Inactivation and injury of Listeria monocytogenes as affected by heating and freezing

Food Microbiology, 1988, 5, 17-23

Inactivation and injury of Listeria monocytogenes as affected by heating and freezing David A. Golden, Larry R. Beuchat and Robed E. Brackett Department of Food Science and Technology, The University of Georgia, Agricultural Experiment Station, Griffin, Georgia 30223-1797, USA Received 20 October 1987 The effects of heating and freezing Listeria monocytogenes in tryptose phosphate broth on inactivation and injury were investigated. Four strains (Scott A, Brie-1, LCDC 81-861, and DA-3) were examined. Only slight decreases in the viable populations of all four strains were detected after 75 min at 50°C. Populations of strains Scott A, LCDC 81-861, and DA-3 were reduced to non-detectable levels after 10 min at 60°C while viable cells of strain Brie-1 were recovered after heating at 60°C for 30 min. Viable populations of aU four strains declined steadily at 52, 54, and 56°C; similar inactivation rates were observed for strains Scott A, LCDC 81-861, and DA-3. Strain Brie-1 was consistently the most heat resistant. Substantial populations of viable cells of all strains heated at 52, 54, and 56°C were injured as evidenced by increased sensitivity to NaCl when plated on tryptose phosphate recovery agar. Strain DA-3 was the most susceptible strain to sublethal heat injury. Viable populations of all four test strains were not appreciably reduced after 14 days at -18°C, although strain LCDC 81-861 lost viability at a more rapid rate at this temperature than did the other test strains. About 72, 82, 73, and 80% of Scott A, Brie-1, LCDC 81-861, and DA-3 cells, respectively, held at -18°C for 14 days were not recovered on tryptose phosphate agar (TPA) supplemented with 8% NaCl compared to populations recovered on TPA not supplemented with NaCl.

Introduction During the summer of 1983, 49 people in Massachusetts acquired listeriosis. Investigations conducted by the Centers for Disease Control, Atlanta, GA revealed that the illness was associated with the consumption of whole or 2% pasteurized milk (Fleming et al. 1985). Inspection of the plant where the milk was processed revealed no evidence of faulty pasteurization and no source of post-pasteurization contamination. As a consequence of this outbreak, questions were raised regarding the ability of Listeria monocytogenes to survive the standard pasteurization process (62-8°C, 0740-0020/88/010017 + 07 $03.00/0

30 min) applied to whole milk in the United States. Several studies have been conducted to determine the heat resistance of L. monocytogenes, although results are conflicting. Bearns and Girard (1958), using the holding technique of pasteurization (61-7°C, 35 min), demonstrated that L. monocytogenes could survive when the initial population exceeded 5 x 103 cells m1-1. Garayzabal et al. (1986) isolated L. monocytogenes from c. 21% of pasteurized (78°C, 15 s) milk samples obtained from a processing plant in Spain. Other studies (Beckers et al. 1987, Bradshaw et al. 1985, Donnelly © 1988 Academic Press Limited

18

D . A . Golden et al.

et al. 1987) h a v e s h o w n t h a t L. monocytogenes can not s u r v i v e s t a n d a r d pasteurization. M o r e r e c e n t i n v e s t i g a t i o n s r e p o r t e d b y Doyle et al. (1987) indicate t h a t s o m e L. monocytogenes cells, w h e n h a r b o r e d w i t h i n p o l y m o r p h o n u c l e a r leukocytes, c a n s u r v i v e h i g h t e m p e r a t u r e , s h o r t - t i m e (71.7°C, 15 s) p a s t e u r i z a t i o n . B u n t i n g et al. (1986, 1988) r e p o r t e d e s t i m a t e d D71.7°c v a l u e s for L. monocytogenes in whole m i l k to r a n g e f r o m 1.6 to 4.1 s. I t h a s also b e e n d e m o n s t r a t e d t h a t L. monocytogenes c a n s u r v i v e t h e r m a l processing t r e a t m e n t s applied to non-fat d r y fnilk (Doyle et al. 1985), cottage cheese (Ryser et al. 1985) a n d m e a t b a l l s ( K a r a i o a n n o g l o u a n d Xenos 1980). S e v e r a l s t u d i e s on t h e r m a l r e s i s t a n c e c h a r a c t e r i s t i c s o f L . monocytogenes h a v e b e e n d e s i g n e d to c o m p a r e s t r a i n s t h a t w e r e isolated f r o m m i l k or h u m a n s , w i t h s t r a i n Scott A e m e r g i n g as t h e m o s t frequently tested strain. However, studies c o m p a r i n g t h e h e a t r e s i s t a n c e of s t r a i n s isolated f r o m a r a n g e of n a t u r a l h a b i t a t s a r e limited. V e r y little i n f o r m a tion is a v a i l a b l e d e s c r i b i n g t h e susceptibility Of L. monocytogenes to d e a t h a n d injury a t freezing t e m p e r a t u r e s . T h e p u r p o s e of this i n v e s t i g a t i o n w a s to d e t e r m i n e t h e effects of h e a t i n g a n d freezing on i n a c t i v a t i o n a n d developm e n t of i n j u r y of four s t r a i n s of L. monocytogenes isolated f r o m a n infected h u m a n , cheese, a n d cabbage.

Materials and Methods Strains tested, maintenance of cultures, and preparation of inoculum Four strains of L. monocytogenes (Scott A, a human isolate from the 1983 Massachusetts outbreak involving milk; Brie-1, isolated from Brie cheese; LCDC 81-861, from cabbage implicated in a Canadian outbreak; and DA-3, an isolate from a woman in Dallas, Texas were evaluated. All four strains were serotype 4b. Strains Scott A, Brie-1, and DA-3 were obtained from Dr Joseph Lovett at the United States Food and Drug Adminis-

tration, Cincinnati, Ohio. Strain LCDC 81861 was obtained from Dr Jeffrey Farber at the Sir Frederick G. Banting Research Centre, Health and Welfare Canada, Ottawa, Ontario. Stock cultures were maintained at 5°C on tryptose phosphate agar [TPA, pH 7.3; which contained, per litre of deionized water: 20.0 g of tryptose (Difco), 2.0 g of dextrose, 5.0 g of NaC1, 2.5 g of Na2HPO4, and 15.0 g of agar]. Early stationary phase cultures grown in tryptose phosphate broth (TPB, pH 7.3; which contained the same ingredients as TPA, minus agar) at 30°C for 24 h served as inocula for heat- and freeze-treatment studies.

Procedures for stressing cells (i) Heating. Each test strain was subjected to heating temperatures of 50, 52, 54, 56, 58, and 60°C under constant agitation in a waterbath shaker. One millilitre of a 24-h culture was inoculated into 100 ml of TPB (pH 7.1) in 250-ml Erlenmeyer flasks that had been adjusted at the desired heating temperature for c. 30 min in the waterbath shaker. Heating times for 50-54°C and 5660°C were 0, 15, 30, 45, 60, and 75 min, and 0, 10, 20, 30, 40, and 50 min, respectively. The water level in the bath was adjusted so that the contents of the flasks were completely submerged throughout the heat treatment. Samples were withdrawn after appropriate heating times, serially diluted in sterile 0.1 M potassium phosphate buffer (pH 7.0), and immediately surface plated (0-1 ml) in duplicate on TPA supplemented with 0, 2, or 4% NaC1. Colonies were counted after 72 h of incubation at 30°C. The susceptibility of each strain to injury upon sublethal heat treatment was determined based on the organism's increased sensitivity to 2 or 4% NaC1 compared to TPA recovery media not supplemented with NaC1. Decimal reduction times (D values) were calculated from the linear portions of thermal inactivation curves of test strains heated at 52, 54, and 56°C. Two replicate trials were conducted.

(ii) Freezing. One millilitre of a 24-h culture of each strain was inoculated into 25 × 150-mm test tubes containing 9 ml of TPB (pH 7.3). After thorough mixing, serial dilutions (0.1 M potassium phosphate buffer) of the cell suspensions were surface plated (0.1 ml) in duplicate on TPA supplemented with 0, 2, 4, 6, 7 or 8% NaC1. Tubes were stored at -18°C for 7 or 14 days. At the end of each

Inactivation and injury of L. monocytogenes

!

storage period, tubes were removed from the freezer, the contents were thawed (30-40 s) under cool running tap water, and serial dilutions were made in buffer and surface plated (0.1 ml) on TPA supplemented with 0, 2, 4, 6, 7, or 8% NaC1. All plates were incubated for 72 h at 30°C before colonies were counted. As in studies designed to determine the effects of sublethal heat treatment on cells, injury was demonstrated by increased sensitivity to NaC1 after freezing.

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Results

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g 3 The average standard error of m e a n loglo values was 0.053. (i) Heating. Decreases in viable populations of test strains were generally less t han 90% after 75 min at 50°C. After heating for 10 min at 58°C, viable populations of strains Scott A, LCDC 81-861, and DA-3 were reduced from 1.6 x 107, 5.5 x 106, and 7.5 × 10 s to 4.0 x 104, 4.5 x 103, and 1.4 x 104 cells m1-1, respectively. After h e a t i n g the same t hr ee strains for 30 min at 58°C, no viable cells were detected. The Brie-1 strain exhibited the g r eate s t resistance to t h e r m a l stress; after h e a t i n g for 40 min at 58°C, 3.6 x 104 cells ml -~ were recovered on TPA (1.1 x 107 cells ml - I initially). After heating at 60°C for 10 min, no viable cells of strains Scott A, LCDC 81-861, or DA-3 were detected. However, cells of the Brie-1 strain were still detected after h eatin g for 30 min at 60°C; the viable population was reduced from 1.8 x 107 to 1-0 x 102 cells m1-1 At 52, 54, and 56°C, the rates of t h e r m a l inactivation of all four strains were much more gradual t h a n inactivation rates at 58 and 60°C. Figure 1 illustrates t h e r m a l inactivation curves of L. monocytogenes Scott A. At 52°C, a steady decline in viable population was observed. The decline in viable population was much more rapid at 54 and 56°C. The viable population of Scott A was reduced by about 2 logs during a 75-min period at 52°C whereas reduction

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Fig. 1. Recovery ofL. monocytogenes Scott A heated at 52, 54, and 56°C in TPB (pH 7-1) and plated on TPA supplemented with 0 (O), 2 (A), and 4% NaC1 ([3). at 54°C (75 min) was about 4.5 logs. The viable population after 50 min at 56°C was reduced to non-detectable levels. After heat i ng for 15 min at all t hree temperatures, significant injury, manifested as increased sensitivity to 4% NaC1 in TPA recovery medium, was observed in the surviving population. The degree of injury, i.e. the m a g n i t u d e of increased sensitivity to NaC1, generally increased with increasing temperature of t r e a t m e n t . The rat e of decline in viable population of strain LCDC 81-861 (Fig. 2) at 52°C was not as rapid as t h a t observed for strain Scott A, although the t h e r m a l inactivation curves for both strains at 54 and 56°C were quite similar. Cells of strain LCDC 81-861 also u n d e r w e n t sub-

D . A . Golden et al.

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81-861 heated at 52, 54, and 56°C in TPB (pH 7-1) and plated on TPA supplemented with 0 (O), 2 (A), and 4% NaC1 ([3).

Fig. 3. Recovery of L. monocytogenes DA-3 heated at 52, 54, and 56°C in TPB (pH 7-1) and plated on TPA supplemented with 0 (©), 2 (A), and 4% NaC1 ([D).

lethal injury at all three t r e a t m e n t temperatures. Thermal inactivation curves for L. monocytogenes DA-3 are illustrated in Fig. 3. The susceptibility of the DA-3 strain to heating at 52 and 54°C was similar to t h a t of strains Scott A and LCDC 81-861. However, no viable cells of strain DA-3 were detected after 40 min at 56°C. Although the viable cell populations of strains Scott A, LCDC 81-861, and DA-3 were similar at 52 and 54°C, DA-3 was apparently the most extensively injured by t r e a t m e n t at these temperatures, as evidenced by the degree of sensitivity to 4% NaC1. The development of injury of strain DA-3 was most pronounced at 54°C where, after

heating for 30 min, about 98% of the surviving population was injured. The rate of decline in viable population of strain Brie-1 heated at 52, 54, and 56°C (Fig. 4) was steady but not as great as t h a t for the other test strains. During a 75-min period at 52°C, only about a 1 log reduction in viable population was observed. After heating for 50 min at 56°C, about a 3 log reduction in initial population was observed. In addition, injury at all three temperatures was not as extensive as t h a t observed for strains Scott A, LCDC 81-861, and DA-3. Decimal reduction times (D values) for test strains heated at 52, 54, and 56°C are listed in Table 1. The D52oc value for strain LCDC 81-861 was somewhat higher t h a n t h a t for the Scott A strain,

Inactivation and injury of L. monocytogenes 21

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values for the test strains differed substantially. The Brie-1 strain consistently had the highest D values at 52, 54, and 56°C. (ii) Freezing. The effects of storage at -18°C on the development of injury of L. monocytogenes cells are illustrated in Fig. 5. No appreciable inhibition of colony formation of L. monocytogenes was observed when control cells (no freeze treatment) were plated on TPA supplemented with up to 8% NaC1. Generally, as the storage time increased from 0 to 14 days, a small but steady decrease in viable populations (as determined by plating cells on TPA not supplemented with NaC1) was apparent. Inactivation rates of strains Scott A, Brie-1 and DA-3 were similar. The viable populations of these three strains, as determined by recovering on TPA not supplemented with NaC1, decreased by about 0.1 and

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Fig. 4. Recovery of L. monocytogenes Brie-1 heated at 52, 54, and 56°C in TPB (pH 7.1) and ( (A), plated and O 4% on TPA NaCI ) supplemented (y]). , 2 with 0

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i~ut their D values at 54 and 56°C were similar. The D52oc values for strains DA-3 and Brie-1 were much higher than D52oc values for strains Scott A and LCDC 81-861, although strain DA-3 had the lowest D value of the test strains at 56°C. These observations indicate that z

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Fig. 5. Recovery ofL. monocytogenes Scott A, Brie-1, LCDC 81-861, and DA-3 after freezing at -18°C for 0 (open bar), 7 (diagonal bar), and 14 days (cross-hatched bar) and plated on TPA supplemented with up to 8% NaC1.

22

D.A. Golden et al.

0-2 logs after 7 and 14 days, respectively, while the viable population of the LCDC 81-861 strain decreased by about 0-4 and 0.5 logs during the same periods. All four strains became more sensitive to increased levels of NaC1 in TPA recovery medium after 7 days of storage at -18°C. Appreciable differences in recovery were generally observed between populations recovered on TPA supplemented with 0 and 6% NaC1. More substantial injury was typically demonstrated when TPA supplemented with 8% NaC1 was used as the recovery medium. Freeze treatment for 14 days generally had minimal effect on additional development of injury compared to treatment for 7 days. After 7 days at -18°C about 71, 76, 63, and 62% of the surviving populations of strains Scott A, Brie-1, LCDC 81-861, and DA-3, respectively, were injured (based on sensitivity to 8% NaC1), whereas about 72, 82, 73, and 80% of the respective populations were injured after 14 days. Discussion Results of this investigation indicate that L. monocytogenes is somewhat resistant to inactivation when heated at or below 54°C but rapidly becomes sensitive as the temperature increases. Beuchat et al. (1986) made similar observations on resistance of strains Scott A and LCDC 81-861 to heat inactivation in clarified cabbage juice. Based on results of investigations reported here, it is unlikely that L. monocytogenes, freely suspended in milk, would survive standard pasteurization treatment. However, the heat resistance ofL. monocytogenes may vary depending on the heating menstruum; the susceptibility of bacterial cells to thermal inactivation is known to be influenced by specific components within the heating medium (Moats et al. 1971). Bradshaw et al. (1987) observed this phenomenon with L. monocytogenes

Scott A, noting substantial differences in D values when the organism was heated in skim milk, whole milk, and cream. Interestingly, these researchers also observed that resistance of L. monocytogenes to inactivation appeared to be greater when the organism was heated in the same products which had been previously sterilized. Our results also demonstrate that some strains ofL. monocytogenes exhibit a greater resistance to thermal inactivation than others. In this study, strains Scott A and LCDC 81-861 were quite similar in their resistance to thermal inactivation; the DA-3 strain was slightly more resistant while the Brie-1 strain exhibited the greatest heat resistance. The observation that Brie-1 is appreciably more heat resistant than the other three strains emphasizes the need to test multiple strains for their ability to withstand heat treatment in order to assess thermal stability characteristics ofL. monocytogenes in food products. It is worth noting that the environment from which strain Brie-1 was isolated (Brie cheese) would not normally be expected to select for heat tolerance. Substantial proportions of surviving populations of all four strains heated at 52, 54, and 56°C consisted of injured cells. Thus, media and methods used to detect and enumerate L. monocytogenes in foods previously subjected to thermal treatment should be formulated and designed to resuscitate heat-injured cells. Currently used procedures do not address this need and, in fact, many methods currently in use do not include procedures that promote recovery of injured food-borne micro-organisms in general (Jay 1986). The literature reveals little information concerning the ability of L. monocytogenes to withstand freezing temperatures. Data obtained from this investigation indicate that the organism

Inactivation and injury of L. monocytogenes 23 is quite resistant to inactivation over a 14-day storage period at -18°C. The viable populations of the four strains tested were decreased by only about 3-6%, although up to 82% of the viable population was injured. Further research is needed to det er m i ne the ability of L. monocytogenes to survive freezing t e m p e r a t u r e s and resist sublethal freeze-injury in foods. In addition, the ability of various selective media to recover freeze-injured L. monocytogenes needs to be investigated.

Acknowledgements The authors are grateful to Brenda V. Nail and K a t r i n a D. Benton for technical assistance. This investigation was supported, in part, by contract n u m b e r 223-84-2031 from the United States Food and Drug Administration, Washington, D.C., t hrough Booz, Allen and Hamilton, Inc., Bethesda, Maryland.

References Bearns, R. E. and Girard, K. F. (1958) The effect of pasteurization on Listeria monocytogenes. Can. J. Microbiol. 4, 55-61. Beckers, H. J., Soentoro, P. S. S. and Delfgou-van Asch, E. H. M. (1987) The occurrence of Listeria monocytogenes in soft cheeses and raw milk and its resistance to heat. Int. J. Food Microbiol. 4, 249-256. Beuchat, L. R., Brackett, R. E., Hao, D. Y.-Y. and Conner, D. E. (1986) Growth and thermal inactivation ofListeria monocytogenes in cabbage and cabbage juice. Can. J. Microbiol. 32, 791-795. Bradshaw, J. G., Peeler, J. T., Corwin, J. J., Hunt, J. M., Tierney, J. T., Larkin, E. P. and Twedt, R. M. (1985) Thermal resistance ofListeria monocytogenes in milk. J. FoodProt. 48, 743-745. Bradshaw, J. G., Peeler, J. T., Corwin, J. J., Hunt, J. M. and Twedt, R. M. (1987) Thermal resistance of Listeria monocytogenes in dairy products. J. Food Prot. 50, 543-544. Bunting, V. K., Crawford, R. G., Bradshaw, J. G., Peeler, J. T., Tierney, J. T. and Twedt, R. M. (1986) Thermal resistance of intracellular Listeria monocytogenes cells suspended in raw bovine milk. Appl. Environ. Microbiol. 52, 1398-1402. Bunting, V. K., Donnelly, C. W., Peeler, J. T., Briggs, E. H., Bradshaw, J. G., Crawford, R. G., Beliveau, C. M. and Tierney, J. T. (1988) Thermal inactivation of Listeria monocytogenes within bovine milk phagocytes. Appl. Environ. Microbiol. 54, 364-370. Donnelly, C. W., Briggs, E. H. and Donnelly, L. S. (1987) Comparison of heat resistance of Listeria monocytogenes in milk as determined by two methods. J. Food Prot. 50, 14-17. Doyle, M. P., Glass, K. A., Beery, J. T., Garcia, G. A., Pollard, D. J. and Schultz, R. D. (1987) Survival of Listeria monocytogenes in milk during high-temperature, short-time pasteurization. Appl. Environ. Microbiol. 53, 1433-1438. Doyle, M. P., Meske, L. M. and Marth, E. H. (1985) Survival ofListeria monocytogenes during manufacture and storage of nonfat dry milk. J. Food Prot. 48, 740-742. Fleming, D. W., Cochi, S. L., MacDonald, K. A., Brondum, J., Hayes, P. S., Plikaytis, B. D., Holmes, M. B., Audurier, A., Broome, C. V. and Reinhold, A. L. (1985) Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. N e w Engl. J. Med. 312, 404 407. Garayzabal, J. F. F., Rodriguez, L. D., Boland, J. A. V., Cancelo, J. L. B. and Fernandez, G. S. (1986) Listeria monocytogenes dans le lait pasteuriz6. Can. J. Microbiol. 32, 149-150. Jay, J. M. (1986) Determining microorganisms and their products in foods. In Modern Food Microbiology, Third edn. pp. 97-127. New York, Van Nostrand Reinhold Company. Karaioannoglou, P. G. and Xenos, G. C. (1980) Survival of Listeria monocytogenes in meatballs. Hell. Vet. Med. 23, 111-117. Moats, W. A., Dabbah, R. and Edwards, V. M. (1971) Survival of Salmonella anatum heated in various media. Appl. Microbiol. 21,476-481. Ryser, E. T., Marth, E. H. and Doyle, M. P. (1985) Survival ofListeria monocytogenes during the manufacture and storage of cottage cheese. J. Food Prot. 48, 746-751.