Biofilm Formation and Sporulation by Bacillus cereus on a Stainless Steel Surface and Subsequent Resistance of Vegetative Cells and Spores to Chlorine, Chlorine Dioxide, and a Peroxyacetic Acid–Based Sanitizer

Abstract

Biofilm formation by Bacillus cereus 038-2 on stainless steel coupons, sporulation in the biofilm as affected by nutrient availability, temperature, and relative humidity, and the resistance of vegetative cells and spores in biofilm to sanitizers were investigated. Total counts in biofilm formed on coupons immersed in tryptic soy broth (TSB) at 12 and 22°C consisted of 99.94% of vegetative cells and 0.06% of spores. Coupons on which biofilm had formed were immersed in TSB or exposed to air with 100, 97, 93, or 85% relative humidity. Biofilm on coupons immersed in TSB at 12°C for an additional 6 days or 22°C for an additional 4 days contained 0.30 and 0.02% of spores, respectively, whereas biofilm exposed to air with 100 or 97% relative humidity at 22°C for 4 days contained 10 and 2.5% of spores, respectively. Sporulation did not occur in biofilm exposed to 93 or 85% relative humidity at 22°C. Treatment of biofilm on coupons that had been immersed in TSB at 22°C with chlorine (50 μg/ml), chlorine dioxide (50 μg/ml), and a peroxyacetic acid–based sanitizer (Tsunami 200, 40 μg/ml) for 5 min reduced total cell counts (vegetative cells plus spores) by 4.7, 3.0, and 3.8 log CFU per coupon, respectively; total cell counts in biofilm exposed to air with 100% relative humidity were reduced by 1.5, 2.4, and 1.1 log CFU per coupon, respectively, reflecting the presence of lower numbers of vegetative cells. Spores that survived treatment with chlorine dioxide had reduced resistance to heat. It is concluded that exposure of biofilm formed by B. cereus exposed to air at high relative humidity (≥97%) promotes the production of spores. Spores and, to a lesser extent, vegetative cells embedded in biofilm are protected against inactivation by sanitizers. Results provide new insights to developing strategies to achieve more effective sanitation programs to minimize risks associated with B. cereus in biofilm formed on food contact surfaces and on foods.