Peroxide sensitivity of cold-shocked Salmonella typhimurium and Escherichia coli and its relationship to minimal medium recovery

Cold-shocked Salmonella typhimurium displayed minimal medium recovery (MMR), viable counts on M9 minimal agar being much higher than those on tryptone soya yeast extract agar (TSYA). The addition of catalase to TSYA restored counts to the level found on M9 agar. Peroxide concentrations between 12 and 30 mumol/l were measured in TSYB but none was detected in M9 medium. Cold-shocked cells were sensitive to reagent hydrogen peroxide at a concentration similar to that found in TSYB. The minimal medium recovery phenomenon of cold-shocked cells is thus a manifestation of peroxide sensitivity. Changing the composition of growth media affected both cellular catalase activity and the magnitude of the MMR effect but the two properties were not directly related. Factors additional to cellular catalase activity must therefore affect susceptibility to peroxide following cold shock. Mutational loss of catalase, exonuclease III or recA-dependent DNA repair functions all increased the sensitivity of cold-shocked Escherichia coli to the inhibitory effects of peroxide present in rich medium. The peroxide resistant fraction of a cold-shocked population of Salm. typhimurium (i.e. those cells able to grow on TSYA) was more resistant to gamma radiation than the population as a whole. Cold shock thus sensitizes cells to more than one form of oxidative stress. Prior exposure of growing cells to 30 mumol/l hydrogen peroxide abolished their sensitivity to rich medium following cold shock implying that Salm. typhimurium contains an inducible system protecting against oxidative stress.     

Alcohols protect Escherichia coli against cold shock

Alcohols protect Escherichia coli against cold shock, and the concentration of alcohol which provided optimal protection declined with increasing hydrophobicity of the alcohol. The rate of loss of viability after the chilling transition was decreased by n-octanol, even when it was added after that chilling transition. Cold-shocked cells exhibited a sensitivity toward dioxygen, seen as greater enumeration on anaerobic, rather than on aerobic, trypticase-yeast extract agar plates, and addition of catalase or antioxidants, such as alpha-tocopherol or probucol, to the agar plates did not lessen this dioxygen sensitivity. Respiratory capacity was diminished by cold shock, and cyanide-sensitive respiration was more affected than was cyanide-resistant respiration. Discharging the proton gradient, with the uncoupler carbonyl cyanide trifluoromethoxy-phenylhydrazone, did not change sensitivity to cold shock. There was no evidence for minimal medium recovery after cold shock. The data presented, as well as that already in the literature, are explained on the basis of membrane damage caused by patches of ordering transitions in one membrane leaflet, unmatched by comparable transitions in the mating leaflet.     

The effect of catalase on recovery of heat-injured DNA-repair mutants of Escherichia coli

The apparent sensitivity of Escherichia coli K12 to mild heat was increased by recA (def), recB and polA, but not by uvrA, uvrB or recF mutations. However, addition of catalase to the rich plating medium used to assess viability restored counts of heat-injured recA, recB and polA strains to wild-type levels. E. coli p3478 polA was sensitized by heat to a concentration of hydrogen peroxide similar to that measured in autoclaved recovery medium. The apparent heat sensitivity of DNA-repair mutants is thus due to heat-induced sensitivity to the low levels of peroxide present in rich recovery media. It is proposed that DNA damage in heated cells could occur indirectly by an oxidative mechanism. The increased peroxide sensitivity of heat-injured cells was not due to a decrease in total catalase activity but may be related specifically to inactivation of the inducible catalase/peroxidase (HPI).     

Factors that interact with the antibacterial action of thyme essential oil and its active constituents

The viable counts of Salmonella typhimurium on nutrient agar (NA) decreased upon the addition of either the essential oil of thyme or its constituent thymol, especially under anaerobic conditions. Antagonistic effects of thymol against Staphylococcus aureus were also greater under anaerobic conditions. In contrast to the phenolic constituents of the oil, thymol and carvacrol, the chemically related terpenes p -cymene and y -terpinene had no antagonistic effects against Salm. typhimurium.
The addition of Desferal to NA counteracted the antibacterial effects of both thyme oil and thymol. No support was obtained, however, for a possible role of iron in the oxygen-related antibacterial action of the thyme oil and thymol or for the observed effect of Desferal. In the presence of thymol, the viable counts of Salm. typhimurium obtained on a minimal medium (MM) were lower than those obtained on NA. Addition of bovine serum albumin (BSA) neutralized the antibacterial action of thymol. It is suggested that the effects of BSA or Desferal are due to their ability to bind phenolic compounds through their amino and hydroxylamine groups, respectively, thus preventing complexation reactions between the oil phenolic constituents and bacterial membrane proteins. This hypothesis is supported by the marked decrease in the viable counts of Salm. typhimurium caused by either thyme oil or thymol when the pH of the medium was changed from 6.5 to 5.5 or the concentration of Tween 80 in the medium was reduced.     

Mechanisms of triple stress-mediated damage in stationary phase cells of Salmonella typhimurium exposed to succinate-acidified hypochlorite system at 5° C

Exposure for 20 min of stationary phase cells of Salmonella typhimurium to a combined triple stress system (TSS) treatment comprising hypochlorite derived 5 ppm free available chlorine in solution acidified with 1% succinate (pH 2·5) and at a chill shock temperature of 5°C resulted in symptoms of injury. Cells became sensitive to 40 μg/ml lysozyme, 50 μg/ml actinomycin D and 100 μg/ml ribonuclease B, to which control cells were resistant. Metabolic injury was indicated by reduction in colony forming ability of stressed cells on minimal salts glucose agar M9 medium. There was no detectable leakage loss of 260–280 nm-absorbing materials. This was also confirmed by assay of the cellular RNA material components. Loss of alkaline phosphatase activity was observed in the stressed cells.
The intensity of induced cellular damage as measured by lysozyme sensitivity was greatest in the cells exposed to the complete TSS, followed by those stressed in 1% succinate at 5°C, then 5 ppm chlorine at 5°C and the singular chill shock stress at 5°C, respectively. The magnitudes of cellular damage, however, were suggestive of synergistic interactions among the component stress factors of the TSS.
The findings obtained indicate impairment of the structural integrity and functional capabilities of the permeability barriers and the inactivation of certain periplasmic enzymes. The resultant cumulative cellular damage from the TSS exposure may therefore enhance greater sensitivity of treated cells to subsequent stress factors.     

Cold shock induction of thermal sensitivity in Listeria monocytogenes

Cold shock at 0 to 15 degrees C for 1 to 3 h increased the thermal sensitivity of Listeria monocytogenes. In a model broth system, thermal death time at 60 degrees C was reduced by up to 45% after L. monocytogenes Scott A was cold shocked for 3 h. The duration of the cold shock affected thermal tolerance more than did the magnitude of the temperature downshift. The Z values were 8.8 degrees C for controls and 7.7 degrees C for cold-shocked cells. The D values of cold-shocked cells did not return to control levels after incubation for 3 h at 28 degrees C followed by heating at 60 degrees C. Nine L. monocytogenes strains that were cold shocked for 3 h exhibited D(60) values that were reduced by 13 to 37%. The D-value reduction was greatest in cold-shocked stationary-phase cells compared to cells from cultures in either the lag or exponential phases of growth. In addition, cold-shocked cells were more likely to be inactivated by a given heat treatment than nonshocked cells, which were more likely to experience sublethal injury. The D values of chloramphenicol-treated control cells and chloramphenicol-treated cold-shocked cells were no different from those of untreated cold-shocked cells, suggesting that cold shock suppresses synthesis of proteins responsible for heat protection. In related experiments, the D values of L. monocytogenes Scott A were decreased 25% on frankfurter skins and 15% in ultra-high temperature milk if the inoculated products were first cold shocked. Induction of increased thermal sensitivity in L. monocytogenes by thermal flux shows potential to become a practical and efficacious preventative control method.     

Sperm production and mating potential of males after a cold shock on pupae of the parasitoid wasp Dinarmus basalis (Hymenoptera: Pteromalidae)

For ectothermic species, temperature is a key environmental factor influencing several aspects of their physiology and ecology, acting particularly on reproduction. To measure the consequences of a severe thermal stress during development on male reproduction, a cold shock (1h at -18 degrees C) was tested on Dinarmus basalis pupae. D. basalis (Hymenoptera: Pteromalidae) is a parasitoid wasp in which sperm management in both male and female is of prime importance. After a cold shock, developmental success was reduced, with a quarter of cold-shocked males not emerging correctly. The stress effects were estimated at the level of sperm stock in seminal vesicles of males at different ages and on the ability of 2-day-old males to access females in single and multiple mating and in male-male competition. Cold-shocked males had a reduced sperm stock compared to control males and this difference persisted with age. The rate of sperm production was similar in both groups. The consequences of a cold shock on male reproductive ability were perceptible in multiple mating and male-male competition but not in single mating. Cold-shocked males were at a disadvantage, inseminating fewer females and copulating less frequently. Finally, male pupae of D. basalis were able to withstand severe temperature stresses and their reproductive functions were partially preserved.     

Superoxide and hydrogen peroxide in oxygen damage.

Escherichia coli were damaged and killed by exposure to hyperbaric oxygen. Lethality was measured as the decrease in the number of colonies formed upon plating the exposed cells onto rich agar. Damage was assessed by plating onto both rich and minimal agar. Cells which gave rise to visible colonies on rich but not on minimal agar were considered to be damaged. That this differential colony count was largely due to reparable damage rather than to stable mutagenesis was shown by replica plating from the rich onto the minimal agar. Most of the cells which had been unable to grow when directly plated onto minimal agar regained this ability after growth upon rich agar. Repair of the damage imposed by exposure to oxygen was thus more readily accomplished on a nutritionally rich medium. The enzymes superoxide dismutase, catalase, and peroxidase appeared to protect against oxygen damage. It is thus likely that both O₂^? and H₂O₂ are important agents of oxygen toxicity. In accord with this conclusion were the observations that augmented intracellular levels of these enzymes correlated with increased resistance towards oxygen damage, whereas increased respiratory capacity correlated with increased sensitivity towards hyperbaric oxygen.     

Cold Shock Lethality and Injury in Clostridium perfringens

Several observations have been made in regard to cold shock lethality of Clostridium perfringens: (i) loss of viability was not consequence of exposure of the cells to air; (ii) stationary-phase cells were much more resistant to cold shock at 4 C than exponential-phase cells; (iii) at 4 C 96% of an initial population of exponential-phase cells was killed upon cold shock and 95% of the remaining population was killed within 90 min of continued exposure at 4 C; (iv) the minimal temperature differential for detectable cold shock lethality was between 17 and 23 C, and the maximum beyond which lethality was not appreciably increased was between 28 and 33 C. Up to 75% of viable cold-shocked cells were injured, as demonstrated by cold shocking late exponential-phase cells at 10 C and using differential plating procedure for recovery. Repair of injury was temperature dependent, and occurred in a complex medium and 0.1% peptone but not water. Nalidixic acid, chloramphenicol, and rifampin did not inhibit repair of injury.     

Rapid suppression of multidrug resistance of leukemic cells by oxidative srtess

The effect of a short-time (1 h) oxidative stress on multidrug resistance (MDR) of murine leukemic P388VR cells has been investigated. We studied the production of reactive oxygen species (ROS) in cells depending on the composition of medium and the concentration of cells and hydrogen peroxide, as well as the effect of hydrogen peroxide on MDR of cells. MDR was determined from the transport of calcein acetoxymethyl ester out of the cells and from a change in cell sensitivity to vincristine. The amount of ROS arising in cells was determined using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH₂-DA). It was shown that the rate of ROS formation in cells decreases after the addition of serum to the medium and with an increase of the cell number. By the action of hydrogen peroxide, the amount of ROS increases directly with its concentration. Oxidative stress generated by 30–300 μM hydrogen peroxide decreases the MDR of the cells. The effect of hydrogen peroxide increases with the treatment duration and concentration of hydrogen peroxide. MDR determined by the criterion of the efflux of calcein ester from cells is completely suppressed after 1-h exposure to 300 μM hydrogen peroxide. At a concentration of hydrogen peroxide of 60 μM and treatment duration of 1 h, the sensitivity of P388VR cells to vincristine increases to reach the sensitivity of the wild-type P388 cells. Rapid (about 1 h) suppression of MDR is caused by inhibition of the activity of transport proteins. MDR decrease induced by oxidative stress can be used in therapy of tumors resistant to anticancer drugs.     

Minimal Medium Recovery of Thermally Injured Salmonella senftenberg 4969

Exposure of Salmonella senftenberg 4969 to sublethal heating in phosphate buffer, pH 7·0, at 52· produced thermally injured cells characterized by their relative inability to form colonies on trypticase soy yeast extract agar compared to minimal medium (M9) agar. During subsequent incubation at 37· in liquid media, more injured cells were capable of repair in M9 than in nutrient media used for pre-enrichment purposes. M9 was superior to lactose broth as a liquid holding medium to restore the ability of injured cells to grow on both rich and selective agar media. The addition of food products produced a more favourable environment for the repair of thermally injured cells in M9 rather than lactose broth. Pre-enrichment in M9 was 100 times more effective than using lactose broth as the preliminary step in the detection of S. senftenberg in laboratory pasteurized liquid egg albumen.     

Cold Shock Induction of Thermal Sensitivity in Listeria monocytogenes

Cold shock at 0 to 15°C for 1 to 3 h increased the thermal sensitivity of Listeria monocytogenes. In a model broth system, thermal death time at 60°C was reduced by up to 45% after L. monocytogenes Scott A was cold shocked for 3 h. The duration of the cold shock affected thermal tolerance more than did the magnitude of the temperature downshift. The Z values were 8.8°C for controls and 7.7°C for cold-shocked cells. The D values of cold-shocked cells did not return to control levels after incubation for 3 h at 28°C followed by heating at 60°C. Nine L. monocytogenes strains that were cold shocked for 3 h exhibited D₆₀ values that were reduced by 13 to 37%. The D-value reduction was greatest in cold-shocked stationary-phase cells compared to cells from cultures in either the lag or exponential phases of growth. In addition, cold-shocked cells were more likely to be inactivated by a given heat treatment than nonshocked cells, which were more likely to experience sublethal injury. The D values of chloramphenicol-treated control cells and chloramphenicol-treated cold-shocked cells were no different from those of untreated cold-shocked cells, suggesting that cold shock suppresses synthesis of proteins responsible for heat protection. In related experiments, the D values of L. monocytogenes Scott A were decreased 25% on frankfurter skins and 15% in ultra-high temperature milk if the inoculated products were first cold shocked. Induction of increased thermal sensitivity in L. monocytogenes by thermal flux shows potential to become a practical and efficacious preventative control method.     

Survival of dehydrated cells of Salmonella typhimurium LT2 at high temperatures

Cells of Salmonella typhimurium LT2 were dehydrated on hydrophobic membranes (Millipore FGLP2500) placed in a controlled atmosphere chamber held at 57% equilibrium relative humidity (ERH) and 37 degrees C. Dehydration for 48 h under the above conditions increased the heat resistance of Salm. typhimurium LT2 when measured as the surviving fraction after a heat challenge of 135 degrees C for 30 min. Results also showed that little or no death occurred during heat challenges of 1 h at temperatures of up to 100 degrees C. The survival of Salm. typhimurium LT2 was measured as the ability to form colonies on solid media tryptone soy broth plus 1.2% agar (TSBA) after 24 h at 37 degrees C. Incorporation of sodium pyruvate, at a concentration of (TSBA) after 24 h at 37 degrees C. Incorporation of sodium pyruvate, at a concentration of 0.2% into the recovery medium, did not enhance the recovery of heated Salm. typhimurium LT2. Dehydrated cells of S. typhimurium LT2 showed a triphasic death curve. Increasing the period of dehydration from 48 h to 34 d, reduced initial numbers due to die off but did not alter the shape of the subsequent survival curve.     

Survival of dehydrated cells of Salmonella typhimurium LT2 at high temperatures

Cells of Salmonella typhimurium LT2 were dehydrated on hydrophobic membranes (Millipore FGLP2500) placed in a controlled atmosphere chamber held at 57% equilibrium relative humidity (ERH) and 37°C. Dehydration for 48 h under the above conditions increased the heat resistance of Salm. typhimurium LT2 when measured as the surviving fraction after a heat challenge of 135°C for 30 min. Results also showed that little or no death occurred during heat challenges of 1 h at temperatures of up to 100°C. The survival of Salm. typhimurium LT2 was measured as the ability to form colonies on solid media tryptone soy broth plus 1.2% agar (TSBA) after 24 h at 37°C. Incorporation of sodium pyruvate, at a concentration of 0.2% into the recovery medium, did not enhance the recovery of heated Salm. typhimurium LT2. Dehydrated cells of S. typhimurium LT2 showed a triphasic death curve. Increasing the period of dehydration from 48 h to 34 d, reduced initial numbers due to die off but did not alter the shape of the subsequent survival curve. and accepted 22 June 1989     

Complex medium toxicity to some DNA repair-deficient strains of Salmonella typhimurium.

The recovery of several strains of Salmonella typhimurium LT-2 which had first been grown in minimal medium varies when the organisms are grown on minimal medium agar and complex medium agar. The strains tested included mutants with deficiencies in DNA-repair systems (uvrB-and rec-), a deep rough (rfa-) mutant, and a double mutant carrying both the uvrB- and the rfa-mutation. The uvrB- and rec-mutations imparted sensitivity to complex medium agar. The rfa-mutation suppressed the sensitivity of the uvrB-mutant to complex medium agar. Differences in colony-forming ability were not observed when the bacteria were first grown in the complex medium broth.     

Effects of cold shock and phospholipase A2 on intact boar spermatozoa and sperm head plasma membranes

Head plasma membranes (HPM) isolated from cryopreserved boar spermatozoa show an excessive fluidization, which might be involved in the loss of fertility. The current study assessed the ability of cold shock (5 degrees C) and phospholipase A2 (PA2) to duplicate these effects on membrane structure and to affect 45Ca2+ uptake and gross morphological characteristics of whole, fresh boar-sperm. The HPM from cold-shocked sperm showed a significantly greater rate of fluidization over time than did HPM from control sperm. Addition of PA2 (bee or snake venom, 0.1 or 10.0 ng/ml) to HPM from control sperm caused fluidization similar to cold shocking, but to a lesser degree (P less than 0.05). Cold-shocked intact sperm exhibited severe acrosomal disruption, loss of motility, and increased 45Ca2+ uptake relative to control sperm. Addition of PA2 (bee or snake venom, 0.1, 1.0., 10.0, and 1,000 ng/ml) to control sperm had no effect on gross morphology or motility while maintaining or increasing sperm extrusion of 45Ca2+. Therefore, although PA2 can, to some extent, duplicate the effects of cold shock on HPM molecular organization, its lipid hydrolytic action is insufficient to cause all the gross disruptions of severe thermal shock. Both PA2 and cold shock disrupted HPM structure, but only cold shock increased 45Ca2+ uptake, suggesting that cold shock may be increasing 45Ca2+ uptake in areas other than the head. Cold shock disrupts sperm on three levels; membrane molecular organization, intracellular Ca2+ regulation, and gross morphology/motility.     

Regeneration of doubled haploid plants by androgenesis of cucumber (<Emphasis Type="Italic">Cucumis sativus </Emphasis>L.)

Six experiments (including pretreatment, embryonic callus induction media, preculture conditions, embryo induction media, embryo germination media, and genotypic effects) were conducted to develop an efficient cucumber (Cucumis sativus L., 2n = 2x = 14) anther culture protocol. Pretreatment and embryo induction were key factors for successful anther culture. Suitable temperature stress depended on the ecotype, i.e., cucumbers from cold areas responded well to cold shock whereas those from temperate areas responded well to heat treatment. The best medium for embryonic callus induction was MS medium supplemented with 4.44 μM BA, 2.26 μM 2, 4-D, 4.64 μM KIN, 3% sucrose and 0.8% agar. For embryo induction, MS medium supplemented with 0.54 μM NAA, 13.32 μM BA, 3% sucrose and 0.8% agar was optimal, and for embryo germination MS medium containing 2.22 μM BA, 6% sucrose and 1.2% agar was best. Using this protocol, we produced callus from 16 genotypes and regenerated plants from three of 20 evaluated. Three embryos per anther and 42 DH per 45 anthers (93% success) were obtained for cv. Ningjia No. 1, which was an improved result over a previous report. The origin of regenerants from microspores was determined by cytological, morphological and AFLP analyses.     

Enumeration of Escherichia coli in Frozen Samples After Recovery from Injury

More than 90% of the surviving cells of Escherichia coli NCSM were injured after freezing in water at -78 C. Injury was determined by the ability of cells to form colonies on Trypticase soy agar with yeast extract but not on violet red-bile agar and deoxycholate-lactose agar. Exposure of the injured cells to Brilliant Green-bile broth and lauryl sulfate broth prevented subsequent colony formation on Trypticase soy agar with yeast extract. The freeze-injury could be repaired rapidly in a medium such as Trypticase soy broth with yeast extract (TSYB). The repaired cells formed colonies on violet red-bile agar and deoxycholate-lactose agar and were not inhibited by Brilliant Green-bile broth and lauryl sulfate broth. At least 90% of the cells repaired in TSYB within 30 min at 20 to 45 C and began multiplication within 2 h at 25 C. When the cells were frozen in different foods, 60 to 90% of the survivors were injured. Repair of the injured cells occurred in foods during 1 h at 25 C, but generally repair was greater and more reproducible when the foods were incubated in TSYB. The study indicated that the repair of freeze-injured coliform bacteria should be accomplished before such cells are exposed to selective media for their enumeration.     

Mechanisms of triple stress-mediated damage in stationary phase cells of Salmonella typhimurium exposed to succinate-acidified hypochlorite system at 5 degrees C

Exposure for 20 min of stationary phase cells of Salmonella typhimurium to a combined triple stress system (TSS) treatment comprising hypochlorite derived 5 ppm free available chlorine in solution acidified with 1% succinate (pH 2.5) and at a chill shock temperature of 5 degrees C resulted in symptoms of injury. Cells became sensitive to 40 micrograms/ml lysozyme, 50 micrograms/ml actinomycin D and 100 micrograms/ml ribonuclease B, to which control cells were resistant. Metabolic injury was indicated by reduction in colony forming ability of stressed cells on minimal salts glucose agar M9 medium. There was no detectable leakage loss of 260-280 nm-absorbing materials. This was also confirmed by assay of the cellular RNA material components. Loss of alkaline phosphatase activity was observed in the stressed cells. The intensity of induced cellular damage as measured by lysozyme sensitivity was greatest in the cells exposed to the complete TSS, followed by those stressed in 1% succinate at 5 degrees C, then 5 ppm chlorine at 5 degrees C and the singular chill shock stress at 5 degrees C, respectively. The magnitudes of cellular damage, however, were suggestive of synergistic interactions among the component stress factors of the TSS. The findings obtained indicated impairment of the structural integrity and functional capabilities of the permeability barriers and the inactivation of certain periplasmic enzymes. The resultant cumulative cellular damage from the TSS exposure may therefore enhance greater sensitivity of treated cells to subsequent stress factors.     

Serum survival and plasmid possession by strains of Salmonella enteritidis, Salm. typhimurium and Salm. virchow

Strains of Salmonella enteritidis, Salm. typhimurium and Salm. virchow , carrying different numbers of plasmids, were examined for the ability to multiply in sera. Viable counts were performed to monitor the kinetics of growth of bacteria when in human, chicken and turkey sera. The presence of plasmids in Salm. enteritidis, Salm. typhimurium and Salm. virchow reduced considerably the ability of strains of these serotypes to multiply in serum. SDS-PAGE was used to show that growth of Salm. enteritidis in serum did not involve changes in outer membrane proteins or lipopolysaccharide. It was concluded that the carriage of plasmids may be disadvantageous for the survival in serum of certain common salmonella serotypes.