TY - JOUR
AU - FRCS, John R. Nicholls,
AB - Background Pouchitis is believed to occur as a reaction to dysbiosis. In this study we assessed differences between mucosal bacterial communities cultured from noninflamed and inflamed ileal pouches. Methods Thirty-two ileal pouch patients, 22 with ulcerative colitis (UC) and 10 with familial adenomatous polyposis (FAP), underwent symptomatic, endoscopic, and histological assessment. The Objective Pouchitis Score (OPS) and the Pouch Disease Activity Index (PDAI) were used to diagnose pouchitis. Seven UC patients had pouchitis (UC+), 15 had a noninflamed pouch (UC−), 9 had a noninflamed pouch (FAP−), and 1 FAP patient had pouchitis (FAP+). Biopsies taken from the ileal mucosa of the pouch were cultured under aerobic and anaerobic conditions. Following standardized DNA extraction a polymerase chain reaction (PCR) was performed to generate 16S rRNA gene products. A “fingerprint” of the bacterial community within each sample was created using terminal-restriction fragment length polymorphism (T-RFLP) profiling. Species richness and evenness were determined using T-RF band lengths and relative band intensities. Results From the 64 DNA samples, 834 bands were detected, of which 179 represented different species (operational taxonomic units [OTUs]). The average species richness for the FAP−, FAP+, UC−, and UC+ groups was 26, 35, 23.9, and 29.6 per patient, with the average species diversity within the groups of 10.6, 29, 8.3, and 11.4, respectively. Similar trends were observed when the anaerobic and aerobic-derived bacterial groups were analyzed separately. Conclusions No significant differences were found between the bacterial cultures derived from any of the clinical groups or between pouchitis and nonpouchitis patients. bacteria, familial adenomatous polyposis, inflammatory bowel disease, pathogenesis, pouchitis, ulcerative colitis The prevalence of ulcerative colitis (UC) has been estimated at over 30 cases per 10,000 in the UK.1 Approximately 20% of these patients will at some point require colectomy for medically refractory disease or dysplasia.2 Restorative procto-colectomy (RPC) with ileal pouch anal anastomosis has become the most common elective surgical procedure for UC patients and in selected patients with familial adenomatous polyposis (FAP). The ileal pouch mucosa can become acutely inflamed. This condition, termed “pouchitis,” has a prevalence of 10%3 and a cumulative incidence of ≈50% over a 5-10-year period.4 Pouchitis was thought to be rare in FAP patients but recent studies have suggested a prevalence of 5%.5,6 After RPC the ileal pouch mucosa undergoes morphological changes characterized by villous shortening and increased leukocyte infiltration. This process, termed “colonization,” may be a reflection of ileal mucosal adaptation to the new fecal environment within the ileal reservoir.7 There is a million-fold increase in the number of bacteria present and a transformation of the flora toward a spectrum more typical of the large bowel.8,9 The cause of pouchitis is unknown but the effectiveness of antibiotic treatment to provide symptomatic,10 endoscopic, and histological11,–13 benefit suggests that bacteria are involved. Dysbiosis, defined as an abnormality of the host's gastrointestinal microflora, is thought to play a key role in the pathogenesis of inflammatory bowel disease14,15 and this partly explains the clinical improvements seen with antibiotic therapy in UC16,–21 and Crohn's disease.21,–24 Thirteen observational studies have been performed to investigate dysbiosis in ileal pouches and these vary considerably in their conclusions. Most studies have used traditional microbiological techniques to identify bacterial species. This methodology is time-consuming and labor-intensive, with the number of isolates characterized in any 1 study being limited by the complexity of the technique. In this study a molecular microbiological approach was used to characterize the dominant bacterial species through analysis of the 16S ribosomal RNA (rRNA) gene. This phylogenetically informative gene is found in all bacteria and contains certain regions that are conserved with other more variable regions specific for individual bacterial species.25 Based on differences in the amplified 16S rRNA gene sequences, terminal-restriction fragment length polymorphism (T-RFLP) profiling can be used to resolve electrophoretically the bacterial species within complex communities.26 In this way T-RFLP profiling provides a “fingerprint” of the dominant members of the bacterial community derived from each patient. This technique has been extensively used in environmental microbiology and is being increasingly used in clinical microbiology, with over 500 citations in the literature to date.27,–29 T-RFLP profiling allows the number of dominant species to be estimated (species richness) and the degree to which these species are present in equal numbers (species evenness). Furthermore, a rank-abundance curve can be derived that is able to identify important differences in community structure variation. In this study bacterial communities were obtained from UC and FAP pouch mucosal biopsies and characterized using T-RFLP profiling in order to test the hypothesis that pouchitis is associated with dysbiosis. The bacterial samples derived from pouchitis and nonpouchitis patients were then compared. To date this is the largest study of pouchitis to use molecular microbiological techniques and it is the first time that T-RFLP profiling has been used to assess potential dysbiosis in ileal pouch patients. Materials and Methods Ethical Consideration Ethical approval for this work was granted by the Ethics Committee of the North West London Hospitals NHS Trust. Written consent to participate in this research was obtained from 32 patients on long-term follow-up, with demographic details shown in Table 1. Table 1 Pouch Patient Demographics     View Large Table 1 Pouch Patient Demographics     View Large Diagnosis of Pouchitis Patients were excluded if they had taken antibiotics in the 2 months prior to assessment. All patients underwent a flexible pouchoscopy during which the number of macroscopic features of inflammation were identified and scored 0-6 following the scale put forward by the Moskowitz et al.3 Four biopsies were taken from the upper and lower pouch and scored by an experienced histopathologist who was blind to the patients' clinical details using a modification of Moskowitz's scoring system.3 Clinical features were recorded and a symptomatic score was determined. The Objective Pouchitis Score (OPS)30 and the Pouch Disease Activity Index (PDAI)31 were calculated for each patient. Unlike the PDAI, the OPS does not contain a subjective symptom score and a diagnosis of pouchitis can only be made when a histological score of >2 out of 6 is seen in conjunction with an endoscopic score of >3 out of 6. Harvesting Bacteria from the Biopsies Two biopsies were placed directly into aerobic (Brain Heart Infusion, BHI) culture medium (Media Unit, St. Thomas' Hospital, London) and 2 were placed at the bottom of an anaerobic (cooked meat) culture medium (Media Unit, St. Thomas' Hospital). The aerobic broths were incubated for 48 hours and the anaerobic broths for 72 hours each at 37°C to allow enough time for the slow-growing bacteria to reach the peak of their lag phase multiplication. Four milliliters of the resultant growth were removed and mixed in a 2:1 ratio with a 10% glycerol solution (Media Unit, St. Thomas' Hospital) and stored at −70°C in cryovials (Corning, Corning, NY). DNA Extraction and Polymerase Chain Reaction (PCR) Amplification The methodology used for DNA extraction and quantification and the subsequent PCR amplification has previously been described at length by Rogers et al.32,–34 The oligonucleotide primers used to amplify a region of the 16S rRNA gene for members of the Domain Bacteria were 8f700 (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 926r (5′-CCG TCA ATT CAT TTG AGT TT-3′).34 The primer 8f700 was labeled at the 5′ end with IRD700 (TAGN, Gateshead, UK); the primer 926r was unlabeled. T-RFLP Profiling Restriction endonuclease digestion and T-RFLP analysis were performed as previously described.35 Approximately 200 ng of each digested PCR product was placed in lanes within a 25 cm SequagelXR denaturing polyacrylamide gel (National Diagnostics, Manville, NJ) prepared in accordance with the manufacturer's instructions. The products within each sample were separated in length using a LI-COR IR2 automated DNA sequencer (LI-COR Biosciences, Lincoln, NE) at 55°C and 1200V. T-RFLP Profile Analysis T-RFLP profiles were analyzed using Phoretix 1D Advanced software v. 5.10 (Nonlinear Dynamics, Newcastle upon Tyne, UK). The sizes of the T-RFLP bands were determined by comparing their relative position with known size markers of single-stranded DNA (microSTEP 15a [700 nm]) from Microzone (Lewes, UK). Phoretix 1D Advanced software was also used to determine the volume of each band and intensity of signal. Band volume was expressed as a percentage of the total volume of bands detected in a given electrophoretic profile. T-RFLP bands were resolved over the region between 50 and 958 bases. No bands shorter than 50 bases in length were recorded, as they were in the region susceptible to high levels of signal stemming from the IR tag on unattached 8f700IR primer. In this study the threshold used to detect bands was 0.01% of the total signal between the 50 and 958 base region. Patient Groups Patients were separated by disease status into the following groups: UC nonpouchitis (UC−) and UC pouchitis (UC+) and FAP nonpouchitis (FAP−) and FAP pouchitis (FAP+). The species richness for each group was calculated by dividing the total number of bands found within that group by the number of patients within the same group, i.e., the average number of bands per sample within the group in question. An estimate of the species diversity for each group was calculated by dividing the number of unique bands found in a group by the number of patients within the same group. Statistical Analysis Species rank-abundance plots are valuable assays of community structure36 and were used in this study to assess the differences or similarities between the bacteria in the clinical groups. The area under the curve, or size of the T-RFLP peaks, was used to estimate the relative abundance (%) of each peak within a given sample. The abundances were converted to logarithmic form (Log10 %) and plotted in rank order to give rank-abundance curves. Linear regression lines were fitted to these curves and their slopes were noted for comparisons. As T-RFLP peaks may not always be identified to a specific species, these were considered OTUs, referred to as “species” to ease understanding. Tests of normality and homogeneity of variances, the t-test, and linear regressions were performed with SPSS 12.0.2 for Windows (Chicago, IL). A 2-sample 2-tailed t-test was later used to assess variation between the UC and FAP patient groups and between the pouchitis and nonpouchitis groups. Since there was only 1 patient with FAP and pouchitis (FAP+), this case was not considered in the statistical analysis, although the results are given in the text and tables. Hierarchical cluster analysis and the production of dendrograms were performed using Microsoft Excel software. Results Patient Groups For the 32 patients the OPS classification was as follows: 9 FAP−, 1 FAP+, 15 UC−, and 7 UC+. Using the PDAI system this was: 9 FAP−, 1 FAP+, 17 UC−, and 5 UC+ (Table 1). The median acute histological, endoscopic, and symptomatic scores for the OPS-defined patient groups are shown in Table 1, along with the median OPS and PDAI scores. Bacterial Characterization The T-RFLP profiles for all 32 patients were distinct (sample seen in Fig. 1). In total, 834 bands were identified from the 64 (32 aerobic; 32 anaerobic) samples studied. Of these, 179 represented unique bacterial species referred to as OTUs. By in silico analysis, the individual band lengths formed were compared with sequences deposited at GenBank (Bethesda, MD). Tentative assignments at the genus level were made with the most common band sizes being consistent with the genera Enterobacter, Citrobacter, or Shewanella in 39 cases, Clostridium in 16 cases, Lactobacillus in 13 cases, and Prevotella in 7 cases. Figure 1 View largeDownload slide T-RFLP profiles generated from pouch-derived bacterial samples. SM indicated lanes in which known sizes markers have been run. Automated comparison of the position of bands with these standards allows determination of band length. Position “a,” indicated here by an arrow, represents the band size that would be produced by the species Escherichia coli. Figure 1 View largeDownload slide T-RFLP profiles generated from pouch-derived bacterial samples. SM indicated lanes in which known sizes markers have been run. Automated comparison of the position of bands with these standards allows determination of band length. Position “a,” indicated here by an arrow, represents the band size that would be produced by the species Escherichia coli. Comparison of Species Richness and Diversity Among the Bacterial Communities Derived from the Different Clinical Groups For each clinical group the total number of bacterial bands, the number of unique species, the species richness, and the species diversity were determined (Table 2). A summary of the statistical analysis of the species richness between aerobic and anaerobic-derived cultures acquired from the 4 clinical groups is given in Table 3. Although not statistically significant, the inflamed pouches showed a trend toward increased species richness and diversity. In the UC+ patients this appeared largely to be due to higher numbers of aerobic species when compared with the UC− group. Similar numbers of aerobic and anaerobic species were found in all the groups apart from those taken from the 1 FAP+ patient. Table 2 T-RFLP Profile Data for the 4 Clinical Groups     View Large Table 2 T-RFLP Profile Data for the 4 Clinical Groups     View Large Table 3 Statistical Analysis of Species Richness and Rank-Abundance Slopes     View Large Table 3 Statistical Analysis of Species Richness and Rank-Abundance Slopes     View Large Comparison of Species Evenness Among the Bacterial Communities Derived from the Different Clinical Groups The rank-abundance curves for the anaerobic and aerobically derived bacterial communities are shown in Figures 2 and 3, with fitted linear regressions summarized in Table 3. The shapes of the curves generated were similar, with the dominant member of the community frequently accounting for more than a third of the total bacterial content. The evenness of the species distribution was also assessed and no significant differences were found when comparing the aerobic and anaerobic slopes derived for the FAP−, UC+, and UC− clinical groups. The data obtained from the 1 FAP+ patient did reveal a marked reduction in the magnitude of the aerobic and anaerobic rank-abundance slopes. Flat rank-abundance curves reflect a more even distribution of species among the groups' bacterial communities. Figure 2 View largeDownload slide Rank-abundance curves for the bacterial community samples derived from anaerobic conditions. The y-axis plots the proportion (%) of the bands within the samples. On the x-axis the bacterial taxa are organized most abundant to least abundant from left to right. Placement of a rank-abundance curve along this axis is arbitrary. Figure 2 View largeDownload slide Rank-abundance curves for the bacterial community samples derived from anaerobic conditions. The y-axis plots the proportion (%) of the bands within the samples. On the x-axis the bacterial taxa are organized most abundant to least abundant from left to right. Placement of a rank-abundance curve along this axis is arbitrary. Figure 3 View largeDownload slide Rank-abundance curves for the bacterial community samples derived from aerobic conditions. The y-axis plots the proportion (%) of the bands within the samples. On the x-axis the bacterial taxa are organized most abundant to least abundant from left to right. Placement of a rank-abundance curve along this axis is arbitrary. Figure 3 View largeDownload slide Rank-abundance curves for the bacterial community samples derived from aerobic conditions. The y-axis plots the proportion (%) of the bands within the samples. On the x-axis the bacterial taxa are organized most abundant to least abundant from left to right. Placement of a rank-abundance curve along this axis is arbitrary. Comparison of the Species Richness and Rank-Abundance Between the Aerobic and Anaerobic Samples Derived from UC and FAP Patients and Between the Pouchitis and Nonpouchitis Patient Groups The results of the clinical groups were combined to allow assessment between UC37 and FAP16 patients and between pouchitis38 and nonpouchitis39 patients. A 2-sample 2-tailed t-test was used to compare variations between these groups (Table 4). No statistically significant differences were found except when comparing the rank abundance curves generated from the UC and FAP aerobically derived bacteria. Here the magnitude of the slopes for the UC patients (−0.229) were significantly steeper than the slopes for the FAP (−0.145) patients (P = 0.035). Table 4 Statistical Analysis of Species Richness and Rank-Abundance Slopes     View Large Table 4 Statistical Analysis of Species Richness and Rank-Abundance Slopes     View Large Assessment of the Hierarchical Cluster Analysis of the Bacterial Communities Derived from the Different Clinical Groups Hierarchical cluster analysis (HCA) was used to compare flora derived from the pouchitis and nonpouchitis groups (Fig. 4). In HCA, the more similar 2 communities are the more 2 samples will cluster together. No marked clustering was observed, suggesting that the bacterial species derived from the 4 clinical groups were broadly similar. Figure 4 View largeDownload slide Hierarchical cluster analysis. Cluster dendrograms generated from the T-RFLP profile data showed that there were no clear distinctions between the profiles generated from 4 disease groups analyzed (UC−, UC+, FAP−, and FAP+). Figure 4 View largeDownload slide Hierarchical cluster analysis. Cluster dendrograms generated from the T-RFLP profile data showed that there were no clear distinctions between the profiles generated from 4 disease groups analyzed (UC−, UC+, FAP−, and FAP+). Discussion There are several theories with regard to the pathogenesis of pouchitis but presently the true cause is yet to be elucidated. The effectiveness of antibiotic treatment to provide symptomatic,10 endoscopic, and histological benefit11,–13 suggests that bacteria play a key role in this process. The 2 leading theories of pathogenesis include dysbiosis14,15 and loss of immune tolerance toward otherwise normal bowel flora.40,41 There have been 13 observational studies assessing dysbiosis within ileal pouches. Ten of these studies concentrated on the fecal bacteria flora,9,37,38,42,–48 and only 3 concentrated on the mucosa-adherent flora.39,49,50 These 2 groups differ markedly, the latter being more likely to influence the host immune response.50,–52 The results of these studies differed due to variations in the diagnostic criteria, methodologies, and the clinical groups used for comparison. Two studies suggested an increase in anaerobic counts,53,54 while others report a reduction in number and diversity.39,46,55,56 Increases in aerobic bacterial counts have been noted with an overall reduction in the anaerobic:aerobic ratio.42,43,46 Interestingly, some studies have suggested higher counts of Clostridium perfringens38 with reductions in the probiotic bacteria flora, such as lactobacilli and bifidobacteria.42,46 Three studies found no difference in the bacterial flora of pouchitis and nonpouchitis patients.37,44,48 In summary, it is therefore far from clear what, if any, dysbiotic changes occur in pouchitis. To date only 2 studies of pouchitis have used molecular microbiological techniques. One explored the potential mechanism by which VSL3 maintains clinical remission in pouchitis-prone patients.39 The other included 20 UC patients post-RPC, 5 of whom had been diagnosed with pouchitis.49 One of these pouchitis patients had received a course of antibiotics 2 weeks prior to the trial and should have been excluded using their own criteria. The study used a 16S rRNA length heterogeneity PCR (LH-PCR) technique and suggested dysbiosis in the pouchitis group with 3 unique amplicons, all of which were within 1 basepair of each other, raising the possibility of electrophoretic anomaly. Previous work directly comparing this technique with T-RFLP found the LH-PCR limited in its ability to determine the species present or the overall diversity, due to the relatively small differences in the amplicon lengths generated.34 We tested the hypothesis that pouchitis is associated with demonstrable dysbiosis. Biopsies were taken from 32 patients who had undergone RPC and IPAA and used 16srRNA gene amplification and T-RFLP profiling. Previous studies have used this technique to identify patients with known pathogens.57,58 Comparisons were made between the bacterial communities derived from the pouchitis and nonpouchitis patients in order to detect the presence or absence of bacterial species within the different clinical groups, from which one could imply causative association or susceptibility. To minimize any subjective bias we used the OPS rather than the PDAI, as it has recently been shown to be more sensitive and specific in the diagnosis of pouchitis.30,59 Previous studies using similar molecular techniques to study mucosal-adherent bacteria found no difference in washed and unwashed biopsies.60 The pouch biopsies taken in the present study should reflect the general adherent pouch bacterial community, although it is inevitable that some bacteria specific to the fecal stream would also have been included. Small (3 mm) biopsy forceps were used to minimize the risks of perforation, although it was not initially clear whether enough bacterial DNA could be extracted from these smaller biopsy samples. For this reason we used an enrichment process to enhance the growth of viable bacteria. This also has the benefit of preserving the DNA of those bacteria that fail to thrive or die in the culture conditions, allowing later amplification and identification.33 No attempt was made to quantify the bacteria using dynamic real-time PCR techniques, as the enrichment process did not allow proportional growth. We were, however, able to make inferences on the proportional representation of any 1 bacterial species within the total culture growth. The enteric microflora forms an extremely complex ecosystem which is thought to include ≈400 different bacterial species, 75% of which are unclassified.61,62 In this study, 834 T-RFLP bands were detected from the 64 samples collected. Of these, 179 were unique and represented 179 different bacterial species or OTUs. Such a high species diversity lends support for the analytical system used. Despite this, our results did not demonstrate any statistically significant evidence to support the hypothesis that dysbiosis is associated with pouchitis. The hierarchical cluster analysis did not show any obvious clustering among the bacteria derived from either the inflamed or uninflamed pouches, and the type and variety of bacteria seen among the 4 clinical groups were essentially similar. Rank-abundance curve analysis did not identify any significant differences in the community structure between the FAP−, UC+, and UC− bacterial flora. Both the species richness and diversity were slightly higher in the pouchitis groups, due to an increase in the number and variation of aerobic-derived bacteria, but these differences did not prove to be statistically significant. It is possible that a study with a larger patient number may have revealed more marked differences. The rank-abundance curves obtained were surprisingly steep, with the most prevalent bacterial species accounting for approximately one-third of the total culture growth. This generally indicates harsher growth conditions, such as that seen with postsurgical ischemia. In the present study 1 FAP patient had OPS- and PDAI-defined pouchitis with a flatter rank-abundance curve, increased species richness and diversity, with an “equitably distributed community” suggestive of established stable flora with good nutrient supply. It is possible, therefore, that this patient's pouchitis may have had a different etiological origin than that seen in the UC group. The rank-abundance curves generated from the UC-derived aerobic bacterial flora was considerably steeper, suggesting a “less equitably distributed community” than that seen with the FAP aerobically derived flora (P = 0.035). Theoretically it is possible that an increased dominance of otherwise normal aerobic bacteria within the “colonized” pouches of some UC patients is poorly tolerated, leading to a breakdown in immune tolerance and secondary inflammation. Antibiotic therapy may temporarily readjust this balance by allowing the pouch to be repopulated by anaerobic flora transmitted from the small bowel, until aerobic species again start to dominate. This may account for the cyclical or episodic nature of pouchitis, and explain why on stopping antibiotic and probiotic therapy many relapse shortly afterwards. This is the largest study of its kind to use molecular microbiological techniques in the investigation of pouch flora. No clear evidence of dysbiosis could be demonstrated in the UC pouchitis group. This is the first time that T-RFLP profiling has been used in pouchitis research. The technique is accurate and relatively simple, lending itself to use within the clinical situation and allowing useful assessment of the impact of therapeutic interventions. One of its strengths over other diagnostic tools is that it allows novel or “unexpected” species to be detected and is not limited by preconceptions as to what bacteria are important within a given system. References 1. Ehlin AG, Montgomery SM, Ekbom A, et al.   Prevalence of gastrointestinal diseases in two British national birth cohorts. Gut.  2003; 52: 1117- 1121. CrossRef Search ADS PubMed  2. Katz JA. Prevention is the best defense: probiotic prophylaxis of pouchitis. Gastroenterology.  2003; 124: 1535- 1538. CrossRef Search ADS PubMed  3. Moskowitz RL, Shepherd NA, Nicholls RJ. An assessment of inflammation in the reservoir after restorative proctocolectomy with ileoanal ileal reservoir. Int J Colorectal Dis.  1986; 1: 167- 174. CrossRef Search ADS PubMed  4. Mahadevan U, Sandborn WJ. 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TI - Bacterial Community Diversity in Cultures Derived from Healthy and Inflamed Ileal Pouches After Restorative Proctocolectomy
JF - Inflammatory Bowel Diseases
DO - 10.1002/ibd.21022
DA - 2009-12-01
UR - https://www.deepdyve.com/lp/oxford-university-press/bacterial-community-diversity-in-cultures-derived-from-healthy-and-gSsfiof9zG
SP - 1803
EP - 1811
VL - 15
IS - 12
DP - DeepDyve
ER -