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Curr Opin Infect Dis. Author manuscript; available in PMC Jun 1, 2010.
Published in final edited form as:
PMCID: PMC2763597
NIHMSID: NIHMS111122
Commensal Bacteria, Traditional and Opportunistic Pathogens, Dysbiosis and Bacterial Killing in Inflammatory Bowel Diseases
Christopher Dennis Packey1 and R. Balfour Sartor1,2
1 Department of Medicine, Center for Gastrointestinal Biology and Disease, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
2 Department of Immunology and Microbiology, Center for Gastrointestinal Biology and Disease, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
Correspondence: R. Balfour Sartor, MD, UNC Department of Medicine/GI, CB 7032, Room 7309 MBRB, 103 Mason Farm Road, Chapel Hill, NC 27599-7032, rbs/at/med.unc.edu, Phone: (919) 966-0149, Fax: (919) 843-6899
Purpose of review
The authors present evidence published during the past two years of the roles of commensal and pathogenic bacteria in the pathogenesis of the inflammatory bowel diseases (IBD).
Recent findings
Rodent models conclusively implicate commensal enteric bacteria in chronic, immune-mediated, experimental colitis, and genetically determined defects in bacterial killing by innate immune cells are found in a subset of Crohn’s disease patients. There is no evidence that a single pathogen, including Mycobacterium avium subspecies paratuberculosis, causes Crohn’s disease or ulcerative colitis. However, adherent/invasive E. coli (AIEC) are associated with ileal Crohn’s disease, with the mechanisms and genetics of AIEC virulence being elucidated. Molecular characterization of the microbiota in patients with IBD reveals decreased biodiversity of commensal bacteria, most notably the phyla Bacteroidetes and Firmicutes, including the clinically-relevant Faecalibacterium prausnitzii, and increased E. coli concentrations. VSL#3 is one probiotic preparation shown to be efficacious in certain clinical situations in small clinical trials.
Summary
Further characterization of altered microbiota in patients with IBD and linking dysbiosis with host genetic alterations in immunoregulation, innate microbial killing and barrier function are critical, so that individualized treatments to increase beneficial commensals and their metabolic products (probiotic and prebiotic administration) and diminish deleterious species such as AIEC can be tailored for defined patient subsets.
Keywords: IBD, bacteria, commensals, dysbiosis, probiotics
Chronic idiopathic inflammatory bowel diseases (IBD), which affect 1.0 to 1.5 million Americans, [1*] are a heterogeneous group of disorders resulting from continuous enteric microbial antigenic stimulation of pathogenic T cell responses in individuals with genetic defects in mucosal barrier function, innate bacterial killing or immunoregulation. [2*] Altered microbial composition, defective clearance of bacteria and enhanced mucosal uptake in Crohn’s disease (CD) and ulcerative colitis (UC) increase immune stimulation. CD and UC preferentially occur in areas of highest intestinal bacterial concentrations and fecal flow sustains inflammation of CD. [3] Ciprofloxacin and metronidazole treat colonic CD and experimental colitis in a number of rodent models. [4] This review describes evidence of the roles of commensal bacteria in the pathogenesis of IBD in articles published January 2007 to December 2008.
Defective bacterial killing in Crohn’s disease
Pathophysiologic similarities between CD and chronic granulomatous diseases associated with defective phagocyte function, polymorphisms of genes regulating clearance of intracellular pathogens and observed defects in innate antimicrobial function have led to the hypothesis that a subset of CD is caused by defective clearance of commensal, opportunistic or pathogenic bacteria with subsequent initiation of compensatory antibacterial effector T cells that cause tissue damage. [2*, 5] Secretion of both α- and β-defensins is defective in CD, [6, 7] with decreased Paneth cell α-defensin production due to reduced expression of Tcf-4, a WNT-signaling pathway transcription factor. [8] The truncation mutation of NOD2, which is an intracellular receptor for the peptidoglycan component muramyl dipeptide, results in decreased ileal α-defensin production. [6] Furthermore, NOD2 deficient mice exhibit decreased α-defensin defcr-4, [9] while deletion of the autophagy gene ATG16L1 or the endoplasmic reticulum stress protein XBP-1 results in Paneth cell morphologic changes and decreased expression of antimicrobial peptides. [10**, 11*] CD is associated with genetic polymorphisms of at least 2 autophagy pathway components, ATG16L1 and IRGM, which, like NOD2 and NCF2, regulate intracellular bacterial killing. [12*, 13*, 14*] Decreased antimicrobial peptide secretion could lead to overgrowth, increased mucosal adherence and translocation of commensal bacteria, while defective clearance of invasive or phagocytosed bacteria promotes persistence of viable intracellular bacteria. (Figure 1.) Both mechanisms could result in excessive antigenic stimulation of pathogenic TH1/TH17 cells, chronic granulomatous inflammation and susceptibility to infection by traditional and opportunistic pathogens.
Figure 1
Figure 1
Defective containment of commensal bacteria in IBD
Microbial pathogens
Comprehensive culture and molecular-based analyses of the microbiota of IBD patients fail to identify consistent enrichment of individual pathogenic species in IBD tissues. However, bacterial pathogens continue to reap attention because of similarities between CD, UC and enteric infections and the hope of finding a cure analogous to Helicobacter pylori in peptic ulcers.
Mycobacterium avium subspecies paratuberculosis
Mycobacterium avium subspecies paratuberculosis (MAP) causes spontaneous granulomatous enterocolitis (Johne’s disease) with diarrhea and wasting in ruminants, making this obligate intracellular pathogen a credible etiologic agent of Crohn’s disease. [15] After considerable investigation, the link between MAP and CD remains neither substantiated nor invalidated. Many investigators continue to measure the MAP-specific insertion element IS900 DNA in the tissue and/or blood of IBD patients using polymerase chain reaction (PCR) with mixed results. (Table 1.)
Table 1
Table 1
MAP-specific IS900 DNA PCR data from peer-reviewed articles published in 2007–2008
A study conducted at the University of Colorado reported no detectable MAP DNA in 190 tissue samples from CD, UC and control patients. [16**] Baumgart et al similarly reported lack of detectable IS900 DNA in ileal CD biopsies. [17**] Contradictory results were reported by Scanu et al, who detected MAP DNA in 87% of CD tissues and 15% of controls in a small cohort. [18] Results are also inconsistent in PCR assays for MAP DNA in blood samples. [2022]
One recent, blinded study offered an alternative method to PCR for identifying MAP in CD patients. [25] Coded, paraffin-embedded surgical resections from CD and control subjects at two centers subjected to acid-fast staining and rRNA in situ hybridization (ISH) for MAP were visualized with oil-immersion microscopy. Both methods provided positive results in 10/17 CD subjects, (59%, CI: 36–78), contrasted with 5/35 control subjects, (OR for CD versus controls = 8.6, p = 0.002). Agreement between the two methods was good, but the time-consuming ISH probes could not discriminate between mycobacterial subspecies.
Defective bacterial killing by innate immune cells could increase risk of infection by intracellular pathogens. Two recent studies showed no association between MAP and NOD2 mutations. No significant association was seen in New Zealand CD patients for carriage of heterozygous or homozygous NOD2 mutations and MAP status, [21] or between MAP serologies and NOD2 polymorphisms in a large, population-based study in Manitoba. [26*] A recent epidemiologic report also failed to support exposure to MAP by contaminated milk or water. [27*] Furthermore, consumption of pasteurized milk and fruit was associated with a reduced risk of CD, whereas meat intake was associated with an increased risk. Finally, a well-designed, 2 year prospective trial of clarithromycin, rifabutin, and ethambutol failed to show sustained clinical response in CD patients. [28*]
Recent studies possibly linking MAP to pathogenic mechanisms of CD fuel the ongoing debate. 235 patients with CD, UC, irritable bowel syndrome (IBS) and no known disease were tested for presence of MAP IS900 DNA and cytokine secretion in intestinal biopsy samples. [23] Greater TNF levels correlated with the presence of MAP in CD patients (p < 0.05). However, there was no correlation of MAP with IL-2, IL-12, IL-10, and IFN-γ secretion. A separate study linked MAP to active CD via IL-4 and IL-2 cytokine profiles in peripheral blood with no differences in IFN-γ or TNF levels. [24]
Finally, MAP was recently reported to induce experimental colitis in gnotobiotic IL-10−/− mice. [29] Germ-free IL-10−/− mice receiving a single oral dose of milk containing 10^4 CFU of live MAP obtained from a CD patient developed more severe and aggressively progressive colitis, had higher serum amyloid A, IFN-γ and TNF levels, and lost more weight than did IL-10−/− mice that received heat-killed MAP.
Despite continued suggestions of a link between MAP and IBD, it remains doubtful that MAP is the causative agent of most CD patients, although infection of a subset of patients with intracellular killing defects caused by ATG16L1, IGRM or NCF4 needs to be investigated. CD-related NOD2 polymorphisms do not appear to be risk factors for MAP infection.
Functional changes in Escherichia coli
Darfeuille-Marchaud reported that adherent/invasive E. coli (AIEC) that persist within macrophages and epithelial cells selectively colonize the ileum of CD patients. [30, 31] At least 2 separate groups have confirmed these observations. Baumgart et al demonstrated AIEC in the ileum of CD patients, documented in vivo mucosal adherence with fluorescent in situ hybridization (FISH) and identified AIEC virulence factors common to uropathic E. coli strains and E. coli strains isolated from boxer dogs with spontaneous granulomatous colitis. [17**] In a separate study, E. coli comprised 99% of invasive bacterial isolates in mucosal biopsies of CD patients as opposed to 42% in UC patients and 2% in normal controls. [32*] A prototypic AIEC strain, LF82, induced in vitro granulomas using blood-derived mononuclear cells. [33*] Serum antibodies directed against E. coli outer membrane protein C (OmpC) are present in 37–55% of patients with CD, in contrast to ≤ 5% of UC patients and subjects without IBD. High serum reactivity to E. coli OmpC is associated with severe CD with longer disease duration, frequent disease progression, small bowel involvement, and increased resections. [34]
Mechanisms of epithelial adherence and invasion of AIEC are being elucidated. Studies utilizing isogenic mutants of LF82 that lack OmpC and other genes suggest that increased expression of OmpC, particularly at high osmolarities reflecting the GI tract, and/or induction of the σE regulatory pathway promote adherence and invasion of epithelial cells by LF82 independent of the transcriptional regulator OmpR. [35*]
Flagellin is necessary for LF82’s ability to exacerbate DSS-induced murine colitis. [36*] Nonflagellated LF82 mutants behaved like the nonpathogenic E. coli strain K-12 in this model. An LF82 mutant lacking dsbA, a gene that encodes a periplasmic oxidoreductase that determines virulence for several pathogens, expressed neither flagella nor type 1 pili, and displayed decreased survival ability. [37*] In contrast, decreased epithelial adhesion and invasion of the LF82 OmpC and OmpR mutants were not associated with decreased expression of flagella and type 1 pili. In a separate study, E. coli 083:H1 was dependent upon flagellin for its adherent and invasive phenotype. [38*] Increased expression of carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), which acts as a receptor for AIEC, by ileal epithelial cells in active Crohn’s disease may mediate increased mucosal adherence of AIEC. [39*] Increased CEACAM6 expression by cultured colonic epithelial cells after IFN-γ or TNF stimulation and after exposure to LF82 indicate that AIEC may promote its own colonization.
Monocytes from CD patients carrying homozygous or heterozygous NOD2 polymorphisms displayed reduced secretion of IL-1β, IL-6, and IL-10 to LF82 in vitro compared with monocytes from CD patients without NOD2 polymorphisms. [40**] TLR4 polymorphisms did not influence monocyte response. In preliminary studies, we showed that macrophages from NOD2 deficient mice display defective clearance of a murine AIEC strain with prolonged secretion of IL-12/23 p40 and TNF. [41] These findings demonstrate the importance of genetically programmed host immune responses to disease-related bacteria in the pathogenesis of CD.
Enteric pathogens as environmental triggers
A case-control study examining medical records of 3,019 U.S. soldiers who developed IBD and 11,646 matched controls compared data using a conditional logistic regression model to determine that a single episode of infectious gastroenteritis increased the risk of IBD, (OR: 1.40, CI: 1.19–1.66). [42] These results suggest that a self-limited enteric pathogen could trigger IBD, possibly by breaking the mucosal barrier and initiating an early inflammatory response in a genetically susceptible host with immunoregulatory abnormalities. [2*] Clostridium difficile can reactivate quiescent IBD [43*] and induce acute experimental epithelial injury by Fas-mediated apoptosis. [44] Likewise enterotoxigenic Bacteroides fragilis can induce experimental colitis and IL-17 production. [45] A role for non-pylori Helicobacter in IBD pathogenesis is unlikely based on detection of species-specific serum antibodies in only 6/137 IBD patients. [46]
Dysbiosis
Recent studies in rodents confirm that bacterial composition changes with colonic inflammation and/or infection. [47] Use of molecular techniques is clarifying changes in the composition of the mucosally associated and fecal microbiota in patients with CD and UC and vastly extending previous culture based studies. (Table 2.)
Table 2
Table 2
Changes in the composition of the mucosa-associated microbiota (MAM) and/or fecal microbiota in patients with CD and UC compared with normal controls, as published in 2007–2008 in peer-reviewed articles
Examining DNA libraries of mucosal-associated microbiota, which may be more relevant than the fecal microbiota to the pathogenesis of IBD, reveals that patients with CD and UC have decreased complexity of commensal bacteria. [16**] Most notably, members of the phyla Bacteroidetes and Firmicutes are decreased in CD and UC. These organisms promote GI health in multiple ways. [59] Reduced Faecalibacterium prausnitzii, a major member of the family Firmicutes, in CD patients [16**, 50**] was confirmed and associated with a higher risk of post-resection recurrence of ileal CD. [49**] In vitro peripheral blood mononuclear cell stimulation by F. prausnitzii decreased IL-12 and IFN-γ production and stimulated secretion of IL-10. Oral administration of either live F. prausnitzii or its supernatant reduced the severity of TNBS colitis and corrected the associated dysbiosis. In parallel, the abundance of E. coli is increased in IBD, particularly the B2+D phylogenetic group. [17**, 48*] E coli isolated from CD patients express uropathic-like virulence factors that are postulated to facilitate mucosal invasion. [17**] The number of mucosal E. coli in situ correlates with the severity of ileal disease and invasive E. coli are restricted to inflamed mucosa. Compositional changes of the microbiota (dysbiosis) in IBD subsets may contribute to disease severity, since abnormal microbiotas correlated with the occurrence of abscesses in CD patients, and IBD patients with dysbiosis underwent surgery at a younger age than those with normal microbiotas. [16**] Finally, fecal and mucosally associated microbial communities of patients with CD and UC are consistently less diverse with increased temporal instability. [16**, 53*, 54*, 55*, 56*]
Nonpathogenic bacteria can cause colitis in hosts with immunoregulatory and mucosal barrier deficits. When germ-free IL-10−/− and wild type (WT) mice were inoculated with nonpathogenic E. faecalis and/or E. coli, dual-associated IL-10−/− (but not WT) mice developed aggressive TH1/TH17-mediated colitis within 3 weeks that progressed to severe pancolitis by 7 weeks. [60*] A separate study revealed that uptake of nonpathogenic E. coli strains K-12 and HB101 by specialized follicle-associated epithelial cells overlying Peyer’s patches, (the site of the earliest observable microscopic lesions of recurrent CD), is increased in CD patients, but not in UC subjects. [61*] Increased E. coli localized within dendritic cells of CD mucosa correlated with augmented tissue release of TNF.
Metabolic products of the microbiota have important effects on mucosal epithelial cell and immune function. [2*] Butyrate and other short-chain fatty acids are the primary metabolic substrates of colonocytes, with rectal epithelial cells dependent on this fuel source, while hydrogen sulfide (HS), nitric oxide (NO) and serine proteases produced by a subset of commensal microbiota can injure epithelial cells and matrix components. [62*] HS and NO block butyrate metabolism and could lead to a state of epithelial starvation relevant to the pathogenesis of UC.
Probiotics
Animal models and human studies suggest that therapeutically manipulating the balance between beneficial and detrimental intestinal bacterial species can influence health and disease. [63] Use of probiotics, (viable, nonpathogenic microorganisms that exert health benefits beyond basic nutrition), to shift this balance to favor protective species and treat IBD, has been extensively reviewed [63, 64*, 65, 66, 67*], yet the role for probiotics in treating CD and UC remains undetermined because most trials are underpowered and therefore not definitive.
In a TNBS colitis model, oral Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus casei reduced intestinal inflammation. [68] L. casei had more modest protection than the other probiotic species, yet in a separate in vitro study, live L. casei decreased TNF, IFN-γ, IL-2, IL-6, IL-8, and CXCL1 secretion by explanted CD mucosa and counteracted the proinflammatory effects of commensal E. coli ATCC 35345. [69] This and other studies emphasize host-specific responses to probiotics. Other probiotic species control aberrant immune responses in intestinal tissue. Bifidobacterium bifidum (BGN4) prevented colitis in a murine CD4+ CD45RBhigh T cell transfer model. [70*] Lactobacillus gasseri expressing manganese superoxide dismutase ameliorated colitis in IL-10−/− mice, demonstrating the therapeutic potential of recombinant bacteria engineered to secrete biologically active molecules. [71*] Orally administered Lactobacillus suntoryeus HY7801 ameliorated TNBS-induced colitis, decreased colonic IL-1β, IL-6, and TNF expression and inhibited TLR4-linked NFκB activation. [72*] Bifidobacterium-fermented milk enhanced IL-10 production in peripheral blood mononuclear cells from UC patients and inhibited IL-8 secretion by intestinal epithelial cells. [73] Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 fed in yogurt to IBD patients had peripheral blood anti-inflammatory effects. In an in vitro study, L. reuteri potently suppressed human TNF production by lipopolysaccharide-activated monocytes and monocyte-derived macrophages from children with CD in a strain-dependent manner by inhibiting activation of MAP kinase-regulated c-Jun and the transcription factor AP-1. [74*]
Several groups have investigated the probiotic combination VSL#3 as a treatment for IBD based on its ability to maintain remission in relapsing pouchitis. [75, 76] This combination of 4 live Lactobacillus strains, (L. casei, bulgaricus, plantarium and acidophilus), 3 live Bifidobacterium strains, (B. longum, breve and infantis), and 1 live Streptococcus strain, (thermophilus), dose-dependently ameliorated DSS-induced colitis in weanling rats. [77] In a small (N=18) open-label study, VSL #3 induced remission in 56% of pediatric patients with mild to moderate UC. 6% showed a response, and 39% had no response or did worse with treatment. [78*] Daily oral VSL #3 significantly reduced the pouchitis disease activity index and expanded the number of mucosal regulatory T cells following colectomy with ileal pouch anal anastomosis. [79*] Two components of VSL #3, B. longum and B. breve, in combination with the prebiotic psyllium, were well-tolerated at high doses and induced remission in 6/10 patients with Crohn’s disease who had failed a regimen of aminosalicylates and prednisolone. [80*]
Similar to Sokol et al’s studies with F. prausnitzii, [49**] an Israeli group isolated and characterized a new putative protective bacterial species, Enterococcus durans, from the fecal microbiota of a healthy human vegetarian. [81*] In a DSS model of colitis, oral administration of E. durans significantly ameliorated colitis compared to controls and mice fed Lactobacillus delbrueckii. To properly note negative studies, oral administration of Lactobacillus johnsonii failed to prevent early postoperative endoscopic recurrence of CD 12 weeks after ileo-caecal resection. [82]
To date, probiotics appear to be more effective in preventing relapse of UC and pouchitis, with poor results in CD. Examining F. prausnitzii in preventing postoperative recurrence of CD will be quite interesting and could lay the foundation for targeted correction of dysbiotic microbiota in IBD. Use of prebiotics, which are poorly absorbed oligosaccharides that stimulate growth and metabolic activity of beneficial microbiota, could be a low cost, nontoxic strategy to correct dysbiosis in IBD patients.
There remains no evidence that MAP is a causative agent in the pathogenesis of IBD. A more likely scenario involves specific E. coli strains and other species with virulence factors that allow them to adhere to and invade epithelial cells and persist within macrophages. These strains could be viewed as opportunistic pathogens in genetically susceptible hosts with innate killing, mucosal barrier or immunoregulatory defects, with no disease induced in normal hosts. Identifying and then eliminating these strains, blocking expression of their virulence factors or altering their metabolism and identifying host susceptibility factors could be essential keys to treating IBD. Likewise, identifying changes in microbiota composition, gene expression and metabolic profiles in different IBD patient subsets using rapidly evolving molecular tools will provide important new insights into the pathogenesis and novel treatment of IBD using the example of F. prausnitzii. [49**] Meanwhile, using probiotics and prebiotics to enhance concentrations of beneficial species remains promising, although clinical trials lack statistical power due to small numbers. Treatments need to be individualized based on compositional alterations in patient subsets. Understanding bacterial and host mutalistic interactions will determine success in this area.
Acknowledgments
The authors thank Susie May for expert secretarial assistance. Original data discussed in this review were supported by UPHS grants R01 DK40249, R01 DK53347, P40 RR018603, P30 DK34987 and a grant from the Crohn’s and Colitis Foundation of America.
Papers of particular interest, published within the annual period of review, are highlighted as:
* Of special interest
** Of outstanding interest
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12 *. Prescott NJ, Fisher SA, Franke A, et al. A nonsynonymous SNP in ATG16L1 predisposes to ileal Crohn’s disease and is independent of CARD15 and IBD5. Gastroenterology. 2007;132(5):1665–71. The T300A variant of ATG16L1 was genotyped in two cohorts in the U.K. totaling over 1,200 CD patients and 1,200 controls, and was found to confer a 1.65-fold risk of Crohn’s disease, with a 2.2-fold risk of ileal disease. [PubMed]
13 *. Rioux JD, Xavier RJ, Taylor KD, et al. Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis. Nat Genet. 2007;39(5):596–604. A strong and significantly replicated association was made between ileal CD and a coding variant in ATG16L1 in this study, which included 988 patients with ileal CD and over 1,000 controls. ATG16L1 was also shown to be expressed in intestinal epithelial cell lines and involved in killing an intracellular pathogen. [PMC free article] [PubMed]
14 *. Barrett JC, Hansoul S, Nicolae DL, et al. Genome-wide association defines more than 30 distinct susceptibility loci for Crohn’s disease. Nat Genet. 2008;40(8):955–62. This study combined data from three previous studies on CD, including 3,230 cases and 4,829 controls, and confirmed 11 previously reported loci while providing genome-wide significant evidence for 21 additional loci. This study is important because it provides the largest set of subjects for CD genome-wide association studies. [PMC free article] [PubMed]
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17 **. Baumgart M, Dogan B, Rishniw M, et al. Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn’s disease involving the ileum. ISME J. 2007;1:403–18. Invasive E. coli were linked to ileal CD in this study, with enrichment of their sequences in inflamed ileal mucosa. The number of E. coli in situ correlated with the severity of ileal disease. [PubMed]
18. Scanu AM, Bull TJ, Cannas S, et al. Mycobacterium avium subspecies paratuberculosis infection in cases of irritable bowel syndrome and comparison with Crohn’s disease and Johne’s disease: common neural and immune pathogenicities. J Clin Microbiol. 2007;45(12):3883–90. [PMC free article] [PubMed]
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26 *. Bernstein CN, Wang MH, Sargent M, et al. Testing the interaction between NOD-2 status and serological response to Mycobacterium paratuberculosis in cases of inflammatory bowel disease. J Clin Microbiol. 2007;45(3):968–71. This study reports a lack of association between MAP serologies and NOD2 polymorphisms. [PMC free article] [PubMed]
27 *. Abubakar I, Myhill DJ, Hart AR, et al. A case-control study of drinking water and dairy products in Crohn’s Disease--further investigation of the possible role of Mycobacterium avium paratuberculosis. Am J Epidemiol. 2007;165(7):776–83. This epidemiological survey refuted any association between MAP and contamination of water sources in the United Kingdom. This is important because of the historical concern regarding potential transmission of MAP through water sources. [PubMed]
28 *. Selby W, Pavli P, Crotty B, et al. Two-year combination antibiotic therapy with clarithromycin, rifabutin, and clofazimine for Crohn’s disease. Gastroenterology. 2007;132:2313–2319. This large, placebo-controlled trial reported no sustained benefit of triple anti-mycobacterial therapy. [PubMed]
29. Singh UP, Singh S, Singh R, et al. Influence of Mycobacterium avium subsp. paratuberculosis on colitis development and specific immune responses during disease. Infect Immun. 2007;(8):3722–8. [PMC free article] [PubMed]
30. Darfeuille-Michaud A, Boudeau J, Bulois P, et al. High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn’s disease. Gastroenterology. 2004;127(2):412–21. [PubMed]
31. Barnich N, Darfeuille-Michaud A. Adherent-invasive Escherichia coli and Crohn’s disease. Curr Opin Gastroenterol. 2007;23(1):16–20. [PubMed]
32 *. Sasaki M, Sitaraman SV, Babbin BA, et al. Invasive Escherichia coli are a feature of Crohn’s disease. Lab Invest. 2007;87(10):1042–54. Invasive strains of E. coli were strongly associated with CD by gentamicin protection assays and biochemical profiling. [PubMed]
33 *. Meconi S, Vercellone A, Levillain F, et al. Adherent-invasive Escherichia coli isolated from Crohn’s disease patients induce granulomas in vitro. Cell Microbiol. 2007;9(5):1252–61. Prototypical AIEC LF82 selectively induced the aggregation of infected human macrophages to form granulomas. [PubMed]
34. Mow WS, Vasiliauskas EA, Lin YC, et al. Association of antibody responses to microbial antigens and complications of small bowel Crohn’s disease. Gastroenterology. 2004;126(2):414–24. [PubMed]
35 *. Rolhion N, Carvalho FA, Darfeuille-Michaud A. OmpC and the σE pathway are involved in adhesion and invasion of the Crohn’s disease-associated Escherichia coli strain LF82. Mol Microbiol. 2007;63(6):1684–700. One of a series of important studies by Darfeuille-Michaud’s group investigating the pathways utilized by the prototype AIEC strain to adhere to and invade epithelial cells. [PubMed]
36 *. Carvalho FA, Barnich N, Sauvanet P, et al. Crohn’s disease-associated Escherichia coli LF82 aggravates colitis in injured mouse colon via signaling by flagellin. Inflamm Bowel Dis. 2008;14(8):1051–60. Mechanisms by which nonpathogenic E. coli strains acquire virulence are elucidated in this animal model. [PubMed]
37 *. Bringer MA, Rolhion N, Glasser AL, Darfeuille-Michaud A. The oxidoreductase DsbA plays a key role in the ability of the Crohn’s disease-associated adherent-invasive Escherichia coli strain LF82 to resist macrophage killing. J Bacteriol. 2007;189(13):4860–71. Molecular mechanisms by which LF82 resist macrophage killing are elucidated. AIEC can replicate intracellularly within macrophages. [PMC free article] [PubMed]
38 *. Eaves-Pyles T, Allen CA, Taormina J, et al. Escherichia coli isolated from a Crohn’s disease patient adhere, invade, and induce inflammatory responses in polarized intestinal epithelial cells. Int J Med Microbiol. 2008;298(5–6):397–409. In this study, AIEC strains isolated from CD patients demonstrated the ability to induce inflammatory responses in Caco-2BBe and T-84 intestinal epithelial cells in in vitro experiments. [PubMed]
39 *. Barnich N, Carvalho FA, Glasser AL, et al. CEACAM6 acts as a receptor for adherent-invasive E. coli, supporting ileal mucosa colonization in Crohn’s disease. J Clin Invest. 2007;117(6):1566–74. The adhesion characteristics of AIEC that are implicated in the pathogenesis of IBD are investigated in this study by Darfeuille-Michaud’s group. Ileal epithelial cells of CD patients express higher levels of CEACAM 6, which is further upregulated by AIEC exposure. [PMC free article] [PubMed]
40 **. Peeters H, Bogaert S, Laukens D, et al. CARD15 variants determine a disturbed early response of monocytes to adherent-invasive Escherichia coli strain LF82 in Crohn’s disease. Int J Immunogenet. 2007;34(3):181–91. This is one of the first, important attempts to investigate associations between polymorphisms linked to CD and the adhesion and invasion characteristics of AIEC. [PubMed]
41. Liu B, Schmitz JM, Holt LC, et al. Increased intracellular survival of E. coli NC101 within macrophages may contribute to its ability to induce colitis. Gastroenterology. 2009 in press (abstract)
42. Porter CK, Tribble DR, Aliaga PA, et al. Infectious gastroenteritis and risk of developing inflammatory bowel disease. Gastroenterology. 2008;135(3):781–6. [PubMed]
43 *. Issa M, Vijayapal A, Graham MB, et al. Impact of Clostridium difficile on inflammatory bowel disease. Clin Gastroenterol Hepatol. 2007;5:345–351. The role of a traditional triggering pathogen in the pathogenesis of IBD is explored. [PubMed]
44. Kim H, Rhee SH, Pothoulakis C, et al. Inflammation and apoptosis in Clostridium difficile enteritis is mediated by PGE(2) up-regulation of Fas ligand. Gastroenterology. 2007;133:875–886. [PubMed]
45. Rabizadeh S, Rhee KJ, Wu S, et al. Enterotoxigenic Bacteroides fragilis: a potential instigator of colitis. Inflamm Bowel Dis. 2007;13:1475–1483. [PMC free article] [PubMed]
46. Veijola L, Nilsson I, Halme L, et al. Detection of Helicobacter species in chronic liver disease and chronic inflammatory bowel disease. Ann Med. 2007;39(7):554–60. [PubMed]
47. Lupp C, Robertson ML, Wickham ME, et al. Host-mediated inflammation disrupts the intestinal microbiota and promotes the overgrowth of Enterobacteriaceae. Cell Host Microbe. 2007;2:119–129. [PubMed]
48 *. Kotlowski R, Bernstein CN, Sepehri S, Krause DO. High prevalence of Escherichia coli belonging to the B2+D phylogenetic group in inflammatory bowel disease. Gut. 2007;56(5):669–75. This study corroborates findings from previous studies that E. coli are increased in tissues of patients with IBD. A novel finding of this study is the higher prevalence of E. coli from the B2 + D phylogenetic group in these tissues. In addition, serine protease autotransporters were also found to be more prevalent in isolates from patients with IBD. [PMC free article] [PubMed]
49 **. Sokol H, Pigneur B, Watterlot L, et al. Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients. Proc Natl Acad Sci U S A. 2008;105(43):16731–6. F. prausnitzii decreased IL-12 and IFN-γ production in stimulated peripheral blood mononuclear cells, and reduced the severity of TNBS colitis after being fed to mice. These results suggest that counterbalancing dysbiosis using F. prausnitzii could be a means of reducing inflammation in IBD patients. [PubMed]
50 **. Swidsinski A, Loening-Baucke V, Vaneechoutte M, Doerffel Y. Active Crohn’s disease and ulcerative colitis can be specifically diagnosed and monitored based on the biostructure of the fecal flora. Inflamm Bowel Dis. 2008;14(2):147–61. In this study, FISH was used to compare the MAM of patients with IBD and healthy controls. F. prausnitzii were found to be depleted in the tissues of CD patients and increased in the tissues of UC patients. [PubMed]
51 *. Andoh A, Sakata S, Koizumi Y, et al. Terminal restriction fragment length polymorphism analysis of the diversity of fecal microbiota in patients with ulcerative colitis. Inflamm Bowel Dis. 2007;13(8):955–62. Dysbiosis was seen in the feces of patients with UC using T-RFLP. [PubMed]
52. Takaishi H, Matsuki T, Nakazawa A, et al. Imbalance in intestinal microflora constitution could be involved in the pathogenesis of inflammatory bowel disease. Int J Med Microbiol. 2008;298(5–6):463–72. [PubMed]
53 *. Dicksved J, Halfvarson J, Rosenquist M, et al. Molecular analysis of the gut microbiota of identical twins with Crohn’s disease. ISME J. 2008;2(7):716–27. This study is novel in that it analyzes the microbiotas of monozygotic twin pairs with and without CD. [PubMed]
54 *. Nishikawa J, Kudo T, Sakata S, et al. Diversity of mucosa-associated microbiota in active and inactive ulcerative colitis. Scand J Gastroenterol. 2008;30:1–7. Using T-RFLP, this study demonstrated in a small cohort that the MAM of patients with active UC differs from that of patients with inactive disease. Microbial compositions were more diverse in patients with inactive UC when compared to subjects with active disease. [PubMed]
55 *. Ott SJ, Plamondon S, Hart A, et al. Dynamics of the mucosa-associated flora in ulcerative colitis patients during remission and clinical relapse. J Clin Microbiol. 2008;46(10):3510–3. Patients with UC were found to have less biodiversity in their MAM than healthy controls. [PMC free article] [PubMed]
56 *. Martinez C, Antolin M, Santos J, et al. Unstable composition of the fecal microbiota in ulcerative colitis during clinical remission. Am J Gastroenterol. 2008;103(3):643–8. PCR and DGGE revealed that patients with UC who do not have active disease demonstrate low biodiversity and temporal instability in their fecal microbiota. [PubMed]
57. Zhang M, Liu B, Zhang Y, et al. Structural shifts of mucosa-associated Lactobacilli and Clostridium leptum subgroup in patients with ulcerative colitis. J Clin Microbiol. 2007;45(2):496–500. [PMC free article] [PubMed]
58. Vasquez N, Mangin I, Lepage P, et al. Patchy distribution of mucosal lesions in ileal Crohn’s disease is not linked to differences in the dominant mucosa-associated bacteria: a study using fluorescence in situ hybridization and temporal temperature gradient gel electrophoresis. Inflamm Bowel Dis. 2007;13(6):684–92. [PubMed]
59. Backhed F, Ley RE, Sonnenburg JL, et al. Host-bacterial mutualism in the human intestine. Science. 2005;307(5717):1915–20. [PubMed]
60 *. Kim SC, Tonkonogy SL, Karrasch T, et al. Dual-association of gnotobiotic IL-10-/- mice with 2 nonpathogenic commensal bacteria induces aggressive pancolitis. Inflamm Bowel Dis. 2007;13(12):1457–66. Demonstration that in the setting of immunoregulatory defects, non-pathogenic bacteria have additive effects in causing immune-mediated colitis. [PubMed]
61 *. Keita AV, Salim SY, Jiang T, et al. Increased uptake of non-pathogenic E. coli via the follicle-associated epithelium in longstanding ileal Crohn’s disease. J pathol. 2008;215(2):135–44. This is an important study because it demonstrates that in IBD, typically non-pathogenic bacteria such as E. coli strains K-12 and HB101 can cause inflammation through increased intercellular and transcellular uptake. This was demonstrated by E. coli strains isolated from the tissue of CD patients, but not in strains isolated from UC patient specimens. [PubMed]
62 *. Roediger WE. Review article: nitric oxide from dysbiotic bacterial respiration of nitrate in the pathogenesis and as a target for therapy of ulcerative colitis. Aliment Pharmacol Ther. 2008;27(7):531–41. This review summarizes novel studies investigating metabolic properties of bacteria that may play key roles in the pathogenesis of UC, which should stimulate further investigations. [PubMed]
63. Sartor RB. Therapeutic manipulation of the enteric microflora in inflammatory bowel diseases: antibiotics, probiotics, and prebiotics. Gastroenterology. 2004;126(6):1620–33. [PubMed]
64 *. Boirivant M, Strober W. The mechanism of action of probiotics. Current Opinion in Gastroenterology. 2007;23(6):679–692. This review offers background on the use of probiotics, the mechanisms of action of probiotics, and the effects of probiotics on epithelial barrier function, lymphoid cells, development of regulatory cells, inflammatory responses and IBD. [PubMed]
65. Sheil B, Shanahan F, O’Mahony L. Probiotic effects on inflammatory bowel disease. J Nutr. 2007;137:S819–S824. [PubMed]
66. Hedin C, Whelan K, Lindsay JO. Evidence for the use of probiotics and prebiotics in inflammatory bowel disease: a review of clinical trials. Proc Nutr Soc. 2007;66:307–315. [PubMed]
67 *. Isaacs K, Herfarth H. Role of probiotic therapy in IBD. Inflammatory Bowel Diseases. 2008;14(11):1597–1605. This is an excellent, comprehensive review of past clinical trials investigating the use of probiotics to treat CD, UC, and pouchitis. [PubMed]
68. Peran L, Camuesco D, Comalada M, et al. A comparative study of the preventative effects exerted by three probiotics, Bifidobacterium lactis, Lactobacillus casei and Lactobacillus acidophilus, in the TNBS model of rat colitis. J Appl Microbiol. 2007;103(4):836–44. [PubMed]
69. Llopis M, Antolin M, Carol M, et al. Lactobacillus casei downregulates commensals’ inflammatory signals in Crohn’s disease mucosa. Inflamm Bowel Dis. 2009;15(2):275–83. [PubMed]
70 *. Kim N, Kunisawa J, Kweon MN, et al. Oral feeding of Bifidobacterium bifidum (BGN4) prevents CD4(+) CD45RB(high) T cell-mediated inflammatory bowel disease by inhibition of disordered T cell activation. Clin Immunol. 2007;123(1):30–9. This study identifies a probiotic that can inhibit disordered T cell activation in mice. In humans, dysregulation of T cell activation is an important component of IBD. [PubMed]
71 *. Carroll IM, Andrus JM, Bruno-Barcena JM, et al. Anti-inflammatory properties of Lactobacillus gasseri expressing manganese superoxide dismutase using the interleukin 10-deficient mouse model of colitis. Am J Physiol Gastrointest Liver Physiol. 2007;293(4):G729–38. This study demonstrates the intricate connection between regulation of the immune system and metabolic properties of commensal bacteria. [PubMed]
72 *. Lee JH, Lee B, Lee HS, et al. Lactobacillus suntoryeus inhibits pro-inflammatory cytokine expression and TLR-4-linked NF-kappaB activation in experimental colitis. Int J Colorectal Dis. 2009;24(2):231–7. This study investigates the effects of probiotics on the NF-κB (an important transcription regulator) pathway, which is intimately linked to inflammation. [PubMed]
73. Imaoka A, Shima T, Kato K, et al. Anti-inflammatory activity of probiotic Bifidobacterium: Enhancement of IL-10 production in peripheral blood mononuclear cells from ulcerative colitis patients and inhibition of IL-8 secretion in HT-29 cells. World J Gastroenterol. 2008;14(16):2511–6. [PMC free article] [PubMed]
74 *. Lin YP, Thibodeaux CH, Pena JA, et al. Probiotic Lactobacillus reuteri suppresses proinflammatory cytokines via c-Jun. Inflamm Bowel Dis. 2008;14(8):1068–83. This study demonstrates that secreted factors of probiotics can possess immunoregulatory capabilities. [PubMed]
75. Gionchetti P, Rizzello F, Venturi A, et al. Oral bacteriotherapy as maintenance treatment in patients with chronic pouchitis: a double-blind, placebo-controlled trial. Gastroenterology. 2000;119(2):305–9. [PubMed]
76. Gionchetti P, Rizzello F, Helwig U, et al. Prophylaxis of pouchitis onset with probiotic therapy: a double-blind, placebo-controlled trial. Gastroenterology. 2003;124(5):1202–9. [PubMed]
77. Fitzpatrick LR, Hertzog KL, Quatse AL, et al. Effects of the probiotic formulation VSL#3 on colitis in weanling rats. J Pediatr Gastroenterol Nutr. 2007;44(5):561–70. [PubMed]
78 *. Huynh HQ, Debruyn J, Guan L, et al. Probiotic preparation VSL#3 induces remission in children with mild to moderate acute ulcerative colitis: A pilot study. Inflamm Bowel Dis. 2009 (epub ahead of print.) The probiotic preparation VSL#3 induced remission in children with UC in a small clinical trial. [PubMed]
79 *. Pronio A, Montesani C, Butteroni C, et al. Probiotic administration in patients with ileal pouch-anal anastomosis for ulcerative colitis is associated with expansion of mucosal regulatory cells. Inflamm Bowel Dis. 2008;14(5):662–8. VSL#3 was found to treat pouchitis in this open-label clinical trial involving 31 patients. [PubMed]
80 *. Fujimori S, Tatsuguchi A, Gudis K, et al. High dose antibiotic and prebiotic cotherapy for remission induction of active Crohn’s disease. J Gastroenterol Hepatol. 2007;22(8):1199–204. In a small cohort, this study showed that high-dose probiotics are well-tolerated by human subjects, and that two components of the much-studied probiotic preparation VSL#3 may hold promise for inducing remission in UC patients, even in a subset that has failed other treatments. [PubMed]
81 *. Raz I, Gollop N, Polak-Charcon S, Schwartz B. Isolation and characterization of new putative probiotic bacteria from human colonic flora. Br J Nutr. 2007;97(4):725–34. Enterococcus durans was found to reduce inflammation in Balb/c mice in the DSS model of colitis. More importantly, this study emphasizes the importance of characterizing various commensal bacteria in the hope of discovering a novel probiotic species to treat UC or CD. [PubMed]
82. Van Gossum A, Dewit O, Louis E, et al. Multicenter randomized-controlled clinical trial of probiotics (Lactobacillus johnsonii, LA1) on early endoscopic recurrence of Crohn’s disease after ileo-caecal resection. Inflamm Bowel Dis. 2007;13(2):135–42. [PubMed]