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Decontamination of Bacillus thuringiensis spores on selected surfaces by chlorine dioxide gas.

Introduction

From October 4 to November 2, 2001, the Centers for Disease Control and Prevention (CDC) and state and local public health authorities reported l0 confirmed cases of inhalational anthrax caused by intentional delivery of Bacillus anthracis spores through mailed letters or packages in the United States (Jernigan et al., 2001). One of these mailings resulted in the Hart Senate Office Building being contaminated with B. anthracis spores when a Senate aide opened a contaminated letter on October 15, 2001. In the effort to decontaminate the office suite of Senate Majority Leader Tom Daschle, U.S. Environmental Protection Agency (U.S. EPA) officials fumigated the 3,000-square-foot office with 500-800 parts per million chlorine dioxide (Cl[O.sub.2]) gas under 70-80 percent relative humidity for 12 to 20 hours (Heilprin, 2001; "Anthrax Contamination of Hart Building Finished," 2001). This was the first reported successful attempt to use Cl[O.sub.2] gas to decontaminate a large room ("Hart Senate Office Building to Reopen Friday," 2002).

Aqueous Cl[O.sub.2] has been used as a disinfectant in drinking-water plants and in the food industry. The use of gaseous Cl[O.sub.2], however, has been primarily limited to pulp bleaching in the paper industry. Studies on the use of gaseous Cl[O.sub.2] for disinfection of surfaces are not as extensive as those on the use of aqueous Cl[O.sub.2]. Cl[O.sub.2] gas has been reported to have high bactericidal activity on epoxy-coated stainless steel surfaces (Han, Guentert, Smith, Linton, & Nelson, 1999) and green-pepper surfaces (Hart, Floros, Linton, Nielsen, & Nelson 2001: Han. Linton, Nielsen, & Nelson, 2000), and sporicidal activity on paper and aluminum foil surfaces (Jeng & Woodworth, 1990: Rosenblatt, Rosenblatt, & Knapp, 1987). The effectiveness of utilizing Cl[O.sub.2] gas as a sporicide on different kinds of surfaces has not, however, been compared and evaluated. To better understand Cl[O.sub.2] as a disinfection agent, more information on the inactivation of Bacillus spores on different surfaces, such as envelope paper, wood, and plastic, is needed.

In this study, a Bacillus thuringiensis strain designated BT1 was chosen as a surrogate for B. anthracis and utilized for the preparation of spores. B. thuringiensis was chosen for this work because of its non-pathogenicity and because it is a member of the Bacillus subgroup I, to which B. anthracis also belongs. The two Gram-positive spore-forming organisms share morphology, have similar DNA, and have similar spore composition, as demonstrated with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI MS) (Ryzhov, Hathout, & Fenselau, 2000). On the basis of results from previous studies of Cl[O.sub.2] gas (Han et al., 1999, 2000, 2001), this study performed treatments with 5, 10, 15, 20, 25, or 30 milligrams per liter (mg/L) Cl[O.sub.2] gas (1 mg/L Cl[O.sub.2] gas = 332 parts per million under standard conditions) for 12 hours at 85-92 percent relative humidity (RH) and 22 [+ or -] 1[degrees]C. The purpose was to simulate a fumigation using Cl[O.sub.2] gas to inactivate B. anthracis spores in a closed environment, where the contamination might be on a variety of surfaces, including paper, wood, and plastic materials.

Methods

Preparation of Bacillus thuringiensis Spores The authors prepared suspensions of B. thuringiensis spores by growing BT1 on nutrient agar (DIFCO Laboratories, Detroit, Michigan) plates overnight at 34[degrees]C. The overnight culture then was used to inoculate Roux bottles containing nutrient agar supplemented with manganese sulfate (3 mg/L). The cultures were incubated at 34 [degrees] C and periodically examined microscopically to detect sporulation. Once sporulation values of 90 percent to 95 percent were obtained (after five days), spore stocks were prepared according to a modification of a protocol previously described by Couvert and co-authors (Couvert, Leguerinel, & Mafart, 1999). Spores were scraped from the surface of the agar, re-suspended in sterile deionized water, washed (three times), and re-suspended in a 50 percent ethanol solution. The spore suspension was stored at 4[degrees]C for 20 hours to eliminate vegetative cells, washed (three times), and re-suspended to a working concentration of approximately [10.sup.8] spores per milliliter in deionized water.

Inoculation of Spores on Paper, Wood, Plastic, and Coated Stainless-Steel Strips

Four different materials were used in this study: regular white envelope paper (2 x 6 centimeters [cm], envelopes purchased from the local post office), wood strips (2 x 15 cm, tongue depressors from Fisher Scientific Company), polyethylene plastic strips (2.5 x 12 cm, strips purchased from a local market), and stainless-steel strips with epoxy coating (2.5 x 8 cm, from Enerfab Company). The purpose of using the epoxy surface in this study was to compare the results with those from the authors' previous study of Cl[O.sub.2] gas inactivation of spoilage microbes on the epoxy surface of orange-juice storage tanks (Han et al., 1999). The four surface materials were autoclaved for 30 minutes at 121[degrees]C before inoculation (no damage to the paper surface was visible after the autoclave). No viable microorganisms, including spores, were found on these autoclaved materials according to the end-point determination method described below. A 0.1-mL suspension of B. thuringiensis spores ([10.sup.7] spores per mL) was spotted onto the four different surfaces (approximately 2 x 2 [cm.sup.2]). The inoculated samples were dried for three hours in a biosafety hood and then treated with Cl[O.sub.2] gas as described below.

Cl[O.sub.2] Gas Treatment

Cl[O.sub.2] gas treatment was carried out in a 10-L Irvine Plexiglass cylinder with a stainless-steel shelf (Han et al., 2001). The inoculated envelope paper, wood, epoxy-coated stainless steel, and polyethylene plastic were randomly placed on three layers of a stainless-steel shelf inside the cylinder. Cl[O.sub.2] gas was generated from a CDG laboratory generator (CDG Technology, Inc., New York) as 4 percent chlorine in nitrogen gas. A 60-mL plastic gas-sampling syringe was used to deliver specific volumes of Cl[O.sub.2] gas into the cylinder. A diaphragm vacuum pump (KNF Neuberger, Inc., Trenton, New Jersey) was used to circulate the gas mixture inside the cylinder. A Thermo-Hydro recorder (Control Company, Friendwood, Texas) was used to monitor relative humidity (RH) and temperature inside the treatment cylinder. Before injection of Cl[O.sub.2] gas, the air inside the cylinder was circulated through a 125-mL washing bottle with 100 mL of deionized water for 10 to 15 minutes in order to achieve an RH of 85-92 percent. The experiments were performed in the dark to prevent photo inactivation of the Cl[O.sub.2]. A DPD (N, N-diethy-r-phenylenediamine) colorimetric analysis kit (CHEMetrics, Inc., Calverton, Virginia) was used to measure the Cl[O.sub.2] gas concentration (Greenberg, Clesceri, & Eaton, 1992). The inoculated samples were treated with 5. 10, 15, 20, 25, or 30 mg/L Cl[O.sub.2] gas (initial concentrations) for 12 hours under 85-92 percent RH at 22 [+ or -] 1 [degree] C. The Cl[O.sub.2] gas concentrations in the cylinder were measured at 0, 6, and 12 hours. The results are shown in Table 1; the concentration reported for each treatment was the initial concentration.

Enumeration and Detection of Spores

After the treatments, the populations of surviving spores on each surface were recovered and enumerated. Each Cl[O.sub.2]-treated sample was mixed with 100 mL of sterile neutralizing buffer containing 0.5 percent thiosulfate (DIFCO Laboratories) in a sterile stomaching bag to eliminate the residual effects of Cl[O.sub.2]. The neutralizing buffer was replaced with 100 mL of 0.1 percent peptone solution for the samples not treated with Cl[O.sub.2] gas because both solutions produced the same bacterial recovery for the Cl[O.sub.2]-untreated samples (data not shown). To recover spores from each surface, the sample bag was placed in a plastic beaker and agitated for 15 minutes at 260 rpm on an Innova [TM] 2100 flat-form shaker (New Brunswick Scientific Co., Inc., Edison, New Jersey). Serial dilution of the recovered spores was followed by surface-plating on tryptic soy agar (TSA) (DIFCO Laboratories).

To enumerate low concentrations ([less than or equal to] [10.sup.2] CFU/mL) of surviving spores, a filtration method was used to concentrate the sample. A volume of 1, 10, or 50 mL of fluid was filtered through a 47-millimeter (47-mm) polycarbonate membrane (0.22-pm pore size) (Millipore, Inc., Bedford, Massachusetts) with a 47-mm Sterifil Aseptic System (Millipore Inc.) After filtration, the membrane was transferred onto TSA plates and incubated for 24 hours at 37[degrees]C before counting.

An end-point determination method was used to detect if all the inoculated spores were completely inactivated by Cl[O.sub.2] gas (Han et al., 1999). Bottles (300 mL each) of tryptic soy broth (DIFCO Laboratories) supplemented with 0.6 percent yeast extract were autoclaved for 30 minutes at 121[degrees]C. After each Cl[O.sub.2] gas treatment, the sample was immediately aseptically transferred into the 300-mL sterile tryptic soy broth and incubated for three days at 37[degrees] C. Turbid samples after incubation indicated bacterial growth, while clear samples indicated no growth and suggested a complete inactivation of the inoculated spores.

Statistical Analysis

All experiments were completed in triplicate, and the mean values of bacterial populations were calculated and reported with a 95 percent confidence interval. Data were subjected to analysis of variance and Student Newman Keuls' (SNK) multiple range tests (SAS Inc., Cary, North Carolina) to determine if significant differences (p = .05) in mean populations of B. thuringiensis spores existed after the different treatments.

Results

Twelve-Hour Treatments with 5, 10, and 15 mg/L Cl[O.sub.2] Gas After 5, 10, and 15 mg/L Cl[O.sub.2] gas treatments for 12 hours, the populations of spores on the four different surfaces decreased to different levels from an initial inoculation level of 6.1-6.3 log CFU per strip (Figure 1). For each Cl[O.sub.2] gas treatment, the highest population of surviving spores was found on the paper surface, and the lowest was on the plastic surface (p < .05). No significant difference (p > .05) was seen between the surviving spore populations on the wood and epoxy surfaces. The 5 mg/L Cl[O.sub.2] gas treatment inactivated 2.5, 3.6, 4.0, and 4.9 log spores on paper, wood, epoxy, and plastic surfaces, respectively. With increased gas concentration, log reductions of the spores increased. After the 10 mg/L Cl[O.sub.2] gas treatment, 3.6, 5.0, 5.7, and 5.8 log spores were inactivated on the paper, wood, epoxy, and plastic surfaces, respectively. A more than 5-log reduction of spores was achieved on each surface after 15 mg/L Cl[O.sub.2] gas treatment; no surviving spores could be detected on the plastic surface with the surface plating method.

[FIGURE 1 OMITTED]

Twelve-Hour Treatments with 15, 20, 25, and 30 mg/L Cl[O.sub.2] Gas

Using the end-point method, each surface was examined for complete inactivation of the spores after 12 hours of 15, 20, 25, and 30 mg/L Cl[O.sub.2] gas treatments (Table 2). The 15 mg/L Cl[O.sub.2] gas treatment did not inactivate all the inoculated spores on any of the four surfaces; all the strips showed microbial growth after incubation. After the 20 mg/L Cl[O.sub.2] gas treatment, surviving spores were found on the paper, wood, and epoxy surfaces, but not on the plastic surface. The epoxy and plastic surfaces showed complete inactivation at 25 mg/L Cl[O.sub.2] gas treatment, and no viable spores were detected on any of the four surfaces after 30 mg/L Cl[O.sub.2] gas treatment, which resulted in more than a 6-log reduction.

Discussion

The experimental results described above made it possible to evaluate how effective different initial concentrations of Cl[O.sub.2] gas were in inactivating B. thuringiensis spores on different surfaces under the selected conditions. The spores on the envelope paper showed the most resistance to inactivation by Cl[O.sub.2] gas. A complete inactivation of 6 log B. thuringiensis spores on the paper was achieved by 30 mg/L of Cl[O.sub.2] gas for 12 hours under 85-92 percent RH. Jeng and Woodworth (1990) reported that 30 mg/L for 30 minutes at ambient humidity (30-40 percent) and room temperature inactivated more than 5 log B. subtilis subsp. niger biological indicators on analytical paper disk blanks. Rosenblatt and co-authors (1987) also reported that at least 40 mg/L Cl[O.sub.2] gas treatment for one hour at 60 percent RH effectively sterilized Whatman 3-mm filter paper strips (7 x 35 mm) that were contaminated with 6 log B. subtilis spores. These results suggested that a relatively high Cl[O.sub.2] gas concentration (>1,000 ppm or 30 mg/L) might be necessary to decontaminate a high load of spores on paper surfaces.

As indicated in Table 1, the Cl[O.sub.2] gas concentrations decreased rapidly over time. In the first six hours, the concentrations were reduced by more than 80 percent. This reduction might be due to rapid decomposition, to absorption by or reaction with experimental materials, or both. It was reported that about 800 ppm Cl[O.sub.2] gas quickly dropped to 16 ppm during the fumigation of the Senate office building ("Anthrax Decontamination of Hart Building Finished," 2001). To overcome this problem, it may be necessary, in the decontamination of a large environment, to add Cl[O.sub.2] gas after a short exposure time, such as 0.5 to 6 hours. Under all the tested conditions, words printed in black ink on the envelope were not bleached or discolored. Thus, Cl[O.sub.2] gas at 30 mg/L concentration is feasible for decontamination of letters or paper documents without damage to the contents.

It is not known why the spores had higher survival rates on paper surfaces than on plastic surfaces. One of the possible explanations relates to the different surface structures of the materials. Paper surfaces are visibly coarser than plastic surfaces and contain large fiber networks that might provide crevices in which spores may be able to lodge, affecting the efficacy of Cl[O.sub.2] gas. Rosenblatt and co-authors (1987) found that Cl[O.sub.2] gas treatment of at least 35 mg/L for 1 hour at 60 percent RH was needed to inactivate 6 log B. subtilis spores on aluminum foil strips, while at least 40 mg/L gas was needed for paper surfaces. Hart et al. (1999) reported that a 10 mg/L Cl[O.sub.2] gas treatment for 30 minutes at high relative humidity (RH > 90 percent) and at temperatures of 9-28[degrees]C could inactivate more than 6 log spoilage microorganisms on epoxy-lined stainless-steel strip surfaces. In other words, the concentration of Cl[O.sub.2] gas needed to inactivate the Bacillus spores was at least three times higher than the concentration needed for bacteria. The wood surface was not discolored after Cl[O.sub.2] gas treatments. Cl[O.sub.2] gas treatment might therefore be suitable for the decontamination of an environment containing furniture or other wooden structures, like doors and windows. Because of differences in surface structures, however, decontamination of a room containing various other materials, such as rugs, cloths, and computers, presents numerous challenges.

Conclusion

The efficacy of Cl[O.sub.2] gas for the inactivation of Bacillus thuringiensis spores inoculated on paper, wood, epoxy, and plastic surfaces increased with increasing concentrations of the gas. Under the experimental conditions reported here, the highest population of surviving spores was found on the paper surface, and the lowest was on the plastic surface (p < .05). No significant difference (p > .05) was seen between the surviving spore populations on the wood and epoxy surfaces. The minimum Cl[O.sub.2] gas concentration needed to completely inactivate an inoculum of 6 log spores was 30 mg/L for paper and wood surfaces, 25 mg/L for epoxy surfaces, and 20 mg/L for plastic surfaces. These results may provide insight into the parameters of effective decontamination procedures for Bacillus spores. In particular, the data from this study suggest that when strips are used to verify the effectiveness of Cl[O.sub.2] gas treatments, a battery of different material surfaces should be used for the strips to provide adequate information on treatment efficacy.
TABLE 1
Changes in Chlorine Dioxide Gas Concentration Over a 12-Hour Period (a)

Treatment Time (hours) Chlorine Dioxide Gas Concentration (mg/L)

 0 (initial) 5 10 15 20 25 30

 6 0.8 1.2 1.8 3.0 4.5 6

 12 0 0.2 0.3 0.8 1.5 2

(a) In a 10-liter cylinder treatment vessel.

TABLE 2
Inactivation of Bacillus thuringiensis Spores on Different Surfaces
by 12-Hour Treatment with Different Concentrations of CI[O.sub.2]
Gas (a)

CI[O.sub.2] Gas Concentration (mg/L) Spore Viability on Surfaces (b)

 Paper Wood Epoxy Plastic

 15 3/3 3/3 3/3 3/3
 20 3/3 3/3 3/3 0/3
 25 2/3 1/3 0/3 0/3
 30 0/3 0/3 0/3 0/3

(a) Treatment conditions: 85-92% relative humidity, 22 [+ or -]
1[degrees]C.

(b) Number of turbid samples in trypticase soy broth after three days
incubation at 37[degrees]C using an end-point determination method.


Acknowledgement: This paper is journal article #16920 of the Purdue University Agricultural Research Program.

Corresponding Author: Bruce Applegate. Assistant Professor, Department of Food Science, 745 Agriculture Mall Dr., West Lafayette, IN 47907-2009. E-mail: applegate@foodsci.purdue.edu.

REFERENCES

Couvert, O., Leguerinel, L., & Mafart, P. (1999). Modeling the overall effect of pH on the apparent heat resistance of Bacillus cereus spores. International Journal of Food Microbiology. 49, 57-62.

Greenberg, A.E., Clesceri. L.S., & Eaton, A.D. (1992). Standard methods for the examination of water and wastewater: 4500-ClO2 C. In Amperometric Method I (pp. 4-56). Washington. DC: American Public Health Association.

Han, Y., Guentert, A.M., Smith, R.S., Linton, R.H., & Nelson, P.E. (1999). Efficacy of chlorine dioxide gas as a sanitizer for tanks used for aseptic juice storage. Food Microbiology, 16(1) 53-61.

Han, Y., Floros, J.D., Linton, R.H., Nielsen, S.S., & Nelson, P.E. (2001). Response surface modeling for the inactivation of Escherichia coli O157:H7 on green peppers (Capsicum annum L.) by chlorine dioxide gas. Journal of Food Protection. 64, 1128-1133.

Han, Y., Linton. R.H., Nielsen, S.S., & Nelson, P.E. (2000). Inactivation of Escherichia coli O157: H7 on surface-uninjured and -injured green pepper (Capsicum annum L.) by chlorine dioxide gas as demonstrated by confocal laser scanning microscopy. Food Microbiology, 17, 643-655.

Heilprin J. (2001, December 2). Fumigation to kill final anthrax spores. Daily Camera, p. 9A.

Jernigan, J.A., Stephens, D.S., Ashford. D.A., Omenaca. C., Topiel, M.S., Galbraith, M., Tapper, M., Fisk, T.L., Zaki, S., Popovic, T., Meyer, R.F., Quinn, C.P., Harper, S.A., Fridkin, S.K., Sejvar, J.J., Shepard, C.W., McConnell, M., Guarner, J., Shieh, W., Malecki, J.M., Gerberding, J.L., Hughes, J. M., Perkins, B.A., & members of the Anthrax Bioterrorism Investigation Team. (2001). Bioterrorism-related inhalational anthrax: The first 10 cases reported in the United States. Emerging Infectious Diseases, 7(6). http://www.cdc.gov/ncidod/EID/vol7no6/jernigan.htm (3 April 2002).

Jeng, D.K., & Woodworth, A.G. (1990). Chlorine dioxide gas sterilization under square-wave conditions. Applied Environmental Microbiology, 56, 514-519.

Rosenblatt, D.H., Rosenblatt, A.A., & Knapp, J.E. (1987). Use of chlorine dioxide gas as a chemosterilizing agent. U.S. Patent 4,681,739.

Ryzhov, V., Hathout, Y., & Fenselau, C. (2000). Rapid characterization of spores of Bacillus cereus group bacteria by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry Applied Environmental Microbiology, 66, 3828-3834.

Hart Senate Office Building to reopen Friday (16 Jan. 2002). USA Today. http://www.usatoday.com/news/washdc/2002/01/16/hartsenate.htm (3 April 2002).

Anthrax decontamination of Hart building finished. (2 Dec. 2001). USA Today. http://www.usatoday.com/news/attack/2001/12/02/anthrax-congress.htm (3 April 2002).
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Author:Nelson, P.E.
Publication:Journal of Environmental Health
Geographic Code:1USA
Date:Nov 1, 2003
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