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STRAIN DIFFERENCES OF HEAT ADAPTED Listeria monocytogenes CELLS EXPOSED TO CARVACROL, ALKALI, [H.sub.2][O.sub.2] AND LAURIC ARGINATE (LAE).

INTRODUCTION

Listeria monocytogenes is a foodborne pathogen with ubiquitous nature and capable of surviving at diverse conditions including temperature range of 2-45[degrees]C, pH 3.5-12 and salt up to 10% which makes them highly prevalent in food processing areas (Farber and Peterkin 1991). L. monocytogenes is commonly isolated from meat products, dairy products, delicatessen products and seafood's and fruits and vegetables (Swaminathan and Gerner-Smidt 2007). L. monocytogenes causes listeriosis which is a deadly disease with a high mortality rate of 25-30% (Ramaswamy et al. 2007). Thirteen serotypes of L. monocytogenes are known, of which 1/2a, 1/2b, and 4b are responsible for most listeriosis outbreaks in the United States. Interestingly, L. monocytogenes serotype 1/2a is most often isolated from food but the majority of reported foodborne outbreaks have been caused by serotype 4b (Gandhi and Chikindas 2007).

Heating is one of the general practices carried out to control the growth of microorganisms in the food-processing plants and households. However, it was reported that pathogens might develop enhanced resistance to heat and other environmental stresses after exposure to sublethal heat which was known as heat tolerance response (Doyle et al. 2001; Skandamis et al. 2008). L. monocytogenes subjected to sublethal heat at 45[degrees]C for 60 min were more heat tolerant at 60[degrees]C compared to non-sublethally heated cells. In addition, a high diversity of heat tolerance within strains of L. monocytogenes serotypes were reported (Shen et al. 2014a). L. monocytogenes that undergone sublethal heat stress at 45[degrees]C for 10 min or 48[degrees]C for 60 min had higher resistance to other environmental stresses such as ethanol, chlorine and osmotic stresses. On the otherhand, sublethal heat stressed L. monocytogenes cells were not resistant to acid and QAC treatments (Doyle et al. 2001; Lin and Chou 2004; Lianou et al. 2006).

The use of essential oils (EOs) and lauric arginate (LAE) as antimicrobial agents in food processing industries are attracting much attention because they are considered as GRAS (generally recognized as safe) compounds (Burt 2004; Martin et al. 2009). However, it is likely that L. monocytogenes cells that encountered heat stress can induce cross-resistance to subsequent treatments with essential oils. One study demonstrated that L. monocytogenes heat shocked at 45[degrees]C for 1 h showed increased resistance to 200 ppm carvacrol (Ait-Ouazzou et al. 2013). The high stability of L. monocytogenes heat-stress adaptation after cooling step to 4[degrees]C could mainly result from the absence of active growth in such cold environment. Even though refrigeration temperature normally may delay the growth of L. monocytogenes, it may successfully preserve the acquired heat-stress adaptation resulting from the initial sublethal heat-stress treatment if occurred prior to cold storage (Pagan et al. 1997). However, there is limited data on the cross protection of L. monocytogenes sublethal heat stressed cells towards other environmental stresses at cold storage temperature.

The extensive diversity in nature of L. monocytogenes strains indicates that processing conditions cannot be standardized based on a particular standard strain alone. Hence, it is imperative to understand the characteristics of strain variation in L. monocytogenes. Therefore, in the present study the three L. monocytogenes strains that were categorized from low, medium and high heat tolerant groups were considered to study the survival of sublethal heat stress at 48[degrees]C for 1 h when exposed to carvacrol, [H.sub.2][O.sub.2], NaOH and Lauric arginate (LAE) stresses.

MATERIAL AND METHODS

Bacterial strains and growth conditions: Three L. monocytogenes strains that were categorized from our previous study as low, medium and high heat tolerant namely, ScottA, NRRL B-33157, and F4260 respectively belonged to 4b and 1/2a serotypes were used in this study. The strains was stored in -80[degrees]C in tryptic soy broth containing 0.6% yeast extract (TSBYE, pH 7.2; BD Bio sciences, San Jose, CA) supplemented with 16% glycerol. Working stock culture of this strain was maintained at 4[degrees]C in TSBYE. Ten ml of TSBYE was inoculated with a single colony of L. monocytogenes from the working stock culture, and incubated overnight in a shaker (C24 Classic series incubator shaker, New Brunswick Scientific, Inc., Edison, NJ, USA) at 37[degrees]C to reach stationary phase.

Induction of heat stress adaptation: To prepare the heat adapted cells of L. monocytogenes, one ml of stationary-phase culture was mixed with 10 ml of TSBYE that was pre-heated in to a reciprocal water bath shaker (model R76, New Brunswick Scientific, Inc., Edison, NJ, USA). The cells were then heated at 48 C for 60 min. After heat adaptation, the cell suspension was immediately used for studying the cross-protection studies. The non-adapted control cells were kept at room temperature for 1 h without exposure to sublethal stress.

Preparation of disinfectant solutions: The pH 12.0 lethal alkali treatment was prepared by adding 380 [micro]l of 4M NaOH (Fisher Scientific, New Jersey, USA) to 10 ml of TSBYE. The lethal 1000 ppm of [H.sub.2][O.sub.2] (Acros Organics, New jersey, USA) was prepared by adding 800 [micro]l of [H.sub.2][O.sub.2] (1.5%) to 10 ml of TSBYE. Carvacrol (>98%) was purchased from Sigma Aldrich (St. Louis, Mo., U.S.A.). It was solubilized by diluting (1:1) in propylene glycol (PGMP Biochemicals LLC, Solon, Ohio). The lethal concentration of carvacrol (428 ppm) was prepared by adding 70 [micro]l of working stock concentration in TSBYE respectively. PG is a food additive approved by FDA with both solvent and emulsifying properties and L. monocytogenes is able to grow in concentrations up to 12.5% PG. The LAE solution obtained from Vedeqsa (Vedeqsa inc., New York, NY 10001) was approved by FDA at 200 ppm in food products (USFDA, 2005). The original solution (10%) of LAE was diluted by adding 100 [micro]l to 900 [micro]l in saline (0.85%) solution to obtain 1% LAE solution. Then for 33 and 41 ppm LAE solutions, 35 and 41 [micro]l of the prepared 1% solution was added to 9 ml TSB-YE broth respectively. Then 900[micro]l of these LAE solutions were distributed in 1.5 ml eppendorf tubes for post-exposure treatments and allowed to be at either room temperature (RT) or 4[degrees]C depending upon the treatment temperatures. Carvacrol solution was purchased from Sigma Aldrich (St. Louis, Mo., U.S.A.). The concentrations of alkali and [H.sub.2][O.sub.2] were initially standardized for the controls (non-heated) cells and to compare the difference with the heat-stressed cells after post-exposure to alkali-stress or [H.sub.2][O.sub.2].

Survival of heat adapted and non-adapted L. monocytogenes cells to disinfectants and antimicrobials: To perform the survival of heat stress adapted and non-adapted (control) L. monocytogenes cells in lethal disinfectants and antimicrobials, 1 ml of heat stressed or control cells were added to 9 ml of TSBYE containing disinfectants to yield 7 log CFU/ml. The post-exposure times of LAE and carvacrol were for 30 min each at room temperature or for up to 2 and 4 h, respectively, at 4[degrees]C. Survivors were enumerated by serial dilutions of the cell suspensions and by plating out on Tryptic soy agar containing yeast extract, esculin and ferric ammonium citrate (TSAYE-EF).

Statistical analysis: All experiments were performed in three replicates with three individual trials. Student t-test (P < 0.05) was performed using Microsoft excel to determine significant mean difference between survival of heat stress adapted and non-adapted control cells in lethal disinfectants or essential oils.

RESULTS

After sublethal heating at 48[degrees]C for 60 min, three strains of L. monocytogenes ScottA, NRRL B 33157 and F4260 showed greater survival (P < 0.05) by 1-2 log CFU/ml in 33 ppm of LAE exposure at room temperature (Figure. 1A, 1B and 1C). The sublethal heat-stressed and non-heated control cells of all three strains of L. monocytogenes did not show any significant difference at 4[degrees]C with 44 ppm LAE dose in this assay. After sublethal heating at 48[degrees]C for 60 min, three strains of L. monocytogenes ScottA, NRRL-B 33157 and F4260 showed greater survival (P < 0.05) by 1.5-2.5 log CFU/ml in 428 ppm of carvacrol exposure at room temperature and 2.5-4.5 log CFU/ml in 535 ppm of carvacrol at 4[degrees]C (Figure. 2A, 2B and 2C).

Strain differences were observed when exposed to lethal alkali pH 12.5 NaOH. After sublethal heating at 48[degrees]C for 60 min, L. monocytogenes ScottA and NRRL B-33157 showed greater survival (P < 0.05) by approximately 2.5 log CFU/ml in pH 12.5 NaOH for 30 min at room temperature (Figure. 3A and B). On the other hand, L. monocytogenes F4260 did not show any difference in survival between sublethal heat stress and non-heat stressed cells (Figure 3C). In addition sublethal heat stressed L. monocytogenes ScottA cells showed greater survival (P < 0.05) by 1.5 log CFU/ml in pH 12.5 NaOH for 4 h at 4[degrees]C. Under the same conditions, other two strains NRRL B-33157 and F4260 did not show any significant difference between sublethal heat stress and non-heat stressed cells. Similarly, after exposing to 1000 ppm of [H.sub.2][O.sub.2] for 30 min at room temperature and 1200 ppm of [H.sub.2][O.sub.2] for 4 h at 4[degrees]C, the sublethal heat stressed cells of all three L. monocytogenes strains were sensitive with approximately 2 log CFU/ml lesser survival (P < 0.05) as compared to non-heat stressed cells (Figure. 4A, 4B and 4C).

DISCUSSION

In our previous studies, a significant increase in heat tolerance was observed after L. monocytogenes cells were exposed to sublethal heat stress at 48[degrees]C for 1 h (Shen et al. 2014b). In the present study, the cross-protection of L. monocytogenes sublethal heat stressed cells exposed to various disinfectants and essential oils at lethal levels was studied. Under room temperature, the limited lethal inactivation time was within 1 h as heat-stress adaptation was partially reversed within 1 h at room temperature depending on the strain. For those assays performed under 4[degrees]C, lethal inactivation time was not a limiting factor since up to 24 h L. monocytogenes cells were able to maintain acquired heat-stress adaptation. Since commonly used cleaners are either alkali- or oxidative-stress based, the survival responses of heat-stress adapted cells of L. monocytogenes in lethal concentration of alkali-stress and hydrogen peroxide were determined. Heat-stress adaptation conferred alkali-stress resistance appears to be strain dependent indicating the antimicrobial efficacy of alkali disinfectants could be undermined when heat-stress adapted cells are present. Similar observations were also reported by others which proposed that heat-stress adaptation in L. monocytogenes induces cross resistance to alkali based cleaners (Taormina and Beuchat 2001; Novak and Yuan 2003). For oxidative stress, a reverse pattern was observed that heat-stress adaptation rendered impaired survival in lethal concentration of hydrogen peroxide. [H.sub.2][O.sub.2] generates oxygen-free radicals that damages the cell membrane and disrupts the electron transport system. Present findings are in agreement with Lin and Chou, 2001, whereas Lou and Yousef observed increased survival in lethal [H.sub.2][O.sub.2]. These distinct observations may be due to the differences in bacterial strains (Bug600 verses ScottA), physiological state of L. monocytogenes (stationary verses exponential phase) and heat adaptation conditions (45[degrees]C verses 48[degrees]C). Also, a reasonable explanation should be heat-stress adaptation caused down-regulation of oxidative related gene expression. However, so far no published data is available on how does heat-stress adaptation in L. monocytogenes modulates the oxidative stress related genes. Interestingly, in the presence of oxidative stress, survival of L. monocytogenes cells from low, medium and high groups exhibited the same order as their heat-stress resistance. According to our findings, oxidative chemical agents are more efficient in eliminating the heat-stress resistant phenotypes of L. monocytogenes. Heat-stress adapted cells survived slightly higher at room temperature as compared to 4[degrees]C in LAE treatment whereas enhanced carvacrol resistance in heat-stress adapted cells was evident at both temperatures tested. According to the literature, both compounds exhibit similar antimicrobial mechanism through interacting with the bacteria cell membrane (Kanazawa et al. 1995; Ultee et al. 2002). However, for control cells when the temperature was lowered from 22[degrees]C to 4[degrees]C it diminished antimicrobial efficacy of LAE while this type of efficacy reduction did not occur for carvacrol. Therefore, LAE and carvacrol might have different mode of action at 4[degrees]C which could be responsible for different cross resistance response of heat-stress adapted L. monocytogenes at 4[degrees]C.

CONCLUSIONS

The outcome of this study indicates that the heat stressed cells of L. monocytogenes are not easily killed by LAE, carvacrol and alkali based antimicrobials. These compounds should be carefully considered when different strains of sublethal heat stressed cells of L. monocytogenes may be present.

ACKNOWLEDGEMENT

This research was supported in part by Strategic Research Initiative and Food Safety Initiative awards to R. Nannapaneni from the Mississippi Agricultural and Forestry Experiment Station under project MIS-401160.

CONFLICT OF INTEREST

There is no conflict of interest to declare

LITERATURE CITED

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Nitin Dhowlaghar, Mark W. Schilling and Ramakrishna Nannapaneni *

Department of Food Science, Nutrition and Health Promotion, Mississippi State University, Mississippi State, MS 39762, USA

Corresponding Author: Ramakrishna Nannapaneni, E-mail: nannapaneni@fsnhp.msstate.edu

Caption: Figure 1: Effect of sublethal heat-stress at 48[degrees]C/30 min on survival in LAE treatment at room temperature (35 ppm/30 min) or 4[degrees]C (41ppm/2h) in three L. monocytogenes serotypes: (A) Bug600 (serotype 1/2a); (B) NRRL B-33157 (serotype 4b); and (C) F4260 (serotype 1/2b). No sublethal heating ([]) or sublethal heating at 48[degrees]C ([??]). Sublethal heating treatments showing statistically higher survival are marked by asterisk (P < 0.05).

Caption: Figure 2: Effect of sublethal heat-stress at 48[degrees]C/30 min on survival in carvacrol treatment at room temperature (428 ppm/30 min) or 4[degrees]C (535ppm/4h) in three L. monocytogenes serotypes: (A) Bug600 (serotype 1/2a); (B) NRRL B-33157 (serotype 4b; and (C) F4260 (serotype 1/2b). No sublethal heating ([]) or sublethal heating at 48[degrees]C ([??]). Sub-lethal heating treatments showing statistically higher survival are marked by asterisk (P < 0.05).

Caption: Figure 3: Effect of sublethal heat-stress at 48[degrees]C/30 min on survival in NaOH treatment at room temperature (12.5 pH/30 min) or 4[degrees]C (pH 12.5/4h) in three L. monocytogenes serotypes: (A) Bug600 (serotype 1/2a); (B) NRRL B- 33157 (serotype 4b; and (C) F4260 (serotype 1/2b). No sublethal heating ([]) or sublethal heating at 48[degrees]C ([??]). Sub lethal heating treatments showing statistically higher survival are marked by asterisk (P < 0.05).

Caption: Figure 4: Effect of sublethal heat-stress at 48[degrees]C/30 minon survival in [H.sub.2][O.sub.2] treatment at room temperature (12.5 pH/30 min) or 4[degrees]C (pH 12.5/4h) in three L. monocytogenes serotypes: (A) Bug600 (serotype l/2a); (B) NRRL B- 33157 (serotype 4b; and (C) F4260 (serotype l/2b). No sublethal heating ([]) or sublethal heating at 48[degrees]C ([??]). Sublethal heating treatments showing statistically higher survival are marked by asterisk (P < 0.05).
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Author:Dhowlaghar, Nitin; Schilling, Mark W.; Nannapaneni, Ramakrishna
Publication:Journal of the Mississippi Academy of Sciences
Date:Jul 1, 2018
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