Interaction between potential probiotic lactic acid bacteria and Listeria monocytogenes in functional human gut models

Listeria (L.) monocytogenes is one of the most important foodborne pathogen causing severe systemic infections posing a significant health risk for pregnant women, newborns and other immunocompromised individuals. In order to maintain intracellular lifestyle this pathogen has evolved a number of mechanisms to exploit host process to grow and spread cell to cell without damaging the host cell. The traditional vaccination is not affordable for the treatment and control of listeriosis, therefore an intake of probiotic bacteria could be considerate as a novel and effective strategies to prevent it in susceptible people. In relation to the pathogenesis of L. monocytogenes the inhibition of its penetration into the epithelial human cells could be considered as the most desirable feature. In this contest, in vitro gut models are fundamental tools to investigate the potential inhibitory action of probiotic bacteria versus L. monocytogenes infection mechanism. Therefore, the aim of this study was the identification of lactic acid bacteria (LAB) strains capable of reducing L. monocytogenes invasion on in vitro human gut model. With this purpose, 17 strains of Lactobacillus (Lb.) plantarum, Lb. pentosus and Leuconostoc mesenteroides isolated from olives fermentations and resistance to a simulation of human digestion were tested in 2D and 3D models. In vitro 3D models were built using foetal human epithelial cells grown in microporous inserts and human macrophages placed under the epithelial layer, called H4 and TLT respectively. The 2D model was prepared using H4 cells only in the undifferentiated status. After a preliminary assay of adhesion in 3D model, all strains were tested in a simulation of L. monocytogenes infection performed in the 2D model. Inhibition of pathogen adhesion and invasion was assessed and the strains with the greater inhibition potentiality were selected and tried in 3D models. Invasion, translocation assays and the evaluation of macrophage viability were performed on 3D model, both treated or not with the selected strains. In order to enhance the knowledge on their L. monocytogenes inhibition action, the macrophages secretion of interleukin (IL)-10 and IL-12 was quantified by sandwich ELISA. Concerning the results, the Lb. plantarum S11T3E, together with Lb. plantarum S2T10D and Lb. pentosus S3T60C showed the ability to inhibit the L. monocytogenes invasion in 2D epithelial model (P<0.05). The L. pentosus strain was also able to reduce the pathogen adhesion (P<0.05). However, the three strains did not confirm in a significant way (P>0.05) the reduction of pathogen invasion when tested in 3D model, despite the S11T3E decreased the number of recovered L. monocytogenes by more than 50%. Moreover this strain reduced the stress conditions in the macrophages infected with L. monocytogenes but, concerning the modulation activity on the human cells, neither IL-12 nor IL-10 were detected. In conclusion, the strain Lb. plantarum S11T3E has shown the ability to significantly inhibit the invasion of L. monocytogenes in 2D model, but the mechanisms of inhibition did not seem related to its immunomodulation activity.