Catalase and superoxide dismutase activities after heat injury of Listeria monocytogenes

Four strains of Listeria monocytogenes were examined for catalase (CA) and superoxide dismutase (SOD) activities. The two strains having the highest CA activities (LCDC and Scott A) also possessed the highest SOD activities. The CA activity of heated cell extracts of all four strains examined decreased sharply between 55 and 60 degrees C. SOD was more heat labile than CA. Two L. monocytogenes strains demonstrated a decline in SOD activity after heat treatment at 45 degrees C, whereas the other two strains demonstrated a decline at 50 degrees C. Sublethal heating of the cells at 55 degrees C resulted in increased sensitivity to 5.5% NaCl. Exogenous hydrogen peroxide was added to suspensions of L. monocytogenes; strains producing the highest CA levels showed the greatest H2O2 resistance.     

Catalase and superoxide dismutase activities after heat injury of Listeria monocytogenes.

Four strains of Listeria monocytogenes were examined for catalase (CA) and superoxide dismutase (SOD) activities. The two strains having the highest CA activities (LCDC and Scott A) also possessed the highest SOD activities. The CA activity of heated cell extracts of all four strains examined decreased sharply between 55 and 60 degrees C. SOD was more heat labile than CA. Two L. monocytogenes strains demonstrated a decline in SOD activity after heat treatment at 45 degrees C, whereas the other two strains demonstrated a decline at 50 degrees C. Sublethal heating of the cells at 55 degrees C resulted in increased sensitivity to 5.5% NaCl. Exogenous hydrogen peroxide was added to suspensions of L. monocytogenes; strains producing the highest CA levels showed the greatest H2O2 resistance.     

Effect of prior heat shock on heat resistance of Listeria monocytogenes in meat

The effect of prior heat shock on the thermal resistance of Listeria monocytogenes in meat was investigated. A sausage mix inoculated with approximately 10(7) L. monocytogenes per g was initially subjected to a heat shock temperature of 48 degrees C before being heated at a final test temperature of 62 or 64 degrees C. Although cells heat shocked at 48 degrees C for 30 or 60 min did not show a significant increase in thermotolerance as compared with control cells (non-heat shocked), bacteria heat shocked for 120 min did, showing an average 2.4-fold increase in the D64 degrees C value. Heat-shocked cells shifted to 4 degrees C appeared to maintain their thermotolerance for at least 24 h after heat shock.     

Effect of prior heat shock on heat resistance of Listeria monocytogenes in meat.

The effect of prior heat shock on the thermal resistance of Listeria monocytogenes in meat was investigated. A sausage mix inoculated with approximately 10(7) L. monocytogenes per g was initially subjected to a heat shock temperature of 48 degrees C before being heated at a final test temperature of 62 or 64 degrees C. Although cells heat shocked at 48 degrees C for 30 or 60 min did not show a significant increase in thermotolerance as compared with control cells (non-heat shocked), bacteria heat shocked for 120 min did, showing an average 2.4-fold increase in the D64 degrees C value. Heat-shocked cells shifted to 4 degrees C appeared to maintain their thermotolerance for at least 24 h after heat shock.     

Temperature- and surfactant-induced membrane modifications that alter Listeria monocytogenes nisin sensitivity by different mechanisms

Nisin interacts with target membranes in four sequential steps: binding, insertion, aggregation, and pore formation. Alterations in membrane composition might influence any of these steps. We hypothesized that cold temperatures (10 degrees C) and surfactant (0.1% Tween 20) in the growth medium would influence Listeria monocytogenes membrane lipid composition, membrane fluidity, and, as a result, sensitivity to nisin. Compared to the membranes of cells grown at 30 degrees C, those of L. monocytogenes grown at 10 degrees C had increased amounts of shorter, branched-chain fatty acids, increased fluidity (as measured by fluorescence anisotropy), and increased nisin sensitivity. When 0.1% Tween 20 was included in the medium and the cells were cultured at 30 degrees C, there were complex changes in lipid composition. They did not influence membrane fluidity but nonetheless increased nisin sensitivity. Further investigation found that these cells had an increased ability to bind radioactively labeled nisin. This suggests that the modification of the surfactant-adapted cell membrane increased nisin sensitivity at the binding step and demonstrates that each of the four steps can contribute to nisin sensitivity.     

Survival and heat resistance of Listeria monocytogenes after exposure to alkali and chlorine

A strain of Listeria monocytogenes isolated from a drain in a food-processing plant was demonstrated, by determination of D values, to be more resistant to the lethal effect of heat at 56 or 59 degrees C following incubation for 45 min in tryptose phosphate broth (TPB) at pH 12.0 than to that of incubation for the same time in TPB at pH 7.3. Cells survived for at least 6 days when they were suspended in TPB at pHs 9.0, 10.0, and 11.0 and stored at 4 or 21 degrees C. Cells of L. monocytogenes incubated at 37 degrees C for 45 min and then stored for 48 or 144 h in TPB at pH 10.0 were more resistant to heat treatment at 56 degrees C than were cells stored in TPB at pH 7.3. The alkaline-stress response in L. monocytogenes may induce resistance to otherwise lethal thermal-processing conditions. Treatment of cells in 0.05 M potassium phosphate buffer (pH 7.00 +/- 0.05) containing 2.0 or 2.4 mg of free chlorine per liter reduced populations by as much as 1.3 log(10) CFU/ml, while treatment with 6.0 mg of free chlorine per liter reduced populations by as much as 4.02 log(10) CFU/ml. Remaining subpopulations of chlorine-treated cells exhibited some injury, and cells treated with chlorine for 10 min were more sensitive to heating at 56 degrees C than cells treated for 5 min. Contamination of foods by L. monocytogenes cells that have survived exposure to processing environments ineffectively cleaned or sanitized with alkaline detergents or disinfectants may have more severe implications than previously recognized. Alkaline-pH-induced cross-protection of L. monocytogenes against heat has the potential to enhance survival in minimally processed as well as in heat-and-serve foods and in foods on holding tables, in food service facilities, and in the home. Cells surviving exposure to chlorine, in contrast, are more sensitive to heat; thus, the effectiveness of thermal processing in achieving desired log(10)-unit reductions is not compromised in these cells.     

Real-time measurements of the interaction between single cells of Listeria monocytogenes and nisin on a solid surface

A method to obtain real-time measurements of the interactions between nisin and single cells of Listeria monocytogenes on a solid surface was developed. This method was based on fluorescence ratio-imaging microscopy and measurements of changes in the intracellular pH (pH(i)) of carboxyfluorescein succinimidyl ester-stained cells during exposure to nisin. Immobilized cells were placed in a chamber mounted on a microscope and attached to a high-precision peristaltic pump which allowed rapid changes in the nisin concentration. In the absence of nisin, the pH(i) of L. monocytogenes was almost constant (approximately pH 8.0) and independent of the external pH in the pH range from 5.0 to 9.0. In the presence of nisin, dissipation of the pH gradient (DeltapH) was observed, and this dissipation was both time and nisin concentration dependent. The dissipation of DeltapH resulted in cell death, as determined by the number of CFU. In the model system which we used the immobilized cells were significantly more resistant to nisin than the planktonic cells. The kinetics of DeltapH dissipation for single cells revealed a variable lag phase depending on the nisin concentration, which was followed by a very rapid decrease in pH(i) within 1 to 2 min. The differences in nisin sensitivity between single cells in a L. monocytogenes population were insignificant for cells grown to the stationary phase in a liquid laboratory substrate, but differences were observed for cells grown on an agar medium under similar conditions, which resulted in some cells having increased resistance to nisin.     

Catalase, superoxide dismutase, and hemolysin activities and heat susceptibility of Listeria monocytogenes after growth in media containing sodium chloride

The activities of catalase, superoxide dismutase, and a thiol-activated hemolysin produced by four strains of Listeria monocytogenes propagated in media containing various concentrations of sodium chloride were examined. L. monocytogenes 7644 showed an increase in catalase, superoxide dismutase, and thiol-activated hemolysin activities when grown in a medium containing 2.5% (wt/vol) NaCl followed by a decrease in activities when propagated in media containing salt concentrations higher than 2.5%. L. monocytogenes LCDC 81-861 demonstrated enhanced catalase activity when grown in media containing NaCl ranging from 1.5 to 4.6% and increased superoxide dismutase activity when propagated in media containing 1.5 to 3.5% NaCl. L. monocytogenes LCDC 81-861 did not exhibit any detectable hemolysin activity under the conditions tested. After growth in various NaCl-containing media, both strains were subjected to sublethal heat injury for 30 min at 55 degrees C. L. monocytogenes LCDC 81-861 showed increased sensitivity to the heat treatment when grown in media containing 4.6 and 6.5% NaCl, whereas L. monocytogenes 7644 did not exhibit enhanced heat lability.     

Catalase, superoxide dismutase, and hemolysin activities and heat susceptibility of Listeria monocytogenes after growth in media containing sodium chloride.

The activities of catalase, superoxide dismutase, and a thiol-activated hemolysin produced by four strains of Listeria monocytogenes propagated in media containing various concentrations of sodium chloride were examined. L. monocytogenes 7644 showed an increase in catalase, superoxide dismutase, and thiol-activated hemolysin activities when grown in a medium containing 2.5% (wt/vol) NaCl followed by a decrease in activities when propagated in media containing salt concentrations higher than 2.5%. L. monocytogenes LCDC 81-861 demonstrated enhanced catalase activity when grown in media containing NaCl ranging from 1.5 to 4.6% and increased superoxide dismutase activity when propagated in media containing 1.5 to 3.5% NaCl. L. monocytogenes LCDC 81-861 did not exhibit any detectable hemolysin activity under the conditions tested. After growth in various NaCl-containing media, both strains were subjected to sublethal heat injury for 30 min at 55 degrees C. L. monocytogenes LCDC 81-861 showed increased sensitivity to the heat treatment when grown in media containing 4.6 and 6.5% NaCl, whereas L. monocytogenes 7644 did not exhibit enhanced heat lability.     

Pulsed electric field alters molecular chaperone expression and sensitizes Listeria monocytogenes to heat

Pulsed electric field (PEF)-resistant and PEF-sensitive Listeria monocytogenes strains were sublethally treated with electric pulses at 15 kV/cm for 29 micro s and held at 25 degrees C for 5 to 30 min prior to protein extraction. The levels of the molecular chaperones GroEL, GroES, and DnaJ were determined by immunoblotting. After 10 to 20 min after sublethal PEF treatment, a transient decrease in molecular chaperone expression was observed in the PEF-sensitive strain (Scott A). The levels of GroEL and DnaJ increased back to the basal expression level within 30 min. A substantial decrease in GroES expression persisted for at least 30 min after PEF treatment. Chaperone expression was suppressed after PEF treatment to a smaller extent in the PEF-resistant (OSY-8578) than in the PEF-sensitive strain, and no clear expression pattern was identified in OSY-8578. Inactivation of Scott A and OSY-8578 in phosphate buffer was compared when lethal PEF (27.5 kV/cm, 144 micro s) and heat (55 degrees C, 10 min) were applied in sequence. When PEF and heat treatments were applied separately, the populations of L. monocytogenes Scott A and OSY-8578 decreased 0.5 to 0.6 log CFU/ml. Cells treated first with PEF and incubated at 25 degrees C for 10 min showed substantial sensitivity to subsequent heat treatment; the decrease in counts for Scott A and OSY-8578 was 6.1 and 2.8 log CFU/ml, respectively. The sequence and time lapse between the two treatments were crucial for achieving high inactivation rates. It is concluded that PEF sensitized L. monocytogenes to heat and that maximum heat sensitization occurred when chaperone expression was at a minimum level.     

Synergistic antibacterial action of heat in combination with nisin and magainin II amide

Lactobacillus plantarum has been exposed to mild heat at temperatures between 48 and 56 °C in combination with low concentrations of the lantobiotic nisin in different sequential set-ups. Exposure to heat and nisin caused synergistic reductions of Lact. plantarum viability. Efficient antimicrobial action was dependent on the growth state of the culture as well as on levels and sequences of treatment applications. Listeria monocytogenes and Escherichia coli were treated at 55 °C in the presence of magainin II amide. Synergistic reductions in viable counts could be observed for L. monocytogenes and, after prolonged exposure, also for E. coli . The bacterial membrane could be identified by fluorometry and flow cytometry as an important target of applied treatment combinations.     

Elevation of the heat resistance of Salmonella typhimurium by sublethal heat shock

The survival of Salmonella typhimurium after a standard heat challenge at 55 degrees C for 25 min increased by several orders of magnitude when cells grown at 37 degrees C were pre-incubated at 42 degrees, 45 degrees or 48 degrees C before heating at the higher temperature. Heat resistance increased rapidly after the temperature shift, reaching near maximum levels within 30 min. Elevated heat resistance persisted for at least 10 h. Pre-incubation of cells at 48 degrees C for 30 min increased their resistance to subsequent heating at 50 degrees, 52 degrees, 55 degrees, 57 degrees or 59 degrees C. Survival curves of resistant cells were curvilinear. Estimated times for a '7D' inactivation increased by 2.6- to 20-fold compared with cells not pre-incubated before heat challenge.     

Carbon dioxide and nisin act synergistically on Listeria monocytogenes

This paper examines the synergistic action of carbon dioxide and nisin on Listeria monocytogenes Scott A wild-type and nisin-resistant (Nis(r)) cells grown in broth at 4 degrees C. Carbon dioxide extended the lag phase and decreased the specific growth rate of both strains, but to a greater degree in the Nis(r) cells. Wild-type cells grown in 100% CO(2) were two to five times longer than cells grown in air. Nisin (2.5 microg/ml) did not decrease the viability of Nis(r) cells but for wild-type cells caused an immediate 2-log reduction of viability when they were grown in air and a 4-log reduction when they were grown in 100% CO(2). There was a quantifiable synergistic action between nisin and CO(2) in the wild-type strain. The MIC of nisin for the wild-type strain grown in the presence of 2.5 microg of nisin per ml increased from 3.1 to 12.5 microg/ml over 35 days, but this increase was markedly delayed for cultures in CO(2). This synergism between nisin and CO(2) was examined mechanistically by following the leakage of carboxyfluorescein (CF) from listerial liposomes. Carbon dioxide enhanced nisin-induced CF leakage, indicating that the synergistic action of CO(2) and nisin occurs at the cytoplasmic membrane. Liposomes made from cells grown in a CO(2) atmosphere were even more sensitive to nisin action. Liposomes made from cells grown at 4 degrees C were dramatically more nisin sensitive than were liposomes derived from cells grown at 30 degrees C. Cells grown in the presence of 100% CO(2) and those grown at 4 degrees C had a greater proportion of short-chain fatty acids. The synergistic action of nisin and CO(2) is consistent with a model where membrane fluidity plays a role in the efficiency of nisin action.     

Resistance of heat-shocked cells of Listeria monocytogenes to mano-sonication and mano-thermo-sonication

Heat shocks did not increase the resistance of Listeria monocytogenes to an ultrasonication treatment under pressure (Mano-Sonication; MS). While heat-shocked cells (180 min, 45 degrees C) became sixfold more heat resistant than native cells (D62 = 1.8 min vs D62 = 0.24 min), the resistance of native and heat-shocked cells to MS (200 kPa, 117 microns) was the same (DMS = 1.6 min). The inactivation rate of non-heat-shocked cells of L monocytogenes by a combined heat/ultrasonication treatment under pressure (Mano-Thermo-Sonication; MTS) was an additive effect. On the contrary, on heat-shocked cells, the inactivation rate of MTS was greater than that of heat added to the inactivation rate of MS (synergistic effect) in the range 62-68 degrees C.     

The effect of temperature and growth rate on the susceptibility of Listeria monocytogenes to environmental stress conditions

R.A. PATCHETT, N. WATSON, P.S. FERNANDEZ AND R.G. KROLL. 1996. The effect of growth temperature and growth rate on the susceptibility to heat and pH stress were investigated in Listeria monocytogenes grown in continuous culture where these two growth variables could be varied independently of each other, and in batch culture. After growth at 30°C or 10°C at constant growth rate, or at 30°C at different growth rates, cells did not differ in their resistance to heat at 55°C. Cells grown at 30°C were more resistant to acid stress at pH 2.5 than cells grown at the same growth rates at 10°C. Cells grown at low growth rate at 30°C gave greater resistance to acid stress than those grown at high growth rate. Growth temperature and growth rate had independent effects on the susceptibility of L. monocytogenes to acid stress conditions. This may have implications for the survival of L. monocytogenes in acidic foods.     

The effect of acid shock on the heat resistance of Listeria monocytogenes

The effect of acid shock on the heat resistance of Listeria monocytogenes was investigated. After growth for 24 h at 30°C in tryptic soy broth containing 0.6% yeast extract, cell culture suspensions of L. monocytogenes were acidified with HCl or acetic acid over various time periods before being heated in whole milk to a temperature of 58°C. When cells were acid-shocked immediately with HCl for 1, 2 or 4 h, those acid-shocked for 1 h demonstrated the largest increase in thermotolerance as compared to control cells, when heated at 58°C in whole milk. In fact, cells acid-shocked for longer than 1 h with HCl demonstrated in some instances a decreased recovery as compared to control cells. Other types of acid-shock treatments included lowering the pH gradually either over a 4 h or a 24 h period. However, regardless of the type of acid-shock treatment, cells acid-shocked with HCl (but not acetic acid) prior to heating had significantly greater heat resistance as compared to control (non-acid-shocked) cells. It appears that acidification with HCl prior to final heating can enhance the heat resistance of L. monocytogenes.     

Growth condition-related response of Listeria monocytogenes 412 to bacteriocin inactivation

Bacteriocin inactivation of Listeria monocytogenes 412 was studied as a function of growth phase. Cells were treated with nisin (300 IU ml-1) or pediocin (320 or 2560 AU ml-1) for 20 min at 30 degrees C. Inactivation with nisin or the low concentration of pediocin was growth phase dependent, with exponentially growing cells being more susceptible than stationary cells. No effect of growth phase was observed for the high pediocin concentration. Pediocin inactivation (320 AU ml-1) of L. monocytogenes 412 exposed to osmotic (6.5% NaCl) or low-temperature (5 degrees C) stress was investigated. Pediocin failed to inactivate osmotically stressed cultures and was unable to inhibit cold-stressed cells to the same degree as unstressed cells.     

Combined action of S-carvone and mild heat treatment on Listeria monocytogenes Scott A

The combined action of the plant-derived volatile, S-carvone, and mild heat treatment on the food-borne pathogen, Listeria monocytogenes, was evaluated. The viability of exponential phase cultures grown at 8 degrees C could be reduced by 1.3 log units after exposure to S-carvone (5 mmol l-1) for 30 min at 45 degrees C, while individual treatment with S-carvone or exposure to 45 degrees C for 30 min did not result in a loss in viability. Other plant-derived volatiles, namely carvacrol, cinnamaldehyde, thymol and decanal, were also found to reduce the viability of L. monocytogenes in combination with the same mild heat treatment at concentrations of 1.75 mmol l-1, 2.5 mmol l-1, 1.5 mmol l-1 and 2 mmol l-1, respectively. These findings show that essential oil compounds can play an important role in minimally processed foods, and can be used in the concept of Hurdle Technology to reduce the intensity of heat treatment or other individual hurdles.     

Thermotolerance of Listeria monocytogenes and Salmonella typhimurium after sublethal heat shock.

The effect of prior heat shock on thermotolerance of Listeria monocytogenes and Salmonella typhimurium in broth culture was determined. Bacteria were grown at the permissive temperature of 35 degrees C, sublethally heated at 35 (control), 42, 48, and 52 degrees C (nonpermissive control) for various times, and inactivated at either 57.8 or 52 degrees C. The induction of increased thermotolerance by heat shock, although consistent within each experiment, was generally not significant for L. monocytogenes; the increase was significant for S. typhimurium. Temperature shift experiments with L. monocytogenes suggested that induced thermotolerance was not long lived unless the shock temperature was maintained.     

Combined action of nisin and carvacrol on Bacillus cereus and Listeria monocytogenes

Nisin, a small antimicrobial protein, was tested for its bactericidal action against Listeria monocytogenes and Bacillus cereus and a typical biphasic reduction of the viable count was observed. The reduction was most fast during the first 10 min of exposure, while the viable count remained stable in the last part of the exposure period. Bacillus cereus was more sensitive towards nisin than L. monocytogenes and the inhibitory effect of nisin was stronger towards cells cultivated and exposed at 8 degrees C than towards cells cultivated and exposed at 20 degrees C. Combining nisin with sublethal doses of carvacrol resulted in an increased reduction in the viable count of both organisms, indicating synergy between nisin and carvacrol. Addition of lysozyme as a third preservative factor increased the synergistic effect between nisin and carvone, especially in the last part of the exposure period.