UV resistance of Bacillus anthracis spores revisited: validation of Bacillus subtilis spores as UV surrogates for spores of B. anthracis Sterne

Abstract

Recent bioterrorism concerns have prompted renewed efforts towards understanding the biology of bacterial spore resistance to radiation with a special emphasis on the spores of Bacillus anthracis. A review of the literature revealed that B. anthracis Sterne spores may be three to four times more resistant to 254-nm-wavelength UV than are spores of commonly used indicator strains of Bacillus subtilis. To test this notion, B. anthracis Sterne spores were purified and their UV inactivation kinetics were determined in parallel with those of the spores of two indicator strains of B. subtilis, strains WN624 and ATCC 6633. When prepared and assayed under identical conditions, the spores of all three strains exhibited essentially identical UV inactivation kinetics. The data indicate that standard UV treatments that are effective against B. subtilis spores are likely also sufficient to inactivate B. anthracis spores and that the spores of standard B. subtilis strains could reliably be used as a biodosimetry model for the UV inactivation of B. anthracis spores.

FIG. 1.

UV inactivation curves of spores of B. subtilis ATCC 6633 (A) (triangles), B. subtilis WN624 (B) (circles), and B. anthracis Sterne (C) (squares). For comparison, the heavy dashed line in the background of each panel represents the UV inactivation curve of spores of B. subtilis ATCC 6633, as determined by Hoyer (5). The data points and error bars are the averages and standard deviations of results from four independent trials. S/S₀ represents the fraction calculated by dividing the viable spore titer at any given UV dose (S) by the spore titer obtained from the nonirradiated suspension (S₀).

FIG. 2.

Microscopic appearance of B. anthracis Sterne spores prepared by the method described in references (A) and (used in this study) (B). The images are of wet mounts prepared in water and photographed with phase-contrast optics. Original magnification, ×1,000. Several representative fields are presented in each panel.

FIG. 3.

Effect of turbidity of, and absorbance of UV by, buffer on the apparent UV resistance of B. anthracis Sterne spores. Spores were prepared and irradiated as described previously (7) and in the text. Survival curves were plotted based on dosimetry calculated by using UVCalc with no correction (circles) or with correction for the A₂₅₄ of the buffer used (triangles) or for the A₂₅₄ values of both the buffer and the cell suspension (squares). For comparison, a heavy dashed line represents the UV inactivation curve of spores of B. subtilis ATCC 6633, as determined by Hoyer (5).