Clinical-alimentary tractHigh prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn’s disease☆
Section snippets
Patients and controls
E. coli strains included in this study were isolated from ileal specimens of patients with CD or of controls, and from colonic specimens of patients with CD, UC, or of controls. All patients and controls gave their informed consent, and approval from the local ethics committee was obtained.
Microbiologic methods
The intestinal specimens were collected into sterile vials containing either phosphate-buffered saline (PBS) or cysteinated one-quarter strength Ringer’s solution. One biopsy specimen weighing approximately 5–10 mg was tested for each patient or control. The specimens were crushed and 10-fold dilutions were plated on blood agar medium. Suspicious E. coli colonies were identified by the API system (bioMérieux sa, Marcy l’Etoile, France). Up to 3 colonies for each patient or control were
Reference bacterial strains
Enteropathogenic E. coli E2348/69,35 enterotoxigenic E. coli H10407,36 and AFA A3037E. coli strains, which belong to different pathogenic groups that are responsible for gastrointestinal or urinary tract infections, were used as sources of the eae, tia, and afaD genes. Shigella flexneri strain SC30138 was used as a source of the ipaC gene. E. coli strain K-12 C600 was used as a noninvasive control.
Cell lines and cell culture
The following epithelial cell models were used: the Caco-2 cell line established from a human colonic adenocarcinoma that exhibits structural and functional differentiation patterns in postconfluence culture characteristic of mature enterocytes of the small intestine, the Intestine-407 cell line derived from human embryonic jejunum and ileum as a model of undifferentiated intestinal epithelial cells, and the HEp-2 cell line derived from a human laryngeal carcinoma. All cell lines were purchased
Invasion assays
Intestine-407 and HEp-2 cells were seeded in 24-well tissue culture plates (Polylabo, Strasbourg, France) at a density of 4.105 cells/well and incubated for 20 hours. The cell monolayers were washed twice with PBS (pH 7.2). Bacterial invasion of epithelial cells was measured using the gentamicin protection assay.39 Each monolayer was infected in 1 mL of the cell culture medium lacking antibiotics with a multiplicity of infection of 10 bacteria per epithelial cell. After a 3-hour incubation
Adhesion assays
Adhesion to differentiated Caco-2 and undifferentiated Intestine-407 intestinal cells was measured as reported previously20 with minor modifications. Briefly, Caco-2 cells were seeded at a density of 2.104 cells/well and used at postconfluence after 15 days of culture. Intestine-407 cells seeded at 4.105 cells/well were incubated for 20 hours. The cells were washed with PBS before the adhesion test and a suspension of 108 bacteria/mL in the cell line culture medium was added to the tissue
Effect of eukaryotic cytoskeletal inhibitors
HEp-2 cells were preincubated for 30 minutes before the invasion assay in cell culture medium lacking antibiotics with 1 μg/mL of cytochalasin D or 0.5 μg/mL of colchicine (Sigma). The inhibitors were present throughout a 3-hour bacterial infection period. The inhibitory effect of each inhibitor on bacterial uptake was evaluated against a control assay without inhibitor, which was defined as 100% of bacterial uptake. All of the assays were performed at least 3 times in separate experiments.
Bacterial survival and replication within macrophages
Bacterial uptake, survival, and replication were measured by the gentamicin protection assay. J774-A1 macrophages were seeded in 24-well tissue culture plates (Polylabo) at a density of 2.105 cells per well and were grown for 24 hours. Before infection, the cell monolayers were washed twice with PBS and the medium was replaced with 1 mL of RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum. Each monolayer was infected with a multiplicity of infection of 10 bacteria per macrophage
Transmission electron microscopy
Cross-sections of infected cultured cells were prepared as follows. After infection, cells were fixed with 3% glutaraldehyde in 0.2 mol/L cacodylate buffer at 4°C for 2 hours and postfixed in 1% OsO4 in cacodylate buffer at 4°C for 1 hour. After dehydration in a graded series of ethanol, the cultures were embedded in a 2-mm thick Epon coating (Resolution Performance Product, Houston, TX) in the tissue culture well and polymerized for 3 days at 60°C. Suitable areas were oriented parallel to the
Hybridization experiments
DNA fragments used as nucleic probes were generated by polymerase chain reaction. Oligonucleotides used for amplification of ipaC-specific, eae-specific, tia-specific, and afaD-specific sequences were synthesized on the basis of published nucleotide sequences (Table 1). Polymerase chain reaction protocols were performed as described previously.30 Polymerase chain reaction fragments obtained from each specific amplification of DNA from reference E. coli strains were used as nucleic probes for
Definition of AIEC
Determination of E. coli strains as belonging to the AIEC pathovar was performed using the following criteria: (1) the ability of the bacteria to invade Intestine-407 and HEp-2 epithelial cells with an invasion index equal or superior to 0.1% of the original inoculum, (2) the involvement of host cell actin polymerization and microtubule recruitment in bacterial uptake, (3) the absence of known invasive determinants, (4) the ability to adhere to differentiated Caco-2 and/or undifferentiated
Statistical analysis
The data were analyzed by the χ2 test unless the variables needed a 2-tailed Fisher exact test. A P value <0.05 was considered statistically significant.
Search for invasive E. coli strains associated with intestinal mucosa of CD and UC patients and controls
The invasion level of the noninvasive reference E. coli strain K-12 C600 was 0.0005% ± 0.0005% of the original inoculum (Table 2). Reference strain LF82, included in all of the assays as a positive invasive control, gave a mean invasion level of 1.29% ± 0.55%. E. coli strains were considered invasive when the mean invasion level was superior or equal to 0.1% of the original inoculum. Based on this criteria, invasive E. coli strains isolated from patients with IBD or controls are listed in
Discussion
The main result of this study was that pathogenic AIEC are associated with ileal CD. AIEC strains were isolated from almost one third of ileal specimens in CD as compared with 6% in ileal controls and less than 5% in colonic samples from both IBD patients and controls. AIEC strains were found more frequently in early recurrent lesions after surgery. This led us to propose that AIEC are not only secondary invaders but that they could be involved in the initiation of the inflammatory process.
Acknowledgements
The authors thank Michael Donnenberg for providing enteropathogenic E. coli strain E2348/69, Dolores Evans for providing enterotoxigenic E. coli strain H10407, Chantal Le Bouguénec for providing AFA strain A30, and Philippe Sansonetti for providing Shigella flexneri strain SC301. The authors also thank Chantal Rich and Laurence Nakusi for their technical help, and Herbert Van Kruiningen for helpful discussion and critical reading of the manuscript.
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Supported by a grant from the Ministère de la Recherche et de la Technologie (PRFMMIP 2000) and grants from the Association F. Aupetit, Société Nationale Française de Gastro-Entérologie, and the Institut de Recherche des Maladies de l’Appareil Digestif (to J.B.).