Comparison of Crohn's disease-associated adherent-invasive Escherichia coli (AIEC) from France and Hong Kong: results from the Pacific study

Abstract

Association between ileal colonization by Adherent-Invasive Escherichia coli (AIEC) and Crohn's disease (CD) has been widely described in high-incidence Western countries but remains unexplored in Asian countries with a fast increase in CD incidence. In the PACIFIC study, we compared the characteristics of AIEC pathobionts retrieved from ileal biopsies of CD patients enrolled in France (FR) and Hong Kong (HK). The prevalence of AIEC was similar in France (24.5%, 25/102) and Hong Kong (30.0%, 18/60) (p = 0.44). No difference was observed between the two populations of AIEC regarding adhesion and invasion levels. When tested for antibiotic resistance, the proportion of AIEC strains resistant to ampicillin, piperacillin, tobramycin, and gentamicin was significantly higher in HK AIEC strains compared to French strains. AIEC strains from FR or HK population were both able to persist in the mice intestine (DSS-treated CEABAC10 mice model). Moreover, genomic analysis of 25 FR and 17 hK AIEC strains using next-generation sequencing revealed the co-existence of several virulence factors associated with enteric E. coli pathotypes, although no single virulence factor was significantly associated with either country of origin or AIEC status. In vitro, all AIEC strains (FR and HK) were sensitive to the EcoActive™ phage cocktail, suggesting that it could be a promising option to target AIEC in CD across the world.

Keywords: Crohn’s disease; adherent-invasive E. coli; bacterial comparison; trans-ethnic study.

Figure 1.

Adhesion and invasion of AIEC strains isolated from ileum of CD patients in France (n = 29) and Hong Kong (n = 17) into I-407 epithelial intestinal cell line. The number of adhered (a) or internalized (b) bacteria, to assess adhesion and invasion, respectively, was determined as described in section “materials and methods.” Results were expressed as numbers of CFU/cell and represented as median with interquartile range of triplicate experiments. Data were analyzed by Mann Whitney test, ns.

Figure 2.

Clinical symptoms of colitis in CEABAC10 transgenic mice after an oral challenge with four FR strains (CEA614S, CEA501S, CEA303S, CEA615S), four HK strains (1162d, 1186IFc, 1133a, 1222a) and AIEC LF82 reference strain. (a) Schema of the dss-treated CEABAC10 transgenic mouse model infection protocol. AUC values represent the evolution of mice body weight (b) and DAI score (c) during 10 days after infection. n = 5-8 mice per group. Results are presented as mean with 95% confidence interval; Kruskal–Wallis test, *p < .05.

Figure 3.

AIEC gut colonization after an oral challenge with four FR strains (CEA614S, CEA501S, CEA303S, CEA615S), four HK strains (1162d, 1186IFc, 1133a, 1222a) and AIEC LF82 reference strain in CEABAC10 transgenic mice. (a) Levels of fecal AIEC bacteria at days 1, 3, 6, 8 and 10 postinfection (Cfu/g of feces). (b) AIEC bacteria associated with the intestinal mucosa at day 10 postinfection (Cfu/g of tissue). n = 5-8 mice per group. Results are presented as box-plot with whiskers from minimum to maximum (red box = LF82 reference, purple boxes = hK AIEC strains, green boxes = FR AIEC strains); Kruskal-Wallis test, *p < .05, **p < .01.

Figure 4.

Phylogenetic analysis of AIEC isolated from French and Hong Kong Chinese CD patients. Concatenated core genome SNPs sequences were aligned and phylogenetic inferences obtained using Gubbins method and RAxML program. Sequence types (ST) correspond to Achtman’s MLST scheme. Countries where the sample collection originated are indicated by two different colors on the outer ring. All isolates were clustered into five phylogroups represented by five colors on the inner ring.

Figure 5.

Analysis of virulence genes detected in the Illumina gene sequencing dataset of the 46 AIEC strains of this study. (a) Dendrogram analysis was performed for AIEC strains phylogenic relationships and virulence factors (VFs) relatedness using blastx DIAMOND. Black boxes indicate the presence of the gene listed on the right and white boxes, their absence. The scale on the right indicate the phylogroups and the family of the listed gene, identified by colors. (b) t-sne plot visualizing cluster assignments of AIEC strains. Strains are projected into t-sne space, with the first two t-sne components as the axes of the plot. Phylogroups denoted as distinct colors were assigned according to the Clermont method and cgSNP phylogeny.

Figure 5.

Continued.

Figure 6.

Minimum spanning trees of ompA, fimH, fliC and chiA genes variants deduced from the whole-genome sequences of the 46 AIEC strains of this study. The length of the edges is proportional to the number of amino acid substitutions (scale: substitution number), and the size of nodes to the number of strains (number of sectors). The geographic origin is indicated by the node color.