Mycobacterial infection has been postulated in the aetiology of Crohn’s disease since its first description in 1913. The association stems from the observed similarity between Crohn’s disease and the bovine condition Johne’s disease, a condition caused by Mycobacterium avium paratuberculosis (MAP) infection leading to granulomatous enterocolitis. There have been vast numbers of studies in this area but the role of MAP remains uncertain[50,82,83].

MAP can be widely isolated from meat, dairy products and water, indicating sources of infection and supporting its role[82,83]. However, a large study found a lack of epidemiological support for environmental exposure[84]. Over the last decade research into the prevalence of MAP in IBD patients has been inconclusive. A large number of researchers have successfully shown a higher prevalence of MAP in Crohn’s patients compared to controls but, it seems for each of these there has been an equivalent study yielding no association[85-97].

In support of its role the ability of MAP to invade gut epithelial cells, inducing tissue damage and inflammation, has been shown[98]. A dominant T-cell response to MAP has also been seen in CD patients and macrophages infected with viable MAP are associated with high production of tumour necrosis factor-alpha (TNF-α), a marker for CD[95,99,100]. Using mouse models, MAP has been found to induce full-thickness necrotizing colitis after subcutaneous and transluminal injection[101].

The discovered association between CD and the autophagy gene ATG16L1 lends further credence to the theory as it is known that autophagy is essential for inhibition of mycobacterium tuberculosis in infected macrophages[102,103]. Defective innate immune killing mechanisms in patients with NOD2 polymorphisms at first also seem to support the idea, and indeed it has been found that monocytes heterozygous for a NOD2 polymorphism are more permissive to the growth of MAP[104]. Beyond contemplation, however, evidence for this hypothesis is limited. MAP has been detected most commonly in colonic disease; this is in direct contrast with the prevalence of NOD2 mutation in ileal disease[105,106]. In fact a study directly looking at the relationship between NOD-2 and MAP serology found no association[107]. Combining this with the response of CD to immunosuppressant and anti-TNF therapy, known to cause MAP proliferation and a lack of success of anti-mycobacterial therapy, the role of MAP is clearly still in doubt[108].

Helicobacters, as human gastrointestinal pathogens, have assumed great research interest since the discovery by Robin Warren and Barry Marshall of Helicobacter pylori as the infectious agent in gastric and duodenal ulceration. Also, similar to MAP, one of the main prompters of research into the role of Helicobacter in IBD has been their propensity to cause colitis in animal models like Cotton-top tamarin monkeys (Saguinus oedipus). Despite this, there has been a lack of success in the last decade establishing presence of Helicobacter in IBD patients. The findings of studies looking into the molecular evidence of Helicobacter presence are varied and studies aimed at culturing viable Helicobacter from IBD tissue have failed[109-121]. Interestingly the only seemingly universally accepted action of Helicobacter in IBD is the apparent protective effect of Helicobacter pylori which has convincingly been found to be negatively correlated with IBD[113,118-122]. This may conform to the “hygiene hypothesis” for the development of IBD[123].

The evidence for an association is much stronger with enterohepatic Helicobacter species. Non-pylori Helicobacter organisms have been shown to induce colitis in a number of rodent models; Helicobacter hepaticus and Helicobacter bilis (H. bilis) most prominently but, also Helicobacter trogontum, Helicobacter rodentium and Helicobacter typhlonius with cytokine patterns which were very similar to that of human IBD[124-128]. When studying the response to H. bilis, Jergens et al[126] showed that there was an IgG mediated response to the microbiota prior to the development of colitis, suggesting the ability of H. bilis to induce the hosts immune response to commensal bacteria, leading to the observed immune-mediated intestinal inflammation of IBD. In human subjects, enterohepatic Helicobacter species prevalence was significantly higher in colonic biopsy samples from patients with UC group compared to control subjects[118].

Campylobacter is a relatively new and important player in IBD. Unlike the other IBD suspects, Campylobacter does not have a suitable animal disease model; instead interest stems from the recognition of Campylobacter jejuni (C. jejuni) as the leading cause of gastroenteritis worldwide[129].

The role of C. jejuni in human disease has been long recognised and its prevalence in IBD investigated[130]. The main advance in the last decade has been the recognition of the importance of non-jejuni Campylobacter as human pathogens. Zhang et al[131] found a higher prevalence of Campylobacter concisus (C. concisus) DNA and IgG levels in newly diagnosed paediatric patients with Crohn’s disease, even managing to culture C. concisus from a biopsy sample, indicating viability. Another study using faecal samples from newly diagnosed CD patients also found a significant association with C. concisus, 35 of 54 CD patients testing positive and only 11 of 33 healthy controls. This study also found that C. hominis was present in 13% of Crohn’s samples, Campylobacter ureolyticus in 9%, Campylobacter showae (C. showae) in 4%, Campylobacter gracilis (C. gracilis) in 2% and C. rectus in 2%. Interestingly C. gracilis, Campylobacter rectus and C. showae were only detected in patient samples[132]. Similar results have been obtained in a number of studies in adult patients[119,133-136]. Mahendran et al[134] also showed an increased prevalence in UC, a finding supported by Mukhopadhya et al[135] who found C. concisus DNA in biopsy samples in 23/69 (33.3%) of UC patients compared to 7/65 (10.8%) of controls. This study also found C. ureolyticus to be in higher prevalence in UC patients. The most recent study found that although Campylobacter appear to be surprisingly common, with positive PCR in 33/44 IBD patients and 32/42 controls, there was no association with IBD[119]. A dominant serological antibody response to C. concisus has been documented in IBD patients indicating the prevalence of infection[137,138]. Specifically CD patients have been shown to recognise flagellin B, ATP synthase F α subunit and outer membrane protein 18 of C. concisus[137].

The origins of Campylobacter have led to a few researchers looking into the risks of developing IBD after acute gastroenteritis. A long term study published in 2009 documents the risk of developing IBD after acute infection with Campylobacter (C. jejuni) or Salmonella[130]. The findings indicated a significant increased risk in the exposed group for subsequently developing IBD, which has been supported by similar studies[139-141].

The pathogenesis of C. jejuni had been fairly well established prior to the last decade. C. jejuni has been used to induce colitis in rodent models and previous exposure correlated with disease severity[142]. The ability of C. jejuni to attach and invade the gut epithelium is well documented[143]. The newest discovery has been that C. jejuni can promote translocation of commensal luminal bacteria. This is a natural process thought to be essential for immunological tolerance and mucosal surveillance in the GI tract. Up regulation could affect the normal mucosal response to the intestinal microbiota leading to the chronic immune-mediated intestinal inflammation of IBD[144]

A number of studies have demonstrated the ability of C. concisus to colonise and adhere to intestinal epithelial cells, causing cell damage and microvillus degradation[146]. Man et al[145] comprehensively described the method of C. concisus attachment and invasion. They showed C. concisus to attach to the intracellular junction, disrupting barrier function - increasing permeability by causing a loss of tight junction proteins and decreasing transepithelial electrical resistance and to invade by a process mediated by polar flagellum[146]. Other non-jejuni Campylobacters have also been shown to be invasive and induce pro-inflammatory cytokines as well as producing a number of virulence factors such as haemolysins, cytolethal distending toxin and zonula occludens toxin[129,146-150]. These mechanisms could have an important bearing when one considers a causative role for this group of pathogens in IBD.

A specific pathogenetic group of Escherichia coli (E. coli), adherent-invasive E. coli (AIEC) have recently been extensively implicated in human IBD and are currently one of the most exciting players in the pathogen story. This group are characterised by their ability to adhere and invade epithelial cells using actin microfilaments and microtubule recruitment. AIEC strains have been shown to be the cause of granulomatous colitis in boxer dogs and to induce granulomas, similar to early epithelioid granulomas, in vitro[151-153]. Similarly to the previously discussed bacteria, they have been documented to induce colitis in infected animals.

There is a growing body of evidence supporting the prevalence of AIEC in human disease. A number of studies initially showed a disproportionate increase in Enterobacteria as a whole[36,47]. When looking at AIEC organisms specifically, Darfeuille-Michaud et al[154] found them to be more prevalent in ileal Crohn’s lesion tissue (36.4%) then controls (6.2%). This study also found that AIEC seemed to be rarely found in colonic tissue with 3.7% detected from Crohn’s patients and 1.9% from controls, and none in UC specimens. This suggests a specific association of AIEC with ileal Crohn’s. The findings of this initial study have been backed up by many researchers obtaining similar results[28,155-161]. Additionally, antibodies to the E. coli membrane protein C and the CD associated bacterial sequence I2 have been shown to not only be more prevalent in CD but also to be associated with more severe disease, with small bowel involvement, faster disease progression and increased need for surgical intervention[157,162].

The mechanism by which AIEC might induce colitis has been fairly well established towards the end of the decade. AIEC have type one pili and flagella that can bind to host adhesion receptor carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6)[163,164]. CEACAM6 has been shown to be more highly expressed in ileal CD tissue, to be increased after-γ or TNF-α stimulation and to be upregulated by AIEC itself[163,164]. AIEC have also been shown to possess long polar fimbriae and so can cross the mucosal barrier to access lymphoid cells[165]. They can then invade macrophages without inducing cell death, allowing them to replicate and continuously activate immune cells, triggering TNF-alpha release and granuloma formation, which are hallmarks of Crohn’s disease. In fact the use of TNF-alpha antibodies has been shown to decrease the number of intramacrophagic bacteria, relating this to the success of anti-TNF therapy and further supporting the role of AIEC[166].

In the last decade, there has been renewed focus in studying the role of various other bacterial strains in the aetiopathogenesis of IBD as well. The role of Fusobacterium was studied in mucosal biopsies of patients with IBD and was found to be significant compared to controls in a number of studies prior to this review period. More recently seropositivity to Fusobacterium varium (F. varium) infection was found to be higher in UC patients as opposed to controls with increased severity of disease in seropositive UC patients[167]. Further study has revealed the ability of F. varium to adhere to and invade colonic epithelial cells, increasing IL-8 and TNF-α secretion, providing a mechanism whereby F. varium infection may induce inflammation similar to that seen in IBD[168].

A similar association has also been found with Klebsiella infection, with Anti-Klebsiella antibodies found more commonly in IBD patients than in controls with the bacteria being implicated in disease relapses[169]. Klebsiella pneumoniae has recently been shown to increase the severity of colitis in mouse models, increasing COX-2, IL-1β, IL-6 and TNF-α expression and reducing tight junction associated proteins[170]. Colitis has been shown to be induced even in wild type mice highlighting the high pathogenic potential of this bacteria[171].

The role of Salmonella infection has been postulated from numerous studies that have documented the risk of developing IBD after acute Salmonella gastroenteritis[130,139-141]. When searching for a mechanism it has been found that a Salmonella virulence factor, the invasion-associated type Darfeuille-Michaud secretion system induces inflammation by activation of the NOD1/NOD2 signalling pathways[172]. This ties Salmonella nicely to the growing body of research into the genetics of IBD, supporting the role of the pathogen.

The potential role of Yersinia was proposed at the beginning of the last decade based upon the observed parallel increase in IBD and refrigeration, “the cold chain hypothesis”[173]. Similarly to Salmonella and Campylobacter there has been evidence that acute Y. enterocolitica infection increases the short and long term risk of developing IBD[174]. Despite some later successes in isolation Yersinia from IBD patients there has yet to be a compelling body of evidence coupling the prevalence of Yersinia with established IBD[175-177].

Probiotics are beneficial microorganisms that, when ingested, may influence the gut microbiota composition, metabolic activity and immunomodulation to confer benefit to the host[178]. They can alter microbial diversity through competitive inhibition of other microbes, increase mucosal barrier function through the production of short chain fatty acids (SCFA) and interact with intestinal DC to stimulate an anti-inflammatory response[179-182]. These probiotic strains must be of human origin, be non-pathogenic and have the intrinsic ability to survive the gastrointestinal transit in order to confer maximal benefit[183]. The most common probiotics used in the treatment of IBD have been Lactobacillus sp, Bifidobacterium sp, Sacchromyces bouladrii, E. coli Nissle 1917 and the probiotic combination VSL#3[184-191]. The strongest indication for the use of probiotics in IBD has been in the treatment of pouchitis in the post-operative setting in UC patients[192]. E. coli Nissle 1917 and VSL#3 have been found to be effective in preventing relapse and inducing remission in this setting[193-198]. The data is not very robust with respect to the role of probiotics maintaining remission in UC and a recent Cochrane Database System Review has not recommended its use[197]. The current body of evidence does not show any demonstrable benefit in patients with CD[198,199].

Prebiotics are non-digestible oligosaccharides that are selectively fermented in the colon into SCFAs and can alter microbial composition and activity and confer benefit to the host[200]. Examples include inulin, fructooligosaccharide (FOS), galactooligosaccharides and lactulose[201-206]. Prebiotics can selectively stimulate the growth of certain probiotics such as Lactobacillus and Bifidobacterium, decrease intraluminal pH and increase the production of SCFA, such as acetate and butyrate, which play an important role in epithelial and DC function[207,208]. SCFAs have also been found to have an anti-inflammatory effect[209]. In an open labelled trial FOSuse decreased the disease activity index in patients with active CD and resulted in increased faecal Bifidobacterium, but this benefit was not demonstrated in a subsequent randomized placebo controlled trial[205,206]. Another prebiotic inulin showed some promise in a randomized controlled trial in patients with UC and was also found to decrease inflammation in patients with pouchitis[202,203]. A couple of studies have also found a potential role of germinated barley foodstuff in maintaining remission in patients with active UC[210,211]. Lastly, some benefit was also accrued with the use of Ispaghula husk in patients with UC[212]. The important studies and their brief outcomes are summarized in Table 2.

A couple of recent meta-analyses on antibiotics in IBD found that the use of antibiotics improved clinical outcomes of patients with IBD[213,214]. There is evidence that metronidazole and ciprofloxacin are useful in the treatment of CD and pouchitits[215,216]. Support for the use of antibiotics as the primary treatment in UC is less convincing, however, there are some studies which suggest that rifaximin and ciprofloxacin could be useful as an adjunctive treatment for UC[213]. The mechanisms through which antibiotics are thought to benefit patients with CD are through the inhibition of pathogenic bacteria or through reducing overall bacterial numbers. The main issues with antibiotic treatment include lack of understanding of which bacteria may be involved in the initiation of inflammation, lack of specificity and the potential for antibiotic resistance. There have been several trials in the past studying the specific role of anti-mycobacterials in the treatment of CD and this has been summarized in a European consensus document which has deemed the futility of such treatment[217].

Faecal transplantation or faecal microbial therapy (FMT) as it is more commonly known is a technique in which stool is taken from a healthy surrogate and inserted into an unhealthy person, with curative intent[218]. The origins of faecal transplantation as a method of treating enteric pathology can be traced back for more than two millennia, when it was used as a traditional Chinese medicine to treat diarrhoea[219].

In recent times, FMT is perhaps best known for its potential role in treating Clostridium difficile (C. difficile) infectious diarrhoea[220]. After donor-faeces infusion in a group of patients infected with C. difficile, there was an alteration in the gut microflora with an increased faecal bacterial diversity, similar to that in healthy donors, with an increase in Bacteroidetes species and Clostridium clusters and a decrease in Proteobacteria species. This therapeutic benefit by FMT as documented in this trial, would theoretically have a beneficial effect in patients with IBD as well. The use of this form of intervention is still restricted to few exploratory trials. Donor faecal enemas were given to a group of ten children over five days with moderate to severe UC and resulted in 78% clinical response after a week and 67% with sustained response after a month in the nine children who could tolerate the treatment[221]. This was similarly documented in a subset of six adult UC patients who were treated over a period of 5 d. Complete reversal of symptoms was achieved in all patients by 4 mo, by which time all other UC medications had been ceased and at 1 to 13 years post FMT and without any UC medication, there was no clinical, colonoscopic, or histologic evidence of UC in any patient[222]. However, a single infusion in six adult patients with severe UC did not have a similar beneficial effect and the faecal microbiota changed to the donor phenotype in only 50% of those treated, suggesting that as opposed to treatment of C. difficile a prolonged treatment is indicated in IBD[223]. Although the data described above is certainly promising, there is clearly a need to move on from individual case reports and conduct more large scale randomised control trials before any benefit of FMT can be claimed with any certainty. Some concerns have also been raised regarding safety and side effects, with some IBD patients suffering mild side effects following FMT, and the obvious issues surrounding potential transmission of host infectious disease[224]. The efficacies of different administration techniques and dosing regimens for FMT also need to be refined and investigated. Literature to date describes a range of methods including colonoscopy, duodenal or gastric tubes and self-administered enema yet to date there is no clear evidence to support one method over any other. There is no doubt that manipulation of the gut microbiota could have enormous therapeutic potential and FMT will play an important role in its future.