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- MATERIALS AND METHODS
- Supporting Information
We have analyzed the bacterial content (microbiota) present in the stool of FAP patients and UC patients with normal pouches (NP) and chronic or relapsing pouchitis (CP). We assumed that the immune system of FAP patients was not abnormally reactive to the pouch microbiota. Therefore, we postulated that if the composition of the microbiota were the same in FAP and CP pouches, then immunological factors would be predominant in the etiology of pouchitis. Another strength of including FAP pouches is that they have less mucosal inflammation. This limits a potential confounding factor in comparing normal UC pouches and pouches with pouchitis, as some “normal” pouches created for UC may be considered to have “mild” or “subclinical” pouchitis. There is certainly ongoing inflammation at a mucosal level.3 If the composition of the microbiota differed between FAP, NP, or CP pouches, then the nature of the bacterial inhabitants of CP pouches would be implicated as having a key role in the etiology of the pouchitis. Further, by comparing the CP microbiota in the absence (CP-off) or presence (CP-on) of antibiotic administration, a correlation between particular types of bacteria and pouchitis might be revealed.
The results of FISH analysis showed that bacteria not commonly present in human feces, nor in the stool of FAP patients, comprised about 50% of the stool microbiota of CP-off patients. Antibiotic treatment reduced the proportion of these unknown bacteria in the stool of pouchitis patients. Therefore, chronic or recurrent pouchitis was found to be associated with a microbiota that contained bacteria not commonly associated with human feces or FAP pouches. The uncommon bacteria that formed large proportions of the CP-off, but not of CP-on, microbiota were found to be members of the Caulobacteraceae, Sphingomonadaceae, Comamonadaceae, Peptostreptococcaceae, and Clostridiaceae. At least some of these groups may therefore be linked to the pathogenesis of pouchitis. Of additional note was the large diversity of clostridial OTU in CP-off microbiota, which presumably denotes particularly favorable conditions in the pouch for expansion of clostridial populations. NP stool also had an expanded phylogenetic gap relative to FAP subjects. In contrast to the CP-off microbiota, Ruminococcaceae, and particularly Bifidobacteriaceae, seemed responsible for the phylogenetic gap of NP stool. This knowledge may be useful in developing strategies to colonize newly formed pouches with innocuous bacteria.
Clostridium perfringens (comprising about 30% of the total bacterial community) was detected by qPCR (targeting the phospholipase gene sequence) in symptomatic pouches of 10 out of 17 (58.8%) CP-off patients. This species was not detected by qPCR in the same pouches when asymptomatic. However, 16S rRNA gene sequences of C. perfringens were detected in asymptomatic pouches, albeit as very small proportions of the total microbiota. The discrepancy between the results obtained by the two assay methods probably reflects their relative sensitivities of detection. The number of target sequences per cell differed greatly between assays. C. perfringens was detected in some normal pouches by qPCR, but in numbers about 30-fold lower than in pouchitis. Other authors have reported the detection of C. perfringens in pouchitis samples from UC patients.30–32 Taken together, the results suggest that C. perfringens may have an etiological role in pouchitis in some patients. Nevertheless, C. perfringens was present in similar numbers in CP-off and FAP stool, although of significantly lower prevalence among patients in the latter. There is thus an intriguing relationship between C. perfringens, a well-known opportunist pathogen, and pouchitis. Future research could compare the relative toxicity of isolates of these bacteria obtained from FAP and CP-off patients because CP isolates might have higher virulence than those normally detected in feces of healthy subjects. This would be reminiscent of the detection of adherent, invasive E. coli strains in patients with ileal CD that are more virulent than commensal strains.33
Clostridium difficile was rarely detected in our subjects. We did not obtain evidence, therefore, to invoke C. difficile as a common cause of pouchitis, although it is clear that in certain circumstances, it can have devastating effects on pouch patients.34
Duffy et al35 and Ohge et al36 reported the detection of sulfate-reducing bacteria in pouches, especially in patients with pouchitis. However, we did not detect any sequences representative of sulfate-reducing bacteria in the stool microbiota.
There is a growing consensus that inflammatory bowel diseases (IBDs), including pouchitis, are associated with a decrease in the number of phylotypes (reduced biodiversity) comprising the stool microbiota.37–43 The results of our study clearly support this consensus. The results of studies to date show a quantitative and a qualitative (biodiversity) reduction in representation of the Firmicutes phylum, and particularly clostridial cluster IV members in the stool of CD patients. This phylogenetic group contains several butyrate-producing bacteria, such as Faecalibacterium prausnitzii. Butyrate and other short chain fatty acids are believed to be important sources of energy for colonic epithelial cells and may have antiinflammatory properties, as well as improving barrier function of the bowel epithelium. Hence, the decrease in butyrate-producing bacteria in the colon or pouch might have an overall detrimental effect on the colonic mucosa.44–48F. prausnitzii was not detected in CP stool microbiota, but was detected at low levels in FAP and NP stool. Sequencing results were consistent with FISH results targeting these same bacteria (clostridial cluster IV). Moreover, members of the Lachnospiraceae, some of which produce butyric acid, were depleted in CP microbiota (average 6.93% CP-off sequences, 9.78% CP-on) relative to FAP and NP (average 33.61% and 21.86%, respectively). It is not clear from other studies whether the reduced biodiversity initiates IBD, perpetuates the diseases, or is a result of the diseases. NP and FAP pouches had similar proportions of Lachnospiraceae and clostridial cluster IV, indicating that the proportions of these bacterial groups in the microbiota might anyway be used as a biomarker of pouch health.
VSL#3 is a probiotic that contains viable lactobacilli, bifidobacteria, and streptococci. Kuehbacher et al49 carried out a double-blind, randomized, placebo-controlled trial to study the impact of VSL#3 on the microbiota of chronic pouchitis patients in remission induced by antibiotics. The authors focused mainly on the Enterobacteriaceae group. However, Lactobacillus and Bifidobacterium clone libraries generated from the VSL#3 group displayed a diverse spectrum of species in comparison with the two other experimental groups (pretreatment remission, n = 15; placebo group, n = 5). Some of these species were those included in the probiotic preparation. CP-on subjects in our study had larger proportions of Lactobacillaceae compared to CP-off patients. Bifidobacteriaceae were present in a higher proportion in NP stool compared to CP pouches. Therefore, pouches formed in UC patients provide a suitable habitat for Bifidobacteriaceae. Why they are in reduced proportions in CP stool poses another interesting topic for investigation. Since both Lactobacillaceae and Bifidobacteriaceae have the capacity to populate pouches and are associated with absence of inflammation, future research could investigate the physiological characteristics of these kinds of bacteria in relation to the pouch habitat. This could lead to interventions that would result in the population of newly constructed pouches with these bacteria, certain species of which may be more suitable for colonization of pouches than others.
As stated earlier, we hypothesized that if the composition of the microbiota differed between NP/FAP and CP pouches, then the nature of the bacterial inhabitants of CP pouches would be implicated as having a key role in the etiology of the pouchitis. Our detailed analysis of the bacterial content of ileo–anal pouches showed that differences in microbiota composition clearly existed between patient groups. The principal outcome was slightly incongruous: CP pouches had a microbiota of reduced biodiversity but expanded phylogenetic gap. In other words, our data showed that the CP microbiota was composed of less than 200 OTU representing uncommon fecal inhabitants. Further, by comparing the CP microbiota in the absence (CP-off) or presence (CP-on) of antibiotic administration, we hoped to associate particular types of bacteria with pouchitis. Possible associations exist but there is clearly a need to build on our detailed phylogenetic study by investigating the physiology of cultured bacteria representative of the phylogenetic groups that we have identified, and by testing them for potential to cause inflammation in experimental animals. This will help to explain the bacteriology of pouchitis in functional, ecological, and pathological terms.