Adherent-invasive Escherichia coli (AIEC) in pediatric Crohn's disease patients: phenotypic and genetic pathogenic features

Abstract

Background: Adherent-invasive Escherichia coli (AIEC) have been implicated in the ethiopathogenesis of Crohn's disease (CD). In this study, we analyzed a collection of intestinal mucosa-associated E. coli isolates, presenting AIEC phenotypes, isolated from biopsies of CD pediatric patients and non-inflammatory bowel diseases (IBD) controls, in order to investigate their genetic and phenotypic pathogenic features.

Results: A total of 616 E. coli isolates from biopsies of four pediatric CD patients and of four non-IBD controls were collected and individually analyzed. For AIEC identification, adherent isolates were assayed for invasiveness, and the capacity of the adhesive-invasive isolates to survive and replicate intracellularly was determined over macrophages J774. In this way we identified 36 AIEC-like isolates. Interestingly, their relative abundance was significantly higher in CD patients (10%; 31/308) than in non-IBD controls (1%; 5/308) (χ2 = 38.96 p < 0.001). Furthermore pulsed field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) techniques were applied to analyze the clonality of the 36 AIEC-like isolates. The results obtained allowed us to identify 27 distinct genotypes (22 from CD patients and 5 from non-IBD controls). As for the AIEC prototype strain LF82, all 27 AIEC genotypes presented an aggregative pattern of adherence (AA) that was inhibited by D-mannose, indicating that adhesiveness of AIEC is likely mediated by type 1 pili. PCR analisys was used to investigate presence of virulence genes. The results indicated that among the 27 AIEC isolates, the incidence of genes encoding virulence factors K1 (χ2 = 6.167 P = 0.013), kpsMT II (χ2 = 6.167 P = 0.013), fyuA (χ2 = 6.167 P = 0.013), and ibeA (χ2 = 8.867 P = 0.003) was significantly higher among AIEC strains isolated from CD patients than non-IBD controls.

Conclusions: The identification of AIEC strains in both CD and non-IBD controls, confirmed the "pathobiont" nature of AIEC strains. The finding that AIEC-like isolates were more abundant in CD patients, indicates that a close association of these strains with CD may also exists in pediatric patients.

Figure 1

Genetic variability among the 36 AIEC isolates from CD patients CD1, CD2 and CD3, and from non-IBD controls Control-1 and Control-2. Consensus UPGMA dendrogram, generated on Dice coefficients of XbaI PFGE and RAPD-PCR combined profiles (similarity cut-off level 95%) identified 27 genetic variants (22 from CD patients and 5 for controls). Genotypes of AIEC strains isolated from the same patient tended to cluster.

Figure 2

Similarity analysis between AIEC genotypes isolated from CD patients and non-IBD controls. Percentage values were based on Dice similarity index. A P value ≤0.05 was considered statistically significant.

Figure 3

Adhesion phenotypes assay. Phase contrast fields showing Hep-2 and Caco-2 cell monolayers infected with two representative AIEC strains isolated in this work (PP45 and DF6) and with reference AIEC LF82 strain. All 27 AIEC strains showed an aggregative (AA) patterns of adherence. Magnification, 1000 X.