Ileal Pouch–Anal Anastomosis and Pouchitis: The Role of the Microbiota in the Pathogenesis and Therapy

Inflammatory bowel diseases, Crohn’s disease and ulcerative colitis, are life-long disorders characterized by the chronic relapsing inflammation of the gastrointestinal tract with the intermittent need for escalation treatment and, eventually, even surgery. The total proctocolectomy with ileal pouch–anal anastomosis (IPAA) is the surgical intervention of choice in subjects affected by ulcerative colitis (UC). Although IPAA provides satisfactory functional outcomes, it can be susceptible to some complications, including pouchitis as the most common. Furthermore, 10–20% of the pouchitis may develop into chronic pouchitis. The etiology of pouchitis is mostly unclear. However, the efficacy of antibiotics in pouchitis suggests that the dysbiosis of the IPAA microbiota plays an important role in its pathogenesis. We aimed to review the role of the microbiota in the pathogenesis and as a target therapy in subjects who develop pouchitis after undergoing the surgical intervention of total proctocolectomy with IPAA reconstruction.


Introduction
In humans, a wide number of different microbial species are located in the bowel, which hosts several trillion microbial cells [1]. This ensemble of microbial species is generally called gut microbiota [2]. There is a profound interplay between the gut microbiota and the human biology [3]. Indeed, the gut microbiota is important for various physiological functions such as eliciting immune maturation [4], defending against pathogens colonization and overgrowth [5], influencing epithelial proliferation [6] and intestinal vascular density [7], modifying bile acids in the large bowel [8], promoting metabolic homeostasis [9] and hormone modulation [10], synthesizing vitamins [11] and neurotransmitters [12], supplying energy [1], and regulating bone metabolism [13]. The intestinal microbiota of a healthy subject is composed predominantly by Bacteroidetes and Firmicutes, with also other smaller sections comprised by Actinobacteria, Proteobacteria, Verrucomicrobia, methanogenic archaea, Eucarya, and various phages [1]. Modifications in the constitution and function of gut microbes lead to dysbiosis [1], and several diseases are associated with gut dysbiosis [1,14]. In particular, intestinal dysbiosis is an important feature of inflammatory bowel diseases (IBD) [14,15], playing a crucial role in the onset of the disease in predisposed subjects [16]. Indeed, some important alterations of intestinal microbiota

Methods
We performed a literature search in the PubMed database. The key words used were: "ileal pouch-anal anastomosis", "pouchitis", "microbiota", and "dysbiosis". Furthermore, references of original articles and relevant reviews were screened to find further publications.

IPAA Microbiota Evolution over Time
Although derived from the small intestinal tissue, the microbiota of the IPAA changes over time into a microbiota with a colonic profile [39][40][41][42]. These modifications can arise as early as two months after surgery and achieve a more stable composition as the years go by after the creation of the IPAA [40,42]. Clostridium coccoides, Clostridium leptum, Bacteroides fragilis, Atopobium, E. coli, Klebsiella, Veillonella, Staphylococcus (coag-), and Enterobacter are the more counted bacterial species in functional IPAA [40,41,43]. In particular, it seems that the microbiota of healthy IPAAs try to recover to a composition comparable to that observed prior to surgery [43], and it has been hypothesized that the presence of Veillonella, Lachnospiraceae, Ruminococcus gnavus, and clostridial cluster IV (i.e., Faecalibacterium prausnitzii) might be a marker of regularity of the IPAA flora [37,43,44]. Furthermore, a comparison between the microbiota of IPAA in subjects with UC and subjects with familial adenomatous polyposis (FAP), which exhibit a low incidence of pouch inflammation, might help to understand the microbial families potentially implicated in the pathogenesis of pouchitis [37]. Indeed, a higher presence of sulfate-reducing bacteria (SRB) in UC-IPAA has been observed compared to FAP-IPAA [45,46]. SRB produce hydrogen-sulfide, which inhibits butyrate oxidation and prevents its utilization by the intestinal epithelial cells, potentially resulting in the damage of the mucosa of IPAA [37,46]. Other findings confirm the presence of differences between UC-IPAA and FAP-IPAA observing less bacterial diversity, an increased proportion of Proteobacteria, and decreased levels of Bacteroidetes and Faecalibacterium prausnitzii in the UC-IPAA group [47,48]. Table 1 shows the most common microorganisms and the main differences between healthy adults, IBD patients, UC-IPAA, and FAP-IPAA patients.

IPAA Microbiota and Pouchitis
It has been shown that patients with IPAA experiencing pouchitis exhibit a lower bacterial diversity [47,[49][50][51] with an increased amount of aerobes and a decrease in anaerobes [36,37]. In particular, subjects with pouchitis display decreased levels of Bacteroidetes [50,52,53], Lachnospiraceae [44,49], Ruminococcaceae [49], Streptococci [47,54], and Faecalibacterium [49], while incremented levels of Enterobacteriaceae (including E.Coli) [47,49] Fusobacterium [54,55], and Propionibacterium acnes [56] have been observed. Furthermore, Lim et al. identified seventeen operational taxonomic units that were seen exclusively in subjects with inflamed pouches including Desulfosporosinus, Leptospira, Microcystis, Methylobacter, and Pseudoalteromonas [57], while Pawelka et al. observed that the presence of Staphylococcus aureus correlated with a higher degree of chronic inflammation [58]. Ruminococcaceae and Lachnospiraceae have an important role in preserving the host's health due to their ability to hydrolyze starch and other sugars producing butyrate and other short-chain fatty acids (SCFAs), which are considered the principal nutrients of the colonic epithelial cells [59]. When pouchitis occurs, lower levels of SCFAs have been observed in contrast to uninflamed IPAA [37,60], and antibiotic therapy during pouchitis is associated with an increment of SCFAs [61]. Furthermore, Lachnospiraceae and Ruminococcaceae families are the only few bacteria with the ability to produce secondary bile acids (deoxycholic acid-DCA, lithocholic acid-LCA) through the 7α-hydroxylation of the primary bile acids (PBAs). Indeed, lower levels of Ruminococcaceae and secondary bile acids (SBAs) have been demonstrated in UC-IPAA patients in comparison to FAP-IPAA patients. These findings could partially explain the role of dysbiosis in pouchitis as it is known that SBAs can reduce both acute and chronic colitis in animal models and may be essential in preserving immune homeostasis in the IPAA [48].
Another difference that has been observed between UC-IPAA and FAP-IPAA patients is that the former group displays a higher presence of SRBs, which are known to reduce butyrate oxidation and intestinal nutrients, potentially damaging the IPAA mucosa. Indeed, SRBs have been linked to pouchitis [37,62]. The presence of SRBs may be due to the degradation of sulfomucin by commensal bacteria, resulting in the production of free sulfate and SRB colonization [37]. Additionally, in this case, treatment with antibiotics during pouchitis is associated with a reduction in hydrogen sulfide and SRBs [62]. These studies show that the microbiota undoubtedly plays a role in the inflammation of the IPAA. However, future longitudinal studies in the same subjects could reduce the impact of other factors on microbiota alteration (i.e., diet, drugs) [37,42]. Table 2 summarizes the studies that investigated the microbiota changes in UC-IPAA patients with pouchitis.  Reshef [49] 131 UC-IPAA (83 with pouchitis and 10 with unstable pouch behavior), 9 FAP-IPAA 16S rRNA gene amplicon pyrosequencing SDI was higher in normal UC-IPAA compared with UC-IPAA with pouchitis. Faecalibacterium was reduced in pouchitis compared to normal pouch (P 0.021). Lachnospiraceae and Ruminococcaceae were significantly decreased in pouchitis compared to normal UC-IPAA and FAP-IPAA. Healthy controls displayed a higher microbial biodiversity compared to UC-IPAA (p < 0.001). UC-IPAA with pouchitis showed fewer Eubacterium rectale (a butyrate-producing bacteria) and more Clostridium perfringens compared to UC-IPAA with normal pouch and healthy controls.

Antibiotics
Antibiotics represent the mainstay for the treatment of pouchitis [63] as they can induce remission by 74% in chronic pouchitis [64]. Ciprofloxacin and metronidazole are the first-line recommended antibiotics, although the best modality of treatment is still unclear [65]. A randomized clinical trial showed that both antibiotics produced a reduction in the total Pouchitis Disease Activity Index (PDAI) score in patients with acute pouchitis; however, ciprofloxacin produced a greater reduction in both symptom score and endoscopic score with fewer adverse events compared to metronidazole (0% vs. 33%, respectively) [66]. Furthermore, it has been shown that a combination of the two antibiotics can be effective also in patients with refractory/recurrent pouchitis [67]. The mechanisms implicated in the pouch microbiota's changes after antibiotic therapy that are responsible for their favorable effects are not clearly understood [38]. In the study of Gosselink et al., ciprofloxacin could eradicate pathogens that are significantly increased during pouchitis (Clostridium perfringens, hemolytic Escherichia coli) while not disrupting most of the anaerobic bacteria that contribute to the stability of the IPAA's flora [36]. Subsequently, Dubinsky et al. observed that the effectiveness of antibiotic therapy in pouchitis may be ascribed to the establishment of an intestinal microbiota with non-pathogenic, antibiotic-resistant bacteria with low inflammatory potential. This newly established microbiota may prevent more aggressive inflammatory bacteria from colonizing the pouch [68]. However, within three months after therapy discontinuation, most subjects relapsed, requiring additional antibiotic treatment [68]. Indeed, 7-20% of subjects that experience a first episode of pouchitis will eventually develop chronic pouchitis [69]. Therefore, further interventions following treatment with antibiotics should be contemplated (i.e., probiotics, diet) to support beneficial bacteria and prevent subsequent colonization by pathogenic species [68].

Probiotics
Randomized placebo-controlled trials (RCTs) showed that a probiotic mixture of Lactobacilli (four strains), Bifidobacteria (three strains), and Streptococcus thermophilus was effective in maintaining remission in subjects that suffered previous pouchitis. The treatment was generally well tolerated without significant serious adverse events (only one patient stopped medication complaining of abdominal cramps, vomiting, and diarrhoea) [70,71]. The same probiotic mixture led to increased fecal levels of lactobacilli, bifidobacteria, and Streptococcus salivarius in comparison to patients treated with placebo (p < 0.001) [70]. Similar findings were observed also in the RCT of Mimura et al. [71]. One of the potential mechanisms of the beneficial effect of the probiotic mixture could be its ability to increase tissue levels of the anti-inflammatory interleukin (IL)-10 and to reduce levels of tumor necrosis factor (TNF)-α, interferon-γ, and IL-1α. IL-10 may increase the tolerance of the intestinal immune system to resident pouch bacteria. Nevertheless, other mechanisms are probably involved in the anti-inflammatory effects of probiotics [72]. Indeed, Persborn et al. demonstrated that maintenance treatment with Ecologic 825 (another probiotic mixture containing strains of Lactobacilli and Bifidobacterium) after induction therapy with antibiotics restored the mucosal barrier to E.Coli in subjects with pouchitis [73].
Interestingly, some probiotics have been shown to be effective also as a prophylaxis therapy after surgery, reducing the rate of the first episode of pouchitis [74,75]. Therefore, some Authors suggest prescribing prophylaxis treatment with probiotics in subjects with high-risk factors of pouchitis after surgery (i.e., primary sclerosing cholangitis and extraintestinal manifestations) [69].
In addition to probiotics, other treatments have been studied to rebalance the intestinal flora in patients with IPAA.

Fecal Microbiota Transplantation
Fecal microbiota transplantation (FMT) has been shown to be a successful treatment in other conditions of microbiota alteration, such as recurrent Clostridioides difficile infection [76,77]. As a result, there is increasing interest in the use of FMT to treat pouchitis. In the study of Kousgaard et al., FMT could increase the microbial diversity in subjects with chronic pouchitis and obtain clinical remission in 33% of the patients at 6 months of follow-up [78]. However, a recent systematic review observed that FMT seems ineffective in treating chronic pouchitis [79]. Indeed, two recent randomized controlled trials observed a low efficacy of FMT in chronic pouchitis. Interestingly, the majority of the relapses occurred during or shortly after the completion of FMT [80,81]. Overall, only a few studies have explored the role of FMT in chronic pouchitis so far, exhibiting some pitfalls such as the heterogeneity in study design and type of fecal transplant delivery [38]. Future specifically dedicated RCTs with large sample-sizes and standardized protocols (i.e., disease definitions, type of FMT delivery, dose, or duration) will help to ensure reproducible data and provide higher quality of evidence on the real efficacy of FMT in the treatment of chronic pouchitis [82].

Diet and Prebiotics
Finally, it is important to keep in mind that diet and prebiotics may also help in modifying the gut microbial composition [83,84]. Prebiotics are substrates that are selectively utilized by host microorganisms conferring health benefits [85]. Welters et al. showed that three weeks of dietary supplementation with 24g of inulin increased IPAA butyrate concentration and reduced the inflammation of the pouch mucosa with a significant decrease in both endoscopic and histologic PDAI scores [86]. Fruit consumption may also be protective against pouch inflammation. Ianco et al. observed that a decreased consumption in fruit and vegetables may be associated with pouchitis [87]. Accordingly, in the prospective study of Godny et al. the reduction in fruit consumption over time was associated with pouchitis recurrence and with reduced microbial diversity. Interestingly, the consumption of 1.5 or more servings/day of fruit was associated with a reduced risk of developing pouchitis in the following year [88]. Fruits are a source of dietary fibers that can be fermented in SCFA and can increase the levels of fiber-degrading bacteria (i.e., Faecalibacterium), balancing the mucus-degrading bacteria and supporting the intestinal barrier function [88]. Dietary intervention in chronic pouchitis has also been investigated by McLaughlin et al. In particular, treatment with 28 days of an exclusive elemental diet improved the median clinical PDAI and changed the microbiota with a trend towards improved levels of Clostridium Coccoides and Eubacterium rectale, both producers of butyrate. However, there was no reduction in endoscopic or histologic inflammation, and the study comprised a small sample size, so the authors did not recommend an elemental diet as a therapy for pouchitis but only for the temporary reduction of symptoms [89]. Based on this evidence, fruits and vegetables seem to have protective effects against pouchitis, and physicians should encourage pouch patients to include them in their daily diet. The abundant supply of micronutrients in fruits and vegetables may play a role by modulating the microbiota and exercising antiinflammatory effects [90]. In the future, more interventional trials are needed to clarify the role and the impact of diet as a treatment and prophylaxis of pouchitis. Figure 1 and Table 3 summarizes the possible therapeutic interventions involving microbiota for the treatment of pouchitis.

Figure 1.
Interventions in the microbiota for the treatment of pouchitis. Antibiotic treatment (i.e., ciprofloxacin) may reduce the aerobic and pathogen bacteria (which are increased during pouchitis) while leaving undisturbed the larger part of the anaerobic flora, which is present in high numbers in subjects free of pouchitis [36]. Treatment with probiotics can increase fecal concentrations of anaerobes [70,75] and can decrease the levels of the Escherichia subgroup [75]. Pouch microbial diversity is also positively correlated with fruit consumption. In addition, the intake of fruit is positively correlated with the presence of Faecalibacterium and Lachnospira [88]. Finally, the treatment of pouchitis with fecal microbiota transplantation may increase the microbial diversity with a switch towards the donor's microbiota [78]. Table 3. Studies evaluating the interventions on the microbiota as a therapeutic target of pouchitis.

osselink [36] Observational
Ciprofloxacin (daily, 2 × 500 mg) or Metronidazole (daily, 3 × 500 mg) for two weeks In subjects with pouchitis, metronidazole eradicated the anaerobic flora, including C. perfringens but not E. coli. Ciprofloxacin eradicated C. perfringens and E. coli, while the large part of the anaerobic flora was not changed. Ciprofloxacin produced a larger reduction in the PDAI score compared to metronidazole (p = 0.04).
Shen [  while leaving undisturbed the larger part of the anaerobic flora, which is present in high numbers in subjects free of pouchitis [36]. Treatment with probiotics can increase fecal concentrations of anaerobes [70,75] and can decrease the levels of the Escherichia subgroup [75]. Pouch microbial diversity is also positively correlated with fruit consumption. In addition, the intake of fruit is positively correlated with the presence of Faecalibacterium and Lachnospira [88]. Finally, the treatment of pouchitis with fecal microbiota transplantation may increase the microbial diversity with a switch towards the donor's microbiota [78]. Table 3. Studies evaluating the interventions on the microbiota as a therapeutic target of pouchitis.

Authors Study Design Intervention Results
Gosselink [36] Observational Ciprofloxacin (daily, 2 × 500 mg) or Metronidazole (daily, 3 × 500 mg) for two weeks In subjects with pouchitis, metronidazole eradicated the anaerobic flora, including C. perfringens but not E. coli. Ciprofloxacin eradicated C. perfringens and E. coli, while the large part of the anaerobic flora was not changed. Ciprofloxacin produced a larger reduction in the PDAI score compared to metronidazole (p = 0.04).
Shen [ In total, 79% of the antibiotic-treated subjects achieved clinical response. Antibiotics established an antibiotic-resistant microbiome with low inflammatory characteristics, which may confer resistance against colonization by bacteria that stimulate inflammation.
placebo for 9 months All subjects with chronic pouchitis were in clinical and endoscopic remission at the start of the study. During the follow-up, 15% had relapses in the probiotic group compared with 100% in the placebo group (p < 0.001). Fecal concentration of lactobacilli, bifidobacteria, and S. thermophilus increased from baseline levels only in the probiotic group (p < 0.01).

Mimura [71] RCT
Probiotic mixture (6 g/day) of lactobacilli (4 strains), bifidobacterial (3 strains), and Streptococcus thermophilus vs. placebo for 12 months or until relapse Patients with refractory or recurrent pouchitis that achieved remission with a four week course of ciprofloxacin plus metronidazole were included at the start of the study. The cumulative maintained remission rate over the 12 months period was 85% in the probiotic group and 6% in the placebo group (p < 0.0001).
placebo for one year The study included subjects with IPAA and started within 1 week after ileostomy closure. During the follow-up, 10% of patients in the probiotic group experienced an episode of acute pouchitis compared with the 40% of patients treated with placebo (p < 0.05).
The subjects included had not developed previous pouchitis after surgery; 11% in the probiotic group and 50% in the placebo group developed pouchitis (p = 0.07).
Kousgaard [78] Observational Fecal microbiota transplantation by enemas (20 g) for 14 consecutive days Patients with chronic pouchitis were included, and 44% of patients were in clinical remission at 30-day of follow-up; 33% were in remission until 6 months of follow-up. FMT increased microbial diversity in 67% of subjects.
Herfarth [80] RCT Fecal microbiota transplantation by both enemas (24 g) and oral capsules (4.2 g) vs. placebo Patients with antibiotic-dependent pouchitis were enrolled. The study was terminated prematurely due to a small clinical remission rate (6 subjects included). Only 1 patient (17%) clinically responded to FMT and remained off antibiotics for the study period of 16 weeks.
Karjalainen [81] RCT Fecal microbiota transplantation by endoscopy (30 g) and via transanal catheter (30 g) vs. placebo Patients with chronic pouchitis were included and were followed up for 52 weeks; 34.6% patients in the FMT group and 30.8% in the placebo group relapsed during the follow-up (p = 0.183) Welters [86] RCT 24 g of inulin daily for three weeks vs. placebo Butyrate concentrations were significantly higher in the intervention group compared to placebo. The endoscopic and histologic scores were lower in the inulin group compared with placebo. Table 3. Cont.

Authors Study Design Intervention Results
Ianco [87] Observational Diet Subjects with normal pouch consumed more fruits than subjects with pouchitis (3.6 s/d vs 1.8 s/d, p = 0.015) and tended to consume more vegetables (4.5 s/d vs 3.3 s/d, p = 0.06).
Godny [88] Observational Diet Subjects in the lower tertile of fruit consumption (<1.45 s/d) had a higher rate of pouchitis within 1 year of follow-up, compared with patients in the upper two tertiles (30.8% vs 3.8%, p = 0.03). Fruit consumption was associated with an improved microbial diversity (p = 0.003).
McLaughlin [89] Observational Elemental diet (E028, SHS, UK) for 28 days Elemental diet reduced the PDAI symptom score (from 4 to 1, p = 0.039). There was no reduction in endoscopic or histological signs of inflammation.

Conclusions
The pathogenesis of pouchitis remains mostly unclear. However, evidence has shown that the microbiota plays a fundamental role and that dysbiosis with a reduced microbial diversity is associated with pouch inflammation. Nevertheless, a clear understanding of whether inflammation causes an alteration in IPAA microbiota or vice versa is still lacking. However, active interventions based on rebalancing the pouch microbiota are currently the key strategy for the treatment of pouchitis. Antibiotics represent the gold standard treatment for inducing remission. However, due to the high rate of relapse and the risk of developing chronic pouchitis after a first episode of acute pouchitis, prophylactic treatment with probiotics in order to maintain remission after antibiotics treatment appears to be a successful strategy. High-evidence studies are warranted to understand the role of FMT and diet intervention to maintain the remission of the disease.