Endospore appendages enhance adhesion of Bacillus cereus sensu lato spores to industrial surfaces, modulated by physicochemical factors

ABSTRACT Spores of species belonging to the Bacillus cereus sensu lato (s.l.) group 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 investigated the role of ENAs in spore adhesion to abiotic surfaces using a wild-type (WT) Bacillus paranthracis strain 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 (Δ exsY ), whereas adhesion to polystyrene (PS) and glass was not significantly affected by the presence of ENAs. The Δ exsY mutant also showed the lowest adhesion across all tested surfaces, a pattern similarly observed for vegetative cells. The strongest adhesion to PP was observed when both fiber types were present. A clear trend also emerged: on PP, WT remained adhered for at least an hour, while bald spores tended to detach within that time. Under saline conditions and at different pH levels, bald spores adhered strongly to SS. However, in the presence of a non-ionic surfactant or a concentrated protein solution, WT spores adhered more. Our results highlight the crucial role of ENAs in B. cereus spp. spore adhesion to industrially relevant surfaces, providing mechanistic insight into spore persistence. These insights support the design of surface treatments to prevent contamination, spoilage, and foodborne illnesses. IMPORTANCE Bacteria belonging to the Bacillus cereus sensu lato group represent a persistent challenge in food production due to their highly resilient endospores (spores), which withstand cleaning, disinfection, and food processing. Understanding spore adhesion 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 assays 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. paranthracis spore adhesion dynamics, guiding the development of tailored cleaning protocols to improve contamination control and sustainability.