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Background: Pathogenic adherent-invasive Escherichia coli have been isolated from ileal lesions of Crohn's disease., Aim: : To investigate the non-pathogenic E. coli strain Nissle 1917 (Mutaflor) as possible maintenance therapy in inflammatory bowel disease by testing its ability to prevent adherent-invasive E. coli strains from adhering to and invading human intestinal epithelial cells in vitro., Methods: Bacterial adhesion to and invasion of intestinal epithelial cells (Intestine-407) were assessed by counting the colony-forming units. The inhibitory effect of E. coli Nissle 1917 was determined after co-incubation with adherent-invasive E. coli strains or after pre-incubation of the intestinal epithelial cells with this probiotic strain prior to infection with adherent-invasive E. coli strains., Results: Strain Nissle 1917 exhibited dose- and time-dependent adherence to intestinal epithelial cells and inhibited the adhesion and invasion of various adherent-invasive E. coli strains. In co-infection experiments, the inhibitory effect on adherent-invasive E. coli adhesion reached 78-99.9%. Pre-incubation of intestinal epithelial cells with strain Nissle 1917 reduced adherent-invasive E. coli adhesion by 97.2-99.9%. The inhibitory effect on adherent-invasive E. coli invasion paralleled that on adhesion., Conclusion: As strong and significant inhibitory effects on adherent-invasive E. coli adhesion and invasion were observed in co-infection and pre-infection experiments, E. coli Nissle 1917 could be efficient for preventive or curative probiotic therapy in patients with Crohn's disease.

Genetic determinants that co-operate with type 1 pili to mediate invasion were sought for in adherent-invasive Escherichia coli strain LF82 isolated from a patient with Crohn's disease. Two mutants selected for their impaired ability to invade epithelial cells carried insertions of a TnphoA transposon within genes of the flagellar regulon. An isogenic mutant LF82-DeltafliC deleted for the flagellin-encoding gene did not adhere, did not invade and, surprisingly, expressed only a few type 1 pili. Type 1 pili downregulation resulted from a preferential switch towards the off-position of the invertible DNA element located upstream of the fim operon. This was also correlated with a decrease in the flagellar regulator flhDC mRNA levels, suggesting that the transcriptional regulator FlhD2C2 could control type 1 pili expression directly or indirectly. Transformation with a cloned fim operon allowed bypass of the type 1 pili downexpression in the LF82-DeltafliC mutant. Thus, we showed that flagella play a direct role in the adhesion process via active motility. In addition to downregulating type 1 pili expression, flagella also play an undefined role in strain LF82 invasion, which is not restricted to motility or flagellar structure, but could be related to co-ordinate expression of invasive determinants.

Escherichia coli strains recovered from Crohn's disease (CD) lesions are able to adhere to and invade cultured intestinal epithelial cells. We analyzed the behavior within macrophages of adherent invasive E. coli (AIEC) strains isolated from patients with CD. All the 15 AIEC strains tested were able to replicate extensively within J774-A1 cells: the numbers of intracellular bacteria increased 2.2- to 74.2-fold at 48 h over that at 1 h postinfection. By use of murine peritoneal macrophages and human monocyte-derived-macrophages, the reference AIEC strain LF82 was confirmed to be able to survive intracellularly. Transmission electron micrographs of AIEC LF82-infected macrophages showed that at 24 h postinfection, infected cells harbored large vacuoles containing numerous bacteria, as a result of the fusion of several vacuoles occurring after 8 h postinfection. No lactate dehydrogenase (LDH) release, no sign of DNA fragmentation or degradation, and no binding to fluorescein isothlocyanate-labeled annexin V were observed with LF82-infected J774-A1 cells, even after 24 h postinfection. LF82-infected J774-A1 cells secreted 2.7-fold more tumor necrosis factor alpha (TNF-alpha) than cells stimulated with 1 microg of lipopolysaccharide (LPS)/ml. No release of interleukin-1beta was observed with LPS-prestimulated J774-A1 cells infected with AIEC LF82. These findings showed that (i) AIEC strains are able to survive and to replicate within macrophages, (ii) AIEC LF82 replication does not induce any cell death of the infected cells, and (iii) LF82-infected J774-A1 cells release high levels of TNF-alpha. These properties could be related to some features of CD and particularly to granuloma formation, one of the hallmarks of CD lesions.

We previously characterized the invasive ability of Escherichia coli strain LF82, isolated from an ileal biopsy of a patient with Crohn's disease. In the present study, we performed TnphoA insertion mutagenesis to identify genes involved in LF82 invasion of intestinal epithelial cells. Most of the non-invasive mutants had an insertion mutation within the type 1 pili-encoding operon. Two non-invasive fim mutants, which harboured an insertion within the fimI and fimF genes, still adhered but had lost the ability to induce host cell membrane elongations at the sites of contact with the epithelial cells. Transcomplementation experiments with a fim operon cloned from E. coli K-12 restored both invasive ability and the ability to induce host cell membrane elongations. Expression of the cloned LF82 or K-12 fim operon into the non-invasive laboratory strain JM109 did not confer invasive properties. Thus, these findings showed that: (i) type 1 pili-mediated adherence is involved in LF82-induced perturbation of host cell signalling responsible for membrane elongations; (ii) native shafts are required for type 1 pilus-mediated induction of membrane elongations; (iii) this active phenomenon is a key step in the establishment of the invasive process; and (iv) type 1 pili alone are not sufficient to trigger bacterial internalization.