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Food Research International

journal homepage: www.elsevier.com/locate/foodres

Quantitative assessment of tolerance response to stress after exposure to oregano and rosemary essential oils, carvacrol and 1,8-cineole in Salmonella Enteritidis 86 and its isogenic deletion mutants Δdps, ΔrpoS and ΔompR

Myrella Lira Cariria, Adma Nadja Ferreira de Meloa, Luke Mizzib, Ana Carolina Ritterc, Eduardo Tondoc, Evandro Leite de Souzad, Vasilis Valdramidisb, Marciane Magnania,⁎

a Laboratory of Microbial Process in Foods, Department of Food Engineering, Center of Technology, Federal University of Paraíba, João Pessoa, Brazil b Department of Food Sciences and Nutrition, Faculty of Health Sciences, Msida MSD 2080, University of Malta, Malta c Laboratory of Food Microbiology, Food Science and Technology Institute, Federal University of Rio Grande do Sul, Porto Alegre, Brazil d Laboratory of Food Microbiology, Department of Nutrition, Health Sciences Center, Federal University of Paraíba, João Pessoa, Brazil

A R T I C L E I N F O

Keywords: Salmonella enterica Stress response Essential oils rpoS dps ompR

A B S T R A C T

This study assessed the influence of rpoS, dps and ompR genes on the tolerance response of Salmonella Enteritidis 86 (SE86) to homologous and heterologous stressing agents after exposure to essential oils (EOs) from Origanum vulgare L. (oregano; OVEO) and Rosmarinus officinalis L. (rosemary; ROEO) and their major constituents (ICs), carvacrol (CAR) and 1,8-cineole (CIN), respectively, by modelling the log reduction over time. Minimum in- hibitory concentration values of OVEO (1.25 μL/mL), CAR (0.62 μL/mL), ROEO (20 μL/mL) and CIN (10 μL/mL) against SE86 were always one-fold higher than those against Δdps, ΔrpoS and ΔompR mutants. Exposure to the same concentration of OVEO, CAR, ROEO or CIN caused higher reductions (up to 2.5 log CFU/mL) in Δdps, ΔrpoS and ΔompR mutants than in SE86 in chicken broth. In assays with homologous stressing agents, ompR, dps and rpoS influenced the tolerance to OEs or ICs. After adaptation to OVEO, CAR, ROEO and CIN, osmotolerance and acid tolerance of SE86 were influenced by rpoS gene, while thermotolerance of SE86 was influenced by ompR. Tolerance of SE86 to sodium hypochlorite after adaptation to OEs or ICs was influenced by rpoS and dps. These findings quantitatively describe for the first time the influence of rpoS, dps and ompR genes on the tol- erance of Salmonella Enteritidis to OVEO, CAR, ROEO and CIN.

1. Introduction

Among approximately 2600 serotype subspecies of Salmonella en- terica subsp. enterica, S. Enterititis is reported as one of the major etiological agents of salmonellosis outbreaks worldwide (Center for Disease Control and Prevention, 2018; Yadav, Saxena, Saxena, & Kataria, 2016). Epidemic strains of S. Enteritidis present a dynamic interaction within the host and environment (Melo et al., 2017). S. Enteritidis can activate a complex regulatory system of stress responses when exposed to unfavorable conditions, such as low or high tem- peratures, acidic pH, osmotic pressure and oxidative stress, commonly applied during food processing or storage (Ritter et al., 2014; Yadav et al., 2016; Yang, Khoo, Zheng, Chung, & Yuk, 2014).

Stress responses of S. Enteritidis to harsh conditions have been tentatively associated with gene regulation through the activation of

sigma factor RpoS (σS) encoded by the rpoS gene (Yang et al., 2014). σS- activated genes are involved in repairing damage in S. enterica cells caused by osmotic stress, acidic pH, high temperatures and anti- microbials (Álvarez-Ordóñez, Prieto, Bernardo, Hill, & López, 2012; Fang, Frawley, Tapscott, & Vazquez-Torres, 2016; Trastoy et al., 2018). The dps gene, which imparts the σS regulatory system, is involved in response of S. enterica to oxidative damage (Farizano, Torres, Pescaretti, & Delgado, 2014; Pacello et al., 2008; Ritter et al., 2012). The reg- ulatory ompR gene, which is independent of σS regulation, is related to the adaptive response of S. enterica during exposure to acidic pH or high temperature (Chakraborty, Mizusaki, & Kenney, 2015; Ritter et al., 2014).

Development of tolerance to homologous (direct-tolerance) or het- erologous (cross-tolerance) stressors during food processing has been a major concern about the stress responses of S. Enteritidis (Melo et al.,

https://doi.org/10.1016/j.foodres.2019.01.046 Received 23 October 2018; Received in revised form 17 January 2019; Accepted 20 January 2019

⁎ Corresponding author at: Laboratory of Microbial Processes in Foods, Department of Food Engineering, Technology Center, Federal University of Paraíba, Campus I, 58051-900 João Pessoa, Brazil.

E-mail address: [email protected] (M. Magnani).

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0963-9969/ © 2019 Published by Elsevier Ltd.

Please cite this article as: Cariri, M.L., Food Research International, https://doi.org/10.1016/j.foodres.2019.01.046

2017; Shah, Desai, Chen, Stevens, & Weimer, 2013). Cross-tolerance markedly enhances the survival of S. Enteritidis during multiple hur- dles, posing a risk from a food safety perspective (Álvarez-Ordóñez et al., 2012; Humphrey, 2004). Increased direct- and cross-tolerance of S. enterica to classical antimicrobial food preservatives (e.g., acids and salts) has supported research on the use of alternative antimicrobials in foods, such as essential oils (EOs) and their individual constituents (ICs) (Adelakun, Oyelade, & Olanipekun, 2016; Mahmoudi, 2014; Stojanović-Radić et al., 2018).

Among the EOs Generally Recognized as Safe (GRAS) (FDA, 2009), those obtained from Origanum vulgare L. (oregano; OVEO) and Ros- marinus officinalis L. (rosemary; ROEO), and their major ICs carvacrol (CAR) and 1,8-cineole (CIN), respectively, are considered candidates for use as antimicrobials in foods (Honório et al., 2015; De Souza, Almeida, & de Sousa, 2016; Stojanović-Radić et al., 2018). ROEO and CIN present lower efficacy against S. enterica compared to OVEO and CAR (Gomes-Neto, Luz, Franco, Magnani, & Souza, 2014; Govaris, Solomakos, Pexara, & A., & Chatzopoulou, P.S., 2010; Luz et al., 2012). However, the use of ROEO combined at synergistic concentrations with other EOs has been suggested in food preservation systems (Barbosa et al., 2016; Stojanović-Radić et al., 2018). Similarly, the combined use of CIN with CAR in food based-systems is proposed as a strategy to decrease the concentration required of each IC (Oliveira et al., 2015; Honório et al., 2015). In addition, ROEO has been studied in combi- nation with non-thermal technologies, such as pulsed electric fields (Gomes-Neto et al., 2015), incorporated in edible coatings (Andrade et al., 2017; de Sousa et al., 2013) or in ecofriendly films applied for meat packaging (Souza et al., 2019).

Previous studies reported both that exposure to sub-lethal amounts of OVEO and CAR induced an adaptive response in S. Typhimurium with alterations in membrane fatty acid composition (Luz et al., 2014), and that RpoS influences tolerance of E. coli to OVEO and ROEO (Gomes-Neto et al., 2015). However, the influence of stress response genes on the tolerance of S. Enteritidis after adaptation, i.e. the survival and/or growth under exposure to stressful conditions (Dubois- Brissonnet, 2012; Hernández, Cota, Ducret, Aussel, & Casadesús, 2012), including those imposed by sublethal concentration of OVEO, ROEO, CAR and CIN remains unknown.

A specific strain of Salmonella Enteritidis, named SE86, was identi- fied in > 95% of the salmonellosis outbreaks notified in the State of Rio Grande do Sul (Brazil) in the last decade (Oliveira, Pasqualotto, da Silva, & Tondo, 2012; Ritter et al., 2012). This high frequency increased the interest of researchers to use this strain as a target organism in studies with different environmental stress conditions. SE86 presents increased acid and thermal resistance (Perez, Ceccon, Malheiros, Jong, & Tondo, 2010; Ritter et al., 2014) and increased tolerance to sodium hypochlorite (NaClO) (Ritter et al., 2012).

This study assessed the influence of rpoS, dps and ompR genes in tolerance of SE86 to OVEO, CAR, ROEO and CIN through determination of the minimum inhibitory concentrations and survival curves. The influence of these genes on the tolerance response of SE86 to homo- logous or heterologous stressing agents, namely, temperature, lactic acid, sodium chloride and sodium hypochlorite, after exposure to OVEO, ROEO, CAR or CIN, through modelling the bacterial log re- duction over time was also assessed.

2. Materials and methods

2.1. Test strains and inoculum

Test strains used in this study were Salmonella Enteritidis 86 (SE86) and its isogenic deletion mutants Δdps, ΔrpoS and ΔompR. SE86 was first isolated from cabbage involved in a salmonellosis outbreak that occurred in Rio Grande do Sul State (Brazil) in 1999 (Oliveira et al., 2012). Mutants of SE86 (Δdps, ΔrpoS and ΔompR) were constructed in the Laboratorio di Microbiologia (Universita degli Studi di Sassari,

Sassari, Italy) using the knockout method, as previously described (Datsenko & Wanner, 2000). The construct was verified by PCR. SSM5327 (dps::Kan), SSM5333 (rpoS::Kan) and SSM5337 (ompR::Kan) mutations were transferred into a clean SE86 background by P22 transduction. Growth media of mutants was supplemented with kana- mycin (5 μL/mL). Stock cultures were maintained in cryovials at −20 °C.

Inocula of each strain were obtained after preparing suspensions in sterile saline solution (0.85% NaCl, w/v) from cultures grown in brain heart infusion (BHI) broth (HiMedia, India) at 37 °C for 18 h and har- vested through centrifugation (4500 ×g, 10 min, 4 °C). Harvested cells were washed twice in sterile saline solution and re-suspended in sterile saline solution to obtain standard cell suspensions. The optical density of 0.08 at 625 nm (OD625) provided viable counts of 8 ( ± 0.2) log CFU/ mL for all strains. Assays were performed with stationary phase cells because bacterial resistance to stress is usually maximal at this growth stage (Melo et al., 2017).

2.2. EOs and ICs

OVEO (batch OREORG01; density at 20 °C, 0.90; refractive index at 20 °C, 1.47) and ROEO (batch ROSTUN04; density at 20 °C: 0.94; re- fractive index at 20 °C: 1.51) were purchased from Ferquima Ind. Com. Ltda. (São Paulo, Brazil). The EOs were obtained from leaves by com- mercial scale steam distillation using the Clevenger apparatus (Clevenger, 1928). Constituents of OVEO and ROEO were identified using gas chromatography coupled with mass spectrometry (CGMS- QP2010 Ultra Shimadzu, Kyoto, Japan). Analysis was performed under the following conditions: an RTX-5 MS capillary column (30 m × 0.25 mm × 0.25 μm); program temperature: 60–240 °C (3 °C/ min); injector temperature: 250 °C; detector temperature: 220 °C; car- rier gas: helium adjusted to 0.99 mL/min speed; ionizing energy: 70 eV; and mass range (m/z): 40–500. Samples were co-injected with a homologous series of n-alkanes (C9-C31). Identification of each com- ponent was performed by comparing its mass spectrum with the NIST/ EPA/NIH Mass Spectral Database (National Institute of Standards Technology, Norwalk, CT) and FFNSC1.3 (Flavour and Fragrance Nat- ural and Synthetic Compounds) libraries as well as the Kovats retention index (Adams, 2001). Some structures (camphene, pseudolimonene, thymol methyl ether, tricyclene, α-terpinene, verbenone, eugenol α- muurolene, β-bisabolene, γ-cadienene, and caryophyllene oxide) were further confirmed by available authentic standards analyzed under the conditions described above. Quantification was computed as the per- centage contribution of each compound to the total amount present.

Because the antimicrobial properties of OVEO and ROEO have been attributed primarily to their major constituents, which are present in fairly high concentrations (20–70%) (Bakkali, Averbeck, Averbeck, & Idaomar, 2008; Burt, 2004), assays were also performed with CAR (47.01% of the assayed OVEO) and CIN (32.07% of the assayed ROEO). CAR (batch 0656-810; density at 20 °C: 0.976; refractive index at 20 °C: 1.522) and CIN (batch 0723-683; density at 20 °C: 0.99; refractive index at 20 °C: 1.419) were purchased from Sigma Aldrich (Sigma, France). Emulsions of OVEO, ROEO, CAR and CIN were prepared in BHI broth at a range of concentrations (80–0.312 μL/ mL) using Tween 80 (1%, v/v; Sigma–Aldrich, USA) as an emulsifier. Tween 80, at the higher assayed concentration (1%, v/v), did not present inhibitory effects against the tested Salmonella strains or interfere with the activity of OVEO, ROEO, CAR and CIN.

2.3. Preparation of chicken broth

Assays to evaluate the antibacterial activity of OVEO, CAR, ROEO and CIN and changes in tolerance of SE86 and its Δdps, ΔrpoS and ΔompR mutants to homologous or heterologous stressing agents were performed using chicken broth. Chicken breast samples were obtained from a slaughterhouse located in the city of Guarabira (Paraíba, Brazil).

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Before the assays, chicken breast meat samples were checked for compliance of current microbiological standards (Brazilian Legislation, Normative Instruction NI 12, 2001), which include the absence of Salmonella spp. For preparation of the chicken broth, chicken breast samples were cut in pieces of uniform sizes (3 × 3 × 3 cm) and boiled in water for 30 min at 110 °C. The obtained chicken broth was vacuum- filtered using Whatman no. 1 filter paper, and the filtrate was sterilized in an autoclave (121 °C, 1.1 atm, 15 min). Chicken broth was stored at −20 °C in aliquots of 50 mL, and when required, one aliquot was thawed under refrigeration (7 ± 1 °C) and used for the assays (Luz et al., 2014). The average gross composition of the chicken broth, de- termined using standard procedures (AOAC, 2016), was 95.5 g/100 g moisture, 0.65 g/100 g carbohydrates, 1.5 g/100 g protein, 0.19 g/ 100 g ash, and 0.31 g/100 g fat, with a final pH of 6.9 ± 0.2.

2.4. Evaluation of antimicrobial activity of OVEO, CAR, ROEO and CIN

2.4.1. Determination of the minimum inhibitory concentration (MIC) MIC values of OVEO, CAR, ROEO and CIN against SE86 and its Δdps,

ΔrpoS and ΔompR mutants were determined using a microtiter plate assay (CLSI, 2012), with minor modifications related to the cultivation media, inoculum size and MIC reading. Ninety-six-well plates were prepared by dispensing 50 μL of OVEO (40 to 0.312 μL/mL), CAR (20 to 0.15 μL/mL), ROEO (80–0.625 μL/mL) or CIN (80–0.625 μL/mL) in chicken broth. Then, 50 μL of bacterial suspension (6 log CFU/mL) were added to each well. The microplates were covered with lids and wrapped loosely with cling wrap to ensure that the EOs or ICs would not volatilize (Melo et al., 2017). Each plate included controls without the antimicrobials. The system was incubated at 37 °C for 24 h. After the incubation time, a 20 μL aliquot of resazurin aqueous solution (0.01 g/ 100 L, w/v) (Inlab, Brazil) was added to each well. Color changes were visually assessed after 20 min at 37 °C. Resazurin (7-Hydroxy-3H-

phenoxazin-3-one 10-oxide) is a blue-purple dye and cell permeable redox indicator used to monitor cell viability when added directly to cells in culture in a homogeneous format. Viable cells with active me- tabolism can reduce resazurin into the resorufin product, which is pink. Thus, bacterial growth is indicated by color changes from blue-purple to pink (or colorless). MIC values were confirmed as the lowest con- centration capable of inhibiting bacterial growth (Melo et al., 2017; Sarker, Nahar, & Kumarasamy, 2007). Because the objective was to work with sub-inhibitory concentrations, once the color reading was performed, the lowest concentration that indicated growth and the following well were plated on BHI agar (HiMedia, India) (containing 5 μL/mL kanamycin for mutants) to check the growth after incubation and to ensure that the minimum bactericidal values were correct. Based on the MIC values, sub-inhibitory concentrations of each EO or IC were selected to obtain survival curves of SE86 and its mutants when exposed to the same concentration of OVEO, CAR, ROEO or CIN.

2.4.2. Effects of EOs and ICs on counts of test strains in chicken broth The effect of OVEO (0.62 and 0.31 μL/mL), CAR (0.31 and 0.15 μL/

mL), ROEO (10 and 5 μL/mL) or CIN (5 and 2.5 μL/mL) on the counts of SE86 and its Δdps, ΔrpoS and ΔompR mutants in chicken broth was evaluated by a viable cell count procedure. An aliquot (5 mL) of the tested bacterial suspension (viable counts of approximately 7 log CFU/ mL) was inoculated into 45 mL of chicken broth samples containing EOs or ICs at the desired final concentrations. The different systems (final viable cell counts of approximately 6 log CFU/mL) were gently hand- shaken for 30 s and subsequently incubated at 37 °C. Just after the mixing (time zero) and each 15 min interval during 90 min of exposure time (Gomes-Neto et al., 2015), an aliquot of 100 μL of each system was serially diluted (101 – 108) in sterile saline solution. Subsequently, 20 μL of each dilution were plated on BHI agar (HiMedia, India) using the microdrop inoculation technique (Herigstad, Hamilton, & Heersink,

Table 1 Constituents identified in the essential oils from Origanum vulgare L. and Rosmarinus officinalis L.

Essential oils from Origanum vulgare L. Essential oils from Rosmarinus officinalis L

Constituenta Kovats indexb (%) Identification c Constituenta Kovats indexb (%) Identification c

α-thujene 926 0.83 RI, MS Tricyclene 923 0.20 RI, MS, PC α-pinene 934 2.57 RI, MS α -phellandrene 925 0.53 RI, MS Camphene 948 0.50 RI, MS, PC α-pinene 936 9.44 RI, MS β-pinene 977 0.87 RI, MS Camphene 950 3.78 RI, MS Myrcene 991 2.32 RI, MS β-pinene 981 7.71 RI, MS Pseudolimonene 1005 0.17 RI, MS, PC Myrcene 990 1.72 RI, MS α-terpinene 1017 1.68 RI, MS α-terpinene 1017 0.60 RI, MS, PC p-cymene 1030 11.93 RI, MS σ-cymol 1023 0.30 RI, MS γ-terpinene 1063 9.90 RI, MS 1,8-cineole 1035 32.07 RI, MS 1,2,3- octatriene, 2,7-dimethyl- 1088 0.18 RI, MS γ-terpinene 1059 0.84 RI, MS Linalol 1103 5.09 RI, MS Terpinolene 1089 0.45 RI, MS Thymol methyl ether 1243 0.34 RI, MS, PC Linalol 1104 2.66 RI, MS Thymol 1294 3.30 RI, MS Camphor 1154 18.00 RI, MS Carvacrol 1309 47.01 RI, MS Isoborneol 1170 3.86 RI, MS p-thymol 1327 5.59 RI, MS Terpinen-4-ol 1179 0.93 RI, MS β-caryophyllene 1424 1.79 RI, MS α -terpineol 1194 2.67 RI, MS Cyclohexene, 1-methyl-4-(5-methyl-1-methyle-4-hexenyl) 1509 0.19 RI, MS Verbenone 1211 0.14 RI, MS, PC Caryophyllene oxide 1587 1.11 RI, MS Bornyl acetate 1287 1.17 RI, MS

Eugenol 1359 0.35 RI, MS, PC Copaene 1376 0.46 RI, MS Caryophyllene 1425 6.37 RI, MS α -humulene 1455 0.70 RI, MS γ-muurolene 1477 0.29 RI, MS α-muurolene 1501 0.10 RI, MS, PC β-bisabolene 1508 0.15 RI, MS, PC γ-cadienene 1515 0.20 RI, MS, PC δ-cadinene 1524 0.49 RI, MS Caryophyllene oxide 1584 0.20 RI, MS, PC

Total 95.37 total 96.38

a Results expressed as percentage (%) of the total area for constituents with percentage area > 0.1; b Identification by Kovats index (Adams, 2001); MS: Identification by NIST/EPA/NIH; PC: Identification by authentic standards analyzed by mass spectrometry.

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2001). The results were expressed as log CFU/mL, and the detection limit was 1.5 log CFU/mL.

2.5. Evaluation of tolerance response after exposure to OVEO, CAR, ROEO and CIN

2.5.1. Assays for adaptation of strains and further exposure to homologous stressing agents

The concentration of each EO or IC used for adaptation was defined considering their effects on counts of SE86, Δdps, ΔrpoS and ΔompR strains in chicken broth. The tolerance responses of SE86, Δdps, ΔrpoS and ΔompR strains to OVEO, CAR, ROEO or CIN after exposure to these same substances in chicken broth were determined as previously de- scribed (Gomes-Neto et al., 2015) with adaptations. Briefly, the bac- terial suspension (viable counts of approximately 7 log CFU/mL) was inoculated in chicken broth containing 0.31 μL/mL of OVEO, 0.15 μL/ mL of CAR, 10 μL/mL of ROEO or 5 μL/mL of CIN, which corresponds to the MIC/2 of the OEs or ICs against SE86 and MIC/4 against its mu- tants, allowing the exposure of the parental and its mutant strains to the same concentrations. The mixtures were placed in screw-cap tubes (in 50 mL aliquots) and vortexed for 30 s. The systems were incubated statically for 18 h at 37 °C. After the incubation time, cells were

harvested by centrifugation (4500 g × 10 min, 4 °C), washed twice in sterile saline solution, and re-suspended in the same diluent to obtain cell suspensions with an OD625 that provided counts of approximately 7 log CFU/mL. Cells were inoculated in fresh chicken broth (final viable counts of 6 log CFU/mL) containing OVEO (0.62 μL/mL), CAR (0.30 μL/ mL), ROEO (20 μL/mL) or CIN (10 μL/mL) followed by incubation at 37 °C. Viable cells were enumerated at each 10 min interval during 240 min by serial dilution in sterile saline and plating in BHI agar. Cells of SE86 and its mutants not exposed to EOs or ICs were assayed simi- larly as controls.

2.5.2. Assays for adaptation of strains and further exposure to heterologous stressing agents

Preliminary experiments were performed to evaluate the thermo- tolerance, acid tolerance, osmotolerance and tolerance to the oxidizing agent NaClO of SE86 and ΔrpoS, ΔDps and ΔompR strains. Untreated cultures were inoculated in chicken broth containing NaCl (1–10 g/ 100 mL, at 37 °C), lactic acid (pH 4.5–6.0, at 37 °C), NaClO (200–500 ppm at room temperature: 25 °C) or chicken broth incubated at different temperatures (40–60 °C). The range for each stressing agent was selected considering the results of previous investigations (Luz et al., 2014; Ritter et al., 2012; Ritter et al., 2014), and the values 52 °C,

Fig. 1. Survival curves of SE86 and its deletion isogenic mutants Δdps, ΔrpoS and ΔompR in chicken broth containing (a-d) OVEO (Origanum vulgare L. essential oil): (●) 0 μL/mL; (■) 0.31 μL/mL and (▲) 0.62 μL/mL; (e-h) CAR: (carvacrol): (●) 0 μL/mL; (■) 0.15 μL/mL; and (▲) 0.31 μL/mL; (i-m); ROEO (Rosmarimus officinalis L. essential oil): (●) 0 μL/mL; (■) 5 μL/mL; and (▲)10 μL/mL; (n-q) CIN: (1,6-cineole) (●) 0 μL/mL; (■) 2.5 μL/mL and (▲) 5 μL/mL. The * symbol denotes the beginning of significant differences (p < .05) from the control (0 μL/mL).

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5 g/100 mL NaCl, pH 5.2 and 200 ppm NaClO were selected for eva- luation of tolerance response to heterologous stressors because they allow the growth of all strains.

The assays to evaluate the tolerance responses of SE86 and its Δdps, ΔrpoS and ΔompR mutants to the heterologous stressing agents (tem- perature, NaCl, acidic pH and NaClO) were performed following the same procedures described in section 2.5.1. After exposure to 0.31 μL/ mL of OVEO, 0.15 μL/mL of CAR, 10 μL/mL of ROEO or 5 μL/mL of CIN in chicken broth, cell suspensions were inoculated in fresh chicken broth (final viable counts of approximately 6 log CFU/mL) containing 5 g/100 mL NaCl, 200 ppm NaClO or pH 5.2 by addition of lactic acid. Viable cells were enumerated as described in Section 2.5.1 at each 10 min interval during 240 min of incubation at 37 °C to assess acid tolerance and osmotolerance, at 52 °C to assess thermotolerance or at room temperature to assess NaClO tolerance.

2.5.3. Modelling the tolerance response using a biphasic model The values (log CFU/mL) obtained for evaluation of tolerance re-

sponse to homologous and heterologous stressing agents were plotted against time, and different inactivation models (previously presented and reviewed by Geeraerd, Valdramidis, & Van Impe, 2005) were evaluated for their performance on fitting these data. Based on the re- sults of this preliminary study, the microbial inactivation kinetics were described by a biphasic model (Cerf, 1977), as follows:

= + ∙ + − ∙ − ∙ − ∙N N f e f elog ( ) log ( (0) ) log ( (1 ) )k t k tmax max1 2

For this model, N represents the microbial cell density expressed in CFU/mL, N(0) represents the initial microbial cell density [CFU/mL], while f is the fraction of the initial population in a major subpopulation, (1-f) is the fraction of the initial population in a minor subpopulation, and kmax1 and kmax2 [1/min] are the specific inactivation rates of the two populations, respectively. Its linear log linear version was also considered when two phases were not observed (i.e., kmax2 = 0). This model was used to numerically determine the time needed to achieve a 3-log reduction (t-3D) of the bacteria count. The model was not extra- polated; therefore, cases where the 3-log reduction was reached after 240 min are only presented as having a t-3D value > 240 min. t-3D values were used to describe the tolerance of each strain to the tested homologous or heterologous stressing agents. The average values of the estimated parameters along with the standard deviation were then calculated.

2.6. Reproducibility and statistical analysis

The assays were performed in three independent experimental re- plicates, and three samples (triplicate) were analyzed in each replicate. MIC values were expressed as modal values because no variation was observed between the replicated results. Viable counts in survival curves were expressed as the mean ± standard deviation. Statistical analyses using SAS 9.1 software were performed to determine sig- nificant differences (p < .05) using ANOVA followed by post hoc Tukey test. In order to determine significant differences in t-3D values

Fig. 2. Time needed to achieve a 3-log reduction (t-3D) of viable counts of SE86 and its deletion isogenic mutants Δdps, ΔrpoS and ΔompR determined using a biphasic model that described the tolerance of each strain to the tested homologous stressing agents. (a) Origanum vulgare L. (OVEO); (b) carvacrol (CAR); (c) Rosmarimus

officinalis L. (ROEO); and (d) 1,6-cineole (CIN); ( ) no exposed cells to stressing agents; ( ) exposed cell to stressing agents. Significant differences (p < .05)

between them is reported by a * symbol.

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among the treatments, t-test was carried out using two-tailed distribu- tion and two samples unequal variance. Statistical significance was reported for p < .05.

3. Results

3.1. Identification of OVEO and ROEO constituents

A total of 18 and 28 constituents were identified in OVEO and ROEO used in this study, respectively (Table 1). The constituents detected at the highest amounts in OVEO were CAR (47.01%), p-cymene (11.93%), γ-terpinene (9.90%), p-thymol (5.59%), linalol (5.09%), and thymol (3.30%). Other constituents, such as α-pinene (2.57%), myrcene (2.32%), β-caryophyllene (1.79%) and α-terpinene (1.68%) were de- tected in minor amounts. The major constituent detected at the highest amounts in ROEO was CIN (32.07%), camphor (18.00%), α-pinene (9.44%), β-pinene (7.71%), and caryophyllene (6.37%). Other con- stituents, such as isoborneol (3.86%), camphene (3.78%), α -terpineol (2.67%), and linalol (2.66%) were detected in minor amounts.

3.2. Inhibitory effects of EOs and ICs against SE86 and mutants

The MIC of OVEO against SE86 was 1.25 μL/mL and against Δdps, ΔrpoS and ΔompR strains was 0.62 μL/mL. The MIC of CAR against SE86 0.62 μL/mL and against Δdps, ΔrpoS and ΔompR strains was 0.31 μL/ mL. The MIC of ROEO against SE86 was 20 μL/mL and against Δdps, ΔrpoS and ΔompR strains was 10 μL/mL. The MIC of CIN against SE86 was 10 μL/mL and against Δdps, ΔrpoS and ΔompR strains was 5 μL/mL. Survival curves of SE86 and Δdps, ΔrpoS and ΔompR strains in chicken broth containing OVEO (0.62 and 0.31 μL/mL), CAR (0.31 and 0.15 μL/ mL), ROEO (10 and 5 μL/mL) or CIN (5 and 2.5 μL/mL) showed a linear decrease in counts of all target strains over time (Fig. 1a-q). After a 90 min exposure, the same concentrations of OVEO, CAR, ROEO or CIN caused higher reductions (up to 2.5 log CFU/mL) in counts of Δdps, ΔrpoS and ompR strains compared to SE86 (Fig. 1a-q). Survival curves

of Δdps, ΔrpoS and ΔompR strains exposed to OVEO, CAR, ROEO or CIN showed a dose response effect, while SE86 presented similar decreases when exposed to distinct concentrations of these substances.

3.3. Modelling the tolerance response of test strains to homologous stressing agents in chicken broth

Cells of SE86 not adapted to OVEO, CAR, ROEO or CIN displayed higher (p < .05) tolerance than Δdps, ΔrpoS or ΔompR cells not adapted to the same EO or IC (t-3D ≥103 min vs t-3D ≤ 82.55) (Fig. 2a-d). Cells of SE86, Δdps, ΔrpoS and ΔompR adapted to OVEO did not show any significant change on their tolerance to OVEO (Fig. 2a). Similar results were obtained for SE86, Δdps, ΔrpoS or ΔompR cells adapted to CAR (Fig. 2b). Although cells of SE86 adapted to ROEO increased their ROEO tolerance (t-3D increase of approximately 9 min) the changed was not significant. Similar results were obtained for Δdps, ΔrpoS or ΔompR adapted to ROEO. A significant difference was only observed for cells of SE86 adapted to CIN whose tolerance decreased (p < .05) when further exposed to the same IC (t-3D decrease > 10 min) (Fig. 2d).

3.4. Tolerance response of test strains to heterologous stressing agents

Adaptation to OVEO, CAR, ROEO or CIN did not change the os- motolerance of SE86, Δdps or ΔompR cells (t-3D > 240 min), but de- creased (p < .05) the osmotolerance of ΔrpoS cells adapted to EOs or ICs (t-3D decrease of approximately 5 min) (Fig. 3a). SE86 cells adapted or not adapted to EOs or ICs showed higher (p < .05) osmotolerance than adapted or non-adapted ΔrpoS cells (t-3D > 240 min vs t- 3D ≤ 54).

Adaptation to OVEO, CAR, ROEO or CIN did not change the acid tolerance in SE86, Δdps or ΔompR (t-3D > 240 min), but decreased (p < .05) the acid tolerance in ΔrpoS cells (t-3D decrease from 41 to 78 min) (Fig. 3b). Overall, SE86 cells adapted or not adapted to OVEO, CAR, ROEO or CIN showed higher (p < .05) acid tolerance than ΔrpoS

Fig. 3. Time needed to achieve a 3-log reduction (t-3D) of viable counts of SE86 and its deletion isogenic mutants Δdps, ΔrpoS e ΔompR determined using a biphasic model that described the tolerance of each strain to the tested heterologous stressing agents. (a) 5 g/100 mL NaCl (osmotic stress); (b) pH 5.2 (acid stress); (c)

200 ppm NaClO (oxidative stress); (d) temperature of 52 °C (thermal stress); ( ) control (no exposed cells to stressing agents); ( ) exposed cells to OVEO; ( )

exposed cells to ROEO; ( ) exposed cells to CAR and ( ) exposed cells to CIN. Significant differences (p < .05) between them are reported with different letters.

Values that reach t-3D > 240 min (y axis) were not included in statistical analysis as they exceed the time of the experimental range.

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cells adapted or not adapted to the same EO or IC, respectively (t- 3D > 240 min vs t-3D ≤ 178) (Fig. 3b).

SE86 cells adapted or not adapted to OVEO, CAR, ROEO or CIN showed higher (p < .05) NaClO tolerance than Δdps, and ΔrpoS cells adapted or non-adapted to the same EO or IC (t-3D > 240 min vs t- 3D ≤ 120). SE86 adapted to OVEO, CAR, ROEO or CIN did not appear to affect their NaClO tolerance. Similarly, adaptation to OVEO, CAR, ROEO or CIN did not change the NaClO tolerance in Δdps cells (t-3D of approximately 20 min). In ΔrpoS or ΔompR cells adapted to EOs or IC, a tendency of decreased NaClO tolerance (t-3D decrease of up to 60 min) was observed, however differences were not significant (Fig. 3c).

Cells of SE86 not adapted to OVEO, CAR, ROEO or CIN showed higher (p < .05) thermotolerance (t-3D up to 68 min higher) than ΔrpoS and ΔompR cells not adapted to EOs or ICs. Adaptation to OVEO, CAR, ROEO or CIN decreased (p < .05) the thermotolerance in ΔompR cells (t-3D decrease up to maximum of 68 min) (Fig. 3d).

4. Discussion

Previous studies have also reported CAR, thymol and p-cymene as the predominant constituents in OVEO (Govaris et al., 2010; Khan et al., 2018). Similarly, CIN, camphor, and α-pinene have been de- scribed as the major constituents in ROEO (Pesavento et al., 2015; Zaouali, Bouzaine, & Boussaid, 2010). However, the quantities of the major constituents detected in EOs can vary due to environmental conditions (e.g., altitude, temperature, rainfall, and geographical dis- tribution) on the plant source (Burt, 2004; Khan et al., 2018). Thus, knowledge of the chemical composition may help to understand simi- larities or differences in biological activities observed in distinct EOs from the same plant species.

MIC values of OEs against SE86 or its Δdps, ΔrpoS and ΔompR mu- tants were one double dilution higher than those displayed by ICs. Interestingly, in our previous study, both OVEO and CAR displayed a MIC value of 1.25 μL/mL against S. Typhimurium ATCC 14028 (Luz et al., 2012), which was the same as what we observed in the present study only for OVEO. Otherwise, Gomes-Neto et al. (2014) reported a MIC value of ROEO (80 μL/mL) two double dilutions lower than CIN (20 μL/mL) against S. Typhimurium ATCC 14028. Overall, the MIC values of OVEO or ROEO against SE86 or its Δdps, ΔrpoS and ΔompR mutants were lower than those previously reported against S. En- teritidis (Penalver et al., 2005; Stojanović-Radić et al., 2018).

These differences were most likely related to the different compo- sitions of OVEO and ROEO used in the distinct studies. Constituents present in smaller amounts in EOs may also play a critical role for achieving these antimicrobial effects and, consequently, contribute to modulating the antimicrobial efficacy of the major constituents present in these substances (Hossain et al., 2016). Overall, the distinct MIC values observed could be also related to the intrinsic tolerance of S. enterica strains assayed, particularly when they belong to distinct ser- ovars (Moraes et al., 2018).

In the present study, counts of Δdps, ΔrpoS, and ΔompR displayed concentration-dependent decreases when exposed to the OEs or ICs tested, and this behavior was not observed for SE86. A previous study assessing the efficacy of OVEO (0.1, 0.3, and 0.5%) to inactivate S. Newport on organic leafy greens also observed that the increase of the tested concentration was related to the achieved inactivation by time (Moore-Neibel et al., 2013). Similarly, Govaris et al. (2010) reported a dose-response inhibitory effect of OVEO (0.6 and 0.9%) against S. En- teritidis in mine meat during storage at 10 °C. Probably specific features of SE86 were involved in the similar response to distinct concentrations of EOs or ICs.

MIC values of OVEO, CAR, ROEO and CIN against SE86 were higher than those against Δdps, ΔrpoS, and ΔompR mutants, and the same concentrations of these substances caused greater decreases in viable counts of the mutant strains than of parental SE86. A previous study suggested that the lower resistance of E. coli ΔrpoS to OVEO, CAR,

ROEO and CIN (measured by MIC values and survival curves) compared to its parental strain was associated with the lower resistance of cyto- plasmic and outer membranes in mutants or with impaired cell ability to repair injuries caused by these EOs or ICs (Gomes-Neto et al., 2015). Considering the diversity of stress rpoS-regulated genes, the deletion of rpoS results in a pleiotropic phenotype (i.e., multiple phenotype im- plications resulting from one genetic change) (McMeechan et al., 2007; Guiney & Fierer, 2011). Consequently, ΔrpoS cells demonstrate in- creased susceptibility to a variety of environmental stresses (Shah et al., 2013), such as those caused by exposure to EOs or ICs.

When exposed to EOs or ICs, bacterial cells may react by increasing the expression of stress-response proteins (de Souza, 2016) to repair damage in membranes that affects cell wall structure and increases membrane permeability. EOs or ICs, even at sub-lethal concentrations, are capable of inducing these effects on bacterial cells (de Souza et al., 2016). These substances also interfere with cell wall proteins involved in transport of molecules into cells (Di Pasqua et al., 2007; Nazzaro, Fratianni, De Martino, Coppola, & De Feo, 2013). Because ompR is the regulator of systems that involve major outer membrane proteins (Bang, Audia, Park, & Foster, 2002), the absence of this gene might increase the susceptibility of SE86 to OVEO, CAR, ROEO or CIN. Si- milarly, the lower survival rates of SE86 isogenic deletion Δdps mutant in chicken broth containing these substances was probably due to the lack of antioxidant proteins encoded by dps in S. enterica to tolerate environmental stresses (Farizano et al., 2014; Ritter et al., 2012). Overall, despite the knowledge that the major constituents typically define the antimicrobial activity of EOs, constituents present in smaller amounts also contribute to these effects (Burt, 2004; de Souza, 2016), as observed by the higher MIC values of OVEO and ROEO compared to CAR and CIN, respectively.

Assays of tolerance to homologous stressing agents showed the in- fluence of ompR, dps and rpoS in increased tolerance of SE86 to OVEO, CAR, ROEO and CIN. EOs are mixtures of constituents (phenolics, ter- penes, ketones, alcohols, ethers and hydrocarbons), and their inhibitory effects against S. enterica are primarily attributed to the hydrophobic nature of these constituents (Bajpai, Baek, & Kang, 2012; Burt, 2004). By accumulation in the cell membrane, ICs cause a disturbance of the phospholipid bilayer and compromise membrane function (Nazzaro et al., 2013). However, having distinct chemical nature, EOs con- stituents elicit different antimicrobial responses in foodborne pathogens (Bajpai et al., 2012). This can explain the decreased tolerance of SE86 to CIN after adaptation to this same IC.

The rpoS gene influenced the osmotolerance and acid tolerance of SE86. In Salmonella, rpoS acts as a master regulator required for survival under harsh conditions (McMeechan et al., 2007; Shah et al., 2013). Thus, despite the lack of induction of cross protection because SE86 did not increase its osmotolerance or acid tolerance after adaptation to EOs or ICs, the absence of rpoS could have reduced the ability of SE86 to address the unfavorable conditions imposed by the addition of NaCl or lactic acid in chicken broth.

rpoS, and dps influenced the NaClO tolerance of SE86 after adap- tation to EOs or ICs. A previous study reported that the expression of oxidative stress response genes (oxyR, soxR and rpoS) increased in re- sponse to exposure to cinnamon EO in Escherichia coli O157:H7 (Sheng, Rasco, & Zhu, 2016). Upregulation of rpoS or the other studied genes could explain the increased NaClO tolerance of SE86. According to Chen and Jiang (2017), rpoS is involved in cross-protection of de- siccation-adapted S. Typhimurium by upregulation of rpoS. The influ- ence of dps on NaClO tolerance of SE86 after exposure to EOs or ICs was probably related to the protective effects of this gene against oxidative damage (Pacello et al., 2008). The deletion of dps increased the sensi- tivity of SE86 to NaClO (Ritter et al., 2012), indicating a possible det- rimental role of this gene after sub-lethal injuries caused by exposure to OVEO, CAR, ROEO and CIN (Gomes-Neto et al., 2015; Luz et al., 2014). Further studies should be performed to clarify the underlying me- chanisms behind the decreased NaClO tolerance of SE86 isogenic

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deletion mutants ΔrpoS and ΔompR, but not in Δdps after exposure to EOs or ICs.

The results of thermotolerance assays after adaptation to EOs or ICs suggest the involvement of ompR in thermotolerance of SE86 because greater decreases in cell counts occurred when this gene was absent. ompR is also involved in the decreased thermotolerance of acid-adapted SE86 cells (Ritter et al., 2014), as well as is one of the genes involved in porin gene expression (Begic & Worobec, 2006). Porins are outer membrane aqueous channels used in the diffusion of various com- pounds across the membrane that can also aid in adapting bacteria to various environmental conditions and confer antibacterial drug re- sistance (Gil et al., 2009). A previous study showed that porin ompS1 regulated by the EnvZ/OmpR system is down-regulated in S. Enteritidis by trans-cinnamaldehyde and eugenol (Johny et al., 2017). Eugenol was a constituent of the EOs tested in the present study.

5. Conclusions

The results of this study show quantitatively the influence of ompR or dps, rpoS and ompR genes on increased tolerance of SE86 to OVEO or ROEO after adaptation to these same agents and the influence of dps, rpoS and ompR genes on increased NaClO tolerance of SE86 after ex- posure to OVEO. Further investigation of the effects of OVEO, CAR, ROEO and CIN toward gene expression regulation would help to elu- cidate the underlying mechanisms behind the observed tolerance re- sponse.

Acknowledgements

The authors thank CNPq-Brazil for the financial support grants #302763/2014-7 and CAPES for partial funding of this research (Finance code 001) and for a PhD scholarship awarded to the first au- thor (M.L. Cariri).

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