William R. Richter

Vapour‐phase hydrogen peroxide inactivates Yersinia pestis dried on polymers, steel, and glass surfaces

Aims:  This study evaluated the inactivation of virulent Yersinia pestis dried on polymers, steel, and glass surfaces using vapour‐phase hydrogen peroxide.

Bacillus Anthracis Spore Inactivation by Fumigant Decontamination

In 2001, envelopes containing virulent Bacillus anthracis spores were placed into the U.S. mail, resulting in contamination of mail processing and distribution facilities and office buildings. These spore-contaminated facilities were subsequently decontaminated primarily by fumigation with hydrogen peroxide, chlorine dioxide, and formaldehyde. These highly-publicized incidents resulted in increased public awareness of the threat to human health posed by Bacillus anthracis, and increased interest in sampling, detection, and decontamination of indoor surfaces, rooms, and buildings. Fumigants offer advantages over liquid application for decontaminating rooms or buildings due to the increased coverage of large surface areas and ease of cleanup. From 2001 to the present, however, no decontamination technology has been registered by the U.S. Environmental Protection Agency (EPA) for use against B. anthracis spores; rather, decontamination has been performed through the EPAs issuance of Crisis Exemptions. Since 2001, research and testing efforts have assessed the efficacy and maturity of sporicidal fumigants as well as the maturity, chemical toxicity, material compatibility, and ventilation requirements of these technologies. Several studies have been published in the scientific literature regarding fumigant inactivation of Bacillus spores; however, most decontamination studies with fumigants have utilized surrogates for B. anthracis surrogates, which can be more resistant to a specific type of fumigant (e.g., Geobacillus stearothermophilus and vaporous hydrogen peroxide) than virulent B. anthracis. Therefore, the purpose of this review is to summarize current knowledge available in the open scientific literature describing the inactivation of virulent B. anthracis spores by various fumigating agents with respect to key operational variables that can affect fumigant decontamination efficacy, such as fumigant concentration, contact time, operational temperature, and relative humidity.

Large-scale Inactivation of Bacillus anthracis Ames, Vollum, and Sterne Spores Using Vaporous Hydrogen Peroxide

This study evaluated the inactivation of Bacillus anthracis Ames, Vollum, and Sterne spores on various materials (glass, Hypalon® rubber glove, and stainless steel) using vaporous hydrogen peroxide fumigation of a ∼15 m3 aerosol research and component assessment (ARCA) chamber. Suspensions of each spore type (∼1 × 108 CFU) were dried on coupons made from each type of test surface and exposed to vaporous hydrogen peroxide fumigation for a decontamination time of 5.5 hours. For these three materials, the log reductions ranged from 7.7 to 7.9, 8.2 to 8.5, and 7.6 to 7.8 for B. anthracis Ames, Vollum, or Sterne spores, respectively. The effectiveness of vaporous hydrogen peroxide fumigation on the growth of Geobacillus stearothermophilus biological indicators (BI) was evaluated in parallel as a qualitative assessment of decontamination. At 1 and 7 days post-exposure, all decontaminated BI exhibited no growth. This study provides information for using vaporous hydrogen peroxide fumigation as an approach for the surface decontamination of B. anthracis spores within a large-scale chamber.

Inactivation of Brucella Suis, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis using Vaporous Hydrogen Peroxide

This study evaluated the inactivation of Brucella suis, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis on glass, Hypalon® rubber glove, and stainless steel using vaporous hydrogen peroxide fumigation of a ∼15 m3 chamber. A suspension of approximately 1 × 108 colony forming units (CFU) of each organism was dried on coupons of each type of test surface and exposed to vaporous hydrogen peroxide. A significant reduction in the log10 CFU of each organism on all test materials was observed between the controls evaluated after a 1-hour drying time and unexposed controls evaluated after decontamination. For all organisms, qualitative growth assessments showed that vaporous hydrogen peroxide exposure completely inactivated bacterial viability on all replicates of the test materials incubated up to 7 days post-exposure. In parallel, all Geobacillus stearothermophilus biological indicators (BI) exposed to vaporous hydrogen peroxide exhibited no growth after 1 and 7 days incubation. This study provides information on using a combination of quantitative and qualitative growth assessments to evaluate vaporous hydrogen peroxide for the surface decontamination of B. suis, B. pseudomallei, F. tularensis, and Y. pestis within a large-scale chamber.

A Novel Approach for Conducting Room-scale Vaporous Hydrogen Peroxide Decontamination of Virulent Bacillus Anthracis Spores

Studies have been conducted to determine the efficacy of various decontamination technologies against virulent B. anthracis and surrogate spores within small, bench-scale chambers. This study assessed an approach for evaluating room-scale (∼2,700 ft3) decontamination efficacy of vaporous hydrogen peroxide fumigation against B. anthracis Ames and B. subtilis spores deposited onto porous and non-porous indoor surface materials. Approximately 1times108 colony-forming units (CFU) of B. anthracis and B. subtilis spores were dried onto galvanized metal and ceiling tile coupons and then exposed to vaporous hydrogen peroxide. The materials contaminated with B. anthracis spores were placed inside a Class III biosafety cabinet (BSC III) that circulated vaporous hydrogen peroxide from within the decontaminated room, into and out of the BSC III. Identical materials inoculated in the same manner and at the same density with B. subtilis were placed both inside and outside of the BSC III to compare decontamination efficacy. Three fumigations were conducted using two sets of cycle parameters. The first set of cycle parameters for vaporous hydrogen peroxide exposure (10 minutes of conditioning at 12 g/min; 75 minutes of decontamination at 11 g/min) yielded log reductions in viable B. anthracis and B. subtilis spores ranging from 6.1 to 7.0 on all materials, while only 76% of the commercial biological indicators (1times106 CFU) evaluated in parallel were completely inactivated. The second set of cycle parameters (12 minutes of conditioning at 12 g/min; 104 minutes of decontamination at 8 g/min) yielded log reductions in viable B. anthracis and B. subtilis spores ranging from 6.7 to 7.4 on all materials and complete inactivation of biological indicators. These results demonstrate this method as a viable approach to assess room-scale fumigant decontamination efficacy against B. anthracis Ames spores.

A dynamic system for delivering controlled bromine and chlorine vapor exposures to weanling swine skin.

Abstract Context: Assessing the hazards of accidental exposure to toxic industrial chemical (TIC) vapors and evaluating therapeutic compounds or treatment regimens require the development of appropriate animal models. Objective: The objective of this project was to develop an exposure system for delivering controlled vapor concentrations of TICs to the skin of anesthetized weanling pigs. Injury levels targeted for study were superficial dermal (SD) and deep dermal (DD) skin lesions as defined histopathologically. Materials and methods: The exposure system was capable of simultaneously delivering chlorine or bromine vapor to four, 3-cm diameter exposure cups placed over skin between the axillary and inguinal areas of the ventral abdomen. Vapor concentrations were generated by mixing saturated bromine or chlorine vapor with either dried dilution air or nitrogen. Results: Bromine exposure concentrations ranged from 6.5 × 10−4 to 1.03 g/L, and exposure durations ranged from 1 to 45 min. A 7-min skin exposure to bromine vapors at 0.59 g/L was sufficient to produce SD injuries, while a 17-min exposure produced a DD injury. Chlorine exposure concentrations ranged from 1.0 to 2.9 g/L (saturated vapor concentration) for exposures ranging from 3 to 90 min. Saturated chlorine vapor challenges for up to 30 min did not induce significant dermal injuries, whereas saturated chlorine vapor with wetted material on the skin surface for 30–60 min induced SD injuries. DD chlorine injuries could not be induced with this system. Conclusion: The vapor exposure system described in this study provides a means for safely regulating, quantifying and delivering TIC vapors to the skin of weanling swine as a model to evaluate therapeutic treatments.

Use of superabsorbent polymer gels for surface decontamination of Bacillus anthracis spores

Aims:  This study evaluated the inactivation of Bacillus anthracis Vollum spores dried on a nonporous surface using a superabsorbent polymer (SAP) gel containing commercially available liquid decontaminants.

Evaluation of the Efficacy of Methyl Bromide in the Decontamination of Building and Interior Materials Contaminated with Bacillus anthracis Spores

ABSTRACT The primary goal of this study was to determine the conditions required for the effective inactivation of Bacillus anthracis spores on materials by using methyl bromide (MeBr) gas. Another objective was to obtain comparative decontamination efficacy data with three avirulent microorganisms to assess their potential for use as surrogates for B. anthracis Ames. Decontamination tests were conducted with spores of B. anthracis Ames and Geobacillus stearothermophilus, B. anthracis NNR1Δ1, and B. anthracis Sterne inoculated onto six different materials. Experimental variables included temperature, relative humidity (RH), MeBr concentration, and contact time. MeBr was found to be an effective decontaminant under a number of conditions. This study highlights the important role that RH has when fumigation is performed with MeBr. There were no tests in which a ≥6-log10 reduction (LR) of B. anthracis Ames was achieved on all materials when fumigation was done at 45% RH. At 75% RH, an increase in the temperature, the MeBr concentration, or contact time generally improved the efficacy of fumigation with MeBr. This study provides new information for the effective use of MeBr at temperatures and RH levels lower than those that have been recommended previously. The study also provides data to assist with the selection of an avirulent surrogate for B. anthracis Ames spores when additional tests with MeBr are conducted.

Influence of environmental conditions on the attenuation of ricin toxin on surfaces

Ricin is a highly-toxic compound derived from castor plant beans. Several incidents involving contamination of residences and buildings due to ricin production or dissemination have occurred in recent years. The goal of this study was to determine whether ricin bioactivity could be attenuated in reasonable time via simple modifications of the indoor environment. Attenuation was assessed on six different materials as a function of temperature, relative humidity (RH), and contact time, using both a pure and crude preparation of the toxin. Ricin bioactivity was quantified via a cytotoxicity assay, and attenuation determined as the difference in ricin recovered from test and positive controls. The results showed that pure ricin could be attenuated successfully, while the crude ricin was generally more persistent and results more variable. We found no significant attenuation in crude ricin after two weeks at typical indoor environmental conditions, except on steel. Attenuation mostly improved with increasing temperature, but the effect of RH varied. For pure ricin, heat treatments at 40°C for 5 days or 50°C for 2–3 days achieved greater than 96% attenuation on steel. In contrast, appreciable recovery of the crude ricin preparation still occurred at 40°C after two weeks.

Caenorhabditis elegans Predation on Bacillus anthracis: Decontamination of Spore Contaminated Soil with Germinants and Nematodes

Remediation of Bacillus anthracis-contaminated soil is challenging and approaches to reduce overall spore levels in environmentally contaminated soil or after intentional release of the infectious disease agent in a safe, low-cost manner are needed. B. anthracis spores are highly resistant to biocides, but once germinated they become susceptible to traditional biocides or potentially even natural predators such as nematodes in the soil environment. Here, we describe a two-step approach to reducing B. anthracis spore load in soil during laboratory trials, whereby germinants and Caenorhabditis elegans nematodes are applied concurrently. While the application of germinants reduced B. anthracis spore load by up to four logs depending on soil type, the addition of nematodes achieved a further log reduction in spore count. These laboratory based results suggest that the combined use of nematodes and germinants could represent a promising approach for the remediation of B. anthracis spore contaminated soil. Originality-Significance Statement: This study demonstrates for the first time the successful use of environmentally friendly decontamination methods to inactivate Bacillus anthracis spores in soil using natural predators of the bacterium, nematode worms.