POLYPHASIC IDENTIFICATION OF CLOSELY RELATED BACILLUS SUBTILIS AND BACILLUS AMYLOLIQUEFACIENS ISOLATED FROM DAIRY FARMS AND MILK POWDER

Abstract

A polyphasic approach was done to definitively distinguish isolates into two closely related species of the Bacillus subtilis group collected from the dairy farm environment or commercial powdered milk. A hundred and fifty six isolates and type strains B. subtilis (1A337) and B. amyloliquefaciens (10A18) were classified according to phenotypic and molecular characteristics. Only differences in growth temperature could be used to distinguish isolates among the phenotypic traits tested, and these distinctions were supported by molecular analysis. Randomly amplified polymorphic DNA analysis (RAPD) analysis was shown to be a friendly, technically simple and accurate method for rapid screening and identification of B. subtilis and B. amyloliquefaciens. Further analysis of 16S rRNA, rpoB and gyrA gene sequences of the isolates was done to confirm species identification. Sequences from the isolates and type strains showed between 96.5-100% (16S rRNA), 94.8-100% (rpoB) and 80.6-99.6% (gyrA) similarity, thus allowing for more refined distinction using the rpoB and gyrA genes. In addition, gyrA gene sequences had greater discrimination potential in having higher divergence between species (18.2 ± 0.7 %) than did rpoB sequences (4.9 ± 0.3 %). BOX-PCR fingerprinting was shown to have the potential for analysis of genotypic diversity of these species at the strain level.