Methyl bromide fumigant lethal to Bacillus anthracis spores.

Introduction

Methyl bromide (MB) is a broad-spectrum fumigant that serves as an insecticide, nematicide, herbicide, and fungicide for agricultural soils (Spreen, VanSickle, Moseley, Deepak, & Mathers, 1995); raw and processed foodstuffs (Bond, 1989); and structures (Mueller, 1990). It is widely used for quarantine fumigations because the gas is highly penetrating and most vegetables and fruits are tolerant of concentrations effective against target pests (Bond, 1989). According to current labels, the quarantine rates at which MB should be used to control insect pests in warehouses, ships, railcars, and containers range from 16 to 144 mg/L for exposures of 10-72 hours. Some studies have indicated that Bacillus anthracis spores can be killed with MB in the context of commodity and soil fumigation (Kolb & Schneiter, 1950; Pilipenko, 1976; Polyakov, Kulikovskii, & Pilipenko, 1976; Polyakov, Pilipenko, Volkovskii, & Kulikovskii, 1980). The study reported here found that MB applied at a concentration of 80 mg/L for 48 hours is capable of reducing Bacillus anthracis spores by at least 7 lo[g.subub.10] in a structural environment; while its toxicity in relation to other Bacillus species is highly variable.

Materials and Methods

Bacillus anthracis (ANR-1), Bacillus atrophaeus, Bacillus thuringiensis, and Geobacillus stearothermophilus were grown on nutrient-sporulating agar (Trypticase soy agar plus 50 mg/L manganese sulfate [MnS[O.sub.4]]) for 3 to 5 days to encourage sporulation. The spores were harvested into individual tubes of sterile deionized water containing 0.1 percent Tween 80, heat-shocked at approximately 65[degrees]C for approximately 5 minutes, and stored at 4[degrees]C. The final spore population was titered and reached approximately 1 x [10.sup.9] spores per milliliter for each of the organisms. Gram stains of the suspensions were performed during titering to calculate the percentage of spores in each suspension. Individual sterile glass slides (approximately 22 mm x 30 mm) were placed in sterile Petri dishes, up to five per dish, with no overlap. Aliquots (100 [micro]L) of the spore suspensions were placed on the slides and allowed to dry under aseptic conditions overnight; approximately 1 x [10.sup.6] to 1 x [10.sup.8] spores per slide resulted. Four uncovered Petri dishes containing five inoculated slides each, with one dish for each organism, were placed into 9-L glass fumigation chambers. Spores were exposed to MB gas (Scheffrahn & Su, 1992) at 0, 16, 32, 48, 64, 80, 96, and 112 mg/L for 48 hours at 37[degrees]C.

After exposure, spores were removed from slides by sonication (5 minutes) and vortexing (2 minutes) in sterile water containing 0.1 percent Tween-80. The spore suspensions were serially diluted and plated on TSA. All plates were incubated at 37[degrees]C and counted at 24 hours and 7 days.

Results

Bacillus anthracis ANR-1 proved susceptible to MB, and spore deposits were reduced by over 7 lo[g.subub.10] to below the limit of detection, at MB concentrations [greater than or equal to]80 mg/L (Table 1). Dose-dependent mortality occurred at 64 and 48 mg/L, with concentrations lower than 48 mg/L MB showing no effect. Geobacillus stearothermophilus was slightly more sensitive to MB than was Bacillus anthracis, being reduced by 7 log at 64 mg/L and showing a dose-dependant effect to 32 mg/L. All Bacillus thuringiensis and Bacillus atrophaeus spores tested showed a dose-dependant reduction in spore numbers, but they were not reduced below detection level by any MB concentration tested. In a separate experiment under the same MB exposure conditions, 10 strains of Bacillus anthracis (ATCC 10, ATCC 937, ATCC 4728, ATCC 9660, ATCC 11966, ATCC 14187, AMES-1-RIID, AMES-RIID, ANR-1, and STERNE) were equally susceptible to MB and were not dependent upon virulence factors; at the described conditions, 80 mg/L of MB reduced to below detection level fully virulent strains containing both plasmids (toxin+, capsule+), attenuated strains containing one of two plasmids (+, -) or (-, +), and avirulent strains containing neither virulence plasmid (-, -).

A field validation of MB sporicidal activity was conducted in September 2004. Two hundred surrogate spore coupons on paper and stainless steel, each containing 1 x [10.sup.6] spores of either Bacillus atrophaeus, Bacillus thuringiensis, or Geobacillus stearothermophilus, were deposited in 50 different locations within a 850-[m.sup.3] structure in Big Pine Key, Florida. Locations included electronics, wall voids, appliances, attic insulation, carpeting, and furniture fabric. After fumigation with 312 mg/L of MB for 48 hours at 35.5[degrees]C, only one location contained viable spores of Bacillus atrophaeus on a single coupon (data not shown). That location was inside a magnetically sealed refrigerator that had been turned off to prevent air exchange, creating a barrier to diffusion of MB gas. All untreated controls had expected levels of growth. None of the fumigated electronic equipment (computers, plasma displays, LCD displays, CRT displays, and environmental sensors) and none of the materials showed any visible or functional signs of damage. There was no lingering odor in the structure. No MB residues were detected 3 days after the 48-hour fumigation.

Conclusions

The results reported here indicate that MB is an efficient, cost-effective fumigant for bacterial spore decontamination. MB is a U.S. Environmental Protection Agency (U.S. EPA)-registered structural fumigant, and it is capable of clearing Bacillus anthracis spores from buildings, vehicles, and commodities in the aftermath of a bioterrorism attack. Use of MB to decontaminate areas affected by bioterrorist attacks has several potential advantages. First, because MB is a registered structural fumigant, personnel trained in its use are available nationally. Additional training in decontamination procedures would be minimal for these professionals. Second, decontamination is rapid, occurring within 48 hours. Extensive preparation of the contaminated item is not required, and all furnishings or other internal structures or items may remain in place. Third, MB leaves no residue. Finally, MB is a noncorrosive alkylating agent and does not damage commodities (e.g., food supplies), furnishings, documents, or even sensitive electronic equipment.

The results also indicate, however, that common Bacillus anthracis surrogates are inappropriate for demonstrating the efficacy of MB fumigation. The sensitivity of Geobacillus stearothermophilus to MB, for example, would lead to a possible assumption of total clearing that may not exist, while the increased resistance seen in Bacillus thuringiensis would lead to an assumption of continued contamination when no contamination exists. More research in this area is required to obtain an accurate surrogate for use in ensuring effective removal of Bacillus anthracis spores after fumigation.

Acknowledgments: The study was supported in 2004 by Small Business Innovation Research Grant No. 68-D-03-056 from U.S. EPA under Topic H, Environmental Bioterrorism Detection and Decontamination.

Corresponding Author: Margaret Juergensmeyer, Research Microbiologist, IIT Research Institute, 10 W. 35th Street, Chicago, IL 60616. E-mail: [e-mail].

REFERENCES

Bond, E.J. (1989). Manual of fumigation for insect control. Rome: Food and Agriculture Organization of the United Nations. Retrieved September 20, 2006, from http://www.fao.org/docrep/X5042E/X5042E00.htm.

Kolb, R.W., & Schneiter, R. (1950). The germicidal and sporicidal efficacy of methyl bromide for Bacillus anthracis. Journal of Bacteriology 59(3), 401-412.

Mueller, D.K. (1990). Fumigation. In A. Mallis (Ed.), Handbook of pest control (7th ed.) (pp. 901-939). Cleveland, OH: Franzak & Foster.

Pilipenko, V.N. (1976). Disinfection of soil sources of Bacillus anthracis with methyl bromide (USSR) [Abstract]. Trudy, Vsesoyuzniy Nauchno-Issledovatelskiy Institut Veterinarnoy Sanitarii, 54, 138-144.

Polyakov, A.A., Kulikovskii, V., & Pilipenko, V.N. (1976). Submicroscopic structure of Bacillus anthracis spores subjected to methyl bromide treatment [Abstract]. Doklady Vsesoyuznoy Akademii Selsko-khozyastvennyh Nauk im. V.I. Lenina, 12, 23-24.

Polyakov, A.A., Pilipenko, V.N., Volkovskii, G.D., & Kulikovskii, A.V. (1980). Sporicide properties of methyl bromide and its use for disinfection [Abstract]. Doklady Vsesoyuznoy Akademii Selsko-khozyastvennyh Nauk im. V.I. Lenina, 12, 27-29.

Scheffrahn, R.H., & Su, N.Y. (1992). Comparative toxicity of methyl bromide against ten Nearctic termite species (Isoptera: Termopsidae, Kalotermitidae, Rhinotermitidae). Journal of Economic Entomology, 85, 845-847.

Spreen, T.H., VanSickle, J.J., Moseley, A.E., Deepak, M.S., & Mathers, L. (1995). Use of methyl bromide and the economic impact of its proposed ban (Technical Bulletin No. 898). Gainesville, FL: University of Florida.

Margaret A. Juergensmeyer, Ph.D.

Bruce A. Gingras, Ph.D.

Rudolf H. Scheffrahn, Ph.D.

Mark J. Weinberg

TABLE 1 Recovery of Viable Bacteria After a 48-hour Exposure to Methyl
Bromide at 37[degrees]C*

                            Bacterial Strains
MB Concentration   B. anthracis          G. stearothermophilus
in mg/L (lbs/Mcf)  ANR-I                 ATCC 7953

  0 (0)            7.236 [+ or -] 0.452  5.208 [+ or -] 0.164
 16 (1)            7.742 [+ or -] 0.110  5.300 [+ or -] 0.038
 32 (2)            7.046 [+ or -] 0.204  4.943 [+ or -] 0.138
 48 (3)            6.992 [+ or -] 0.151  3.860 [+ or -] 0.178
 64 (4)            4.876 [+ or -] 0.745  0 [+ or -] 0
 80 (5)            0 [+ or -] 0          0 [+ or -] 0
 96 (6)            0 [+ or -] 0          0 [+ or -] 0
112 (7)            0 [+ or -] 0          0 [+ or -] 0

                                 Bacterial Strains
MB Concentration   B. atrophaeus ATCC
in mg/L (lbs/Mcf)  49337                   B. thuringiensis ATCC 33679

  0 (0)              8.518 [+ or -] 0.184   8.907 [+ or -] 0.688
 16 (1)            >73                     >7
 32 (2)             >7                     >7
 48 (3)             >7                     >7
 64 (4)              7.944 [+ or -] 0.062   7.856 [+ or -] 0.066
 80 (5)             >5                     >5
 96 (6)             >5                     >5
112 (7)              4.939 [+ or -] 0.184   5.21 [+ or -] 0.154

*The mean lo[g.subub.10] per slide [+ or -] the standard deviation is
reported, with a total of five spare-bearing slides used for each
species. Values reported with a ">" showed growth on plates, but were
too numerous to count on all dilutions plated. Colonies were counted 24
hours after plating; counts were unchanged 6 days later.