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The Intestinal Microbiota in the Irritable Bowel Syndrome
CHAPTER TWELVE
The Intestinal Microbiota in the Irritable Bowel Syndrome S.M. Collins1 The Farncombe Family Digestive Health Research Centre, The Michael G DeGroote School of Medicine, McMaster University, Hamilton, ON, Canada 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. The General Appeal of the Microbiota as Putative Pathogenetic Factor in IBS 3. Factors Known to Precipitate or Exacerbate IBS also Induce Intestinal Dysbiosis 3.1 Antibiotic Exposure 3.2 Enteric Infection 3.3 Psychological Stress Including Early Life Stress 3.4 Dietary Factors 4. Evidence of Dysbiosis in IBS Patients 5. Proof of Principle that Intestinal Dysbiosis Alters Function in the Gut and Brain 5.1 Is There a Causal Link Between Dysbiosis and Symptom Expression in IBS? 6. Future Directions References
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Abstract The irritable bowel syndrome (IBS) is a chronic abdominal symptom complex occurring in a bowel devoid of discernible relevant pathology. There is growing interest in the role of the intestinal microbiota as a basis for the intestinal and possibly behavioral manifestations of this condition. Molecular-based microbial profiling has revealed compositional changes in the microbiota of at least a subset of IBS patients but the data are often conflicting and no microbial signature for this condition has yet been defined. Animal studies in which a previously stable intestinal microbiota is perturbed, by antibiotics or dietary change, results in alterations in intestinal function reminiscent of that seen in IBS patients. These include visceral sensitivity to painful stimuli, altered motility and intestinal barrier function as well as immune activation, and low-grade inflammation. More recent studies have shown that perturbation of the microbial composition of the gut alters brain chemistry and behavior. In a step toward establishing a causal link between an altar microbiota and gut–brain manifestations of IBS, colonization of germfree mice with microbiota from IBS patients results in an IBS-like phenotype, including alterations and behavior if the donor exhibited psychiatric comorbidity, such as high levels of anxiety. This model provides an opportunity for exploring the mechanisms
International Review of Neurobiology, Volume 131 ISSN 0074-7742 http://dx.doi.org/10.1016/bs.irn.2016.08.003
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2016 Elsevier Inc. All rights reserved.
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underlying host–microbe interactions relevant to the pathogenesis of IBS and for developing novel therapeutic targets.
1. INTRODUCTION The irritable bowel syndrome (IBS) is a chronic abdominal symptom complex in which pain or discomfort related to altered bowel habit are the clinical hallmarks. These symptoms arise in the absence of any demonstrable pathology and the disorder is considered to be one of function rather than structure. Psychiatric comorbidity is very common and there is a strong link between psychological stress and the expression of IBS. It is therefore considered to be a disorder of the gut–brain axis. While, the IBS is the most common intestinal disorders in our society, our understanding of its underlying pathogenesis remains far from clear. IBS is heterogeneous in terms of its clinical manifestation and much effort has gone into defining clinically homogeneous IBS subgroups. However, there is unlikely to be homogeneity of the underlying pathogenesis even within these subgroups. IBS is simply a clinical descriptor for which there are likely to be several underlying etiological mechanisms—mechanisms that are not necessarily congruous with the clinical presentation. The appeal of a microbiota-based etiology lies in the fact that it is a dynamic system that can affect every aspect of intestinal function and can also impact on the brain and behavior. Despite this appeal, it is already evident that establishing a causal linkage between IBS and the intestinal microbiota represents a daunting and ongoing challenge.
2. THE GENERAL APPEAL OF THE MICROBIOTA AS PUTATIVE PATHOGENETIC FACTOR IN IBS IBS is a chronic relapsing condition and its clinical expression may change over time within an individual sufferer. Thus, the underlying driving mechanism must have inherent flexibility in terms of its ability to alter various aspects of intestinal physiology, as well as to potentially influence the brain and behavior. The intestinal microbiota has these properties; it influences all aspects of gut function, can influence behavior and is susceptible to many of the environmental factors that are linked to the expression of IBS, including psychological stress and dietary factors. “Dysbiosis” is a term of convenience that is a widely used to describe a situation in which the
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composition or metabolism of the microbiota is disturbed; there is an implicit assumption that this is accompanied by changes in one or more host systems. Intestinal dysbiosis is an attractive mechanism to explain many aspects of the natural history of IBS and several lines of reasoning support this popular proposition.
3. FACTORS KNOWN TO PRECIPITATE OR EXACERBATE IBS ALSO INDUCE INTESTINAL DYSBIOSIS 3.1 Antibiotic Exposure Antibiotic exposure clearly has the capacity to alter the intestinal microbiota at least on a temporary, if not a long-term basis in some individuals (Blaser, 2016; Rashid, Weintraub, & Nord, 2015). Several studies have shown an association between antibiotic exposure and a risk for developing chronic functional gastrointestinal (GI) symptoms. In a case–control study, there was a threefold increase in the development of IBS and those who had received antibiotics in the previous year (Maxwell, Rink, Kumar, & Mendall, 2002). In a recent nested case–control study, 83% of those reporting new functional GI symptoms had received antibiotics, generating an odds ratio of 1.95 (95% CI: 1.21–2.98; p ¼ 0.005) (Paula et al., 2015). A recent Swedish study showed that antibiotic exposure at a very young age (0–2 years) was associated with a higher prevalence of abdominal pain in young women at the age of 12 years. Interestingly, antibiotic exposure in years 9–12 did not have a similar effect. The difference in timing of exposure most likely relates to the impact of the antibiotic on the colonization process by intestinal bacteria (Uusijarvi et al., 2014). While acute enteric infection is a recognized risk factor for the development of IBS, Gwee et al. found that those who received antibiotics have a higher risk of developing functional symptoms but the relationship did not reach statistical significance (Gwee et al., 1999). While the apparent benefit of the nonabsorbable antibiotic rifamixin in some IBS patients may be construed as evidence supporting underlying dysbiosis in IBS, the interpretation is undermined by some uncertainty regarding the influences of the drug on the colonic microbiota vs those on host inflammatory responses (Pimentel, 2016).
3.2 Enteric Infection Acute gastroenteritis, whether caused by bacterial, viral, or parasitic infection, is a recognized risk factor for the development or exacerbation of
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preexisting IBS (Spiller & Garsed, 2009). Studies in human subjects recovering from acute gastroenteritis have demonstrated, not surprisingly, changes in the intestinal microbial composition that usually recover within a few weeks. Longer-term studies reveal that those patients who go on to develop postinfectious IBS have compositional changes in their microbiota, compared to healthy subjects. For example, one study, using a combination of a phylogenetic microarray and (Spiller & Garsed, 2009) selected qPCR approach, characterized the microbiome in a total of 57 individuals and found that the microbiota of those patients with postinfectious IBS exhibited differences demonstrable at the phylum level compared to healthy controls but similar in profile to that seen in patients with diarrhea predominant IBS without a previous history of enteric infection. Differences between symptomatic and healthy groups were predominant in the Bacteroidetes phylum, where there was overexpression, and in the reduced expression of mainly uncultured Clostridia compared to the healthy group (Jalanka-Tuovinen et al., 2014), findings that were extended in a subsequent study (Jalanka, Salonen, Fuentes, & de Vos, 2015). Interestingly, this group found evidence of increased expression of host genes relevant to intestinal barrier function and inflammatory responses, findings previously observed in those patients who developed IBS following the outbreak of water poisoning in Walkerton Ontario in May 2000 (Villani et al., 2010). Thus, while enteric infection acts as the trigger for the onset of IBS, longer-term dysfunction may result from a combination of host factors and intestinal display analysis to produce the chronicity of postinfectious IBS (Marshall et al., 2010).
3.3 Psychological Stress Including Early Life Stress There is a relationship between early life stress and the development of IBS later in life (Bradford et al., 2012) and recent studies in animals have shown that maternal separation produces long-lasting anxiety- and depression-like behavior in the offspring and this is associated with a compositional change in the microbiota (De Palma et al., 2015) and a susceptibility to inflammatory stimuli (Varghese et al., 2006). The changes in the microbiota, together with host factors that include altered colonic function, converge to induce the altered behavioral phenotype that characterizes this model (De Palma et al., 2015). A recent study showed that long-term supplementation of an eicosapentaenoic acid/docosahexaenoic acid (Pusceddu et al., 2015) n-3 PUFAs mixture not only improved behavior in maternal separated animals but also normalized the compositional changes in the microbiota.
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Specifically, there was restoration of the relative abundance of the bacterium Akkermansia in the fatty acid-treated animals (Pusceddu et al., 2015). This bacterium is mucolytic and would promote translocation and a susceptibility to inflammation. By inference, these results describe a linkage between early life stress, dysbiosis, altered microbiome function, low-grade inflammation, and anxiety–depression-like behavior reminiscent of changes that have been described in some IBS patients. Stress is a well-accepted factor in the expression of IBS (Qin, Cheng, Tang, & Bian, 2014). Stress has also been shown to alter the composition of the microbiota in adult mice. Using a model of social stress, Bailley et al. demonstrated a shift in microbiota composition and this was accompanied by an increase in circulating interleukin-6 (IL-6), suggesting the induction of a proinflammatory microbiota (Bailey et al., 2011). Thus, stress, in early or later life, may be a factor contributing to dysbiosis in IBS.
3.4 Dietary Factors Diet has always been implicated in the generation or exacerbation of symptoms such as abdominal pain or bloating in IBS. Erratic eating habits are common among IBS patients and this and fat are commonly reported to provoke symptoms such as bloating or abdominal pain. Recently, attention has focused on the role of wheat in symptom generation in IBS patients in whom there is an absence of features of classic celiac disease and this remains a work in progress (Pinto-Sanchez, Bercik, & Verdu, 2015). In addition, much attention has focused recently on the role of FODMAPS in symptoms generation in IBS but the data, that were initially promising, remain controversial (De Giorgio, Volta, & Gibson, 2016). For the purposes of this review, the question is whether these dietary components induce symptoms expression via changes in microbiota composition or metabolism or whether they reflect direct interactions between food components and host physiological or immunological systems. Studies that have attempted to link diet with altered microbiota profiles have often used comparisons of communities in which there are stable dietary differences among culturally or geographically distinct populations (De Filippo et al., 2010). It is difficult to reconcile these findings with the less stable and often erratic food intake patents characteristically seen in IBS patients. Intervention studies have added little to our understanding as they often involve large and dramatic changes in dietary composition that bare little relevance to the fluctuations in diet seen in IBS patients.
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4. EVIDENCE OF DYSBIOSIS IN IBS PATIENTS Despite a large number of studies using molecular-based approaches to profile the microbiome in IBS patients, the field remains unclear, and no microbial signature currently exists that distinguishes IBS or its subtypes from healthy controls. There are several factors the contribute to this and they include the inherent variability in the intestinal microbiota among healthy individuals, the confounding factor of diet, and differences in the technology used to profile the microbiome, rendering comparisons between studies difficult. In addition, and perhaps most important, the fact is that the majority of studies rely on a snapshot of the microbiome at a single point in time and therefore do not accommodate the many lifestyle factors that contribute to variability in both healthy subjects and controls. Commensal bacteria most relevant to the expression of IBS are likely those that reside in the distal small bowel, cecum, and colon. We are just beginning to characterize the small intestinal microbiome but it is clear that this is more likely to be subject to short-term influences of dietary variation than the colonic microbiome, due to the influence of simple dietary carbohydrates on bacterial species such as Streptococcus spp. (Zoetendal et al., 2012). The question as to whether bacterial overgrowth of the small intestine is a component of the pathophysiology of IBS remains controversial due to uncertainties regarding the methodologies used in many of these studies and the mechanism of action of the antibiotic rixaminin (Pimentel, 2016; Spiegel, 2011). With respect to the colonic microbiome, there is general agreement there is a reduction in richness/diversity as well as some evidence of temporal instability. In addition, not all IBS patients exhibit discernible compositional differences in their microbiome compared to healthy controls, although this conclusion is subject to the caveats described earlier (Jeffery, Claesson, O’Toole, & Shanahan, 2012). It is beyond the scope of this review to detail the often conflicting microbiome profiles that have been associated with IBS patients and with symptom expression and IBS subgroups. This is referred to recent excellent reviews on this subject (Rajilic-Stojanovic et al., 2015; Simren et al., 2013). Given the characteristic fluctuation in symptom type and severity seen in IBS patients, this author believes that documentation of temporal instability of the microbiota is more important than the compositional profiles reported to date in IBS subjects. In a study using culture techniques and denaturing gradient gel electrophoresis, Matto et al. found only small compositional
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differences between the IBS and control groups. However, significantly more temporal instability was evident in the IBS group, some of whom were followed up 6 months (Matto et al., 2005). Unfortunately, the impact of antibiotic usage in some patients undermined the impact of the study. Using an RNA-based approach, Maukonen et al. extended these findings, excluding patients that had received antibiotics 2 months prior to the study and confirmed greater instability of the microbiota in the IBS patients compared to healthy controls (Maukonen et al., 2006). A more recent study involving only two diarrhea predominant IBS patients and two healthy controls (including the husband of one of the patients) followed over 6–8 weeks used metagenomic and metatranscriptomic approaches. The IBS in these two cases was characterized by frequent relapses and episodes of remission and enabled the investigators to correlate changes in composition and activity with symptoms. They were able to identify a fraction of the microbiota whose transcriptomic based activity was related to IBS symptoms and showed considerable instability over time. The compositional shifts, particularly in the less active microbiota population of the IBS patients were less prominent. These findings provide encouragement in terms of providing a strategy to better understand host–microbial interactions in this condition. Clearly, while it is tempting to speculate that instability in the microbiota induced fluctuations in symptom profile, as has been implied in a probiotic study that improved both microbiota stability and IBS symptoms (Kajander et al., 2008), the possibility that the community instability simply reflects reactions to changes in intestinal physiology cannot be ruled out. In all likelihood, the findings represent a combination of factors of bacterial and host origin and constitute the mechanism for perpetuating intestinal dysfunction in this chronic condition (Durban et al., 2013). There is growing interest in correlating the products of microbial metabolism with symptom generation in IBS. Increased fecal levels of acetic and propionic acid have been found in IBS patients compared to healthy controls in the findings correlated with the severity of IBS symptoms (Tana et al., 2010). Intestinal bacteria determine the bioavailability of dietary tryptophan to the host and consequently to the synthesis of serotonin. High levels of circulating serotonin have been found in patients with diarrhea predominant IBS and low levels in constipation predominant IBS (Dunlop et al., 2005) in these differences could reflect alterations in the intestinal microbiota across these IBS subgroups. The intestinal microbiota could also influence dietary tryptophan metabolism along a pathway that results in the formation of neurotoxic metabolites that may in turn contribute to behavioral changes in
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those patients with psychiatric comorbidity (Jenkins, Nguyen, Polglaze, & Bertrand, 2016).
5. PROOF OF PRINCIPLE THAT INTESTINAL DYSBIOSIS ALTERS FUNCTION IN THE GUT AND BRAIN There is extensive evidence derived from animal studies showing that the intestinal microbiota can alter parameters of gut dysfunction that have been demonstrated in IBS patients. These include changes in intestinal motility, changes in visceral pain responses, alterations in epithelial secretory and barrier function, and in inflammatory and/or immunological function. Many of these studies are based on comparisons between germ-free and colonized animals and while they demonstrate the importance of the influence of the microbiota on these systems, they have limited relevance to the implied dysbiosis implicated in IBS. In addition, virtually all of the host systems relevant to IBS, including the intestinal physiological apparatus, the mucosal immune system including barrier function, and the brain are immature in germ-free animals. A strategy that is more relevant to the implied dysbiosis of IBS is to experimentally disrupt the intestinal microbiota in an otherwise healthy animal with a previously stable microbiota. This can be achieved through diet or through the use of nonabsorbable orally administered antimicrobial agents. The first study to evaluate changes in host following experimental despite doses utilized a cocktail of antimicrobials that included bacitracin, neomycin, and pimaricin administered in the drinking water for a total of 10 days two specific pathogen free mice. The study was aimed at determining whether experimental dysbiosis could induce changes in visceral pain responses elicited by balloon distention of the colorectum. Antimicrobial treatment resulted in a significant increase in responses to colorectal distention and this was accompanied by an increase in immunoreactive substance P and CGP in the myenteric plexus (Verdu et al., 2006). Antimicrobial therapy was associated with depletion of lactobacilli, Bacteroides and enterococci but, in a separate experiment, administration of lactobacillus paracaseii prevented the dysbiosisinduced changes in visceral pain responses. The sensory changes induced by dysbiosis where accompanied by a small low-grade inflammatory response. Treatment with dexamethasone prevented the inflammatory response as well as the changes in visceral pain responses. Taken together, these findings provided proof of principle that experimental dysbiosis induces low-grade inflammation and increases visceral pain responses—findings reminiscent of IBS
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(Verdu et al., 2006). A subsequent study extended these findings by incorporating a mild stressor with nonabsorbable antibiotics to induce dysbiosis, which was monitored in mucosal adherent bacterial populations rather than in the feces. While total bacterial counts were reduced, adherence was enhanced and there was a corresponding increase in luminal s-IgA. Stress alone increased visceral pain responses and upregulated expression of cannabinoid receptor 2 and this increase was abrogated by the concomitant use of antimicrobials (Aguilera, Vergara, & Martinez, 2013). In a later study, the same authors used intraperitoneal acetic acid to induce visceral hypersensitivity and used neomycin and bacitracin to induce dysbiosis. There was an increase in the expression of the cannabinoid receptor 2 and downregulation of the cannabinoid 1 and the μ-opioid receptors. Visceral pain responses were substantially reduced in the presence of dysbiosis, although colonic muscle contractility was enhanced (Aguilera, Cerda-Cuellar, & Martinez, 2015). The study showing the induction of visceral hypersensitivity in the presence of intestinal dysbiosis and the studies showing that dysbiosis was accompanied by an inhibition of enhanced visceral pain responses were performed in completely different laboratories in which the endogenous microbiota was likely different. This illustrates two points. First, it is difficult to extrapolate results involving experimental dysbiosis between laboratories. Second, they show that depending on the composition of the microbiota, experimental dysbiosis can produce diametrically opposite effects on a given host parameter. This may have bearing on the fluctuations often seen in the nature of symptoms reported by an individual IBS patient over time. A recent study examined the consequences of antibiotic exposure early in life, a subject of considerable current interest in the natural history of IBS. Vancomycin was administered from the 4th to the 13th postnatal days in rat pups. This resulted in transient dysbiosis with restoration of the normal microbiome by 8 weeks. Nevertheless, these mice displayed visceral hypersensitivity later in life (O’Mahony et al., 2014). A similar phenomenon was observed when the combination of neomycin, bacitracin, and pimaricin was used in the postnatal period. Thus, disruption of the bacterial colonization process early in life impacts on enteric sensory function later in life. An interesting phenomenon observed in this study was that the effect on sensory function did not occur in female rats. As mentioned earlier, experimental dysbiosis can lead to immune activation and low-grade inflammation, which in turn led to changes in gut physiology. A study examined the role of the innate immune system in a model of antibiotic induced dysbiosis. In this study, investigators used mice lacking
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critical components of the innate immune system. Antibiotic induced dysbiosis resulted in a significant delay of GI motility and this was accompanied by a reduction in the number of nitrergic neurons in the colon. Because a similar profile was observed in mice with neural crest-specific deletion of Myd88 (Wnt1Cre(+/)/Myd88(fl/fl)) as well as TLR4, the authors concluded that the microbiota influence GI motor function via activation of the innate immune system via TLR4 receptors (Anitha, Vijay-Kumar, Sitaraman, Gewirtz, & Srinivasan, 2012). There is increasing interest in the ability of the intestinal microbiota to influence the brain and behavior, prompting consideration of a microbiota– gut–brain axis (Collins, Surette, & Bercik, 2012) and this may be relevant to the psychiatric comorbidity that is common among IBS patients (Fond et al., 2014). Several strategies have been used to explore microbiota–brain interactions and they include comparisons between germ-free and colonized mice (Bercik et al., 2011; Diaz Heijtz et al., 2011; Neufeld, Kang, Bienenstock, & Foster, 2011; Sudo et al., 2004), the adoptive transfer of behavioral phenotype between murine strains via the intestinal microbiota (Bercik et al., 2011; Collins, Kassam, & Bercik, 2013), and the effect of experimental dysbiosis on brain and behavior (Bercik et al., 2011; Li, Dowd, Scurlock, Acosta-Martinez, & Lyte, 2009). For reasons already mentioned, the latter strategy is more relevant to IBS and will be addressed here. In one study, mice were fed on either a standard rodent chow or a chow containing 50% lean beef for 3 months. This resulted in a significant increase in diversity of the microbiome, as assessed by bacterial tag-encoded FLX amplicon pyrosequencing, in mice fed the beef enriched diet. Performance in memory and learning behavioral testing was enhanced in the mice fed the beef enriched diet and they also exhibited less anxiety like behavior (Li et al., 2009). While it was concluded that the diet-induced dysbiosis was responsible for the behavioral change, the authors acknowledge that direct contributions from the beef enriched diet could have contributed to the changes in brain function. In a more recent study involving antibiotic-induced dysbiosis, Bercik et al. found oral but not intraperitoneal administration of nonabsorbable antibiotics increased exploratory behavior as well as increased levels of brain-derived neurotropic factor in the hippocampus. The changes in neurochemistry and behavior were independent of the integrity of the vagus nerve and the sympathetic nervous system (Bercik et al., 2011). A recent study by Frohlich et al. showed that antibiotic induced dysbiosis in mice reduced microbiota-derived metabolites in the colon and their metabolic derivatives in the plasma. This was accompanied by
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cognitive impairment and changes in chemistry, indicating that intestinal dysbiosis may lead to behavioral changes by influencing host metabolism (http://www.sciencedirect.com/science/article/pii/S088915911630040X).
5.1 Is There a Causal Link Between Dysbiosis and Symptom Expression in IBS? With limited therapeutic or investigative tools, coupled with the absence of confidently identified “bacteria of interest,” it is almost impossible to establish causal links between the intestinal microbiota and symptom generation in IBS. The benefit of selected probiotics in improving symptoms and quality of life in IBS patients provides insufficient support for a causal role of the microbiota in the expression of this condition, as the underlying modes of action and impact on the microbiota community are poorly understood. Currently, our best strategy toward establishing the functional relevance of the IBS microbiota in producing gut dysfunctions is the use of human microbiota-associated animals. This strategy involves colonizing germ-free mice with microbiota taken from IBS patients or healthy controls. Crouzet et al. use this strategy to demonstrate the ability of the microbiota to induce visceral sensitivity in rats. The colonization of germ-free rats with microbiota from IBS patients with demonstrable visceral hypersensitivity, but not healthy controls, resulted in increased responses to colorectal distention in the recipient animals (Crouzet et al., 2013). This was not accompanied by changes in intestinal permeability or in mast cell density in the recipient mice. In a recent preliminary study (De Palma et al., 2014), germ-free mice were colonized with microbiota from diarrhea predominant IBS patients with or without anxiety, or from healthy controls and gut function and behavior were assessed. IBS microbiota-associated mice developed rapid transit, increased intestinal permeability in and secretion, and evidence of innate immune activation. Mice colonized with microbiota from patients with anxiety showed evidence of anxiety-like behavior whereas mice receiving microbiota from IBS patients without anxiety showed normal behavior (De Palma et al., 2014). These results provide strong support for the notion that the intestinal microbiota of IBS-d patients contributes to both the intestinal and behavioral manifestations of this condition. Zheng et al. recently provided further support for the notion that the intestinal microbiota contributes to psychopathology by showing that fecal microbiota from depressed patients altered brain chemistry and behavior following colonization of germ-free animals (Zheng et al., 2016).
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6. FUTURE DIRECTIONS Much evidence now points toward a role of the intestinal microbiota in the expression of IBS, the most common intestinal disorder in our society today. Clinical studies must move away from single time point comparisons of microbiome profiles between IBS patients and healthy controls. More emphasis should be placed on longitudinal studies in which patients serve as their own controls, thus minimizing the problem of inter-subject variation and microbiota composition. The field must now move forward toward establishing evidence of causality between the microbiota and symptom expression, including psychiatric comorbidity. Increased use of human microbiota-associated animals should generate important new information regarding a functional role of the microbiota in this condition. Our increasing ability to culture the “noncultivable” intestinal microbial population will enhance our ability to comprehensively profile the bacterial community, to understand community dynamics, and to adopt prescribed colonization strategies using germ-free mice in order to better understand microbe–host interactions. This in turn will help deliver novel therapeutic targets and the strategies for improving this common condition. Last, but by no means least, the influence of the intestinal virome on the intestinal microbiota must now be studied and exploited for therapeutic gain in IBS and other conditions.
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Jalanka-Tuovinen, J., Salojarvi, J., Salonen, A., Immonen, O., Garsed, K., Kelly, F. M., … de Vos, W. M. (2014). Faecal microbiota composition and host-microbe cross-talk following gastroenteritis and in postinfectious irritable bowel syndrome. Gut, 63(11), 1737–1745. http://dx.doi.org/10.1136/gutjnl-2013-305994. Jeffery, I. B., Claesson, M. J., O’Toole, P. W., & Shanahan, F. (2012). Categorization of the gut microbiota: Enterotypes or gradients? Nature Reviews. Microbiology, 10(9), 591–592. Jenkins, T. A., Nguyen, J. C., Polglaze, K. E., & Bertrand, P. P. (2016). Influence of tryptophan and serotonin on mood and cognition with a possible role of the gut-brain axis. Nutrients, 8(1), 56. http://dx.doi.org/10.3390/nu8010056. http://www.mdpi.com/ 2072-6643/8/1/56. Kajander, K., Myllyluoma, E., Rajilic-Stojanovic, M., Kyronpalo, S., Rasmussen, M., Jarvenpaa, S., … Korpela, R. (2008). Clinical trial: Multispecies probiotic supplementation alleviates the symptoms of irritable bowel syndrome and stabilizes intestinal microbiota. Alimentary Pharmacology & Therapeutics, 27(1), 48–57. http://dx.doi.org/10.1111/ j.1365-2036.2007.03542.x. Li, W., Dowd, S. E., Scurlock, B., Acosta-Martinez, V., & Lyte, M. (2009). Memory and learning behavior in mice is temporally associated with diet-induced alterations in gut bacteria. Physiology & Behavior, 96(4–5), 557–567. http://dx.doi.org/10.1016/j. physbeh.2008.12.004. Marshall, J. K., Thabane, M., Garg, A. X., Clark, W. F., Moayyedi, P., & Collins, S. M. (2010). Eight year prognosis of postinfectious irritable bowel syndrome following waterborne bacterial dysentery. Gut, 59(5), 605–611. http://dx.doi.org/10.1136/gut.2009.202234. Matto, J., Maunuksela, L., Kajander, K., Palva, A., Korpela, R., Kassinen, A., & Saarela, M. (2005). Composition and temporal stability of gastrointestinal microbiota in irritable bowel syndrome—A longitudinal study in IBS and control subjects. FEMS Immunology and Medical Microbiology, 43(2), 213–222. Maukonen, J., Satokari, R., Matto, J., Soderlund, H., Mattila-Sandholm, T., & Saarela, M. (2006). Prevalence and temporal stability of selected clostridial groups in irritable bowel syndrome in relation to predominant faecal bacteria. Journal of Medical Microbiology, 55(Pt. 5), 625–633. http://dx.doi.org/10.1099/jmm.0.46134-0. Maxwell, P. R., Rink, E., Kumar, D., & Mendall, M. A. (2002). Antibiotics increase functional abdominal symptoms. The American Journal of Gastroenterology, 97(1), 104–108. http://dx.doi.org/10.1111/j.1572-0241.2002.05428.x. Neufeld, K. M., Kang, N., Bienenstock, J., & Foster, J. A. (2011). Reduced anxiety-like behavior and central neurochemical change in germ-free mice. Neurogastroenterology and Motility, 23(3), 255–264. http://dx.doi.org/10.1111/j.1365-2982.2010.01620.x. e119. O’Mahony, S. M., Felice, V. D., Nally, K., Savignac, H. M., Claesson, M. J., Scully, P., … Cryan, J. F. (2014). Disturbance of the gut microbiota in early-life selectively affects visceral pain in adulthood without impacting cognitive or anxiety-related behaviors in male rats. Neuroscience, 277, 885–901. http://dx.doi.org/10.1016/j.neuroscience.2014.07.054. Paula, H., Grover, M., Halder, S. L., Locke, G. R., 3rd, Schleck, C. D., Zinsmeister, A. R., & Talley, N. J. (2015). Non-enteric infections, antibiotic use, and risk of development of functional gastrointestinal disorders. Neurogastroenterology and Motility, 27(11), 1580–1586. http://dx.doi.org/10.1111/nmo.12655. Pimentel, M. (2016). Review article: Potential mechanisms of action of rifaximin in the management of irritable bowel syndrome with diarrhoea. Alimentary Pharmacology & Therapeutics, 43(Suppl. 1), 37–49. http://dx.doi.org/10.1111/apt.13437. Pinto-Sanchez, M. I., Bercik, P., & Verdu, E. F. (2015). Motility alterations in celiac disease and non-celiac gluten sensitivity. Digestive Diseases, 33(2), 200–207. http://dx.doi.org/ 10.1159/000371400. Pusceddu, M. M., Kelly, P., Ariffin, N., Cryan, J. F., Clarke, G., & Dinan, T. G. (2015). n-3 PUFAs have beneficial effects on anxiety and cognition in female rats: Effects of
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early life stress. Psychoneuroendocrinology, 58, 79–90. http://dx.doi.org/10.1016/j. psyneuen.2015.04.015. Qin, H. Y., Cheng, C. W., Tang, X. D., & Bian, Z. X. (2014). Impact of psychological stress on irritable bowel syndrome. World Journal of Gastroenterology, 20(39), 14126–14131. http://dx.doi.org/10.3748/wjg.v20.i39.14126. Rajilic-Stojanovic, M., Jonkers, D. M., Salonen, A., Hanevik, K., Raes, J., Jalanka, J., … Penders, J. (2015). Intestinal microbiota and diet in IBS: Causes, consequences, or epiphenomena? The American Journal of Gastroenterology, 110(2), 278–287. http://dx.doi. org/10.1038/ajg.2014.427. Rashid, M. U., Weintraub, A., & Nord, C. E. (2015). Development of antimicrobial resistance in the normal anaerobic microbiota during one year after administration of clindamycin or ciprofloxacin. Anaerobe, 31, 72–77. http://dx.doi.org/10.1016/j.anaerobe.2014.10.004. Simren, M., Barbara, G., Flint, H. J., Spiegel, B. M., Spiller, R. C., Vanner, S., … Zoetendal, E. G. (2013). Intestinal microbiota in functional bowel disorders: A Rome foundation report. Gut, 62(1), 159–176. http://dx.doi.org/10.1136/gutjnl-2012-302167. Spiegel, B. M. (2011). Questioning the bacterial overgrowth hypothesis of irritable bowel syndrome: An epidemiologic and evolutionary perspective. Clinical Gastroenterology and Hepatology, 9(6), 461–469. http://dx.doi.org/10.1016/j.cgh.2011.02.030. quiz e459. Spiller, R., & Garsed, K. (2009). Postinfectious irritable bowel syndrome. Gastroenterology, 136(6), 1979–1988. http://dx.doi.org/10.1053/j.gastro.2009.02.074. Sudo, N., Chida, Y., Aiba, Y., Sonoda, J., Oyama, N., Yu, X. N., … Koga, Y. (2004). Postnatal microbial colonization programs the hypothalamic–pituitary–adrenal system for stress response in mice. The Journal of Physiology, 558(Pt. 1), 263–275. http://dx.doi. org/10.1113/jphysiol.2004.063388. Tana, C., Umesaki, Y., Imaoka, A., Handa, T., Kanazawa, M., & Fukudo, S. (2010). Altered profiles of intestinal microbiota and organic acids may be the origin of symptoms in irritable bowel syndrome. Neurogastroenterology and Motility, 22(5), 512–519. http://dx.doi. org/10.1111/j.1365-2982.2009.01427.x. e114–515. Uusijarvi, A., Bergstrom, A., Simren, M., Ludvigsson, J. F., Kull, I., Wickman, M., … Olen, O. (2014). Use of antibiotics in infancy and childhood and risk of recurrent abdominal pain—A Swedish birth cohort study. Neurogastroenterology and Motility, 26(6), 841–850. http://dx.doi.org/10.1111/nmo.12340. Varghese, A. K., Verdu, E. F., Bercik, P., Khan, W. I., Blennerhassett, P. A., Szechtman, H., & Collins, S. M. (2006). Antidepressants attenuate increased susceptibility to colitis in a murine model of depression. Gastroenterology, 130(6), 1743–1753. http://dx.doi.org/ 10.1053/j.gastro.2006.02.007. Verdu, E. F., Bercik, P., Verma-Gandhu, M., Huang, X. X., Blennerhassett, P., Jackson, W., … Collins, S. M. (2006). Specific probiotic therapy attenuates antibiotic induced visceral hypersensitivity in mice. Gut, 55(2), 182–190. http://dx.doi.org/10.1136/ gut.2005.066100. Villani, A. C., Lemire, M., Thabane, M., Belisle, A., Geneau, G., Garg, A. X., … Marshall, J. K. (2010). Genetic risk factors for post-infectious irritable bowel syndrome following a waterborne outbreak of gastroenteritis. Gastroenterology, 138(4), 1502–1513. http://dx.doi.org/10.1053/j.gastro.2009.12.049. Zheng, P., Zeng, B., Zhou, C., Liu, M., Fang, Z., Xu, X., … Xie, P. (2016). Gut microbiome remodeling induces depressive-like behaviors through a pathway mediated by the host’s metabolism. Molecular Psychiatry, 21(6), 786–796. http://dx.doi.org/10.1038/ mp.2016.44. Zoetendal, E. G., Raes, J., van den Bogert, B., Arumugam, M., Booijink, C. C., Troost, F. J., … Kleerebezem, M. (2012). The human small intestinal microbiota is driven by rapid uptake and conversion of simple carbohydrates. The ISME Journal, 6(7), 1415–1426. http://dx.doi.org/10.1038/ismej.2011.212.
The Intestinal Microbiota in the Irritable Bowel Syndrome S.M. Collins1 The Farncombe Family Digestive Health Research Centre, The Michael G DeGroote School of Medicine, McMaster University, Hamilton, ON, Canada 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. The General Appeal of the Microbiota as Putative Pathogenetic Factor in IBS 3. Factors Known to Precipitate or Exacerbate IBS also Induce Intestinal Dysbiosis 3.1 Antibiotic Exposure 3.2 Enteric Infection 3.3 Psychological Stress Including Early Life Stress 3.4 Dietary Factors 4. Evidence of Dysbiosis in IBS Patients 5. Proof of Principle that Intestinal Dysbiosis Alters Function in the Gut and Brain 5.1 Is There a Causal Link Between Dysbiosis and Symptom Expression in IBS? 6. Future Directions References
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Abstract The irritable bowel syndrome (IBS) is a chronic abdominal symptom complex occurring in a bowel devoid of discernible relevant pathology. There is growing interest in the role of the intestinal microbiota as a basis for the intestinal and possibly behavioral manifestations of this condition. Molecular-based microbial profiling has revealed compositional changes in the microbiota of at least a subset of IBS patients but the data are often conflicting and no microbial signature for this condition has yet been defined. Animal studies in which a previously stable intestinal microbiota is perturbed, by antibiotics or dietary change, results in alterations in intestinal function reminiscent of that seen in IBS patients. These include visceral sensitivity to painful stimuli, altered motility and intestinal barrier function as well as immune activation, and low-grade inflammation. More recent studies have shown that perturbation of the microbial composition of the gut alters brain chemistry and behavior. In a step toward establishing a causal link between an altar microbiota and gut–brain manifestations of IBS, colonization of germfree mice with microbiota from IBS patients results in an IBS-like phenotype, including alterations and behavior if the donor exhibited psychiatric comorbidity, such as high levels of anxiety. This model provides an opportunity for exploring the mechanisms
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underlying host–microbe interactions relevant to the pathogenesis of IBS and for developing novel therapeutic targets.
1. INTRODUCTION The irritable bowel syndrome (IBS) is a chronic abdominal symptom complex in which pain or discomfort related to altered bowel habit are the clinical hallmarks. These symptoms arise in the absence of any demonstrable pathology and the disorder is considered to be one of function rather than structure. Psychiatric comorbidity is very common and there is a strong link between psychological stress and the expression of IBS. It is therefore considered to be a disorder of the gut–brain axis. While, the IBS is the most common intestinal disorders in our society, our understanding of its underlying pathogenesis remains far from clear. IBS is heterogeneous in terms of its clinical manifestation and much effort has gone into defining clinically homogeneous IBS subgroups. However, there is unlikely to be homogeneity of the underlying pathogenesis even within these subgroups. IBS is simply a clinical descriptor for which there are likely to be several underlying etiological mechanisms—mechanisms that are not necessarily congruous with the clinical presentation. The appeal of a microbiota-based etiology lies in the fact that it is a dynamic system that can affect every aspect of intestinal function and can also impact on the brain and behavior. Despite this appeal, it is already evident that establishing a causal linkage between IBS and the intestinal microbiota represents a daunting and ongoing challenge.
2. THE GENERAL APPEAL OF THE MICROBIOTA AS PUTATIVE PATHOGENETIC FACTOR IN IBS IBS is a chronic relapsing condition and its clinical expression may change over time within an individual sufferer. Thus, the underlying driving mechanism must have inherent flexibility in terms of its ability to alter various aspects of intestinal physiology, as well as to potentially influence the brain and behavior. The intestinal microbiota has these properties; it influences all aspects of gut function, can influence behavior and is susceptible to many of the environmental factors that are linked to the expression of IBS, including psychological stress and dietary factors. “Dysbiosis” is a term of convenience that is a widely used to describe a situation in which the
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composition or metabolism of the microbiota is disturbed; there is an implicit assumption that this is accompanied by changes in one or more host systems. Intestinal dysbiosis is an attractive mechanism to explain many aspects of the natural history of IBS and several lines of reasoning support this popular proposition.
3. FACTORS KNOWN TO PRECIPITATE OR EXACERBATE IBS ALSO INDUCE INTESTINAL DYSBIOSIS 3.1 Antibiotic Exposure Antibiotic exposure clearly has the capacity to alter the intestinal microbiota at least on a temporary, if not a long-term basis in some individuals (Blaser, 2016; Rashid, Weintraub, & Nord, 2015). Several studies have shown an association between antibiotic exposure and a risk for developing chronic functional gastrointestinal (GI) symptoms. In a case–control study, there was a threefold increase in the development of IBS and those who had received antibiotics in the previous year (Maxwell, Rink, Kumar, & Mendall, 2002). In a recent nested case–control study, 83% of those reporting new functional GI symptoms had received antibiotics, generating an odds ratio of 1.95 (95% CI: 1.21–2.98; p ¼ 0.005) (Paula et al., 2015). A recent Swedish study showed that antibiotic exposure at a very young age (0–2 years) was associated with a higher prevalence of abdominal pain in young women at the age of 12 years. Interestingly, antibiotic exposure in years 9–12 did not have a similar effect. The difference in timing of exposure most likely relates to the impact of the antibiotic on the colonization process by intestinal bacteria (Uusijarvi et al., 2014). While acute enteric infection is a recognized risk factor for the development of IBS, Gwee et al. found that those who received antibiotics have a higher risk of developing functional symptoms but the relationship did not reach statistical significance (Gwee et al., 1999). While the apparent benefit of the nonabsorbable antibiotic rifamixin in some IBS patients may be construed as evidence supporting underlying dysbiosis in IBS, the interpretation is undermined by some uncertainty regarding the influences of the drug on the colonic microbiota vs those on host inflammatory responses (Pimentel, 2016).
3.2 Enteric Infection Acute gastroenteritis, whether caused by bacterial, viral, or parasitic infection, is a recognized risk factor for the development or exacerbation of
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preexisting IBS (Spiller & Garsed, 2009). Studies in human subjects recovering from acute gastroenteritis have demonstrated, not surprisingly, changes in the intestinal microbial composition that usually recover within a few weeks. Longer-term studies reveal that those patients who go on to develop postinfectious IBS have compositional changes in their microbiota, compared to healthy subjects. For example, one study, using a combination of a phylogenetic microarray and (Spiller & Garsed, 2009) selected qPCR approach, characterized the microbiome in a total of 57 individuals and found that the microbiota of those patients with postinfectious IBS exhibited differences demonstrable at the phylum level compared to healthy controls but similar in profile to that seen in patients with diarrhea predominant IBS without a previous history of enteric infection. Differences between symptomatic and healthy groups were predominant in the Bacteroidetes phylum, where there was overexpression, and in the reduced expression of mainly uncultured Clostridia compared to the healthy group (Jalanka-Tuovinen et al., 2014), findings that were extended in a subsequent study (Jalanka, Salonen, Fuentes, & de Vos, 2015). Interestingly, this group found evidence of increased expression of host genes relevant to intestinal barrier function and inflammatory responses, findings previously observed in those patients who developed IBS following the outbreak of water poisoning in Walkerton Ontario in May 2000 (Villani et al., 2010). Thus, while enteric infection acts as the trigger for the onset of IBS, longer-term dysfunction may result from a combination of host factors and intestinal display analysis to produce the chronicity of postinfectious IBS (Marshall et al., 2010).
3.3 Psychological Stress Including Early Life Stress There is a relationship between early life stress and the development of IBS later in life (Bradford et al., 2012) and recent studies in animals have shown that maternal separation produces long-lasting anxiety- and depression-like behavior in the offspring and this is associated with a compositional change in the microbiota (De Palma et al., 2015) and a susceptibility to inflammatory stimuli (Varghese et al., 2006). The changes in the microbiota, together with host factors that include altered colonic function, converge to induce the altered behavioral phenotype that characterizes this model (De Palma et al., 2015). A recent study showed that long-term supplementation of an eicosapentaenoic acid/docosahexaenoic acid (Pusceddu et al., 2015) n-3 PUFAs mixture not only improved behavior in maternal separated animals but also normalized the compositional changes in the microbiota.
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Specifically, there was restoration of the relative abundance of the bacterium Akkermansia in the fatty acid-treated animals (Pusceddu et al., 2015). This bacterium is mucolytic and would promote translocation and a susceptibility to inflammation. By inference, these results describe a linkage between early life stress, dysbiosis, altered microbiome function, low-grade inflammation, and anxiety–depression-like behavior reminiscent of changes that have been described in some IBS patients. Stress is a well-accepted factor in the expression of IBS (Qin, Cheng, Tang, & Bian, 2014). Stress has also been shown to alter the composition of the microbiota in adult mice. Using a model of social stress, Bailley et al. demonstrated a shift in microbiota composition and this was accompanied by an increase in circulating interleukin-6 (IL-6), suggesting the induction of a proinflammatory microbiota (Bailey et al., 2011). Thus, stress, in early or later life, may be a factor contributing to dysbiosis in IBS.
3.4 Dietary Factors Diet has always been implicated in the generation or exacerbation of symptoms such as abdominal pain or bloating in IBS. Erratic eating habits are common among IBS patients and this and fat are commonly reported to provoke symptoms such as bloating or abdominal pain. Recently, attention has focused on the role of wheat in symptom generation in IBS patients in whom there is an absence of features of classic celiac disease and this remains a work in progress (Pinto-Sanchez, Bercik, & Verdu, 2015). In addition, much attention has focused recently on the role of FODMAPS in symptoms generation in IBS but the data, that were initially promising, remain controversial (De Giorgio, Volta, & Gibson, 2016). For the purposes of this review, the question is whether these dietary components induce symptoms expression via changes in microbiota composition or metabolism or whether they reflect direct interactions between food components and host physiological or immunological systems. Studies that have attempted to link diet with altered microbiota profiles have often used comparisons of communities in which there are stable dietary differences among culturally or geographically distinct populations (De Filippo et al., 2010). It is difficult to reconcile these findings with the less stable and often erratic food intake patents characteristically seen in IBS patients. Intervention studies have added little to our understanding as they often involve large and dramatic changes in dietary composition that bare little relevance to the fluctuations in diet seen in IBS patients.
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4. EVIDENCE OF DYSBIOSIS IN IBS PATIENTS Despite a large number of studies using molecular-based approaches to profile the microbiome in IBS patients, the field remains unclear, and no microbial signature currently exists that distinguishes IBS or its subtypes from healthy controls. There are several factors the contribute to this and they include the inherent variability in the intestinal microbiota among healthy individuals, the confounding factor of diet, and differences in the technology used to profile the microbiome, rendering comparisons between studies difficult. In addition, and perhaps most important, the fact is that the majority of studies rely on a snapshot of the microbiome at a single point in time and therefore do not accommodate the many lifestyle factors that contribute to variability in both healthy subjects and controls. Commensal bacteria most relevant to the expression of IBS are likely those that reside in the distal small bowel, cecum, and colon. We are just beginning to characterize the small intestinal microbiome but it is clear that this is more likely to be subject to short-term influences of dietary variation than the colonic microbiome, due to the influence of simple dietary carbohydrates on bacterial species such as Streptococcus spp. (Zoetendal et al., 2012). The question as to whether bacterial overgrowth of the small intestine is a component of the pathophysiology of IBS remains controversial due to uncertainties regarding the methodologies used in many of these studies and the mechanism of action of the antibiotic rixaminin (Pimentel, 2016; Spiegel, 2011). With respect to the colonic microbiome, there is general agreement there is a reduction in richness/diversity as well as some evidence of temporal instability. In addition, not all IBS patients exhibit discernible compositional differences in their microbiome compared to healthy controls, although this conclusion is subject to the caveats described earlier (Jeffery, Claesson, O’Toole, & Shanahan, 2012). It is beyond the scope of this review to detail the often conflicting microbiome profiles that have been associated with IBS patients and with symptom expression and IBS subgroups. This is referred to recent excellent reviews on this subject (Rajilic-Stojanovic et al., 2015; Simren et al., 2013). Given the characteristic fluctuation in symptom type and severity seen in IBS patients, this author believes that documentation of temporal instability of the microbiota is more important than the compositional profiles reported to date in IBS subjects. In a study using culture techniques and denaturing gradient gel electrophoresis, Matto et al. found only small compositional
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differences between the IBS and control groups. However, significantly more temporal instability was evident in the IBS group, some of whom were followed up 6 months (Matto et al., 2005). Unfortunately, the impact of antibiotic usage in some patients undermined the impact of the study. Using an RNA-based approach, Maukonen et al. extended these findings, excluding patients that had received antibiotics 2 months prior to the study and confirmed greater instability of the microbiota in the IBS patients compared to healthy controls (Maukonen et al., 2006). A more recent study involving only two diarrhea predominant IBS patients and two healthy controls (including the husband of one of the patients) followed over 6–8 weeks used metagenomic and metatranscriptomic approaches. The IBS in these two cases was characterized by frequent relapses and episodes of remission and enabled the investigators to correlate changes in composition and activity with symptoms. They were able to identify a fraction of the microbiota whose transcriptomic based activity was related to IBS symptoms and showed considerable instability over time. The compositional shifts, particularly in the less active microbiota population of the IBS patients were less prominent. These findings provide encouragement in terms of providing a strategy to better understand host–microbial interactions in this condition. Clearly, while it is tempting to speculate that instability in the microbiota induced fluctuations in symptom profile, as has been implied in a probiotic study that improved both microbiota stability and IBS symptoms (Kajander et al., 2008), the possibility that the community instability simply reflects reactions to changes in intestinal physiology cannot be ruled out. In all likelihood, the findings represent a combination of factors of bacterial and host origin and constitute the mechanism for perpetuating intestinal dysfunction in this chronic condition (Durban et al., 2013). There is growing interest in correlating the products of microbial metabolism with symptom generation in IBS. Increased fecal levels of acetic and propionic acid have been found in IBS patients compared to healthy controls in the findings correlated with the severity of IBS symptoms (Tana et al., 2010). Intestinal bacteria determine the bioavailability of dietary tryptophan to the host and consequently to the synthesis of serotonin. High levels of circulating serotonin have been found in patients with diarrhea predominant IBS and low levels in constipation predominant IBS (Dunlop et al., 2005) in these differences could reflect alterations in the intestinal microbiota across these IBS subgroups. The intestinal microbiota could also influence dietary tryptophan metabolism along a pathway that results in the formation of neurotoxic metabolites that may in turn contribute to behavioral changes in
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those patients with psychiatric comorbidity (Jenkins, Nguyen, Polglaze, & Bertrand, 2016).
5. PROOF OF PRINCIPLE THAT INTESTINAL DYSBIOSIS ALTERS FUNCTION IN THE GUT AND BRAIN There is extensive evidence derived from animal studies showing that the intestinal microbiota can alter parameters of gut dysfunction that have been demonstrated in IBS patients. These include changes in intestinal motility, changes in visceral pain responses, alterations in epithelial secretory and barrier function, and in inflammatory and/or immunological function. Many of these studies are based on comparisons between germ-free and colonized animals and while they demonstrate the importance of the influence of the microbiota on these systems, they have limited relevance to the implied dysbiosis implicated in IBS. In addition, virtually all of the host systems relevant to IBS, including the intestinal physiological apparatus, the mucosal immune system including barrier function, and the brain are immature in germ-free animals. A strategy that is more relevant to the implied dysbiosis of IBS is to experimentally disrupt the intestinal microbiota in an otherwise healthy animal with a previously stable microbiota. This can be achieved through diet or through the use of nonabsorbable orally administered antimicrobial agents. The first study to evaluate changes in host following experimental despite doses utilized a cocktail of antimicrobials that included bacitracin, neomycin, and pimaricin administered in the drinking water for a total of 10 days two specific pathogen free mice. The study was aimed at determining whether experimental dysbiosis could induce changes in visceral pain responses elicited by balloon distention of the colorectum. Antimicrobial treatment resulted in a significant increase in responses to colorectal distention and this was accompanied by an increase in immunoreactive substance P and CGP in the myenteric plexus (Verdu et al., 2006). Antimicrobial therapy was associated with depletion of lactobacilli, Bacteroides and enterococci but, in a separate experiment, administration of lactobacillus paracaseii prevented the dysbiosisinduced changes in visceral pain responses. The sensory changes induced by dysbiosis where accompanied by a small low-grade inflammatory response. Treatment with dexamethasone prevented the inflammatory response as well as the changes in visceral pain responses. Taken together, these findings provided proof of principle that experimental dysbiosis induces low-grade inflammation and increases visceral pain responses—findings reminiscent of IBS
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(Verdu et al., 2006). A subsequent study extended these findings by incorporating a mild stressor with nonabsorbable antibiotics to induce dysbiosis, which was monitored in mucosal adherent bacterial populations rather than in the feces. While total bacterial counts were reduced, adherence was enhanced and there was a corresponding increase in luminal s-IgA. Stress alone increased visceral pain responses and upregulated expression of cannabinoid receptor 2 and this increase was abrogated by the concomitant use of antimicrobials (Aguilera, Vergara, & Martinez, 2013). In a later study, the same authors used intraperitoneal acetic acid to induce visceral hypersensitivity and used neomycin and bacitracin to induce dysbiosis. There was an increase in the expression of the cannabinoid receptor 2 and downregulation of the cannabinoid 1 and the μ-opioid receptors. Visceral pain responses were substantially reduced in the presence of dysbiosis, although colonic muscle contractility was enhanced (Aguilera, Cerda-Cuellar, & Martinez, 2015). The study showing the induction of visceral hypersensitivity in the presence of intestinal dysbiosis and the studies showing that dysbiosis was accompanied by an inhibition of enhanced visceral pain responses were performed in completely different laboratories in which the endogenous microbiota was likely different. This illustrates two points. First, it is difficult to extrapolate results involving experimental dysbiosis between laboratories. Second, they show that depending on the composition of the microbiota, experimental dysbiosis can produce diametrically opposite effects on a given host parameter. This may have bearing on the fluctuations often seen in the nature of symptoms reported by an individual IBS patient over time. A recent study examined the consequences of antibiotic exposure early in life, a subject of considerable current interest in the natural history of IBS. Vancomycin was administered from the 4th to the 13th postnatal days in rat pups. This resulted in transient dysbiosis with restoration of the normal microbiome by 8 weeks. Nevertheless, these mice displayed visceral hypersensitivity later in life (O’Mahony et al., 2014). A similar phenomenon was observed when the combination of neomycin, bacitracin, and pimaricin was used in the postnatal period. Thus, disruption of the bacterial colonization process early in life impacts on enteric sensory function later in life. An interesting phenomenon observed in this study was that the effect on sensory function did not occur in female rats. As mentioned earlier, experimental dysbiosis can lead to immune activation and low-grade inflammation, which in turn led to changes in gut physiology. A study examined the role of the innate immune system in a model of antibiotic induced dysbiosis. In this study, investigators used mice lacking
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critical components of the innate immune system. Antibiotic induced dysbiosis resulted in a significant delay of GI motility and this was accompanied by a reduction in the number of nitrergic neurons in the colon. Because a similar profile was observed in mice with neural crest-specific deletion of Myd88 (Wnt1Cre(+/)/Myd88(fl/fl)) as well as TLR4, the authors concluded that the microbiota influence GI motor function via activation of the innate immune system via TLR4 receptors (Anitha, Vijay-Kumar, Sitaraman, Gewirtz, & Srinivasan, 2012). There is increasing interest in the ability of the intestinal microbiota to influence the brain and behavior, prompting consideration of a microbiota– gut–brain axis (Collins, Surette, & Bercik, 2012) and this may be relevant to the psychiatric comorbidity that is common among IBS patients (Fond et al., 2014). Several strategies have been used to explore microbiota–brain interactions and they include comparisons between germ-free and colonized mice (Bercik et al., 2011; Diaz Heijtz et al., 2011; Neufeld, Kang, Bienenstock, & Foster, 2011; Sudo et al., 2004), the adoptive transfer of behavioral phenotype between murine strains via the intestinal microbiota (Bercik et al., 2011; Collins, Kassam, & Bercik, 2013), and the effect of experimental dysbiosis on brain and behavior (Bercik et al., 2011; Li, Dowd, Scurlock, Acosta-Martinez, & Lyte, 2009). For reasons already mentioned, the latter strategy is more relevant to IBS and will be addressed here. In one study, mice were fed on either a standard rodent chow or a chow containing 50% lean beef for 3 months. This resulted in a significant increase in diversity of the microbiome, as assessed by bacterial tag-encoded FLX amplicon pyrosequencing, in mice fed the beef enriched diet. Performance in memory and learning behavioral testing was enhanced in the mice fed the beef enriched diet and they also exhibited less anxiety like behavior (Li et al., 2009). While it was concluded that the diet-induced dysbiosis was responsible for the behavioral change, the authors acknowledge that direct contributions from the beef enriched diet could have contributed to the changes in brain function. In a more recent study involving antibiotic-induced dysbiosis, Bercik et al. found oral but not intraperitoneal administration of nonabsorbable antibiotics increased exploratory behavior as well as increased levels of brain-derived neurotropic factor in the hippocampus. The changes in neurochemistry and behavior were independent of the integrity of the vagus nerve and the sympathetic nervous system (Bercik et al., 2011). A recent study by Frohlich et al. showed that antibiotic induced dysbiosis in mice reduced microbiota-derived metabolites in the colon and their metabolic derivatives in the plasma. This was accompanied by
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cognitive impairment and changes in chemistry, indicating that intestinal dysbiosis may lead to behavioral changes by influencing host metabolism (http://www.sciencedirect.com/science/article/pii/S088915911630040X).
5.1 Is There a Causal Link Between Dysbiosis and Symptom Expression in IBS? With limited therapeutic or investigative tools, coupled with the absence of confidently identified “bacteria of interest,” it is almost impossible to establish causal links between the intestinal microbiota and symptom generation in IBS. The benefit of selected probiotics in improving symptoms and quality of life in IBS patients provides insufficient support for a causal role of the microbiota in the expression of this condition, as the underlying modes of action and impact on the microbiota community are poorly understood. Currently, our best strategy toward establishing the functional relevance of the IBS microbiota in producing gut dysfunctions is the use of human microbiota-associated animals. This strategy involves colonizing germ-free mice with microbiota taken from IBS patients or healthy controls. Crouzet et al. use this strategy to demonstrate the ability of the microbiota to induce visceral sensitivity in rats. The colonization of germ-free rats with microbiota from IBS patients with demonstrable visceral hypersensitivity, but not healthy controls, resulted in increased responses to colorectal distention in the recipient animals (Crouzet et al., 2013). This was not accompanied by changes in intestinal permeability or in mast cell density in the recipient mice. In a recent preliminary study (De Palma et al., 2014), germ-free mice were colonized with microbiota from diarrhea predominant IBS patients with or without anxiety, or from healthy controls and gut function and behavior were assessed. IBS microbiota-associated mice developed rapid transit, increased intestinal permeability in and secretion, and evidence of innate immune activation. Mice colonized with microbiota from patients with anxiety showed evidence of anxiety-like behavior whereas mice receiving microbiota from IBS patients without anxiety showed normal behavior (De Palma et al., 2014). These results provide strong support for the notion that the intestinal microbiota of IBS-d patients contributes to both the intestinal and behavioral manifestations of this condition. Zheng et al. recently provided further support for the notion that the intestinal microbiota contributes to psychopathology by showing that fecal microbiota from depressed patients altered brain chemistry and behavior following colonization of germ-free animals (Zheng et al., 2016).
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6. FUTURE DIRECTIONS Much evidence now points toward a role of the intestinal microbiota in the expression of IBS, the most common intestinal disorder in our society today. Clinical studies must move away from single time point comparisons of microbiome profiles between IBS patients and healthy controls. More emphasis should be placed on longitudinal studies in which patients serve as their own controls, thus minimizing the problem of inter-subject variation and microbiota composition. The field must now move forward toward establishing evidence of causality between the microbiota and symptom expression, including psychiatric comorbidity. Increased use of human microbiota-associated animals should generate important new information regarding a functional role of the microbiota in this condition. Our increasing ability to culture the “noncultivable” intestinal microbial population will enhance our ability to comprehensively profile the bacterial community, to understand community dynamics, and to adopt prescribed colonization strategies using germ-free mice in order to better understand microbe–host interactions. This in turn will help deliver novel therapeutic targets and the strategies for improving this common condition. Last, but by no means least, the influence of the intestinal virome on the intestinal microbiota must now be studied and exploited for therapeutic gain in IBS and other conditions.
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