Sporulation of Bacillus spp. within biofilms: A potential source of contamination in food processing environments

• Sporulation by Bacillus spp. can occur at high levels in single- and mixed-biofilms.
• Biofilm-associated Bacillus spores are resistant to cleaning.
• Biofilm-associated Bacillus spores may be transferred to foods in contact.
• Biofilms may serve as a major source of food contamination with Bacillus spores.

Bacillus strains are often isolated from biofilms in the food industries. Previous works have demonstrated that sporulation could occur in biofilms, suggesting that biofilms would be a significant source of food contamination with spores. In this study, we investigated the properties of mono-species and mixed Bacillus biofilms and the ability of Bacillus strains to sporulate inside biofilms. Bacillus strains were able to form mono-species biofilms on stainless steel coupons, with up to 90% spores after a 48 h-incubation. These spores were highly resistant to cleaning but were easily transferred to agar, mimicking the cross-contamination of food, thereby suggesting that biofilms would be of particular concern due to a potential for Bacillus spore food contamination. This hypothesis was strengthened by the fact that Bacillus strains were able to form mixed biofilms with resident strains and that sporulation still occurred easily in these complex structures.