The normal microbiota: an essential factor in health

The human gastrointestinal (GI) microflora (now more usually referred to as the microbiota) is a complex ecosystem of approximately 300 to 500 bacterial species comprising nearly 2 million genes (the microbiome). Indeed, the number of bacteria within the gut is about 10 times that of all of the cells in the human body. At birth, the entire intestinal tract is sterile; bacteria enter the gut with the first feed. Following infancy, the composition of the intestinal microbiota remains relatively constant thereafter. When disturbed, the microbiota has a remarkable capacity to restore itself and to return to exactly the same state as it was in before.

Because of the normal motility of the intestine (peristalsis) and the antimicrobial effects of gastric acid, the stomach and proximal small intestine contain relatively small numbers of bacteria in healthy subjects; jejunal cultures may not detect any bacteria in as many as 33% of subjects. The microbiology of the terminal ileum represents a transition zone between the jejunum containing predominantly facultative anaerobes and the dense population of anaerobes found in the colon. Bacterial colony counts may be as high as 10 ⁹ colony-forming units (CFU)/mL in the terminal ileum immediately proximal to the ileocecal valve, with a predominance of Gram-negative organisms and anaerobes. On crossing into the colon, the bacterial concentration and variety of the enteric microbiota change dramatically. Concentrations as high as 10 ¹² CFU/mL may be found, comprised mainly of anaerobes such as Bacteroides, Porphyromonas, Bifidobacterium, Lactobacillus, and Clostridium , with anaerobic bacteria outnumbering aerobic bacteria by a factor of 100 to 1000 to 1. The predominance of anaerobes in the colon reflects the fact that oxygen concentrations in the colon are very low; the microbiota has simply adapted to survive in this hostile environment.

It must be emphasized, however, that the true size and diversity of the human microbiota are largely unknown. The application of modern technologies—genomics, metagenomics, and metabolomics—to the study of the colonic microbiota has the potential to expose the true diversity and metabolic profile of the microbiota and the real extent of changes in disease. Techniques based on 16S rDNA sequences have revealed that the diversity of the human microbiota is much greater than previously thought and that most bacterial sequences correspond to unculturable sequences and novel bacteria. At any given level of the gut, the composition of the microbiota also demonstrates variation along its diameter, with certain bacteria tending to be adherent to the mucosal surface while others predominate in the lumen; studies that rely on the analysis of the fecal microbiota alone may miss the impact of an important population of organisms, those closely adherent to the mucosa. In people, the composition of the microbiota is also influenced by age, diet, socioeconomic conditions and, above all, the use of antibiotics. Studies purporting to identify variations in the microbiota in disease states must, accordingly, be interpreted with great care and some degree of skepticism.

The normal enteric bacterial microbiota influences various intestinal functions and plays a key role in nutrition, maintaining the integrity of the epithelial barrier, and the development of mucosal immunity. The relationship between the host’s immune system and nonpathogenic constituents of the microbiota is important in protecting the host from colonization by pathogenic species. In this regard, intestinal bacteria produce various substances, ranging from relatively nonspecific fatty acids and peroxides to highly specific bacteriocins, which can inhibit or kill other, potentially pathogenic, bacteria.

The gut microbiota in disease

The key role of the microbiota in health is only beginning to be understood and it has only been in very recent years that the true extent of the consequences of disturbances in the microbiota, or in the interaction between the microbiota and the host, to health has been recognized. Some of these are relatively obvious; for example, when many components of the normal microbiota are eliminated or suppressed by a course of broad-spectrum antibiotics, the stage is set for other organisms that may be pathogenic to step in and cause disease. The classical example of this is antibiotic-associated diarrhea and its deadliest manifestation, Clostridium difficile colitis. Similar perturbations in the microbiota are thought to be involved in a devastating form of intestinal inflammation that may occur in newborn and, especially, premature, infants: necrotizing enterocolitis. In other situations, bacteria may simply be where they should not be; if intestinal motility is impaired and/or gastric acid secretion abolished, an environment conducive to the proliferation—in the small intestine—of organisms that are normally confined to the colon results. Small intestinal bacterial overgrowth (SIBO) can significantly disturb both the digestion and absorption of food and the products of digestion. Alternatively, when the immunologic interaction between the microbiota is disturbed, the host may, for example, begin to recognize the constituents of the normal microbiota, not as friend, but as foe and may mount an inappropriate inflammatory response that ultimately may lead to conditions such as inflammatory bowel disease. Injury to the intestinal epithelium, regardless of cause, renders the gut wall leaky and permits luminal bacteria (in whole or in part) to gain access to the submucosal compartments or even to the systemic circulation with the associated potential to cause catastrophic sepsis. This mechanism is thought to account for many of the infections that occur in the critically ill patient in the intensive care unit, for example. Most recently, qualitative changes in the microbiota have been invoked in the pathogenesis of a global epidemic: obesity.

Probiotics

Probiotics, derived from the Greek and meaning “for life,” are defined as live organisms that, when ingested in adequate amounts, exert a health benefit to the host. There are several commercially available supplements containing viable microorganisms with probiotic properties. The most commonly used probiotics are lactic acid bacteria and nonpathogenic yeasts. Although probiotics have been proposed for use in inflammatory, infectious, neoplastic, and allergic disorders, the ideal probiotic strain, for use in any of these indications, has yet to be identified. The interpretation of available data on probiotics is further confounded by variability in strain selection, dose, delivery vehicle, and evaluation of viability and efficacy.

Probiotics were first described by Metchnikoff in 1908 based on his observations on the longevity of individuals who lived in a certain part of Bulgaria and which he attributed to their ingestion, on a regular basis, of a fermented milk product. Over the years since then, many products have appeared on health food store and supermarket shelves throughout the world that include the term probiotic in their label. Very few fulfill the definition provided:

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  They may not contain live organisms or have not been adequately tested to ensure that the organisms will survive in the conditions (eg, room temperature) or for the length of time (days, weeks, or months) that is claimed.

  
  
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  They may not confer health benefit, because either, they have never been tested on people or because what tests have been preformed have been inadequate or even negative.

Other issues of quality control continue to complicate the probiotic area. Does the product actually contain the organism and the dose of that organism that the label claims that it contains? Unfortunately, when researchers have analyzed some store products, they have found, not only that organisms claimed to be alive were actually dead, but that the product contained organisms (including pathogens) that it was not supposed to contain.

Some probiotic companies have gone to considerable efforts to ensure that their products do contain the very organisms and in the precise dose that are claimed. These products can guarantee the survival of live organisms over the time and in the conditions specified on the label. Whether these same products can provide the health benefits that they claim can only be deduced from a critical assessment of the medical literature. Fortunately, more and higher quality clinical trials are being performed and can guide the consumer on the optimal product for a given condition. This latter point is critically important; no two probiotics are the same! Even within the same species, different strains may have vastly different and even contrasting effects. Although probiotics have been proposed for use in inflammatory, infectious, neoplastic, and allergic disorders, the ideal probiotic strain for many of these indications has yet to be defined, although progress continues in this area. While probiotic cocktails also have been advocated to maximize effect, it should be noted that some probiotic combinations have been shown to prove antagonistic, rather than synergistic, in certain situations.

Right now the consumer finds it impossible to assess the validity of the many claims made for probiotics. This dilemma stems, in large part, from the manner in which they are regulated. Despite the fact that probiotics are advocated for the management of disease, they are regulated, in most jurisdictions, in a manner that is more akin to a food than a pharmaceutical. This situation may change very soon, as agencies in both the United States and in Europe are currently re-evaluating the status of these products. Deliberations by the European Food Safety Authority on health claims for probiotics pursuant to European Union Regulation 1924/2006 will undoubtedly set a much higher standard for all health claims relating to nutritional products, including probiotics.

One of the areas of most active research pertains to the mechanism of action of probiotics. The interaction between a probiotic strain or cocktail of strains/species must be examined in the context of those interactions that normally take place between the microbiota and the host, as well as between individual components of the microbiota. How does the host differentiate between friend and foe? What interactions between the constituents of the microbiota favor the growth of some bacteria but not others? The complexities of microbiota–host and microbe–microbe interactions continue to be unraveled. With regard to the former, the important roles of pattern recognition receptors [PRRs, such as Toll-like receptors [TLRs], signaling pathways, immune responses, and the secretion of antimicrobial peptides such as defensins and chemokines by the epithelium all appear to play important roles. Administered probiotics appear to be able, if only transiently and for the duration of their administration, to influence the composition and function of the intestinal microbiota. It appears plausible to suggest that effects that are primarily metabolic in nature should occur primarily in that site of greatest microbial metabolic activity, the colon, whereas probiotic effects that are mediated through immune engagement should take place in those areas where immune tissue is most dense, the distal small intestine. Using novel reporter systems, the locations of niche environments for administered probiotics are beginning to be defined in animal models. While many of the factors, such as secretion of antimicrobial peptides, quorum sensing, possession of certain metabolic pathways, competition for resources, bacterial motility, and adherence properties, to name but a few, which have been identified as relevant to competition within the microbiota, are undoubtedly directly relevant to the survival and proliferation of an administered probiotic, very little direct research has been preformed on these issues in people.

If the probiotic survives its hazardous journey through the intestine and manages to establish a niche within the microbiota, how does it exert its health-benefiting effects? For answers, one again must rely largely on animal work, with some limited data from people. Possible modes of action revealed in such studies include competitive metabolic interactions with pathogens, production of chemical products (bacteriocins) that directly inhibit other bacteria or viruses, inhibition of bacterial movement across the gut wall (translocation), enhancement of mucosal barrier function, and signaling with the epithelium and immune system to modulate the inflammatory/immune response. Probiotics also may produce other chemicals, including neurotransmitters that are normally found in the bowel, which can modify other gut functions, such as motility or sensation. The whole area of the production of biologically active products by probiotic organisms promises to be one of the most exciting areas of research in this field and may yet prove relevant to their efficacy in IBS. It is evident that various probiotics have different potency in relation to any one of these actions; some are avid producers of antibacterial peptides and may become active participants in the fight against certain infections, while others are potent anti-inflammatory agents. Still other probiotics have been shown to enhance epithelial barrier function through direct effects on mucin expression, proteins of the cytoskeleton, and intercellular tight junctions and indirect effects emanating from interactions between the bacterium, the mucosa and the mucosa-associated lymphoid tissue. The potent anti-inflammatory effects of some probiotics have clearly emphasized how the therapeutic potential of these agents may extend beyond their ability to displace other organisms and has led to their evaluation in inflammatory bowel disease. In an experimental animal (interleukin [IL]-10 knockout) model of colitis, for example, one group of researchers found that both a Lactobacillus and a Bifidobacterium produced a marked and parallel reduction in inflammation in the colon and cecum and in the production of the proinflammatory cytokines interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and IL-12, while levels of the anti-inflammatory cytokine transforming growth factor (TGF)-β were maintained. Similar effects have been demonstrated for the probiotic cocktail VSL#3 in experimental models of colitis; these anti-inflammatory effects could, indeed, be transmitted by bacterial DNA alone. What is very exciting is the observation, again in an animal model, of the ability of orally administered probiotics to exert anti-inflammatory effects at sites well distant from the gut such as in an inflamed joint. While the focus of this article is on the gut and on digestive disorders, the latter experiments illustrate the ability of strategies that manipulate the microbiota to alter immune function at distal sites and provide an experimental basis for clinical observations on the efficacy of probiotic therapy in allergic disorders, for example. For many probiotics, their mechanism of action in a given disease state, or in health, is likely to be multifactorial.

Probiotics in IBS

While experimental observations suggest potential benefits for probiotics in various GI, pancreatic, and liver disorders, solid clinical data are confined to three main areas: infection, inflammatory bowel disease, and IBS. The latter will be discussed here.

Reflecting, perhaps, the paucity of truly disease-modifying therapies that are available to relieve the disorder, irritable bowel sufferers commonly have recourse to the use of complimentary and alternative medical remedies and practices. Foremost among such approaches have been various dietary manipulations, including exclusion diets, and various dietary supplements. In Europe, in particular, where several such products are advertised widely for their general immune-boosting and health-enhancing properties, probiotics have been widely used as dietary supplements by IBS patients. Recently, based on data from the experimental laboratory, as well as some evidence from clinical trials, the concept of probiotic use in IBS has begun to wind its way into the realm of conventional medicine. While probiotics have been used on an empiric basis for some time in the management of IBS, several recent developments provide a more logical basis for their use in this context. These include the clear recognition that IBS may be induced by bacterial gastroenteritis (postinfectious IBS) and that qualitative and quantitative changes in the microbiota, as well as immune dysfunction, may be prevalent in IBS, in general.

Up to the year 2000, a small number of studies evaluated the response of IBS to probiotic preparations, and, while results between studies were difficult to compare because of differences in study design, probiotic dose and strain, there was some, but by no means consistent, evidence of symptom improvement.

Further studies, since then, have assessed the response to a number of well-characterized organisms and have produced discernible trends. Thus, several organisms, such as Lactobacillus GG, L plantarum, L acidophilus, L casei, the probiotic cocktail VSL#3, and Bifidobacterium animalis , have been shown to alleviate individual IBS symptoms, such as bloating, flatulence, and constipation. Only a few products, however, have been shown to affect pain and global symptoms in IBS. Among these, B infantis 35,624 has attracted particular attention. In the first study with this organism, superiority was shown over both a Lactobacillus and placebo for each of the cardinal symptoms of the IBS (abdominal pain/discomfort, distension/bloating, and difficult defecation), as well as for a composite score. A larger, 4-week duration, dose-ranging study of the same Bifidobacterium in over 360 community-based subjects with IBS confirmed efficacy for this organism in a dose of 10 ⁸ . Again, all of the primary symptoms of IBS were significantly improved, and a global assessment of IBS symptoms at the end of therapy revealed a greater than 20% therapeutic gain for the effective dose of the probiotic over placebo ( Fig. 1 ).

Subjects global assessment of relief of irritable bowel syndrome (IBS) symptoms in response to Bifidobacterium infantis 35,624 in doses of 10 ⁶ or 10 ⁸ or placebo during a 6-week study (4 weeks treatment and 2 weeks wash-out). Note significant increase in percentage of positive responders to the question “Do you feel better now compared with before treatment?” among those randomized to B infantis 35,624 in a dose of 10 ⁸ in comparison to the other 2 groups.

( Data from Whorwell PJ, Altringer L, Morel J, et al. Efficacy of an encapsulated probiotic Bifidobacterium infantis 35,624 in women with irritable bowel syndrome. Am J Gastroenterol 2006;101:326–33.)

Further large, long-term, randomized controlled trials of this Bifidobacterium and other strains are warranted in IBS, and detailed explorations of its mechanism(s) of action are indicated.

Several factors, including a reduction in gas production, changes in bile salt conjugation, an antibacterial or antiviral effect (in the case of postinfectious IBS), the promotion of motility, effects on mucus secretion, or even an antiinflammatory effect could be relevant to the benefits of specific probiotic strains in IBS.

Safety

Many different species and strains and preparations of probiotics have been used for decades and by millions of healthy and diseased individuals, yet definitive data on safety are scanty. In a review in 2006, Boyle and colleagues concluded that although probiotics have an excellent overall safety record, they should be used with caution in certain patient groups, particularly neonates born prematurely or with immune deficiency. They reviewed case reports of instances of abscesses and endocarditis in relation to probiotic use; in many instances the probiotic cultured from the infected tissue was most likely an innocent contaminant rather than the real pathogen. Fears that live probiotic organisms might translocate across the gut and lead to systemic sepsis have also been allayed by the absence of such reports from studies among patients with inflammatory bowel disease and other situations where the intestinal barrier may be compromised. Two notes of caution must be mentioned. The first relates to reports of septicemia occurring among infants with short bowel syndrome, and the second to instances of increased mortality among patients with severe acute pancreatitis who had been administered a probiotic cocktail through a nasoenteric tube. These deaths were associated, not with sepsis, but with intestinal ischemia whose etiology remains unclear.

Prebiotics and synbiotics

Prebiotics are defined as nondigestible, but fermentable, foods that beneficially affect the host by selectively stimulating the growth and activity of one species or a limited number of species of bacteria in the colon. Compared with probiotics, which introduce exogenous bacteria into the human colon, prebiotics stimulate the preferential growth of a limited number of health-promoting species already residing in the colon and, especially, but not exclusively, lactobacilli and bifidobacteria. The oligosaccharides in human breast milk are considered the prototypic prebiotic as they facilitate the preferential growth of bifidobacteria and lactobacilli, in the colon, among exclusively breast-fed neonates; this phenomenon may well account for some of the immunologic and other benefits that accrue to breast-fed infants.

The only prebiotics for which sufficient data have been generated to allow an evaluation of their possible classification as functional food ingredients are the inulin-type fructans, which are linked by β (2–1) bonds that limit their digestion by upper intestinal enzymes, and fructo-oligosaccharides. Both are present in significant amounts in many edible fruits and vegetables, including wheat, onion, chicory, garlic, leeks, artichokes, and bananas. Because of their chemical structure, prebiotics are not absorbed in the small intestine but are fermented, in the colon, by endogenous bacteria to energy and metabolic substrates, with lactic and short-chain carboxylic acids as end products of the fermentation.

Most of the evidence regarding the potential health benefits of prebiotics is derived from experimental animal studies and human trials in small numbers of subjects; there are insufficient, prospective, adequately powered studies in GI disease to permit definitive conclusions to be drawn; some recent studies suggest that prebiotics that have been designed to produce quite selective changes in the composition of the microbiota may have benefits in IBS. It must also be remembered that substances, such as fiber, fiber supplements and lactulose, for example, which have been widely employed in the treatment of constipation, exert prebiotic effects.

Synbiotics, defined as a combination of a probiotic and a prebiotic, aim to increase the survival and activity of proven probiotics in vivo, as well as stimulating indigenous bifidobacteria and lactobacilli. Again, data for efficacy in human disease are scanty, although there have been some small trials in IBS that suggest some promise.

Antibiotics

Although antibiotics have been identified in population studies as a risk factor for the development of IBS, the suggestion that some IBS subjects might harbor SIBO led to clinical trials of these agents in this disorder. In a subsequent study that did not document bacterial overgrowth, Pimentel and colleagues treated IBS patients with the poorly absorbed antibiotic rifaximin. Some IBS patients demonstrated a prolonged response (up to 10 weeks) to a short course of this antibiotic. As pointed out in an accompanying editorial, there were, however, several limitations to this study that reduced its impact. In a recently reported multicenter phase 2 study, 388 diarrhea-associated IBS subjects were randomized to either rifaximin in a dose of 550 mg twice a day or placebo for 14 days, followed by another 14 days on placebo alone and then 2 weeks follow-up. During treatment, at 4 weeks and at 12 weeks, those randomized to rifaximin had a modest 8% to 13% therapeutic gain for adequate relief of global IBS symptoms and a rather disappointing 4% to 8% gain for relief of bloating. These encouraging results have been reproduced and even bettered in two large multicenter trials which have been recently published. These trials, showed a 9% therapeutic gain for adequate relief of IBS symptoms and a 9.9% therapeutic gain for adequate relief of bloating for rifaximin in a dose of 550 mg three times daily over placebo among a total of 1260 patients with nonconstipated IBS.

The eradication of SIBO, as initially proposed, may not be the sole explanation for these responses, which could also be explained on the basis of a suppression of fermenting bacteria in the colon, or indeed, by a multitude of other consequences of altering the colonic microbiota. These results notwithstanding, one must remain reluctant, pending long-term studies, to recommend a prolonged course of antibiotic therapy to any population regardless of the safety profile of a given antibiotic. It also needs to be remembered that relapse rates following discontinuation of rifaximin therapy are high among patients with SIBO (not necessarily associated with IBS), and while such recurrences can be treated successfully by further courses of antibiotic, the advisability of repeated courses of antibiotics in any condition must questioned.

Nevertheless, the consistency of this effect of this antibiotic on IBS symptomatology, albeit modest, is compelling and demands that one address how it is mediating these benefits. Who are the responders, and why do they respond? What is the mechanism of action, and where is this action exerted? The answers to these questions must provide valuable insights into IBS, in general.