Pulsed electric fields cause sublethal injuries in the outer membrane of Enterobacter sakazakii facilitating the antimicrobial activity of citral

Aims: The objective was to evaluate the relation of sublethal injury in the outer membrane of Enterobacter sakazakii to the inactivating effect of the combination of pulsed electric fields (PEF) treatments and citral. Methods and Results: The occurrence of sublethal injury in the outer membrane was measured using selective recovery media containing bile salts. Loss of membrane integrity was measured by the increased uptake of the fluorescent dye propidium iodide (PI). PEF caused nonpermanent and permanent envelope permeabilization of Ent. sakazakii at pH 4·0. After PEF, most surviving cells showed transient cell permeabilization and sublethal injury in their outer membranes. The simultaneous application of a mild PEF treatment (100 pulses, 25 kV cm^?1) and 200 μl l^?1 of citral to cells suspended in pH 4·0 buffer at a final concentration of 10⁷ cells per ml showed an outstanding synergistic lethal effect, causing the inactivation of more than two extra log₁₀ cycles. Conclusions: Our results confirm that the detection of sublethal injury in the outer membrane after PEF may contribute to the identification of the treatment conditions under which PEF may act synergistically with hydrophobic compounds such as citral. Significance and Impact of the Study: Knowledge about the mechanism of microbial inactivation by PEF will aid the establishment of successful combined preservation treatments.     

Campylobacter jejuni: a study on environmental conditions affecting culturability and in vitro adhesion/invasion

Aims:  Nongrowing cultures of Campylobacter jejuni lose their culturability. It is unclear whether this loss in culturability also affects their ability to interact with host cells. The purpose of this study was to determine the relevance of the number of culturable cells to the ability to adhere/invade in Caco-2 cells.
Methods and Results:  For C. jejuni C356, culturability and adhesion/invasion were monitored in time (days) under different storage conditions (temperature, medium, atmosphere). Decrease rates of both culturability and adhesion/invasion were dependent on the conditions used, but the number of adhering/invading cells per culturable cell was not affected by the environmental conditions. Furthermore five strains were monitored at one condition. The culturability and adhesion/invasion decrease rates did not significantly differ per strain; however the number of adhering/invading cells per culturable cell was strain dependent.
Conclusions:  Culturability and adhesion/invasion of C. jejuni are linearly related. The number of adhering/invading cells per culturable C. jejuni cell is strain dependent, but is not affected by environmental conditions.
Significance and Impact of the Study:  It was shown that the number of culturable cells is a good measure for the in vitro adhesion/invasion of. C. jejuni.     

Heat resistance of Cronobacter species (Enterobacter sakazakii) in milk and special feeding formula

Aim:  To determine D - and z -values of Cronobacter species ( Enterobacter sakazakii ) in different reconstituted milk and special feeding formula and the effect of reconstitution of powdered milk and special feeding formula with hot water on the survival of the micro-organism.
Methods and Results:  Five Cronobacter species (four C. sakazakii isolates and C. muytjensii ) were heated in reconstituted milk or feeding formula pre-equilibrated at 52–58°C for various times or mixed with powdered milk or feeding formula prior to reconstitution with water at 60–100°C. The D -values of Cronobacter at 52–58°C were significantly higher in whole milk (22·10–0·68 min) than in low fat (15·87–0·62 min) or skim milk (15·30–0·51 min) and significantly higher in lactose-free formula (19·57–0·66 min) than in soy protein formula (17·22–0·63 min). The z -values of Cronobacter in reconstituted milk or feeding formula ranged from 4·01°C to 4·39°C. Water heated to ≥70°C and added to powdered milk and formula resulted in a > 4 log₁₀ reduction of Cronobacter .
Conclusions:  The heat resistance of Cronobacter should not allow the survival of the pathogen during normal pasteurization treatment. The use of hot water (≥70°C) during reconstitution appears to be an effective means to reduce the risk of Cronobacter in these products.
Significance and Impact of the Study:  This study supports existing data available to regulatory agencies and milk producers that recommended heat treatments are sufficient to substantially reduce risk from Cronobacter which may be present in these products.     

Role of the alternative sigma factor σB on Staphylococcus aureus resistance to stresses of relevance to food preservation

Aims:  To examine the role of the alternative general stress sigma factor σ^B on the resistance of Staphylococcus aureus to stresses of relevance to food preservation, with special emphasis on emerging technologies such as pulsed electric fields (PEF) and high hydrostatic pressure (HHP).
Methods and Results:  S. aureus strain Newman and its isogenic Δ sigB mutant were grown to exponential and stationary growth phases and its resistance to various stresses was tested. The absence of the σ^B factor caused a decrease in the resistance to heat, PEF, HHP, alkali, acid and hydrogen peroxide. In the case of heat, the influence of the σ^B factor was particularly important, and decreases in decimal reduction time values of ninefold were observed as a result of its deficiency. The increased thermotolerance of the parental strain as compared with the sigB mutant could be attributed to a better capacity to sustain and repair sublethal damages caused by heat.
Conclusions:  σ^B factor provides S. aureus cells with resistance to multiple stresses, increasing survival to heat, PEF and HHP treatments.
Significance and Impact of the Study:  Results obtained in this work help in understanding the physiological mechanisms behind cell survival and death in food-processing environments.     

Physiological and behavioral basis for the successful adaptation of goats to severe water restriction under hot environmental conditions

Among domestic ruminants, goats are renowned for their ability to tolerate water deprivation, water restriction and energy restriction. However, some basic questions regarding their ability to endure water restriction under heat stress are still open. Three levels of water restriction (56%, 73% and 87% of the ad libitum) were imposed on 20 cross-bred 3-year-old female goats (75% German Fawn and 25% Hair Goat) distributed into four groups, with five animals per treatment. The experiment was conducted from the beginning of July to the end of August in a farm located in the Eastern Mediterranean region of Turkey (40 m in altitude; 36 59' N, 35 18'E), in which subtropical weather conditions prevail. The average daily temperature during the experiment was 34.2°C, whereas the highest and lowest temperatures were 42°C and 23.1°C, respectively. The average relative humidity was 68.2% and wind speed was 1.2 km/h. Weekly average thermal heat indexes during the experiment were 78.3 (week 1), 79.1 (week 2), 80.1 (weak 3), 79.8 (weak 4), 81.3 (weak 5) and on average 79.7. Feed intake, heart rate, thermoregulatory responses (rectal temperature, respiration rate), blood plasma concentrations of ions (Na, K), antidiuretic hormone (ADH), metabolites (glucose, cholesterol, creatinine and urea) and behavioral aspects (standing, walking, lying) were studied over 30 days. The responses to water restriction were proportional to the level of restriction. The reductions in feed intake (up to 13%), BW (up to 4.6%) and the increases in rectal temperature (0.5°C) and breath rate (10 respirations/min) were moderate and also were far from responses encountered under severe heat and water stresses. The increase in plasma Na (from 119 to 140 mM) and ADH concentrations (from 12.6 to 17.4 pg/ml) indicates that the physiological response to water restriction was in response to mild dehydration, which also explains the increase in blood plasma concentrations of glucose, cholesterol, creatinine and urea. Behavioral responses (reduction in walking from 226 to 209 min/day and increase in lying from 417 to 457 min/day) were associated with conservation of energy or thermoregulation (reducing the exposure to direct radiation).     

The glutamate decarboxylase acid resistance mechanism affects survival of Listeria monocytogenes LO28 in modified atmosphere-packaged foods

AIMS: The contribution of the glutamate decarboxylase (GAD) acid resistance system to survival and growth of Listeria monocytogenes LO28 in modified atmosphere-packaged foods was examined. METHODS AND RESULTS: The survival and growth of the wild-type LO28 and four GAD deletion mutants (DeltagadA, DeltagadB, DeltagadC, DeltagadAB) in packaged foods (minced beef, lettuce, dry coleslaw mix) during storage at 4, 8 and 15 degrees C were studied. Survival and growth patterns varied with strain, product type, gas atmosphere and storage temperature. In minced beef, the wild-type LO28 survived better (P < 0.05) than the GAD mutant strains at 8 and 15 degrees C. In both packaged vegetables at all storage temperatures, the wild-type strain survived better (P < 0.05) than the double mutant DeltagadAB. The requirement for the individual gad genes varied depending on the packaged food. In the case of lettuce, gadA played the most important role, while the gadB and gadC genes played the greatest role in packaged coleslaw (at 15 degrees C). CONCLUSIONS: This work demonstrates that elements of the GAD system play significant roles in survival of L. monocytogenes LO28 during storage in modified atmosphere-packaged foods. SIGNIFICANCE AND IMPACT OF THE STUDY: A better understanding of how L. monocytogenes behaves in modified atmosphere-packaged foods, and how it responds to elevated carbon dioxide atmospheres.     

The DUF1996 and WSC domain‐containing protein Wsc1I acts as a novel sensor of multiple stress cues in Beauveria bassiana

Wsc1I homologues featuring both an N‐terminal DUF1996 (domain of unknown function 1996) and a C‐terminal WSC (cell wall stress‐responsive component) domain exist in filamentous fungi but have never been functionally characterized. Here, Wsc1I is shown to localize in the vacuoles and cell wall/membrane of the insect mycopathogen Beauveria bassiana and hence linked to cell membrane‐ and vacuole‐related cellular events. In B. bassiana, deletion of Wsc1I resulted in marked increases of hyphal and conidial sensitivities to hyperosmotic agents, oxidants, cell wall perturbing chemicals, and metal cations (Cu²⁺, Zn²⁺, Fe²⁺, and Mg²⁺) despite slight impact on normal growth and conidiation. Conidia produced by the deletion mutant showed not only reduced tolerance to both 45°C heat and UVB irradiation but also attenuated virulence to a susceptible insect through normal cuticle infection or cuticle‐bypassing infection. Importantly, phosphorylation of the mitogen‐activated protein kinase Hog1 was largely attenuated or nearly abolished in the Wsc1I‐free cells triggered with hyperosmotic, oxidative, or cell wall perturbing stress. All changes were well restored by targeted gene complementation. Our findings highlight a novel role of Wsc1I in sensing multiple stress cues upstream of the Hog1 signalling pathway and its pleiotropic effects in B. bassiana.     

Natural adaptation to environmental stress via physiological clock-regulation of stress resistance in Drosophila

In the present study we describe a novel mechanism by which populations, in an interplay between physiology and behaviour, can evolve adaptation to environmental extremes. Comparing Drosophila populations from different climate zones, we found diel shifts in high temperature resistance after maintenance at 25 °C for several generations. Resistance changes that co-occurred with field and laboratory activity of the populations are controlled by the physiological clock and appear to be a consequence of local adaptation to the thermal profiles of the environment of origin.     

Bioheat modeling of elderly and young for prediction of physiological and thermal responses in heat-stressful conditions

Exposure to hot and humid conditions results in physiological changes in metabolism, cardiac output and thermoregulation of the young adult and these changes deviate with elderly due to aging. The elderly population is more vulnerable than the healthy and young population due to age-weakened physiology and thermoregulatory functions. There are, however, limited bioheat models addressing such changes due to hot exposure in the young and the elderly. This paper develops robust bioheat models for young and elderly while incorporating the physiological changes under exposure to heat-stressful conditions for both age groups the age-related changes in physiology and thermoregulation to an elderly human. However, due to a large variability of thermoregulation among the elderly population, a sensitivity analysis revealed that the average elderly is characterized by metabolic rate and cardiac output, which are lower than those of the young by 21% and 14.4%, respectively. Moreover, the thresholds of the onset of vasodilation and sweating are delayed from those of young adults by 0.5 °C and 0.21 °C, respectively.The elderly and young bioheat models were validated with number of independent published experimental studies under hot exposures in steady and transient conditions. Model predictions of core and mean skin temperatures showed good agreement with published experimental data with a discrepancy of 0.1 °C and 0.5 °C, respectively.     

Assimilation and allocation of carbon and nitrogen of thermal and nonthermal Agrostis species in response to high soil temperature

We studied whether changes in the assimilation and allocation of carbon and nitrogen are associated with plant tolerance to high soil temperatures. Two Agrostis species, thermal Agrostis scabra, a species adapted to high-temperature soils in geothermal areas in Yellowstone National Park (USA), and two cultivars of a cool-season species, Agrostis stolonifera, L-93 and Penncross, were exposed to soil temperatures of 37 or 20 degrees C, while shoots were exposed to 20 degrees C. Net photosynthesis rate, photochemical efficiency, NO(3) (-)-assimilation rate and root viability decreased with increasing soil temperatures in both species. However, the decreases were less pronounced for A. scabra than for both A. stolonifera cultivars. Carbon investment in growth of plants exposed to 37 degrees C decreased more dramatically in both A. stolonifera cultivars than in A. scabra. Nitrogen allocation to shoots was greater in A. scabra than in both creeping bentgrass cultivars at 37 degrees C soil temperature. Our results demonstrate that plant tolerance to high soil temperature is related to efficient expenditure and adjustment of C- and N-allocation patterns between growth and respiration.     

AtHsfA2 modulates expression of stress responsive genes and enhances tolerance to heat and oxidative stress in Arabidopsis

In nature, plants are subject to changes of tempera-ture. Thus, like other organisms, plants have evolved strategies for preventing damage caused by rapid changes in temperature and for repairing what damage is unavoidable. Heat stress responses have been well documented in a wide range of organisms. In all spe-cies studied, the heat shock (HS) response is charac-terized by a rapid production and a transient accumu-lation of specific families of proteins known as heat shock proteins (Hsps) th…     

Direct and correlated effects of selection on flight after exposure to thermal stress in Drosophila melanogaster

To demonstrate how insects may adapt to ecologically relevant levels of heat stress, we performed artificial selection on the ability of Drosophila melanogaster to fly after an exposure to a high but non-lethal thermal stress. Both tolerance and intolerance to heat stress arose very quickly, as only a few generations of selection were necessary to cause significant separation between high and low lines for heat tolerance. Estimates of heritability based on the lines artificially selected for increased flight ability ranged from 0.024 to 0.052, while estimates of heritability based on the lines selected for the inability to fly after heat stress varied between 0.035 and 0.091. Reciprocal F₁ crosses among these lines revealed strong additive effects of one or more autosomes and a weaker X-chromosome effect. This variation apparently affected flight specifically; neither survival to a more extreme stress nor knockdown by high temperature changed between lines selected for high and low heat tolerance as measured by flight ability. As the well-studied heat-shock response is associated with heat tolerance as measured by survival and knockdown, the aspects of the stress physiology that actually affect flight ability remains unknown.