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DIFFERENCES IN SURVIVAL OF HEAT STRESS ADAPTED CELLS OF Listeria monocytogenes EGD (BUG 600) IN DISINFECTANTS AND ESSENTIAL OILS.

INTRODUCTION

Listeria monocytogenes is a Gram-positive, facultative anaerobic foodborne pathogen associated with a variety of food products such as meat, poultry, fresh produce and dairy products (Farber and Brown, 1990; Gandhi and Chikindas, 2007). L. monocytogenes was also isolated from various environment settings such as soil, ground water and decaying vegetation (Gray et al., 2006). Ingestion of L. monocytogenes via contaminated foods leads to listeriosis, a severe disease that primarily affects immunocompromised individuals, pregnant women, senior individuals and newborns. The fatality rate of listeriosis ranges from 20 to 30 % (Hamon et al., 2006). L. monocytogenes has a potential to persist for extended periods of time under mild processing environments such as heat, acid and alkaline conditions (Vasseur et al., 1999). Exposure to these mild sublethal conditions subsequently induces stress tolerance response in which these initial mild stress shocks provide edge to L. monocytogenes cells in subsequent survival under lethal stress conditions (Ramaswamy et al., 2007; Yousef and Courtney, 2003). There are several reports indicating that exposure to particular mild stress can also lead to enhanced protection against other lethal stress which was defined as cross protection (Soni et al., 2011).

Heating is the most reliable end point preservation technique used in food industries for inactivation of microbes. L. monocytogenes heat stress adaptation is defined as pre-exposing cells at a sublethal heat stress which confers increased heat tolerance at lethal heating temperature (Farber and Brown, 1990). This increased thermal tolerance is partially due to the activation of conserved heat shock proteins (Hsps) (e.g., DnaK and GroEL) under sublethal heat temperatures (Doyle et al., 2001; Ferreira et al., 2001; Hill et al., 2002).

Chemical disinfectants such as chlorine, quaternary ammonium compounds (QACs) and alkali containing compounds are frequently applied in cleaning and sanitation to inactivate undesirable microorganisms. In the food processing environment, contaminated L. monocytogenes may encounter sublethal heat stress that activates its intracellular stress responses and become persistent in the subsequent lethal inactivation by these disinfectants (Taormina and Beuchat, 2001). Studies show that L. monocytogenes cells heat shocked at 45[degrees]C for 1 h had increased tolerance to 25% NaCl, 18% ethanol and 0.01% crystal violet (Lin and Chou, 2004). Lin et al. (2012) observed that L. monocytogenes cells heat stressed at 48[degrees]C for 10 min were more tolerant to 0.128 ppm of chlorine dioxide and 1.384 ppm of QACs compared to non-adapted control cells (Lin et al., 2012). The viability of heat stressed cells of L. monocytogenes in other disinfectant is not known.

Plant essential oils (EOs) are gaining interest for their potential use as antimicrobials in the food industries as they are recognized as generally recognized as safe (GRAS). Many studies show that EOs can efficiently kill pathogenic Escherichia coli, Salmonella Spp. and L. monocytogenes in standard microbiology growth media or in various food substrates (Burt, 2004; Skandamis and Nychas, 2001; Smith-Palmer et al., 2001). It is likely that if the initial treatments which fail to kill the L. monocytogenes cells can provide them with cross protection against EOs. 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). There is no information on the sensitivity of heat stressed L. monocytogenes cells to other EOs. Therefore, the objective of this study was to determine the effect of heat stress adaptation on the survival of L. monocytogenes Bug600 in various disinfectants and essential oils.

MATERIALS AND METHODS

Bacterial strains and growth conditions: L. monocytogenes EGD (Bug600, serotype 1/2a (Institut Pasteur, Paris, France) was used in this study. The strain 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: The sublethal heat stress adaptation was performed by adding 1 ml of stationary-phase culture to 9 ml of TSBYE and heating at 48[degrees]C for 1 h. A reciprocal water bath shaker (model R76, New Brunswick Scientific, Inc., Edison, NJ, USA) was used for heating. Inoculum was directly added into the pre-heated broth and mixed so that the inoculum did not adhere to non-heated part of inner tube wall and cap. The non-adapted control cells were kept at room temperature for 1 h without exposure to sublethal stress.

Preparation of disinfectant solutions: Disinfectants used in this study was shown in Table 1. The working stock concentrations for [H.sub.3]P[O.sub.4] (5,625 ppm), QAC-1 (187 ppm), QAC-2 (525 ppm) and CPC (400 ppm) were prepared by diluting the original stock solution by 1:100 in deionized water. The working stock concentration of [H.sub.2][O.sub.2] (15,000 ppm) was prepared by diluting 428 [micro]l of the original stock solution in 10 ml deionized water. Carvacrol (>98%), Bay oil (100%), Red thyme oil (100%) and cinnamon leaf oil (>99%) were purchased from Sigma Aldrich (St. Louis, Mo., U.S.A.). These essential oils were solubilized by diluting (1:1) in propylene glycol (PGMP Biochemicals LLC, Solon, Ohio) and required concentrations were then prepared in TSBYE as described in Table 2. 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.

Exposure of heat adapted and non-adapted L. monocytogenes cells to disinfectants and essential oils: To determine the survival of heat stress adapted and non-adapted (control) L. monocytogenes Bug600 cells in lethal disinfectants and essential oils, 1 ml of heat stressed or control cells were added to 9 ml of TSBYE containing disinfectants at 22[degrees]C to yield an initial cell concentration of 7 log CFU/ml. Except for the QACs and NaOH, incubation time for all the disinfectants and essential oils was 60 min and survivors were enumerated every 15 min by plating on Tryptic soy agar containing yeast extract, esculin and ferric ammonium citrate (TSAYE-EF). Cells were exposed to QACs for 30 min and to NaOH for 120 min and survivors were enumerated every 10 min or 30 min on 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

The survival of heat stress adapted cells of L. monocytogenes Bug600 in lethal HCl and [H.sub.3]P[O.sub.4] is shown in Fig. 1. The heat stress adapted L. monocytogenes cells were sensitive to lethal HCl and H3PO4 compared to non-adapted control cells. The survival of heat stress adapted cells was significantly decreased by 2 log CFU/ml in pH 2.5 by HCl after 60 min compared to control cells (P < 0.05Fig. 1A). Similarly, in the presence of pH 2.5 by [H.sub.3]P[O.sub.4], the heat stress adapted cells were non-detectable after 60 min while control cells had a survival of 4.7 log CFU/ml under those conditions (P < 0.05)(Fig. 1B).

The survival of heat stress adapted cells of L. monocytogenes Bug600 in lethal NaOH and KOH is shown in Fig. 2. The heat stress adapted L. monocytogenes cells had significantly higher survival in lethal NaOH and KOH compared to control cells. The survival of heat stress adapted L. monocytogenes cells was significantly increased by 4.3 and 2.2 log CFU/ml in lethal NaOH and KOH (pH 12) respectively (P < 0.05Fig. 2A and B) after 120 min compared to control cells which were non-detectable under those conditions.

The survival of heat stress adapted cells of L. monocytogenes Bug600 in lethal Lysol, D-trol and CPC is shown in Fig. 3-4. The heat stress adapted L. monocytogenes cells were sensitive to Lysol, D-trol and CPC compared to non-adapted control cells. The survival of heat stress adapted cells were significantly decreased by about 3.0 log CFU/ml in Lysol or D-trol (3.5 ppm) after 30 min compared to control cells (P < 0.05). Also, the survival of heat stress adapted cells was significantly decreased by 4.4 log CFU/ml in 2.5 ppm CPC compared to control cells which were nondetectable at 60 min (P < 0.05Fig. 4).

The survival of heat stress adapted cells of L. monocytogenes Bug600 in lethal NaOCl and [H.sub.2][O.sub.2] is shown in Fig. 5. The heat stress adapted L. monocytogenes cells were sensitive to NaOCl and [H.sub.2][O.sub.2] compared to non-adapted control cells. The survival of heat stress adapted cells was significantly decreased by 3.0 log CFU/ml in lethal NaOCl (800 ppm) after 60 min compared to control cells (P < 0.05) (Fig. 5A). Also, the survival heat stress adapted cells was significantly decreased by 2.0 log CFU/ml in lethal [H.sub.2][O.sub.2] (1200 ppm) compared to control cells (P < 0.05 Fig. 5B).

The survival of heat stress adapted cells of L. monocytogenes Bug600 in lethal essential oils is shown in Fig. 6. L. monocytogenes heat stress adapted cells had significantly increased in survival in lethal carvacrol and bay essential oils compared to nonadapted control cells (Fig. 6A and B). The survival of heat stress adapted cells was significantly increased by 2.5 log CFU/ml in lethal carvacrol (428 ppmFig. 6A) or by 3.5 log CFU/ml in lethal bay oil (1100 ppm) (Fig. 6B) compared to control cells (P < 0.05). By contract, the heat stress adapted L. monocytogenes cells were sensitive to red thyme oil (300 ppm) where the survival of L. monocytogenes was significantly decreased by 1.4 log CFU/ml compared to control cells (P < 0.05Fig. 6C). On the other hand, there was no significant difference in survival of L. monocytogenes heat stress adapted and non-adapted control cells in lethal cinnamon oil (1050 ppmFig. 6D).

DISCUSSION

Despite the routine use of antimicrobials and disinfectants, L. monocytogenes may still persist in some food processing environments due to its tolerance to various antimicrobial compounds (Davidson and Harrison, 2002). Heating is a reliable end point preservation technique followed in food industries for inactivation of foodborne pathogens. However, heat stress adaptation due to insufficient heat inactivation may allow L. monocytogenes cells to survive during the second round of heat inactivation or in mild heat treatments (e.g., microwave) prior to consumption (Doyle et al., 2001).

Our findings show that the heat stress adapted L. monocytogenes Bug600 cells were more sensitive to lethal acid stress by HCl and H3PO4 compared to control cells. Similar phenomenon has been reported by Lou and Yousef (Lou and Yousef, 1997) and Lee et al.(Lee et al., 1995) in L. monocytogenes and S. Typhimurium where heat adapted cells were more sensitive to lethal acidic pH than non-adapted control cells. Although no molecular mechanisms have been elucidated on how heat adaptation in L. monocytogenes resulted in increased sensitivity to lethal acid challenge, our previous work found that L. monocytogenes cells appeared to be injured after incubation at 48[degrees]C for 1 h as those heat adapted cells grew much slower compared to non-adapted control cells at 37[degrees]C (Shen et al., 2014). Therefore, the reduced survival of heat adapted cells may result from the injury triggered by sublethal heat treatment. We also found that the heat stress adapted L. monocytogenes cells exhibited cross protection to lethal alkaline stress by NaOH and KOH. Similar observations were also reported by others which demonstrated that heat stress adaptation in L. monocytogenes induces cross-resistance to alkali based cleaners (Novak and Yuan, 2003; Taormina and Beuchat, 2001). Therefore, our data suggest that KOH and NaOH might not be suitable to be used to inactivate heat adapted L. monocytogenes cells.

[H.sub.2][O.sub.2] generates oxygen-free radicals that damages the cell membrane and disrupts the electron transport system. We observed stationary phase grown heat adapted L. monocytogenes cells were more sensitive to a lethal concentration of [H.sub.2][O.sub.2] compared to non-adapted cells which was similar to that observed by Lin and Chou (Lin and Chou, 2004). In contrast, Lou and Yousef observed increased survival in 1000 ppm [H.sub.2][O.sub.2] in exponential-phase L. monocytogenes Scott A cells after pre-exposure to 45[degrees]C for 1 h (Lou and Yousef, 1997). These observations may be due to the differences in bacterial strains (Bug600 versus ScottA), the physiological state of L. monocytogenes (stationary phase versus exponential phase) or the differences in heat adaptation conditions (45[degrees]C versus 48[degrees]C). NaOCl is one of the most highly used disinfectants in food industry in which HOCl and H[Cl.sup.-] ions are the main active components responsible for creating oxidative stress (FUKUZAKI, 2006). Since our data showed that heat adaptation in L. monocytogenes resulted in increased susceptibility to [H.sub.2][O.sub.2], it is not surprising to see the increased susceptibility to another oxidizing agent NaOCl.

We observed that the heat stress adapted L. monocytogenes cells had greater sensitivity to quaternary ammonium compound-Lysol. Similar to these findings, Moorman et al. (Moorman et al., 2005) observed that heat adaptation in L. innocua resulted in increased sensitivity to a mixture of QACs. However, Lin et al. (2012) reported that L. monocytogenes heat adapted cells survived greater than non-adapted control cells in QAC (Lin et al., 2012). It is important to notice that Lin and co-workers prepared the heat adapted cells in PBS where the cells may be exposed to starvation instead of bacterial growth medium. Therefore, the observed increased resistance to QAC might result from starvation rather than heat adaptation. QACs exhibits the killing efficacy by interacting with bacterial cell membrane (Ioannou et al., 2007). Our previous study showed that L. monocytogenes cell envelope were thickened after being treated at 48[degrees]C for 60 min suggesting that modified cell membrane resulted from sublethal heat treatment could protect L. monocytogenes against QACs (Saha et al., 2015). However, Moorman et al. (Moorman et al., 2005) found that no membrane fluidity was changed after heat adaptation at 45[degrees]C in L. innocua. Therefore, the change of cell membrane proteins might contribute to the decrease survival to QACs. This hypothesis needs further investigation by comparing the proteome of cell membrane before and after heat stress adaptation.

Our findings show that the sublethal heat treatment at 48[degrees]C for 1 h enhanced the survival of L. monocytogenes cells in lethal concentrations of carvacrol and bay oil. Similarly, Ait-Ouazzou et al. (Ait--Ouazzou et al., 2013) reported that mild heat treatment at 45[degrees]C for 1 h protected L. monocytogenes cells against carvacrol inactivation. Several studies proposed that carvacrol exhibits bactericidal effect by damaging the cell membrane (Helander et al., 1998). Hence, the heat stress adapted L. monocytogenes may change the cell membrane composition during heat treatment which may minimize the interaction between carvacrol and cell membrane. In order to fully understand the heat stress conferred cross protection against carvacrol in L. monocytogenes, it is necessary to perform a comparative lipid composition analysis of the cell membrane before and after heat treatment at 48[degrees]C for 1 h. In addition, we noticed that heat stress adapted cells were still sensitive to red thyme and cinnamon. These distinct responses of heat adapted L. monocytogenes to different essential oils may be due to the different composition of these agents (Burt, 2004). Our data suggest that compared to carvacrol and bay oil, thyme and cinnamon may be better antimicrobial agents during food processing where heat adapted L. monocytogenes are present.

CONCLUSIONS

In conclusion, our findings demonstrate that the heat stress adaptation in L. monocytogenes did not result in increased resistance to lethal acid, oxidative agents, QAC, red thyme and cinnamon. However, NaOH, KOH, carvacrol and bay oil exhibited reduced killing efficacy when L. monocytogenes cells acquired heat stress adaptation. Therefore, the use of NaOH or KOH based alkaline disinfectants, and essential oils containing carvacrol and bay oil should be carefully considered when heat adapted L. monocytogenes cells may be present.

ACKNOWLEDGEMENTS

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

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Nitin Dhowlaghar (1), Qian Shen (1), Piumi De. A. Abeysundara (1), Amruta Udaysinh Jadhav, Ramakrishna Nannapaneni (1) *, Mark W. Schilling, Wen-Hsing Cheng, and Chander S. Sharma (2)

(1) Department of Food Science, Nutrition and Health Promotion, Mississippi State University, Mississippi State, MS 39762, USA and (2) Poultry Science Department, Mississippi State University, Mississippi State, MS 39762, USA.

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

Caption: Figure 1. Survival of L. monocytogenes Bug600 in pH 2.5 HCl (A) and pH 2.5 [H.sub.3]P[O.sub.4] (B) at 22[degrees]C after 1 h preexposure to 48[degrees]C ([??]) or no sublethal heating ([]). Symbols marked with an asterisk indicate significant survival differences between heat adapted ([??]) and non-adapted ([]) cells.

Caption: Figure 2. Survival of L. monocytogenes Bug600 in pH 12.0 NaOH (A) and pH 12.0 KOH (B) at 22[degrees]C after 1 h pre-exposure to 48[degrees]C ([??]) or no sublethal heating ([]). Symbols marked with an asterisk indicate significant survival differences between heat adapted ([??]) and non-adapted ([]) cells.

Caption: Figure 3. Survival of L. monocytogenes Bug600 in 3.5 ppm QAC-1 (A) and 3.5 ppm QAC-2 (B) at 22[degrees]C after 1 h pre-exposure to 48[degrees]C ([??]) or no sublethal heating ([]). Symbols marked with an asterisk indicate significant survival differences between heat adapted ([??]) and non-adapted ([]) cells.

Caption: Figure 4. Survival of L. monocytogenes Bug600 in 2.5 ppm CPC at 22[degrees]C after 1 h pre-exposure to 48[degrees]C ([??]) or no sublethal heating ([]). Symbols marked with an asterisk indicate significant survival differences between heat adapted ([??]) and non-adapted ([]) cells.

Caption: Figure 5. Survival of L. monocytogenes Bug600 in 800 ppm NaOCl (A) and 1200 ppm [H.sub.2][O.sub.2] (B) at 22[degrees]C after 1 h pre-exposure to 48[degrees]C ([??]) or no sublethal heating ([]). Symbols marked with an asterisk indicate significant survival differences between heat adapted ([??]) and non-adapted ([]) cells.

Caption: Figure 6. Survival of L. monocytogenes Bug600 in 428 ppm Carvacrol (A), 1100 ppm Bay oil (B), 300 ppm Red thyme (C) and 1050 ppm Cinnamon (D) at 22[degrees]C after 1 h pre-exposure to 48[degrees]C ([??]) or no sublethal heating ([]). Symbols marked with an asterisk indicate significant survival differences between heat adapted ([??]) and non-adapted ([]) cells.
Table 1. Preparation of disinfectants.

Disinfectant group   Active ingredient     Manufacturers

Acid                 HCl                   Fisher Scientific
                     [H.sub.3]P[O.sub.4]   Sigma Aldrich
Alkaline             NaOH                  Fisher Scientific
                     KOH                   Diversey
Oxidative            NaOCl                 The Clorox company
                     [H.sub.2][O.sub.2]    Acros Organics
Quaternary
ammonium             QAC-1 (1)             Lysol
compounds
                     QAC-1 (2)             Diversey
                     CPC (3)               Safe foods corporation

Disinfectant group   Concentration of    Lethal
                     active ingredient   concentration
                     (ppm)               tested

Acid                 330,000             pH 2.5
                     224,000             pH 12.0
Alkaline             160,000             pH 12.0
                     5,625               pH 2.5
Oxidative            60,000              800 ppm
                     350,000             1200 ppm
Quaternary
ammonium             18,700              3.5 ppm
compounds
                     52,500              3.5 ppm
                     4,000               2.5 ppm

(1) QAC-1 (Lysol) contains dimethylbenzyl ammonium chloride
([C.sub.14] 60%, [C.sub.16] 30%, [C.sub.12] 5%, [C.sub.18] 5%)

(2) QAC-2 (D-trol) contains dimethylbenzyl ammonium chloride
([C.sub.14] 60%, [C.sub.16] 30%, [C.sub.12] 5%, [C.sub.18] 5%) and
dimethylbenzyl ammonium chloride ([C.sub.12] 68%, [C.sub.14] 32%)

(3) Cetylpyridinium chloride (Cecure) contains 1-Hexadecylpyridinium
chloride

Table 2. Preparation of essential oils.

Essential    Active Ingredients    Manufacturers   Working   Lethal
oils                                               stock     conc.
             5-isopropyl-2-                        conc.     tested
                                                   (ppm)     (ppm)

Carvacrol    methylphenol          Sigma Aldrich   61,250    428

             a-pinene, b-pinene,
             myrcene, limonene,
Bay oil      linalool, methyl      Sigma Aldrich   62,500    1100
             chavicol, neral,
             a-terpineol,
             geranyl acetate,
             eugenol and
             chavicol

Red thyme    (a) N/A               Sigma Aldrich   62,500    300
oil

Cinnamon
leaf         (a) N/A               Sigma Aldrich   61,875    1050
oil

(a) N/A represents that the composition of the essential oil is
unknown.
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Author:Dhowlaghar, Nitin; Shen, Qian; Abeysundara, Piumi De. A.; Jadhav, Amruta Udaysinh; Nannapaneni, Rama
Publication:Journal of the Mississippi Academy of Sciences
Date:Jul 1, 2018
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