Characterization of Adherent Bacteroidales from Intestinal Biopsies of Children and Young Adults with Inflammatory Bowel Disease

There is extensive evidence implicating the intestinal microbiota in inflammatory bowel disease [IBD], but no microbial agent has been identified as a sole causative agent. Bacteroidales are numerically dominant intestinal organisms that associate with the mucosal surface and have properties that both positively and negatively affect the host. To determine precise numbers and species of Bacteroidales adherent to the mucosal surface in IBD patients, we performed a comprehensive culture based analysis of intestinal biopsies from pediatric Crohn's disease [CD], ulcerative colitis [UC], and control subjects. We obtained biopsies from 94 patients and used multiplex PCR or 16S rDNA sequencing of Bacteroidales isolates for species identification. Eighteen different Bacteroidales species were identified in the study group, with up to ten different species per biopsy, a number higher than demonstrated using 16S rRNA gene sequencing methods. Species diversity was decreased in IBD compared to controls and with increasingly inflamed tissue. There were significant differences in predominant Bacteroidales species between biopsies from the three groups and from inflamed and uninflamed sites. Parabacteroides distasonis significantly decreased in inflamed tissue. All 373 Bacteroidales isolates collected in this study grew with mucin as the only utilizable carbon source suggesting this is a non-pathogenic feature of this bacterial order. Bacteroides fragilis isolates with the enterotoxin gene [bft], previously associated with flares of colitis, were not found more often at inflamed colonic sites or within IBD subjects. B. fragilis isolates with the ability to synthesize the immunomodulatory polysaccharide A [PSA], previously shown to be protective in murine models of colitis, were not detected more often from healthy versus inflamed tissue

ADAMTS13 Deficiency Worsens Colitis and Exogenous ADAMTS13 Administration Decreases Colitis Severity in Mice.

Background: Inflammatory bowel disease (IBD) affects 1.6 million people in the United States. IBD is associated with an increased risk of thrombosis, which rises with disease activity. The pathogenesis of IBD and its increased thrombotic risk is not completely understood. Ultra large von Willebrand factor (ULVWF) multimers are secreted from activated endothelium, leading to recruitment of platelets and leukocytes. A disintegrin and metalloproteinase with thrombospondin type I repeats motif 13 (ADAMTS13) cleaves highly adhesive ULVWF into smaller, less bioactive, multimers, releasing them into circulation. Mice deficient in ADAMTS13 (ADAMTS13-/-) have heightened inflammatory and thrombotic responses. Objectives: We hypothesized that upon colitis induction, ADAMTS13-/- mice would have more severe symptoms compared with wild-type (WT) mice, and rhADAMTS13 administration to mice with colitis would improve their condition. Results: Dextran sodium sulfate-induced colitis was worse in ADAMTS13-/- mice than WT. ADAMTS13-/- showed increased weight loss, worse anemia, and increased clinical and histologic colitis severity, compared with WT mice. ADAMTS13-/- mice had increased VWF release, with accumulation at inflamed colonic sites. Also, the majority of mice showed one or more submucosal colonic thrombi. ADAMTS13 deficiency worsened colitis and propagated intestinal inflammation, most likely through increased platelet-leukocyte recruitment by VWF. Treatment of WT mice with rhA-DAMTS13 decreased colitis severity without worsening anemia. Additionally, several immune-mediated chronic murine colitis models, and inflamed colon tissue specimens from IBD patients, showed increased VWF release at inflamed sites, suggesting a generalizability of our findings. Conclusion: Measuring VWF/ADAMTS13 levels could have clinical utility. When applicable, the administration of ADAMTS13, in addition to primary treatment, may improve outcomes for IBD patients.status: publishe

Total Bacteroidales [cfu/biopsy) and number of different Bacteroidales species detected per biopsy by diagnoses and degree of inflammation in entire cohort.

<p><i>P</i>-value from generalized estimating equation, controlling for within-subject correlation.</p

PCR analysis of <i>bft</i> from <i>B. fragilis</i> isolates.

<p>Lane one contains a <i>bft</i> containing strain (086 care of C. Sears, MD). Lane two contains a <i>B. fragilis</i> strain without <i>bft</i> (9343). Lane three contains an isolate from the terminal ileum (TI) of subject 34. Lanes four and five contain isolates from TI and ascending colon biopsies from control subject 43. Lane 6 contains an isolate from the non-inflamed terminal ileum of a subject 71 with CD. Lane 7 contains an isolate from an inflamed TI biopsy of subject 64 with CD. Lanes 8 and 9 contains isolates from non-inflamed rectal and inflamed ascending colon biopsies of a subject 74 with CD. Lanes 10 and 11 are isolates from non-inflamed TI and inflamed descending colon biopsies of a subject 39 with UC. Lanes 12 and 13 are isolates from an non-inflamed cecal biopsy and inflamed rectal biopsy from subject 62 with UC.</p

PSA analysis of <i>B. fragilis</i> isolates.

<p>A Western blot of PSA from <i>B. fragilis</i> isolates grown <i>in vitro</i> after isolation from control, CD and UC (inflamed (I) a non-inflamed (NI) biopsies. Panel A: Lanes 1–6 contain <i>B. fragilis</i> isolates from the biopsies control subjects (4, 6, 13, 34, 68, and 1). Lanes 7–8 contain isolates from non-inflamed and inflamed biopsies from subject 23 with UC. Lanes 9–11 contain isolates from inflamed and uninflamed biopsies from subjects with UC: 32, 29 and 77. Panel B: Lane 1 contains <i>B. fragilis</i> type strain 9343 as a positive control. Lanes 2 and 3 contain isolates from a non-inflamed and then an inflamed biopsy from subject 47 with CD. Lanes 4–8 contain <i>B. fragilis</i> isolates from inflamed biopsies of subjects 26, 56, 64, 14, and 85 with CD.</p

Total Bacteroidales (cfu/biopsy) by cohort and degree of inflammation.

<p>Biopsies were grouped by cohort: CD (Crohn Disease), UC (ulcerative colitis) and Control and degree of inflammation: no and mild inflammation (no/mild) or moderate to severe inflammation (mod/svr). N is equivalent to the number of biopsies in each group. Column level represents mean Bacteroidales log cfu/biopsy. Error bars represent inter-quartile ranges <b>A.</b> Entire cohort. There were no significant differences detected between cohorts. <b>B</b> Includes only biopsies from newly diagnosed subjects.</p

Number of biopsies with <i>B. fragilis</i> where <i>bft</i> or PSA was present.

<p>Abbreviations: CD =  Crohn Disease, UC =  Ulcerative Colitis, I =  Inflamed biopsy tissue, NI =  Non-Inflamed biopsy tissue where isolate originated.</p><p>Denominator represents all biopsies in a cohort where <i>B. fragilis</i> was detected and numerators indicate:</p><p>1. <i>bft</i> detected by PCR. (No statistically significant differences were detected between groups).</p><p>2. PSA1 detected by immunoblot. (No statistically significant differences were detected between groups).</p

Bacteroidales species diversity by degree of inflammation.

<p>The median number of species per IBD biopsy detected from biopsies with no or mild inflammation (No/Mild) compared to IBD biopsies with moderate to severe inflammation (Mod/Svr). Error bars represent inter-quartile ranges. N equals the number of biopsies in each group. *<i>P</i> = 0.02.</p