Oxidative stress in E. coli cells upon exposure to heat treatments

Abstract

Heat treatments are widely used by the food industry to inactivate microorganisms, however their mode of action on microbial cells is not fully known. In the last years, it has been proposed that the generation of oxidative species could be an important factor contributing to cell death by heat and by other stresses; however, investigations in this field are scarce. The present work studies the generation of reactive oxygen species (ROS) upon heat treatment in E. coli, through the use of cell staining with specific fluorochromes. Results obtained demonstrate that ROS are detected in E. coli cells when they are subjected to heat exposure, and the amount of fluorescence increases with temperature and time, as does the cellular inactivation. The addition of glutathione or tiron, a potent antioxidant and a superoxide quencher, respectively, to the heating medium protected E. coli against heat inactivation and concurrently reduced the detection of ROS, especially in the case of glutathione. Finally, recovery of heated cells under conditions that relief oxidative stress produced an increase in cell survival. Data presented in this work support the view that ROS generation and subsequent control in bacterial cells could be an essential factor determining inactivation and survival upon exposure to heat, and it could be a potential target to increase the efficacy of current treatments.

The Synergistic Bactericidal Mechanism of Simultaneous Treatment with a 222-Nanometer Krypton-Chlorine Excilamp and a 254-Nanometer Low-Pressure Mercury Lamp

“…Intracellular superoxide was detected using hydroethidine (HDE) oxidized to produce ethidium bromide by reacting with superoxide within the cells. Since this oxidative product becomes fluorescent when it intercalates into DNA, it can quantitatively assess the extent of intracellular superoxide generation through measurement of fluorescence value (82)(83)(84)(85).…”

Protective effect of glutathione on Escherichia coli cells upon lethal heat stress

“…MM-cys consists of minimal medium M9 glucose-salts agar supplemented with FeSO 4 (10 mg/L) (Gerhardt, 1994;Stanier & Villanueva, 1996), and enriched with 3 mM L-cysteine HCl to create a low redox potential environment (Gerhardt, 1994;Suh & Knabel, 2000). This agar was chosen to improve the recovery of cells (Marcén et al, 2017). In this latter case, anaerobic atmosphere was used in order to further reduce the oxidative component of the recovery conditions, and 72 h were allowed for full colony development.…”

“…The presence of ROS and the permeabilization of the membrane in cells were studied through staining with specific fluorochromes followed by epifluorescence microscopy (Marcén et al, 2017). Treated cells, at approximately 10 8 cells, were centrifuged (6708 g/5 min), resuspended in PBS and incubated with the desired fluorescence dye.…”

“…One of the physiological aspects that is gaining attention in the last years is the involvement of an oxidative component in the inactivation and damage of cells exposed to a variety of agents used in the food industry, such as heat, acid or high hydrostatic pressure (Aertsen, De Spiegeleer, Vanoirbeek, Lavilla, & Michiels, 2005;Marcén, Ruiz, Serrano, Condón, & Mañas, 2017;Mols, Van Kranenburg, Van Melis, Moezelaar, & Abee, 2010). It has been suggested that exposure to agents of several nature may cause an unbalance in the intracellular redox homeostasis (Mols & Abee, 2011) that could contribute to cell inactivation and sublethal injury.…”

“…In a previous work carried out in our laboratory, we observed that the addition of glutathione to the heating medium protected E. coli against inactivation (Marcén et al, 2017). The objective of this work was to further study the effect of glutathione on bacterial survival to heat, using E. coli as a model microorganism, with the final aim of gaining insight into the physiological mechanism of cell protection, and obtaining a deeper knowledge about the mode of action of heat on bacterial cells.…”