to eukaryotic cells by such a strain at a dose as low as 1 bacterium per cell can lead also to genomic instability,19 an event associ- ated with the predisposition to colorectal carcinogenesis develop- ment. We can hypothesize that the long-term use of E. coli Nissle 1917 to treat patients may significantly increase their risk to develop colorectal cancer. Conversely, the reduced inflammation conferred by clbA-dependent probiotic activity of E. coli Nissle 1917 treatment could decrease this risk, similarly to the reduced colitis-associated cancer observed for patients treated with other anti-inflammatory drugs such as mesalazine.24 To date, there is no report of short-term adverse side effects of this probiotic, but there is certainly a need for long-term studies evaluating the safety within these populations. A clear understanding of the benefits and risks of E. coli Nissle 1917 to help healthcare profes- sionals to reach a decision on the most suitable treatment for their patients is needed. The endogenous gastrointestinal microbiota is known to play a pivotal role in the development and maintenance of gut physi- ology and immune homeostasis.25 Alterations in its composition referred as dysbiosis play an important part in the pathogenesis of IBD.26,27 E. coli is an Enterobacteriaceae and bacteria of this taxonomic family are increasingly being recognized as microbial “inflammatory allies” contributing to the dysbiosis in IBD.28,29 As previously observed in other experimental models of IBD,28,30 the onset of colitis was followed by a spontaneously increase of endogenous Enterobacteriaceae feces levels in comparison with healthy control untransferred mice (Fig. S4). In addition, we have shown that the loss of the biosynthetic pathway encoded by the pks island not only abolishes probiotic properties of E. coli Nissle 1917 but also leads to the exacerbation of colitis severity (Figs. 2 and 3). Similar observations have been noticed in DSS-treated mice orally challenged with the reference adherent-invasive E. coli strain LF82.31 These pathogenic adherent-invasive strains of E. coli (AIEC) are abnormally predominant in ileal lesions of Crohn disease and seem to contribute to perpetuate colitis. Interestingly, LF82 stain belongs to the same B2 phylogenetic group than E. coli Nissle 1917. Similarly to E. coli Nissle 1917, E. coli strain LF82 does not harbor any known virulence factors expressed by pathogenic E. coli,32 but in contrast to E. coli Nissle 1917, LF82 strain does not harbor the pks island. Other studies have also suggested that certain commensal E. coli strains could potentiate the aggressiveness of intestinal inflammation by using E. coli monoassociated murine models of experimental colitis such as germ-free IL-10 deficient mice33 or IL-2 deficient mice.34 These opposite effects encountered among closely bacteria from the same species highlights that the crosstalk between probiotics, intestinal microbiota and host is probably more complex than previously supposed. In conclusion, although intervention studies with probiotics have yielded promising results,3,12,34 it remains difficult to draw general conclusions. This is probably due to the highly variable nature of the probiotic used since health benefit of one probi- otic strain cannot be extrapolated to others35 and bacteria from the same species may have opposite effects. Deciphering the precise molecular mechanisms supporting probiotic action, and potential side effects, are required to delineate the optimal use of probiotics as supplementary therapy. From this perspective, it is time to connect probiotic researches with approaches used for pathogens in the field of infectious disease. Materials and Methods Bacterial strains and mutagenesis procedures. In order to inves- tigate the potential role of Colibactin production in protection from colitis, we generated chromosomal isogenic mutants of E. coli Nissle 1917 (Mutaflor, DSM 6601, serotype O6:K5:H1) by using the lambda Red recombinase method as described previ- ously.36 Procedures, primers and templates used for directed- mutagenesis and resultant mutants are given in Supplemental Material and Table S1. Successful mutagenesis was confirmed by PCR using flanking primers and sequencing. Cell culture, in vitro infection and genotoxicity assay. Non transformed rat intestinal epithelial IEC-6 cells (ATCC CRL- 1592) were maintained in Dulbecco’s modified Eagle’s medium (DMEM GlutaMax; Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen), 80 μg ml–1 gentamicin, and bovine insulin (0.1 unit ml–1; Sigma) at 37°C in a 5% CO2 atmo- sphere. IEC-6 (~75% confluent) were washed four times and incubated in infection medium based on DMEM supplemented with 25 mM Hepes and 10% FBS (Invitrogen). Bacteria were pre-grown in infection medium to the midlogarithmic phase, and then the infection dose was calculated to a multiplicity of infection of 20 or 100 bacteria per cell. After a 4 h infection at 37°C and 5% CO2, the cells were washed and grown until analysis in cell culture medium supplemented with 200 μg/ml gentamicin. DNA double strand breaks were demonstrated by γ-H2AX immunofluorescence analysis. Mouse anti-γ-H2AX (Millipore) was diluted 1:500 in blocking solution and incubated overnight at 4°C. FITC-conjugated goat anti mouse secondary antibody (Invitrogen) was diluted 1:100 in blocking solution and incu- bated for 1 h. DNA was conterstained for 5 min with TO-PRO-3 (Invitrogen). Slides were mounted in Vectashield containing DAPI (Vectorlabs). Images were acquired with an Olympus IX70 laser scanning confocal microscope, in sequential mode, with the Fluoview FV500 software. At least 15 random fields have been evaluated blindly for γ-H2AX foci counting under 60× apochro- mic objective for each experiment. Pan-nuclear γ-H2AX staining consisted of nuclei with more than 100 γ-H2AX foci. Metaphasic chromosome spreading, visualization of chro- mosomal abnormalities and polyploid cells were done as previ- ously described.19 Chromosomes were stained with TO-PRO-3 (Invitrogen) and examined with an Olympus IX70 laser scanning confocal microscope under a 60× objective. Images were decon- voluted with Iris 5.59 software (Christian Buil, Iris software). Flow cytometry analyses were done with a FACScalibur flow cytometer (Bekton Dickinson). At least 2.104 cells were acquired per sample and analyzed with FlowJo software (Tree Star). Animals. Male Wistar rats (180–200 g body weight; Janvier) or male SCID mice (25 g body weight; Charles River) were main- tained under specific pathogen-free condition in Animal Care Facility of Neuro-gastroenterology group with free access to food 506 Gut Microbes Volume 3 Issue 6