Identification by quantitative carrier test of surrogate spore-forming bacteria to assess sporicidal chemicals for use against Bacillus anthracis

Abstract

The spores of six strains of Bacillus anthracis (four virulent and two avirulent) were compared with those of four other types of spore-forming bacteria for their resistance to four liquid chemical sporicides (sodium hypochlorite at 5,000 ppm available chlorine, 70,000 ppm accelerated H2O2, 1,000 ppm chlorine dioxide, and 3,000 ppm peracetic acid). All test bacteria were grown in a 1:10 dilution of Columbia broth (with manganese) incubated at 37 degrees C for 72 h. The spore suspensions, heat treated at 80 degrees C for 10 min to rid them of any viable vegetative cells, contained 1 x 10(8) to 3 x 10(8) CFU/ml. The second tier of the quantitative carrier test (QCT-2), a standard of ASTM International, was used to assess for sporicidal activity, with disks (1 cm in diameter) of brushed and magnetized stainless steel as spore carriers. Each carrier, with 10 microl (> or = 10(6) CFU) of the test spore suspension in a soil load, was dried and then overlaid with 50 microl of the sporicide being evaluated. The contact time at room temperature ranged from 5 to 20 min, and the arbitrarily set criterion for acceptable sporicidal activity was a reduction of > or = 10(6) in viable spore count. Each test was repeated at least three times. In the final analysis, the spores of Bacillus licheniformis (ATCC 14580(T)) and Bacillus subtilis (ATCC 6051(T)) proved to be generally more resistant than the spores of the strains of B. anthracis tested. The use of one or both of the safe and easy-to-handle surrogates identified here should help in developing safer and more-effective sporicides and also in evaluating the field effectiveness of existing and newer formulations in the decontamination of objects and surfaces suspected of B. anthracis contamination.

FIG. 1.

Comparative resistance of Bacillus spores to sodium hypochlorite (5,000 ppm FAC). Error bars show standard deviations.

FIG. 2.

Comparative resistance of Bacillus spores to AHP (70,000 ppm H₂O₂). Error bars show standard deviations.

FIG. 3.

Comparative resistance of Bacillus spores to aqueous chlorine dioxide (1,000 ppm available chlorine). Error bars show standard deviations.

FIG. 4.

Comparative resistance of Bacillus spores to 0.3% PAA. Error bars show standard deviations.