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The cytolethal distending toxin (CDT) is produced by several Gram-negative pathogenic bacteria. In addition to inflammation, experimental evidences are in favor of a protumoral role of CDT-harboring bacteria such as Escherichia coli, Campylobacter jejuni, or Helicobacter hepaticus. CDT may contribute to cell transformation in vitro and carcinogenesis in mice models, through the genotoxic action of CdtB catalytic subunit. Here, we investigate the mechanism of action by which CDT leads to genetic instability in human cell lines and colorectal organoids from healthy patients’ biopsies. We demonstrate that CDT holotoxin induces a replicative stress dependent on CdtB. The slowing down of DNA replication occurs mainly in late S phase, resulting in the expression of fragile sites and important chromosomic aberrations. These DNA abnormalities induced after CDT treatment are responsible for anaphase bridge formation in mitosis and interphase DNA bridge between daughter cells in G1 phase. Moreover, CDT-genotoxic potential preferentially affects human cycling cells compared to quiescent cells. Finally, the toxin induces nuclear distension associated to DNA damage in proliferating cells of human colorectal organoids, resulting in decreased growth. Our findings thus identify CDT as a bacterial virulence factor targeting proliferating cells, such as human colorectal progenitors or stem cells, inducing replicative stress and genetic instability transmitted to daughter cells that may therefore contribute to carcinogenesis. As some CDT-carrying bacterial strains were detected in patients with colorectal cancer, targeting these bacteria could be a promising therapeutic strategy.

Inflammatory Bowel Diseases (IBD) are chronic inflammatory disorders, where epithelial defects drive, at least in part, some of the pathology. We reconstituted human intestinal epithelial organ, by using three-dimension culture of human colon organoids. Our aim was to characterize morphological and functional phenotypes of control (non-IBD) organoids, compared to inflamed organoids from IBD patients. The results generated describe the epithelial defects associated with IBD in primary organoid cultures, and evaluate the use of this model for pharmacological testing of anti-inflammatory approaches. Human colonic tissues were obtained from either surgical resections or biopsies, all harvested in non-inflammatory zones. Crypts were isolated from controls (non-IBD) and IBD patients and were cultured up to 12-days. Morphological (size, budding formation, polarization, luminal content), cell composition (proliferation, differentiation, immaturity markers expression), and functional (chemokine and tight junction protein expression) parameters were measured by immunohistochemistry, RT-qPCR or western-blot. The effects of inflammatory cocktail or anti-inflammatory treatments were studied in controls and IBD organoid cultures respectively. Organoid cultures from controls or IBD patients had the same cell composition after 10 to 12-days of culture, but IBD organoid cultures showed an inflammatory phenotype with decreased size and budding capacity, increased cell death, luminal debris, and inverted polarization. Tight junction proteins were also significantly decreased in IBD organoid cultures. Inflammatory cytokine cocktail reproduced this inflammatory phenotype in non-IBD organoids. Clinically used treatments (5-ASA, glucocorticoids, anti-TNF) reduced some, but not all parameters. Inflammatory phenotype is associated with IBD epithelium, and can be studied in organoid cultures. This model constitutes a reliable human pre-clinical model to investigate new strategies targeting epithelial repair.

Vibrio species have been associated with recurrent mass mortalities of juvenile oysters Crassostrea gigas threatening oyster farming worldwide. However, knowledge of the ecology of pathogens in affected oyster farming areas remains scarce. Specifically, there are no data regarding (i) the environmental reservoirs of Vibrio populations pathogenic to oysters, (ii) the environmental factors favoring their transmission, and (iii) the influence of oyster farming on the persistence of those pathogens. This knowledge gap limits our capacity to predict and mitigate disease occurrence. To address these issues, we monitored Vibrio species potentially pathogenic to C. gigas in 2013 and 2014 in the Thau Lagoon, a major oyster farming region in the coastal French Mediterranean. Sampling stations were chosen inside and outside oyster farms. Abundance and composition of phyto-, microzoo-, and mesozooplankton communities were measured monthly. The spatial and temporal dynamics of plankton and Vibrio species were compared, and positive correlations between plankton species and vibrios were verified by qPCR on isolated specimens of plankton. Vibrio crassostreae was present in the water column over both years, whereas Vibrio tasmaniensis was mostly found in 2013 and Vibrio aestuarianus was never detected. Moreover, V. tasmaniensis and V. crassostreae were found both as free-living or plankton-attached vibrios 1 month after spring mortalities of the oyster juveniles. Overall, V. crassostreae was associated with temperature and plankton composition, whereas V. tasmaniensis correlated with plankton composition only. The abundance of Vibrio species in the water column was similar inside and outside oyster farms, suggesting important spatial dispersion of pathogens in surrounding areas. Remarkably, a major increase in V. tasmaniensis and V. crassostreae was measured in the sediment of oyster farms during cold months. Thus, a winter reservoir of pathogenic vibrios could contribute to their ecology in this Mediterranean shellfish farming ecosystem.