Jean-Marie Perrier-Cornet

Bacterial spores are among the most resistant forms of life on Earth. Their exceptional resistance properties rely on various strategies, among them the core singular structure, organization and hydration. By using elastic incoherent neutron scattering, we probed the dynamics of Bacillus subtilis spores to determine whether core macromolecular motions at the sub-nanosecond timescale could also contribute to their resistance to physical stresses. In addition, in order to better specify the role of the various spore components, we used different mutants lacking essential structure such as the coat (PS4150 mutant), or the calcium dipicolinic acid complex (CaDPA) located in the core (FB122 mutant). PS4150 allows to better probe the core’s dynamics, as proteins of the coat represent an important part of spore proteins, and FB122 gives information about the role of the large CaDPA depot for the mobility of core’s components. We show that core’s macromolecular mobility is not particularly constrained at the sub-nanosecond timescale in spite of its low water content as some dynamical characteristics as force constants are very close to those of vegetative bacteria such as Escherichia coli or to those of fully hydrated proteins. Although the force constants of the coatless mutant are similar to the wild-type’s ones, it has lower mean square displacements (MSDs) at high Q showing that core macromolecules are somewhat more constrained than the rest of spore components. However, no behavior reflecting the glassy state regularly evoked in the literature could be drawn from our data. As hydration and macromolecules’ mobility are highly correlated, the previous assumption, that core low water content might explain spores’ exceptional resistance properties seems unlikely. Thus, we confirm recent theories, suggesting that core water is mostly as free as bulk water and proteins/macromolecules are fully hydrated. The germination of spores leads to a much less stable system with a force constant of 0.1 N/m and MSDs ~2.5 times higher at low Q than in the dormant state. DPA has also an influence on core mobility with a slightly lower force constant for the DPA-less mutant than for the wild-type, and MSDs that are ~ 1.8 times higher on average than for the wild-type at low Q. At high Q, germinated and DPA-less spores were very similar to the wild-type ones, showing that DPA and core compact structure might influence large amplitude motions rather than local dynamics of macromolecules.

Blue light (400–430 nm) is known to induce lethal effects in some species of fungi by photo-oxidation caused by the excitation of porphyrins but the mechanisms involved remain poorly understood. In this work, we exposed the yeast Saccharomyces cerevisiae to a high density light flux with two-photon excitation (830 nm equivalent to a one-photon excitation around 415 nm) and used quasi real-time visualization with confocal microscopy to study the initiation and dynamics of photo-oxidation in subcellular structures. Our results show that the oxidation generated by light treatments led to the permeabilization of the plasma membrane accompanied by the sudden expulsion of the cellular content, corresponding to cell death by necrosis. Moreover, excitation in the plasma membrane led to very fast oxidation and membrane permeabilization (<60 s) while excitation at the center of the cell did not induce permeabilization even after a period exceeding 600 s. Finally, our study shows that the relationship between the laser power used for two-photon excitation and the time required to permeabilize the plasma membrane was not linear. Thus, the higher the power used, the lower the energy required to permeabilize the plasma membrane. We conclude that fungal destruction can be generated very quickly using a high density light flux. Better knowledge of the intracellular processes and the conditions necessary to induce necrosis should make it possible in the future to improve the efficiency of antimicrobial strategies photo-oxidation-based.

Because of the ability of foodborne pathogens to survive in low-moisture foods, their decontamination is an important issue in food protection. This study aimed to clarify some of the cellular mechanisms involved in inactivation of foodborne pathogens after drying and subsequent heating. Individual strains of Salmonella Typhimurium, Salmonella Senftenberg, and Cronobacter sakazakii were mixed into whole milk powder and dried to different water activity levels (0.25 and 0.58); the number of surviving cells was determined after drying and subsequent thermal treatments in closed vessels at 90 and 100°C, for 30 and 120 s. For each condition, the percentage of unculturable cells was estimated and, in parallel, membrane permeability and respiratory activity were estimated by flow cytometry using fluorescent probes. After drying, it was clearly observable that the percentage of unculturable cells was correlated with the percentage of permeabilized cells (responsible for 20–40% of the total inactivated bacteria after drying), and to a lesser degree with the percentage of cells presenting with loss of respiratory activity. In contrast, the percentages of unculturable cells observed after heat treatment were strongly correlated with the loss of respiratory activity and weakly with membrane permeability (for 70–80% of the total inactivated bacteria after heat treatment). We conclude that cell inactivation during drying is closely linked to membrane permeabilization and that heat treatment of dried cells affects principally their respiratory activity. These results legitimize the use of time–temperature scales and allow better understanding of the cellular mechanisms of bacterial death during drying and subsequent heat treatment. These results may also allow better optimization of the decontamination process to ensure food safety by targeting the most deleterious conditions for bacterial cells without denaturing the food product.

Due to the ability of foodborne pathogens to survive in low moisture food, the decontamination of milk powder is an important issue in food protection. The safety of food products is, however, not always insured and the different steps in the processing of food involve physiological and metabolic changes in bacteria. Among these changes, virulence properties may also be affected. In this study, the effect of drying and successive thermal treatments on the invasion capacity of Salmonella Typhimurium, Salmonella Senftenberg, and Cronobacter sakazakii was assessed. Bacteria were dried on milk powder at three different water activity levels (0.25, 0.58 and 0.80) and heated at two different temperatures (90°C and 100°C) for 30 s and 120 s. After recovery, stressed bacterial populations were placed in contact with Caco-2 cells to estimate their invasion capacity. Our results show that drying increases the invasion capacity of foodborne pathogens, but that heat treatment in the dried state did not exert a selective pressure on bacterial cells depending on their invasion capacity after drying. Taken together, our findings add to the sum of knowledge on food safety in dried food products and provide insight into the effects of food processing.