Abstract
The effects of environmental stresses on microorganisms have been well-studied, and cellular responses to stresses such as heat, cold, acids, and salts have been extensively discussed. Although high pressure processing (HPP) is becoming more popular as a preservation method in the food industry, the characteristics of the cellular damage caused by high pressure are unclear, and the microbial response to this stress has not yet been well-explored. We exposed the pathogen Listeria monocytogenes to HPP (400 MPa, 8 min, 8°C) and found that the high pressure created plasma membrane pores. Using a common staining technique involving propidium iodide (PI) combined with high-frequency fluorescence microscopy, we monitored the rate of diffusion of PI molecules into hundreds of bacterial cells through these pores on days 0, 1, 2, 3, and 4 after pressurization. We also developed a mathematical dynamic model based on mass transfer and passive diffusion laws, calibrated using our microscopy experiments, to evaluate the response of bacteria to HPP. We found that the rate of diffusion of PI into the cells decreased over the 4 consecutive days after exposure to HPP, indicating repair of the pressure-created membrane pores. The model suggested a temporal change in the size of pores until closure. To the best of our knowledge, this is the first time that pressure-created membrane pores have been quantitatively described and shown to diminish with time. In addition, we found that the membrane repair rate in response to HPP was linear, and growth was temporarily arrested at the population level during the repair period. These results support the existence of a progressive repair process in some of the cells that take up PI, which can therefore be considered as being sub-lethally injured rather than dead. Hence, we showed that a subgroup of bacteria survived HPP and actively repaired their membrane pores.
Highlights
Bacteria in nature are exposed to various environmental stresses, including changes in temperature or pH, radiation, antimicrobial compounds, and osmotic pressure
We focused on foodborne pathogenic bacteria L. monocytogenes and the effects of high pressure processing (HPP) (400 MPa, 8 min, 8◦C) on its membrane
We used the k-means algorithm to cluster the intensity curves using a certain number of clusters for days 0, 1, 2, 3, and 4 after pressure exposure based on the rate of propidium iodide (PI) diffusion through membrane pores
Summary
Bacteria in nature are exposed to various environmental stresses, including changes in temperature or pH, radiation, antimicrobial compounds, and osmotic pressure. The potential existence of a membrane repair machinery in bacteria that responds to pressure-induced damage has not been well-investigated. Gänzle and Vogel (2001) showed changes in the kinetics of outer and cytoplasmic membrane permeability in Escherichia (E.) coli after exposure to high pressure (300, 500, 600 MPa) by staining of treated cells with PI and 1-N-phenylnaphthylamine. A few studies have detected cellular proteins outside the cell after exposure to high pressure (Smelt et al, 1994; Gänzle and Vogel, 2001; Mañas and Mackey, 2004) One such example is the study of Mañas and Mackey (2004), which detected intracellular proteins outside pressure-treated E. coli cells (200 MPa, 8 min), indicating membrane leakage
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