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Hamouda et al., Sporicidal Activity of BCTP Nanoemulsion ; Page 6 of 12 Figure 3. Time course of nanoemulsion sporicidal activity against Bacillus cereus. Incubation with BCTP diluted 1 : 100 resulted in 95% killing in 4 h. Incubation with BCTP 401 diluted 1 : 1000 resulted in 95% killing in only 30 min. Difference in killing between BCTP diluted 1 : 100 and BCTP 401 diluted 1 : 1000 up to 4-h point was significant (P < .05). Sporicidal activity of BCTP against B. anthracis. After initial in vitro experiments, the sporicidal activity of BCTP was tested against two virulent strains of B. anthracis (Ames and Vollum 1B). We found that BCTP at a 1 : 100 final dilution incorporated into growth medium completely inhibited the growth of 1 x 10° B. anthracis spores. Sporicidal assays in fluid media, after 4 h of incubation with BCTP at dilutions up to 1 : 1000 with either the Ames or the Vollum 1B spores, resulted in 91% sporicidal activity when the mixtures were incubated at 22°C and 96% sporicidal activity when the mixtures were incubated at 37°C (table 1). Table 1. Sporicidal activity of BCTP against 2 different strains of Bacillus anthracis spores as determined by colony reduction assay (“ killing). Sporicidal activity of BCTP 401 against B. anthracis. Because BCTP 401 was effective at higher dilutions and against more species of Bacillus spores than BCTP, it was tested against 4 different strains of B. anthracis at dilutions of up to 1 : 10,000 at 22°C to prevent germination. BCTP 401 showed peak sporicidal activity between ™1 : 1000 and ~1 : 5000 dilutions (table 2). It was less efficient at concentrations >1 : 100. Table 2. _Sporicidal activity of BCTP 401 against 4 different strains of Bacillus anthracis representing different clinical isolates. . Electron microscopic examination of the spores. Weused B. cereus because it is the most closely related to B. anthracis. Transmission electron microscopic examination of B. cereus spores treated with BCTP diluted 1 : 100 in TSB for 4 h revealed physical damage to the B. cereus spores, including extensive disruption of the spore coat and cortex with distortion and loss of density in the core (figure 4). Figure 4. Electron micrographs of Bacillus cereus spores before (top) and after (bottom) treatment with BCTP. Note uniform density in cortex and well-defined spore coat before treatment with BCTP. Spores after 4h of BCTP treatment show disruption in both spore coat and cortex, with loss of core components. http://www .journals.uchicago.edu/JID/journal/issues/v180n6/990281/99028 1 text html 2/18/2005