Construction of Bacillus thuringiensis Simulant Strains Suitable for Environmental Release.

For a surrogate bacterium to be used in outdoor studies, it is important to consider environmental and human safety and ease of detection. Recently, Bacillus thuringiensis, a popular bioinsecticide bacterium, has been gaining attention as a surrogate bacterium for use in biodefense. In this study, we constructed simulant strains of B. thuringiensis with enhanced characteristics for environmental studies. Through transposon mutagenesis, pigment genes were inserted into the chromosome, producing yellow-colored colonies for easy detection. To prevent persistence of spores in the environment, a genetic circuit was designed to produce a spore without sporulation capability. Two loxP sites were inserted, one on each side of the spo0A gene, which encodes a sporulation master regulator, and a sporulation-dependent Cre expression cassette was inserted into the chromosome. This genetic circuit successfully deleted spo0A during sporulation, producing spores that lacked the spo0A gene. In addition, two major α/ß-type small acid-soluble spore protein (SASP) genes, predicted by synteny analysis, were deleted. The spores of the mutant strain showed increased UV-C sensitivity and quickly lost viability when tested in a solar simulator. When the spores of the mutant strain were administered to the lungs of BALB/c mice, cells were quickly removed from the body, suggesting enhanced in vivo safety. All strains constructed in this study contain no antibiotic resistance markers and all heterologous genes were inserted into the chromosome, which are useful features for simulants to be released into the environment.IMPORTANCEB. thuringiensis has recently been receiving increasing attention as a good spore simulant in biodefense research. However, few studies were done to properly address many important features of B. thuringiensis as a simulant in environmental studies. Since spores can persist in the environment for years after release, environmental contamination is a big problem, especially when genetically engineered strains are used. To solve these problems, we report here the development of B. thuringiensis simulant strains that are capable of forming yellow colonies for easy detection, incapable of forming spores more than once due to a genetic circuit, and lacking in two major SASP genes. The genetic circuit to produce a spore without sporulation capability, together with the deletion of SASP genes, ensures the environmental and human safety of the simulant strains developed in this study. All of these features will allow wider use of B. thuringiensis as a simulant for Bacillus anthracis in environmental release studies.

Effects of combinational prophylactics composed of physostigmine and procyclidine on soman-induced lethality, seizures and brain injuries.

The antidotal, anticonvulsant and neuroprotective effects of physostigmine (PhS) and procyclidine (PC), the combinational prophylactics for organophosphate poisoning, were evaluated. For the investigation of dose-response relationship in rats and guinea pigs, various doses (0-6 mg/kg) of PC in combination with a fixed dose (0.1 mg/kg) of PhS were pretreated subcutaneously 30 min prior to subcutaneous poisoning with soman. Procyclidine in combination with PhS exhibited remarkable synergistic effects in a dose-dependent manner, leading to 1.92-5.07 folds of protection ratio in rats and 3.00-4.70 folds in guinea pigs. On the other hand, a low effect (1.65 fold) was achieved with the traditional antidotes atropine (17.4 mg/kg) plus 2-pralidoxime (30 mg/kg) treated immediately after soman poisoning, compared with a marked protection (5.50 fold) with atropine (17.4 mg/kg) plus HI-6 (125 mg/kg) in unpretreated rats. Noteworthy, the combinational prophylactics greatly potentiated the effect of atropine plus 2-pralidoxime to 6.13 or 12.27 folds and that of atropine plus HI-6 to 12.00 or 21.50 folds with 1.0 or 3.0 mg/kg of PC, respectively. A high dose (100 µg/kg, 1.3×LD(50)) of soman induced severe epileptiform seizures in rats pretreated with HI-6 (125 mg/kg), resulting in brain injuries in discrete brain regions under histopathological examination in 24 h. Interestingly, such seizures and excitotoxic brain injuries were fully prevented by pretreatment with PhS (0.1 mg/kg) and PC (1 mg/kg). Taken together, it is proposed that the prophylactics composed of PhS and PC could be a promising regimen for the prevention of lethality, seizures and brain injuries induced by soman poisoning.