Fluorescent amplified fragment length polymorphism analysis of Norwegian Bacillus cereus and Bacillus thuringiensis soil isolates

We examined 154 Norwegian B. cereus and B. thuringiensis soil isolates (col lected from five different locations), 8 B. cereus and 2 B. thuringiensis r eference strains, and 2 Bacillus anthracis strains by using fluorescent amp lified fragment length polymorphism (AFLP). We employed a novel fragment id entification approach based on a hierarchical agglomerative clustering rout ine that identifies fragments in an automated fashion. No method is free of error, and we identified the major sources so that experiments can be desi gned to minimize its effect. Phylogenetic analysis of the fluorescent AFLP results reveals five genetic groups in these group 1 bacilli. The ATCC refe rence strains were restricted to two of the genetic groups, clearly not rep resentative of the diversity in these bacteria. Both B. anthracis strains a nalyzed were closely related and affiliated with a B. cereus milk isolate ( ATCC 4342) and a B. cereus human pathogenic strain (periodontitis). Across the entire study, pathogenic strains, including B. anthracis, were more clo sely related to one another than to the environmental isolates. Eight strai ns representing the five distinct phylogenetic clusters were further analyz ed by comparison of their 16S rRNA gene sequences to confirm the phylogenet ic status of these groups. This analysis was consistent with the AFLP analy sis, although of much lower resolution. The innovation of automated genotyp e analysis by using a replicated and statistical approach to fragment ident ification will allow very large sample analyses in the future.