Comparative Sporicidal Effects of Disinfectants after Release of a Biological Agent

Military Medicine, Jun 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.

Methods

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 �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.