Efeito protetor de L. diolivorans 1Z isolado de grãos de kefir na infecção experimental com Salmonella enterica subsp. enterica sorovar Typhimurium em modelo murino

Abstract

Kefir grains are composed by yeasts and lactic acid bacteria and the genus Lactobacillus make up an important part of the microbiota of the grains. Various beneficial effects have been attributed to ingestion of kefir, for example, improvement of lactose intolerance, hypocholesterolemic and immunomodulatory effects, among others. Part of these beneficial effects can be attributed to the lactobacilli, which makes kefir grains a source of potential probiotic microorganisms. Therefore, the objective of this work was to evaluate the probiotic capacity of lactobacilli isolated from Brazilian kefir grains cultured in milk or brown sugar solution. The survival rate and weight variation of mice treated or not with Lactobacillus strains (L. satsumensis 18P, L. kefiranofaciens 8U, L. casei 25P, L. perolens 17P2 and L. diolivorans 1Z) and challenged orally with Salmonella entericasubsp.entericaserovarTyphimurium were tested. L. diolivorans 1Z was selected for the best results (70% of the mice survived and continued gaining weight during the entire period of infection). After that, germfree mice were monoassociated with L. diolivorans 1Z by an oral single dose of 108 CFU of the lyophilized microorganism and after seven days were challenged orally with S.Typhimurium (PS group). Groups of animals were sacrificed at two, four, six and eight days after infection, and for each day, a control group (germfree mice challenged by the pathogen – CS group) was also sacrificed.At the same time, groupsofmonoassociatedand non-challengedanimals were also sacrificed (CP group) It was taken from the animals the ileum, for histopathological analysis and detection of pro and antiinflamatory cytokine gene expression; feces for antagonistic assay; and liver for enumeration of Salmonella, histopathological and morphometric analysis and intestinal fluid for sIgA detection. The administration of L. diolivorans 1Z didnot induce pathological changes in theileal mucosa andliver, did not increase theexpressionofproinflammatorycytokines in the ileum and liver (P> 0.05) andincreasedthesIgAtiters (P <0.05) when compared to the germ free control group (GF group). The number of Salmonella in feces of animals from PS group was statistically lesser than in CS Group (P < 0.05) from the fourth day of infection. The histopathological analysis revealed less damage of the ileal mucosa in PS group when compared to CS group from the sixth day after the infection. These results are in accordance with the reduction in Salmonella counts and lower expression of proinflammatory cytokines IFN-γ and TNF-α in intestine in PS group mice. The number of Salmonella in liver from group PS was smaller when compared to the CS group, reaching undetectable levels after the sixth day of infection that could be explained by an apparent increase in Küpffer cells and by increasedsecretionofsIgA in intestinal fluid in the PS group. In the liver, the expressionoftheproinflammatorycytokines IFN-γ, TNF-α, IL1-β andtheenzymeiNOSandanti-inflammatory IL-10 and TGF-β increased progressively over the days of infection in the PS and CS. Comparing the two groups, a tendency to decrease the expression of pro-inflammatory cytokines and increase of anti-inflammatory was observed in the PS group in relation to the CS group (P> 0.05). No differences were detected in the number of nuclei / mm2 in the liver between the PS and CS groups (P> 0.05) indicating that treatment with L. diolivorans 1Z wasnot able to decrease the inflammatory infiltrate for the liver. However, the qualitative histological analysis of the liver revealed that the inflammatory cell profile present in theorgan of the CS groupwaspredominantlypolymorphonuclear, while in the PS group it was predominantly mononuclear. As a consequence, the inflammatory response in the liver of the animals of the PS group was faster and more efficient for the decrease of the count of S. Typhimurium in the organ. The results of this work suggest that L.diolivorans 1Z is a potentiallyprobioticline, safe and capable of protecting animals challenged by S. typhimurium, apparentlybyimmunological mechanisms that deserve to be studied in future works.