APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1991, p. 2389-2391
Vol. 57, No. 8
0099-2240/91/082389-03$02.00/0 Copyright C 1991, American Society for Microbiology
NOTES
Pathogenicity of Nonstressed, Heat-Stressed, and Resuscitated Listeria monocytogenes lAl Cells SUSAN A. McCARTHY Fishery Research Branch, Food and Drug Administration, Dauphin Island, Alabama 36528 Received 25 January 1991/Accepted 13 May 1991
The pathogenicity of nonstressed, heat-stressed, and resuscitated cells of Listeria monocytogenes lAl was assayed in immunocompromised mice. Cells were stressed by heating them at 56°C for 20 min and were resuscitated by incubation in tryptic soy broth at 25°C. A dose of 102 nonstressed and resuscitated cells per mouse was required for pathogenicity; a dose of 104 heat-stressed cells per mouse was considerably less pathogenic. Loss of hemolytic activity accompanied the decreased virulence.
Listeria monocytogenes causes 1,700 serious infections annually in the United States (16). Listeriosis usually occurs in pregnant women, newborns, the elderly, and immunosuppressed persons and is often sporadic and of unknown etiology. In the past decade, listeriosis outbreaks in North America have resulted from the consumption of contaminated cole slaw (28), pasteurized milk (15), Mexican-style cheese (19), and turkey franks (1). The implication that pasteurized dairy products are vehicles of infection has led to numerous studies on the heat resistance of L. monocytogenes. Several investigators have suggested that L. monocytogenes does not survive the minimum high-temperature, short-time pasteurization treatment of fluid milk (5, 6, 10, 13, 21). Others, however, have reported that the organism survives the pasteurization process (11, 14). Thermotolerant L. monocytogenes cells have also been isolated from cooked meat (3, 12, 17). In studies to determine the heat resistance of freely suspended and phagocytized L. monocytogenes cells, Northolt et al. (23) found that the number of Listeria cells recovered increased 2 to 34 times if cells were allowed to resuscitate at 4°C before incubation at 37°C. Viable L. monocytogenes cells were recovered from boiled experimentally contaminated shrimp after resuscitation at 4°C (22). In cooking studies with milk curd, Ryser et al. (27) recovered heat-stressed (30 min, 57.2°C) L. monocytogenes only when the test portions were cold-enriched. Hemolysin (listeriolysin), an important virulence factor of L. monocytogenes, is the only major extracellular protein to be synthesized under heat shock conditions (31). It has been reported that hemolytic activity is required for virulence of L. monocytogenes (7, 25, 26). However, it has also been stated that expression of hemolysin is variable, with no direct relationship to pathogenicity (20). Czuprynski et al. (8) reported that when L. monocytogenes EGD was grown at 4°C, its virulence was increased in intravenously injected mice; however, there was no evidence for increased release of hemolysin. L. monocytogenes EGD actually produced more hemolysin when it was grown at 37°C. There is growing concern in the food processing industry about the organism's thermotolerance and the ability of stressed cells to resuscitate at refrigeration temperatures. The present study was conducted to determine the pathoge-
nicity of both heat-stressed and resuscitated heat-stressed L. monocytogenes cells and to compare their lethalities with those of nonstressed cells. In addition, hemolytic activity was determined and compared with pathogenicity. L. monocytogenes lAl (an isolate from shrimp) was inoculated into 250 ml of tryptic soy broth containing 0.6% yeast extract (Difco Laboratories, Detroit, Mich.) and incubated at 37°C for 18 h. Approximately 40 ml of the culture was harvested by centrifugation (12,000 x g) at 40C for 10 min, washed twice with phosphate-buffered saline (PBS; containing the following, in grams per liter: NaCl, 8; KCI, 0.2; Na2HPO4, 0.94; KH2PO4, 0.2; pH 7) and resuspended in PBS to a concentration of 101 to 105 CFU/ml. Concentrations were confirmed by duplicate surface plating of 10-fold serial dilutions on R2A agar (Difco) and lithium chloridephenylethanol-moxalactam (LPM) agar (LPM agar base; BBL, Cockeysville, Md; moxalactam, Eli Lilly & Co., Indianapolis, Ind.). Colonies were counted after incubation at 250C for 48 h. Suspensions of nonstressed cells (108 CFU/ml in PBS) were prepared, and cell concentrations were confirmed by plating as described above. Portions (10 ml) of cell suspensions were heat stressed after transfer to 300-ml Erlenmeyer flasks containing 90 ml of PBS, which was preheated to 56°C. Cultures were incubated at 56°C for 20 min with shaking at 37 rpm in a shaking water bath (model 25; Precision Scientific, Inc., Chicago, Ill.). Cells were collected by centrifugation (12,000 x g) at 40C for 10 min and resuspended in 9 ml of PBS; cell numbers were determined by plate counts. Colonies were counted after incubation at 25°C for 4 days. The number of heat-stressed cells was calculated as the difference between the R2A and LPM agar plate counts. After heat-induced injury, cell suspensions were cooled on ice to 0°C. Cultiures were supplemented with 1% tryptic soy broth (1:1 [vol/vol]) and incubated at 250C for 2 to 14 days; concentrations of resuscitated cells and progeny were then determined by surface plating on R2A and LPM agar plates. Pathogenicity was determined by using a modification of the method of Stelma et al. (32). (The laboratory animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee, Food and Drug Administration [FDA], Washington, D.C.) Female Swiss Webster mice (weight, 18 to 20 g; Charles River Laboratories, Wilm2389
2390
NOTES
ington, Mass.) were immunocompromised by intraperitoneal injection of carrageenan type 11 (200 mg/kg; Sigma Chemical Co., St. Louis, Mo.). At 24 h after this injection, groups of five mice each were challenged by intraperitoneal injections of doses of lO to 104 nonstressed cells, 104 heat-stressed cells, and 100 to 103 resuscitated cells and progeny in PBS (Table 1). Mice injected with 0.1 ml of PBS served as controls; those injected with heat-stressed cells were observed for 28 days to determine involvement of a prolonged incubation-resuscitation period before the onset of disease. The remaining mice were observed for 5 days. Four separate experiments were conducted with nonstressed and resuscitated cells; five experiments were conducted with heatstressed cells. The 50% lethal doses (LD50s) were determined by the probit method (33), which differs from the Spearman-Karber method in that it does not require responses of 0 and 100%. Chi-square analysis was used to determine significant differences (P s 0.05) between LD50s and mortality percentages for nonstressed versus resuscitated cells. For determination of hemolysin titer, L. monocytogenes lAl cells were inoculated into brain heart infusion broth (Difco) and incubated at 37°C for 18 h. Heat-stressed cells were treated as described above. All cultures were centrifuged (10,000 x g) at 4°C for 10 min; the supernatants (1 ml) were treated with 10 mM dithiothreitol (final concentration) (J. T. Baker Chemical Co., Phillipsburg, N.J.) for 10 min and assayed for hemolytic activity by the method of DominguezRodriguez et al. (9). In short, 100 ,ul of the treated sample was transferred to a 96-well microtiter plate (Becton-Dickinson, Oxnard, Calif.) and serially diluted (1:2) in PBS (pH 6). A 100-,ul portion of 2% sheep erythrocytes (Remel, Inc., Lenexa, Kans.) was added to each dilution. The plates were incubated at 370C for 2 h and then incubated at 4°C overnight. The hemolytic activity titer was expressed in complete hemolysis units, the reciprocal of the highest dilution at which complete hemolysis occurred, and minimal hemolysis units, the reciprocal of the highest dilution at which hemolysis was observed. Results of preliminary bioassay experiments indicated that pathogenic effects (death of at least three of five mice per experiment, or 60% mortality) were manifested in immunocompromised mice by day 5. The measurement of pathogenicity was defined in terms of whole bacterial cultures rather than as individual cells or an entire population of equivalent cells. The pathogenicity data for nonstressed, heat-stressed, and resuscitated L. monocytogenes cells are presented in Table 1. There was no significant difference (P < 0.05) between the pathogenicity titers of nonstressed versus resuscitated cells. Challenge suspensions containing
Vol. 57, No. 8
0099-2240/91/082389-03$02.00/0 Copyright C 1991, American Society for Microbiology
NOTES
Pathogenicity of Nonstressed, Heat-Stressed, and Resuscitated Listeria monocytogenes lAl Cells SUSAN A. McCARTHY Fishery Research Branch, Food and Drug Administration, Dauphin Island, Alabama 36528 Received 25 January 1991/Accepted 13 May 1991
The pathogenicity of nonstressed, heat-stressed, and resuscitated cells of Listeria monocytogenes lAl was assayed in immunocompromised mice. Cells were stressed by heating them at 56°C for 20 min and were resuscitated by incubation in tryptic soy broth at 25°C. A dose of 102 nonstressed and resuscitated cells per mouse was required for pathogenicity; a dose of 104 heat-stressed cells per mouse was considerably less pathogenic. Loss of hemolytic activity accompanied the decreased virulence.
Listeria monocytogenes causes 1,700 serious infections annually in the United States (16). Listeriosis usually occurs in pregnant women, newborns, the elderly, and immunosuppressed persons and is often sporadic and of unknown etiology. In the past decade, listeriosis outbreaks in North America have resulted from the consumption of contaminated cole slaw (28), pasteurized milk (15), Mexican-style cheese (19), and turkey franks (1). The implication that pasteurized dairy products are vehicles of infection has led to numerous studies on the heat resistance of L. monocytogenes. Several investigators have suggested that L. monocytogenes does not survive the minimum high-temperature, short-time pasteurization treatment of fluid milk (5, 6, 10, 13, 21). Others, however, have reported that the organism survives the pasteurization process (11, 14). Thermotolerant L. monocytogenes cells have also been isolated from cooked meat (3, 12, 17). In studies to determine the heat resistance of freely suspended and phagocytized L. monocytogenes cells, Northolt et al. (23) found that the number of Listeria cells recovered increased 2 to 34 times if cells were allowed to resuscitate at 4°C before incubation at 37°C. Viable L. monocytogenes cells were recovered from boiled experimentally contaminated shrimp after resuscitation at 4°C (22). In cooking studies with milk curd, Ryser et al. (27) recovered heat-stressed (30 min, 57.2°C) L. monocytogenes only when the test portions were cold-enriched. Hemolysin (listeriolysin), an important virulence factor of L. monocytogenes, is the only major extracellular protein to be synthesized under heat shock conditions (31). It has been reported that hemolytic activity is required for virulence of L. monocytogenes (7, 25, 26). However, it has also been stated that expression of hemolysin is variable, with no direct relationship to pathogenicity (20). Czuprynski et al. (8) reported that when L. monocytogenes EGD was grown at 4°C, its virulence was increased in intravenously injected mice; however, there was no evidence for increased release of hemolysin. L. monocytogenes EGD actually produced more hemolysin when it was grown at 37°C. There is growing concern in the food processing industry about the organism's thermotolerance and the ability of stressed cells to resuscitate at refrigeration temperatures. The present study was conducted to determine the pathoge-
nicity of both heat-stressed and resuscitated heat-stressed L. monocytogenes cells and to compare their lethalities with those of nonstressed cells. In addition, hemolytic activity was determined and compared with pathogenicity. L. monocytogenes lAl (an isolate from shrimp) was inoculated into 250 ml of tryptic soy broth containing 0.6% yeast extract (Difco Laboratories, Detroit, Mich.) and incubated at 37°C for 18 h. Approximately 40 ml of the culture was harvested by centrifugation (12,000 x g) at 40C for 10 min, washed twice with phosphate-buffered saline (PBS; containing the following, in grams per liter: NaCl, 8; KCI, 0.2; Na2HPO4, 0.94; KH2PO4, 0.2; pH 7) and resuspended in PBS to a concentration of 101 to 105 CFU/ml. Concentrations were confirmed by duplicate surface plating of 10-fold serial dilutions on R2A agar (Difco) and lithium chloridephenylethanol-moxalactam (LPM) agar (LPM agar base; BBL, Cockeysville, Md; moxalactam, Eli Lilly & Co., Indianapolis, Ind.). Colonies were counted after incubation at 250C for 48 h. Suspensions of nonstressed cells (108 CFU/ml in PBS) were prepared, and cell concentrations were confirmed by plating as described above. Portions (10 ml) of cell suspensions were heat stressed after transfer to 300-ml Erlenmeyer flasks containing 90 ml of PBS, which was preheated to 56°C. Cultures were incubated at 56°C for 20 min with shaking at 37 rpm in a shaking water bath (model 25; Precision Scientific, Inc., Chicago, Ill.). Cells were collected by centrifugation (12,000 x g) at 40C for 10 min and resuspended in 9 ml of PBS; cell numbers were determined by plate counts. Colonies were counted after incubation at 25°C for 4 days. The number of heat-stressed cells was calculated as the difference between the R2A and LPM agar plate counts. After heat-induced injury, cell suspensions were cooled on ice to 0°C. Cultiures were supplemented with 1% tryptic soy broth (1:1 [vol/vol]) and incubated at 250C for 2 to 14 days; concentrations of resuscitated cells and progeny were then determined by surface plating on R2A and LPM agar plates. Pathogenicity was determined by using a modification of the method of Stelma et al. (32). (The laboratory animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee, Food and Drug Administration [FDA], Washington, D.C.) Female Swiss Webster mice (weight, 18 to 20 g; Charles River Laboratories, Wilm2389
2390
NOTES
ington, Mass.) were immunocompromised by intraperitoneal injection of carrageenan type 11 (200 mg/kg; Sigma Chemical Co., St. Louis, Mo.). At 24 h after this injection, groups of five mice each were challenged by intraperitoneal injections of doses of lO to 104 nonstressed cells, 104 heat-stressed cells, and 100 to 103 resuscitated cells and progeny in PBS (Table 1). Mice injected with 0.1 ml of PBS served as controls; those injected with heat-stressed cells were observed for 28 days to determine involvement of a prolonged incubation-resuscitation period before the onset of disease. The remaining mice were observed for 5 days. Four separate experiments were conducted with nonstressed and resuscitated cells; five experiments were conducted with heatstressed cells. The 50% lethal doses (LD50s) were determined by the probit method (33), which differs from the Spearman-Karber method in that it does not require responses of 0 and 100%. Chi-square analysis was used to determine significant differences (P s 0.05) between LD50s and mortality percentages for nonstressed versus resuscitated cells. For determination of hemolysin titer, L. monocytogenes lAl cells were inoculated into brain heart infusion broth (Difco) and incubated at 37°C for 18 h. Heat-stressed cells were treated as described above. All cultures were centrifuged (10,000 x g) at 4°C for 10 min; the supernatants (1 ml) were treated with 10 mM dithiothreitol (final concentration) (J. T. Baker Chemical Co., Phillipsburg, N.J.) for 10 min and assayed for hemolytic activity by the method of DominguezRodriguez et al. (9). In short, 100 ,ul of the treated sample was transferred to a 96-well microtiter plate (Becton-Dickinson, Oxnard, Calif.) and serially diluted (1:2) in PBS (pH 6). A 100-,ul portion of 2% sheep erythrocytes (Remel, Inc., Lenexa, Kans.) was added to each dilution. The plates were incubated at 370C for 2 h and then incubated at 4°C overnight. The hemolytic activity titer was expressed in complete hemolysis units, the reciprocal of the highest dilution at which complete hemolysis occurred, and minimal hemolysis units, the reciprocal of the highest dilution at which hemolysis was observed. Results of preliminary bioassay experiments indicated that pathogenic effects (death of at least three of five mice per experiment, or 60% mortality) were manifested in immunocompromised mice by day 5. The measurement of pathogenicity was defined in terms of whole bacterial cultures rather than as individual cells or an entire population of equivalent cells. The pathogenicity data for nonstressed, heat-stressed, and resuscitated L. monocytogenes cells are presented in Table 1. There was no significant difference (P < 0.05) between the pathogenicity titers of nonstressed versus resuscitated cells. Challenge suspensions containing
