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The Indigenous Gastrointestinal Microbiota in Health and Disease

Course Authors

John Y. Kao, M.D., Nirmal Kaur, M.D., and Vincent B. Young M.D., Ph.D.

John Y. Kao, M.D., is Assistant Professor and Nirmal Kaur, M.D., is Fellow, Department of Internal Medicine, Division of Gastroenterology, and Vincent B. Young, M.D., Ph.D., is Assistant Professor, Department of Medicine, Infectious Diseases Division, and Department of Microbiology & Immunology, University of Michigan Medical School, Ann Arbor, Michigan.

Within the past 12 months, Drs. Young, Kaur and Kao report no commercial conflicts of interest.

Albert Einstein College of Medicine, CCME staff and interMDnet staff have nothing to disclose.

Estimated course time: 1 hour(s).

Albert Einstein College of Medicine – Montefiore Medical Center designates this enduring material activity for a maximum of 1.0 AMA PRA Category 1 Credit(s)™. Physicians should claim only the credit commensurate with the extent of their participation in the activity.

In support of improving patient care, this activity has been planned and implemented by Albert Einstein College of Medicine-Montefiore Medical Center and InterMDnet. Albert Einstein College of Medicine – Montefiore Medical Center is jointly accredited by the Accreditation Council for Continuing Medical Education (ACCME), the Accreditation Council for Pharmacy Education (ACPE), and the American Nurses Credentialing Center (ANCC), to provide continuing education for the healthcare team.

 
Learning Objectives

Upon completion of this Cyberounds®, you should be able to:

  • Discuss the importance of the indigenous gastrointestinal microbiota in gut health and disease

  • Identify new technologies for studying the indigenous microbiota

  • Discuss how therapeutic manipulation of the microbiota may represent a novel means to prevent and treat GI disease.

 

The gastrointestinal tract of mammals is inhabited by a complex microbial community. This community of microbes, referred to as the microbiota, has gained increased prominence of late. The traditional view of the microbiota regarded them as “commensal,” merely organisms that had found a ready source of food but did not contribute anything of value to their host. More recently, however, evidence has emerged that suggests that the microbiota can play key roles both in maintenance of health as well as the pathogenesis of disease within the gastrointestinal tract.

From interactions with the epithelial lining of the gastrointestinal tract and interactions with the host immune system, the microbiota form part of a complex symbiotic relationship with the host. In its normal state this symbiosis is beneficial for both partners. However, if the normal balance is upset, disease can result. When the imbalance is felt to represent disturbances in the microbial community the term “dysbiosis” is often used.

In this Cyberounds® review we will introduce concepts that pertain to understanding the structure and function of the gut microbiota, provide a broad overview of methods that can be used to study the microbiota and present evidence that implicates the microbiota in the maintenance of G.I. health as well as gastrointestinal pathology.

The indigenous human microbiota includes at least 10,000 different kinds of microbes.

Roles of the Indigenous Microbiota in the Host

The indigenous human microbiota includes at least 10,000 different kinds of microbes with a total genetic complexity (the microbiome) that exceeds twenty billion base pairs and codes for more than forty million genes.(1) Differences in nutrition, host physiology, immunity, state of health and other ecological stressors lead to significantly different microbial population structures and their collective proteome within and between human hosts. The goal of the Human Microbiome Project is to understand how these complex, dynamic consortia shape human health and well−being.(2)

The complex community of microbes that inhabits the mammalian gut represents an assemblage of organisms that exists in a balanced symbiosis with its host.(3) The metabolism of the gut microbiota participates in the catabolism of ingested nutrients(4) and also produces a wide variety of ligands and antigens that can interface with the host metabolism and immune system. Furthermore, the microbiota can have direct influence on the development of the gut.(5) Therefore, alterations in the community structure of this microbial community can have implications on the homeostasis of the host. Changes in the gut microbiome correlate with obesity,(6) inflammatory bowel disease(7) and antibiotic−associated diarrhea,(8) These pathologic conditions likely arise as a result of changes in the metabolic activity of the altered microbial community(9) or alterations in the interaction of the microbiome with the host immune system.(10)

Study of the Microbiota—Molecular Methods as an Alternative to Classical Culture

Initial studies of microbial diversity were dependent on the ability to isolate and culture organisms on artificial media. The inability to culture a majority of the microbes present in complex environmental communities has led to the development of a variety of culture−independent methods for assessing microbial diversity. Thirty years ago, Woese pioneered the use of rRNA sequence comparisons for charting the evolutionary history of microbes.(11)(12)(13) Pace subsequently developed culture independent means for assessing the diversity and ecology of microorganisms by targeting the 16S rRNA−encoding gene directly.(14)(15) These molecular surveys have resulted in the description of 100 major divisions of bacteria—most of which do not include cultured representatives.(16) Sequence analysis of PCR amplicons for rRNA genes is now the “gold standard” for assessing species richness in microbial communities. Databases contain more than 500,000 rRNA sequences that correspond to phylotypes from diverse microbes (http://greengenes.lbl.gov/cgi-bin/nph-index.cgi, http://www.arb-silva.de, http://rdp.cme.msu.edu, http://www.ncbi.nlm.nih.gov/).

Based on surveys using rRNA gene sequences as proxies for the presence of a microbe in a sample, microbial diversity is at least 100−1000 times greater than estimates based upon cultivation−dependent surveys.(14) It has become clear the “first generation” molecular assays, involving the amplification, cloning and sequencing 16S rRNA−encoding genes, have only captured a fraction of microbial diversity and they rarely provide estimates of relative abundance for different kinds of microbes or operational taxonomic units (OTUs).(17)(18) The largest reported surveys using 16S clone library data described between 13,000−18,000 rRNA gene sequences from humans.(19)(20) Multiple smaller surveys of gut flora describe correlations with disease states,(21)(22)(23)(24) the establishment of gut flora(25)(26) and effects of antibiotics.(8)(27) However, the limited sampling effort for these investigations is not sufficient to describe comprehensively the diversity of the human microbiota.

The data available from superficial surveys of microbial communities — from environments as diverse as marine,(28) soil(29)(30) and the human gut (19)(20) — reveal common diversity patterns where a few dominant OTUs mask the presence of many thousands of different kinds of lower abundance microbes. In order to increase our understanding of the organism that compose this “rare biosphere”(31) a number of techniques that leverage the power of the newest high−throughput DNA sequencing technologies have been developed and applied to examination of the gut microbiota.(32)(33)(34)

Specific changes in the composition of the intestinal microbiota have been demonstrated in patients with IBD.

Role of the Microbiota in Gastrointestinal Disease States

Inflammatory Bowel Disease (IBD)

Specific changes in the composition of the intestinal microbiota have been demonstrated in patients with IBD. Analysis using quantitative PCR showed that patients with IBD have significantly decreased fecal concentrations of Bacteroides fragilis and B. vulgatus.(35) B. fragilis has been shown to protect mice from experimental colitis induced by adoptive transfer of CD4+CD45RBhi T cells.(36) Conte et al. studied the mucosa−associated intestinal microflora in colon biopsies in pediatric patients with newly diagnosed inflammatory bowel disease and revealed that patients with IBD demonstrated higher quantities of mucosa−associated aerobic and facultative−anaerobic bacteria in colonic biopsies compared to control subjects.(37) Differences in distribution were also seen: patients with both ulcerative colitis (UC) and Crohn’s disease (CD) demonstrated higher concentrations of bacteria in the ileum than controls. Interestingly, patients with UC and CD both exhibited a lower concentration of bacteria in the cecum compared to the ileum, but cecal bacterial concentrations in IBD patients were still higher than that of controls. Furthermore, rectal concentrations of bacteria were highest in patients with CD, not UC.

Several studies have implicated Escherichia coli in the pathogenesis of CD. One study by Martin and colleagues cultured the mucosa−associated bacteria in patients with Crohn’s disease, ulcerative colitis, non−inflamed controls, and after surgical resection for colon cancer. Mucosa−associated E. coli, as well as intramucosal E. coli and hemagglutinins expressed by E. coli, were most common in patients with CD and less frequent in patients with UC or colon cancer.(38) A second study by Darfeuille−Michaud et al. analyzed the ileal mucosa of patients with CD, UC, and healthy controls, and found adherent−invasive E. coli strains in 22% of CD patients and only 6% of controls.(39) Sasaki et al. studied invasive bacterial strains in patients with IBD; they also found that in CD, 98% of invasive bacteria was E. coli as opposed to 42% in UC and 2% in healthy controls. These invasive E. coli strains induced interleukin−8 and tumor necrosis factor alpha, and also decreased transepithelial resistance and induced disorganization of tight junction complexes.(40) Together, these data indicate that adherent−invasive E. coli plays a role in the pathogenesis and perpetuation of Crohn’s disease.

The association between various antibodies and IBD further points to the role of intestinal microbiota in the disease pathogenesis of IBD. Anti−saccharomyces cerevisiae antibodies (ASCA) are associated with Crohn’s disease and tend to identify patients with ileal and cecal disease, while perinuclear anti−neutrophil cytoplasmic antibodies (p−ANCA) are associated with UC.(41) In addition, anti Omp−C, antibodies to E. coli outer−membrane protein C, have been shown to increase the sensitivity of serologic testing,(42) while anti−CBir1, antibodies to bacterial flagellin CBir1, were associated with the development of fibrostenotic Crohn’s disease.(43) It is unclear whether these immunological responses represent the presence of pathogenic bacteria or a leaky epithelium resulting in bacterial translocation.

Irritable Bowel Syndrome

The pathophysiology of irritable bowel syndrome (IBS) is currently unclear but the absence of histopathology in the intestine of most affected individuals has led to the speculation about an altered microbiota.(44) Several earlier studies attempted to compare the composition of the luminal microbiota in IBS patients. Culturing the stool of IBS patients and normal controls (20 subjects in each group), Balsari et al. showed a lower concentration of coliforms, lactobacilli and bifidobacteria in patients with IBS compared to controls.(45) A lower concentration of bifidobacteria in IBS patients using bacterial cultures was reported by Si et al. in a case−control study of 50 patients (25 IBS and 25 controls).(46) The use of more advanced molecular profiling of intestinal microbiota has begun to shed light on the role of microbiota in IBS. Due to the limitation of bacterial cultures to identify non−culturable species, PCR−denaturing gradient gel electrophoresis (PCR−DGGE) was used in one study to compare the fingerprints of individuals’ microbiota composition, and the results showed that each subject had their own unique profile.(47) Over a 6−month period, a number of subjects were found to have changes in their profiles.

Using the “gold standard” sequencing of bacterial 16S rRNA−encoding genes, Kassinen et al. reported the most comprehensive study to date.(48) Comparisons were made among various subtypes of IBS and demonstrated that IBS patients, compared to controls, have significant alterations in their fecal microbiota. Findings from this and previous studies demonstrated a similar trend of lower lactobacilli and bifidobacteria in fecal samples of patients with IBS.

Antibiotic−associated Diarrhea/C. difficile

The frequency of AAD varies among different antibiotics but can approach 25%.

As noted above, the complex community of microbes that inhabits the mammalian gut represents an assemblage of organisms that exists in a balanced symbiosis with its host.(3) The metabolism of the gut microbiota participates in the catabolism of ingested nutrients and also produces a wide variety of ligands and antigens that can interface with the host metabolism and immune system. Therefore, alterations in the community structure of this microbial community can have implications on the homeostasis of the host. Changes in the gut microbiome correlate with obesity, inflammatory bowel disease and antibiotic−associated diarrhea. These pathologic conditions likely arise as a result of changes in the metabolic activity of the altered microbial community or alterations in the interaction of the microbiome with the host immune system. Antibiotic−associated diarrhea (AAD), defined as diarrhea associated with the administration of antibiotics and without another obvious cause, is felt to represent such a condition.(49)(50) The frequency of AAD varies among different antibiotics but can approach 25%.

Several mechanisms are presumed to underlie the development of AAD. Overgrowth by the toxigenic bacterium Clostridium difficile is a mechanism that has received particular attention, in part because it can occur in nosocomial outbreaks.(51)(52)(53) The administration of the antibiotic clindamycin has traditionally been associated with increased risk of the development of C. difficile−associated diarrhea (CDAD).(54) With the decreased use of clindamycin, the antibiotics currently most associated with the development of CDAD are ampicillin, amoxicillin and the cephalosporins, in particular third−generation cephalosporins.(55) Although C. difficile likely causes only 15 to 25% of all cases of AAD, it is responsible for virtually all cases of antibiotic−associated pseudomembranous colitis, which can lead to complications such as paralytic ileus, colonic dilatation and perforation. A recent study estimated that the cost of nosocomial C. difficile infection in the United States exceeds $1.1 billion per year.(56)

The term “colonization resistance” was coined to refer to the ability of a previously established gut microbial community to resist invasion by an additional microbe.(57)(58)(59) Although colonization resistance initially applied to pathogenic microbes, the concept was derived from concepts of community robustness applied to “classical” ecologic systems (for example, grasslands and lakes) and thus could be applied to any invading microbe.

Current dogma holds that the normal indigenous microbiota is not permissive for colonization of C. difficile.(60) In the minority of cases where healthy individuals are colonized by C. difficile without overt clinical disease, it is further hypothesized that the normal indigenous microbiota can at least limit the production of toxin, perhaps by directly interfering with toxin production or limiting the population size of C. difficile and preventing significant amounts of toxin from accumulating in the gut. According to this model, disruption of the indigenous microbiota by antibiotics leads to a loss of colonization resistance, making the gut vulnerable to colonization by exogenous C. difficile spores or, in previously colonized patients, expansion and toxin production.

Koch’s postulates for C. difficile as a cause of antibiotic−associated colitis were fulfilled by experimental treatment of Syrian hamsters with antibiotics, which induced a lethal, hemorrhagic colitis.(61)(62) When initially described, the colitis was found to result from expansion of indigenous C. difficile and an increase in toxin production. Wilson and colleagues provided evidence for the ability of the normal gut microbiota to inhibit C. difficile by demonstrating that administration of normal SQL homogenates would decrease the number of viable C. difficile and prevent colitis in antibiotic−challenged hamsters.(63)

Subsequent studies in a variety of model systems were initiated to define precisely which members of the indigenous microbiota played a role in mediating colonization resistance. However, the complexity of the gut microbial community and the limitations in the culture−dependent methods that were utilized at the time prevented the performance of more than descriptive studies. The recent development of culture−independent methods of following complex microbial communities and the advent of genomic technologies will now allow us to revisit hypothesis−driven studies of colonization resistance.

Since infection with C. difficile is only responsible for an estimated 25% of all cases of antibiotic−associated diarrhea, researchers believe that in the remainder of cases alteration of the normal indigenous microbiota interferes with the digestion of complex carbohydrates and causes an osmotic diarrhea. In one of our initial studies(27) using culture−independent analysis to profile the gut microbiota, we followed changes in the fecal bacterial community of an individual who developed diarrhea on amoxicillin/clavulanic acid. DNA was purified from fecal samples and the diversity of the bacterial microbiota determined by amplification using broad−range PCR primers that target conserved regions of the 16S rRNA−encoding gene. Our analysis demonstrated that antibiotic administration resulted in dramatic shifts in the composition of the fecal microbiota. Of note, we observed a marked decrease in members of the family Clostridiaceae. These organisms are known to ferment complex carbohydrates to short chain fatty acids including butyrate, which is the preferred energy source for colonic enterocytes. Interestingly, two weeks after the discontinuation of antibiotics the community structure returned largely to baseline.

In a subsequent study, we profiled the bacterial microbiota from the feces of patients diagnosed with AAD due to C. difficile.(8) In particular, we compared patients who presented with an initial episode of C. difficile diarrhea to patients with recurrent disease. We noted that whereas patients with an initial episode of C. difficile−associated diarrhea had a fecal microbiota of similar diversity to matched controls, the microbiota from patients with recurrent disease exhibited significantly decreased diversity. These findings provided support for the hypothesis that recurrent C. difficile−associated diarrhea is at least partially related to persistent abnormalities in the gut microbiota that lead to diminished colonization resistance. Furthermore, these findings suggest that the use of fecal transplants for recurrent C. difficile infection is successful because of the restoration of the normal diversity of the gut microbiota and restoration of colonization resistance.

These two studies from our laboratory indicate that studies of the gut microbiota can lead to hypotheses about the role of the indigenous gut bacteria in the pathogenesis of antibiotic−associated diarrhea.

Probiotics have efficacy in the prevention, maintenance of remission and treatment of pouchitis.

Therapeutic Manipulation of the Microbiota

Given the probable role for the indigenous gut microbiota in the maintenance of health and in the causation of various disease states, it is reasonable to pursue intentional manipulation of the microbiota’s community structure as a therapeutic modality. Antibiotic use is obviously one such modality, although it can lead to other side effects including the development of C. difficile as noted above. However, antibiotics have been used successfully in the acute treatment of IBD, although considerable uncertainty about their precise use remains.(64)(65)(66)

Another treatment that presumably works via alteration of the community structure of the gut microbiota is the administration of probiotics, commonly defined as “live microorganisms, which when administered in adequate amounts, confer a health benefit on the host.”(67) Although the potential advantages of using live microorgansisms to alter the gut microbiota are significant, data are only now being collected that provide some evidence for their efficacy. Key studies for a variety of conditions are summarized below.

Pouchitis

VSL#3 is a mixture of eight different probiotic strains including Lactobacillus and Bifidobacterium, as well as Streptococcus salivarius thermophilus. A double−blind, placebo−controlled trial by Gionchetti and colleagues randomized 40 patients who had recently undergone ileal−pouch anal anastamosis to either VSL#3 or placebo. At one−year follow−up, the VSL#3 group experienced a 10% rate of pouchitis, compared to 40% in the placebo arm.(68) Mimura et al. found similar results in a randomized, double−blinded study which demonstrated that daily high−dose VSL#3 maintains remission in recurrent or refractory pouchitis. In this study, 36 patients with a history of ileal−pouch anal−anastamosis and a history of pouchitis were randomized to daily VSL#3 or placebo. Maintenance was achieved in 85% of the treatment arm and only 6% of the placebo arm.(69) A prospective case series by Gionchetti et al. administered high−dose VSL#3 for the treatment of active pouchitis to 23 patients with mild pouchitis. At four−week follow−up, 16 of 23 patients were in remission.(70) These three studies taken together demonstrate that probiotics have efficacy in the prevention, maintenance of remission and treatment of pouchitis.

Irritable Bowel Syndrome

Since the standard therapy for IBS is either suboptimal or has significant side effects, probiotics offer a very attractive alternative therapeutic approach. One of the most promising probiotics for the treatment of IBS is Bifidobacterium infantis 35624. Preclinical studies indicated B. infantis 35624 attenuated colitis in IL−10 KO mice and was associated with a reduced ability to produce Th1−type cytokines systemically and mucosally, while levels of TGF−β were maintained.(71) Two randomized control studies subsequently validated the promising benefit of B. infantis 35624 in the management of IBS−related symptoms.(72)(73) Other studies with smaller sample size have found other probiotics to be promising but await validation in larger randomized control trials.(74)

Ulcerative Colitis

Kato et al. performed a randomized placebo−controlled trial assessing the effect of Bifidobacteria−fermented milk on active ulcerative colitis (UC).Twenty patients with active UC were given probiotic milk or placebo for 12 weeks. Clinical and endoscopic activity indices demonstrated statistically significant improvements in the probiotic arm but not in the placebo arm.(75) An open label trial by Bibiloni et al. evaluated the efficacy of VSL#3 for the treatment of active UC. Thirty−two patients with active UC received VSL#3 for six weeks and remission was found in 53% of patients.(76)

A randomized, double−blinded, single−center pediatric study by Miele et al. also studied VSL#3 for induction and maintenance of remission in UC. Twenty−nine patients with newly diagnosed UC were given induction with steroids, then maintenance with 5−ASA. Patients were then randomized to VSL#3 or placebo for one year. Remission was seen in 92% of the treatment arm versus 36% of the placebo arm, and relapse rate was 21% in the treatment arm but 73% in the placebo arm.(77) Another pediatric study by Huynh et al. demonstrated efficacy of VSL#3 in UC. This group conducted an open−label trial of VSL#3 in 18 patients with mild−moderate UC and found remission rate of 56% and a combined remission/response rate of 61%.(78) While some of these data are pediatric, these studies all support the efficacy of probiotics for induction and maintenance of remission of UC.

Crohn’s Disease

Evidence favoring the use of probiotics in Crohn’s disease (CD) is less plentiful, probably because practitioners support the theory of colonic dysbiosis as a contributing factor of UC and pouchitis, and because of the regional ileal distribution of Crohn’s disease. Madsen et al. performed a randomized controlled trial of VSL#3 to evaluate for the prevention of endoscopic recurrence after surgery for Crohn’s disease. This group studied 120 CD patients undergoing ileocolonic surgical resection within 30 days and randomized them to receive VSL#3 or placebo. Follow−up at 90 days revealed disease recurrence occurred in 4/58 in the VSL#3 group versus 8/62 in the placebo arm.(79)

[For UC] statistically significant improvements in the probiotic arm but not in the placebo arm [were seen].

C. difficile and Antibiotic−associated Diarrhea

Great enthusiasm followed the publication of a meta−analysis of the use of probiotics for the prevention of antibiotic−associated diarrhea and C. difficile−associated colitis since the analysis indicated that probiotics showed promise as effective therapies.(80) However, a subsequent systematic review failed to find evidence for a role of probiotics in treatment of C. difficile−associated colitis.(81) Given the conflicting evidence, most recent reviewers have been hesitant to recommend routine treatment or prevention with probiotics, although additional study has been encouraged.(82)(83)

Summary

Medical researchers now appreciate that the microbes that normally inhabit the gut are more than mere “freeloaders” that have found an easy meal. Rather, these microorganisms exist within a balanced symbiosis with the host and play a key role in maintaining gastrointestinal homeostasis. Recent culture−independent molecular techniques have led to an increased understanding of the role of the indigenous gut microbiota in health and disease. With additional study, it is hoped that intentional manipulation of the gut microbiota, for example through the rationale use of probiotics, may lead to novel ways to prevent and treat a variety of gastrointestinal disease states.


Footnotes

1Dethlefsen, L., McFall-Ngai, M. & Relman, D.A. An ecological and evolutionary perspective on human-microbe mutualism and disease. Nature 449, 811-818 (2007).
2Turnbaugh, P.J., et al. The human microbiome project. Nature 449, 804-810 (2007).
3Ley, R.E., Peterson, D.A. & Gordon, J.I. Ecological and evolutionary forces shaping microbial diversity in the human intestine. Cell 124, 837-848 (2006).
4Martens, E.C., Koropatkin, N.M., Smith, T.J. & Gordon, J.I. Complex glycan catabolism by the human gut microbiota: the Bacteroidetes Sus-like paradigm. J. Biol. Chem. 284, 24673-24677 (2009).
5Hooper, L.V. Bacterial contributions to mammalian gut development. Trends Microbiol. 12, 129-134 (2004).
6Turnbaugh, P.J., et al. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444, 1027-1031 (2006).
7Sartor, R.B. Microbial influences in inflammatory bowel diseases. Gastroenterology 134, 577-594 (2008).
8Chang, J.Y., et al. Decreased Diversity of the Fecal Microbiome in Recurrent Clostridium difficile-Associated Diarrhea. J. Infect. Dis. 197, 435-438 (2008).
9Waldram, A., et al. Top-down systems biology modeling of host metabotype-microbiome associations in obese rodents. J Proteome Res 8, 2361-2375 (2009).
10Ivanov, II, et al. Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine. Cell host & microbe 4, 337-349 (2008).
11Sogin, S.J., Sogin, M.L. & Woese, C.R. Phylogenetic measurement in procaryotes by primary structural characterization. J Mol Evol 1, 173-184 (1971).
12Woese, C.R. & Fox, G.E. Phylogenetic structure of the prokaryotic domain: the primary kingdoms. Proc Natl Acad Sci U S A 74, 5088-5090 (1977).
13Woese, C.R., Sogin, M.L. & Sutton, L.A. Procaryote phylogeny. I. Concerning the relatedness of Aerobacter aerogenes to Escherichia coli. J Mol Evol 3, 293-299 (1974).
14Pace, N.R. A molecular view of microbial diversity and the biosphere. Science 276, 734-740 (1997).
15Pace, N.R., Stahl, D.A., Lane, D.J. & Olsen, G.J. Analyzing natural microbial populations by rRNA sequences. ASM News 51, 4-12 (1985).
16Ley, R.E., et al. Unexpected diversity and complexity of the Guerrero Negro hypersaline microbial mat. Appl Environ Microbiol 72, 3685-3695 (2006).
17Pedros-Alio, C. Marine microbial diversity: can it be determined? Trends Microbiol 14, 257-263 (2006).
18Pedros-Alio, C. Ecology. Dipping into the rare biosphere. Science 315, 192-193 (2007).
19Eckburg, P.B., et al. Diversity of the human intestinal microbial flora. Science 308, 1635-1638 (2005).
20Ley, R.E., Turnbaugh, P.J., Klein, S. & Gordon, J.I. Microbial ecology: human gut microbes associated with obesity. Nature 444, 1022-1023 (2006).
21Bibiloni, R., Mangold, M., Madsen, K.L., Fedorak, R.N. & Tannock, G.W. The bacteriology of biopsies differs between newly diagnosed, untreated, Crohn's disease and ulcerative colitis patients. J Med Microbiol 55, 1141-1149 (2006).
22Eckburg, P.B. & Relman, D.A. The role of microbes in Crohn's disease. Clin Infect Dis 44, 256-262 (2007).
23Hopkins, M.J., Sharp, R. & Macfarlane, G.T. Age and disease related changes in intestinal bacterial populations assessed by cell culture, 16S rRNA abundance, and community cellular fatty acid profiles. Gut 48, 198-205 (2001).
24Zoetendal, E.G., et al. Mucosa-associated bacteria in the human gastrointestinal tract are uniformly distributed along the colon and differ from the community recovered from feces. Appl Environ Microbiol 68, 3401-3407 (2002).
25Favier, C.F., de Vos, W.M. & Akkermans, A.D. Development of bacterial and bifidobacterial communities in feces of newborn babies. Anaerobe 9, 219-229 (2003).
26Magne, F., et al. Low species diversity and high interindividual variability in faeces of preterm infants as revealed by sequences of 16S rRNA genes and PCR-temporal temperature gradient gel electrophoresis profiles. FEMS Microbiol Ecol 57, 128-138 (2006).
27Young, V.B. & Schmidt, T.M. Antibiotic-associated diarrhea accompanied by large-scale alterations in the composition of the fecal microbiota. J. Clin. Microbiol. 42, 1203-1206 (2004).
28Huber, J.A., et al. Microbial population structures in the deep marine biosphere. Science 318, 97-100 (2007).
29Ashby, M.N., Rine, J., Mongodin, E.F., Nelson, K.E. & Dimster-Denk, D. Serial analysis of rRNA genes and the unexpected dominance of rare members of microbial communities. Appl Environ Microbiol 73, 4532-4542 (2007).
30Roesch, L.F., et al. Pyrosequencing enumerates and contrasts soil microbial diversity. Isme J 1, 283-290 (2007).
31Sogin, M.L., et al. Microbial diversity in the deep sea and the underexplored "rare biosphere". Proc Natl Acad Sci U S A 103, 12115-12120 (2006).
32Antonopoulos, D.A., et al. Reproducible community dynamics of the gastrointestinal microbiota following antibiotic perturbation. Infect. Immun. 77, 2367-2375 (2009).
33Andersson, A.F., et al. Comparative analysis of human gut microbiota by barcoded pyrosequencing. PLoS ONE 3, e2836 (2008).
34McKenna, P., et al. The macaque gut microbiome in health, lentiviral infection, and chronic enterocolitis. PLoS pathogens 4, e20 (2008).
35Takaishi, H., et al. Imbalance in intestinal microflora constitution could be involved in the pathogenesis of inflammatory bowel disease. Int J Med Microbiol 298, 463-472 (2008).
36Mazmanian, S.K., Round, J.L. & Kasper, D.L. A microbial symbiosis factor prevents intestinal inflammatory disease. Nature 453, 620-625 (2008).
37Conte, M.P., et al. Gut-associated bacterial microbiota in paediatric patients with inflammatory bowel disease. Gut 55, 1760-1767 (2006).
38Martin, H.M., et al. Enhanced Escherichia coli adherence and invasion in Crohn's disease and colon cancer. Gastroenterology 127, 80-93 (2004).
39Darfeuille-Michaud, A., et al. High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn's disease. Gastroenterology 127, 412-421 (2004).
40Sasaki, M., et al. Invasive Escherichia coli are a feature of Crohn's disease. Lab Invest 87, 1042-1054 (2007).
41Knight, P., Campbell, B.J. & Rhodes, J.M. Host-bacteria interaction in inflammatory bowel disease. Br Med Bull 88, 95-113 (2008).
42Zholudev, A., Zurakowski, D., Young, W., Leichtner, A. & Bousvaros, A. Serologic testing with ANCA, ASCA, and anti-OmpC in children and young adults with Crohn's disease and ulcerative colitis: diagnostic value and correlation with disease phenotype. Am J Gastroenterol 99, 2235-2241 (2004).
43Austin, G.L., Herfarth, H.H. & Sandler, R.S. A critical evaluation of serologic markers for inflammatory bowel disease. Clin Gastroenterol Hepatol 5, 545-547 (2007).
44Parkes, G.C., Brostoff, J., Whelan, K. & Sanderson, J.D. Gastrointestinal microbiota in irritable bowel syndrome: their role in its pathogenesis and treatment. Am J Gastroenterol 103, 1557-1567 (2008).
45Balsari, A., Ceccarelli, A., Dubini, F., Fesce, E. & Poli, G. The fecal microbial population in the irritable bowel syndrome. Microbiologica 5, 185-194 (1982).
46Si, J.M., Yu, Y.C., Fan, Y.J. & Chen, S.J. Intestinal microecology and quality of life in irritable bowel syndrome patients. World J Gastroenterol 10, 1802-1805 (2004).
47Matto, J., et al. Composition and temporal stability of gastrointestinal microbiota in irritable bowel syndrome--a longitudinal study in IBS and control subjects. FEMS Immunol Med Microbiol 43, 213-222 (2005).
48Kassinen, A., et al. The fecal microbiota of irritable bowel syndrome patients differs significantly from that of healthy subjects. Gastroenterology 133, 24-33 (2007).
49Bartlett, J.G. Clinical practice. Antibiotic-associated diarrhea. N. Engl. J. Med. 346, 334-339 (2002).
50Hogenauer, C., Hammer, H.F., Krejs, G.J. & Reisinger, E.C. Mechanisms and management of antibiotic-associated diarrhea. Clin. Infect. Dis. 27, 702-710 (1998).
51Kelly, C.P. & LaMont, J.T. Clostridium difficile infection. Annu. Rev. Med. 49, 375-390 (1998).
52Mylonakis, E., Ryan, E.T. & Calderwood, S.B. Clostridium difficile--Associated diarrhea: A review. Arch. Intern. Med. 161, 525-533 (2001).
53Johnson, S. & Gerding, D.N. Clostridium difficile--associated diarrhea. Clin. Infect. Dis. 26, 1027-1034; quiz 1035-1026 (1998).
54Lusk, R.H., et al. Gastrointestinal side effects of clindamycin and ampicillin therapy. J. Infect. Dis. 135 Suppl, S111-119 (1977).
55Johnson, S., et al. Epidemics of diarrhea caused by a clindamycin-resistant strain of Clostridium difficile in four hospitals. N. Engl. J. Med. 341, 1645-1651 (1999).
56Kyne, L., Hamel, M.B., Polavaram, R. & Kelly, C.P. Health care costs and mortality associated with nosocomial diarrhea due to Clostridium difficile. Clin. Infect. Dis. 34, 346-353 (2002).
57Freter, R. In vivo and in vitro antagonism of intestinal bacteria against Shigellaflexneri. II. The inhibitory mechanism. J. Infect. Dis. 110, 38-46 (1962).
58Hentges, D.J. & Freter, R. In vivo and in vitro antagonism of intestinal bacteria against Shigella flexneri. I. Correlation between various tests. J. Infect. Dis. 110, 30-37 (1962).
59Vollaard, E.J. & Clasener, H.A. Colonization resistance. Antimicrob. Agents Chemother. 38, 409-414 (1994).
60Wilson, K.H. The microecology of Clostridium difficile. Clin. Infect. Dis. 16 Suppl 4, S214-218 (1993).
61Bartlett, J.G., Chang, T.W., Gurwith, M., Gorbach, S.L. & Onderdonk, A.B. Antibiotic-associated pseudomembranous colitis due to toxin-producing clostridia. N. Engl. J. Med. 298, 531-534 (1978).
62Bartlett, J.G., Onderdonk, A.B., Cisneros, R.L. & Kasper, D.L. Commentary: Bartlett JG, Onderdonk AB, Cisneros RL, Kasper DL. Clindamycin-associated colitis due to a toxin-producing species of Clostridium in hamsters. J Infect Dis 1977; 136:701. J. Infect. Dis. 190, 202-209 (2004).
63Wilson, K.H., Silva, J. & Fekety, F.R. Suppression of Clostridium difficile by normal hamster cecal flora and prevention of antibiotic-associated cecitis. Infect. Immun. 34, 626-628 (1981).
64Isaacs, K.L. & Sartor, R.B. Treatment of inflammatory bowel disease with antibiotics. Gastroenterol. Clin. North Am. 33, 335-345, x (2004).
65Perencevich, M. & Burakoff, R. Use of antibiotics in the treatment of inflammatory bowel disease. Inflamm Bowel Dis 12, 651-664 (2006).
66Sartor, R.B. Therapeutic manipulation of the enteric microflora in inflammatory bowel diseases: antibiotics, probiotics, and prebiotics. Gastroenterology 126, 1620-1633 (2004).
67FAO. Health and nturitional properties of probiotics in food including powder milk with live lactic acid bacteria. (Food and Agriculture Organization of the United Nations, Cordoba, Argentina, 2001).
68Gionchetti, P., et al. Prophylaxis of pouchitis onset with probiotic therapy: a double-blind, placebo-controlled trial. Gastroenterology 124, 1202-1209 (2003).
69Mimura, T., et al. Once daily high dose probiotic therapy (VSL#3) for maintaining remission in recurrent or refractory pouchitis. Gut 53, 108-114 (2004).
70Gionchetti, P., et al. High-dose probiotics for the treatment of active pouchitis. Dis Colon Rectum 50, 2075-2082; discussion 2082-2074 (2007).
71McCarthy, J., et al. Double blind, placebo controlled trial of two probiotic strains in interleukin 10 knockout mice and mechanistic link with cytokine balance. Gut 52, 975-980 (2003).
72O'Mahony, L., et al. Lactobacillus and bifidobacterium in irritable bowel syndrome: symptom responses and relationship to cytokine profiles. Gastroenterology 128, 541-551 (2005).
73Whorwell, P.J., et al. Efficacy of an encapsulated probiotic Bifidobacterium infantis 35624 in women with irritable bowel syndrome. Am J Gastroenterol 101, 1581-1590 (2006).
74Brenner, D.M., Moeller, M.J., Chey, W.D. & Schoenfeld, P.S. The utility of probiotics in the treatment of irritable bowel syndrome: a systematic review. Am J Gastroenterol 104, 1033-1049; quiz 1050 (2009).
75Kato, K., et al. Randomized placebo-controlled trial assessing the effect of bifidobacteria-fermented milk on active ulcerative colitis. Aliment Pharmacol Ther 20, 1133-1141 (2004).
76Bibiloni, R., et al. VSL#3 probiotic-mixture induces remission in patients with active ulcerative colitis. Am J Gastroenterol 100, 1539-1546 (2005).
77Miele, E., et al. Effect of a probiotic preparation (VSL#3) on induction and maintenance of remission in children with ulcerative colitis. Am J Gastroenterol 104, 437-443 (2009).
78Huynh, H.Q., et al. Probiotic preparation VSL#3 induces remission in children with mild to moderate acute ulcerative colitis: a pilot study. Inflamm Bowel Dis 15, 760-768 (2009).
79Madsen K., B.J., Leddin D, Dieleman L, Bitton A, Feagan B, Petrunia DM, Chiba N, Enns RA, Fedorak R. . A Randomized Controlled Trial of VSL#3 for the Prevention of Endoscopic Recurrence Following Surgery for Crohn's Disease. . Gastroenterology 134, A-361: M1207 (2008).
80McFarland, L.V. Meta-analysis of probiotics for the prevention of antibiotic associated diarrhea and the treatment of Clostridium difficile disease. Am. J. Gastroenterol. 101, 812-822 (2006).
81Pillai, A. & Nelson, R. Probiotics for treatment of Clostridium difficile-associated colitis in adults. Cochrane Database Syst Rev, CD004611 (2008).
82Miller, M. The fascination with probiotics for Clostridium difficile infection: Lack of evidence for prophylactic or therapeutic efficacy. Anaerobe (2009).
83Kelly, C.P. & LaMont, J.T. Clostridium difficile--more difficult than ever. N. Engl. J. Med. 359, 1932-1940 (2008).