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To assess the effect of two lactobacilli on the biological activity of enterohaemorrhagic Escherichia coli (EHEC) in vitro.Strains CIDCA 133 (Lactobacillus delbrueckii subsp. lactis) and CIDCA 83114 (Lactobacillus plantarum) were studied. Hep-2 cells were used as an in vitro model to assess the biological effect of a clinical isolate of EHEC. Preincubation of cell monolayers with lactobacilli before EHEC prevented detachment of eukaryotic cells and minimizes both F-actin rearrangements and morphological alterations. Interestingly, the protective effect could not be ascribed to pathogen exclusion. In addition, viability of the lactobacilli was not necessary for protection and other species of the genus Lactobacillus failed to protect eukaryotic cells.Our results suggest that lactobacilli are antagonizing virulence mechanisms of EHEC either by modification of the microenvironment or by interfering with the signalling cascades triggered by the pathogen.Our findings give a rationale basis for the use of specific probiotic strains for the prophylaxis and prevention of intestinal infections due to EHEC.