Effects of levels of purity of spores of two Bacillus species on their resistance, germination, and core CaDPA and water content

Previous work (V. K. Juneja, M. Osoria, E. G. Altuntas, N. K. Taneja, et al., Food Res Int 177:113904, 2024, https://doi.org/10.1016/j.foodres.2023.113904 ) showed that less well-purified spores of Bacillus cereus and Bacillus subtilis were more wet heat resistant than more well-purified spores. Extending the previous work, the current work used (i) B. subtilis spores identical to the one used previously but lacking a plasmid providing kanamycin resistance and (ii) spores of a different B. cereus strain, and with spores made on both plates and in liquid. There were larger differences in spores’ levels of purity in the new work than previously, and differences in spore purity were visible in micrographs of spore preparations purified differently. With spores made on plates, the least purified B. subtilis and B. cereus spores were more wet heat resistant than those purified further, but less so with spores prepared in liquid. These findings suggest that it may be important to consider the purity of spores prepared as indicators for sterilization assurance, especially since large-scale spore preparations are often not extensively purified. However, resistance to UV radiation, hydrogen peroxide, or sodium hypochlorite was relatively similar with the B. cereus or B. subtilis spores purified to different degrees. Thus, these methods for spore inactivation appear not be affected significantly by spore purity. Spores’ purity also had minimal effects on rates of germinant-receptor (GR) dependent spore germination with physiological germinants, or with dodecylamine that opens a channel protein leading to release of spore cores’ large pool of Ca-dipicolinic acid (CaDPA). In addition, contents of water in cores of B. cereus or B. subtilis spores purified to different degrees were almost identical, as were CaDPA levels. All these results do not yet allow obvious assignment of causes for the higher wet heat resistance of cruder B. subtilis or B. cereus spores. However, it was notable that levels of soluble protein in suspensions of spores purified differently were the highest in the crude spores and lowest in the most highly purified spores. IMPORTANCE Spores of many Bacillota give rise to cells and spores, some of which cause food spoilage, food poisoning, and serious human disease. Consequently, there is applied interest in spore killing, something that is difficult because of spores’ resistance to killing regimens including by wet heat, radiation, and toxic chemicals and the many causes of this extreme resistance. The current work shows that for spores of two Bacillus species made on plates or in liquid, spore purity plays no role in the ability of spores to germinate or their resistance to agents other than wet heat. However, the least purified B. subtilis or B. cereus spores, especially those made on plates, were significantly more wet heat resistant than more purified spores. While precise causes of the effects of spore purity on spore wet heat resistance are not clear, this seems to be an additional variable to consider when preparing spores for various uses, including as in sterilization assurance.