Molecular characterization and phylogenetic analysis of archived Bacillus anthracis isolates from Karnataka, India

Bacillus anthracis, the causative agent of anthrax, poses a persistent threat in endemic regions. In this study, eight archived B. anthracis isolates from the year 2018-2019 were successfully revived, highlighting the resilience of spores and the effectiveness of long-term storage in maintaining virulence. Genomic DNA was extracted using three methods: Qiagen DNeasy kit, Zymo bead-based method, and phenol-chloroform extraction. The inclusion of ampicillin during initial incubation in the phenol-chloroform method enhanced DNA yield by selectively eliminating vegetative cells without inducing sporulation. The highest DNA concentration of 3710 ng/μL was obtained using this method. PCR analysis confirmed the presence of pXO1 and pXO2 virulence plasmids, along with the chromosomal rpoB gene, using OIE-recommended primers. The rpoB marker was chosen over 16S rRNA for its superior resolution among closely related strains. Phylogenetic analysis with bootstrap replicates revealed conserved sequences in plasmid genes, while rpoB exhibited notable diversity, suggesting chromosomal variation among the isolates. These findings contribute to ongoing anthrax surveillance and molecular epidemiology efforts in Karnataka, India, and underscore the potential of combining plasmid and chromosomal markers in understanding strain-level variation. Further whole genome sequencing (WGS) will provide deeper insights into the genomic diversity and evolutionary dynamics of B. anthracis.