Aims:  To conduct in vitro and in vivo assessments of the safety of two species of Bacillus, one of which, Bacillus subtilis, is in current use as a food supplement. Methods and Results:  Cultured cell lines, Caco‐2, HEp‐2 and the mucus‐producing HT29‐16E cell line, were used to evaluate adhesion, invasion and cytotoxicity. The Natto strain of B. subtilis was shown to be able to invade and lyse cells. Neither species was able to adhere significantly to any cell line. The Natto strain was also shown to form biofilms. No strain produced any of the known Bacillus enterotoxins. Disc‐diffusion assays using a panel of antibiotics listed by the European Food Safety Authority (EFSA) showed that only Bacillus indicus carried resistance to clindamycin at a level above the minimum inhibitory concentration breakpoints set by the EFSA. In vivo assessments of acute and chronic dosing in guinea pigs and rabbits were made. No toxicity was observed in animals under these conditions. Conclusions:  Bacillus indicus and B. subtilis should be considered safe for oral use although the resistance of B. indicus to clindamycin requires further study. Significance and Impact of the Study:  The results support the use of B. subtilis and B. indicus strains as food supplements.

Probiotics based on Bacillus strains have been increasingly proposed for prophylactic and therapeutic use against several gastro-intestinal diseases. We studied safety for two Bacillus strains included in a popular East European probiotic. Bacillus subtilis strain that was sensitive to all antibiotics listed by the European Food Safety Authority. Bacillus licheniformis strain was resistant to chloramphenicol and clindamycin. Both were non-hemolytic and did not produce Hbl or Nhe enterotoxins. No bceT and cytK toxin genes were found. Study of acute toxicity in BALB/c mice demonstrated no treatment-related deaths. The oral LD(50) for both strains was more than 2 x 10(11) CFU. Chronic toxicity studies were performed on mice, rabbits, and pigs and showed no signs of toxicity or histological changes in either organs or tissues. We demonstrated that while certain risks may exist for the B. licheniformis strain considering antibiotic resistance, B. subtilis strain may be considered as non-pathogenic and safe for human consumption.

Recombinant Bacillus subtilis spores expressing a TB antigen, MPT64, were tested for their ability to protect mice against tuberculosis challenge. A chimeric gene consisting of the spore coat gene cotB fused to mpt64 was constructed, and expression of a stable CotB-MPT64 hybrid protein of the spore coat verified. Spores were evaluated as a live vaccine and also formaldehyde inactivated. Mice were given three doses of spores or alternatively used in a prime-boost regimen with BCG. The results showed that inactivated recombinant spores were able to reduce the bacterial burden in the lungs of mice to comparable levels to that of BCG. In the prime-boost regimen, both live and inactivated spores showed a reduction in bacterial load in comparison with BCG. ELISPOT and polyfunctional T-cell analysis were performed to examine cellular responses and showed that antigen-specific secretion of Th1 cytokines was stimulated after immunisation with inactive recombinant spores and BCG. In summary, recombinant spores can elicit Th1 responses, which are important for protection against TB disease.

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