Characterization Of The Toxin Production And Transferable Tet(45) Gene In Bacillus Cereus

Food safty situation is still grim and caused wide public concern in China. Food poisoning incidents caused by microbial contamination happen frequently and have become a major problem of food safety. Bacillus cereus as a ubiquitous Gram-positive bacterium widely distributed in soil. B. cereus, as an opportunistic foodborne agent, often causes two main symptoms vomiting and diarrhea. Due to that the symptoms in patients are always self-healing and transient, the number of B. cereus incidents is highly under estimated.In mainland China, B. cereus is the second most common foodborne pathogen, following Salmonella caused incidents. However, as probiotics, B cereus has been widely used for medical treatment, animal husbandry and agriculture. Therefore, a systematic investigations on epidemiology and pathogenic characterizations of B. cereus are urgent needed in China.To systematically evaluate the transmission of B. cereus between host and environment, we collected 205 milk and 101 environmental samples from local dairy farms in Beijing, and bought 15 commercial probiotic products in China from 2013 to 2014.110 B. cereus-like strains were isolated, with the isolation rate of 34.5%. The detection rates of four toxin genes nhe, hbl, cytKl and ces were 100%,80%,0 and 0.9%, respectively.96.4% of the isolates could secrete non-hemolytical enterotoxin (Nhe) and 92.7% of the isolates showed cytotoxity to Vero cells. One emetic B. cereus named CAU45 showed high cytotoxicity to HEp-2 cells. Based on antimicrobial susceptibility tests, all isolates were resistant to lincomycin and pleuromulins and 35.5% isolates were insensitive to tetracycline with multidrug resistance rate at 23.6%.The milk origin emetic B. cereus CAU45 harbours both nhe and ces genes, nevertheless, there is no expression of nhe gene. The amount of cereulide secreted by B. cereus CAU45 was 7 times higher than the reference stain B. cereus DSMZ 4312. The cytotoxic titer of cereulide from B. cereus CAU45 was 15.3 times higher than B. cereus DSMZ 4312 in HEp-2 cells as well. Additionally, we established animal models of a single oral administrated mice and a single intravenous injected rabbits, which showed a dose dependent toxic effects of cereulide in mice and gender differences in both mice and rabbits. Lastly, the toxicokinetics of cereulide in rabbit models indicated that cereulide had a short elimination half-life of 10.8±9.1 h, with the mean retention time of 9.6±2.9 h, and acute toxic effects to liver.In addition, we found a novel plamid-borne tetracycline resistance gene tet(45) variant encoding for an efflux pump protein, in probiotic B. cereus CAU17. The plasmid-borne tet(45) gene could be successfully transferred into Staphylococcus aureus RN4220 by electrotransformation, and the electrotransformatant showed resistance to tetracycline as well. However, the flanking regions of tet(45) gene in B. cereus CAU17 showed significant difference to the tet(45) gene environment in other bacteria. The plasmid-borne tet(45) gene might be integrated into B. cereus CAU17 by its accompanying integrases.In conclusion, the pathogenic B. cereus strains show a huge hazard to the public health in China. On one hand, the toxins produced by B. cereus are more urgent to food safety. On the other hand, the transfereable antibiotic resistance gene found in probiotic B. cereus strain constitutes a potential threat to public health.