Molecular characterization of B. anthracis isolates from the anthrax outbreak among cattle in Karnataka, India

Background: Anthrax, a zoonotic disease is caused by the Gram positive bacterium Bacillus anthracis. During January 2013, an anthrax outbreak among cattle was reported in Gundlupet Taluk, neighboring Bandipur National Park and tiger reserve, India. The present study aims at the molecular identification and characterization of 12 B. anthracis isolates from this outbreak by 16S rRNA gene sequencing, screening B. anthracis specific prophages and chromosomal markers, protective antigen (pag) gene and canonical single nucleotide polymorphism (canSNP) analysis to subtype the isolates into one of the twelve globally identified clonal sub-lineages of B. anthracis. Results: These isolates had identical 16S rDNA nucleotide sequences with B. anthracis specific dual peaks showing mixed base pair R (G/A) at position 1139 with visual inspection while the automated basecaller software indicated a G. Alternatively the nucleotide A at 1146 position was indicative of the 16S rDNA type 7. Multiple sequence alignment with additional 170 (16S rDNA) sequences of B. cereus sensu lato group from GenBank database revealed 28 new 16S types in addition to eleven 16S types reported earlier. The twelve B. anthracis isolates were found to harbor the four B. anthracis specific prophages (lambdaBa01, lambdaBa02, lambdaBa03, and lambdaBa04) along with its four specific loci markers (dhp 61.183, dhp 77.002, dhp 73.019, and dhp 73.017). The pag gene sequencing identified the isolates as protective antigen (PA) genotype I with phenylalanine-proline-alanine phenotype (FPA phenotype). However, sequence clustering with additional 34 pag sequences from GenBank revealed two additional missense mutations at nucleotide positions 196 bp and 869 bp of the 2294 bp pag sequence among the 5 B. cereus strains with pXO1 like plasmids. The canSNP analysis showed that the isolates belong to A.Br.Aust94 sublineage that is distributed geographically in countries of Asia, Africa, Europe and Australia. Conclusions: The analysis of 16S rDNA sequences reiterated the earlier findings that visual inspection of electropherogram for position 1139 having nucleotide R could be used for B. anthracis identification and not the consensus sequence from base caller. The canSNP results indicated that the anthrax outbreak among cattle was caused by B. anthracis of A.Br.Aust94 sub-lineage.