◆ SpookStack

Hamouda et al., Sporicidal Activity of BCTP Nanoemulsion Page 4 of 12 were calculated by use of StatView software (Abacus Concepts, Berkeley, CA). Analysis of variance tables and paired f test were used when applicable. Electron microscopy. B. cereus spores were treated with BCTP at a final dilution of 1 : 100 in TSB by means of Erlenmeyer flasks in a 37°C shaker incubator. The spore-BCTP mixture was washed with saline and centrifuged at 2500 g for 20 min, and the supernatant was discarded. The pellet was fixed in 4% glutaraldehyde in 0.1.M cacodylate (pH 7.3). Spore pellets were processed for transmission electron microscopy, and thin sections were examined after staining with uranyl acetate and lead citrate. Germination inhibitors or enhancers. B. cereus spores (final concentration, 10° spores/mL) were suspended in TSB with either the germination inhibitor D-alanine (final concentration, 10 mJ) or the germination enhancer L-alanine (final concentration, 5 mA) [17-19]. This suspension was then immediately mixed with BCTP (final dilution, 1 : 100) and incubated for variable intervals. Then the mixtures were serially diluted, plated, and incubated overnight. The next day, growth on the plates was counted, and the percentage of sporicidal activity was calculated. In vivo toxicity testing. Mice were exposed to vatious concentrations of the different emulsions by means of different routes of administration. The highest concentrations that produced no gross or histopathologic lesions in mice were reported. Exposures included subcutaneous or intramuscular injection of 100 #L, open wound irrigation with 2 mL of the emulsions, and intranasal instillation of 25 HL /naris. The emulsions are relatively viscous when not diluted, so toxicity testing in the nares was conducted at the highest concentration that would not suffocate the animals. Three to four mice were tested for each concentration of each compound, and the experiments were repeated on at least three occasions. In vivo sporicidal activity. Two animal models were developed to confirm the sporicidal activity of the emulsions in vivo. In the first model, B. cereus spores (suspended in sterile saline) were mixed with an equal volume of BCTP to a final emulsion dilution of 1 : 10. As acontrol, the same B. cereus spore suspension was mixed with an equal volume of sterile saline. Next, 100 #L of each of the suspensions, containing 4 x 10’ spores, was then immediately injected subcutaneously into CD-1 mice. Nine mice were inoculated in each group, and the experiment was repeated on three different occasions. In the second model, a simulated wound was created by making an incision in the skin on the back of the mice. The skin was separated from the underlying muscle by blunt dissection. The pocket was inoculated with 200 HL of saline containing 2.5 x 10’ spores and closed by use of wound clips. One hour later, the clips were removed, and the wound was irrigated either with 2 mL of sterile saline or with 2 mL of BCTP (1 : 10 in sterile saline). The wounds were then closed with wound clips. The animals were observed for clinical signs. Gross and histopathologic examination were done when the animals were euthanized 5 days later. The wound size was calculated by the following formula: 3 ax 3 b x x, where aand b-are two perpendicular diameters of the wound. Five mice were used in each group, and the experiment was repeated on three different occasions. Both sets of animal studies were also conducted with BCTP 401 at identical dilutions. http://www journals.uchicago.edu/JID/journal/issues/v180n6/99028 1/990281 text. html 2/18/2005