Sporicidal Activity of the KMT reagent in its vapor phase against Geobacillus stearothermophilus Spores

In an investigation of the sporicidal activity of the KMT reagent, a vapor phase study was performed using five kinds of carriers contaminated with Geobacillus stearothermophilus spores. When 25 ml of the KMT reagent was vaporized in a chamber (capacity; approximately 95 liters), the 2-step heating method (vaporization by a combination of low temperature and high temperature) showed the most effective sporicidal activity in comparison with the 1-step heating method (rapid vaporization). The 2-step heating method appeared to be related to the sporicidal activity of vaporized KMT reagent, i.e., ethanol and iodine, which vaporized mainly when heated at a low temperature such as 55 C, and acidic water, which vaporized mainly when heated at a high temperature such as 300 C. We proposed that the KMT reagent can be used as a new disinfectant not only in the liquid phase but also in the vapor phase in the same way as peracetic acid and hydrogen peroxide.     

Assessing the activity of microbicides against bacterial spores: knowledge and pitfalls

Bacterial endospores (spores) have a higher intrinsic resistance to microbicides as compared to other microbial forms, most likely due to their impermeable outer layers and low water content. Though structural differences between the spores of various bacterial species may account for observed variations in their resistance to microbicides, flaws in methods for testing the sporicidal activity of microbicides often exaggerate the differences. This has major implications when considering the selection of one or more surrogates to assess microbicides against clinically relevant spore‐formers such as Clostridium difficile. The mounting significance of Cl. difficile as a pathogen is leading to a corresponding increase in the number of commercially available microbicidal formulations claiming activity against its spores without proper differentiation between the product's sporistatic and sporicidal actions. In this review we critically assess the situation and the implications of product claims on the field use of microbicidal products.     

Evaluation of the efficacy of electrochemically activated solutions against nosocomial pathogens and bacterial endospores

Aims: Electrochemically activated solutions (ECAS) are generated from halide salt solutions via specially designed electrolytic cells. The active solutions are known to possess high biocidal activity against a wide range of target microbial species, however, literature revealing the kill‐kinetics of these solutions is limited. The aim of the study was to identify the kill‐rate and extent of population kill for a range of target species (including endospores) using ECAS generated at the anode (anolyte). Methods and Results: Standard suspensions of methicillin‐resistant Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus atrophaeus spores and Clostridium difficile spores were treated with anolyte in a quantitative suspension assay. For vegetative cells, all concentrations of anolyte tested reduced the viable population to below the detection limit within 10 s. At a concentration of 99%, anolyte produced a log₁₀ reduction factor of greater than five in viable B. atrophaeus endospores within 90 s and reduced numbers of C. difficile endospores to below the experimental detection limit within 20 s at concentrations of 5% or greater. Conclusions: Anolyte was highly effective in killing test‐bacteria and spores. The bactericidal efficacy was retained against vegetative cells at dilutions as low as 1% and against C. difficile spores as low as 5%. Significance and Impact of Study: The results of this study demonstrate that ECAS are effective at lower concentrations and act more rapidly than previously reported. Potent bactericidal and sporicidal activity coupled with point‐of‐use generation, low production‐costs and environmental compatibility suggest that acidic ECAS has the potential to be a useful addition to the current armoury of disinfectants.     

An effective sporicidal reagent against Bacillus subtilis spores

We developed a reagent which showed significant sporicidal activity against Bacillus subtilis spores. This reagent was composed of ethylenediaminetetraacetic acid, disodium salt (EDTA-2Na), ferric chloride hexahydrate (FeCl3 x 6H2O) and ethanol (tentatively designated as the ethanol reagent). The ethanol reagent showed pH- and temperature-dependent sporicidal activity. At pH 0.3, its activity was almost the same as that of 0.05% sodium hypochlorite at 20 C and was higher at 37 C than at 20 C. The activity of the ethanol reagent was similar both with and without 10% serum. The ethanol reagent might be applicable for disinfecting Bacillus spores.     

An effective iodide formulation for killing Bacillus and Geobacillus spores over a wide temperature range

AIMS: To develop a sporicidal reagent which shows potent activity against bacterial spores not only at ambient temperatures but also at low temperatures. METHODS AND RESULTS: Suspension tests on spores of Bacillus and Geobacillus were conducted with the reagent based on a previously reported agent (N. Kida, Y. Mochizuki and F. Taguchi, Microbiology and Immunology 2003; 47: 279-283). The modified reagent (tentatively designated as the KMT reagent) was composed of 50 mmol l(-1) EDTA-2Na, 50 mmol l(-1) ferric chloride hexahydrate (FeCl(3).6H(2)O), 50 mmol l(-1) potassium iodide (KI) and 50% ethanol in 0.85% NaCl solution at pH 0.3. The KMT reagent showed significant sporicidal activity against three species of Bacillus and Geobacillus spores over a wide range of temperature. The KMT reagent had many practical advantages, i.e. activity was much less affected by organic substances than was sodium hypochlorite, it did not generate any harmful gas and it was stable for a long period at ambient temperatures. The mechanism(s) of sporicidal activity of the KMT reagent was considered to be based on active iodine species penetrating the spores with enhanced permeability of the spore cortex by a synergistic effect of acid, ethanol and generated active oxygen. CONCLUSIONS: The data suggest that the KMT reagent shows potent sporicidal activity over a wide range temperatures and possesses many advantages for practical applications. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate development of a highly applicable sporicidal reagent against Bacillus and Geobacillus spores.     

Destruction of Bacillus licheniformis spores by microwave irradiation

Aims:  To investigate the sporicidal mechanisms of microwave irradiation on Bacillus licheniformis spores.
Methods and Results:  We measured spore viability and the release of DNA and proteins, and performed transmission electron microscopy (TEM). A microwave oven (0·5 kW) was modified to output power at 2·0 kW, which allowed a shorter sterilization cycle. A 2·0 kW microwave treatment at the boiling temperature for 1 min did not kill all spores, but killed most spores. The spore inactivation rate was faster than that of boiling and 0·5 kW microwave oven. In contrast to boiling and 0·5 kW microwave treatments, the 2·0 kW microwave resulted in significant leakage of proteins and DNA from spores due to injury to the spore structure. TEM revealed that 2·0 kW microwave irradiation affected spore cortex hydrolysis and swelling, and ruptured the spore coat and inner membrane.
Conclusions:  These results suggest that 2·0 kW microwave irradiation ruptures the spore coat and inner membrane, and is significantly different from boiling.
Significance and Impact of the Study:  This study provides information on the sporicidal mechanisms of microwave irradiation on B. licheniformis spores.     

Virulent spores of Bacillus anthracis and other Bacillus species deposited on solid surfaces have similar sensitivity to chemical decontaminants

AIMS: To compare the relative sensitivity of Bacillus anthracis and spores of other Bacillus spp. deposited on different solid surfaces to inactivation by liquid chemical disinfecting agents. METHODS AND RESULTS: We prepared under similar conditions spores from five different virulent and three attenuated strains of B. anthracis, as well as spores of Bacillus subtilis, Bacillus atrophaeus (previously known as Bacillus globigii), Bacillus cereus, Bacillus thuringiensis and Bacillus megaterium. As spore-surface interactions may bias inactivation experiments, we evaluated the relative binding of different spores to carrier materials. The survival of spores deposited on glass, metallic or polymeric surfaces were quantitatively measured by ASTM standard method E-2414-05 which recovers spores from surfaces by increasing stringency. The number of spores inactivated by each decontaminant was similar and generally within 1 log among the 12 different Bacillus strains tested. This similarity among Bacillus strains and species was observed through a range of sporicidal efficacy on spores deposited on painted metal, polymeric rubber or glass. CONCLUSIONS: The data obtained indicate that the sensitivity of common simulants (B. atrophaeus and B. subtilis), as well as spores of B. cereus, B. thuringiensis, and B. megaterium, to inactivation by products that contain either: peroxide, chlorine or oxidants is similar to that shown by spores from all eight B. anthracis strains studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The comparative results of the present study suggest that decontamination and sterilization data obtained with simulants can be safely extrapolated to virulent spores of B. anthracis. Thus, valid conclusions on sporicidal efficacy could be drawn from safer and less costly experiments employing non-pathogenic spore simulants.