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Comparative Sporicidal Effects of Disinfectants After Release of a Biological Agent

June 28, 2007

By Kenar, Levent Ortatatli, Mesut; Yaren, Hakan; Karayilanoglu, Turan; Aydogan, Hakan

Because of spore formation, Bacillus anthracis is considered the most resistant biological warfare agent known. The present study aimed to assess and compare well-known decontamination routes to inactivate the spores on daily-use environmental tools contaminated previously. To simulate the agent, Bacillus atrophaeus was used. Various environmental samples (such as tile, fabric clothing, wood, protective suit, glass, paper, soil, water, plastic, and metal) that may be contaminated after a biological incident were used as test carriers and inoculated with B. atrophaeus. Sodium hypochlorite, free chlorine, autoclaving, ethylene oxide, hydrogen peroxide, ultraviolet irradiation, and boiling decontaminated the samples. Glutaraldehyde (2%) and free chlorine solution (10,000 mg/L) were also found to be effective in decontaminating the samples and are recommended as alternatives to the use of sodium hypochlorite solution. Soil, tile, paper, and metal were determined to be the most difficult materials to decontaminate. It was concluded that 5% hypochlorite adjusted with acetic acid might also be used for decontamination. Decontamination strategies to reduce contamination of the environment by biological warfare agents need to be applied to mitigate the number of victims, in terms of prominent characteristics like cost-effectiveness and user-friendliness. Introduction

Biological warfare (BW) agents may cause large number of casualties with minimal cost, compared with chemical, nuclear, or radiological weapons. For these reasons, some countries and terrorist groups continue to manufacture, to stockpile, and to use BW agents. Following the terrorist attacks of September 11,2001, and the subsequent distribution of anthrax by mail, biological weapons have become an even greater concern. The threat for the use of BW agents on military forces and civilian populations is now more likely than at any time in history. BW agents are easy to acquire, to synthesize, and to use. In addition, they are difficult to detect or to protect against because they are odorless and colorless and their dispersal can be performed silently.12

Aerosol dissemination would be the most likely method for a biological attack against large populations. The resultant dis eases and the BW agent used would be more difficult to define, and the proper treatment and prophylaxis would be more costly, compared with chemical weapons. Such a scenario would present serious challenges for patient treatment; therefore, the optimal goal is to prevent the use of such highly destructive biological weapons.

Biological weapons, including spores of Bacillus anihracis, can maintain their viability in the environment for years. It is extremely expensive, difficult, and time-consuming to eliminate spores of B. anthracis from a particular area, as seen in the case of Gruinard Island.1 Only limited data are available on the susceptibility of B. anihracis to current disinfecting and sterilizing agents. For this reason, we studied the susceptibility of the closely related Bociiius atrophaeus (formerly Bociiius subiiiis) to several sterilization methods. B. atrophoeus has been reported to be slightly less susceptible to germicides than B. anihracis and therefore is an excellent surrogate.2″4 However, assessment of sporicidal activities of different inactivators, including disinfecting and sterilizing agents, needs further clarification. The goal was to assess and to compare various disinfection methods for the inactivation of spores on daily-use environmental elements that might be contaminated following a BW attack.


Environmental Materials

A number of environmental materials, including tile, fabric clothing, wood, protective suit coated with activated carbon particles, glass, paper, plastic, and metal, were sampled in a standard dimension of 10 x 10 cm. These were simulated, contaminated elements following a biological attack. In addition, 5 mL of distilled water was taken into a sterile Vacutainer tube (BD Biosciences, Rutherford, New Jersey) and a soil sample of 1-cm thickness was placed into glass Petri dishes (10 cm in diameter). Seventeen specimens were prepared for each of the environmental samples.

Sterilization Methods

Sodium Hypochlorite

Sodium hypochlorite (NaOCl) was used in dilutions of 5%, 0.5%, and 0.05%, at pH 7 (adjusted with acetic acid) and at pH 12 for each dilution. Common household bleach (sodium hypochlorite) has a pH of 12 to prolong its shelf-life. To achieve effective sporicidal activity, bleach must be diluted with water and acetic acid to increase the available free chlorine to change the pH of the solution to 7.

Free Chlorine

Free chlorine solutions of 1,000 mg/L and 10,000 mg/L were prepared by dissolving 5-g tablets of 50% sodium dichloroisocyanurate (NaDCC) (purchased from Johnson and Johnson Medical, Dorset, United Kingdom) in autoclaved, sterile, distilled water. For solutions with 1,000 mg/L, 3.5 tablets were dissolved in 10 L of water; for solutions with 10,000 mg/L, 9 tablets were dissolved in 2.5 L. Hydrogen peroxide (3%, w/v) was purchased from Kim-Pa Drug Laboratory (Istanbul, Turkey). For sterilization with ethylene oxide, the materials were treated for 6 hours, and a common procedure for autoclave-sterilization at 1210C and 15 psi for 15 minutes was applied.

Thirty percent glutaraldehyde was diluted to 2% with water and the pH was adjusted to 8 by using sodium bicarbonate. For ultraviolet (UV) applications, an UV lamp (Philips TUV 30 W/G30T8; Philips, Mainz, Germany) with a dose rate at the level of irradiation of 83 mJ/cm^sup 2^ and a wavelength of 254 nm (UV-C) was used in a class II biosafery cabinet. After the spore suspension was dried, the plates were exposed to UV light at a distance of 50 cm from the source. In addition, each material was kept in boiling water for 10 and 30 minutes.


In the present study, spores of B. atrophaeus were used. To promote sporulation, B. atrophaeus was cultured on tryptose agar at 37[degrees]C for 48 hours, Mowed by 2 weeks at 23[degrees]C. Sporulation was periodically monitored by using the Schaeffer- Fulton stain, with which the vegetative cells appeared red to pink and the spores became green. When sporulation reached 95%, the number of bacterial spores was adjusted to McFarland 0.5 density, with dilution to 108 colony-forming units (CFU) per mL in distilled water. Viability of spores was assessed through the pour-plate method on trypticase soy agar, confirming populations of >10^sup 8^ CFU per mL.

All environmental material pieces were contaminated through pouring of 5 mL of suspension containing bacterial spores. After contamination, a number of sterilization methods were administered (Table I).

For bacterial counts, samples were taken from all materials by using a sterile trypticase soy broth-applied sterilized swab after the application of all decontamination methods. A chemical neutralizing agent was not used to remove the effect of disinfectant, however; 5 mL of trypticase soy broth dilution was used for this purpose. The samples were kept stored at 37[degrees]C for 1 hour under normal atmospheric conditions. They were cultured on blood agar with a sterile loop, and colony counts were scored as CFU per milliliter after 24 hours. The same procedures were used for control samples, which were not sterilized.


No bacterial growth was observed at either pH 7 or pH 12 with sterilization with 5% NaOCl for 30 minutes. No growth was seen at pH 7 for samples sterilized with 0.5% NaOCl, but bacteria were obtained in cultures of paper and soil samples at pH 12. When the concentration of NaOCl was diluted to 0.05%, samples of paper, soil, and metal gave positive results at pH 7. However, specimens from tile, paper, and soil samples had positive results at pH 12 (Table II). Bacteria were also grown in tile and water samples with 6-hour sterilization with ethylene oxide, but only sou samples showed growth with autoclaving at 121[degrees]C for 15 minutes (Table III).

Effectiveness was 100% for 30-minute sterilization with 2% glutaraldehyde solution, because no bacteria were found. No sample gave perfect sterilization, although the number of bacteria was reduced when sterilization with 10-minute boiling was evaluated. When the boiling time was increased to 30 minutes, samples of protective suit, paper, and distilled water provided perfect sterilization; the number of bacteria showed only reduction in other samples (Table IV).

All samples excluding soil were found to be completely decontaminated with the use of 3% hydrogen peroxide. Contaminated samples were irradiated in a laminar flow cabinet equipped with a germicidal UV lamp. When 12-hour UV irradiation was applied, bacteria were totally removed only from fabric clothing. A 24-hour UV exposure provided complete sterilization in samples taken from fabric, wood, protective suit, and paper (Table V).

Soil samples containing bacteria were found at 1,000 mg/L concentrations, but other samples were fully disinfected at both 1,000 mg/L and 10,000 mg/L (Table VI). Figure 1 illustrates the effectiveness of the various sterilization methods for the contaminated samples.


Many biocides are bactericidal or bacteriostatic at low concentrations for nonsporulating microorganisms, including the vegetative cells of Bociiius and Ciostridium species, but high concentrations may be necessary to achieve a sporicidal effect. A typical spore has a complex structure. In brief, the germ cell (protoplast or core) and its membranes are surrounded by the cortex, outside which are the inner and outer spore coats. A thin layer of exosporium may be present in the spores of some species but may surround with just one spore coat. The spore coats constitute a major portion of the spore. These structures consist largely of protein, with an alkali-soluble fraction made of acidic polypeptides found in the inner coat and an alkali-resistant fraction associated with the presence of disulfide-rich bonds found in the outer coat. These issues, especially the functions of the coat and cortex, are all relevant to the mechanism of resistance to antiseptics and disinfectants presented by bacterial spores.5,6 A number of methods may be needed to decontaminate different materials. For surface decontamination, the effectiveness of a decontaminant depends on the concentrations of the decontaminant and the agent, the type of agent, the time of contact, and other environmental conditions. Neutralization of disinfectants is important, because disinfectant carryover can result in a false no-growth result. Common methods for inhibition of residual biocide include dilution or chemical neutralization of the biocide.7 In our study, the effect of disinfectant was neutralized via dilution with trypticase soy broth.

As in a laboratory, where some items are wiped, some items are autoclaved, and some spaces are treated with the agent, more than one method may be required for decontamination.3,8 A number of sterilization methods has been used to disinfect soil, tools, clothes, buildings, and other environmental samples, including tile, fabric clothing, wood, protective suits, glass, paper, water, plastics, and metal, especially after an attack involving biological weapons such as anthrax.

Some reviews on the chemical, physical, and microbiological properties of chlorine-releasing agents have been reported. The most important types of chlorine-releasing agents are sodium hypochlorite, chlorine dioxide, and N-chloro compounds such as NaDCC, with chloramine-T being used to some extent.6,9,10 Sodium hypochlorite solutions (household bleach) are widely used for hard surface disinfection. In water, sodium hypochlorite ionizes to produce Na+ and the hypochlorite ion, OCl-, which establishes an equilibrium with hypochlorous acid, HOCl. Between pH 4 and 7, chlorine exists predominantly as HClO, the active moiety; above pH 9, OC1- predominates. Deleterious effects of chlorine-releasing agents on bacterial DNA, involving the formation of chlorinated derivatives of nucleotide bases, have been described.10,11 Hypochlorous acid has also been found to disrupt oxidative phosphorylation and other membrane-associated activities.11,12

The sporicidal effect of sodium hypochlorite might be changed by the concentration and pH.6,13 Therefore, we also used sodium hypochlorite, which is easily provided and stockpiled at pH 7 and pH 12. All samples were found to be free of bacteria and spores when 5% NaOCl, both at pH 7 and at pH 12, was used.

As the concentration of NaOCl was decreased, the sporicidal effect improved through pH, as also noted by Sagripanti and Bonifacino.13 The same concentration of NaOCl was found to be more effective in sporicidal activity at pH 7, compared with pH 12.

In our study, no bacterial growth was seen on all materials when 5% hypochlorite solution was used. A 0.5% sodium hypochlorite solution also caused no growth at pH 7, but positive results were obtained in cultures of paper and soil samples at pH 12. A NaOCl solution of 0.5% at pH 7 has been found to be very effective especially in skin decontamination of injury-free body areas, because it has neutral pH and effectiveness even at lower concentrations. The present study indicated that a 5% hypochlorite solution can be used confidently for environmental and vehicle decontamination and can be applied on very sensitive layers after the pH becomes neutral or acidic, for use in lower concentrations.

NaDCC releases free available chlorine in the form of HOCl (hypochlorous acid) and OC1~ (hypochlorite). It is generally supposed that the antimicrobial activity of NaDCC is attributable to chlorination of either cell protein or enzyme systems by free HOCl, causing hydrolysis of peptide chains found in the cell membranes of the microorganisms.10,14

Higher concentrations of H^sub 2^O^sub 2^ (10-30%) and longer contact times are required for sporicidal activity,15 although this activity is significantiy increased in the gaseous phase. H^sub 2^O^sub 2^ acts as an oxidant by producing hydroxyl free radicals (OH), which attack essential cell components, including lipids, proteins, and DNA. It has been proposed that exposed sulfhydryl groups and double bonds are particularly targeted.16

Ferma et al.17 found a minimal inhibitory concentration of 5.990 mg/L for NaDCC for B. atrophaeus. However, 1,000 mg/L levels of free chlorine used in our study could not provide complete sterilization, except in soil samples, using 3% hydrogen peroxide. All other samples were shown to be disinfected from bacteria spores.

Ethylene oxide is a flammable explosive gas that is known as both a mutagen and a carcinogen. The microbicidal activity of ethylene oxide is attributable to alkylation of sulfhydryl, arnino, carboxyl, phenolic, and hydroxyl groups in the spores or vegetative cells. The reaction of ethylene oxide with nucleic acids is the primary mechanism of its bactericidal and sporicidal activity. Ethylene oxide is used because of its ability to inactivate most bacteria, molds, yeasts, and viruses, but its use is limited because of the hazards mentioned. It was also observed that it might be used in sterilization of some tools that could be damaged with the use of hypochlorite.6,8,18 From this point of view, this method should be kept in mind for the sterilization of supposedly contaminated medical tools.

Sterilization through autoclaving is emphasized as the most effective and practical method for sterilization of materials containing liquid-like media.19″21 Our study also demonstrated that autoclaving could be safely used for decontamination of small, heat- resistant materials, such as metal and glass materials, contaminated after a possible biological attack. Only sterilization of soil samples was not possible using an autoclave.

Although it has been traditionally noted that disinfection and complete sterilization against bacteria spores are available with 10- minute boiling and 30-minute boiling, respectively,22-24 the single boiling procedure was not a safe method in our study. It is strongly suggested that decontamination of fabric clothing be performed by using the boiling method with water containing NaOCl or free chlorine.

Huber et al.25 reported that UV irradiation was effective in the sterilization of paper films incubated with B. atrophaeus spores, Serratia marcescens, and Mycobacterium tuberculosis. However, Morris26 emphasized that UV rays could not completely sterilize the layers, so the effectiveness of UV light was questioned. According to Schemeister,27 aluminum and glass layers could be sterilized, but wood and rubber could not be disinfected even after 4-hour UV sterilization.

It was also stressed that UV light had no sterilizing effect on a number of goods, including foods and fabrics, because of the preservation of microorganisms by solid bodies on the dust particles.27 Our study also showed that, in any contamination case, sterilization of valuable archives and documents, which are sometimes governmentally critical, might be possible if they were kept under UV rays for at least 24 hours. In addition, fabric, wood, and protective suit samples, which might be damaged during decontamination, were found to be completely sterilized with 24- hour UV irradiation.

Glutaraldehyde has a broad spectrum of activity against bacteria and their spores, fungi, and viruses. Low concentrations (0.1%) of glutaraldehyde inhibit germination, whereas much higher concentrations (2%) are sporicidal, probably as a consequence of strong interaction with outer cell layers. Aldehyde groups, at both acidic and alkaline pH values, interact strongly with the outer spore layers.6,28,29

The sporicidal activity of glutaraldehyde obtained under our test conditions was consistent with previous observations. Dyas and Das30 found that B. subtiiis subsp. globigii survived a 2-hour treatment with 2% glutaraldehyde. Boucher31 found that a 10-hour treatment was necessary for complete spore killing. In our study, 30-minute sterilization with 2% glutaraldehyde showed no growth for all samples.

The concentrations of 2% glutaraldehyde and 10,000 mg/L free chlorine solution were found to sterilize all samples with 100% effectiveness. This could be an alternative sterilization method when sodium hypochlorite solution is not available.


Available methods for disinfection of contaminated layers were discussed, with their effectiveness concerns. The highest levels of sterilization were found in samples of soil, tile, paper, and metal. The methods that might be used to disinfect materials contaminated with BW agents are summarized in Table VII. According to Table VII, we strongly suggest that NaOCl, free chlorine, and 2% glutaraldehyde be used practically, because those may be easily obtained and procured with little expense.

The inactivation of weapons of mass destruction has implications for laboratory-based research and development of fieldbased applications of the various decontamination methods. These issues are likely to become increasingly important in the arena of global politics, and further study is warranted.


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Guarantor: LT COL Levent Kenar

Contributors: LT COL Levent Kenar*; MAJ Mesut Ortatatli*; LIEUT Hakan Yaren*; COL Turan Karayllanoglu* LT COL Hakan Aydogan[dagger]

* Department of Medical Nuclear/Biological/Chemical Defense, Gulhane Military Medical Academy, 06018 Ankara, Turkey.

[dagger] Department of Microbiology and Clinical Microbiology, Gulhane Military Medical Academy, 06018 Ankara, Turkey.

This manuscript was received for review in May 2006. The revised manuscript was accepted for publication in January 2007.

Reprint & Copyright (c) by Association of Military Surgeons of U.S., 2007.

Copyright Association of Military Surgeons of the United States Jun 2007

(c) 2007 Military Medicine. Provided by ProQuest Information and Learning. All rights Reserved.