The aetiology and pathogenesis of pouchitis is still unclear although ulcerative colitis and pouchitis share many similarities.66,67,68,69
This study investigated the influence of the probiotic agent VSL#3 on the bacterial microbiota in a placebo controlled clinical trial52
which was designed to assess the value of VSL#3 in maintaining antibiotic induced remission in patients with pouchitis.
In pouchitis, bacterial overgrowth and a decrease in beneficial bacteria, in particular lactobacilli and bifidobacteria, in comparison with conventional ileostomy flora, has been described.69,70,71
It appears likely that faecal stasis and immune stimulation by stool bacteria play an important role in the development of pouch inflammation. The mucosa associated flora is probably of immediate relevance to the disease process28,35
and differs significantly from the composition of the ileal and midstream/faecal flora.29,72
Morphological data from FISH analysis indicate that normal Enterobacteriaceae
species (mainly E coli
) are found in uninflamed epithelium in ileoanal pouch biopsies (that is, from pouchitis patients who are in remission) in contrast with the normal ileum and colon of healthy volunteers in which no E coli
was found within the epithelium.28
When recurrent pouch inflammation occurred during placebo treatment, low bacterial diversity and a marked reduction in intramucosal Enterobacteriaceae
species were found. The increase in Enterobacteriaceae
within the mucosa during VSL#3 therapy indicates that remission maintenance under probiotics is associated with restoration of parts of the normal pouch flora.
For other relevant groups of intestinal bacteria, such as Bacteroides/Prevotella or the Lactobacilli/Bifidobacteria group, only sporadic signals were detected by FISH. The limited amount of mucosal biopsy material as well as the confined sensitivity of FISH analysis for single bacteria prompted us to complement the FISH results with PCR based molecular techniques.
The FISH results were supported by diversity analysis with SSCP and, at least in part, by taxa specific clone libraries. Successful maintenance of remission after VSL#3 treatment appeared to result in higher diversity of bacterial species that included members of the normal anaerobic enteric flora and not merely colonisation with strains contained only in VSL#3. With the exception of the Bacteroides/Prevotella group, the richness of bacteria was increased in the VSL#3 group compared with pretreatment levels. As diversity was increased during VSL#3 maintained remission, we suggest that this effect may be a component of the therapeutic mechanism of probiotics. The suggestion that low bacterial diversity could be an important mechanism for mucosal inflammation is supported by earlier investigations56
in which mucosal inflammation in IBD was associated with loss of normal anaerobic bacteria such as Bacteroides
species, and Lactobacillus
Restoration of Enterobacteriaceae
species in the mucosa and the increase in bacterial diversity are observed in the setting of the clinical trial, which demonstrated successful remission maintenance by VSL#3.52
The effects were observed on the specific background of remission induction with antibiotics. Antibiotics lead to significant alteration of the enteric bacterial balance. In neonates, antibiotic treatment induces complete eradication of Lactobacillus
species together with a marked reduction in colonic total aerobic and anaerobic bacteria, in particular Enterobacteriaceae
The net effect of antimicrobial therapy therefore results in (i) a decrease in the total number of bacterial cells and (ii) a reduction in specific bacterial populations. Colonisation of the mucosa by Enterobacteriaceae
species and diversification of Lactobacillus
species and Bifidobacterium
species in the VSL#3 group might be facilitated by eradication of complex populations by the preceding antibiotic therapy and the diminished competition by other dominant bacterial consortia, such as Enterobacteriaceae
. Therefore, expansion and diversification of the bacterial microbiota observed in the VSL#3 group could be specific to pretreatment with antibiotics on the background of the dysbiotic flora in IBD.56,74
In fact, this may also be the case for the clinical efficacy of the treatment in pouchitis.52
It is well documented that pouchitis is associated with loss of beneficial intestinal bacteria, especially the subgroups lactobacilli and bifidobacteria.69,70,71
We observed, as a net effect of VSL#3 associated remission, recolonisation and diversification of the potentially beneficial Lactobacillus and Bifidobacterium flora. As most patients in the placebo group relapsed and almost all patients in the VSL#3 group remained in remission, the specific effects of VSL#3 and the effect of an inflammation free mucosa interact and therefore cannot be separated in this study. Additional therapeutic mechanisms induced by VSL#3 treatment are possible that may be either primary or secondary to alteration of the constitution of the pouch microbiota. Specific induction of anti‐inflammatory cytokines by probiotics could decrease the inflammatory activity in the mucosa and thereby provide better growth conditions for diversification of the flora.11
Rachmilewitz et al
demonstrated in Toll‐like receptor and MyD88 deficient mice that the immune stimulatory effect of probiotic bacteria isolated from the VSL#3 compound was dependent on Toll‐like receptor 9 signalling.75
Therefore, direct interaction of probiotic bacteria with mucosal immunoregulation could either induce anti‐inflammatory pathways11
or directly interact with innate immune mechanisms75
as the primary mechanisms altering the growth conditions of the mucosal microbiota, or could be secondary to changes in microbial constitution and diversity. The significant difference between the placebo and the VLS#3 groups indicates that VSL#3 induced remission is associated with reconstruction of the intestinal flora, but investigation of the hierarchy of mechanistic events has to await further exploration in model systems.
Fungi are important elements of the human flora. However, little is known about the composition of comensal fungal intestinal species. Fungal microbiotic diversity was inversely related to bacterial diversity. However, it has to be pointed out that fungal species represent only a small fraction of the total microbiota. Immunocompetent hosts have an efficient phagocytic capacity to prevent fungal invasion.76
It can be hypothesised that alteration in the balance of bacterial and fungal species in the mucosal flora reflects a metabolic dysbalance of the complex microbial ecosystem with pathophysiological consequences for the mucosal barrier. Most importantly, restored balance between bacterial and fungal diversity was seen in VSL#3 treated patients. The question of whether the increase in fungal diversity is a direct result of higher bacterial diversity or an independent parallel effect remains unclear. Although fungi are normal members of the intestinal microbiota to a certain extent, fungal overgrowth is a typical complication of any bacterial imbalance following antibiotic therapy or a specific dietary regimen, as for example in intensive care units. Most likely the fungal overgrowth seen here was a direct consequence of bacterial dysbalance supported by diminished control mechanisms and reduced competition through the total bacterial microbiota. Studies on the mutual dependencies of the different parts of the intestinal microbiota are urgently needed to clarify this point.
Our study demonstrated that VSL#3 maintained remission was accompanied by a higher bacterial and a reduced fungal diversity in comparison with placebo treatment. The increase in bacterial diversity was not caused by colonisation with bacterial strains contained in VSL#3 but represents an independent effect. It is not unclear at present whether the anti‐inflammatory and immunoregulatory effects of probiotics are primary or secondary to induction of changes in the diversity of the mucosal microbiota. Increase in bacterial diversity may be a therapeutic mechanism for the probiotic mixture VSL#3 in maintenance of antibiotic induced remission in pouchitis. Methods for manipulating the complex microecology of the mucosal flora may be an important field for future therapeutic developments, especially addressing the secondary or primary prevention of pouchitis.