Phenotypic, Genetic and Biochemical Characterizations of Global Bacillus anthracis Isolates

Abstract

Thirty-one Bacillus anthracis strains, of world-wide origin, were screened for the presence of plasmids pX01 and pX02. These plasmids encode the known virulence factors associated with anthrax. The tripartite anthrax toxin is encoded by pX01, and the enzymes responsible for capsule synthesis are encoded on pX02. Sixteen virulent strains were chosen for further study and were selectively cured of the plasmids to create delta (Δ) strains which contained only the pX02 plasmid, novobiocin rough (NR) strains which contained only the pX01 plasmid, and ΔNR strains which contained neither of the plasmids. To determine if the loss of plasmid(s) is associated with changes in phenotypic and bichemical characteristics, strains were inoculated on specialized media and API test strips. The polymerase chain reaction was used to amplify the toxin and capsule genes. Variations in phenotypic and biochemical characteristics were observed in this study, but these changes did not seem to be associated with loss of plasmid. Some strains cured of pX02 seemed to produce colony types larger and flatter than typical B. anthracis colonies. However, this seemed to be associated with the actions of novobiocin used to cure the plasmid and not caused by loss of the plasmid. Two strains were found to produce halos on immunoassay agar and capsule on blood agar as well as bicarbonate agar but neither strain contained detectable plasmid elements. Restriction digest patterns of the amplified capsule genes were consistent with sequence data with the exception of the cap c gene. HinfI cleavage of this region did not result in fragments of the size predicted by the DNA sequence and further analysis revealed that oligonucleotide primers to the cap c region did not allow amplification of the entire gene.