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...induction of intracellular heat-shock proteins and the activation of extracellular alarmones in vegetative cells exposed to mildly lethal temperatures are important cell responses. In bacterial spores, several factors contribute to the overall resistance to moist (wet) and dry heat; the latter, but not the former, induces mutations, Heat resistance develops during sporulation, when spore-specific heat-shock proteins are also produced. Heat sensitivity is regained during germination of spores.
Sci. Prog. 86:115-137 [c]2003 Science Reviews
Keywords: lethal temperature, bacterial physiology and structure
Introduction
Bacteria vary considerably in their temperature response (Table I). For every organism, there is minimum, optimum and maximum growth temperature."2 Some organisms (psychrophiles) have the ability to grow at low temperatures (minimum 0[degrees]C or lower, optimal 5[degrees]C or lower, maximum 20[degrees]C and others (thermophiles) at high temperatures (above 45[degrees]C but often of the order of 50--60[degrees]C), whereas the majority of bacteria (mesophiles) grow best at intermediate ones (35--42[degrees]C) Psychrotolerant organisms grow at 0[degrees]C but have optima of 20--40[degrees]C. Hyperthermophile archeae have optimal very high temperatures for growth, above 80[degrees]C but sometimes above the boiling point of water. In this context, an extremoenzyme is an enzyme that either functions at very high temperatures or optimally in any environmental extreme, with organisms producing them being referred to as extremophiles.
When lethal effects of high temperatures are considered, differences are again apparent. Generally, many non-sporulating bacteria are readily inactivated at temperatures of about 50[degrees]C and above, the rate of inactivation increasing as the temperature is raised!-6 By contrast, bacterial spores are much more resistant and moist heat temperatures of at least 1 100[degrees]C and often appreciably higher are usually needed to produce significant decreases in viability. (4-6)
Other aspects need to be mentioned. The first is the nature of the heat shock applied, i.e. whether it is moist heat or dry heat, respectively in the presence or absence of water or moisture, since very different effects on bacterial physiology result. Second, thermal methods of inactivating micro-organisms are of great importance in the canning industry and in the sterilization of parenteral and certain other types of pharmaceutical and medical products. (5,6) Additionally, high moist heat temperatures, below those necessary for sterilization, may provide an effective means of disinfection. (6)
Heat inactivation kinetics
Older terms such as thermal death time (TDT) and thermal death point (TDP) are now of little relevance because they vary greatly with the inoculum size. (4) By contrast, the D-value (decimal reduction time, DRT), which is independent of the inoculum size, is widely employed. This is defined as the time, usually in minutes, necessary to produce a 1-[log.sub.10] reduction in viability, i.e. to reduce the numbers of colony-forming units (cfu) by 90% (to 10%). The D-value is inversely related to temperature.
The z-value is defined as the number of degrees ([degrees]C) to achieve a 10-fold reduction in the D-value. It can be obtained from
(a) the slope of the curve when temperature is plotted against D-value, or
(b) [Q.sub.10] = [10.sup.10/z], from which z = 10/log[Q.sub.10], where [Q.sub.10] is the temperature coefficient per 10[degrees]C rise in temperature.
The terms defined above are widely used in studies involving thermal injury as well as for sterilization purposes. Table 2 presents some examples of D-values and z-values for sporulating bacteria.
Some important conclusions can be reached:
(i) most non-sporulating bacteria are highly susceptible to temperatures that have no effect on the viability of bacterial spores;
(ii) hyperthermophiles /extremophiles are extremely resistant to heat; (3,7,8)
(iii) thermophilic spores, e.g. Bacillus stearothermophilus, are less susceptible to high temperatures than are other spores;
(iv) dry heat is a far less effective inactivation process than moist heat.
The shapes of time-inactivation (time-survival) curves have revealed some interesting findings. (9-11) Different types of response to high temperatures may be shown. Log-linear kinetics are demonstrated in Figure 1A. Deviations from this are known and may be associated with heterogeneity of resistance within a population or with changes in resistance during heating. An initial shoulder (Figure 1B) may denote clumping of cells or demonstrate that inactivation occurs via a 'multihit' process on target sites. Tailing (Figure 1C) indicates the presence of small numbers of large clumps or of heterogeneity of resistance in the population. A sigmoidal curve (Figure 1D) results from a combination of factors. In Escherichia coli O157:H7, Stringer et al. (13) suggested that a shoulder and/or tailing could result from multiple hits on intracellular targets or could be a consequence of heat adaptation during the heating process. Deviations from log-linear kinetics occur more frequently and to a greater degree with vegeta tive bacteria. (12)
Mechanisms of thermal inactivation of non-sporulating bacteria
Several factors influence bacterial inactivation, (13) in particular the composition and pH of the menstruum, the type of organism (there could be a strain-dependent response), the growth conditions (medium, growth phase, temperature), heating method (open systems are less accurate than closed ones) and the recovery conditions.
Every cellular component (outer layers, membrane, enzymes and proteins, DNA, RNA) is likely to be affected to some degree by high temperatures (Table 3, Figure 2). Thus, it is no easy task to ascribe a lethal effect to a single alteration within an organism. Nevertheless, some changes are more pronounced than others, some types of damage may be repairable and all depend upon the intensity of the temperature applied. It is thus advisable to examine a range of temperatures, from mild to severe, for their effects on bacterial cells in order to determine the nature of the lethal effects that occur.
(1) Damage to the cell wall / outer membrane (OM)
High temperatures may produce changes in the outer cell layers of bacteria. In Gram-negative bacteria, damage to the OM occurs when cells are subjected to a mild heat shock. (14) Morphological and structural changes (blebbing) (15) and loss of OM lipopolysaccharide (LPS) (16,17) have been reported, thereby altering the permeability barrier, with the release of periplasmic proteins, entry of otherwise impermeable fluorescent dyes, (16,17) sensitivity to hydrophobic antibiotics (18) and transient sensitivity to nisin. (19) The effect of moderate temperatures is enhanced in the presence of Tris buffer, (16,17) which has long been known to affect the OM in its own right. (20,21)
The cell wall of organisms such as Staphylococcus aureus is much more rigid with a greater amount and extent of cross-linked peptidoglycan. As such, Welker (21) considered it less likely to be affected by high temperatures than the OM of Gram-negative bacteria. Allwood and Russell (22) found cell shrinkage and precipitation of intracellular materials as well as leakage with heated S.aureus cells.
(2) Damage to the cytoplasmic / inner membrane
The cytoplasmic (inner) membrane is a delicate, semipermeable lipoprotein structure situated beneath the cell wall (OM in Gram-negative bacteria). It...
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More articles from Science Progress
Extracellular proteins as enterobacterial thermometers., March 22, 2003
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