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

Abstract

BackgroundAdherent-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.ResultsA 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.ConclusionsThe 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.

Comparative genomics of Crohn's disease-associated adherent-invasiveEscherichia coli

“…We conducted presence/absence and phylogenetic analyses on the following LF82-associated genes: rpoE ;39 htrA (also known as degP and ptd );40 dsbA, 41 hfq, 42 and AIEC/IBD-associated E. coli genes : pduC 29 (propanediol utilisation), fyuA, ibeA, K1, kpsmTII, 17 vgrG , hcp , vasD , vasG , impL , impK (type VI secretion system genes), and vat , insA , insB, 30 fimH, 28 afaC 45 and clb (pks island),46and the combination of gipA and lpfA, 43 or lpfA alone 44 45. Nine of the 41 strains harboured the afaC gene (H504, H305, H020, H296, 61–1 ti1, 52–2 ti10, 52–1 ti3, 18–4 ti12, 18–3 ti5), 4 of which were AIEC (36% of all AIEC strains), belonged to ST95 and had an O18:H7 serotype.…”

“…We used MicroScope to determine the presence/absence of genes identified in previous studies as being important for phenotypical characteristics of LF82 ( rpoE, 39 htrA, 40 dsbA, 41 hfq, 42 fimH, 28 lpfA/gipA 43–45) or AIEC/IBD-associated E. coli ( vgrG , hcp , vasD , vasG, impL , impK (Type VI secretion system genes), vat , insA , insB, 30 afaC, 45 and clb (pks island),46 pduC, 29 K1, kpsmTII, fyuA, ibeA. 17 The nucleotide sequence of each of these genes was also used for phylogenetic analyses. MEGA V.647 was used to align the sequences and generate phylogenetic trees.…”

“…E. coli belonging to this pathovar are defined by their in vitro abilities to adhere to and to invade epithelial cells, and to survive and replicate within macrophages 9 16 17…”

“…Conte et al 17 showed that some virulence genes are more frequent in the E. coli isolates from patients with CD than controls, including: K1 and kpsMT II, both involved in capsule synthesis; fyuA , involved in iron acquisition; and ibeA , involved in invasion. The relative abundance of these strains was significantly higher in patients with CD (10%) compared with controls (1%).…”

Glycolysis and pyrimidine biosynthesis are required for replication of adherent–invasive Escherichia coli in macrophages

“…Indeed recent genomic studies have failed to identify any AIEC-specific genes that can adequately explain the ability of this group of E. coli to invade epithelial cells and replicate in macrophages (Clarke et al, 2011;Krause et al, 2011;Miquel et al, 2010;Nash et al, 2010;Vejborg et al, 2011;Zhang et al, 2015). Nonetheless epidemiological evidence does suggest that E. coli are enriched in the guts and macrophages of the lamina propria of CD patients compared to healthy controls (Conte et al, 2014;Darfeuille-Michaud et al, 2004;de Souza et al, 2012;Elliott et al, 2015;Martinez-Medina et al, 2009).…”