ABSTRACT
BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) is the most enduring infectious candidate that may be associated with inflammatory bowel disease (IBD). It is possible that the inconsistencies in the prevalence studies of MAP in adults reflect clinical differences in adult patients studied, including duration of disease and treatment regimens, and also in lack of specificity of some of the assays used. The aim was to determine the presence of MAP in children with symptoms of Crohn's disease (CD) and ulcerative colitis (UC), using gut biopsy tissue and peripheral blood mononuclear cells (PBMC) collected at initial endoscopic examination prior to clinical treatment. METHODS: Mucosal biopsies and/or PBMC specimens were collected from a total of 142 children, comprising 62 with CD, 26 with UC, and 54 with non-IBD. MAP-specific IS900 polymerase chain reaction (PCR) analysis was performed on all biopsies and PBMC specimens. Conventional MAP culture technique was performed on a subset of 10 CD, 2 UC, and 4 non-IBD patients to isolate MAP. RESULTS: MAP was identified by IS900 PCR significantly more often in mucosal biopsies from CD 39% (22/56) than from non-IBD 15% (6/39) patients (P < 0.05), and in PBMC from CD 16% (8/50) than from non-IBD 0% (0/31) patients (P < 0.05). Viable MAP were cultured from mucosal biopsies from 4/10 CD, 0/2 UC, and 0/4 non-IBD patients, but were not cultured from PBMC specimens. CONCLUSIONS: This unique study on the occurrence of MAP in gut tissue and blood from pediatric IBD patients suggests the possible involvement of MAP in the early stages of development of CD in children.
Subject(s)
Crohn Disease/complications , Crohn Disease/microbiology , Intestinal Mucosa/microbiology , Intestinal Mucosa/pathology , Mycobacterium avium subsp. paratuberculosis/isolation & purification , Paratuberculosis/complications , Adolescent , Age of Onset , Base Sequence , Biopsy , Child , Child, Preschool , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Female , Humans , Leukocytes, Mononuclear/microbiology , Male , Microbiological Techniques , Molecular Sequence Data , Mycobacterium avium subsp. paratuberculosis/genetics , Paratuberculosis/blood , Paratuberculosis/pathology , Polymerase Chain ReactionABSTRACT
Molecular analysis of bacterial 16S rRNA genes has made a significant contribution to the identification and characterisation of bacterial flora in the human gut. In particular, this methodology has helped characterise bacterial families implicated in the aetiology of inflammatory bowel disease (IBD). In this study we have used a genus specific bacterial 16S PCR to investigate the prevalence and diversity of Pseudomonas species derived from the ileum of children with Crohn's disease (CD), and from control children with non-inflammatory bowel disease (non-IBD) undergoing their initial endoscopic examination. Fifty eight percent of CD patients (18/32) were positive using the Pseudomonas PCR, while significantly fewer children in the non-IBD group, 33% (12/36), were PCR positive for Pseudomonas (p<0.05, Fischer's exact test). Pseudomonas specific 16S PCR products from 13 CD and 12 non-IBD children were cloned and sequenced. Five hundred and eighty one sequences were generated and used for the comparative analysis of Pseudomonas diversity between CD and non-IBD patients. Pseudomonas species were less diverse in CD patients compared with non-IBD patients. In particular P.aeruginosa was only identified in non-IBD patients.
