Habituation of Salmonella spp. at reduced water activity and its effect on heat tolerance

The effect of habituation at reduced water activity (a(w)) on heat tolerance of Salmonella spp. was investigated. Stationary-phase cells were exposed to a(w) 0.95 in broths containing glucose-fructose, sodium chloride, or glycerol at 21 degrees C for up to a week prior to heat challenge at 54 degrees C. In addition, the effects of different a(w)s and heat challenge temperatures were investigated. Habituation at a(w) 0.95 resulted in increased heat tolerance at 54 degrees C with all solutes tested. The extent of the increase and the optimal habituation time depended on the solute used. Exposure to broths containing glucose-fructose (a(w) 0.95) for 12 h resulted in maximal heat tolerance, with more than a fourfold increase in D(54) values. Cells held for more than 72 h in these conditions, however, became as heat sensitive as nonhabituated populations. Habituation in the presence of sodium chloride or glycerol gave rise to less pronounced but still significant increases in heat tolerance at 54 degrees C, and a shorter incubation time was required to maximize tolerance. The increase in heat tolerance following habituation in broths containing glucose-fructose (a(w) 0.95) was RpoS independent. The presence of chloramphenicol or rifampin during habituation and inactivation did not affect the extent of heat tolerance achieved, suggesting that de novo protein synthesis was probably not necessary. These data highlight the importance of cell prehistory prior to heat inactivation and may have implications for food manufacturers using low-a(w) ingredients.     

Effects of temperature shift on acid and heat tolerance in Salmonella enteritidis phage type 4.

The transfer of cells of Salmonella enteritidis phage type 4 from 20 to 37-46 degrees C resulted in marked increases in acid and heat tolerance. The former was maximized within 5 to 15 min of the shift and was largely independent of protein synthesis. In contrast, induction of increased heat tolerance was slower, requiring more than 60 min to be completed, and was prevented by inhibition of protein synthesis. When cells were transferred to medium at temperatures between 47 and 50 degrees C, the kinetics of induction of heat tolerance were essentially the same as at the lower temperatures. In contrast, the cells became more acid sensitive. The results of these studies clearly show that although both acid and heat resistance can be enhanced by preexposure to high incubation temperatures, the mechanisms involved are different.     

Mechanisms of heat inactivation in Salmonella serotype Typhimurium as affected by low water activity at different temperatures

AIMS: To determine the effect of reduced water activity (a(w)) on thermal inactivation of Salmonella serotype Typhimurium at different temperatures and its mechanism. METHODS AND RESULTS: D-value determinations at a range of different temperatures showed that heating at reduced a(w) (0.94, produced by addition of glucose or sodium chloride to nutrient broth) was protective at temperatures above 53-55 degrees C but sensitizing below this temperature. Using selective enumeration media to determine injury, it was shown that at lower heating temperatures cells survived at high a(w) with cytoplasmic injury whereas at low a(w) these cells were killed. At higher temperatures ribosome degradation was a more important cause of death and was inhibited by low a(w) heating media thereby providing greater heat resistance. CONCLUSIONS: The observed change in behaviour reflects the different reactions responsible for thermal death at different temperatures and their different response to reduced a(w). SIGNIFICANCE AND IMPACT OF THE STUDY: This work qualifies the previous assumption that reduced a(w) is protective and suggests that the efficacy of low temperature pasteurization regimes may be increased by reduced a(w).     

Life at low water activity

Two major types of environment provide habitats for the most xerophilic organisms known: foods preserved by some form of dehydration or enhanced sugar levels, and hypersaline sites where water availability is limited by a high concentration of salts (usually NaCl). These environments are essentially microbial habitats, with high-sugar foods being dominated by xerophilic (sometimes called osmophilic) filamentous fungi and yeasts, some of which are capable of growth at a water activity (a(w)) of 0.61, the lowest a(w) value for growth recorded to date. By contrast, high-salt environments are almost exclusively populated by prokaryotes, notably the haloarchaea, capable of growing in saturated NaCl (a(w) 0.75). Different strategies are employed for combating the osmotic stress imposed by high levels of solutes in the environment. Eukaryotes and most prokaryotes synthesize or accumulate organic so-called 'compatible solutes' (osmolytes) that have counterbalancing osmotic potential. A restricted range of bacteria and the haloarchaea counterbalance osmotic stress imposed by NaCl by accumulating equivalent amounts of KCl. Haloarchaea become entrapped and survive for long periods inside halite (NaCl) crystals. They are also found in ancient subterranean halite (NaCl) deposits, leading to speculation about survival over geological time periods.     

Survival and filamentation of Salmonella enterica serovar enteritidis PT4 and Salmonella enterica serovar typhimurium DT104 at low water activity

In this study we investigated the long-term survival of and morphological changes in Salmonella strains at low water activity (a(w)). Salmonella enterica serovar Enteritidis PT4 and Salmonella enterica serovar Typhimurium DT104 survived at low a(w) for long periods, but minimum humectant concentrations of 8% NaCl (a(w), 0. 95), 96% sucrose (a(w), 0.94), and 32% glycerol (a(w), 0.92) were bactericidal under most conditions. Salmonella rpoS mutants were usually more sensitive to bactericidal levels of NaCl, sucrose, and glycerol. At a lethal a(w), incubation at 37 degrees C resulted in more rapid loss of viability than incubation at 21 degrees C. At a(w) values of 0.93 to 0.98, strains of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium formed filaments, some of which were at least 200 microm long. Filamentation was independent of rpoS expression. When the preparations were returned to high-a(w) conditions, the filaments formed septa, and division was complete within approximately 2 to 3 h. The variable survival of Salmonella strains at low a(w) highlights the importance of strain choice when researchers produce modelling data to simulate worst-case scenarios or conduct risk assessments based on laboratory data. The continued increase in Salmonella biomass at low a(w) (without a concomitant increase in microbial count) would not have been detected by traditional microbiological enumeration tests if the tests had been performed immediately after low-a(w) storage. If Salmonella strains form filaments in food products that have low a(w) values (0.92 to 0.98), there are significant implications for public health and for designing methods for microbiological monitoring.     

Effect of temperature on freezing and heat stress tolerance of cultured plant cells

The relationship between freezing and heat tolerance was investigated with suspension-cultured pear (Pear cammunis cv. Bartlett) cells. This culture showed considerable capacity for both cold and heat acclimation. Growth at 2 °C (Cold acclimation) and at 30 °C (heat acclimation) both increased the freezing tolerance [measured via triphenyltetrazolium chloride (TTC) reduction]of pear cells. However, heat acclimation induced by heat shock treatment did not significantly effect freezing tolerance. Although growth at 30 °C increased freezing tolerance (relative to 22 °C-grown controls), growth at 2 °C (cold acclimation) decreased heat tolerance substantially. Thus, the only similarity detected between cold and heat acclimation was that both processes conferred freezing resistance to TTC-reducing system(s) in pear cells. The pear suspension culture will be a useful tool to further investigate cold acclimation via comparisons between heat and freezing acclimation and injury.     

Antibiofilm activity of a lytic Salmonella phage on different Salmonella enterica serovars isolated from broiler farms

International Microbiology - Bacteriophages have been mainly used in treating infections caused by planktonic bacterial cells in the veterinary sector. However, their...     

Urease reaction rates at low water activity

Hydrolysis of urea by urease takes place in dry urea-urease mixture exposed to discrete water vapor pressures from 100% to 20% relative humidity and at 2°C to 70°C. A discontinuity in enzymatic activities is observed at the transition of urea from a solid to a deliquescent solution. Urease is inactivated more readily at higher relative humidities in saturated urea solution than at lower relative humidities where water for urea hydrolysis is adsorbed on enzyme-protein only. Hydrolysis of urea by urease proceeds at a measurable rate in concentrated solutions of urea and of urea hydrolysis products, ammonium carbonate and bicarbonate, in the absence of ionic strength or pH stabilizing agents.     

Effect of low ambient temperature on protein turnover and heat production in chicks

1. Effect of low ambient temperature on protein turnover in the liver and whole body was investigated in chicks together with the contribution of protein synthesis to the total heat production. 2. Both protein synthesis and degradation in the whole body were increased, the latter to a larger extent, at low ambient temperature (LT, 22 degrees C) compared with adequate temperature (AT, 30 degrees C). Liver protein synthesis was not significantly altered by the temperature treatment. 3. The total heat production of LT group was as high as 160% of the AT group. 4. The increased heat production due to enhanced whole-body protein synthesis accounted for only 1.4% of the heat increment in thermogenesis at low ambient temperature, suggesting that protein synthesis would contribute little, if any, to cold-induced thermogenesis in chicks.     

Studies on heat tolerance at rest and orthostatic heat tolerance in a hot environment

Twelve young male Japanese were exposed to a hot environment in summer, and changes in systolic blood pressure induced by changing the position from recumbent to sitting were observed. Sweating was induced in a climatic chamber at 30 degree C with 70% relative humidity by immersing both legs up to the knees into a water bath of 42 degree C for 90 min after sitting on a chair at rest for 30 min in the chamber. The mean values of rise in rectal temperature, body weight loss and mean sodium concentration in sweat were 0.62 degree C, 0.67 kg and 46 mEq/l, respectively. The mean values of fall in systolic blood pressure associated with changing posture and its recovery time were 13.7 mm Hg and 77.5 sec, respectively. Heat tolerance at rest was assessed by a numerical heat tolerance index involving relative water loss, relative rise in rectal temperature, and relative salt loss. Values of relative water loss, relative rise in rectal temperature, and the numerical heat tolerance index correlated closely to those of fall in systolic blood pressure and its recovery time.     

Catalytic properties of thermophilic lactate dehydrogenase and halophilic malate dehydrogenase at high temperature and low water activity

Thermophilic lactate dehydrogenases from Thermotoga maritima and Bacillus stearothermophilus are stable up to temperature limits close to the optimum growth temperature of their parent organisms. Their catalytic properties are anomalous in that Km shows a drastic increase with increasing temperature. At low temperatures, the effect levels off. Extreme halophilic malate dehydrogenase from Halobacterium marismortui exhibits a similar anomaly. Increasing salt concentration (NaCl) leads to an optimum curve for Km, oxaloacctate while Km, NADH remains constant. Previous claims that the activity of halophilic malate dehydrogenase shows a maximum at 1.25 M NaCl are caused by limiting substrate concentration; at substrate saturation, specific activity of halophilic malate dehydrogenase reaches a constant value at ionic strengths I greater than or equal to 1 M. Non-halophilic (mitochondrial) malate dehydrogenase shows Km characteristics similar to those observed for the halophilic enzyme. The drastic decrease in specific activity of the mitochondrial enzyme at elevated salt concentrations is caused by the salt-induced increase in rigidity of the enzyme, rather than gross structural changes.     

Survival of Salmonella enteritidis and Salmonella typhimurium in chicken manure at different levels of water activity

Survival of Salmonella enteritidis and Salmonella typhimurium in chicken manure at different levels of water activity (aw) was determined. The aw was adjusted by means of saturated salts with defined equilibrium relative humidity and the manure samples were stored aerobically at 20 degrees C. At aw levels higher than 0.93, a moderate increase in colony-forming units over 8-9 h was found for both strains; at aw levels of 0.89-0.75, there was a thousand-fold reduction. Extended storage resulted in a million-fold reduction of Salmonella enteritidis in 8 days at an aw of 0.89. At higher and lower levels of aw, the reduction was less extensive.     

Effect of low temperature and culture media on the growth and freeze-thawing tolerance of Exiguobacterium strains

Bacteria of the genus Exiguobacterium have been repeatedly isolated from ancient permafrost sediments of the Kolyma lowland of Northeast Eurasia. Here we report that the Siberian permafrost isolates Exiguobacterium sibiricum 255-15, E. sibiricum 7-3, Exiguobacterium undae 190-11 and E. sp. 5138, as well as Exiguobacterium antarcticum DSM 14480, isolated from a microbial mat sample of Lake Fryxell (McMurdo Dry Valleys, Antarctica), were able to grow at temperatures ranging from -6 to 40 degrees C. In comparison to cells grown at 24 degrees C, the cold-grown cells of these strains tended to be longer and wider. We also investigated the effect of growth conditions (broth or surface growth, and temperature) on cryotolerance of the Exiguobacterium strains. Bacteria grown in broth at 4 degrees C showed markedly greater survival following freeze-thawing treatments (20 repeated cycles) than bacteria grown in broth at 24 degrees C. Surprisingly, significant protection to repeated freeze-thawing was also observed when bacteria were grown on agar at either 4 or 24 degrees C.     

Hyperthermophilic archaeal prefoldin shows refolding activity at low temperature

Prefoldin is a molecular chaperone that captures a protein-folding intermediate and transfers it to a group II chaperonin for correct folding. Previous studies of archaeal prefoldins have shown that prefoldin only possesses holdase activity and is unable to fold unfolded proteins by itself. In this study, we have demonstrated for the first time that a prefoldin from hyperthermophilic archaeon, Pyrococcus horikoshii OT3 (PhPFD), exhibits refolding activity for denatured lysozyme at temperatures relatively lower than physiologically active temperatures. The interaction between PhPFD and denatured lysozyme was investigated by use of a surface plasmon resonance sensor at various temperatures. Although PhPFD showed strong affinity for denatured lysozyme at high temperature, it exhibited relatively weak interactions at lower temperature. The protein-folding seems to occur through binding and release from PhPFD by virtue of the weak affinity. Our results also imply that prefoldin might be able to contribute to the folding of some cellular proteins whose affinity with prefoldin is weak.     

桉树枝瘿姬小蜂的耐寒性测定

桉树枝瘿姬小蜂Leptocybe invasa Fisher&LaSalle是一种新入侵的检疫性有害生物,为了明确其对极端低温的耐受性,以了解其适生范围,测定了桉树枝瘿姬小蜂幼虫、蛹、成虫及不同地区、不同寄主条件下雌雄成虫以及广东、广西、海南3省6地越冬幼虫12—3月的过冷却点和冰点。结果表明,不同虫态的过冷却点和冰点由低到高顺序为:蛹<幼虫<成虫。蛹的过冷却点和冰点分别为(-24.93±0.10)℃、(-22.81±0.14)℃,成虫的过冷却点和冰点为(-20.93±0.24)℃和(-17.33±0.27)℃。随着纬度的升高,桉树枝瘿姬小蜂的过冷却点和冰点都呈现降低的趋势。海南地区不同寄主桉树枝瘿姬小蜂过冷却点从低到高的顺序排列为:湛-201<小叶桉<广林9号。在12—3月,桉树枝瘿姬小蜂的越冬幼虫过冷却点和冰点随着环境温度的升高而升高,以广东广州地区1月份的越冬幼虫过冷却点和冰点为最低,其数值分别为(-25.44±0.17)℃和(-24.04±0.21)℃,个体过冷却点的最低值为-26.9℃。由实验结果可知,桉树枝瘿姬小蜂蛹和幼虫的耐寒力最强,以幼虫和蛹越冬。地区、寄主、温度对其耐寒力均有显著的影响,而且其有向现疫区以北的区域扩散的潜能。