Figure 1. E. coli Nissle 1917 but not a clbA isogenic mutant induces DNa double-strand breaks and chromosomal abnormalities in cultured Iec-6 intestinal epithelial cells. Rat intestinal crypt Iec-6 cells were transiently infected for 4 h with live E. coli Nissle 1917 (Nissle WT) or Nissle ΔclbA with a multiplicity of infection of 100 (A) or 20 (B) bacteria per cell, then washed and cultivated with gentamicin for 24–72 h. error bars represent the seM in all cases and data are representative of at least two experiments. (A) c ells were examined by confocal microscopy for DNa (blue), and phosphorylated h2a X (green), a sensitive marker of DNa double strand breaks.40 pan-nuclear γ-h2a X staining is highlighted with a yellow arrow. Bars = 10 μm. (B) Metaphasic chromosomes were visualized 72 h after infection. c hromatid break and chromosomal abnormalities are shown. (C) Quantification of the phosphorylated γ-h2a X foci was expressed both as the mean foci per cell for nuclei with less than 100 foci (left), and the percentage of nuclei with pan-nuclear staining (right). Quantification of chromosomal numerical abnormalities (polyploidy; below) was assessed by flow cytometry. polyploid cells (DNa content > 8 for s er10-phosphorylated histone h 3-positive cells) were gated and expressed as % of total mitotic cells. *p < 0.05, *** p < 0.001, **** p < 0.0001, one-factor a NOVa analysis, Bonferroni’s multiple comparison test. pathway could improve E. coli Nissle 1917 biosafety without altering the health benefits of this probiotic in the management of IBD. Results Construction of an isogenic mutant of E. coli Nissle 1917 (Nissle ΔclbA) unable to induce DNA double strand breaks and chromosomal abnormalities in eukaryotic cells. We have previously shown that the clbA gene localized on the pks island codes for a phosphopantetheinyl transferase that catalyzes the essential post-translational activation of polyketide synthases and non-ribosomal polypeptide synthetases involved in E. coli genotoxicity.18 To generate a non-genotoxic isogenic mutant of E. coli Nissle 1917, we disrupted clbA by allelic exchange. Whereas the Nissle wild-type (WT) strain was able to inflict high levels of DNA double strand breaks to cultured intesti- nal crypt cells, the isogenic Nissle ΔclbA mutant was unable to induce such damage, as revealed by analyzing the phosphory- lation of the histone γH2A X (Fig. 1A–C). We next assessed chromosomal abnormalities in dividing cells 72 h after infec- tion with a low dose of genotoxic bacteria inducing reversible DNA damage response that did not repair all double strand breaks, and may therefore lead to chromosome abnormalities. Whereas Nissle WT induced chromosomal abnormalities (poly- ploidy, chromatid break, translocation or ring chromosomes) as previously observed with other pks+ E. coli strains,19 the iso- genic Nissle ΔclbA mutant was unable to induce such damage (Fig. 1B and C). The non-genotoxic E. coli Nissle 1917 mutant is impaired in prevention of DSS-induced colitis. To examine the probi- otic properties of the non-genotoxic Nissle 1917 mutant, we first used a rat model in which colonic inflammation was chemi- cally induced by the administration of Dextran Sodium Sulfate (DSS) in drinking water. DSS induces acute colitis character- ized by bloody diarrhea, ulcerations and inflammatory infiltra- tion of the colon. Daily oral administration of PBS, Nissle WT or Nissle ΔclbA was initiated one week before DSS initiation and prolonged during the inflammation. Over the course of the experiments, fecal counts of Nissle WT and Nissle ΔclbA did not differ between groups (Fig. S1). As already observed,20-23 oral administration of Nissle WT limited the severity of the DSS-induced colitis. Consistent with a significant reduction of the colitis score (Fig. 2B), oral administration of Nissle WT decreased both colonic myeloperoxidase (MPO) activity and pro-inflammatory cytokine IL-1β production and increased production of the regulatory cytokine IL-10 (Fig. 2C and D). Whereas our results support the claim that administration of E. coli Nissle 1917 can reduce intestinal inflammation, treatment with the Nissle ΔclbA mutant failed to protect the colon from DSS-induced damage. Indeed, as compared with rats fed with Nissle WT, we observed in rats fed with Nissle ΔclbA mutant 502 Gut Microbes Volume 3 Issue 6