Sporicidal activity of hydrogen peroxide and peracetic acid against Bacillus anthracis spores

The aim of the presented study was determined the effectiveness of sporicidal activity the peracetic acid and the hydrogen peroxide against B. anthracis spores. In the investigations was used B. anthracis stain "Sterne" 34F2. As inactivators were applied 0,5 % natriumthiosulphate and catalase. The obtained results show that the sporicidal effect of studied substances depends from their concentration and operates time. 5% water solution of peracetic acid shows the full sporicidal activity after outflow 120 minutes and the hydrogen peroxide about concentration 30% after outflow 180 minutes. However the hydrogen peroxide.     

A note on the effect of sporulation conditions on the resistance of Bacillus spores to heat and chemicals

Spores of Bacillus subtilis SA22 harvested after 22 d incubation on nutrient agar at 30°C were more resistant to 0–04% peracetic acid at 20°C than spores harvested following 2 d incubation. Similarly, spores of B. subtilis globigii B17, harvested after 7 d incubation on a sporulation agar were up to 10 times less resistant to 0.04% peracetic acid at 20°C than spores harvested after 35 d incubation. An increase in resistance to heating at 100°C and to exposure to 17.7% hydrogen peroxide at 20°C occurred as the age of B. subtilis SA22 spores prior to harvesting increased, whereas differences in resistance were not observed with spores of B. subtilis globigii B17.     

Possible overestimation of surface disinfection efficiency by assessment methods based on liquid sampling procedures as demonstrated by in situ quantification of spore viability

The standard test methods used to assess the efficiency of a disinfectant applied to surfaces are often based on counting the microbial survivors sampled in a liquid, but total cell removal from surfaces is seldom achieved. One might therefore wonder whether evaluations of microbial survivors in liquid-sampled cells are representative of the levels of survivors in whole populations. The present study was thus designed to determine the "damaged/undamaged" status induced by a peracetic acid disinfection for Bacillus atrophaeus spores deposited on glass coupons directly on this substrate and to compare it to the status of spores collected in liquid by a sampling procedure. The method utilized to assess the viability of both surface-associated and liquid-sampled spores included fluorescence labeling with a combination of Syto 61 and Chemchrome V6 dyes and quantifications by analyzing the images acquired by confocal laser scanning microscopy. The principal result of the study was that the viability of spores sampled in the liquid was found to be poorer than that of surface-associated spores. For example, after 2 min of peracetic acid disinfection, less than 17% ± 5% of viable cells were detected among liquid-sampled cells compared to 79% ± 5% or 47% ± 4%, respectively, when the viability was evaluated on the surface after or without the sampling procedure. Moreover, assessments of the survivors collected in the liquid phase, evaluated using the microscopic method and standard plate counts, were well correlated. Evaluations based on the determination of survivors among the liquid-sampled cells can thus overestimate the efficiency of surface disinfection procedures.     

Effects of the peracetic acid and sodium hypochlorite on the colour stability and surface roughness of the denture base acrylic resins polymerised by microwave and water bath methods

Objective: This study evaluated the surface roughness (Ra) and color stability of acrylic resin colors (Lucitone 550, QC‐20 and Vipi‐Wave) used for fabricating bases for complete, removable dentures, overdentures and prosthetic protocol after immersion in chemical disinfectants (1% sodium hypochlorite and 2% peracetic acid) for 30 and 60 minutes. Material and Methods: Sixty specimens were made of each commercial brand of resin composite, and divided into 2 groups according to the chemical disinfectants. Specimens had undergone the finishing and polishing procedures, the initial color and roughness measurements were taken (t=0), and after this, ten test specimens of each commercial brand of resin composite were immersed in sodium hypochlorite and ten in peracetic acid, for 30 and 60 minutes, with measurements being taken after each immersion period. These data were submitted to statistical analysis. Results: There was evidence of an increase in Ra after 30 minutes immersion in the disinfectants in all the resins, with QC‐20 presenting the highest Ra values, and Vipi‐Wave the lowest. After 60 minutes immersion in the disinfectants all the resins presented statistically significant color alteration. Conclusions: Disinfection with 1% sodium hypochlorite and peracetic acid altered the properties of roughness and color of the resins.     

The resistance of Bacillus atrophaeus spores to the bactericidal activity of peracetic acid is influenced by both the nature of the solid substrates and the mode of contamination

Aims: To evaluate the impact of the mode of contamination in relation with the nature of solid substrates on the resistance of spores of Bacillus atrophaeus ‐selected as surrogates of Bacillus anthracis‐ to a disinfectant, peracetic acid. Methods and Results: Six materials confronted in urban and military environments were selected for their different structural and physicochemical properties. In parallel, two modes of contamination were examined, i.e. deposition and immersion. Deposition was used to simulate contamination by an aerosol and immersion by an extended contact with liquids. A pronounced difference in the biocontamination levels and spatial organization of spores was observed depending on the mode of contamination and the nature of the solid substrate considered, with consequences on decontamination. Contamination by immersion led to lower efficiency of peracetic acid decontamination than contamination by deposition. Infiltration of spores into porous materials after immersion is one reason. In contrast, the deposition mode aggregates cells at the surface of materials, explaining the similar disinfecting behaviour of porous and nonporous substrates when considering this inoculation route. Conclusions: The inoculation route was shown to be as influential a parameter as material characteristics (porosity and wettability) for decontamination efficacy. Significance and Impact of the Study: These results provide comparative information for the decontamination of B. atrophaeus spores in function of the mode of contamination and the nature of solid substrates.     

Sporicidal Effect of Peracetic Acid Vapor

The sporicidal activity of peracetic acid (PAA) vapor at 20, 40, 60, and 80% relative humidity (RH) and 25 C was determined on Bacillus subtilis var. niger spores on paper and glass surfaces. Appreciable activity occurred within 10 min of exposure to 1 mg of PAA per liter and 40% or higher RH. The sporicidal rate decreased from the optimum at 80% RH to a slight effect at 20% RH. Spores on an impermeable surface were more difficult to kill than those on a porous one, probably because the cells tend to pile up on an impermeable surface and the vapor penetrates poorly through the layer of covering cells.     

Killing bacterial spores by organic hydroperoxides

Killing of wild-type spores of Bacillus subtilis by t-butyl hydroperoxide, cumene hydroperoxide and peracetic acid was not through DNA damage, as shown by the absence of mutations in the survivors and the identical sensitivity of spores of strains with or without a recA mutation. In contrast, B. subtilis spores (termed α⁻β⁻) lacking the DNA protective α/β-type small, acid-soluble spore proteins (SASP) were more sensitive to t-butyl hydroperoxide and cumene hydroperoxide, and their killing was in large part through DNA damage, as shown by the high frequency of mutations in the survivors and the greater sensitivity of α⁻β⁻ recA spores. Analysis of t-butyl hydroperoxide-treated spores showed that generation of DNA damage in α⁻β⁻ spores was more rapid than in wild-type spores; α/β-type SASP also protected against DNA strand breakage in vitro caused by t-butyl hydroperoxide. α/β-Type SASP appeared to play no role in protection of spores from killing by peracetic acid; wild-type and α⁻β⁻ spores exhibited identical peracetic acid sensitivity and their killing by this agent appeared to be not through DNA damage. Received 17 December 1996/ Accepted in revised form 13 March 1997     

Sporicidal Activity of Peracetic Acid and β-Propiolactone at Subzero Temperatures

A method was developed to evaluate and measure the sporicidal activity of peracetic acid (PAA) and beta-propiolactone (BPL) at subzero temperatures as low as -40 C. Bacillus subtilis var. niger spores were used as the test organism. Both PAA and BPL were sporicidal at low temperatures, with PAA the more active. The temperature coefficients of the two chemicals are generally low over a range of 20 to -20 C, but increase significantly at temperatures below this. Results showed an initial lag in the PAA death rates that was directly dependent on the temperature. BPL did not show this lag time.     

The Destruction of Byssochlamys fulva asci by Low Concentrations of Gaseous Methyl Bromide and by Aqueous Solutions of Chlorine, an Iodophor and Peracetic Acid

S ummary : Byssochlamys fulva asci are resistant to high concentrations of aqueous chlorine and iodophor solutions, but are sensitive to peracetic acid. Concentrations of 2% and 4% of peracetic acid gave 99·9% reductions in 2·5 and 1·3 min, respectively. The asci were also sensitive to methyl bromide gas (MeBr), c. 5 × 10⁴ asci/g inoculated into tapioca starch powder ( a _w= 0·69) being killed in 30 days by 90 mg of MeBr/kg of starch. About 180 asci/g were killed by adding 60 mg of MeBr/kg of starch.     

Sporicidal Activity of Peracetic Acid and β-Propiolactone at Subzero Temperatures

A method was developed to evaluate and measure the sporicidal activity of peracetic acid (PAA) and β-propiolactone (BPL) at subzero temperatures as low as -40 C. Bacillus subtilis var. niger spores were used as the test organism. Both PAA and BPL were sporicidal at low temperatures, with PAA the more active. The temperature coefficients of the two chemicals are generally low over a range of 20 to -20 C, but increase significantly at temperatures below this. Results showed an initial lag in the PAA death rates that was directly dependent on the temperature. BPL did not show this lag time.     

The bactericidal, fungicidal and sporicidal properties of hydrogen peroxide and peracetic acid

The antimicrobial properties of aqueous solutions of peracetic acid and hydrogen peroxide have been compared. Peracetic acid exhibited excellent antimicrobial properties, especially under acidic conditions. Reductions by a factor of 10⁶ in the numbers of vegetative bacteria are obtained within 1 min at 25°C using a solution containing 1.3 mmol/l of peracetic acid. Rapid activity against bacterial spores and yeasts also occurs. Hydrogen peroxide is more effective as a sporicide than as a bactericide, with sporicidal action being obtained using a solution containing 0.88 mol/l. Bactericidal action is poor but hydrogen peroxide was bacteriostatic at concentrations above 0.15 mmol/l.     

Comparative sporicidal effects of liquid chemical agents.

We compared the effectiveness of glutaraldehyde, formaldehyde, hydrogen peroxide, peracetic acid, cupric ascorbate (plus a sublethal amount of hydrogen peroxide), sodium hypochlorite, and phenol to inactivate Bacillus subtilis spores under various conditions. Each chemical agent was distinctly affected by pH, storage time after activation, dilution, and temperature. Only three of the preparations (hypochlorite, peracetic acid, and cupric ascorbate) studied here inactivated more than 99.9% of the spore load after a 30-min incubation at 20 degrees C at concentrations generally used to decontaminate medical devices. Under similar conditions, glutaraldehyde inactivated approximately 90%, and hydrogen peroxide, formaldehyde, and phenol produced little killing of spores in suspension. By kinetic analysis at different temperatures, we calculated the rate of spore inactivation (k) and the activation energy of spore killing (delta E) for each chemical agent. Rates of spore inactivation had a similar delta E value of approximately 20 kcal/mol (ca.83.68 kJ/mol) for every substance tested. The variation among k values allowed a quantitative comparison of liquid germicidal agents.     

Inactivation of Bacillus piliformis spores by heat and certain chemical disinfectants

The inactivation of Tyzzer's organism (Bacillus piliformis) spore isolated from rats by heat and various chemical disinfectants was studied. The spores were from B. piliformis-infected rat liver tissues. The spore suspension (10(4) 50% of rat liver lesion producing dose with prednisolone treatment/ml) was treated with heart or disinfectants. Inactivation of the spores was examined in experimentally infected rats. Rats were inoculated perorally with a treated spore suspension and injected subcutaneously with prednisolone. On the sixth day after inoculation, rats were examined grossly for liver lesions. Spores were inactivated at 80 degrees C for 15 min but not at 60 degrees C for 30 min. Spores were inactivated by 0.4% peracetic acid, 0.015% sodium hypochrolite, 1% iodophol, 5% phenol. Alcide and 0.37% formaldehyde solution, but not by 0.037% formaldehyde solution, 70% ethanol, 0.3% benzethonium chloride solution, 3% cresol and soap solution, or 4% chlorhexidine digluconate. These findings suggest that B. piliformis spores are relatively sensitive to heat and certain chemical disinfectants.     

New methods of disinfecting the hands]

An equimolar mixture--9.6 g of 85% formic acid and 19 g of 30% hydrogen peroxide which after 60-minute contact at room temperature contained over 3% and after 120-minute contact--over 5% performic acid was used. Before use the mentioned mixture was diluted with water (to the volume of 1 litre). Exposure period--3 minutes. Comparative tests demonstrated that a mixture with performic acid in hand disinfection provided somewhat better results than 0.2% peracetic acid, and that both peracids produced a much stronger disinfecting action on the skin microbial flora than 80% methyl alcohol.     

Prevention of DNA damage in spores and in vitro by small, acid-soluble proteins from Bacillus species.

The DNA in dormant spores of Bacillus species is saturated with a group of nonspecific DNA-binding proteins, termed alpha/beta-type small, acid-soluble spore proteins (SASP). These proteins alter DNA structure in vivo and in vitro, providing spore resistance to UV light. In addition, heat treatments (e.g., 85 degrees C for 30 min) which give little killing of wild-type spores of B. subtilis kill > 99% of spores which lack most alpha/beta-type SASP (termed alpha - beta - spores). Similar large differences in survival of wild-type and alpha - beta - spores were found at 90, 80, 65, 22, and 10 degrees C. After heat treatment (85 degrees C for 30 min) or prolonged storage (22 degrees C for 6 months) that gave > 99% killing of alpha - beta - spores, 10 to 20% of the survivors contained auxotrophic or asporogenous mutations. However, alpha - beta - spores heated for 30 min at 85 degrees C released no more dipicolinic acid than similarly heated wild-type spores (< 20% of the total dipicolinic acid) and triggered germination normally. In contrast, after a heat treatment (93 degrees C for 30 min) that gave > or = 99% killing of wild-type spores, < 1% of the survivors had acquired new obvious mutations, > 85% of the spore's dipicolinic acid had been released, and < 1% of the surviving spores could initiate spore germination. Analysis of DNA extracted from heated (85 degrees C, 30 min) and unheated wild-type spores and unheated alpha - beta - spores revealed very few single-strand breaks (< 1 per 20 kb) in the DNA. In contrast, the DNA from heated alpha- beta- spores had more than 10 single-strand breaks per 20 kb. These data suggest that binding of alpha/beta-type SASP to spore DNA in vivo greatly reduces DNA damage caused by heating, increasing spore heat resistance and long-term survival. While the precise nature of the initial DNA damage after heating of alpha- beta- spores that results in the single-strand breaks is not clear, a likely possibility is DNA depurination. A role for alpha/beta-type SASP in protecting DNA against depurination (and thus promoting spore survival) was further suggested by the demonstration that these proteins reduce the rate of DNA depurination in vitro at least 20-fold.     

Sporicidal action of peracetic acid and protective effects of transition metal ions

Although peracetic acid (PAA) is used widely for cold sterilization and disinfection, its mechanisms of sporicidal action are poorly understood. PAA at high concentrations (5–10%) can cause major loss of optical absorbance and microscopically-visible damage to bacterial spores. Spores killed by lower levels of PAA (0.02–0.05%) showed no visible damage and remained refractile. Treatment of spores ofBacillus megaterium ATCC 19213 with PAA at concentrations close to the lethal level sensitized the cells to subsequent heat killing. In addition, PAA was found to act in concert with hypochlorite and iodine to kill spores. Antioxidant sulfhydryl compounds or ascorbate protected spores against PAA killing. Trolox, a water-soluble form of -tocopherol, was somewhat protective, while other antioxidants, including -tocopherol, urate, bilirubin, ampicillin and ethanol were not protective. Chelators, including dipicolinate, were not protective, but transition metal ions, especially the reduced forms (Co²⁺, Cu⁺ and Fe²⁺) were highly protective. The net conclusions are that organic radicals formed from PAA are sporicidal and that they may act as reducing agents for spores that are normally in a highly oxidized state, in addition to their well known actions as oxidizing agents in causing damage to vegetative cells.     

A Comparative Study of the Effects of Five Chemicals on the Survival of Chlamydospores of Fusarium oxysporum f. sp. narcissi

Formaldehyde is routinely used in hot water treatment for the control of narcissus basal rot (caused by Fusarium oxysporum f. sp. narcissi) and stem and bulb eelworm (Ditylenchus dipsaci). It does not kill all the thick walled chlamydospores of F. oxysporum. Four chemicals were evaluated for their efficacy against F. oxysporum by comparing them with the standard formaldehyde rate. At rates of 0.25% a.i. or more, a glutaraldehyde formulation (Cidex) killed 100% of spores within 160 min. Peratol, a hydrogen peroxide and peracetic acid containing formulation, gave 100% kill after 80 min at a concentration of 0.5%. A thiabendazole formulation (Storite Clear Liquid) killed all the chlamydospores. Decon 90 failed to control the fungus adequately. Formaldehyde (as 0.5% commercial formalin) gave good, but not total, control of the fungus but, at higher rates (2.5%), caused flower malformation and corkiness of bulb base plates. The results suggest that Cidex and Peratol, as they are more effective than formaldehyde in controlling the fungus and less unpleasant to use, may be suitable replacements.     

Performic acid toxicity studies on stabilized HEp-2 cells

Since performic acid is used as a potent disinfectant, chemical sterilant and/or skin antiseptics, information related to its toxicity for living tissues was needed to evaluate the degree of its biotolerance and compare it to that of peracetic acid. In tests on cultured HEp-2 cells distinct cytotoxic effects occurred after treatments with 0.05% and higher concentrations. The 0.005% to 0.000,05% concentrations had only minor effects on the morphology of cells, if passaged they were capable of further growth, but were clearly inhibited in their growth ability. The lowest concentration tested (0.000005%) had no appreciable effect on HEp-2 cells. At the same concentration levels, the cells were affected to a greater degree with performic acid than with peracetic acid treatment. The use of cell cultures as a tool to study in vitro toxicity of various materials is discussed.     

Spores of microorganisms

The addition of different cysteine or thioproline concentrations (1–5×10^?4M) to the culture at the outset of the formation ofBacillus cereus prespores, i.e. before the commencement of dipicolinic acid synthesis, led to the death of some of the cells and injured the thermoprotection mechanism of the surviving spores. In control spores with a high dipicolinic acid content, inactivation by heating at 85°C was preceded by a lag phase, while in cysteine- and thioproline-treated spores this lag phase was completely absent and the death rate of most of the spores (D-value=17) was actually higher than the final death rate of the control spores (D-value=33). A small proportion of the spores in inhibited cultures (less than 10%) displayed almost the same heat resistance as untreated spores. The heat sensitivity of treated spores was greater than might have been anticipated from their dipicolinic acid content. Their resistance to X-rays was not lowered, but was actually slightly raised. The results are discussed with reference to the differentiation of a possible “basal” and “additional” spore thermoprotection mechanism and to differentiation of the nature of heat and radiation resistance in bacterial spores.