Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) stimulates murine macrophages infected with Citrobacter rodentium.

Citrobacter rodentium is a specific murine enteropathogen which causes diarrheal disease characterized by colonic hyperplasia and intestinal inflammation. Recruitment of neutrophils and macrophages constitute a key step to control the infection. Since modulation of the activity of professional phagocytic cells could contribute to improve host´s defences against C. rodentium, we investigated the effect of Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) on the interaction between murine macrophages (RAW 264.7) and C. rodentium. Phagocytosis, surface molecules and inducible nitric oxide synthase (iNOs) expression were determined by flow cytometry. Reactive oxygen species (ROS) were assessed by fluorescence microscopy. The presence of lactobacilli increased phagocytosis of C. rodentium whereas C. rodentium had no effect on lactobacilli internalization. Survival of internalized C. rodentium diminished when strain CIDCA 133 was present. CD-86, MHCII, iNOs expression and nitrite production were increased when C. rodentium and lactobacilli were present even though strain CIDCA 133 alone had no effect. Strain CIDCA 133 led to a strong induction of ROS activity which was not modified by C. rodentium. Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) is able to increase the activation of murine macrophages infected with C. rodentium. The sole presence of lactobacilli is enough to modify some stimulation markers (e.g. ROS induction) whereas other markers require the presence of both bacteria; thus, indicating a synergistic effect.

Fate of Bacillus cereus within phagocytic cells.

In this study we assessed the interaction of different strains of Bacillus cereus with murine peritoneal macrophages and cultured phagocytic cells (Raw 264.7 cells). Association, internalization, intracellular survival, routing of bacteria to different compartments and expression of MHCII were assessed in cells infected with different strains of B. cereus in vegetative form. Association values (adhering + internalized bacteria) and phagocytosis were higher for strain B10502 than those for strains 2 and M2. However, after 90 min interaction, intracellular survival was higher for strain 2 than for strains M2 and B10502. Acquisition of lysosomal markers by B. cereus containing vacuoles (BcCV), assessed by LAMP1 and Lysotracker labelling occurred shortly after internalization. The highest ratio of LAMP1(+)-BcCV was found for strain M2. This strain was able to survive longer than strain B10502 which routes to LAMP1 containing vacuoles to a lesser extent. In addition, strain M2 stimulated expression of MHCII by infected cells. Confocal analyses 60 or 90 min post-infection showed different percentages of co-localization of bacteria with Lysotracker. Results suggest strain-dependent interaction and intracellular killing of B. cereus by phagocytic cells. These findings could be relevant for the pathogenic potential of Bacillus cereus strains.

Murine model of Bacillus cereus gastrointestinal infection.

Bacillus cereus is a spore-forming micro-organism responsible for foodborne illness. In this study, we focus on the host response following intragastric challenge with a pathogenic B. cereus strain (B10502) isolated from a foodborne outbreak. C57BL/6J female mice were infected by gavage with strain B10502. Controls were administered with PBS. Infection leads to significant modification in relevant immune cells in the spleen, Peyer's patches (PP) and mesenteric lymph nodes (MLN). These findings correlated with an increase in the size of PP as compared with uninfected controls. Histological studies showed that B. cereus infection increased the ratio of intestinal goblet cells and induces mononuclear cell infiltrates in spleen at 5 days post-infection. Evaluation of cytokine mRNA expression demonstrated a significant increase in IFN-γ in MLN after 2 days of infection. The present work demonstrates that infection of mice with vegetative B. cereus is self-limited. Our findings determined relevant cell populations that were involved in the control of the pathogen through modification of the ratio and/or activation.

Interaction between Bacillus cereus and cultured human enterocytes: effect of calcium, cell differentiation, and bacterial extracellular factors.

Bacillus cereus interaction with cultured human enterocytes and the signaling pathways responsible for the biological effects of the infection were investigated. Results demonstrate that calcium depletion increases the ability of strains T1 and 2 to invade cells. Bacteria associated in greater extent to undifferentiated enterocytes and extracellular factors from strain 2 increased its own association and invasion. Inhibitors of signaling pathways related to phosphorylated lipids (U73122 and wortmannin) were able to significantly reduce cytoskeleton disruption induced by B. cereus infection. Adhesion of strain T1 decreased in the presence of U73122 and of wortmannin, as well as when those inhibitors were used together. In contrast, invasion values were diminished only by U73122. Results show that different factors are involved in the interaction between B. cereus and cultured human enterocytes. Following infection, disruption of the cytoskeleton could facilitate invasion of the eukaryotic cells.

Oral administration of kefiran induces changes in the balance of immune cells in a murine model.

The aim of the present study was to evaluate the effect of the oral administration of kefiran on the balance of immune cells in a murine model. Six week old BALB/c mice were treated with kefiran (300 mg/L) for 0, 2 and 7 days. Kefiran treatment increased the number of IgA+ cells in lamina propria after 2 and 7 days. Percentage of B220+/MHCII(high) cells in mesenteric lymph nodes (2 days) and Peyer's patches (7 days) was higher compared to untreated control mice. An increase of macrophages (F4/80+ cells) was observed in lamina propria and peritoneal cavity (2 and 7 days). In contrast, at day 7, macrophage population decreased in Peyer's patches. These results show the ability of kefiran to modify the balance of immune cells in intestinal mucosa. This property could be highly relevant for the comprehension of the probiotic effect attributed to kefir.