Understanding low hydration state of bacteria spores

International audienc

Compréhension des mécanismes de l'inactivation cellulaire par les hautes pressions hydrostatiques et les basses températures

Ce travail est axé sur la compréhension des effets des hautes pressions hydrostatiques (HPH) et du froid sur différents modèles cellulaires : bactéries, levures, cellules leucémiques, sphéroplastes, liposomes. Une première partie a consisté à mettre en évidence les dommages cellulaires occasionnés par le froid jusqu à -20 C en milieu liquide vs milieu congelé lors de traitements avec de longs temps de maintien jusqu à 71 jours. L influence des HPH (jusqu à 600 MPa) à température ambiante est ensuite étudiée en fonction de l état d hydratation cellulaire. Le rôle crucial de l eau a notamment été souligné puisque les cellules faiblement hydratées peuvent acquérir une baro-résistance totale même dans le cas des traitements hyperbares drastiques. L étude s est ensuite focalisée sur l influence des traitements combinant hautes pressions et basses températures. Une interaction synergique entre les HPH et le froid vis-à-vis de l inactivation cellulaire est observée pour les niveaux de pression inférieurs à 300 MPa (pour un niveau d hydratation physiologique : aw=0,992). L amplitude de cette synergie augmente avec le niveau d hydratation cellulaire. Un effet d antagonisme du froid apparait pour les niveaux de pressions supérieurs à 300 MPa, permettant de préserver les cellules contre l action de la pression observée à 25 C. L amplitude de cet antagonisme augmente lorsque le niveau d hydratation cellulaire diminue. Les effets des différents traitements à l échelle subcellulaire sont étudiés en considérant plusieurs aspects liés à la physiologie, l ultrastructure, la morphologie, la structure et l intégrité membranaires de la cellule. Ces effets sont reliés à la compression volumétrique cellulaire, phénomène plus global décrivant l action de la pression sur le système cellulaire.The aim of this work was to understand the effects of high hydrostatic pressure (HHP) and low temperature on different cell models: bacteria, yeast, leukemia cells, spheroplasts, lipid vesicles. The viability of cells subjected to cold stress was first assessed, through long-term supercooling experiments (up to 71 days), versus freeze-thawing stress. Results showed that cells could be inactivated by the only action of sub-zero temperature, that is, without any water crystallization. The effects of HHP at room temperature were then studied as a function of cell hydration. We highlighted the crucial role of water in determining cellular damage under pressure. The combined effects of HHP and low temperature on cells were studied. Results showed a synergistic interaction between cold and HHP in their effects on microbial inactivation at pressures in the range of 100 to 300 MPa with an aw of 0.992. However, at pressures greater than 300 MPa, this trend was reversed, and cold counteracted the inactivation effect of pressure. When the amount of water in the cell suspension was increased, the synergistic effect was enhanced. Conversely, when the aw was decreased by the addition of solute to the cell suspension, the baroprotective effect of subzero temperature increased sharply. These results support the argument that water compression is involved in the mechanisms of HHP inactivation of cells. From a thermodynamic point of view, the mechanical energy transferred to the cell during the pressure treatment can be characterized by the change in volume of the system, which depends on the water quantity in the cytoplasm. The consequences of cell volume compression at the cellular level were characterized through the assessment of metabolic activity, the study of cell membrane structure and integrity and the visualization of cell shape and ultrastructure.DIJON-BU Sciences Economie (212312102) / SudocSudocFranceF

Applications des hautes pressions hydrostatiques en agroalimentaire

Article à vocation de transfer

Compréhension des mécanismes de l inactivation microbienne sous hautes pressions gazeuses

Ce travail est axé sur la compréhension des effets des gaz sous pression sur la survie microbienne en fonction du niveau d hydratation des micro-organismes. Cette étude a été menée sur deux levures Saccharomyces cerevisiae et Candida edax et sur une bactérie sous forme sporulée, Bacillus subtilis. Une phase préliminaire de développement a permis de mettre en place des enceintes Haute Pression compactes permettant de traiter les micro-organismes en milieux gazeux sous pression sur des longs temps de maintien, jusqu à plusieurs mois. Une première partie a consisté à comparer les effets d une pression isostatique transmise par un liquide ou par l azote sur la viabilité microbienne. Lorsque les micro-organismes sont placés à de forts niveaux d hydratation, l inactivation est liée à la compression cellulaire et la présence d azote n a pas d effet spécifique sur la viabilité. Aux faibles niveaux d hydratation, la viabilité des micro-organismes n est pas affectée par une pression transmise par un liquide. Au contraire, l azote sous pression présente un effet spécifique et original, qui conduit à l inactivation des micro-organismes faiblement hydratés. L étude s est ensuite focalisée sur la caractérisation de cet effet spécifique et a montré que les phénomènes de sorption et de désorption des gaz dans les micro-organismes permettent d expliquer les inactivations observées. Les techniques d investigation utilisées (microscopie électronique, microscopie et spectroscopie de fluorescence ) ont permis d identifier certains des mécanismes liés à la mort microbienne en milieux gazeux sous pression. Une explication intégrant l ensemble de ces éléments est enfin proposée et discutée.The effects of high gas pressure on the survival of microorganisms were here investigated at different hydration levels. A preliminary step was dedicated to the development of compact high-pressure cylinders to perform high-pressure treatments in gaseous media over long periods, up to several months. In a first section, the influence of both liquid and nitrogen pressures on microbial viability were compared. At high hydration levels, the presence of nitrogen had no specific effect on viability and the observed inactivation was induced by cell compression. At low hydration levels, liquid pressure did not affect microbial viability. On the contrary, pressurised nitrogen induced cell inactivation due to gas sorption and desorption phenomena. We then focused on this specific effect and identified some of the mechanisms leading to cell death by employing different methods (scanning electron microscopy, fluorescence techniques .). Finally, an explanation is developed and discussed.DIJON-BU Sciences Economie (212312102) / SudocSudocFranceF

Synergistic and Antagonistic Effects of Combined Subzero Temperature and High Pressure on Inactivation of Escherichia coli

The combined effects of subzero temperature and high pressure on the inactivation of Escherichia coli K12TG1 were investigated. Cells of this bacterial strain were exposed to high pressure (50 to 450 MPa, 10-min holding time) at two temperatures (−20°C without freezing and 25°C) and three water activity levels (a(w)) (0.850, 0.992, and ca. 1.000) achieved with the addition of glycerol. There was a synergistic interaction between subzero temperature and high pressure in their effects on microbial inactivation. Indeed, to achieve the same inactivation rate, the pressures required at −20°C (in the liquid state) were more than 100 MPa less than those required at 25°C, at pressures in the range of 100 to 300 MPa with an a(w) of 0.992. However, at pressures greater than 300 MPa, this trend was reversed, and subzero temperature counteracted the inactivation effect of pressure. When the amount of water in the bacterial suspension was increased, the synergistic effect was enhanced. Conversely, when the a(w) was decreased by the addition of solute to the bacterial suspension, the baroprotective effect of subzero temperature increased sharply. These results support the argument that water compression is involved in the antimicrobial effect of high pressure. From a thermodynamic point of view, the mechanical energy transferred to the cell during the pressure treatment can be characterized by the change in volume of the system. The amount of mechanical energy transferred to the cell system is strongly related to cell compressibility, which depends on the water quantity in the cytoplasm

L’imagerie microscopique et la spectroscopie de fluorescence : outils essentiels pour l’étude de modèles cellulaires sous haute pression

National audienc

Are bacterial spores activated by High Pressure treatment at 20°C ?

International audienc

Damage in Escherichia coli Cells Treated with a Combination of High Hydrostatic Pressure and Subzero Temperature▿

The relationship between membrane permeability, changes in ultrastructure, and inactivation in Escherichia coli strain K-12TG1 cells subjected to high hydrostatic pressure treatment at room and subzero temperatures was studied. Propidium iodide staining performed before and after pressure treatment made it possible to distinguish between reversible and irreversible pressure-mediated cell membrane permeabilization. Changes in cell ultrastructure were studied using transmission electron microscopy (TEM), which showed noticeable condensation of nucleoids and aggregation of cytosolic proteins in cells fixed after decompression. A novel technique used to mix fixation reagents with the cell suspension in situ under high hydrostatic pressure (HHP) and subzero-temperature conditions made it possible to show the partial reversibility of pressure-induced nucleoid condensation. However, based on visual examination of TEM micrographs, protein aggregation did not seem to be reversible. Reversible cell membrane permeabilization was noticeable, particularly for HHP treatments at subzero temperature. A correlation between membrane permeabilization and cell inactivation was established, suggesting different mechanisms at room and subzero temperatures. We propose that the inactivation of E. coli cells under combined HHP and subzero temperature occurs mainly during their transiently permeabilized state, whereas HHP inactivation at room temperature is related to a balance of transient and permanent permeabilization. The correlation between TEM results and cell inactivation was not absolute. Further work is required to elucidate the effects of pressure-induced damage on nucleoids and proteins during cell inactivation

A new design intended to relate high pressure treatment to yeast cell mass transfer

International audienceA new optical device has been developed to allow the observation of microorganisms during a high pressure treatment up to 700 MPa. To measure cell volume variation during the high pressure application, an image analysis system was connected with the light microscope. With this device, growth of Saccharomyces cerevisiae was studied at moderate pressure (10 MPa) through the observation of individual cell budding. Cell volume variations were also measured on the yeast Saccharomycopsis fibuligera on fixed cells as well on a population sample and a shrinkage in average cell volume was observed consequently to a pressure increase of 250 MPa. The observed compression rate (25%) under pressure and the partial irreversibility of cell compression (10%) after return to atmospheric pressure lead to the conclusion that a mass transfer between cell and cultivation medium occurred. The causes of this transfer could be explained by a modification of membrane properties, i.e., disruption or increase in permeability