Thermal inactivation of Listeria monocytogenes within bovine milk phagocytes.

Thermal resistance of intracellular and freely suspended Listeria monocytogenes that was associated with a milkborne outbreak of listeriosis was studied by using the sealed tube and slug flow heat exchanger methods. Test temperatures for the former method were 57.8, 62.8, 66.1, and 68.9 degrees C (136, 145, 151, and 156 degrees F, respectively); whereas those for the latter method were 66.1, 68.9, 71.7, and 74.4 degrees C (151, 156, 161, and 166 degrees F, respectively). The heating menstruum was sterile, whole milk. The intracellular inoculum was generated from an in vitro phagocytosis reaction by using endotoxin-induced bovine milk phagocytes. The phagocyte population consisted of 88% neutrophils, 8% macrophages, and 4% lymphocytes. Neutrophils harbored the majority of intracellular L. monocytogenes. The mean level of infectivity in the phagocyte population was 43%, and there were 26.1 +/- 19.3 bacteria per cell (10(4) viable cells per ml of test milk). Initial bacterial counts for the freely suspended and intracellular experiments (the latter was based on a sonically disrupted sample) were 10(6) L. monocytogenes cells per ml. Heat-stressed bacteria were recovered by direct plating in parallel with recovery from an enrichment broth; both methods gave comparable results. The predicted D62.8 degrees C (145 degrees F) value for intracellular sealed tube studies was 53.8 s (ZD = 5.6 degrees C [10.0 degrees F]), indicating a safe 33.4 D margin of inactivation for vat pasteurization (62.8 degrees C for 30 min).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Survival of Listeria monocytogenes in milk during high-temperature, short-time pasteurization

Milk from cows inoculated with Listeria monocytogenes was pooled for 2 to 4 days and then heated at 71.7 to 73.9 degrees C for 16.4 s or at 76.4 to 77.8 degrees C for 15.4 s in a high-temperature, short-time plate heat exchanger pasteurization unit. L. monocytogenes was isolated from milk after heat treatment in six of nine pasteurization trials done at 71.7 to 73.9 degrees C and in none of three trials done at 76.4 to 77.8 degrees C. An average of 1.5 to 9.2 L. monocytogenes cells was seen in each milk polymorphonuclear leukocyte before heat treatment in 11 of 12 pasteurization trials. Noticeable degradation of leukocytes with intracellular listeria was detected in unpasteurized milk after 3 days of storage at 4 degrees C, and by 4 days of storage leukocytes had deteriorated to cellular debris, suggesting that holding unpasteurized milk refrigerated for 4 or more days would eliminate a protective effect leukocytes may provide for increasing heat resistance of L. monocytogenes. Results indicate that under the conditions of this study, L. monocytogenes can survive the minimum high-temperature, short-time treatment (71.7 degrees C, 15 s) required by the U.S. Food and Drug Administration for pasteurizing milk.     

Survival of Listeria monocytogenes in milk during high-temperature, short-time pasteurization.

Milk from cows inoculated with Listeria monocytogenes was pooled for 2 to 4 days and then heated at 71.7 to 73.9 degrees C for 16.4 s or at 76.4 to 77.8 degrees C for 15.4 s in a high-temperature, short-time plate heat exchanger pasteurization unit. L. monocytogenes was isolated from milk after heat treatment in six of nine pasteurization trials done at 71.7 to 73.9 degrees C and in none of three trials done at 76.4 to 77.8 degrees C. An average of 1.5 to 9.2 L. monocytogenes cells was seen in each milk polymorphonuclear leukocyte before heat treatment in 11 of 12 pasteurization trials. Noticeable degradation of leukocytes with intracellular listeria was detected in unpasteurized milk after 3 days of storage at 4 degrees C, and by 4 days of storage leukocytes had deteriorated to cellular debris, suggesting that holding unpasteurized milk refrigerated for 4 or more days would eliminate a protective effect leukocytes may provide for increasing heat resistance of L. monocytogenes. Results indicate that under the conditions of this study, L. monocytogenes can survive the minimum high-temperature, short-time treatment (71.7 degrees C, 15 s) required by the U.S. Food and Drug Administration for pasteurizing milk.     

Thermotolerance of heat-shocked Listeria monocytogenes in milk exposed to high-temperature, short-time pasteurization.

The effect of prior heat shock (48 degrees C for 15 min) on the thermotolerance of Listeria monocytogenes at the minimal high-temperature, short-time (71.7 degrees C for 15 s) parameters required by the Pasteurized Milk Ordinance was examined. The mean D71.7 degrees C value for heat-shocked L. monocytogenes was 4.6 +/- 0.5 s (control D = 3.0 +/- 1.0 s); the ratio of D to control D was 1.5. The increased thermotolerance of heat-shocked Listeria cells was not significant and appeared unlikely to have practical implications, in terms of risk assessment, for the safety of pasteurized milk.     

Thermotolerance of heat-shocked Listeria monocytogenes in milk exposed to high-temperature, short-time pasteurization.

The effect of prior heat shock (48 degrees C for 15 min) on the thermotolerance of Listeria monocytogenes at the minimal high-temperature, short-time (71.7 degrees C for 15 s) parameters required by the Pasteurized Milk Ordinance was examined. The mean D71.7 degrees C value for heat-shocked L. monocytogenes was 4.6 +/- 0.5 s (control D = 3.0 +/- 1.0 s); the ratio of D to control D was 1.5. The increased thermotolerance of heat-shocked Listeria cells was not significant and appeared unlikely to have practical implications, in terms of risk assessment, for the safety of pasteurized milk.     

Comparative recovery of uninjured and heat-injured Listeria monocytogenes cells from bovine milk.

The standard selective enrichment protocols of the Food and Drug Administration (FDA) and U.S. Department of Agriculture (USDA) were compared with an experimental nonselective broth enrichment (NSB) protocol and variations of the standard cold-enrichment (CE) protocol for the recovery of heat-injured Listeria monocytogenes. Bacterial cells (10(7)/ml) were suspended in sterile milk and heated at 71.7 degrees C in a slug-flow heat exchanger for holding times ranging from 1 to 30 s. Surviving cells were determined (50% endpoint) by the given protocols, and the following D values were obtained: NSB, D = 2.0 +/- 0.5 s; FDA, D = 1.4 +/- 0.3 s; USDA, D = 0.6 +/- 0.2 s; CE, D less than or equal to 1.2 s. The respective direct-plating media used in these enrichments were also analyzed for recovery, and the following D values were calculated from the enumeration of surviving cells; NSB, D = 2.7 +/- 0.8 s; FDA, D = 1.3 +/- 0.4 s; USDA, D = 0.7 +/- 0.2 s. The low levels of heat-injured L. monocytogenes cells which were detected at inactivation endpoints on the optimal nonselective media (25 degrees C for 7 days) failed to recover and multiply during experimental CEs (4 degrees C for 28 days). Initial inactivation experiments in which raw whole milk was used as the heating menstruum gave much lower recoveries with all protocols. The detectable limits for uninjured cells that were suspended in raw milk were similar (0.35 to 3.2 cells per ml) for the standard CE, FDA, and USDA protocols.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative recovery of uninjured and heat-injured Listeria monocytogenes cells from bovine milk

The standard selective enrichment protocols of the Food and Drug Administration (FDA) and U.S. Department of Agriculture (USDA) were compared with an experimental nonselective broth enrichment (NSB) protocol and variations of the standard cold-enrichment (CE) protocol for the recovery of heat-injured Listeria monocytogenes. Bacterial cells (10(7)/ml) were suspended in sterile milk and heated at 71.7 degrees C in a slug-flow heat exchanger for holding times ranging from 1 to 30 s. Surviving cells were determined (50% endpoint) by the given protocols, and the following D values were obtained: NSB, D = 2.0 +/- 0.5 s; FDA, D = 1.4 +/- 0.3 s; USDA, D = 0.6 +/- 0.2 s; CE, D less than or equal to 1.2 s. The respective direct-plating media used in these enrichments were also analyzed for recovery, and the following D values were calculated from the enumeration of surviving cells; NSB, D = 2.7 +/- 0.8 s; FDA, D = 1.3 +/- 0.4 s; USDA, D = 0.7 +/- 0.2 s. The low levels of heat-injured L. monocytogenes cells which were detected at inactivation endpoints on the optimal nonselective media (25 degrees C for 7 days) failed to recover and multiply during experimental CEs (4 degrees C for 28 days). Initial inactivation experiments in which raw whole milk was used as the heating menstruum gave much lower recoveries with all protocols. The detectable limits for uninjured cells that were suspended in raw milk were similar (0.35 to 3.2 cells per ml) for the standard CE, FDA, and USDA protocols.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of growth temperature and strictly anaerobic recovery on the survival of Listeria monocytogenes during pasteurization.

Listeria monocytogenes F5069 was suspended in either Trypticase soy broth-0.6% yeast extract (TSBYE) or sterile, whole milk and heated at 62.8 degrees C in sealed thermal death time tubes. Severely heat-injured cells were recovered in TSBYE within sealed thermal death time tubes because of the formation of reduced conditions in the depths of the TSBYE. Also, the use of strictly anaerobic Hungate techniques significantly increased recovery in TSBYE containing 1.5% agar compared with aerobically incubated controls. The exogenous addition of catalase, but not superoxide dismutase, slightly increased the recovery of heat-injured cells in TSBYE containing 1.5% agar incubated aerobically. Growth of cells at 43 degrees C caused a greater increase in heat resistance as compared with cells heat shocked at 43 degrees C or cells grown at lower temperatures. Growth of L. monocytogenes at 43 degrees C and enumeration by the use of strictly anaerobic Hungate techniques resulted in D62.8 degrees C values that were at least sixfold greater than those previously obtained by using cells grown at 37 degrees C and aerobic plating. Results indicate that, under the conditions of the present study, high levels of L. monocytogenes would survive the minimum low-temperature, long-time treatment required by the U.S. Food and Drug Administration for pasteurizing milk. The possible survival of low levels of L. monocytogenes during high-temperature, short-time pasteurization and enumeration of injured cells by recovery on selective media under strictly anaerobic conditions are discussed.     

Effects of growth temperature and strictly anaerobic recovery on the survival of Listeria monocytogenes during pasteurization

Listeria monocytogenes F5069 was suspended in either Trypticase soy broth-0.6% yeast extract (TSBYE) or sterile, whole milk and heated at 62.8 degrees C in sealed thermal death time tubes. Severely heat-injured cells were recovered in TSBYE within sealed thermal death time tubes because of the formation of reduced conditions in the depths of the TSBYE. Also, the use of strictly anaerobic Hungate techniques significantly increased recovery in TSBYE containing 1.5% agar compared with aerobically incubated controls. The exogenous addition of catalase, but not superoxide dismutase, slightly increased the recovery of heat-injured cells in TSBYE containing 1.5% agar incubated aerobically. Growth of cells at 43 degrees C caused a greater increase in heat resistance as compared with cells heat shocked at 43 degrees C or cells grown at lower temperatures. Growth of L. monocytogenes at 43 degrees C and enumeration by the use of strictly anaerobic Hungate techniques resulted in D62.8 degrees C values that were at least sixfold greater than those previously obtained by using cells grown at 37 degrees C and aerobic plating. Results indicate that, under the conditions of the present study, high levels of L. monocytogenes would survive the minimum low-temperature, long-time treatment required by the U.S. Food and Drug Administration for pasteurizing milk. The possible survival of low levels of L. monocytogenes during high-temperature, short-time pasteurization and enumeration of injured cells by recovery on selective media under strictly anaerobic conditions are discussed.     

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.     

Enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus by the lactoperoxidase system.

The lactoperoxidase system (LPS) enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus. After LPS activation, biphasic survival curves were observed for L. monocytogenes at 57.8 degrees C and for S. aureus at 55.2 degrees C. The data were consistent with a model that assumed two bacterial populations differing in heat sensitivity. The more heat-sensitive fractions (93% of the L. monocytogenes, 92% of the S. aureus) were killed almost instantly. For these biphasic survival curves, D values were based on the much smaller, less-heat-sensitive fractions. For L. monocytogenes, the D52.2 degrees C values were 30.2 min (untreated milk) and 10.7 min (LPS activated); corresponding D55.2 degrees C values were 8.2 and 1.6 min; corresponding D57.8 degrees C values were 2.3 and 0.5 min. For S. aureus, the D52.2 degrees C values were 33.3 min (untreated milk) and 2.2 min (LPS activated), and the corresponding D55.2 degrees C values were 7.6 and 1.1 min, respectively. The most rapid killing of L. monocytogenes occurred when samples were heated soon after activation of the LPS. Activation of the LPS followed by heating can increase the margin of safety with respect to milkborne pathogens.     

Enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus by the lactoperoxidase system

The lactoperoxidase system (LPS) enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus. After LPS activation, biphasic survival curves were observed for L. monocytogenes at 57.8 degrees C and for S. aureus at 55.2 degrees C. The data were consistent with a model that assumed two bacterial populations differing in heat sensitivity. The more heat-sensitive fractions (93% of the L. monocytogenes, 92% of the S. aureus) were killed almost instantly. For these biphasic survival curves, D values were based on the much smaller, less-heat-sensitive fractions. For L. monocytogenes, the D52.2 degrees C values were 30.2 min (untreated milk) and 10.7 min (LPS activated); corresponding D55.2 degrees C values were 8.2 and 1.6 min; corresponding D57.8 degrees C values were 2.3 and 0.5 min. For S. aureus, the D52.2 degrees C values were 33.3 min (untreated milk) and 2.2 min (LPS activated), and the corresponding D55.2 degrees C values were 7.6 and 1.1 min, respectively. The most rapid killing of L. monocytogenes occurred when samples were heated soon after activation of the LPS. Activation of the LPS followed by heating can increase the margin of safety with respect to milkborne pathogens.     

A proposed nonpathogenic biological indicator for thermal inactivation of Listeria monocytogenes.

Listeria innocua M1 was developed as a thermal processing indicator organism for L. monocytogenes by selection of a rifampin- and streptomycin-resistant mutant. zetaD values were 5.6 and 5.8 degrees C, and D (68 degrees C) values were 3.8 and 4.9 s for L. monocytogenes and L. innocua, respectively, in skim milk. The advantages of easy selection, similar heat resistance, and nonpathogenicity make L. innocua M1 appropriate for challenge studies designed to evaluate process lethality with respect to L. monocytogenes.     

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.     

Survival of Listeria monocytogenes in raw milk treated in a pilot plant size pasteurizer

The survival of Listeria monocytogenes in raw milk treated in a pilot plant size pasteurizer was investigated. Raw milk was inoculated with different initial concentrations of L. monocytogenes and heated at temperatures ranging from 69 degrees to 73 degrees C. Listerias were not isolated from any of the milk samples immediately after thermal treatment. They were isolated, however, from 46.6% of heated samples (none from samples heated at 73 degrees C) after variable periods at refrigeration temperature. The results suggest that a low number of listerias survive some thermal treatments, but a cold enrichment is necessary to repair the thermally injured cells and detect these organisms in milk. The importance of the isolation technique in the recovery of listerias from pasteurized milk samples is discussed.     

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.     

Hyperthermic killing and hyperthermic radiosensitization in Chinese hamster ovary cells: effects of pH and thermal tolerance

To quantitatively relate heat killing and heat radiosensitization, asynchronous or G1 Chinese hamster ovary (CHO) cells at pH 7.1 or 6.75 were heated and/or X-irradiated 10 min later. Since no progression of G1 cells into S phase occurred during the heat and radiation treatments, cell cycle artifacts were minimized. However, results obtained for asynchronous and G1 cells were similar. Hyperthermic radiosensitization was expressed as the thermal enhancement factor (TEF), defined as the ratio of the D0 of the radiation survival curve to that of the D0 of the radiation survival curve for heat plus radiation. The TEF increased continuously with increased heat killing at 45.5 degrees C, and for a given amount of heat killing, the amount of heat radiosensitization was the same for both pH's. When cells were heated chronically at 42.4 degrees C at pH 7.4, the TEF increased initially to 2.0-2.5 and then returned to near 1.0 during continued heating as thermal tolerance developed for both heat killing and heat radiosensitization. However, the shoulder (Dq) of the radiation survival curve for heat plus radiation did not manifest thermal tolerance; i.e., it decreased continuously with increased heat killing, independent of temperature, pH, or the development of thermotolerance. These results suggest that heat killing and heat radiosensitization have a target(s) in common (TEF results), along with either a different target(s) or a difference in the manifestation of heat damage (Dq results). For clinical considerations, the interaction between heat and radiation was expressed as (1) the thermal enhancement ratio (TER), which is the dose of X rays alone divided by the dose of X rays combined with heat to obtain an isosurvival, e.g., 10(-4), and (2) the thermal gain factor (TGF), the ratio of the TER at pH 6.75 to the TER at pH 7.4. Since low pH reduced the rate of development of thermal tolerance during heating at low temperatures, low pH enhanced heat killing more at 42-42.5 degrees C than at 45.5 degrees C where thermal tolerance did not develop. Therefore, the increase in the TGF after chronic heating at 42-42.5 degrees C was greater than after acute heating at 45.5 degrees C, due primarily to the increase in heat killing causing an even greater increase in heat radiosensitization. These findings agree with animal experiments suggesting that in the clinic, a therapeutic gain for tumor cells at low pH may be greater for temperatures of 42-42.5 degrees C than of 45.5 degrees C.     

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.