Heat resistance, membrane fluidity and sublethal damage in Staphylococcus aureus cells grown at different temperatures
Introduction
Despite the widespread use and proven efficacy of thermal treatments for the inactivation of pathogen and spoilage microorganisms from foods, there is still a lack of knowledge regarding the factors influencing bacterial inactivation by heat, their mode of action, the rationale behind the kinetics of inactivation, and the responses that bacteria may develop to increase their tolerance to heat, among other aspects. This is still more relevant now, since consumer requirements for fresher and less processed foods are currently leading to a decrease in the intensity of treatments, thus requiring improved knowledge of all these factors in order to design processes that ensure food safety (Abee and Wouters, 1999; Raso and Barbosa-Cánovas, 2003).
Growth temperature is generally acknowledged as one of the main factors influencing bacterial resistance to heat (Cebrián et al., 2017; Jay, 1992; Russell, 1984). It is assumed that bacteria display greater heat resistance when grown at higher temperatures (Cebrián et al., 2008; Elliker and Frazier, 1938; Knabel et al., 1990; Mañas et al., 2003; Pagán et al., 1999). Nevertheless, it should be noted that the degree of influence of growth temperature varies widely among species (Pagán et al., 1999). Although several theories have been proposed to explain this behavior, studies specifically designed to elucidate the mechanisms leading to the increased resistance of cells grown at higher temperatures are scarce.
One controversial question is whether cells grown at higher temperatures are more heat-resistant because they have more stable membranes. The cytoplasmic membrane is considered to be one of the targets of heat treatment (Hurst et al., 1973; Mackey, 2000). However, it has hitherto not been possible to establish a direct relationship between membrane damage and bacterial inactivation by heat. It has been suggested that the membrane could play an indirect role in inactivation, since its damage might result in a loss of homeostasis and a loss of cellular components that would lead to cell death (Coote et al., 1994; Kramer and Thielmann, 2016; Mackey et al., 1991; Marcén et al., 2017; Teixeira et al., 1997). Therefore, it is reasonable to suppose that changes in membranes − for instance in their structure, composition, or mechanical behavior − may influence cell survival to a variety of stressing agents, including heat. Beuchat and Worthington (1976) proposed that the increased heat tolerance of cells grown at higher temperatures might be related to a higher degree of saturation of the fatty acids of their membranes. Such increased saturation would lead to less fluid and thus more thermo-stable membranes reduce their fluidity, thus making membranes more thermo-stable. However, it should be noted that fatty acid composition is not the only factor that determines membrane fluidity; other components should be considered in order to determine how membrane fluidity varies with growth temperature (Denich et al., 2003; Stintzi, 2003). Furthermore, to validate this hypothesis, it would be necessary to carry out measurements of membrane fluidity at lethal temperatures, since the complex composition of bacterial membranes makes it difficult to predict a membrane's degree of fluidity at a given temperature.
On the other hand, it has also been observed that variations in growth temperature induce proteomic changes. When bacterial cells are grown close to their maximum growth temperature, a number of so-called Heat-Shock Proteins (HSPs), including chaperones and proteases, are induced (Lim and Gross, 2010; Schumann, 2007). Various authors have hypothesized that these proteins might be responsible for the increased resistance of bacterial cells grown at higher temperatures, which could be attributed to their greater ability to repair damages caused by heat exposure at different cellular levels (Herendeen et al., 1979; Schumann, 2007; Smith et al., 1991). Moreover, it is known that several heat shock proteins also exert direct stabilizing effects on cellular structures including membranes (Coucheney et al., 2005; Török et al., 1997; Tsvetkova et al., 2002).
Staphylococcus aureus is a well-known foodborne pathogen (Baird-Parker, 2000). However, in spite of its frequent isolation as a causative agent of food poisoning in almost every part of the world, little is known about the influence of physiological factors on the susceptibility of this microorganism to heat treatments. In this study we attempt to describe and characterize the influence of growth temperature on heat resistance, along with the occurrence of repairable cellular injuries, in S. aureus cells in both exponential and stationary growth phases. Our ultimate aim was to examine the role that membrane fluidity plays in cell survival, and to gain fundamental knowledge about the physiology of bacterial inactivation by heat treatments.
Section snippets
Bacterial culture and media
Staphylococcus aureus CECT 4459 was used in this investigation. The bacterial culture was maintained frozen at −80 °C in cryovials. Stationary-phase cultures were prepared by inoculating 10 mL of tryptone soya broth (Biolife, Milan, Italy) supplemented with 0.6% yeast extract (Biolife) (TSB-YE) with a loopful of growth from tryptone soy agar supplemented with 0.6% yeast extract (TSA-YE) (Biolife) and incubating the resulting culture for 12 h at 37 °C in a shaking incubator. 50 μL of this
Influence of growth temperature on S. aureus heat resistance: exponential growth phase cells
Fig. 1A shows the survival graphs to heat at 58 °C of S. aureus exponential growth phase cells grown at 10, 20, 30, 37 and 42 °C. As can be observed, survival curves obtained for exponential growth phase cells displayed shoulders of varying length. Cells grown between 10 and 37 °C showed a very similar thermotolerance, and no significant differences (p > 0.05) were found among either the D58 values (0.208–0.138 min) or the sl58 values (0.146–0.090 min). Conversely, cells grown at 42 °C showed
Discussion
In this study, the influence of growth temperature on Staphylococcus aureus survival to heat was investigated. The mechanisms behind the changes in resistance were also explored with special emphasis on the influence of the physiological state of cells, the occurrence of sublethal damages, and the relationship with membrane fluidity.
Survival curves of S. aureus suspensions exposed to heat proved to be non-linear: therefore, a special case of the Baranyi and Roberts model and the DMfit software
Concluding remarks
Results reported herein demonstrate that increasing growth temperature leads to a greater rigidity of membranes at treatment temperatures; moreover, a significant correlation between that parameter and cellular heat resistance was found. We have proven that cells exposed to a fluidizer compound (benzyl alcohol), and thus with a more fluid membrane, are sensitized against the action of heat. According to this data, the physical state of the membrane is an important factor determining the
Acknowledgements
The authors would like to thank the Ministerio de Economía y Competitividad, EU-FEDER (project AGL-2015-69565-P) and the Department of Science, Technology and University of the Aragon Government for the support.
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