Functional grouping of thermophilic Bacillus strains using amplification profiles of the 16S-23S internal spacer region

Molecular and biochemical assays were used to determine the identification of thermophilic bacilli isolated from New Zealand milk powder. One hundred and forty one isolates of thermophilic bacilli were classified into six species using biochemical profiles. Geobacillus stearothermophilus represented 56% of the isolates. All isolates were also analysed by randomly amplified polymorphic DNA (RAPD) analysis, with 45 types identified. Amplification of the 16S-23S rDNA internal spacer region produced two to eight amplification products per strain. The patterns from gel electrophoresis of the internal spacer region amplicons formed two major groupings suggesting the possibility of two distinct species. Partial sequences of 16S rDNA from representatives from each group were compared with sequences in GeneBank and were found to match the 16S rDNA sequences of B. flavothermus and G. thermoleovorans. Primers were designed for these species and used to screen an arbitrary selection of 59 of the dairy isolates. This enabled the identification of 28 isolates as B. flavothermus and 31 isolates as Geobacillus species and these appear to be the predominant isolates in the New Zealand milk powder samples examined. Comparison of the fragment pattern generated by amplification of the 16S-23S rDNA internal spacer region is a simple method to differentiate thermophilic Bacillus species associated with the dairy industry.