Synergy between Nisin and Select Lactates against Listeria monocytogenes Is Due to the Metal Cations

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1 1631 Journal of Food Protection, Vol. 66, No. 9, 2003, Pages Copyright q, International Association for Food Protection Synergy between Nisin and Select Lactates against Listeria monocytogenes Is Due to the Metal Cations JENNIFER CLEVELAND MCENTIRE, THOMAS J. MONTVILLE, AND MICHAEL L. CHIKINDAS* Department of Food Science, New Jersey Agricultural Experiment Station, Rutgers, State University of New Jersey, New Brunswick, New Jersey 08901, USA MS : Received 13 December 2002/Accepted 21 March 2003 ABSTRACT Listeria monocytogenes, a major foodborne pathogen, has been responsible for many outbreaks and recalls. Organic acids and antimicrobial peptides (bacteriocins) such as nisin are produced by lactic acid bacteria and are commercially used to control pathogens in some foods. This study examined the effects of lactic acid (LA) and its salts in combination with a commercial nisin preparation on the growth of L. monocytogenes Scott A and its nisin-resistant mutant. Because of an increase in its activity at a lower ph, nisin was more active against L. monocytogenes when used in combination with LA. Most of the salts of LA, including potassium lactate, at up to 5% partially inhibited the growth of L. monocytogenes and had no synergy with nisin. Zinc and aluminum lactate, as well as zinc and aluminum chloride (0.1%), worked synergistically with 100 IU of nisin per ml to control the growth of L. monocytogenes Scott A. No synergy was observed when zinc or aluminum lactate was used with nisin against nisin-resistant L. monocytogenes. The nisin-resistant strain was more sensitive to Zn lactate than was wild-type L. monocytogenes Scott A; however, the cellular ATP levels of the nisin-resistant strain were not signi - cantly affected. Changes in the intracellular ATP levels of the wild-type strain support our hypothesis that pretreatment with zinc lactate sensitizes cells to nisin. The similar effects of the salts of hydrochloric and lactic acids support the hypothesis that metal cations are responsible for synergy with nisin. Because approximately 76,000,000 cases of foodborne disease occur each year (10) and because of the increased resistance of pathogens to current commercial methods of food preservation, the food industry is constantly challenged to develop and introduce novel preservative combinations that are cost-effective and work synergistically against food pathogens. Recent disease outbreaks (2, 15) and recalls (16) attributable to Listeria monocytogenes have prompted the food industry, the public, and government authorities to question current methods of food preservation. The increased demand for natural and minimally processed food has led to a growing interest in naturally produced antimicrobial agents, including bacteriocins and organic acids. Bacteriocins are ribosomally synthesized peptides that kill closely related bacteria (8). One such bacteriocin, nisin, inhibits sensitive cells by forming pores in the membrane, which results in the disruption of the proton motive force and leakage of cellular materials. Our laboratory has isolated a mutant L. monocytogenes strain that is resistant to nisin (4). This mutant strain, NR 30, has an altered cell membrane that is more rigid than that of the parental strain, L. monocytogenes Scott A, which enables NR 30 cells to resist the formation of pores by nisin. Lactic acid (LA) is a commonly used preservative that exerts its antimicrobial effect primarily by decreasing the ph of a food system. The undissociated (protonated) form * Author for correspondence. Tel: , Ext 218; Fax: ; tchikindas@aesop.rutgers.edu. of the acid crosses the cell membrane and dissociates in the more alkaline cytoplasm. This development causes a decrease in intracellular ph, which disrupts critical cell functions, causing cell death (3). LA salt derivatives such as sodium and potassium lactate are approved for use as avoring agents at up to 2% in meat and poultry products. Because of their preservative effect, they are used at up to 4.8% in some refrigerated ready-to-eat cooked meats (1). In early work, the antimicrobial activity of sodium lactate was attributed to its ability to lower water activity (9). However, others have suggested that lactates act as undissociated acids, exerting their effect by crossing the cell membrane as undissociated species (11). This hypothesis is supported by the fact that lactates are generally active in acidic conditions, under which the amount of undissociated acid would be higher. Hurdle technology combines different preservation methods to inhibit microbial growth. Nisin has been used in combination with LA or its salt derivatives in hurdle technology (11, 13). Ideally, components of hurdle technology exhibit synergy, meaning that the level of inhibition achieved by the combination of the components is greater than the sum of the levels of inhibition achieved by each alone. When the overall level of inhibition is the sum of the individual levels of inhibition, the result is additive. An understanding of the modes of action of individual components in hurdle technology enables us to understand how synergy works mechanistically. Our laboratory has studied the inhibition of gram-positive organisms by nisin. Differences in the mechanisms of

2 1632 MCENTIRE ET AL. the antimicrobial action of nisin and LA prompted us to study the combined effect of these preservatives against L. monocytogenes. We hypothesized that antimicrobial agents with different modes of action are likely to work synergistically to effectively inhibit L. monocytogenes. A nisin-resistant mutant was used to determine whether the presence of lactates sensitized cells to nisin. The differences between the effects of the salts of LA were examined and shown to result from a speci c cation effect, not from an effect of the lactate anion. MATERIALS AND METHODS Bacterial strains and growth conditions. L. monocytogenes Scott A was grown overnight at 378C with agitation in tryptic soy broth (ph 6.5; Difco, Sparks, Md.) supplemented with 0.6% yeast extract and 0.5% glucose (TSB-YEG). Nisin-resistant L. monocytogenes NR 30 (ATCC ), a spontaneous mutant of Scott A, was grown in TSB-YEG with 200 IU of nisin (Novasin, Rhodia, Madison, Wis.) per ml. Cells were diluted 1/100 in fresh broth before they were used in the 96-well assay. Nisin preparation. Nisin (Novasin, Rhodia) was autoclaved in nisin diluent (0.02 N HCl and 0.75% NaCl solution [ph 3.0] (5)) to a stock concentration of IU/ml, and this mixture was stored at 48C until it was used. Dilutions were prepared with nisin diluent immediately before they were used. LA and salts. Commercial preparationsof LA (88%, wt/vol), potassium and sodium lactates (60%, wt/vol), and magnesium, calcium, zinc, and aluminum lactates were supplied by Purac (Lincolnshire, Ill.) and dissolved in sterile water to produce solutions with nal concentrations of 1 to 10%, except for LA, which was used as a control to acidify media. Aluminum chloride and zinc chloride (Fisher Scienti c, Fair Lawn, N.J.) were dissolved in sterile water to a concentration of 1%. Growth assay. A temperature-controlled Dynex 96-well microplate reader with Revelation software was used to monitor absorbance at 630 nm. LA or its salt derivative was added to fresh broth, and the ph of the broth was adjusted to the desired value. The diluted overnight culture was added at a nal concentration of 10 5 to 10 6 CFU/ml. Nisin was added to produce a nal concentration of up to 1,000 IU/ml. The microplate was incubated at 378C for 26 h, with A 630 readings being taken every half hour immediately after 5 s of shaking. Replicate or triplicate wells were used for each treatment, and each experiment was repeated at least twice. ATP assay. Total and extracellular levels of ATP were determined with the ATP Bioluminescence assay kit (Sigma Chemical Co., St. Louis, Mo.). A standard curve was generated by adding 100 ml of the standard ( to mol of ATP per ml) to a cuvette containing 100 ml of a 25-fold diluted enzyme mix. To assess total and extracellular ATP levels, cells were washed in 50 mm 2-[N-Morpholino]ethanesulfonic acid (ph 6.5; Sigma) and resuspended to an A 600 value of 0.6. The washed cells were resuspended in 50 mm 2-[N-Morpholino]ethanesulfonic acid (ph 6.5) supplemented with 10 mm MgSO 4 in half the volume and incubated with 0.2% glucose for 20 min at 30 8C and were then stored on ice until they were used. Cells (0.9 ml) were added to Eppendorf tubes alone (control) or with a treatment solution. For a comparison of acids, 40 mmol of lactic or hydrochloric acid were added. In another set of experiments, 100 ml of concentrated nisin or Zn lactate was added to yield concentrations of 60 IU of nisin per ml and 0.1% Zn lactate, respectively. Nisin diluent was used as the control for nisin, and sterile water was used as the control for acids and Zn lactate. Readings for the control and for cells treated with one preservative were taken immediately after the addition of the preservative (time 0) and after 3, 10, and 20 min. Time 21 indicates the cellular ATP levels before the addition of the preservative. Pretreated cells were incubated with the rst preservative for 7 min before the second preservative was added. ATP levels were determined at the same time intervals mentioned above, and an additional reading was taken immediately following the second treatment. The total ATP level was determined by adding 20 ml of treated cells to 80 ml of dimethylsulfoxide, adding 5 ml of sterile water, and using 100 ml of the mixture in the assay with 100 ml of enzyme mix. For the determination of extracellular ATP levels, 100 ml of treated cells was added to a test tube. Five milliliters of sterile water was added, and a 100-ml sample was added to 100 ml of enzyme mix. For standardization between experiments, cell dry weight was determined by drying 1 ml of cell suspension in 0.1% NaCl in a preweighed aluminum dish overnight at 458C. Statistics. All experiments were conducted at least twice. Signi cant differences (P, 0.05) in ATP levels were determined by Student s t test (Sigma Plot 7). For each preservativetreatment, absorbance values were combined and were tted to the fourparameter Gompertz equation (equation 1) with the use of Sigma Plot 7. The coef cients were used to calculate the maximum growth rate (equation 2) and time to detection (equation 3). [2B(t2M )] 2e N 5 A 1 Ce (1) (t) B 3 C maximum growth rate 5 (2) e 1 time to detection 5 M 2 (3) B where N (t) is the absorbance at time t, A is the absorbance at time 0, C is the difference between absorbance at inoculation and absorbance at the stationary phase, B is the maximum relative growth rate, t is time (h), and M is the time at which the maximum exponential growth rate is attained. RESULTS The inhibition of L. monocytogenes Scott A by nisin when LA was used to acidify the broth to ph 6.1 was similar to that observed when the broth was acidi ed with HCl (Fig. 1). In the absence of nisin, the bacteriocin-resistant L. monocytogenes NR 30 was more sensitive to LA than wild-type L. monocytogenes Scott A was (Fig. 2). Strain NR 30 was also more sensitive to HCl, although at equivalent ph values, LA was more inhibitory than HCl (data not shown). For both organisms, the difference between the level of inhibition achieved with LA and that achieved with HCl was more apparent at a lower ph. HiPure 60 potassium lactate (3%) caused a slight increase in the lag time for L. monocytogenes Scott A (Table 1). In the presence of nisin at 250 IU/ml, potassium lactate treated cells had a lower maximum growth rate and a longer lag time than did cells subjected to only one treatment. This effect was not synergistic, although the calculated time to detection for the combination was longer than the sum of the times for the individual treatments. Time to detection is dependent on the initial inoculum, so the use of an inoculum with a higher concentration results in a shorter de-

3 NISIN AND CATIONS INHIBIT L. MONOCYTOGENES 1633 TABLE 1. Time to detection and maximum growth rate of L. monocytogenes as calculated from Gompertz coef cients (see Materials and Methods for calculations) L. monocytogenes strain Treatment Time to detection (h) Maximum growth rate (A 630 /h) FIGURE 1. Inhibition of L. monocytogenes Scott A by nisin at 100 IU/ml (open symbols) in TSB-YEG acidi ed with HCl ( l ) or with lactic acid ( ). Closed symbols indicate control cells. In all cases, the broth was acidi ed to ph 6.12 with the appropriate acid. tection time. The response of L. monocytogenes to potassium lactate was similar to its responses to calcium and magnesium lactates (Table 1). The levels of inhibitory activity for these salts was low compared with those for equivalent concentrations of LA, and the addition of the salts did not decrease the ph of the medium like LA did. Aluminum and zinc lactates decreased the ph of the broth and were effective against L. monocytogenes Scott A at concentrations of 0.1% (ph ;6.0). Alone, 0.05% Al lactate had a weak effect on the growth of L. monocytogenes Scott A. However, nisin activity increased in the presence of Al lactate, resulting in a greater increase in lag time than would be expected on the basis of the sum of the individual effects. Complete inhibition of L. monocytogenes Scott A was not achieved unless higher concentrations of Al lactate or nisin were used (data not shown). Under these conditions, synergy was more dramatic. The nisin-resistant mutant L. monocytogenes NR 30 was slightly more sensitive to 0.05% Al lactate than L. monocytogenes Scott A was Scott A Nisin (250 IU/ml) 3% K lactate 3% K lactate 1 nisin (250 IU/ml) Nisin (250 IU/ml) 1% Mg lactate 1% Mg lactate 1 nisin (250 IU/ml) 1% Ca lactate 1% Ca lactate 1 nisin (250 IU/ml) Nisin (100 IU/ml) 0.05% Al lactate 0.05% Al lactate 1 nisin (100 IU/ ml) NR 30 Nisin (250 IU/ml) 0.1% Al lactate 0.1% Al lactate 1 nisin (250 IU/ml) (Table 1). However, Al lactate in combination with 250 IU of nisin per ml was no more effective than the lactate alone. As a control, the effects of Al chloride and Zn chloride (with and without nisin) on the growth of L. monocytogenes Scott A were determined. Zn lactate and Zn chloride had similar effects on cell growth (Figs. 3A and 3B), as did Al lactate and Al chloride (data not shown). Synergy with nisin was observed in all cases. The concentration of Zn lactate (0.1%) required to produce synergy with nisin against L. monocytogenes Scott A was higher than the concentra- FIGURE 2. Effect of ph on L. monocytogenes Scott A (closed symbols) and L. monocytogenes NR 30 (open symbols). Lactic acid was used to acidify brain heart infusion broth to ph 5.5 ( l ), ph 5.0 ( m ), or ph 4.8 ( ). Results reported represent averages for three wells per experiment and at least two independent experiments.

4 1634 MCENTIRE ET AL. FIGURE 3. (A) L. monocytogenes Scott A control ( l ) and L. monocytogenes Scott A grown with 0.1% Zn chloride ( ), 100 IU of Novasin per ml ( m ), and a combination of Novasin and 0.1% Zn chloride ( v ). (B) L. monocytogenes Scott A control ( l ) and L. monocytogenes Scott A grown with 0.1% Zn lactate ( ), 100 IU of Novasin per ml ( m ), and a combination of Novasin and 0.1% Zn lactate ( v ). (C) L. monocytogenes Scott A control ( l ) and L. monocytogenes Scott A grown with 0.025% Zn lactate (a lower concentration of Zn lactate was used in this experiment to achieve partial inhibition of growth; strain NR 30 is completely inhibited by 0.1% Zn lactate) ( ), 100 IU of Novasin per ml ( m ), and a combination of Novasin and 0.025% Zn lactate ( v ). Results reported represent averages for three wells per experiment and at least two independent experiments. tion of Al lactate required to produce synergy. Nisin-resistant L. monocytogenes NR 30 was not sensitized to nisin by Zn lactate (Fig. 3C), although lower Zn lactate concentrations were used because of the increased sensitivity of L. monocytogenes NR 30 to Zn lactate. To further study the effect of Zn lactate, nisin, and a combination of the two agents on L. monocytogenes Scott A and L. monocytogenes NR 30, total and extracellular ATP levels were determined. The experiments were performed to better understand the mechanism of synergy between Zn lactate and nisin and to determine why NR 30 is more sensitive to Zn lactate than Scott A is. Although the wild-type and nisin-resistant cells showed different sensitivities to Al lactate, this salt interfered with the ATP assay, preventing the collection of data. For Scott A, there was no signi cant difference between intracellular ATP levels resulting from treatment with nisin, treatment with Zn lactate, and treatment with nisin for 20 min followed by exposure to Zn lactate (Fig. 4). However, the decrease in ATP caused by nisin occurred more rapidly than that caused by Zn lactate. Scott A cells pretreated with Zn lactate showed a rapid decrease in intracellular ATP when nisin was added. Levels of ATP for this cell population were signi cantly lower than those for the control population. NR 30 cells show a similar but not statistically signi cant trend (Fig. 4). The effect of pretreatment with zinc on the intracellular ATP levels of nisin-treated cells was more dramatic and significant for wild-type L. monocytogenes Scott A than for L. monocytogenes NR 30. Since L. monocytogenes NR 30 is more sensitive to acid than L. monocytogenes Scott A is, the ATP assay was also used to study the effects of HCl and LA on cellular ATP levels. L. monocytogenes NR 30 exhibited a signi - cant, rapid decrease in intracellular ATP when acid was added (Fig. 5). This effect was more pronounced for LA than for HCl. The addition of HCl to Scott A cells had very little effect on ATP levels. The decrease in intracellular ATP caused by LA was not statistically signi cant. DISCUSSION Nisin activity is inversely proportional to ph. The use of LA as an acidulant does not result in increased nisin activity compared to HCl at ph 6.1. As the ph decreases, LA is more inhibitory than HCl is, which con rms the results of Vasseur et al. (17). Presumably, at lower phs, more of the acid is in the undissociated form and is able to penetrate the cell membrane. Since no difference between the level of inhibition caused by LA and the level of inhibition caused by HCl at ph 6.1 was observed, inhibition at the higher ph may be solely due to protons, as opposed to undissociated acid. With the exception of Zn and Al lactates, LA salts were ineffective in controlling growth and did not decrease the ph of the medium. In fact, when lactates were added, the broth s ph increased slightly. This development may cause increased microbial spoilage of foods treated with lactates (9). The antimicrobial activity of lactates may be due to the ph of the system in which they are tested. Since the salts completely dissociate, at a low ph the anion may become protonated, resulting in the formation of LA (3). Although other researchers have speculated that organic acids act not only by decreasing the internal phs of cells but also through special effects of the anion (7), our results indicate

5 NISIN AND CATIONS INHIBIT L. MONOCYTOGENES 1635 FIGURE 4. Percentages of change in intracellular ATP levels for L. monocytogenes Scott A (left) and L. monocytogenes NR 30 (right) upon treatment with 60 IU of nisin per ml ( V ) and with 0.1% Zn lactate ( ). After 7 min, the second preservative was added and a reading was taken., nisin-treated cells to which Zn lactate was applied after 7 min; m, 0.1% Zn lactate treated cells to which nisin (60 IU/ml) was applied after 7 min; v, control cells. Results reported represent averages for three experiments. Values with different letters are signi cantly different (P, 0.05). that in the free form the anion is ineffective, presumably because it is unable to enter the cell. Commercial potassium, calcium, and magnesium lactate preparations did not show synergy with nisin. The salts decreased the maximum growth rate of cells, and nisin did not increase the maximum growth rate of cells exposed to any of the salts except potassium lactate (Table 1). Although Phillips (13) states that nisin used in conjunction with sodium lactate is more effective in inhibiting the growth of Arcobacter butzleri than lactate alone is, this effect did not appear to be synergistic. Nykanen et al. (11) demonstrated synergy between 1.8% sodium lactate and nisin at 120 to 180 IU/ml against L. monocytogenes in smoked sh. These authors speculated that the synergy re- FIGURE 5. Changes in intracellular ATP levels for L. monocytogenes Scott A (closed symbols) and L. monocytogenes NR 30 (open symbols) upon addition of HCl (, ) and lactic acid ( m, M ). Circles indicate controls. Results reported represent averages for three experiments. Values with different letters are signi cantly different (P, 0.05). sulted from the chelating effect of lactate. The sh used in their study contained up to 2.1% salt, which increases the activity of sodium lactate (12). Since Zn and Al lactates worked synergistically with nisin but Ca, Mg, and K lactates did not, we hypothesized that the cation was responsible for the synergy. Zn and Al lactates are also effective at much lower concentrations and decrease ph, unlike the other salts. Salts containing aluminum and zinc were found to work synergistically with nisin, regardless of whether they were LA salts or chlorides. We speculate that ionic Zn and Al interact with the membrane, somehow facilitating pore formation by nisin. To test this hypothesis, we used the nisin-resistant mutant L. monocytogenes NR 30. In this strain, nisin resistance results from an altered membrane composition resulting in increased rigidity. In the presence of bivalent cations, L. monocytogenes NR 30 is resistant to nisin; however, in the absence of cations, this strain is sensitive to nisin (4). Since both broth and food systems contain ions, ATP studies were called out in the presence of 10 mm MgSO4. Although cations can interfere with luciferase activity in the ATP assay (Sigma Technical Service), control studies showed that Al decreased the signal and prevented the collection of data but Zn did not. Additional methods of analysis are therefore needed to explain the effects of Al lactate. The increased sensitivity of L. monocytogenes NR 30 to Zn lactate but not to Al lactate was unexpected. Surprisingly, neither intra- nor extracellular ATP levels of NR 30 cells treated with Zn lactate adequately explain the in vivo sensitivity observed (Fig. 4; data for extracellular ATP not shown). This nding indicates that inhibition is due to factors other than simply the depletion of ATP. Treatment with Zn lactate increased the nisin-induced loss of intracellular ATP for both organisms. However, the magnitude of the decrease was not as great for NR 30 cells as it was for Scott A cells. This nding may partially ex-

6 1636 MC ENTIRE ET AL. plain why synergy is observed only for Scott A cells. Pretreatment of cells with Zn lactate followed by the application of nisin causes a more extensive loss of ATP than does pretreatment of cells with nisin followed by the application of Zn lactate. Scott A cells are more affected by the combination than NR 30 cells are. The change in intracellular ATP levels combined with absorbance data supports a model in which ionic zinc interacts with the cell, facilitating the activity of nisin. The results obtained in the present study con rm that LA is more inhibitory than hydrochloric acid at or below ph 5.5 (6, 14). This nding is also supported by the magnitude of the decrease in intracellular ATP levels when acids are added to the cells. Although equal molar concentrations of HCl and LA yielding similar ph values were used, both Scott A and NR 30 cells exhibited more extensive losses of ATP in the presence of LA. L. monocytogenes NR 30 maintains a relatively high level of intracellular ATP compared with the parental strain, L. monocytogenes Scott A. Intracellular ATP levels of Scott A cells were unaffected by HCl, whereas NR 30 cells exhibited a rapid loss of ATP. The drastic decrease in intracellular ATP when acid is added to NR 30 cells helps explain why this nisin-resistant mutant, in comparison with wild-type L. monocytogenes Scott A, is increasingly sensitive to acid in vivo ACKNOWLEDGMENTS 14. The authors thank Rhodia for generously supplying Novasin, and the authors thank Purac for providing lactic acid and lactates. McEntire is supported by a grant from the USDA National Needs Fellowship program. 15. REFERENCES Anonymous Nisin preparation: af rmation of GRAS status as a direct human food ingredient. Fed. Regist. 65: Anonymous Marathon enterprises recalls hotdogs due to possible listeria contamination. Available at: reuters/prof/1999/10/10.28/pb10289b.htm. Cherrington, C. A., M. Hinton, G. C. Mead, and I. Chopra Organic acids: chemistry, antibacterial activity and practical applications. Adv. Microb. Physiol. 32: Crandall, A. D., and T. J. Montville Nisin resistance in Listeria monocytogenes ATCC is a complex phenotype. Appl. Environ. Microbiol. 64: Davies, E. A., H. E. Bevis, R. W. Potter, J. M. Harris, G. C. Williams, and J. Delves-Broughton The effect of ph on the stability of nisin solution during autoclaving. Lett. Appl. Microbiol. 27: Farber, J. M., G. W. Sanders, S. Dun eld, and R. Prescott The effect of various acidulants on the growth of Listeria monocytogenes. Lett. Appl. Microbiol. 9: Ita, P. S., and R. W. Hutkins Intracellular ph and survival of Listeria monocytogenes Scott A in tryptic soy broth containing acetic, lactic, citric and hydrochloric acids. J. Food Prot. 1: Klaenhammer, T. R Genetics of bacteriocins produced by lactic acid bacteria. FEMS Microbiol. Rev. 12: Lamkey, J. W., F. W. Leak, W. B. Tuley, D. D. Johnson, and R. L. West Assessment of sodium lactate addition to fresh pork sausage. J. Food Sci. 56: Mead, P. S., L. Slutsker, V. Dietz et al Food-related illness and death in the United States. Emerg. Infect. Dis. 5: Nykanen, A., K. Weckman, and A. Lapvetelainen Synergistic inhibition of Listeria monocytogenes on cold-smoked rainbow trout by nisin and sodium lactate. Int. J. Food Microbiol. 61: Pelroy, G. A., M. E. Peterson, P. J. Holland, and M. W. Eklund Inhibition of Listeria monocytogenes in cold-process (smoked) salmon by sodium lactate. J. Food Prot. 57: Phillips, C. A The effect of citric acid, lactic acid, sodium citrate and sodium lactate, alone and in combination with nisin, on the growth of Arcobacter butzleri. Lett. Appl. Microbiol. 29: Sorrells, K. M., D. C. Enigl, and J. R. Hat eld Effect of ph, acidulant, time and temperature on the growth and survival of Listeria monocytogenes. J. Food Prot. 52: U.S. Department of Agriculture. 20 November New Jersey rm expands recall of poultry products for possible Listeria contamination. Available at: pr htm. U.S. Department of Agriculture. 9 October Pennsylvania rm expands recall of turkey and chicken products for possible Listeria contamination. Available at: prelease/pr htm. Vasseur, C., L. Baverel, M. Hebraud, and J. Labadie Effect of osmotic, alkaline, acid or thermal stresses on the growth and inhibition of Listeria monocytogenes. J. Appl. Microbiol. 86: