Isolation of adhesive strains and evaluation of the colonization and immune response by Lactobacillus plantarum L2 in the rat gastrointestinal tract

Abstract

Five Lactobacillus strains were tested for their ability to adhere to Caco-2 and IEC-6 cell lines as in vitro models and to induce of the secretion of pro- and anti-inflammatory cytokines by human peripheral blood mononuclear cells (PBMCs). Among the tested strains, Lactobacillus plantarum L2 was the most adhesive strain, approximately 595+/-125 or 704+/-273 of the added bacteria adhered to Caco-2 or IEC-6 cell cultures, respectively. Furthermore, L. plantarum L2 was also found to induce a considerable level of IL-10 from PBMCs, but low levels of all three pro-inflammatory cytokines TNF-alpha, IFN-gamma and IL-12. From these results, one promising strain, L. plantarum L2, was selected for in vivo studies. For 28 days F344 rats were fed a daily dose of 2 x 10(9)L. plantarum L2; for the next 14 days the rats were not fed any Lactobacillus. Intestinal mucosal samples and feces were taken at days 0, 28 and 42 to determine the colonizing ability of the lactobacilli. Recovered Lactobacillus isolates were initially identified by API 50CHL and strain-specific PCR. Intestinal specimen was analyzed using fluorescence in situ hybridization with a strain-specific molecular probe, and immune cell populations were determined by immunostaining for evidence of immune responses at the colonized sites. After intake of L. plantarum L2 for 28 days, a significant increase in live L. plantarum was found in the rats' feces, small intestine and colon. The bacterial levels remained high even after the L. plantarum L2 administration had been stopped for two weeks. Strain-specific PCR and FISH provided clear and direct evidence of colonization of the rat gastrointestinal tract by L. plantarum L2. Additionally, a significant increase in CD19-positive cells in the ileum was observed after intake of L. plantarum L2. In conclusion, dietary supplementation with L. plantarum L2 induced significant colonization of the gastrointestinal tract of rats, and this was associated with significant alteration of the immune response in the gastrointestinal mucosa.