Impact of sorbic acid and other mild preservation stresses on germination and outgrowth of Bacillus cereus spores

Weak organic acids such as sorbic acid, lactate, and acetic acid are widely used by the food industry as preservatives to control growth of micro-organisms. With the current trend towards milder processing of food products, opportunities arise for spore-forming spoilage and pathogenic microorganisms such as Bacillus cereus, that may survive the use of milder heating regimes. Dormant spores produced by B. cereus can survive processing conditions and their subsequent outgrowth increases the risk of premature spoilage and food safety issues. As a consequence, the use of additional preservation hurdles, such as acidification with weak organic acid additives to ensure the quality and safety of a product is important. Sorbic acid is widely used as an antimicrobial compound because of its strong inhibitory properties against bacteria and other spoilage organisms. Its effectivity has also been ascribed to its hydrophobic character, resulting in an additional mode of action not observed with other less lipophilic organic acids such as lactic acid and acetic acid. In this project the impact of sorbic acid on spore germination and outgrowth was studied at transcriptome level and was linked to the distinct phenotypic responses observed for spores exposed to different levels of sorbic acid. The various stages of spore germination and outgrowth could be recognized by distinct gene expression profiles representing either the germination phase, transition state between germination and outgrowth or outgrowing cells, respectively. A subset of genes was specifically expressed in sorbic acid-exposed germinating spores and included functions related to cell envelope, (multi) drug transporters and amino acid metabolism. At high concentrations of sorbic acid (3mM of the undisssociated form, HSA), nutrient-induced germination of B. cereus ATCC 14579 spores was completely blocked. This blockage was shown to be reversible and could be bypassed by known non-nutrient triggers that activate spores in a receptor-independent way, pointing to a possible interference of HSA with the signaling event between germinant receptors and proposed SpoVA-channels, possibly by accumulation into the spore’s inner membrane. Additional experiments with other inhibiting compounds, including organic acids and their structurally similar alcohol counterparts, showed that the lipophilic properties are an important determinant of its efficacy to block germination. Building on current knowledge on the interaction of germination-relevant protein clusters, we discuss a hypothetical model on the mode of action of sorbic acid and other short-chain lipophilic compounds in germination inhibition of B. cereus spores. In addition to the interference or even blockage of germination, sorbic acid may increase outgrowth heterogeneity when applied at lower concentrations (0.25-1.5 mM) that still allow outgrowth. The first stages of outgrowth were shown to specifically occur heterogeneously when spores were exposed to multiple stresses simultaneously. Heterogeneity effects were most pronounced for combined stress-effects where heat-treated spores were also exposed to low pH stress. Under these conditions, a large subpopulation of spores was delayed between initial germination and swelling and further outgrowth. For the food producing industry, it would be desirable to have reliable parameters to predict the behavior of surviving spores in a food product. Data presented in this thesis show that germination rate is not a good predictor for outgrowth heterogeneity when applied as a single indicator. In conclusion, the work described in this thesis strive to obtain a better understanding on the impact that preservation stresses, including (sorbic) acid stress, have on the germination and outgrowth (heterogeneity) of B. cereus spores. The results obtained in this project may contribute to the rational design of new concepts for improved food preservation and safety.