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_ Hamouda et al., Sporicidal Activity of BCTP Nanoemulsion Page 9 of 12 Discussion In these studies, we demonstrated that BCTP and its derivative BCTP 401 have effective sporicidal activity against a variety of Bacillus spores, including B. anthracis. BCTP diluted 1 : 100 has a sporicidal activity against B. cereus, B. circulans, and B. megaterium, whereas 1 : 1000 is effective against B. anthracis in 4h. BCTP 401, aBCTP-P10 mixture, appears to have a more rapid and broader sporicidal activity than BCTP. BCTP 401 diluted 1 : 1000 killed 95% of B. cereus spores in 30 min at 37°C, compared with a 70% reduction achieved by BCTP diluted 1: 100. BCTP 401 diluted 1 : 1000 was also effective in 4 h against B. subtilis spores that were resistant to BCTP for up to 24 h. BCTP 401 did not show effective sporicidal activity against B. anthracis at dilutions of <1 : 100, contrary to the . otiginal BCTP, which showed killing at dilutions between 1 : 10 and 1 : 1000. The fact that BCTP 401 requires dilution to be effective against B. anthracis spores suggests that BCTP 401 needs dispersion by water to minimize its aggregation and to facilitate direct contact with spores. Comparison of the sporicidal activity of BCTP against B. anthracis at 22°C, a temperature that does not promote spore germination, and at 37°C, at which germination occurs (as confirmed by microscopic examination), indicates that complete spore germination (j.e., outgrowth) is not necessary for the bactericidal activity of the emulsion. The small difference observed between the sporicidal activity at 37° C and 22°C may represent the killing of additional organisms from a few germinating spores. Sporicidal activity was also confirmed in water, a condition unsuitable for B. anthracis spore germination (data not shown). The sporicidal effect seems to start almost immediately and occurs within 30 min of incubation with the emulsion. Factors facilitating germination resulted in acceleration of the sporicidal activity of BCTP. Inhibition of the initiation of germination with D-alanine delayed BCTP's sporicidal activity. On the basis of these observations, we hypothesize that the sporicidal action of these emulsions occurs through initiation of germination before complete reversion to the vegetative form, leaving the spore susceptible to disruption by the emulsion. The initiation of germination could be mediated by the action of the emulsion or its components, but the emulsion appears necessary, as spores do not initiate germination in its absence. The results of the electron microscopy studies show disruption of the spore coat and cortex with disintegration of the core contents after BCTP treatment. However, the exact mechanism of killing is unclear and requires future investigation. Sporicidal activity appears to be mediated by both the Triton X-100 and tri-n-butyl phosphate components, because nanoemulsions lacking either component are inactive in vitro (data not shown). This unique sporicidal action of the emulsions, which is similar in efficiency to that of 1% bleach, is interesting because Bacillus spores are generally resistant to most disinfectants, including many commonly used detergents [15]. Animal studies demonstrated the protective and therapeutic effect of BCTP in vivo. B. cereus infection in experimental animals has been used previously as a model system for the study of anthrax [21, 22, 25]. The disease induced in animals experimentally infected with B. cereus is in many respects similar to anthrax [9, 23]. In this study, we demonstrated that mixing BCTP with B. cereus spores before injecting the spores into mice prevented the pathologic effect of B. cereus. We also demonstrated that BCTP treatment of simulated wounds contaminated with B. cereus spores markedly reduced the risk of {infection and mortality in mice. Because the emulsion appeared to lose sporicidal activity when diluted http://www journals.uchicago.edu/JID/journal/issues/v1 80n6/990281/990281 text. html 2/18/2005