Abstract
BackgroundAnthrax, 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.ResultsThese 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 sub-lineage that is distributed geographically in countries of Asia, Africa, Europe and Australia.ConclusionsThe 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.
Highlights
Anthrax, a zoonotic disease is caused by the Gram positive bacterium Bacillus anthracis
Later it was reported that the mixed base pair R (G/A) at position 1139 in B. anthracis 16S rRNA gene owing to the presence of multiple rRNA operons in the genome could be used for its differentiation from other closely related B. cereus group species [6]
16S rDNA sequence analysis B. anthracis genome is characterized by the presence of multiple rRNA operons [5]
Summary
A zoonotic disease is caused by the Gram positive bacterium Bacillus anthracis. A defined set of 13 canSNPs from different loci in the genome has been used for molecular typing to determine the phylogenetic position and distinguish geographically separated B. anthracis isolates owing to their high level of genetic resolution and discriminatory power [9]. The distribution of these canSNPs identified 12 sub-lineages from the B. anthracis clonal lineages A, B and C representing different geographical locations [9]
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