Antibiotic MIC/MBC analysis of Bacillus-based commercial insecticides: use of bioreduction and DNA-based assays.

Minimum inhibitory concentration (MIC) assays, monitored by colony counts, growth (turbidity) and bioreduction of non-toxic XTT [2,3-bis (2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide, inner salt], were used to assess the performance of several types of antibiotics against: (1) commercial BT products made from scale-up sporulation phase cultures of Bacillus thuringiensis subsp israelensis (Bti) and subsp kurstaki (Btk); (2) vegetative cells derived from these BT products; and (3) Gram-positive and Gram-negative bacteria used as controls. The XTT-kinetic assay improved sensitivity and early reading of MIC breakpoints. The conventional colony count method for determining minimal bactericidal concentration (MBC) was used to validate a multi-sample dot-blot assay in which organisms in individual MIC assays are trapped free of residual antibiotic and their viability is estimated by in situ conversion of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] to insoluble formazan. Tolerance (MBC/MIC) for most antibiotics was low (</=4). Resistance to beta-lactams was attributed to beta-lactamase activity in both BT products and cultures derived from them. MIC and MBC breakpoints in spore-based assays were also approximated by changes in genome copy, using delta-endotoxin and beta-lactamase genes as probes. The DNA assays are effective for monitoring and authenticating organisms in microbe-containing biotechnology products.