veronii infection. We used CFS of VR1 to examine its efficacy in amelioration of cytotoxicity caused by A. veronii supernatant. We observed high level of vacuole formation as an indication of cytotoxicity and morphological changes in Vero cells. Earlier, in an enterohaemorrhagic E. coli infection model, it was shown that pre-incubation

with L. plantarum abolished the cytotoxicity caused by enteropathogenic strain [10]. To test whether VR1 had similar effects, we studied the time dependent effects of CFS of A. veronii, VX-661 in vivo VR1, in combination or treatment of A. veronii on VR1 pre-incubated cells. We found that pre-incubation of Vero cells with VR1 CFS delayed cytotoxicity, which was induced by A. veronii. Vacuolating cytotoxic factor from A. veronii was earlier reported to cause cell death [38]. Tight junction disruption is considered to be one of the indicators of morphological damage caused due to cytotoxicity. MDCK cell line infected with V. cholerae cytotoxin and S. typhimurium showed a clear indication of epithelial barrier dysfunction by disruption of tight junction [39, 40]. In fish, pre-incubation with

prospective probiont L. delbrueckii sub sp. lactis could prevent epithelial damage caused by A. salmonicida [36]. To investigate the effect of CFS derived from VR1, and A. veronii on click here epithelial barrier, we selected MDCK cell line over Caco2 cell line mafosfamide because it exhibits similar epithelial characteristics like formation of uniform columnar epithelia, tight junction, and it has an advantage of a short culture period of 5-7 days in comparison to Caco2 which has 21 days of growth period [[41–43]]. We found that A. veronii indeed caused epithelial damage by disruption of ZO-1 and AMPK inhibitor F-Actin in MDCK cell line, which was prevented by pre-incubation with VR1 supernatant for 6 h, whereas co-incubation was not able to restore the epithelial integrity. ZO-1 is a cytoplasmic protein which interacts directly with F-Actin and is very important in structural and functional organisation of tight junction. In this study, microscopic observation of cellular damage is well supported

by immunolocalization of ZO-1 and F-Actin, which give clear evidence of VR1 in ameliorating the epithelial damage caused by A. veronii. This finding is consistent with earlier report that, L. rhamnosus GG treatment ameliorated the redistribution of ZO-1 and claudin in MDCK cell line caused by enterohemorrhagic E. coli [16]. In another study, incubation with CFS of B. lactis 420 has been shown to increase the intestinal epithelial integrity against enteropathogenic E. coli (EPEC) [44]. Cell viability assessed by MTT assay revealed that VR1 CFS treatment was not detrimental to cells and there was no loss in viability when pre-incubated with VR1 CFS. On the other hand, co-incubation could not prevent the loss in cell viability caused by A.