STUDY ON GENETIC VARIABILITY OF BACILLUS CEREUS AND BACILLUS THURINGIENSIS

Raphael Leonardo (FIOCRUZ); Tatiana Brasil (UNICAMP); Viviane Zahner (FIOCRUZ)

Members of the Bacillus cereus group of organisms include Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis among others. Collectively, these organisms represent microbes of high economic, medical and biodefense importance. Given this significance, this group contains the highest number of closely related fully sequenced genomes, giving the unique opportunity for thorough comparative genomic analyses. Bacillus cereus is traditionally considered the most problematic member of the genus Bacillus to the food industry due to the ability of many strains to produce enterotoxins. In this work, heterogeneity of four virulence-associated gene: bceT, nheB, hblA and spH were analyzed from isolates of Bacillus cereus and Bacillus thuringiensis from Brazilian foods and soils, children with diarrhea, arthropods, annelida, ATTC global bioresource center and National Collection of Type Cultures (NCTC). Genomic DNA of isolates was amplified with specific primers for bceT, nheB, hblA and spH genes amplification and submitted for sequencing. Sequences were compared with available databases using BlastN from GenBank. BioEdit and ClustalW were used to sequence alignment and MEGA 4.0 to phylogenetic analysis and Expasy Tools was used in order to search for aminoacid sequence homology in the data bank. Our results show that those four virulence-associated genes are founded in both species and are very conserved among samples independently of their origin. Some point mutations were found in all samples and hblA sequence has the lowest level of heterogeneity. Divergences were found in intergenic region of NHE operon and in one sample isolated from children with diarrhea where bceT sequence codes for a protein with a completely different c-terminal than the expected. As observed in other works, phylogenetic analysis based in those sequences bring controversial results, and it is unclear whether B. cereus and B. thuringiensis are varieties of the same species or different species.