The Role of Adherent and Invasive E.coli in Pediatric Crohn’s Disease. Cause or Effect?

Abstract

Background and aims Crohn’s disease (CD) is a form of inflammatory bowel disease (IBD) that is characterised by a chronic inflammation of the gastrointestinal tract (GIT). A dysbiotic microbiota with an increase in the relative abundance of Enterobacteriaceae is a common feature of CD. The ability of specific strains, adherent-invasive Escherichia coli (AIEC), to adhere and invade intestinal epithelial cells and survive autophagy implicates them in the pathogenesis of CD. Dietary intervention with exclusive enteral nutrition (EEN) is well-established as the preferred treatment to induce and maintain remission in children with active CD for a prolonged time. The mechanisms of action of EEN are still not fully understood. Recent data support several potential mechanisms including, alteration of gut microbiome. Previous studies focused mostly on changes in the abundance of E. coli during EEN in patients with CD. Here, we aimed to study changes in E. coli at strain-level included characterisations of the dominant E. coli strains, and their virulence genes during treatment with EEN, at food reintroduction, and compared with healthy controls. These changes in E. coli have been correlated with disease activity indices and the gut specific inflammatory marker faecal calprotectin (FC). Methods E. coli strains were isolated from culturing stool samples of twelve children with CD before and during induction treatment with EEN and following that, at food reintroduction. Five faecal samples were used from each patient (before EEN; two faecal samples during EEN (30-day & 60-day EEN); and finally, two faecal samples during food reintroduction). Ten healthy children acted as controls and each provided one stool sample. Twenty healthy adult volunteers were recruited and assigned to groups that received EEN for 7 days or on habitual diet. Faecal samples were collected before and after experimental diet or during the habitual diet; and changes in E. coli strains and their metabolic functions were assessed. DNA samples were extracted from all faecal samples and enrichment cultures of patients with CD and healthy controls. The absolute concentration and relative abundance of E. coli were measured with qPCR. Changes in E. coli strains and virulence genes were explored by using metagenome strain-level analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG), and colicin sensitivity spot tests. The Biolog phenotype microarrays were used to investigate the metabolic changes and utilisation of different nutrients by Enterobacteriaceae, from healthy controls and patients with CD during EEN and food reintroduction. Results There was no significant change in the absolute levels and relative abundances of E. coli in patients with CD before or during EEN, and after food reintroduction. However, in comparison to healthy children, E. coli levels by the end of EEN in patients with CD decreased to levels similar to those found in healthy controls, and this change was reversible at food reintroduction. Patients with CD had significantly higher levels of E. coli at baseline and post-EEN during food reintroduction compared to healthy children (p-value= 0.001). There were changes in E. coli at a strain-level in 58% patients with CD (75% patients according to colicin sensitivity tests) as tested using metagenome strain-level analysis before, during EEN, and after food reintroduction. These strains showed variation during EEN in genomic content and phylogenetic origin. Virulence factors fimA and 6-phospho-beta-glucosidase bglA significantly reduced during EEN, which were lower in strains extracted from EEN samples compared to strains at baseline and food reintroduction (fimA: Baseline vs. EEN end p=0.026; EEN end vs. 60d-post p=0.034)(bglA: Baseline vs. EEN end p=0.006; EEN end vs. 60d-post p=0.033). There was a positive correlation between the absolute concentra