Inhibition of Bacillus cereus spore outgrowth and multiplication by chitosan

Bacillus cereus is an endospore-forming bacterium able to cause food-associated illness. Different treatment processes are used in the food industry to reduce the number of spores and thereby the potential of foodborne disease. Chitosan is a polysaccharide with well-documented antibacterial activity towards vegetative cells. The activity against bacterial spores, spore germination and subsequent outgrowth and growth (the latter two events hereafter denoted (out)growth), however, is poorly documented. By using six different chitosans with defined macromolecular properties, we evaluated the effect of chitosan on Bacillus cereus spore germination and (out)growth using optical density assays and a dipicolinic acid release assay. (Out)growth was inhibited by chitosan, but germination was not. The action of chitosan was found to be concentration-dependent and also closely related to weight average molecular weight (M_w) and fraction of acetylation (F_A) of the biopolymer. Chitosans of low acetylation (F_A = 0.01 or 0.16) inhibited (out)growth more effectively than higher acetylated chitosans (F_A = 0.48). For the F_A = 0.16 chitosans with medium (56.8 kDa) and higher M_w (98.3 kDa), a better (out)growth inhibition was observed compared to low M_w (10.6 kDa) chitosan. The same trend was not evident with chitosans of 0.48 acetylation, where the difference in activity between the low (19.6 kDa) and high M_w (163.0 kDa) chitosans was only minor. In a spore test concentration corresponding to 10²-10³ CFU/ml (spore numbers relevant to food), less chitosan was needed to suppress (out)growth compared to higher spore numbers (equivalent to 10⁸ CFU/ml), as expected. No major differences in chitosan susceptibility between three different strains of B. cereus were detected. Our results contribute to a better understanding of chitosan activity towards bacterial spore germination and (out)growth.