Isolation, stability, and characteristics of high-pressure superdormant Bacillus subtilis spores

Bacterial spores are a major challenge in industrial decontamination processes owing to their extreme resistance. High-pressure (HP) of 150 MPa at 37 °C can trigger the germination of spores, making them lose their extreme resistance. Once their resistance is lost, germinated spores can easily be inactivated by a mild decontamination step. The implementation of this gentle germination-inactivation strategy is hindered by the presence of a subpopulation of so-called high-pressure superdormant (HPSD) spores, which resist germination or germinate only very slowly in response to HP. It is essential to understand the properties of HPSD spores and the underlying causes of superdormancy to tackle superdormant spores and further develop germination-inactivation strategies involving HP. This study investigated factors influencing the prevalence of HPSD spores and successfully isolated them by combining buoyant density centrifugation and fluorescence-activated cell sorting, which allowed further characterisation of HPSD spores for the first time. The prevalence of HPSD spores was shown to be strongly dependent on the HP dwell time, with increasing treatment times reducing their prevalence. Spore mutants lacking major germinant receptors further showed a highly increased prevalence of HPSD spores; 93% of the spores remained dormant even after a prolonged HP dwell time of 40 min. In contrast to nutrient germination, sublethal heat treatment of 75 °C for 30 min prior to pressure treatment did not induce spore activation and increase germination. The isolated HPSD spores did not show visible structural differences compared to the initial dormant spores when investigated with transmission electron microscopy. Re-sporulated HPSD spores showed similar germination capacity compared to the initial dormant spores, indicating that HPSD spores are most likely not genetically different from the rest of the population. Moreover, the majority of HPSD spores germinated when exposed a second time to the same germination treatment; however, the germination capacity was lower than that of the initial population. The fact that the majority of spores lost superdormancy when exposed a second time to the same trigger makes it unlikely that there is one factor that determines whether a spore germinates with a certain HP treatment or not. Instead, it seems possible that there are other reversible or cumulative causes. This study investigated the factors influencing spore HP superdormancy to improve the understanding of HPSD spores with regard to their stability, germination capacity, and potential underlying causes of spore HP superdormancy. This knowledge will contribute to the development of HP-based germination-inactivation strategies for gentle but effective spore control.