Safety assessment of Bacillus subtilis CU1 for use as a probiotic in humans.

Bacillus subtilis CU1 is a recently described probiotic strain with beneficial effects on immune health in elderly subjects. The following work describes a series of studies supporting the safety of the strain for use as an ingredient in food and supplement preparations. Using a combination of 16S rDNA and gyrB nucleotide analyses, the species was identified as a member of the Bacillus subtilis complex (B. subtilis subsp. spizizenii). Further characterization of the organism at the strain level was achieved using random amplified polymorphic DNA polymerase chain reaction (RAPD PCR) and pulsed field gel electrophoresis (PFGE) analyses. B. subtilis CU1 did not demonstrate antibiotic resistance greater than existing regulatory cutoffs against clinically important antibiotics, did not induce hemolysis or produce surfactant factors, and was absent of toxigenic activity in vitro. Use of B. subtilis CU1 as a probiotic has recently been evaluated in a 16-week randomized, double-blind, placebo-controlled, parallel-arm study, in which 2 × 109 spores per day of B. subtilis CU1 were administered for a total 40 days to healthy elderly subjects (4 consumption periods of 10 days separated by 18-day washouts). This work describes safety related endpoints not previously reported. B. subtilis CU1 was safe and well-tolerated in the clinical subjects without undesirable physiological effects on markers of liver and kidney function, complete blood counts, hemodynamic parameters, and vital signs.

Secreted Compounds of the Probiotic Bacillus clausii Strain O/C Inhibit the Cytotoxic Effects Induced by Clostridium difficile and Bacillus cereus Toxins.

Although the use of probiotics based on Bacillus strains to fight off intestinal pathogens and antibiotic-associated diarrhea is widespread, the mechanisms involved in producing their beneficial effects remain unclear. Here, we studied the ability of compounds secreted by the probiotic Bacillus clausii strain O/C to counteract the cytotoxic effects induced by toxins of two pathogens, Clostridium difficile and Bacillus cereus, by evaluating eukaryotic cell viability and expression of selected genes. Coincubation of C. difficile and B. cereus toxic culture supernatants with the B. clausii supernatant completely prevented the damage induced by toxins in Vero and Caco-2 cells. The hemolytic effect of B. cereus was also avoided by the probiotic supernatant. Moreover, in these cells, the expression of rhoB, encoding a Rho GTPase target for C. difficile toxins, was normalized when C. difficile supernatant was pretreated using the B. clausii supernatant. All of the beneficial effects observed with the probiotic were abolished by the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Suspecting the involvement of a secreted protease in this protective effect, a protease was purified from the B. clausii supernatant and identified as a serine protease (M-protease; GenBank accession number Q99405). Experiments on Vero cells demonstrated the antitoxic activity of the purified protease against pathogen supernatants. This is the first report showing the capacity of a protease secreted by probiotic bacteria to inhibit the cytotoxic effects of toxinogenic C. difficile and B. cereus strains. This extracellular compound could be responsible, at least in part, for the protective effects observed for this human probiotic in antibiotic-associated diarrhea.

Probiotic strain Bacillus subtilis CU1 stimulates immune system of elderly during common infectious disease period: a randomized, double-blind placebo-controlled study.

BACKGROUND: Bacillus probiotics health benefits have been until now quite poorly studied in the elderly population. This study aimed to assess the effects of Bacillus subtilis CU1 consumption on immune stimulation and resistance to common infectious disease (CID) episodes in healthy free-living seniors. RESULTS: One hundred subjects aged 60-74 were included in this randomized, double-blind, placebo-controlled, parallel-arms study. Subjects consumed either the placebo or the probiotic (2.10(9) B. subtilis CU1 spores daily) by short periodical courses of 10 days intermittently, alternating 18-day course of break. This scheme was repeated 4 times during the study. Symptoms of gastrointestinal and upper/lower respiratory tract infections were recorded daily by the subjects throughout the study (4 months). Blood, saliva and stool samples were collected in a predefined subset of the first forty-four subjects enrolled in the study. B. subtilis CU1 supplementation did not statistically significantly decrease the mean number of days of reported CID symptoms over the 4-month of study (probiotic group: 5.1 (7.0) d, placebo group: 6.6 (7.3) d, P = 0.2015). However, in the subset of forty-four randomized subjects providing biological samples, we showed that consumption of B. subtilis CU1 significantly increased fecal and salivary secretory IgA concentrations compared to the placebo. A post-hoc analysis on this subset showed a decreased frequency of respiratory infections in the probiotc group compared to the placebo group. CONCLUSION: Taken together, our study provides evidence that B. subtilis CU1 supplementation during the winter period may be a safe effective way to stimulate immune responses in elderly subjects.

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.

Safety characterization and antimicrobial properties of kefir-isolated Lactobacillus kefiri.

Lactobacilli are generally regarded as safe; however, certain strains have been associated with cases of infection. Our workgroup has already assessed many functional properties of Lactobacillus kefiri, but parameters regarding safety must be studied before calling them probiotics. In this work, safety aspects and antimicrobial activity of L. kefiri strains were studied. None of the L. kefiri strains tested caused α- or ß-hemolysis. All the strains were susceptible to tetracycline, clindamycin, streptomycin, ampicillin, erythromycin, kanamycin, and gentamicin; meanwhile, two strains were resistant to chloramphenicol. On the other hand, all L. kefiri strains were able to inhibit both Gram(+) and Gram(-) pathogens. Regarding the in vitro results, L. kefiri CIDCA 8348 was selected to perform in vivo studies. Mice treated daily with an oral dose of 10(8) CFU during 21 days showed no signs of pain, lethargy, dehydration, or diarrhea, and the histological studies were consistent with those findings. Moreover, no differences in proinflammatory cytokines secretion were observed between treated and control mice. No translocation of microorganisms to blood, spleen, or liver was observed. Regarding these findings, L. kefiri CIDCA 8348 is a microorganism isolated from a dairy product with a great potential as probiotic for human or animal use.

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.