Adherent-invasive Escherichia Coli Strains Research Articles

NOD2 induces autophagy to control AIEC bacteria infectiveness in intestinal epithelial cells.

The importance of autophagy in mechanisms underlying inflammation has been highlighted. Downstream effects of the bacterial sensor NOD2 include autophagy induction. Recently, a relationship between defects in autophagy and adherent/invasive Escherichia coli (AIEC) persistence has emerged. The present study aims at investigating the interplay between autophagy, NOD2 and AIEC bacteria and assessing the expression level of autophagic proteins in intestinal biopsies of pediatric patients with inflammatory bowel disease (IBD). A human epithelial colorectal adenocarcinoma (Caco2) cell line stably over-expressing NOD2 was produced (Caco2NOD2). ATG16L1, LC3 and NOD2 levels were analysed in the Caco2 cell line and Caco2NOD2 after exposure to AIEC strains, by western blot and immunofluorescence. AIEC survival inside cells and TNFα, IL-8 and IL-1βmRNA expression were analysed by gentamicin protection assay and real time PCR. ATG16L1 and LC3 expression was analyzed in the inflamed ileum and colon of 28 patients with Crohn's disease (CD), 14 with ulcerative colitis (UC) and 23 controls by western blot. AIEC infection increased ATG16L1 and LC3 in Caco2 cells. Exposure to AIEC strains increased LC3 and ATG16L1 in Caco2 overexpressing NOD2, more than in Caco2 wild type, while a decrease of AIEC survival rate and cytokine expression was observed in the same cell line. LC3 expression was increased in the inflamed colon of CD and UC children. The NOD2-mediated autophagy induction is crucial to hold the intramucosal bacterial burden, especially towards AIEC, and to limit the resulting inflammatory response. Autophagy is active in inflamed colonic tissues of IBD pediatric patients.

Carrageenan Gum and Adherent Invasive Escherichia coli in a Piglet Model of Inflammatory Bowel Disease: Impact on Intestinal Mucosa-associated Microbiota.

Inflammatory bowel diseases (IBD) including Crohn's disease (CD), and ulcerative colitis (UC), are chronic conditions characterized by chronic intestinal inflammation. Adherent invasive Escherichia coli (AIEC) pathotype has been increasingly implicated in the etiopathogenesis of IBD. In a 21-day study, we investigated the effects of AIEC strain UM146 inoculation on microbiota profile of the ileal, cecal, ascending and descending colon in a pig model of experimental colitis. Carrageenan gum (CG) was used to induce colitis in weaner piglets whereas AIEC strain UM146 previously isolated from a CD patient was included to investigate a cause or consequence effect in IBD. Treatments were: (1) control; (2) CG; (3) AIEC strain UM146; and (4) CG+UM146. Pigs in groups 2 and 4 received 1% CG in drinking water from day 1 of the study while pigs in groups 3 and 4 were inoculated with UM146 on day 8. Following euthanization on day 21, tissue mucosal scrapings were collected and used for DNA extraction. The V4 region of bacterial 16S rRNA gene was then subjected to Illumina sequencing. Microbial diversity, composition, and the predicted functional metagenome were determined in addition to short chain fatty acids profiles in the digesta and inflammatory cytokines in the intestinal tissue. CG-induced colitis decreased bacterial species richness and shifted community composition. At the phylum level, an increase in Proteobacteria and Deferribacteres and a decrease in Firmicutes, Actinobacteria, and Bacteroidetes were observed in CG and CGUM146 compared to control and UM146. The metabolic capacity of the microbiome was also altered in CG and CGUM146 compared to UM146 and control in the colon. We demonstrated that CG resulted in bacterial dysbiosis and shifted community composition similar to what has been previously observed in IBD patients. However, AIEC strain UM146 alone did not cause any clear changes compared to CG or control in our experimental IBD pig model.

Adherent-invasive Escherichia coli Exacerbates Antibiotic-associated Intestinal Dysbiosis and Neutrophil Extracellular Trap Activation

Antibiotic-associated disruption of the gut microbiota is a known risk factor for Crohn's disease. This chronic inflammatory disorder results from aberrant host immune responses to subsets of the gut microbiota, and is characterized by intense neutrophil recruitment to the lamina propria, surface and crypt epithelium. Importantly, adherent-invasive Escherichia coli (AIEC) is abundant in ileal biopsies, highlighting a possible etiological role. In this study, we investigated the impact of antibiotics and AIEC challenge on murine intestinal dysbiosis and neutrophil extracellular trap activation, which is a critical component of the neutrophil antimicrobial repertoire. Male C57BL/6 mice were administered vancomycin and gentamicin (once daily, 3 days), and subsequently challenged with AIEC strain LF82 (once daily, 2 days). Perturbation of the gut microbiota was monitored using a combination of molecular and phylogenetic analyses. The impact of commensal and dysbiotic gut bacterial communities on neutrophil extracellular trap mobilization and intestinal redox balance was also quantified. Exposure of neutrophils to murine commensal gut microbial communities activated neutrophil extracellular trap formation. The capacity of neutrophils to cast these web-like structures was exacerbated following antibiotic and AIEC-associated intestinal dysbiosis, highlighting the possible overgrowth of immune-activating intestinal pathobionts. Intestinal dysbiosis was associated with an elevated capacity of the cultivated gut bacteria to produce reactive oxygen species in vitro, and increased colonic oxidative stress in vivo. Together, these data provide new insights into the detrimental effects of antibiotics on the gut microbiota, with clinically relevant implications for intestinal dysbiosis on neutrophil function and intestinal redox balance.

The Vat-AIEC protease promotes crossing of the intestinal mucus layer by Crohn's disease-associated Escherichia coli.

The aetiology of Crohn's disease (CD) involves disorders in host genetic factors and intestinal microbiota. Adherent-invasive Escherichia coli (AIEC) are receiving increased attention because in studies of mucosa-associated microbiota, they are more prevalent in CD patients than in healthy subjects. AIEC are associated both with ileal and colonic disease phenotypes. In this study, we reported a protease called Vat-AIEC from AIEC that favours the mucosa colonization. The deletion of the Vat-AIEC-encoding gene resulted in an adhesion-impaired phenotype in vitro and affected the colonization of bacteria in contact with intestinal epithelial cells in a murine intestinal loop model, and also their gut colonization in vivo. Furthermore, unlike LF82Δvat-AIEC, wild-type AIEC reference strain LF82 was able to penetrate a mucus column extensively and promoted the degradation of mucins and a decrease in mucus viscosity. Vat-AIEC transcription was stimulated by several chemical conditions found in the ileum environment. Finally, the screening of E. coli strains isolated from CD patients revealed a preferential vat-AIEC association with AIEC strains belonging to the B2 phylogroup. Overall, this study revealed a new component of AIEC virulence that might favour their implantation in the gut of CD patients.

Structural and adhesive properties of the long polar fimbriae protein LpfD from adherent-invasive Escherichia coli.

Crohn's disease (CD) is an inflammatory bowel disease characterized by an exaggerated immune response to commensal microbiota in the intestines of patients. Metagenomic studies have identified specific bacterial species and strains with increased prevalence in CD patients, amongst which is the adherent-invasive Escherichia coli (AIEC) strain LF82. AIEC strains express long polar fimbriae (LPF), which are known to target Peyer's patches in a mouse CD model. Here, the recombinant production of a soluble, self-complemented construct of the LpfD protein of E. coli LF82 is reported and it is demonstrated that it forms the adhesive tip subunit of LPF. The LpfD crystal reveals an N-terminal adhesin domain and a C-terminal pilin domain that connects the adhesin to the minor pilus subunit LpfE. Surface topology and sequence conservation in the adhesin domain hint at a putative receptor-binding pocket as found in the Klebsiella pneumoniae MrkD and E. coli F17-G (GafD) adhesins. Immunohistostaining of murine intestinal tissue sections revealed that LpfD specifically binds to the intestinal mucosa and submucosa. LpfD binding was found to be resistant to treatment with O- or N-glycosidases, but was lost in collagenase-treated tissue sections, indicating the possible involvement of an intestinal matrix-associated protein as the LpfD receptor. LpfD strongly adhered to isolated fibronectin in an in vitro assay, and showed lower levels of binding to collagen V and laminin and no binding to collagens I, III and IV.

Identification of Candidate Adherent-Invasive E. coli Signature Transcripts by Genomic/Transcriptomic Analysis.

Adherent-invasive Escherichia coli (AIEC) strains are detected more frequently within mucosal lesions of patients with Crohn’s disease (CD). The AIEC phenotype consists of adherence and invasion of intestinal epithelial cells and survival within macrophages of these bacteria in vitro. Our aim was to identify candidate transcripts that distinguish AIEC from non-invasive E. coli (NIEC) strains and might be useful for rapid and accurate identification of AIEC by culture-independent technology. We performed comparative RNA-Sequence (RNASeq) analysis using AIEC strain LF82 and NIEC strain HS during exponential and stationary growth. Differential expression analysis of coding sequences (CDS) homologous to both strains demonstrated 224 and 241 genes with increased and decreased expression, respectively, in LF82 relative to HS. Transition metal transport and siderophore metabolism related pathway genes were up-regulated, while glycogen metabolic and oxidation-reduction related pathway genes were down-regulated, in LF82. Chemotaxis related transcripts were up-regulated in LF82 during the exponential phase, but flagellum-dependent motility pathway genes were down-regulated in LF82 during the stationary phase. CDS that mapped only to the LF82 genome accounted for 747 genes. We applied an in silico subtractive genomics approach to identify CDS specific to AIEC by incorporating the genomes of 10 other previously phenotyped NIEC. From this analysis, 166 CDS mapped to the LF82 genome and lacked homology to any of the 11 human NIEC strains. We compared these CDS across 13 AIEC, but none were homologous in each. Four LF82 gene loci belonging to clustered regularly interspaced short palindromic repeats region (CRISPR)—CRISPR-associated (Cas) genes were identified in 4 to 6 AIEC and absent from all non-pathogenic bacteria. As previously reported, AIEC strains were enriched for pdu operon genes. One CDS, encoding an excisionase, was shared by 9 AIEC strains. Reverse transcription quantitative polymerase chain reaction assays for 6 genes were conducted on fecal and ileal RNA samples from 22 inflammatory bowel disease (IBD), and 32 patients without IBD (non-IBD). The expression of Cas loci was detected in a higher proportion of CD than non-IBD fecal and ileal RNA samples (p <0.05). These results support a comparative genomic/transcriptomic approach towards identifying candidate AIEC signature transcripts.

Subversion of Human Intestinal Mucosa Innate Immunity by a Crohn's Disease‐associated E. coli

Adherent‐invasive Escherichia coli (AIEC), associated with Crohn's disease, are likely candidate contributory factors in the disease. However, signaling pathways involved in human intestinal mucosa innate host response to AIEC remain unknown. Here we use a 3‐D model of human intestinal mucosa explant culture to explore the effects of the AIEC strain LF82, on two innate immunity platforms, i.e. the inflammasome through evaluation of caspase‐1 status, and NFkB signaling. We showed that LF82 bacteria enter and survive within a few intestinal epithelial cells and lamina propria macrophages, without altering the mucosa overall architecture. While 4h infection with a Salmonella strain caused crypt disorganization, caspase‐1 activation and mature IL‐18 production, LF82 bacteria were unable to activate caspase‐1 and induce IL‐18 production. In parallel, LF82 bacteria activated NFkB signaling in epithelial cells through IkBα phosphorylation, NFkBp65 nuclear translocation and TNFα secretion. In addition, pharmacological blockade of NFkB activation using the SN50 peptide showed that NFkB activation was crucial for the maintenance of epithelial homeostasis upon LF82 infection. In conclusion, here we decipher at the whole mucosa level the mechanisms of the LF82‐induced subversion of innate immunity that, by maintaining host cell integrity, ensure intracellular bacteria survival.

Analysis of the σE regulon in Crohn's disease-associated Escherichia coli revealed involvement of the waaWVL operon in biofilm formation.

Ileal lesions of patients with Crohn's disease are colonized by adherent-invasive Escherichia coli (AIEC), which is able to adhere to and to invade intestinal epithelial cells (IEC), to replicate within macrophages, and to form biofilms on the surface of the intestinal mucosa. Previous analyses indicated the involvement of the σ(E) pathway in AIEC-IEC interaction, as well as in biofilm formation, with σ(E) pathway inhibition leading to an impaired ability of AIEC to colonize the intestinal mucosa and to form biofilms. The aim of this study was to characterize the σ(E) regulon of AIEC strain LF82 in order to identify members involved in AIEC phenotypes. Using comparative in silico analysis of the σ(E) regulon, we identified the waaWVL operon as a new member of the σ(E) regulon in reference AIEC strain LF82. We determined that the waaWVL operon is involved in AIEC lipopolysaccharide structure and composition, and the waaWVL operon was found to be essential for AIEC strains to produce biofilm and to colonize the intestinal mucosa. An increased prevalence of adherent-invasive Escherichia coli (AIEC) bacteria was previously observed in the intestinal mucosa of Crohn's disease (CD) patients, and clinical observations revealed bacterial biofilms associated with the mucosa of CD patients. Here, analysis of the σ(E) regulon in AIEC and commensal E. coli identified 12 genes controlled by σ(E) only in AIEC. Among them, WaaWVL factors were found to play an essential role in biofilm formation and mucosal colonization by AIEC. In addition to identifying molecular tools that revealed a pathogenic population of E. coli colonizing the mucosa of CD patients, these results indicate that targeting the waaWVL operon could be a potent therapeutic strategy to interfere with the ability of AIEC to form biofilms and to colonize the gut mucosa.

Vitamin D deficiency predisposes to adherent-invasive Escherichia coli-induced barrier dysfunction and experimental colonic injury.

Adherent-invasive Escherichia coli (AIEC) colonization has been strongly implicated in the pathogenesis of Crohn's disease. Environmental triggers such as vitamin D deficiency have emerged as key factors in the pathogenesis of inflammatory bowel diseases. The aim of this study was to investigate the effects of 1,25(OH)2D3 on AIEC infection-induced changes in vivo and in vitro. Barrier function was assessed in polarized epithelial Caco-2-bbe cells grown in medium with or without vitamin D and challenged with AIEC strain LF82. Weaned C57BL/6 mice were fed either a vitamin D-sufficient or -deficient diet for 5 weeks and then infected with AIEC, in the absence and presence of low-dose dextran sodium sulphate. Disease severity was assessed by histological analysis and in vivo intestinal permeability assay. Presence of invasive bacteria was assessed by transmission electron microscopy. Caco-2-bbe cells incubated with 1,25(OH)2D3 were protected against AIEC-induced disruption of transepithelial electrical resistance and tight-junction protein redistribution. Vitamin D-deficient C57BL/6 mice given a course of 2% dextran sodium sulphate exhibited pronounced epithelial barrier dysfunction, were more susceptible to AIEC colonization, and showed exacerbated colonic injury. Transmission electron microscopy of colonic tissue from infected mice demonstrated invasion of AIEC and fecal microbiome analysis revealed shifts in microbial communities. These data show that vitamin D is able to mitigate the deleterious effects of AIEC on the intestinal mucosa, by maintaining intestinal epithelial barrier homeostasis and preserving tight-junction architecture. This study highlights the association between vitamin D status, dysbiosis, and Crohn's disease.

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

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.

Subversion of human intestinal mucosa innate immunity by a Crohn's disease-associated E. coli.

Adherent-invasive Escherichia coli (AIEC), associated with Crohn's disease, are likely candidate contributory factors in the disease. However, signaling pathways involved in human intestinal mucosa innate host response to AIEC remain unknown. Here we use a 3D model of human intestinal mucosa explant culture to explore the effects of the AIEC strain LF82 on two innate immunity platforms, i.e., the inflammasome through evaluation of caspase-1 status, and NFκB signaling. We showed that LF82 bacteria enter and survive within a few intestinal epithelial cells and macrophages, without altering the mucosa overall architecture. Although 4-h infection with a Salmonella strain caused crypt disorganization, caspase-1 activation, and mature IL-18 production, LF82 bacteria were unable to activate caspase-1 and induce IL-18 production. In parallel, LF82 bacteria activated NFκB signaling in epithelial cells through IκBα phosphorylation, NFκBp65 nuclear translocation, and TNFα secretion. In addition, NFκB activation was crucial for the maintenance of epithelial homeostasis upon LF82 infection. In conclusion, here we decipher at the whole-mucosa level the mechanisms of the LF82-induced subversion of innate immunity that, by maintaining host cell integrity, ensure intracellular bacteria survival.

AIEC pathobiont instigates chronic colitis in susceptible hosts by altering microbiota composition

BackgroundInflammatory bowel disease (IBD) is driven by a seemingly aberrant immune response to the gut microbiota with disease development dictated by genetics and environmental factors. A model exemplifying this notion...

Point Mutations in FimH Adhesin of Crohn's Disease-Associated Adherent-Invasive Escherichia coli Enhance Intestinal Inflammatory Response

Adherent-invasive Escherichia coli (AIEC) are abnormally predominant on Crohn's disease (CD) ileal mucosa. AIEC reference strain LF82 adheres to ileal enterocytes via the common type 1 pili adhesin FimH and recognizes CEACAM6 receptors abnormally expressed on CD ileal epithelial cells. The fimH genes of 45 AIEC and 47 non-AIEC strains were sequenced. The phylogenetic tree based on fimH DNA sequences indicated that AIEC strains predominantly express FimH with amino acid mutations of a recent evolutionary origin - a typical signature of pathoadaptive changes of bacterial pathogens. Point mutations in FimH, some of a unique AIEC-associated nature, confer AIEC bacteria a significantly higher ability to adhere to CEACAM-expressing T84 intestinal epithelial cells. Moreover, in the LF82 strain, the replacement of fimH LF82 (expressing FimH with an AIEC-associated mutation) with fimH K12 (expressing FimH of commensal E. coli K12) decreased the ability of bacteria to persist and to induce severe colitis and gut inflammation in infected CEABAC10 transgenic mice expressing human CEACAM receptors. Our results highlight a mechanism of AIEC virulence evolution that involves selection of amino acid mutations in the common bacterial traits, such as FimH protein, and leads to the development of chronic inflammatory bowel disease (IBD) in a genetically susceptible host. The analysis of fimH SNPs may be a useful method to predict the potential virulence of E. coli isolated from IBD patients for diagnostic or epidemiological studies and to identify new strategies for therapeutic intervention to block the interaction between AIEC and gut mucosa in the early stages of IBD.

Crohn's Disease-Associated Adherent-Invasive Escherichia coli Adhesion Is Enhanced by Exposure to the Ubiquitous Dietary Polysaccharide Maltodextrin

Crohn's disease (CD) is associated with intestinal dysbiosis evidenced by an altered microbiome forming thick biofilms on the epithelium. Additionally, adherent-invasive E. coli (AIEC) strains are frequently isolated from ileal lesions of CD patients indicating a potential role for these strains in disease pathogenesis. The composition and characteristics of the host microbiome are influenced by environmental factors, particularly diet. Polysaccharides added to food as emulsifiers, stabilizers or bulking agents have been linked to bacteria-associated intestinal disorders. The escalating consumption of polysaccharides in Western diets parallels an increased incidence of CD during the latter 20th century. In this study, the effect of a polysaccharide panel on adhesiveness of the CD-associated AIEC strain LF82 was analyzed to determine if these food additives promote disease-associated bacterial phenotypes. Maltodextrin (MDX), a polysaccharide derived from starch hydrolysis, markedly enhanced LF82 specific biofilm formation. Biofilm formation of multiple other E. coli strains was also promoted by MDX. MDX-induced E. coli biofilm formation was independent of polysaccharide chain length indicating a requirement for MDX metabolism. MDX exposure induced type I pili expression, which was required for MDX-enhanced biofilm formation. MDX also increased bacterial adhesion to human intestinal epithelial cell monolayers in a mechanism dependent on type 1 pili and independent of the cellular receptor CEACAM6, suggesting a novel mechanism of epithelial cell adhesion. Analysis of mucosa-associated bacteria from individuals with and without CD showed increased prevalence of malX, a gene essential for MDX metabolism, uniquely in the ileum of CD patients. These findings demonstrate that the ubiquitous dietary component MDX enhances E. coli adhesion and suggests a mechanism by which Western diets rich in specific polysaccharides may promote dysbiosis of gut microbes and contribute to disease susceptibility.

Adherent-invasiveEscherichia coliblocks interferon-γ-induced signal transducer and activator of transcription (STAT)-1 in human intestinal epithelial cells

Adherent-invasive Escherichia coli (AIEC) is a pathogen isolated from the ileum of patients with Crohn disease. IFNγ is a key mediator of immunity, which regulates inflammatory responses to microbial infections. Previously, we showed enterohemorrhagic E. coli prevents STAT1 activation. The aim of this study was to determine whether activation of STAT1 by IFNγ was prevented by AIEC infection, and to define the mechanisms used. Human epithelial cells were infected with three different AIEC strains or other pathogenic and commensal E. coli strains. Following infection, cells were stimulated with IFNγ, and STAT1 activation was monitored by immunoblotting. Our data show that live AIEC with active protein synthesis machinery is able to prevent IFNγ-mediated STAT1 phosphorylation, and that a secreted factor may be involved. We conclude that the suppression of epithelial cell STAT1 signal transduction by AIEC strains isolated from patients with Crohn disease represents a novel mechanism by which the pathogen evades host immune responses to the infection.

Bile salts induce long polar fimbriae expression favouring Crohn's disease‐associated adherent‐invasive Escherichia coli interaction with Peyer's patches

Ileal lesions of patients with Crohn's disease are colonized by adherent-invasive Escherichia coli (AIEC). The earliest lesions of recurrent Crohn's disease are erosions of Peyer's patches (PP). We recently reported the presence of a functional lpf operon in AIEC, encoding long polar fimbriae (LPF), that allows AIEC bacteria to interact with PP and to translocate across M cells. The aim of this study was to analyse the effect of gastrointestinal conditions on LPF expression in AIEC strains. The LF82 bacterial growth in an acid pH medium or at high osmolarity medium had no effect on lpf transcription level, in contrast to bacterial growth in the presence of bile salts, which promoted activation of lpf transcription. When cultured in the presence of bile salt, LF82 wild-type bacteria, but not the isogenic mutant deleted for lpfA, exhibited a higher level of interaction with PP and a higher level of translocation through M cell monolayers. The FhlA transcriptional factor was found to be a key bacterial regulator at the origin of LPF expression in the presence of bile salts.

Multidrug Resistance Is Common in Escherichia coli Associated with Ileal Crohnʼs Disease

Escherichia coli is increasingly implicated in the pathogenesis of ileal Crohn's disease (ICD), offering a potential therapeutic target for disease management. Empirical antimicrobial targeting of ileal E. coli has advantages of economy and speed of implementation, but relies on uniform susceptibility of E. coli to routinely selected antimicrobials to avoid apparent treatment failure. Therefore, we examined the susceptibility of ileal E. coli to such antimicrobials. E. coli from 32 patients with ICD and 28 with normal ileum (NI) were characterized by phylogroup, pathotype, antimicrobial susceptibility, and presence of antimicrobial resistance genes. In all, 17/32 ICD and 12/28 NI patients harbored ≥ 1 E. coli strain; 10/24 E. coli strains from ICD and 2/14 from NI were nonsuscepti-ble to ≥ 1 antimicrobial in ≥ 3 categories (multidrug-resistant). Resistance to amoxicillin/clavulanic-acid, cefoxitin, chloramphenicol, ciprofloxa-cin, gentamicin, and rifaximin was restricted to ICD, with 10/24 strains from 8/17 patients resistant to ciprofloxacin or rifaximin (P < 0.01). Adherent-invasive E. coli (AIEC) were isolated from 8/32 ICD and 5/28 NI, and accounted for 54% and 43% of E. coli strains in these groups. In all, 8/13 AIEC strains from ICD (6/8 patients) versus 2/6 NI (2/5 patients) showed resistance to the macrophage-penetrating antimicrobials ciprofloxacin, clarithromycin, rifampicin, tetracycline, and trimethoprim/sulfamethoxazole. Resistance was associated with tetA, tetB, tetC, bla-(TEM), bla(oxa)-1, sulI, sulII, dhfrI, dhfrVII, ant(3″)-Ia, and catI genes and prior use of rifaximin (P < 0.01). ICD-associated E. coli frequently manifest resistance to commonly used antimicrobials. Clinical trials of antimicrobials against E. coli in ICD that are informed by susceptibility testing, rather than empirical selection, are more likely to demonstrate valid outcomes of such therapy.

Characterization of adherent-invasive Escherichia coli isolated from pediatric patients with inflammatory bowel disease

Crohn's disease (CD) and ulcerative colitis (UC), known as inflammatory bowel diseases (IBD), are characterized by an abnormal immunological response to commensal bacteria colonizing intestinal lumen and mucosa. Among the latter, strains of adherent-invasive Escherichia 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. 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-α) and interleukin (IL)-8 mRNA expression was detected by real-time polymerase chain reaction (PCR), after bacterial infection. Transmission electron microscopy and trans-epithelial electric resistance assays were performed on biopsies to assess bacteria-induced morphological and functional epithelial alterations. 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-α, 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. 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.

Role of Meprins to Protect Ileal Mucosa of Crohn's Disease Patients from Colonization by Adherent-Invasive E. coli

Ileal lesions in Crohn's disease (CD) patients are colonized by pathogenic adherent-invasive Escherichia coli (AIEC) able to adhere to and invade intestinal epithelial cells (IEC), and to survive within macrophages. The interaction of AIEC with IEC depends on bacterial factors mainly type 1 pili, flagella, and outer membrane proteins. In humans, proteases can act as host defence mechanisms to counteract bacterial colonization. The protease meprin, composed of multimeric complexes of the two subunits alpha and beta, is abundantly expressed in IECs. Decreased levels of this protease correlate with the severity of the inflammation in patients with inflammatory bowel disease. The aim of the present study was to analyze the ability of meprin to modulate the interaction of AIEC with IECs. In patients with ileal CD we observed decreased levels of meprins, in particular that of meprin β. Dose-dependent inhibition of the abilities of AIEC strain LF82 to adhere to and invade intestinal epithelial T84 cells was observed when bacteria were pre-treated with both exogenous meprin α and meprin β. Dose-dependent proteolytic degradation of type 1 pili was observed in the presence of active meprins, but not with heat-inactivated meprins, and pretreatment of AIEC bacteria with meprins impaired their ability to bind mannosylated host receptors and led to decreased secretion of the pro-inflammatory cytokine IL-8 by infected T84 cells. Thus, decreased levels of protective meprins as observed in CD patients may contribute to increased AIEC colonization.

Crohn disease--associated adherent-invasive E. coli bacteria target mouse and human Peyer's patches via long polar fimbriae.

Crohn disease (CD) is a multifactorial disease in which an abnormal immune response in the gastrointestinal (GI) tract leads to chronic inflammation. The small intestine, particularly the ileum, of patients with CD is colonized by adherent-invasive E. coli (AIEC)--a pathogenic group of E. coli able to adhere to and invade intestinal epithelial cells. As the earliest inflammatory lesions are microscopic erosions of the epithelium lining the Peyer's patches (PPs), we investigated the ability of AIEC bacteria to interact with PPs and the virulence factors involved. We found that AIEC bacteria could interact with mouse and human PPs via long polar fimbriae (LPF). An LPF-negative AIEC mutant was highly impaired in its ability to interact with mouse and human PPs and to translocate across monolayers of M cells, specialized epithelial cells at the surface of PPs. The prevalence of AIEC strains harboring the lpf operon was markedly higher in CD patients compared with controls. In addition, increased numbers of AIEC, but not LPF-deficient AIEC, bacteria were found interacting with PPs from Nod2(-/-) mice compared with WT mice. In conclusion, we have identified LPF as a key factor for AIEC to target PPs. This could be the missing link between AIEC colonization and the presence of early lesions in the PPs of CD patients.