The differential effects of heat-shocking on the viability of spores from Bacillus anthracis, Bacillus subtilis, and Clostridium sporogenes after treatment with peracetic acid- and glutaraldehyde-based disinfectants.

This study investigated (1) the susceptibility of Bacillus anthracis (Ames strain), Bacillus subtilis (ATCC 19659), and Clostridium sporogenes (ATCC 3584) spores to commercially available peracetic acid (PAA)- and glutaraldehyde (GA)-based disinfectants, (2) the effects that heat-shocking spores after treatment with these disinfectants has on spore recovery, and (3) the timing of heat-shocking after disinfectant treatment that promotes the optimal recovery of spores deposited on carriers. Suspension tests were used to obtain inactivation kinetics for the disinfectants against three spore types. The effects of heat-shocking spores after disinfectant treatment were also determined. Generalized linear mixed models were used to estimate 6-log reduction times for each spore type, disinfectant, and heat treatment combination. Reduction times were compared statistically using the delta method. Carrier tests were performed according to AOAC Official Method 966.04 and a modified version that employed immediate heat-shocking after disinfectant treatment. Carrier test results were analyzed using Fisher's exact test. PAA-based disinfectants had significantly shorter 6-log reduction times than the GA-based disinfectant. Heat-shocking B. anthracis spores after PAA treatment resulted in significantly shorter 6-log reduction times. Conversely, heat-shocking B. subtilis spores after PAA treatment resulted in significantly longer 6-log reduction times. Significant interactions were also observed between spore type, disinfectant, and heat treatment combinations. Immediately heat-shocking spore carriers after disinfectant treatment produced greater spore recovery. Sporicidal activities of disinfectants were not consistent across spore species. The effects of heat-shocking spores after disinfectant treatment were dependent on both disinfectant and spore species. Caution must be used when extrapolating sporicidal data of disinfectants from one spore species to another. Heat-shocking provides a more accurate picture of spore survival for only some disinfectant/spore combinations. Collaborative studies should be conducted to further examine a revision of AOAC Official Method 966.04 relative to heat-shocking.

The differential susceptibility of spores from virulent and attenuated Bacillus anthracis strains to aldehyde- and hypochlorite-based disinfectants.

This study compared the sensitivity of spores from virulent and attenuated Bacillus anthracis strains in suspension to inactivation by various chemical disinfectants. Spore suspensions from two virulent strains (A0256 and A0372) and two attenuated strains (Sterne and A0141) of B. anthracis were tested against two aldehyde-based disinfectants and one hypochlorite-based disinfectant. A novel statistical model was used to estimate 4-log(10) reduction times for each disinfectant/strain combination. Reduction times were compared statistically using approximate Z and χ(2) tests. Although there was no consistent correlation between virulence and increased sporicidal resistance across all three disinfectants, spores from the two virulent and two attenuated strains did display significantly different susceptibilities to different disinfectants. Significant disinfectant-strain interactions were observed for two of the three disinfectants evaluated. The comparative results suggest that the use of surrogate organisms to model the inactivation kinetics of virulent B. anthracis spores may be misleading. The accuracy of such extrapolations is disinfectant dependent and must be used with caution.

A quadruplex real-time PCR assay for rapid detection and differentiation of the Clostridium botulinum toxin genes A, B, E and F.

Clostridium botulinum is the aetiological agent of botulism, a disease marked by flaccid paralysis that can progress to asphyxiation and death. This species is defined by the production of one of the botulinum neurotoxins (BoNTs), which are the most potent toxins known. Because of their potency, these toxins have the potential to be used as biological weapons, and therefore C. botulinum has been classified as a category A select agent. There are four related but antigenically distinct BoNT types that cause disease in humans, A, B, E and F. The mouse bioassay is the current gold standard by which BoNTs are confirmed. However, this method is expensive, slow and labour-intensive. Although PCR-based assays have been used extensively for the detection of BoNT-producing bacteria in food, animals and faecal samples, and recently to help diagnose disease in humans, no real-time quantitative PCR (qPCR) assay has yet been developed that can identify and differentiate all four BoNTs that cause disease in humans. This report describes the development of a qPCR single-tube assay that uniquely identifies these four BoNTs responsible for human disease. A total of 79 C. botulinum isolates with varying toxin types was evaluated in this study, as well as numerous near-neighbours and other bacterial species. The results showed that this quadruplex assay was capable of detecting any of the four toxin genes in a given sample at a sensitivity of about 130-840 fg genomic DNA and could detect the presence of up to all four BoNT genes simultaneously in a given sample. The assay was also functional in the presence of extraneous organic matter commonly found in various environmental samples.