Transfer of Bacillus cereus spores from packaging paper into food.

Food packaging papers are not sterile, as the manufacturing is an open process, and the raw materials contain bacteria. We modeled the potential transfer of the Bacillus cereus spores from packaging paper to food by using a green fluorescent protein-expressing construct of Bacillus thuringiensis Bt 407Cry(-) [pHT315Omega(papha3-gfp)], abbreviated BT-1. Paper (260 g m(-2)) containing BT-1 was manufactured with equipment that allowed fiber formation similar to that of full-scale manufactured paper. BT-1 adhered to pulp during papermaking and survived similar to an authentic B. cereus. Rice and chocolate were exposed to the BT-1-containing paper for 10 or 30 days at 40 or 20 degrees C at relative air humidity of 10 to 60%. The majority of the spores remained immobilized inside the fiber web; only 0.001 to 0.03% transferred to the foods. This amount is low compared with the process hygiene criteria and densities commonly found in food, and it does not endanger food safety. To measure this, we introduced BT-1 spores into the paper in densities of 100 to 1,000 times higher than the amounts of the B. cereus group bacteria found in commercial paper. Of BT-1 spores, 0.03 to 0.1% transferred from the paper to fresh agar surface within 5 min of contact, which is more than to food during 10 to 30 days of exposure. The findings indicate that transfer from paper to dry food is restricted to those microbes that are exposed on the paper surface and readily detectable with a contact agar method.

Toxicological profile of cereulide, the Bacillus cereus emetic toxin, in functional assays with human, animal and bacterial cells.

Some strains of the endospore-forming bacterium Bacillus cereus produce a heat-stable ionophoric peptide, cereulide, of high human toxicity. We assessed cell toxicity of cereulide by measuring the toxicities of crude extracts of cereulide producing and non-producing strains of B. cereus, and of pure cereulide, using cells of human, animal and bacterial origins. Hepatic cell lines and boar sperm, with cytotoxicity and sperm motility, respectively, as the end points, were inhibited by 1 nM of cereulide present as B. cereus extract. RNA synthesis and cell proliferation in HepG2 cells was inhibited by 2 nM of cereulide. These toxic effects were explainable by the action of cereulide as a high-affinity mobile K+ carrier. Exposure to cereulide containing extracts of B. cereus caused neither activation of CYP1A1 nor genotoxicity (comet assay, micronucleus test) at concentrations below those that were cytotoxic (0.6 nM cereulide). Salmonella typhimurium reverse mutation (Ames) test was negative. Exposure of Vibrio fischeri to extracts of B. cereus caused stimulated luminescence up to 600%, independent on the presence of cereulide, but purified cereulide inhibited the luminescence with an IC(50% (30 min)) of 170 nM. Thus the luminescence-stimulating B. cereus substance(s) masked the toxicity of cereulide in B. cereus extracts to V. fischeri.