OVERALL ASSESSMENT OF A MODEL PROBIOTIC BACTERIUM: FROM GUT COLONIZATION TO CLINICAL EFFICACY

Probiotics are “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”. This definition is inclusive of a broad range of microbes and applications, whilst capturing the essence of probiotics (microbial, viable and beneficial to health). Specific guidelines describe the minimal requirement for the probiotic status. In particolar, the conventional process of selecting novel potential probiotic strains includes the assessment of: 1) the ability of the strains to survive during the gastrointestinal transit and reach alive the intestine, 2) the actual impact of the probiotic bacterium on the intestinal microbial ecosystem (IME). To evaluate these two aspects, in my PhD project we first carried out two recovery studies (in children and adults) with a selected probiotic bacterial strain, named Lactobacillus paracasei DG. Then, to evaluate the impact of the strain DG on IME we partecipated in a multicenter, randomized, double-blind, cross-over, placebo-controlled, pilot trial in irritable bowel syndrome (IBS) and determined the overall structure of the intestinal microbial communities by 16S rRNA gene profiling. Specifically, to demonstrate the capability of the selected bacterial strain to survive the gastrointestinal transit when consumed by healthy subjects, we developed and adopted in the recovery studies a strategy that combined culture-based methods and molecular methods for strain specific enumeration of viable cells in fecal samples. The results showed that the L. paracasei DG was re-isolated from at least one fecal sample of all the volunteers, survived the gastrointestinal transit and proliferated in the intestine, and persisted after the interruption of the probiotic intake up to 5 days in adults and 3 days in children. The results of the pilot study in IBS showed that Lactobacillus paracasei DG is able to modulate gut microbiota structure/function and reduce immune activation in IBS. Specifically, the strain induced a significant reduction in genus Ruminococcus, a significant increase in the short chain fatty acids (SCFAs) acetate and butyrate, and a significant reduction in the pro-inflammatory cytokine interleukin-15. Finally, we also investigated on mice the site of colonization of the probiotic bacterium in the intestine (animal study). The obtained results demostrated that L. paracasei DG colonized preferentially caecum and colon compared to ileum, suggesting a specific use of this probiotic in case of pathological situations with a localization at colonic level, such as diverticular disease and IBD, which are conditions including dysbiosis in their etiopathogenesis. At the end of my PhD, we focused on another very important point in the probiotic world, i.e. the “neglected” bacterial components of commercial probiotic formulations. In fact, it is quite clear that not only live, but also dead cells are present in probiotic products and they can generate beneficial biological responses. This can have several implications for the production and application of probiotics, influencing the potential health promoting effects since the relative proportions of live and dead cells in a probiotic formulation is usually unkwnon. This aspect can be very important, even while conducting clinical trials aiming at studying the efficacy of a probiotic product.