DOP06 Non-invasive identification of adherent-invasive E. coli in patients with Crohn’s disease

Abstract Background Medications limiting the adhesion of ‘adherent and invasive E. coli’ (AIEC) represent potential strategies to treat Crohn’s disease (CD). However, the ileal AIEC identification is a time-consuming procedure, and the number of AIEC strains which colonise ileal CD mucosa remains unknown. There is an unmet need for non-invasive biomarkers to identify patients colonised by AIEC. We aimed to evaluate non-invasive biomarker of ileal AIEC colonisation in patients with CD. Methods This prospective and multi-centre study included CD patients requiring ileocoloscopy. Saliva, serum, stools and ileal biopsies were collected. Abundance and global invasive ability of ileal or faecal E. coli were performed. Isolated E. coli were characterised as AIEC or non-AIEC on I407 epithelial cells and THP1 macrophages. The ERIC-PCR profiles of ileal E. coli were performed. Ileal E. coli/CEACAM6 interaction was analysed by a yeast aggregation test and T84 assays (CEACAM6 protein expression, adhesion inhibition test with D-mannose). Quantification of serum anti-E. coli and ileal or salivary CEACAM6 was realised by ELISA. Results Overall, 102 CD patients were enrolled in this study and 25.8% of them exhibited ileal AIEC colonisation (AIEC+). The abundance and global invasive ability of ileal mucosa-associated E. coli were higher in AIEC+ CD patients compared with CD patients without AIEC (AIEC−) (p = 0.0065 and p = 0.0007, respectively). There was no difference between faecal abundance and invasive ability of E. coli between AIEC+ and AIEC− patients. The ERIC-PCR profiles of ileal E. coli showed that CD AIEC+ were for 78% of them colonised by not more than 2 clonal AIEC strains. In addition, AIEC were able to interact with CEACAM6 by binding D-mannose residues and to induce CEACAM6 expression in T84 cells (p = 0.0009 and p = 0.0185, vs. non-AIEC; respectively). This was also supported by adhesion inhibition test. Serum anti-E. coli level was higher for CD AIEC+ (vs. CD AIEC-). Ileal CEACAM6 level were positively correlated with abundance of ileal associated E. coli in AIEC+ patients (r = 0.4000; p = 0.0362) and with salivary CEACAM6 level (r = 0.4690; p < 0.0001). The non-invasive biomarker ‘serum anti-E.coli/salivary CEACAM6’ index was higher for CD AIEC+ (p = 0.0174; vs. CD AIEC-). A cut-off value < 1.34 × 10−6 eliminated the presence of ileal AIEC with a high negative predictive value (90% CI95% [69%–95%]). Conclusion Our study reported that identification of faecal AIEC cannot replace identification of AIEC from ileal biopsies, most of AIEC infection are mono or bi-clonal (≤ 2 strains) and that non-invasive biomarker such as ‘serum anti-E.coli/salivary CEACAM6’ index could be helpful to screen CD patients for AIEC infection.

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Invasive Ability of an Escherichia coliStrain Isolated from the Ileal Mucosa of a Patient with Crohn’s Disease

Infection and Immunity ◽

10.1128/iai.67.9.4499-4509.1999 ◽

1999 ◽

Vol 67 (9) ◽

pp. 4499-4509 ◽

Cited By ~ 278

Author(s):

Jerome Boudeau ◽

Anne-Lise Glasser ◽

Estelle Masseret ◽

Bernard Joly ◽

Arlette Darfeuille-Michaud

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Host Cell ◽

Bacterial Invasion ◽

Intestinal Epithelial ◽

Invasive Ability ◽

Ileal Mucosa ◽

Gastrointestinal Infections ◽

E Coli ◽

Host Cell Membranes

ABSTRACT Crohn’s disease (CD) is an inflammatory bowel disease in whichEscherichia coli strains have been suspected of being involved. We demonstrated previously that ileal lesions of CD are colonized by E. coli strains able to adhere to intestinal Caco-2 cells but devoid of the virulence genes so far described in the pathogenic E. coli strains involved in gastrointestinal infections. In the present study we compared the invasive ability of one of these strains isolated from an ileal biopsy of a patient with CD, strain LF82, with that of reference enteroinvasive (EIEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enteraggregative (EAggEC), enterohemorrhagic (EHEC), and diffusely adhering (DAEC)E. coli strains. Gentamicin protection assays showed thatE. coli LF82 was able to efficiently invade HEp-2 cells. Its invasive level was not significantly different from that of EIEC and EPEC strains (P > 0.5) but significantly higher than that of ETEC (P < 0.03), EHEC (P < 0.005), EAggEC (P < 0.004) and DAEC (P < 0.02) strains. Strain LF82 also demonstrated efficient ability to invade intestinal epithelial cultured Caco-2, Intestine-407, and HCT-8 cells. Electron microscopy examination of infected HEp-2 cells revealed the presence of numerous intracellular bacteria located in vacuoles or free in the host cell cytoplasm. In addition, the interaction of strain LF82 with epithelial cells was associated with the elongation of microvillar extensions that extruded from the host cell membranes and engulfed the bacteria. This internalization mechanism strongly resembles Salmonella- orShigella-induced macropinocytosis. The use of cytochalasin D and colchicine showed that the uptake of strain LF82 by HEp-2 cells was mediated by both an actin microfilament-dependent mechanism and microtubule involvement. In addition, strain LF82 survived for at least 24 h in HEp-2 and Intestine-407 cells and efficiently replicated intracellularly in HEp-2 cells. PCR and hybridization experiments did not reveal the presence of any of the genetic determinants encoding EIEC, EPEC, or ETEC proteins involved in bacterial invasion. Thus, these findings show that LF82, which colonized the ileal mucosa of a patient with CD, is a true invasive E. coli strain and suggest the existence of a new potentially pathogenic group of E. coli, which we propose be designated adherent-invasive E. coli.

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Enhanced E. coli LF82 Translocation through the Follicle-associated Epithelium in Crohn’s Disease is Dependent on Long Polar Fimbriae and CEACAM6 expression, and Increases Paracellular Permeability

Journal of Crohn s and Colitis ◽

10.1093/ecco-jcc/jjz144 ◽

2019 ◽

Vol 14 (2) ◽

pp. 216-229 ◽

Cited By ~ 7

Author(s):

Åsa V Keita ◽

Lina Yakymenko Alkaissi ◽

Elin B Holm ◽

Stéphanie D S Heil ◽

Benoit Chassaing ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Paracellular Permeability ◽

Ileal Mucosa ◽

Clinical Recurrence ◽

Ussing Chambers ◽

E Coli ◽

Follicle Associated Epithelium ◽

Vitro Model

Abstract Background and Aims Patients with Crohn’s disease [CD] harbour an increased number of adherent-invasive E. coli [AIEC]. The strain LF82, identified in the ileal mucosa of CD patients, has been extensively studied for pathogenic mechanisms. However, understanding of the interaction of LF82 with the intestinal mucosa of CD patients is lacking. Methods Here, we investigated the importance of long polar fimbriae [LPF] type 1 pili and the carcinoembryonic antigen-related cell-adhesion molecule 6 [CEACAM6] for translocation of LF82 in an in vitro model of follicle-associated epithelium [FAE], and in the FAE and villus epithelium [VE] of patients with CD and controls, using Ussing chambers. Results Significantly greater LF82 passage occurred in the FAE model compared with in the VE Caco-2cl1 mono-culture. Moreover, bacterial translocation was inhibited by either LPF disruption or pre-incubation with anti-CEACAM6 antibody. Tissue mounted in Ussing chambers showed significantly higher LF82 passage in FAE from patients with CD compared with control FAE, that was diminished in LF82 lacking LPF and by blocking host CEACAM6. Interestingly, addition of LF82 to the CD FAE tissues significantly increased paracellular permeability [of 51Chromium-EDTA] compared with baseline, and the increase was inhibited by anti-CEACAM6. Immunofluorescence and immunoblots showed higher expression of CEACAM6 in FAE of patients with CD compared with in FAE from controls. Conclusions These data suggest that the FAE of CD patients is a site of vulnerability for invasion by LF82 via a mechanism that requires both bacterial LPF and host CEACAM6. Further, LF82 has the ability to increase paracellular passage through the FAE of patients with CD. These data can help define novel therapeutic targets in CD for the prevention of clinical recurrence.

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Invasive ability of escherichia coli strains isolated from ileal mucosa in Crohn's disease (CD)

Gastroenterology ◽

10.1016/s0016-5085(00)83467-8 ◽

2000 ◽

Vol 118 (4) ◽

pp. A342 ◽

Cited By ~ 3

Author(s):

Jerome Boudeau ◽

Anne L. Glasser ◽

Christelle Neut ◽

Pierre Desreumaux ◽

Antoine Cortot ◽

...

Keyword(s):

Escherichia Coli ◽

Crohn’S Disease ◽

Crohn's Disease ◽

Invasive Ability ◽

Ileal Mucosa

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

PLoS ONE ◽

10.1371/journal.pone.0021199 ◽

2011 ◽

Vol 6 (6) ◽

pp. e21199 ◽

Cited By ~ 29

Author(s):

Emilie Vazeille ◽

Marie-Agnès Bringer ◽

Aurélie Gardarin ◽

Christophe Chambon ◽

Christoph Becker-Pauly ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Ileal Mucosa ◽

E Coli

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A non-invasive comparison between NASH and Crohn's disease

Zeitschrift für Gastroenterologie ◽

10.1055/s-0036-1597384 ◽

2016 ◽

Vol 54 (12) ◽

pp. 1343-1404

Author(s):

K Willuweit ◽

M Brandenburg ◽

S Sydor ◽

S Aßmuth ◽

A Wegehaupt ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Non Invasive

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Macrophages behaviour against Adherent and Invasive E. coli (AIEC) isolated from Crohn's disease patients in relation with host factors susceptibility

10.26226/morressier.57c5383ed462b80296c9b40e ◽

2016 ◽

Author(s):

Anthony Buisson

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Host Factors ◽

E Coli

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The Order in Which Antibiotics are Administered Affects the Fitness Costs of Adherent-Invasive E. coli (AIEC) Strains

10.31219/osf.io/46fae ◽

2019 ◽

Author(s):

Jordan B Gregg

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Combination Therapy ◽

Agar Plate ◽

Sequential Therapy ◽

Negative Control ◽

Clinical Settings ◽

Fitness Costs ◽

Inflammatory Gene Expression ◽

E Coli

AIEC-LF82 is a strain of bacteria that is surmised to have a role in causing IBD and Crohn’s disease by activating pro-inflammatory gene expression in organisms. Using antibiotics via combination therapy has been a technique used in clinical settings in an attempt to treat the strains, however, the attempts have not been that effective nor efficient in terms of completely halting the growth and colonization of AIEC to treat IBD and Crohn's disease patients. Research has shown that regarding hindering or preventing the colonization bacterial colonies, sequential therapy tends to be more effective and time-efficient than combination therapy, with fewer adverse effects. To test if this is also the case with the AIEC-LF82 strain of bacteria, I first tested AIEC’s response to combination therapy using the Penicillin-Streptomycin, Kanamycin-Chloramphenicol, antimicrobial peptide (AMP), Kanamycin, SPE phase and LB agar plates, all of which were experimental plates other than the LB agar plate that acted as the negative control. I then tested AIEC-LF82’s response to sequential therapy using the LB+ Kan + Spe, LB + AMP + Spe, LB+ Kan/Cam + Spe, LB + P/S + Spe, LB + P/S + Kan and LB + P/S + AMP and one LB agar plate acting as the negative control. The only differences between sets a and b were the order in which antibiotics were administered in the six aforementioned treatment sets. Ultimately, I found that set b of sequential therapy, strong-weak antibiotic treatments, was the most effective treatment but that set a regarding sequential therapy was actually the least effective of all of the treatments. In conclusion, using strong-weak sequential antibiotic therapy treatments appears to be a potentially promising option to treat patients suffering from Crohn's disease and IBD.

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Expression profiling of ileal mucosa in asthma reveals upregulation of innate immunity and genes characteristic of Paneth and goblet cells

Allergy Asthma & Clinical Immunology ◽

10.1186/s13223-021-00584-9 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Jan K. Nowak ◽

Marzena Dworacka ◽

Nazgul Gubaj ◽

Arystan Dossimov ◽

Zhumabek Dossimov ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Differential Expression ◽

Expression Profiles ◽

Differential Expression Analysis ◽

Goblet Cells ◽

Ileal Mucosa ◽

Significant Differential Expression ◽

Immune Genes ◽

Asthma Patients

Abstract Background The expression profiles of the intestinal mucosa have not been comprehensively investigated in asthma. We aimed to explore this in the Correlated Expression and Disease Association Research (CEDAR) patient cohort. Methods Differential expression analysis of ileal, transverse colon, and rectal biopsies were supplemented by a comparison of transcriptomes from platelets and leukocytes subsets, including CD4+, CD8+, CD14+, CD15+, and CD19+ cells. Asthma patients (n = 15) and controls (n = 15) had similar age (p = 0.967), body mass index (p = 0.870), similar numbers of females (80%) and smoking rates (13.3%). Results Significant differential expression was found in the ileum alone, and not in any other cell/tissue types. More genes were found to be overexpressed (1,150) than under-expressed (380). The most overexpressed genes included Fc Fragment of IgG Binding Protein (FCGBP, logFC = 3.01, pFDR = 0.015), Mucin 2 (MUC2, logFC = 2.78, pFDR = 0.015), and Alpha 1B Defensin (DEFA1B, logFC = 2.73, pFDR = 0.024). Gene ontology implicated the immune system, including interleukins 4 and 13, as well as antimicrobial peptides in this overexpression. There was concordance of gene over- (STAT1, XBP1) and underexpression (NELF, RARA) in asthma and Crohn’s disease ileum when our results were compared to another dataset (p = 3.66 × 10–7). Conclusion Ileal mucosa in asthma exhibits a specific transcriptomic profile, which includes the overexpression of innate immune genes, mostly characteristic of Paneth and goblet cells, in addition to other changes that may resemble Crohn’s disease.

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Antagonism of Adherent Invasive E. coli LF82 With Human α-defensin 5 in the Follicle-associated Epithelium of Patients With Ileal Crohn’s Disease

Inflammatory Bowel Diseases ◽

10.1093/ibd/izaa315 ◽

2020 ◽

Author(s):

Lina Y Alkaissi ◽

Martin E Winberg ◽

Stéphanie DS Heil ◽

Staffan Haapaniemi ◽

Pär Myrelid ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Ex Vivo ◽

Ussing Chambers ◽

E Coli ◽

Follicle Associated Epithelium ◽

Vitro Model ◽

Set Up

Abstract Background The first visible signs of Crohn’s disease (CD) are microscopic erosions over the follicle-associated epithelium (FAE). The aim of the study was to investigate the effects of human α-defensin 5 (HD5) on adherent-invasive Escherichia coli LF82 translocation and HD5 secretion after LF82 exposure in an in vitro model of human FAE and in human FAE ex vivo. Methods An in vitro FAE-model was set up by the coculture of Raji B cells and Caco-2-cl1 cells. Ileal FAE from patients with CD and controls were mounted in Ussing chambers. The effect of HD5 on LF82 translocation was studied by LF82 exposure to the cells or tissues with or without incubation with HD5. The HD5 secretion was measured in human FAE exposed to LF82 or Salmonella typhimurium. The HD5 levels were evaluated by immunofluorescence, immunoblotting, and ELISA. Results There was an increased LF82 translocation across the FAE-model compared with Caco-2-cl1 (P < 0.05). Incubation of cell/tissues with HD5 before LF82 exposure reduced bacterial passage in both models. Human FAE showed increased LF82 translocation in CD compared with controls and attenuated passage after incubation with sublethal HD5 in both CD and controls (P < 0.05). LF82 exposure resulted in a lower HD5 secretion in CD FAE compared with controls (P < 0.05), whereas Salmonella exposure caused equal secretion on CD and controls. There were significantly lower HD5 levels in CD tissues compared with controls. Conclusions Sublethal HD5 reduces the ability of LF82 to translocate through FAE. The HD5 is secreted less in CD in response to LF82, despite a normal response to Salmonella. This further implicates the integrated role of antimicrobial factors and barrier function in CD pathogenesis.

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