Multilocus Sequence Typing of close Neighbours to Bacillus anthracis isolated from Soil Samples

The Bacillus cereus group comprises of B. anthracis, B. cereus, B. thuringiensis, B. mycoides, B. pseudomycoides, B. weihenstephanensis and B. cytotoxicus. B. anthracis is the causative agent of anthrax, whilst B. cereus and B. thuringiensis (B. anthracis close neighbours) are mostly associated as food and insect pathogens respectively. During the last years, strains of B. cereus and B. thuringiensis have been the cause of serious infections in both humans and animals, and both phylogenetic and phenotypic characteristics associated with B. anthracis have been identified in some of these close neighbours. This has led to an increased interest for the different species and strains of B. anthracis close neighbours. The aim of this study was to evaluate the genetic variation among strains of the Bacillus cereus group, with a particular focus on isolates closely related to B. anthracis. Soil samples collected in Etosha National Park, Namibia, near a carcass of a B. anthracis infected Zebra were used to isolate 169 B. cereus group members using a selective growth medium. Isolation was followed by DNA extraction and the DNA was used in real time qPCR using six B. anthracis specific markers. One of the PCR primer pairs amplified a VNTR (variable number of tandem repeat) region and 52 B. cereus group isolates were selected for MLST (multi locus sequence typing) due to having a VNTR region identical to B. anthracis. In addition, 8 isolates were selected for MLST due to the generation of PCR products when using B. anthracis plasmid markers. Phylogenetic analyses were performed using an 11 loci MLST scheme (adk, ccpA, ftsA, glpF, glpT, panC, pta, pycA, pyre, recF and sucC) on 125 B. cereus group members (46 isolates from this study, 67 from Helgason et al. (2004), 5 in-house isolates and 7 from MLST Oslo), which resulted in 96 STs (sequences types). 39 B. cereus group members analysed during this study clustered in clade I. The two isolates (FFIBCgr36 and FFIBCgr46) that clustered closest to B. anthracis revealed only 10 and 12 point mutations that differentiating them from B. anthracis.