Endospore Appendages Enhance Bacillus cereus Spore Adhesion to Industrial Surfaces, Modulated by Physicochemical Factors

Abstract

Spores of species belonging to the Bacillus cereus group (B. cereus spp.) are common contaminants in food processing environments due to their ability to adhere to surfaces and resist cleaning procedures. These spores are equipped with pilus-like endospore appendages (ENAs), which are believed to promote surface adhesion. We have investigated the role of ENAs in spore adhesion to abiotic surfaces using wild-type (WT) B. cereus spp. strains and isogenic mutants lacking ENAs or an intact exosporium. WT spores expressing both short and long ENAs (S+L+) adhered significantly more to stainless steel (SS) and polypropylene (PP) compared to bald spores (S-L-) and spores of an exosporium-deficient mutant ({Delta}exsY), while adhesion to polystyrene (PS) and glass was not significantly affected by the presence of ENAs. The {Delta}exsY mutant showed the lowest adhesion across all tested surfaces, a pattern also observed for vegetative cells. The individual roles of S-ENA and L-ENA were also assessed, where the strongest adhesion occurred when both fibers were present on PP. A trend also emerged on PP; WT remained adhered for an hour, while bald spores tended to detach within that time. For saline conditions and pH, bald spores adhered strongly to SS, while for non-ionic surfactant and concentrated protein solutions, WT spores exhibited the strongest adhesion. These results highlight the crucial role of ENAs in B. cereus spp. spore adhesion to industrial relevant surfaces, providing mechanistic insight into spore persistence. This understanding supports the design of surface treatments to prevent contamination, spoilage, and foodborne illness. ImportanceBacteria belonging to the Bacillus cereus group represent a persistent challenge in food production due to their highly resilient endospores (spores), which resist cleaning and disinfection as well as food processing. Understanding the adhesion properties of these spores is essential for designing effective surface treatments that reduce chemical use, enhance food safety and quality, and minimize environmental impact. This study underscores the important role of endospore appendages (ENAs) in spore adhesion to common materials in food processing and laboratory environments. Wild-type spores expressing both S-ENA and L-ENA adhered significantly more than mutants lacking ENAs or the exosporium, highlighting ENAs as potential targets for disrupting spore adhesion. Time-dependent adhesion tests on polypropylene revealed strong, sustained attachment by wild-type spores, contrasting with weaker, transient adhesion by ENA-depleted mutants. These findings offer valuable insights into B. cereus spore adhesion dynamics, guiding the development of tailored cleaning protocols to improve contamination control and sustainability.