Project description:Bacillus and Clostridium species form spores, which pose a challenge to the food industry due to their ubiquitous nature and extreme resistance. Pressurization at <300 MPa triggers spore germination by activating germination receptors (GRs), while pressurization at >300 MPa likely triggers germination by opening dipicolinic acid (DPA) channels present in the inner membrane of the spores. In this work, we expose spores of Bacillus licheniformis, a species associated with food spoilage and occasionally with food poisoning, to high pressure (HP) for holding times of up to 2 h. By using mutant spores lacking one or several GRs, we dissect the roles of the GerA, Ynd, and GerK GRs in moderately HP (mHP; 150 MPa)-induced spore germination. We show that Ynd alone is sufficient for efficient mHP-induced spore germination. GerK also triggers germination with mHP, although at a reduced germination rate compared to that of Ynd. GerA stimulates mHP-induced germination but only in the presence of either the intact GerK or Ynd GR. These results suggests that the effectiveness of the individual GRs in mHP-induced germination differs from their effectiveness in nutrient-induced germination, where GerA plays an essential role. In contrast to Bacillus subtilis spores, treatment with very HP (vHP) of 550 MPa at 37°C did not promote effective germination of B. licheniformis spores. However, treatment with vHP in combination with elevated temperatures (60°C) gave a synergistic effect on spore germination and inactivation. Together, these results provide novel insights into how HP affects B. licheniformis spore germination and inactivation and the role of individual GRs in this process.IMPORTANCE Bacterial spores are inherently resistant to food-processing regimes, such as high-temperature short-time pasteurization, and may therefore compromise food durability and safety. The induction of spore germination facilitates subsequent inactivation by gentler processing conditions that maintain the sensory and nutritional qualities of the food. High-pressure (HP) processing is a nonthermal food-processing technology used to eliminate microbes from food. The application of this technology for spore eradication in the food industry requires a better understanding of how HP affects the spores of different bacterial species. The present study provides novel insights into how HP affects Bacillus licheniformis spores, a species associated with food spoilage and occasionally food poisoning. We describe the roles of different germination receptors in HP-induced germination and the effects of two different pressure levels on the germination and inactivation of spores. This study will potentially contribute to the effort to implement HP technology for spore inactivation in the food industry.

Project description:UnlabelledWhen nutrients are scarce, Bacillus species form metabolically dormant and extremely resistant spores that enable survival over long periods of time under conditions not permitting growth. The presence of specific nutrients triggers spore germination through interaction with germinant receptors located in the spore's inner membrane. Bacillus licheniformis is a biotechnologically important species, but it is also associated with food spoilage and food-borne disease. The B. licheniformis ATCC 14580/DSM13 genome exhibits three gerA family operons (gerA, gerK, and ynd) encoding germinant receptors. We show that spores of B. licheniformis germinate efficiently in response to a range of different single l-amino acid germinants, in addition to a weak germination response seen with d-glucose. Mutational analyses revealed that the GerA and Ynd germination receptors function cooperatively in triggering an efficient germination response with single l-amino acid germinants, whereas the GerK germination receptor is essential for germination with d-glucose. Mutant spores expressing only GerA and GerK or only Ynd and GerK show reduced or severely impaired germination responses, respectively, with single l-amino acid germinants. Neither GerA nor Ynd could function alone in stimulating spore germination. Together, these results functionally characterize the germination receptor operons present in B. licheniformis We demonstrate the overlapping germinant recognition patterns of the GerA and Ynd germination receptors and the cooperative functionalities between GerA, Ynd, and GerK in inducing germination.ImportanceTo ensure safe food production and durable foods, there is an obvious need for more knowledge on spore-forming bacteria. It is the process of spore germination that ultimately leads to food spoilage and food poisoning. Bacillus licheniformis is a biotechnologically important species that is also associated with food spoilage and food-borne disease. Despite its importance, the mechanisms of spore germination are poorly characterized in this species. This study provides novel knowledge on germination of B. licheniformis spores. We characterize the germinant recognition profiles of the three germinant receptors present in B. licheniformis spores and demonstrate that the GerA germinant receptor cooperates with the Ynd and GerK germinant receptors to enable an effective germination response to l-amino acids. We also demonstrate that GerK is required for germination in response to the single germinant glucose. This study demonstrates the complex interactions between germinant receptors necessary for efficient germination of B. licheniformis spores.

Project description:The major challenge in employing high pressure (HP) at moderate temperature for sterilization is the remarkable resistance of bacterial spores. High isostatic pressure can initiate spore germination, enabling subsequent inactivation under mild conditions. However, not all spores could be triggered to germinate under pressure at temperatures ≤80°C so far. In this study, germination treatment combinations were evaluated for Bacillus spores involving moderate HP (150 MPa, 37°C, 5 min), very HP (vHP, 550 MPa, 60°C, 2.5 or 9 min), simple and complex nutrient germinants [L-valine, L-alanine, and tryptic soy broth (TSB)], nisin, and incubation at atmospheric pressure (37°C). The most effective combinations for Bacillus subtilis resulted in a reduction of culturable dormant spores by 8 log10 units. The combinations involved nisin, a nutrient germinant (L-valine or TSB), a first vHP treatment (550 MPa, 60°C, 2.5 min), incubation at atmospheric pressure (37°C, 6 h), and a second vHP treatment (550 MPa, 60°C, 2.5 min). Such treatment combination with L-valine reduced Bacillus amyloliquefaciens spores by only 2 log10 units. B. amyloliquefaciens, thus, proved to be substantially more HP-resistant compared to B. subtilis, validating previous studies. Despite combining different germination mechanisms, complete germination could not be achieved for either species. The natural bacteriocin nisin did seemingly not promote HP germination initiation under chosen HP conditions, contrary to previous literature. Nevertheless, nisin might be beneficial to inhibit the growth of HP-germinated or remaining ungerminated spores. Future germination experiments might consider that nisin could not be completely removed from spores by washing, thereby affecting plate count enumeration.ImportanceExtremely resistant spore-forming bacteria are widely distributed in nature. They infiltrate the food chain and processing environments, posing risks of spoilage and food safety. Traditional heat-intensive inactivation methods often negatively affect the product quality. HP germination-inactivation offers a potential solution for better preserving sensitive ingredients while inactivating spores. However, the presence of ungerminated (superdormant) spores hampers the strategy's success and safety. Knowledge of strategies to overcome resistance to HP germination is vital to progress mild spore control technologies. Our study contributes to the evaluation and development of mild preservation processes by evaluating strategies to enhance the HP germination-inactivation efficacy. Mild preservation processes can fulfill the consumers' demand for safe and minimally processed food.

Project description:Isostatic high pressure (HP) of 150 MPa can trigger the germination of bacterial spores, making them lose their extreme resistance to stress factors, and increasing their susceptibility to milder inactivation strategies. However, germination response of spores within a population is very heterogeneous, and tools are needed to study this heterogeneity. Here, classical methods were combined with more recent and powerful techniques such as flow cytometry (FCM) and fluorescence activated cell sorting (FACS) to investigate spore germination behavior under HP. Bacillus subtilis spores were treated with HP at 150 MPa and 37°C, stained with SYTO16 and PI, and analyzed via FCM. Four sub-populations were detected. These sub-populations were for the first time isolated on single cell level using FACS and characterized in terms of their heat resistance (80°C, 10 min) and cultivability in a nutrient-rich environment. The four isolated sub-populations were found to include (1) heat-resistant and mostly cultivable superdormant spores, i.e., spores that remained dormant after this specific HP treatment, (2) heat-sensitive and cultivable germinated spores, (3) heat-sensitive and partially-cultivable germinated spores, and (4) membrane-compromised cells with barely detectable cultivability. Of particular interest was the physiological state of the third sub-population, which was previously referred to as "unknown". Moreover, the kinetic transitions between different physiological states were characterized. After less than 10 min of HP treatment, the majority of spores germinated and ended up in a sublethally damaged stage. HP treatment at 150 MPa and 37°C did not cause inactivation of all geminated spores, suggesting that subsequent inactivation strategies such as mild heat inactivation or other inactivation techniques are necessary to control spores in food. This study validated FCM as a powerful technique to investigate the heterogeneous behavior of spores under HP, and provided a pipeline using FACS for isolation of different sub-populations and subsequent characterization to understand their physiological states.

Project description:UnlabelledRaman spectroscopy and phase-contrast microscopy were used to examine calcium dipicolinate (CaDPA) levels and rates of nutrient and nonnutrient germination of multiple individual Bacillus subtilis spores treated with cold atmospheric plasma (CAP). Major results for this work include the following: (i) >5 logs of spores deposited on glass surfaces were inactivated by CAP treatment for 3 min, while deposited spores placed inside an impermeable plastic bag were inactivated only ∼2 logs in 30 min; (ii) >80% of the spores treated for 1 to 3 min with CAP were nonculturable and retained CaDPA in their core, while >95% of spores treated with CAP for 5 to 10 min lost all CaDPA; (iii) Raman measurements of individual CAP-treated spores without CaDPA showed differences from spores that germinated with l-valine in terms of nucleic acids, lipids, and proteins; and (iv) 1 to 2 min of CAP treatment killed 99% of spores, but these spores still germinated with nutrients or exogenous CaDPA, albeit more slowly and to a lesser extent than untreated spores, while spores CAP treated for >3 min that retained CaDPA did not germinate via nutrients or CaDPA. However, even after 1 to 3 min of CAP treatment, spores germinated normally with dodecylamine. These results suggest that exposure to the present CAP configuration severely damages a spore's inner membrane and key germination proteins, such that the treated spores either lose CaDPA or can neither initiate nor complete germination with nutrients or CaDPA. Analysis of the various CAP components indicated that UV photons contributed minimally to spore inactivation, while charged particles and reactive oxygen species contributed significantly.ImportanceMuch research has shown that cold atmospheric plasma (CAP) is a promising tool for the inactivation of spores in the medical and food industries. However, knowledge about the effects of plasma treatment on spore properties is limited, especially at the single-cell level. In this study, Raman spectroscopy and phase-contrast microscopy were used to analyze CaDPA levels and kinetics of nutrient- and non-nutrient-germinant-induced germination of multiple individual spores of Bacillus subtilis that were treated by a planar CAP device. The roles of different plasma species involved in spore inactivation were also investigated. The knowledge obtained in this study will aid in understanding the mechanism(s) of spore inactivation by CAP and potentially facilitate the development of more effective and efficient plasma sterilization techniques in various applications.

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Inactivation Effect of Germination Combined with Cold Plasma Treatment on Bacillus licheniformis Spores

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