Combination of genetic tools to discern Bacillus species isolated from hot springs in South Africa

Using phylogenetic analysis of the 16S rRNA gene 43 Gram-positive, spore-forming bacteria of the phylum Firmicutes were isolated, cultured and identified from five hot water springs in South Africa. Thirty-nine isolates belonged to the family Bacillaceae, genus Bacillus (n = 31) and genus Anoxybacillus (n = 8), while four isolates belonged to the family Paenibacillaceae, genus Brevibacillus. The majority of isolates fell into the Bacillus Bergey’s Group A together with Bacillus subtilis and Bacillus licheniformis. One isolate matched Bacillus panaciterrae which has not previously been described as a hot-spring isolate. Three unknown isolates from this study (BLAST <95% match) and three “uncultured Bacillus” clones of isolates from hot springs in India, China and Indonesia listed in NCBI Genbank, were included in the analysis. When bioinformatic tools: Basic Local Alignment Search Tool (BLAST), in silico amplified rDNA restriction analysis (ARDRA), guanine-cytosine (GC) percentage and phylogenetic analysis are used in combination, but not independently, differentiation between the complex Bacillus and closely related species was possible. Identification that relies solely on BLAST of the 16S rRNA sequence can be misleading.