B acillus thuringiensis HD ‐1 C ry − : development of a safe, non‐insecticidal simulant for B acillus anthracis

Aims A representative simulant for spores of B acillus anthracis is needed for field testing. B acillus thuringiensis is gaining recognition as a suitable organism. A strain that does not form the insecticidal, parasporal crystals that are characteristic of this species is a more accurate physical representative of B . anthracis spores . We developed noninsecticidal derivatives of two isolates of B . thuringiensis HD‐1. Methods and Results Two plasmid‐cured derivatives of B . thuringiensis HD‐1, unable to make crystal toxins (‘Cry − ’), were isolated. These isolates and the existing Cry − strain, B . thuringiensis Al Hakam, were probed with PCR assays against the known insecticidal genes cry , vip and cyt . Their genomic DNA was sequenced to demonstrate a lack of insecticidal genes. This was confirmed by bioassays against a number of invertebrate species. Real‐time PCR assays were developed to identify the B . thuringiensis HD‐1 Cry − derivatives and an effective differential and selective medium was assessed. Conclusions All three Cry − isolates are devoid of known insecticidal determinants. The B . thuringiensis HD‐1 Cry − derivatives can easily be recovered from soil and identified by PCR with some selectivity. Significance and Impact of the Study The B . thuringiensis HD‐1 Cry − derivatives represent accurate, nongenetically manipulated simulants for B . anthracis with excellent human and environmental safety records.