and lactobacilli compared to controls (Balsari et al., 1982). The first study to apply a specifically designed quantitative polymerase chain reaction (qPCR) assay covering 14 species was published in 2005 (Malinen et al., 2005). The authors found lower amounts of Bifidobacterium catenulatum and Clostridium coccoides, amongst others, in faecal samples of IBS patients. When categorising samples according to the IBS subtypes, they found lower amounts of lactobacilli in connection with Beneficial Microbes 5(3)

diarrhoea-predominant IBS (n=12) than with constipationpredominant IBS (n=9). In addition, counts for Veillonella spp. were significantly higher within the constipationpredominant IBS group than in healthy controls (n=22). The same group was also the first to apply high-throughput 16S rRNA gene cloning and sequencing, and they detected changes especially in the phyla Firmicutes and Actinobacteria (Kassinen et al., 2007). A follow-up study of only the diarrhoea-predominant IBS subtype (n=10) found high numbers of Proteobacteria and Firmicutes and low numbers of Actinobacteria and Bacteroidetes compared to controls (Krogius-Kurikka et al., 2009). In a further study, this group applied additional qPCR assays to samples classified according to IBS subgroups (Lyra et al., 2009). Amongst others, they detected that a Clostridium thermosuccinogenes-like phylotype was significantly less abundant in diarrhoea-predominant IBS (n=8) compared to controls (n=15) and to mixed symptom-subtype IBS (n=4). Furthermore, a Ruminococcus bromii-like phenotype was more prevalent in the diarrhoea subgroup compared to the control group. Rajilic-Stojanovic et al. (2011) analysed the microbial composition of faecal samples of 62 IBS patients and 46 controls using a high-throughput phylogenic microarray (HITChip) that enables the unbiased detection of over one thousand phylotypes. In accordance with previous studies, they found that the intestinal microbiota of IBS patients differed significantly from controls, and detected an increased Firmicutes to Bacteroidetes ratio. This ratio was also found to be increased in a recent study using pyrosequencing of 16S rRNA (Jeffery et al., 2012). In this study, the IBS patients clustered into three different groups based on their faecal microbiota composition. Two groups clustered very differently from the healthy controls, whereas the so-called ‘normal-like IBS group’ consisting of 15 of the 37 included patients was indistinguishable from the healthy controls. Most studies so far have focused on investigating faecal microbiota, and not many results can be found on mucosaassociated bacteria. Kerckhoffs et al. (2009) used real-time PCR to analyse the composition of bifidobacteria in faecal and duodenal mucosa brush samples of IBS patients, and found lower values of B. catenulatum compared to controls. Carroll et al. (2011) applied a molecular fingerprinting technique to investigate faecal and unprepared colon mucosal samples of patients with diarrhoea-predominant IBS. They found differences in microbial communities between IBS patients and healthy controls in both faecal and mucosa samples, with a diminished microbial biodiversity only in faecal samples. Parkes et al. (2012) investigated the mucosa-associated microbiota of IBS patients’ rectal biopsies, using in situ hybridisation of selected bacterial groups. They found that IBS patients had a significantly higher number of mucosa-associated bacteria in general, 249



consisting predominantly of Bacteroides and Clostridium spp. In addition, when dividing samples according to IBS subgroups, they detected that bifidobacteria were less abundant in diarrhoea-predominant IBS (n=27) compared to constipation-predominant IBS (n=20) and controls (n=26). An earlier study using fluorescence in situ hybridisation also reported a higher number of mucosal bacteria in IBS patients (Swidsinski et al., 2005). Our own preliminary data showed that IBS patients have a lower proportion of butyrate-producing microbiota both in faecal and unprepared mucosal samples compared to healthy controls. Butyrate is the dominant short-chain fatty acid produced by microbial fermentation of undigested dietary carbohydrates in the gut. It is an important energy source for epithelial cells and has many beneficial effects on colonic mucosal function including inhibition of inflammation and carcinogenesis, and it promotes the colonic mucosal defence barrier (Hamer et al., 2008, 2009, 2010). Further studies analysing the microbiota composition of mucosal biopsies are essential to elucidate the pathophysiology behind IBS. The mucosa-associated bacteria are in close contact with the host and are likely to have a major impact on human health. The differences between faecal and mucosal bacteria composition indicate that faecal samples do not necessarily represent the mucosal bacteria composition. Several studies have correlated the abundance of specific microbiota with IBS symptoms. Rajilic-Stojanovic et al. (2011) reported that several members of Firmicutes and Proteobacteria correlated with IBS symptom scores, for instance pain scores correlated negatively with Bifidobacterium spp. In the study by Jeffery et al. (2012), Cyanobacteria were associated with satiety, bloating and gastrointestinal symptom scores, whereas Proteobacteria correlated with an increased mental component and pain threshold. Parkes et al. (2012) detected a negative correlation of bifidobacteria and lactobacilli with stool frequency, amongst others. A study using qPCR found that the amount of Ruminococcus torques was positively correlated with the severity of self-reported IBS symptoms (Malinen et al., 2010). Interestingly, an increased number of this species in IBS patients has been reported in other studies (Kassinen et al., 2007; Rajilic-Stojanovic et al., 2011). The association of specific bacteria with specific IBS symptoms is a promising tool to provide insight into factors contributing to IBS. However, it needs to be taken into account that in IBS identical symptoms are not necessarily related to the same pathophysiology. Nevertheless, it would be desirable in future studies to investigate the gut microbiota of IBS patients according to their symptom subtypes. An additional aberration in IBS seems to be an altered microbial species variety, also referred to as microbial diversity or heterogeneity. However, results described 250

in literature are inconsistent, and both a loss of diversity (Carroll et al., 2012; Codling et al., 2010) as well as an increased heterogeneity have been reported (RajilicStojanovic, 2007). Both conditions might be indicative of the inability of the IBS ecosystem to maintain its normal composition. A loss of diversity is usually associated with the outgrowth of certain species, while a high degree of variability could refer to a disturbed community trying to recover and re-establish its previous state (Salonen et al., 2010).

4. Perturbations of the intestinal microbiota promoting the development of irritable bowel syndrome Various exogenous factors can bring the normal healthy gut ecosystem out of balance, hence contributing to development of IBS (Figure 1).

Infections and antibiotics The so-called post-infectious IBS develops after infections, such as giardiasis or bacterial and viral gastroenteritis, which Healthy microbiota

Infections

Antibiotics x x x

xxxx

Bowel cleansing

Figure 1. Perturbations of the gut microbiota promoting the development of irritable bowel syndrome. Infections can lead to the overgrowth of pathogenic bacteria, whereas antibiotics and bowel cleansing might reduce the diversity of the gut microbiota by depleting both harmful as well as beneficial bacteria, resulting in a disproportional number of pathogens. Beneficial bacteria (e.g. bifidobacteria, lactobacilli, butyrate producers) are shown as horizontal rods, and pathogens as vertical rods (e.g. Ruminococcus torques), respectively. Beneficial Microbes 5(3)



disturb the intestinal microbiota. Especially the duration of the initial disease correlates with a high risk to develop IBS (relative risk, 11.5) (Spiller and Lam, 2012). In addition, antibiotics are well known to have both short-term and longterm effects on the composition of the intestinal microbiota. In most individuals, the gut ecosystem appears to recover within days or weeks after cessation of antibiotic treatment (De La Cochetiere et al., 2005; Lode et al., 2001). However, in some cases these alterations can result in a persistent depletion of beneficial bacteria and/or overgrowth of harmful ones (De La Cochetiere et al., 2005; Dethlefsen et al., 2008; Jernberg et al., 2007), as is the case in, for example, Clostridium difficile infection. Attention should be paid to the fact that even if in some cases no microbial changes have been detected, deeper analyses might be necessary to reveal more subtle alterations, as the sensitivity of the methods used is limited by the techniques applied. Antibiotic treatment early in life may be related to the development of certain disorders later on, and has been especially associated with immunological disorders, such as asthma and allergies (Droste et al., 2000). There is some evidence that the use of antibiotics is connected to an increased risk of developing IBS. A retrospective survey including 421 subjects (48 with IBS) found a correlation of antibiotic use with IBS (Mendall and Kumar, 1998). Another retrospective review of 26,107 medical records of patients exposed to broad-spectrum antibiotics showed a higher prevalence of IBS among patients who previously were administered macrolide or tetracycline (Villarreal et al., 2012). In a prospective case-control study by Maxwell et al. (2002), subjects receiving antibiotic treatment were more likely to suffer from functional bowel symptoms (according to Rome criteria) than controls during a 4-month follow-up period. Even though further studies are necessary to investigate this in detail, it is possible that a change in the intestinal microbiota caused by antibiotics could contribute to the development of IBS. However, it needs to be considered that also the infection leading to the prescription of antibiotics could be the underlying cause of the increased risk to suffer from IBS. Later in this review, we will discuss the use of antibiotics as a potential therapeutic option for IBS.

A recent study has shown that a standard bowel cleansing procedure using a polyethylene glycol-based preparation led to changes in the composition of the mucosa-adherent intestinal microbiota in healthy individuals (Harrell et al., 2012). A similar effect was observed in another study with regard to the faecal microbiota composition (Mai et al., 2006). The impact of such a bowel cleansing on IBS symptoms has never been investigated, but persisting harmful alterations on the intestinal microbiota are possible. Especially in individuals whose microbial balance has been challenged before, for instance by recent infections or the use of antibiotics, such a bowel cleansing could be a final trigger leading to a sustained aberrant intestinal ecosystem.

5. Therapeutic options to re-establish a healthy gut microbiota in irritable bowel syndrome Against the background that a disturbed gut microbiota might contribute to IBS development, several therapeutic options aim at re-establishing a healthy, beneficial ecosystem (Figure 2).

Microbiota in IBS

Probiotics and prebiotics

Antibiotics x x x

Faecal transplantation

Bowel cleansing Apart from infections and antibiotics, bowel cleansing might lead to harmful alterations of the gut ecosystem as well. This routine procedure is performed to prepare the colon for colonoscopy by washing out faecal residues and likely also leads to the loss of mucosa-adherent bacteria. Some IBS patients use it as a self-treatment in the form of oral laxatives or self-administered enemas with the aim to clean or detoxify the colon, and thereby to improve symptoms such as diarrhoea, constipation or flatulence.

Beneficial Microbes 5(3)

Figure 2. Potential therapies to re-establish a healthy gut microbiota in irritable bowel syndrome (IBS). In IBS, the intestinal microbiota shows an aberrant diversity and is characterised by low numbers of beneficial bacteria (shown as horizontal rods). In addition, abundance of specific harmful bacteria (shown as vertical rods) has been reported. Probiotics and prebiotics might act by increasing the numbers of beneficial bacteria, while antibiotics predominantly deplete harmful ones. Faecal transplantation introduces a healthy, diverse microbiota. 251



Antibiotics Apart from being a possible trigger of IBS, antibiotics have also been successfully used to treat IBS. The first antibiotic investigated in a clinical study was neomycin, an antibiotic that is not absorbed in the gastrointestinal tract. It was demonstrated to be effective in improving IBS symptoms (35.0% improvement in a composite score compared to 11.4% improvement in placebo treatment) (Pimentel et al., 2003). However, its rather severe adverse effects and the fact that it provokes a rapid clinical resistance limit its clinical use (Sachdev and Pimentel, 2012). Nowadays, the antibiotic of choice in treating IBS is rifaximin. It is a semisynthetic derivative of rifamycin with a beneficial side effect profile and no demonstrable systemic absorption (Basseri et al., 2011). It is approved by the US Food and Drug Administration (FDA) for the treatment of traveller’s diarrhoea and hepatic encephalopathy, but still lacks approval in several other countries. Its efficacy for IBS treatment has been tested in various clinical trials, the largest being the TARGET 1 and TARGET 2 studies (Pimentel et al., 2011a). Here, a total of 1,258 subjects with nonconstipated IBS were included. Treatment with rifaximin (550 mg/day) for 14 days resulted in a significantly higher percentage of patients reporting relief of global IBS symptoms compared to the placebo-treated group (41 versus 31%), and effects were sustained for at least 10 weeks. In addition, bloating, abdominal pain, and loose or watery stools were improved. These results are supported by a meta-analysis that included five studies and found improvement of IBS symptoms using rifaximin with a modest therapeutic gain similar to other currently available IBS therapies (Menees et al., 2012). The American College of Gastroenterology Task Force rated rifaximin as a strong drug with moderate evidence for the treatment of IBS with diarrhoea (ACG Task Force on IBS, 2009). A study evaluating the safety of rifaximin reported its so-called ‘number to harm’ to be 846, meaning that 846 patients would benefit from it before one harmful event would occurs (Shah et al., 2012). In addition, rifaximin was effective in retreating patients that presented with a relapse after the first antibiotic treatment, and it does not seem to provoke clinical resistance (Pimentel et al., 2011b; Yang et al., 2008). Hardly any studies have examined the modes of action of antibiotics with regard to IBS treatment. The aforementioned study from Pimentel et al. (2003) investigating the effect of neomycin on IBS, found that subjects with IBS often presented abnormal values in the lactulose breath test (LBT), and antibiotic treatment resulted in normalisation of the LBT along with symptom improvement. The authors suggested that the excessive gas production might be caused by the presence of small intestinal bacterial overgrowth (SIBO), a condition where an abnormal number of bacteria fermenting undigested 252

carbohydrates is present in the small bowel, and that antibiotics were able to reverse this aberrant colonisation. However, the use of LBT to diagnose SIBO is controversial, and several other studies did not find any association between IBS and SIBO (Ford et al., 2009; Posserud et al., 2007). Instead, it might be possible that rifaximin reduces the total number of bacteria, especially in the large intestine, which could lead to a decreased amount of gas produced by bacteria, resulting in less flatulence and bloating. In conclusion, it has been demonstrated that non-absorbable antibiotics are able to – at least – partially improve IBS symptoms, confirming that alterations in the gut microbiota play an important role in the pathophysiology of IBS. However, even though rifaximin seems to be safe, it does not have a very high efficacy and its long-term effects have not been investigated yet. It is still unclear which bacteria are specifically targeted by the antibiotics and whether this or a decrease in the total bacterial number is responsible for their beneficial effects. It further needs to be determined which IBS patients would benefit from a treatment with rifaximin, as it probably is not equally efficient for all subgroups. Especially the large clinical trials have tested its effect only in non-constipated IBS so far, and more information on its effect in constipation-predominant IBS is needed. In addition, a possible harmful effect of antibiotics on the intestinal microbiota composition also needs to be considered. Further studies investigating the effect of rifaximin on both the mucosa-adherent and faecal microbiota are essential.

Bowel cleansing As mentioned before, the impact of a routine or selfadministered bowel cleansing on IBS symptoms has never been investigated, and similar to antibiotic use, also beneficial effects seem possible. In IBS patients, it could be that bowel cleansing leads to a reduction in the overall numbers of bacteria and thereby gives the intestinal microbiota an opportunity to re-establish a healthy balance. This effect could hypothetically be enforced by a concomitant administration of probiotics. However, at least to our knowledge, there are no reports in the literature about a relief of symptoms after performing colonic cleansing in IBS patients, indicating that this procedure is probably lacking positive long-term effects.

Probiotics Probiotics are defined as living microorganisms, usually consumed as food products, which upon ingestion have beneficial effects on human health. Even though recent research has shown that even non-viable probiotic bacteria may possess strong bioactivity in the small bowel (Van Baarlen et al., 2009), it is suggested that probiotics should be resistant to gastric acid and digestive enzymes in Beneficial Microbes 5(3)



order to reach the colon in viable condition. The most commonly administered types are part of the genera Lactobacillus or Bifidobacterium, and they can be applied alone (monospecies) or in combination with several other species (multispecies). Strong evidence for the effectiveness of probiotics has been demonstrated regarding the treatment of antibioticassociated traveller’s and paediatric diarrhoea (McFarland, 2006, 2007; Szajewska et al., 2006). A meta-analysis could show that probiotics are very effective in preventing severe necrotising enterocolitis in preterm infants (Alfaleh et al., 2011). In infants with infantile colic, Lactobacillus reuteri significantly improved symptoms, as demonstrated by a clearly reduced daily crying time (Savino et al., 2007). The mechanisms behind the beneficial effects of probiotics are still not completely understood. Some probiotics are able to adhere to the intestinal mucosa and act as antagonists against pathogenic species by replacing existing pathogens or by inhibiting their adherence (Servin, 2004). Another important function of probiotics is their ability to induce beneficial immune responses. These can take effect by direct interaction with immune or epithelial cells, or via secreted molecules (O’Mahony et al., 2005; Preidis and Versalovic, 2009; Van Baarlen et al., 2009). Probiotics can also act against harmful bacteria via the secretion of bacteriocins (Corr et al., 2007). In addition, probiotics are able to enhance epithelial barrier function by activating signalling pathways that lead to increased expression of tight junction proteins or enhanced mucus production (Karczewski et al., 2010; Spiller, 2008). By inducing the expression of opioid and cannabinoid receptors, some probiotics might also be able to modulate the perception of visceral pain (Rousseaux et al., 2007). In mice, administration of Lactobacillus rhamnosus (JB-1) reduced anxiety and depression-related behaviour by modulating gamma-aminobutyric acid receptor expression in the brain (Bravo et al., 2011). All these mechanisms suggest that probiotics could be a promising treatment option in IBS, and numerous controlled clinical trials testing the effect of a wide selection of probiotic strains on IBS have been performed (Simren et al., 2013). In general, most of the higher-quality clinical trials so far yielded positive results. Some, however, showed no beneficial effects in IBS and one study even reported symptom deterioration using Lactobacillus plantarum MF1298 (Ligaarden et al., 2010). Several meta-analyses came to the conclusion that probiotic use improves IBS symptoms and might be a promising treatment option (Hoveyda et al., 2009; McFarland and Dublin, 2008; Nikfar et al., 2008). Meta-analyses combining the results of studies using different probiotic strains carry the risk of masking the success or failure of a specific strain. Accordingly, the authors agreed that it needs to be further investigated which Beneficial Microbes 5(3)

strains and which doses are most effective. Probiotics that demonstrated IBS symptom improvement in more than one controlled clinical trial with a substantial number of patients include Bifidobacterium infantis 35624 (O’Mahony et al., 2005; Whorwell et al., 2006) and the so-called ‘Finnish combination’ consisting of Lactobacillus rhamnosus GG, L. rhamnosus Lc705, Propionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium breve Bb99 or Bifidobacterium animalis ssp. lactis Bb12, respectively (Kajander et al., 2005, 2008). It is known that different probiotics have distinct functional effects in the human intestine (Van Baarlen et al., 2011). Some strains have been shown to improve total symptom scores in IBS patients, while others primarily seem to affect bloating and flatulence or stool frequency (Simren et al., 2013). Several studies did not distinguish between the different subtypes of IBS, such as diarrhoea or constipationpredominant IBS, discounting the fact that most strains are probably more effective in treating one kind than the other. As mentioned earlier, IBS symptoms are not necessarily predictive of the underlying pathophysiology. Hence, it would be ideal to administer a probiotic that specifically targets the respective pathophysiology instead of applying a treatment based on symptoms. Future studies should thus attempt to discover a link between the probiotic treatment and IBS pathophysiology in addition to IBS symptoms. An additional factor to be considered is that clinical trials are often conducted in a hospital setting, which may give rise to an inclusion bias in comparison to subjects suffering from IBS in the general population. These groups may differ in the proportion of the various pathophysiologic mechanisms contributing to IBS symptoms. Only a few probiotic intervention studies have looked deeper into the underlying mechanisms and evaluated for instance the impact of the tested probiotics on the microbiota composition in IBS. Kajander et al. (2007) investigated the effect of the multispecies ‘Finnish combination’ (see above) on the faecal microbiota composition of IBS patients using qPCR assays specifically designed to detect the ingested probiotic strains, and additional qPCR assays covering about 14 different species or groups. These assays are limited by the fact that not the full microbiota composition is assessed, but only those genera or species for which primers are available. They did not detect any differences in the investigated strains and genera, apart from an increase in Bifidobacterium spp. in the placebo and a decrease in the treatment group. In addition, no changes in the presence of short-chain fatty acids and bacterial enzymes in faecal samples were found. They concluded that other mechanisms besides an increased colonisation with the administered bacteria must have been responsible for the beneficial effects on IBS symptoms, probably involving a direct interaction with the intestinal epithelium. Another explanation could be a 253



more dominant effect of some probiotics in the small bowel rather than in the colon, as probiotics have been shown to provoke a direct metabolic or immunologic effect in the small bowel (Troost et al., 2008; Van Baarlen et al., 2009, 2011). Furthermore, the applied techniques were probably not sufficient to detect the underlying microbial changes. In a subsequent study, the same group applied a similar qPCR assay with a broader target of phylotypes to evaluate the effect of the same probiotic combination on the faecal microbiota in 42 IBS patients. Here, they reported that a phylotype with 94% similarity to Ruminococcus torques was decreased and Clostridium thermosuccinogenes was increased by 85% in the probiotic compared to the placebo group (Lyra et al., 2010). The effect of probiotic treatment on the mucosa-adherent bacteria has not been reported in IBS patients yet. So far, it is still not known if probiotics have a higher efficacy when administered as monospecies or multispecies. As several pathophysiologic mechanisms are involved in IBS, and in addition, patients show different aberrations along the microbe-gut-brain axis, a probiotic multistrain combination could provide a more comprehensive treatment covering various needs. In a multispecies mixture, one strain could for example deliver a beneficial immune effect, while another strain could improve intestinal barrier function. A multispecies probiotic could also be more effective in the various segments of the intestine. Furthermore, it was shown in an in vitro human intestinal mucus model that individual strains may strongly enhance each other’s adherence if combined with other strains, with some combinations being more effective than others (Collado et al., 2007). However, besides a potential synergistic effect, probiotics could also exert antagonistic effects against each other if administered in combination. Even though further research is necessary, some probiotic strains seem to be beneficial in the treatment of IBS. According to the current knowledge, their efficacy is similar to antibiotics. One of the clear advantages of probiotics over conventional pharmacological medication is their favourable adverse effect profile, which enables chronic administration and preventive treatment.

Prebiotics Prebiotics are food compounds that are selectively fermented by specific desirable bacteria in the intestine. They confer favourable health effects on the host by stimulating the metabolism and growth of beneficial bacteria (Roberfroid, 2007). The most commonly administered prebiotics specifically act on the health-promoting bifidobacteria and lactobacilli. Many compounds with prebiotic effects belong to the group of non-digestible carbohydrates, more precisely oligo- or polysaccharides, and include oligofructose (inulin) and trans-galactooligosaccharides. 254

They naturally occur in many edible cereals, fruits and vegetables, such as wheat, bananas, onion, garlic, chicory, and artichokes, in which they function as carbohydrate stores (Quigley, 2012; Seifert and Watzl, 2007). In addition, oligosaccharides with prebiotic effects are found in human mother’s milk and are thought to contribute to the high amount of bifidobacteria and lactobacilli detected in the faeces of breast-fed compared to formula-fed babies. Supplementation of infant formulas with human milklike prebiotics appears to have a beneficial immunological effect resulting in lower incidence of allergies and infections (Arslanoglu et al., 2008). In adults, suggested health benefits of prebiotics include protection against traveller’s and antibiotic-associated diarrhoea (Drakoularakou et al., 2010; Lewis et al., 2005). A few studies have investigated the effect of prebiotics on IBS symptoms. In a double-blind crossover trial with 21 IBS patients no effect of oligofructose (Raftilose 95) administration over a 4-week time course could be observed, even after separate analysis of the diarrhoeaand constipation predominant subgroups (Hunter et al., 1999). The authors speculated that the administered dose of 6 g/day might have been too low to show demonstrable effects. Another clinical trial tested the effect of a novel trans-galactooligosaccharide in a 12-week parallel crossover design (Silk et al., 2009). 44 IBS patients were included and two different doses of the prebiotic were used (3.5 and 7 g/ day). Both doses significantly increased bifidobacteria and lactobacilli numbers. The lower dose was able to improve stool consistency, flatulence and bloating as well as total symptom score and subjective global assessment values of the IBS patients, whereas the higher dose only improved subjective global assessment and anxiety scores. It needs to be pointed out, however, that also the placebo showed positive effects regarding flatulence, abdominal pain and total symptoms. An important readout of these studies could be the absence of reported side effects, which is not necessarily expected. Even though bifidobacteria and most lactobacilli themselves do not produce gases as part of their metabolism, the rapid fermentation of the prebiotics in the proximal bowel often causes increased intestinal gas production. This can lead to enhanced flatulence and bloating even in healthy subjects, and would be an especially unfavourable feature in IBS, where patients already suffer from these symptoms and often experience visceral hypersensitivity (Macfarlane et al., 2006). An ideal prebiotic to be used in IBS should therefore ferment slowly throughout the entire colon, so that the produced gases are evenly distributed, thus causing fewer complaints. Prebiotics have mostly been investigated regarding their effect on bifidobacteria and lactobacilli. As described before, IBS patients tend to have a lower proportion of butyrateBeneficial Microbes 5(3)



producing microbiota. The administration of butyrate via enemas resulted in a substantial decrease of visceral perception in healthy volunteers, suggesting a possible beneficial effect in disorders associated with visceral hypersensitivity such as IBS (Vanhoutvin et al., 2009a,b). The interaction of butyrate with the GPR43 receptor expressed on immune cells seems to play an important role in the regulation of immune response in the gut (Le Poul et al., 2003; Maslowski et al., 2009). Hence, prebiotic compounds that specifically serve as substrates for butyrateproducing bacteria could be beneficial in improving IBS symptoms. The modes of actions of prebiotics are not only limited to the stimulation of growth of beneficial bacteria and their metabolic products. Their favourable immune effect might also result from a direct interaction with carbohydrate receptors on immune cells. Animal studies suggest that mostly cells from the gut-associated lymphoid tissue are involved (Seifert and Watzl, 2007). Clear evidence for this exists for other members of the group of non-digestible carbohydrates, the β-glucans. These compounds can be found in various grains, mushrooms and yeasts. By definition they do not qualify as prebiotics, as they have not been shown to specifically affect certain beneficial bacteria. However, their direct receptor-mediated effects on various immune cells have been demonstrated (Vos et al., 2007).

Synbiotics Prebiotics and probiotics can also be administered in combination, and are then denoted synbiotics. The addition of the prebiotic aims at enhancing the viability and activity of the administered probiotic and resident beneficial bacteria, at best resulting in a synergistic effect. So far, there is only one placebo-controlled trial evaluating the effect of synbiotics on IBS symptoms. It included 68 IBS patients and reported improvement of abdominal pain and bowel habits using a novel synbiotic known as SCMIII, and successfully increased lactobacilli, eubacteria and bifididobacteria (Tsuchiya et al., 2004). Further beneficial effects have been described in several open-label studies, however, those results need to be assessed with caution as the placebo response in IBS is high (Quigley, 2012).

Faecal transplantation Faecal transplantation is a relatively old treatment that has regained interest recently. It aims at re-establishing a healthy intestinal microbiota in patients with a disturbed gut ecosystem by infusing suspended stool from a healthy donor into the intestine of the patients. Its use in Chinese medicine goes back to the 4th century when it was applied to treat food poisoning and severe diarrhoea (Zhang et al., 2012). Nowadays, faecal transplantation is established as a highly efficient treatment for recurrent C. difficile Beneficial Microbes 5(3)

infection, where perturbations of the intestinal microbiota seem to be responsible for the overgrowth of pathogenic C. difficile strains (Hell et al., 2013). In this disorder, faecal transplantation has a cure rate of over 90% (Gough et al., 2011), and has been proven to be a durable and safe method according to a recent multicentre long-term follow-up study (Brandt et al., 2012). In addition, faecal transplantation in this study was highly acceptable to patients. 97% of the treated patients would be willing to undergo another transplantation in case of recurrent C. difficile infection, and 53% would choose it as their first treatment option before antibiotics. Nowadays, faecal transplantations are usually performed via nasoduodenal tubes or colonoscopy. Depending on the affected location in the colon, also less invasive retention enemas can be applied. Faecal transplantation might be a promising treatment for other diseases that are causally linked to alterations in the gut microbiota. Vrieze et al. (2012) demonstrated that the transfer of faecal microbiota from healthy lean donors into patients with metabolic syndrome increased their insulin sensitivity and reduced triglyceride levels. This procedure positively changed the gut microbiota of the recipients, resulting in a higher proportion of health-promoting butyrate-producing bacteria. The successful application of faecal transplantation has also been reported in other disorders, including inflammatory bowel disease, multiple sclerosis, autism, and chronic fatigue syndrome, mainly reflecting case studies (Aroniadis and Brandt, 2013; Borody and Campbell, 2012; Borody and Khoruts, 2011; Borody et al., 2003). No randomised clinical trials investigating the impact of faecal transplantation on IBS have been published so far. Only one study reported possible positive effects in patients with IBS symptoms (Borody et al., 1989). The same group applied a mixture of 18 cultured, non-pathogenic bacteria resembling normal gut microbiota into the caecum of IBS patients and reported improved symptoms in 25 out of 33 (Andrews and Borody, 1993). The successful improvement of IBS symptoms using antibiotics, prebiotics and probiotics suggests that alterations in the gut microbiota are involved in the underlying pathophysiology. However, these treatments are usually not very efficient and tend to provide only moderate and transient effects, probably due to the fact that only a small part of the complex microbial ecosystem is affected. Faecal transplantation, however, results in durable changes of the colonic microbiota that can still be detected six months after the treatment (Grehan et al., 2010). Exchanging the microbiota of an IBS patient with the microbiota of a healthy donor holds the potential to be a lot more efficient in re-establishing a normal, healthy microbiota. Faecal transplantation could therefore be a promising novel treatment option for IBS.

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Potential risks also need to be considered, and donors and their faeces should be carefully screened for pathogens. In the USA, faecal transplants are considered as drugs and regulated by the FDA (Mole, 2013). Until the method is considered completely safe, faecal transplantation should only be performed in a research setting and not in clinical routine. A promising approach to avoid the risks associated with the infusion of an uncharacterised faecal mixture could be the application of so-called synthetic stool, which is composed of large numbers of well-defined microbial communities selected by their beneficial effect on human health (Petrof et al., 2013).

6. Conclusions Many IBS patients show an altered intestinal microbiota composition, and, at least in a subgroup of patients, symptoms can be improved by treatments that aim at re-establishing a healthy microbiota. However, we need to keep in mind that not all microbiota perturbations in IBS are necessarily associated with antibiotic use, prior infection or bowel cleansing. IBS is a multifactorial, complex disease with a subject-specific pathophysiology along the microbe-gut-brain axis, and many contributing factors are not well understood yet. The microbiota in the adult is shaped throughout the first three years of life, and persisting gut microbiota perturbations can be initiated by an insufficient exposure to the microbial diversity (hygiene theory), amongst others. It is still unknown how diet, genetic factors, and factors related to brain function, e.g. stress and depression can influence the individual gut microbiota towards an increased susceptibility for IBS. Further elucidation of the mucosal and faecal microbiota composition in IBS in relation to these endogenous and exogenous factors is required in order to identify optimal personalised therapeutic approaches for the affected patients.

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