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.     

Relationship between variations in pathogenicity and lag phase at 37°C of Listeria monocytogenes previously stored at 4°C

s. BUNCIC AND S.M. AVERY. 1996. Three haemolytic, pathogenic strains of Listeria monocytogenes (a reference strain, a food-derived strain and a human strain) were held at 4°C for 4 weeks in phosphate-buffered saline pH 5.5 or 7.0, with and without 0.2% potassium sorbate or 0.3% sodium acetate. The number of viable cells did not change significantly during this storage. Pathogenicity of non-growing L. monocytogenes cells for 14-d-old chick embryos was determined before and after storage. Storage at 4°C resulted in decreased pathogenicity, but effects were strain-, pH- and substrate-dependent. After 4 weeks storage at 4°C non-growing bacterial cells were transferred to Brain Heart Infusion broth and growth characteristics were determined during incubation at 37°C. Strains that showed decreased pathogenicity had significantly longer lag phases at 37°C than strains that maintained pathogenicity. It is concluded that decreased pathogenicity of L. monocytogenes stored without growth at 4°C for 4 weeks and subsequent long lag phase at 37°C are correlated.     

Production of exotoxins by Aeromonas spp. at 5°C

The ability of 60 strains of Aeromonas to produce enterotoxin and haemolysin after cultivation at 5°C for 7–10 d was investigated. The strains were isolated from lamb meat, offal, carcasses and faeces, and had previously been tested for their ability to produce these exotoxins at 37°C. The results showed that some strains of Aeromonas hydrophila and A. sobria were capable of producing enterotoxin and haemolysin at 5°C, but none of the A. caviae strains tested produced these two factors. Of the 30 A. hydrophila strains investigated 25 and 27 were enterotoxigenic and haemolytic respectively. Likewise, of the 24 A. sobria strains investigated 16 and 18 were enterotoxigenic and haemolytic respectively. The results indicate that certain strains of Aeromonas species, in particular A. hydrophila and A. sobria , are of potential public health significance in meats stored at refrigeration temperature.     

Esterified glucomannan improves aflatoxin-induced damage of sperm parameters during liquid storage of ram semen at 5 °C

The aim of the present work was to study the effects of aflatoxin (AF) on sperm parameters in rams, and to determine the protective efficiency of esterified glucomannan (EG) co-administered with AF up to 96 h of the liquid storage of ram semen at 5 °C. Thirty-two Merino rams (12–14 months old) were used. The animals were examined for their general health status. To ensure their adaptation to the environment and the new feeding regimen, a 15-day acclimatization programme was applied to the animals, prior to the start of the study. Experimental feeding was continued for ninety-two days. The experimental design consisted of four dietary treatments. The control group (C) was fed with commercial feed. The AF group was fed with commercial feed plus 250 μg/day of total AF. The EG group received commercial feed plus 2 g/day of EG. The AF + EG group was given commercial feed plus 250 μg/day of total AF and 2 g/day of EG. In the study, ejaculates were obtained from rams twice a week for 12 weeks, using an electro-ejaculator. After collected, the ejaculates were diluted with a skimmed milk extender, and stored at 5 °C. Sperm motility and rates of abnormal and nonviable spermatozoa were determined for the different treatment groups at 5 °C at 0, 24, 48, 72 and 96 h of liquid storage.     

Loss of Viability and Metabolic Injury of Staphylococcus aureus Resulting from Storage at 1°, 3°, 5° and 7°C

Staphylococcus aureus incubated in Tryptic Soy Broth at 1°, 3°, 5° and 7°C became increasingly sensitive to Mannitol Salt Agar. Injury, as measured by salt sensitivity, decreased with increasing temperature from 1° to 7°C.     

Production of cell wall dissolving enzymes by Erwinia carotovora subsp. atroseptica in vitro at 27°C and 30°5C

Cultures of Erwinia carotovora subsp. atroseptica , grown at 27°C and 30·°C in different liquid media were assayed for activities of pectate lyase, polygalacturonase and cellulase. Total production of both pectate lyase and of polygalacturonase was 3–6 times less at 30·5°C than at 27°C; secretion of pectate lyase was similarly affected. Cellulase was cell bound and its production was not affected by the temperatures investigated. Growth, protein synthesis and protease activity were similar at the two temperatures and production of enzyme activity at 27°C and 30·5°C was independent of the growth medium.     

Lipid changes in mature-green bell pepper fruit during chilling at 2°C and after transfer to 20°C subsequent to chilling

Lipid composition and pigment content in bell pepper ( Capsicum annuum L. cv. Bell Tower) fruit that were freshly harvested, chilled 14 days at 2° C. or chilled and then transferred to 20 °C for 3 days ("rewarmed") were determined. There was slight to moderate loss of membrane glycerolipids during chilling, with much greater losses after chilled fruit was rewarmed. Galactolipid (GL) loss exceeded that of phospholipid (PL). The ratio of monogalactosyl -to digalactosyl-diacylglycerol did not change in chilled or in rewarmed fruit, and there was no chlorophyll loss, but the amount of neutral carotenes declined during chilling and dropped further alter rewarming. Only minor changes in total membrane sterols (TMS = free sterols + steryl glycosides + acylated steryl glycosides) were noted in chilled and in rewarmed fruit (a small increase followed by a small decrease), but major changes occurred in sterol glycosylation and esterification. The ratio of stigmasterol to sitosterol increased during chilling and rose further after rewarming. Due to PL loss, the ratios of TMS and free sterols to PL increased in rewarmed fruit. The ratio of linolenate (18:3) to linoleate (18:2) rose during chilling and after rewarming in all fatty-acyl lipids (GL. PL. and acylated steryl glycosides), but the unsaturation index increased only in GL. These results indicate that most membrane damage occurs after rewarming of chilled fruit and that the chloroplasts are especially chilling sensitive.     

Photoinhibition and recovery of photosynthesis in intact barley leaves at 5 and 20°C

Photoinhibition of photosynthesis and its recovery were studied in intact barley ( Hordeum vuigare L. cv. Gunilla) leaves grown in a controlled environment by exposing them to two temperatures, 5 and 20°C, and a range of photon flux densities in excess of that during growth. Additionally, photoinhibtion was examined in the presence of chloramphenicol (CAP, an inhibitor of chloroplast protein synthesis) and of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Susceptibility to photoinhibition was much higher at 5 than at 20°C. Furthermore, at 20°C. CAP exacerbated photoinhibition strongly, whereas CAP had little additional effect (10%) at 5°C. These results support the model that net photoinhibition is the difference between the inactivation and repair of photosystem II (PSII); i.e. the degradation and synthesis of the reaction centre protein, Dl. Furthermore, the steady-state extent of photoinhibition was strongly dependent on temperature and the results indicated this was manifested through the effects of temperature on the repair process of PSII. We propose that the continuous repair of PS II at 20°C conferred at least some protection from photoinhibition. At 5°C the repair process was largely inhibited, with increased photoinhibition as a consequence. However, we suggest where repair is inhibited by low temperature, some protection is alternatively conferred by the photoinhibited reaction centres. Providing they are not degraded, such centres could still dissipate excitation energy non-radiatively, thereby conferring protection of remaining photochemically active centres under steady-state conditions.
A fraction of PS II centres were capable of resisting photoinhibition when the repair process was inhibited by CAP. This is discussed in relation to PS II heterogeneity. Furthermore, the repair process was not apparently activated within 3 h when barley leaves were transferred to photoinhibitory light conditions at 20°C.     

The N4-hydroxycytidine reduction system in toluenized cells of Salmonella typhimurium.

1. Enzymatic reduction of N4-hydroxycytidine to cytidine in Salmonella typhimurium is highly specific. The reaction occurs only at the nucleoside level. Free base or its 1-methyl analogue is not reduced. 2. The pH optimum shows a broad plateau with a maximum at pH 7.0. The apparent Km value, estimated in the toluene-treated cells, is 4.8 mM and Vmax 1.4 nmoles/min/mg of wet bacterial weight. The reaction is NADH-dependent, although in toluenized bacterial cells it can occure without addition of any exogenous factor.     

Respiration of mitochondria isolated from tulip bulbs stored at 5 and 17°C

For the production of good quality flowers, tulip ( Tulipus gesneriana L.) bulbs need a period of low temperature. In cultivar Apeldoom a treatment of 12 weeks at 5 C can be used. In the bulb scales the respiratory metabolism has to adjust to this low temperature. Mitochondria isolated from the bulb scales are able to use succinate. NADH and pyruvate as respiratory substrates. Respiratory characteristics of these mitochondria changed after transfer of the bulbs to 5°C and the adaptation was complete within 2 weeks. Both state 3 respiration and respiratory control increased. Alternative pathway capacity was constitutively present at both 5°C and 17°C: it was low and substrate dependent at both temperatures. During the following weeks of the treatment no significant changes took place. However, when bulbs were transferred to 17°C after storage at 5°C. various responses could be demonstrated. In bulbs only cooled for a short period no readjustment to this higher temperature occurred. In bulbs stored for longer periods the change depended on the duration of the 5°C treatment. The nature of the re-adaptation is discussed     

Effects of essential oil from mint (Mentha piperita) on Salmonella enteritidis and Listeria monocytogenes in model food systems at 4° and 10°C

The effect of mint ( Mentha piperita ) essential oil (0·5, 1·0, 1·5 and 2·0%, v/w) on Salmonella enteritidis and Listeria monocytogenes in a culture medium and three model foods; tzatziki (pH 4·5), taramosalata (pH 5·0) and pâté (pH 6·8), inoculated at 10⁷ cfu g^-1, at 4° and 10°C for ca 1 week was studied. In the culture medium supplemented with the essential oil, no growth was observed over 2 d at 30°C determined by a conductance method with a Malthus 2000 growth analyser. Salmonella enteritidis died in tzatziki in all treatments and declined in the other foods except for pâté at 10°C as judged with viable counts. Listeria monocytogenes populations showed a declining trend towards the end of the storage period but was increased in pâté. Mint essential oil antibacterial action depended mainly on its concentration, food pH, composition, storage temperature and the nature of the micro-organism.     

Multimeric C9 within C5b-9 deposits in unique locations in the cell wall of Salmonella typhimurium

We have shown previously that multimeric C9 within C5b-9 (C9:C5b-8 greater than 3:1) is needed for killing of a rough strain of Escherichia coli. We now extend these studies using serum sensitive, rough (R) and serum resistant, wild type (WT) strains of Salmonella typhimurium as well as a mutant S. typhimurium strain (TS) with a temperature sensitive mutation in synthesis of keto-deoxy-octulosonate, a constituent within the deep core structure of Salmonella LPS. Both R and TS required multimeric C9 within C5b-9 to be killed. Addition at 37 degrees C of increasing inputs of C9 to TS or R bearing C5b-9 led to a dose-related increase in C9 binding and killing. In contrast, addition of high inputs of C9 to the same strains at 4 degrees C, a procedure that limits the C9:C5b-8 ratio to 1:1, resulted in low C9 binding and minimal killing. Bactericidal C5b-9 formed at 37 degrees C on R and TS with high inputs of C9 co-sedimented with the bacterial outer membrane on sucrose density gradient analysis. Non-bactericidal C5b-9 on R, WT, and TS co-sedimented near the inner membrane, despite the presumed lack of association between these constituents. Whereas 125I C9 within the non-bactericidal pools immunoprecipitate with anti-C5, 125I C9 within bactericidal pools did not immunoprecipitate with anti-C5, anti-C7, or anti-C9. These findings suggest that bactericidal C5b-9 may be deposited in a unique location or configuration within the bacterial cell wall.     

Nisin induces changes in membrane fatty acid composition of Listeria monocytogenes nisin-resistant strains at 10°C and 30°C

Listeria monocytogenes isolates resistant to 10⁵ IU ml^-1 nisin were obtained at 30°C (NR30) and at 10°C (NR10). Nisin prolonged the lag phase of isolate NR30 at 10°C. Isolates NR30 and NR10 did not produce a nisinase. Protoplasts of isolate NR30 were unaffected by exposure to nisin. The fatty acid composition from the wild-type strain and NR isolates was determined. As expected, temperature-induced differences in the C15/C17 fatty acid ratios were found. Growth of the NR strains in the presence of nisin resulted in significantly different C15/C17 ratios and a significant increase in the percentage of C16:0, C16: 1, C18:0 and C18: 1 fatty acids at 10°C and 30°C. Both the NR10 and NR30 isolates had similar growth rates at low temperatures, but these were slower than the wild-type strain. These results indicate that 'nisin resistance'is an environmentally defined phenotype and that nisin induces changes in the fatty acid composition of the membrane in L. monocytogenes nisin-resistant isolates regardless of the growth temperature.     

Measurement of DNA damage in mammalian cells exposed in vitro to radiofrequency fields at SARs of 3-5 W/kg

In the present study, we determined whether exposure of mammalian cells to 3.2-5.1 W/kg specific absorption rate (SAR) radiofrequency fields could induce DNA damage in murine C3H 10T(1/2) fibroblasts. Cell cultures were exposed to 847.74 MHz code-division multiple access (CDMA) and 835.62 frequency-division multiple access (FDMA) modulated radiations in radial transmission line (RTL) irradiators in which the temperature was regulated to 37.0 +/- 0.3 degrees C. Using the alkaline comet assay to measure DNA damage, we found no statistically significant differences in either comet moment or comet length between sham-exposed cells and those exposed for 2, 4 or 24 h to CDMA or FDMA radiations in either exponentially growing or plateau-phase cells. Further, a 4-h incubation after the 2-h exposure resulted in no significant changes in comet moment or comet length. Our results show that exposure of cultured C3H 10T(1/2) cells at 37 degrees C CDMA or FDMA at SAR values of up to 5.1 W/kg did not induce measurable DNA damage.     

Loss of Viability and Metabolic Injury of Staphylococcus aureus Resulting from Storage at 5°C

S ummary . A culture of Staphylococcus aureus in trypticase-soy broth incubated at 5° became increasingly sensitive to mannitol-salt agar. The sensitivity was affected by the pH value of the suspending medium. On transfer to 37° the culture rapidly regained the ability to form colonies on the selective medium.