ORIGINAL ARTICLE Characterization of Adherent-invasive Escherichia coli Isolated from Pediatric Patients with Inflammatory Bowel Disease Anna Negroni, PhD,* Manuela Costanzo, PhD,* Roberta Vitali, PhD,* Fabiana Superti, PhD, † Lucia Bertuccini, PhD, † Antonella Tinari, MSc, † Fabio Minelli, ‡ Giovanni Di Nardo, MD, § Federica Nuti, MD, § Maria Pierdomenico, PhD,* Salvatore Cucchiara, PhD, MD, § and Laura Stronati, PhD* Background: Crohn’s disease (CD) and ulcerative colitis (UC), known as inflammatory bowel diseases (IBD), are characterized by an abnor- mal immunological response to commensal bacteria colonizing intestinal lumen and mucosa. Among the latter, strains of adherent-invasive Esch- erichia coli (AIEC), capable of adhering to and invading epithelium, and to replicate in macrophages, have been described in CD adults. We aimed at identifying and characterizing AIEC strains in pediatric IBD. Methods: In all, 24 CD children, 10 UC, and 23 controls were investigated. Mucosal biopsies, taken during colonoscopy, were analyzed for the presence of AIEC strains by an adhesive-invasive test. Protein expression of the specific AIEC receptor, the carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), was evaluated by western blot and immunohistochemistry, while tumor necrosis factor alpha (TNF-a) and interleukin (IL)-8 mRNA expression was detected by real-time polymerase chain reaction (PCR), after bacterial infection. Transmission elec- tron microscopy and trans-epithelial electric resistance assays were performed on biopsies to assess bacteria-induced morphological and func- tional epithelial alterations. Results: Two bacterial strains, EC15 and EC10, were found to adhere and invade the Caco2 cell line, similar to the well-known AIEC strain LF82 (positive control): they upregulated CEACAM6, TNF-a, and IL-8 gene/protein expression, in vitro and in cultured intestinal mucosa; they could also survive inside macrophages and damage the epithelial barrier integrity. Lesions in the inflamed tissues were associated with bacterial infection. Conclusions: This is the first study showing the presence of adhesive-invasive bacteria strains in the inflamed tissues of children with IBD. Collective features of these strains indicate that they belong to the AIEC spectrum, suggesting their possible role in disease pathogenesis. (Inflamm Bowel Dis 2012;18:913–924) Key Words: Crohn’s disease, ulcerative colitis, intestinal microbiota, Escherichia coli, adherent-invasive strains I nflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), are relatively common chronic disorders, thought to result from inappro- priate and continual activation of intestinal mucosa immune system due to a complex interplay between genetic, immu- nological, and microbial factors. 1,2 Clinical and experimen- tal studies indicate that intestinal bacteria are involved in the initiation and amplification of IBD: indeed, colitis does not occur in most gene knockout models of IBD when ani- mals are kept in a germfree environment, 3,4 and both ani- mal models of colitis and patients with IBD respond to antibiotics and probiotics. 5 In addition, most genes associ- ated with susceptibility to IBD, including NOD2/CARD15, ATG16L1, and IRGM, encode proteins involved in host– microbial interactions. 6 Immunological studies have also shown that the majority of patients with IBD develop sero- logical and T-cell response to their own enteric flora. 7 The role of bacteria in the pathogenesis of IBD is further sup- ported by the observation that surgical diversion of fecal stream or treatment with bowel rest and total parenteral nutrition have successfully been used to control enteric inflammation. 8 Recent advances in molecular techniques in IBD patients have shown a reduction in beneficial bacteria groups such as members of the phyla Firmicutes and Received for publication August 12, 2011; Accepted August 22, 2011. From the *ENEA, Italian National Agency for new Technologies, Energy and Sustainable Economic Development, Rome, Italy; † Department of Technology and Health, Istituto Superiore di Sanita `, Rome, Italy, ‡ Department of Food Safety and Veterinary Public Health, Istituto Superiore di Sanita `, Rome, Italy, § Department of Pediatrics, Pediatric Gastroenterology and Liver Unit, Sapienza University Hospital Umberto I, Rome, Italy. Supported by a grant from: Broad Medical Research Program (BMRP), Inflammatory Bowel Disease Grants, Broad Foundation (No. IBD-0225R3). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the article. Reprints: Laura Stronati, PhD, Section of Toxicology and Biomedical Sciences, Italian National Agency for New Technologies, Energy and Sustainable Economic Development (ENEA), Via Anguillarese 301, 00123 Rome Italy (e-mail: laura.stronati@enea.it). Copyright V C 2011 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1002/ibd.21899 Published online 12 October 2011 in Wiley Online Library (wileyonlinelibrary.com). Inflamm Bowel Dis  Volume 18, Number 5, May 2012 913