Habituation of enterotoxigenic Staphylococcus aureus toOriganum vulgare L. essential oil does not inducedirect-tolerance and cross-tolerance to salts and organic acids
Abstract
Enterotoxigenic Staphylococcus aureus strains that were isolatedfrom foods were investigated for their ability to develop direct-tolerance andcross-tolerance to sodium chloride (NaCl), potassium chloride (KCl), lactic acid (LA)and acetic acid (AA) after habituation in sublethal amounts (1/2 of the minimuminhibitory concentration - 1/2 MIC and 1/4 of the minimum inhibitory concentration -1/4 MIC) of Origanum vulgare L. essential oil (OVEO). Thehabituation of S. aureus to 1/2 MIC and 1/4 MIC of OVEO did notinduce direct-tolerance or cross-tolerance in the tested strains, as assessed bymodulation of MIC values. Otherwise, exposing the strains to OVEO at sublethalconcentrations maintained or increased the sensitivity of the cells to the testedstressing agents because the MIC values of OVEO, NaCl, KCl, LA and AA against thecells that were previously habituated to OVEO remained the same or decreased whencompared with non-habituated cells. These data indicate that OVEO does not have aninductive effect on the acquisition of direct-tolerance or cross-tolerance in thetested enterotoxigenic strains of S. aureus to antimicrobial agentsthat are typically used in food preservation.
Introduction
Food processing exposes spoilage and pathogenic food-related bacteria to variousstress-inducing conditions, including low pH, salts or treatments with cleaners anddisinfecting agents (Cebrián et al.,2010). However, the use of stressing factors in food processing can causesublethal damage to bacterial cells, and during the injury repair process, these cellscould acquire new abilities to adapt to these stress-inducing agents (direct-tolerance),leading to impacts on food safety and preservation (Silva-Angulo et al., 2014). These responses can alsoactivate the intrinsic resistance mechanisms that concomitantly decrease thesusceptibility of cells to other unrelated antimicrobial compounds or procedures(cross-tolerance), meaning major implications for food processing in which multiplestresses are often applied to control microbial growth and survival (Greenacre and Brocklehurst, 2006).
Staphylococcus aureus is one of the most common causes of foodbornediseases worldwide, causing a typical intoxication through the ingestion of enterotoxinsthat have been pre-formed in foods by enterotoxigenic strains (Wang et al., 2013). Previous studies haveshown that S. aureus is capable of developing tolerance to heat, acidicpH and salts when exposed to sublethal stress conditions (Bikels-Goshen et al., 2010; Cebrián et al., 2010). The tolerance acquiredby S. aureus to many procedures used by the food industry to controlbacterial growth and survival has motivated the research and development of noveltechniques to control this bacterium in foods (GomesNeto et al., 2012; Luzet al., 2013).
In this context, essential oils and their active components have received attention asalternative anti-S. aureus compounds to use in foods (Bakkali et al., 2008). Earlier investigationsrevealed that Origanum vulgare L. essential oil (OVEO) possessesbroad-spectrum antimicrobial activity even at low concentration, with interestingresults in inhibiting the growth of a variety of bacteria and food-related fungi whenassayed alone (Nostro et al.,2004; Sousa et al.,2013; Souza et al.,2009; Gomes Neto et al.,2012) or in combination with other antimicrobial compounds or procedures usedby food industry (Barros et al.,2009b; Oliveira et al.,2010). Studies have also revealed that OVEO possesses a strong capacity toinhibit S. aureus in synthetic, food based-broth and in food models,besides to suppress the action of some related virulence factors, including enterotoxin,biofilm production and synthesis of the enzymes lipase, protease and coagulase (Nostro et al., 2007; Barros et al., 2009a). Although theanti-S. aureus activity of OVEO has already been reported, littleattention has been paid to the response of this bacterium when exposed to sublethalamounts of this substance.
The aim of this study was to assess the effects of exposing enterotoxigenic S.aureus strains that were isolated from foods to sublethal OVEOconcentrations for different time points on the development of bacterialdirect-tolerance and cross-tolerance to salts and organic acids typically used by thefood industry. To the best of our knowledge, this is the first study on the induction ofdirec-tolerance or cross-tolerance in enterotoxigenic S. aureus strainsfrom foods in which the strains were subjected to OVEO habituation and further assessedfor modulation of the Minimum Inhibitory Concentration (MIC) values.
Materials and Methods
Antimicrobial agents
The antimicrobial agents used in this study were OVEO (Laszlo Aromaterapia Indústriae Comércio Ltda., Minas Gerais, Brazil), sodium chloride (NaCl P.A.), potassiumchloride (KCl), glacial acetic acid (AA) and lactic acid 85% (LA). The NaCl, KCl, AAand LA were obtained from Vetec Química Fina Ltda. (Rio de Janeiro, Brazil). The OVEOassayed in this study present carvacrol as the most prevalent compound (66.1 g/100mL), followed for p-cymene (12.4 g/100g) and γ-terpinene (8.3g/100g), according to the technical report presented by the supplier.
OVEO solutions (40-0.3 μL mL−1) were prepared in sterile brain heartinfusion (BHI) broth (Himedia, India) with Tween 80 (1%) (Sigma Aldrich, USA) as anemulsifier. Preliminary test to ensure that the antibacterial activity was due to theOVEO and not to Tween 80 was performed, and the results demonstrated that Tween 80 atthe given concentration (1%) did not inhibit the growth of the assayed bacterialstrains cultivated in BHI broth. Solutions of NaCl (600-50 mg mL−1), KCl(600-50 mg mL−1), AA (160-1.25 μL mL−1) and LA (160-1.25 μLmL−1) were prepared in sterile BHI broth.
Bacterial strains
The test organisms used in this study included enterotoxigenic S. aureus strainsisolated from foods (S. aureus FRI-S-6, producing staphylococcalenterotoxins (SE) A and B, which were isolated from frozen shrimp; S.aureus FRI-196-E, producing SEA and D, which were isolated from anunknown food; and S. aureus FRI-326, producing SEE, which wasisolated from a chicken-based meal) (Bergdollet al., 1971; Wu andBergdoll, 1971) and were generously provided by Food Research Institute(Madison, Wisconsin, USA). A standard type strain (S. aureus ATCC13565, producing SEA, isolated from ham) (Johnsonet al., 1991) was also used as a test strain. Stockcultures were kept at 4 °C, and prior to being used in the assay, each strain wasgrown in BHI broth at 37 °C for 18 h (later exponential growth phase), harvested bycentrifugation (4500 g, 15 min, 4 °C), washed twice in sterilesaline solution (NaCl, 0.85%) and resuspended in sterile saline solution to obtainstandard cell suspensions at which the OD reading at 660 nm (OD660) was0.1 (c.a. 107 cfu mL−1) (McMahon et al., 2008).
Determining the Minimum Inhibitory Concentration (MIC)
A modified microtiter plate assay was used to determine the MIC of OVEO, NaCl, KCl,acetic acid (AA) and lactic acid (LA) (17). The 96-well plates were prepared bydispensing 90 μL of OVEO (40 to 0.3 μL mL−1), salt (600-50 mgmL−1) or acid (160 to 1.25 mL mL−1) solutions into 90 μL ofdoubly concentrated BHI broth in each well. Finally, 10 μL of a bacterial suspension(c.a. 107 cfu mL−1) was added to each well. The microplate waswrapped loosely with cling film to ensure the bacteria would not become dehydratedand the OVEO would not volatilize. Each plate included a set of controls without theantimicrobial test agents. The plates were prepared in triplicate, and they wereincubated statically at 37 °C for 24 h in a microplate incubator/reader (EON model,Biotek Inc., USA). After the incubation period, MIC values were confirmed as thelowest concentrations of OVEO, NaCl, KCl, AA or LA at which the OD660 was< 0.01 (McMahon et al.,2008).
Assaying the induction of direct-tolerance
The induction of direct-tolerance was performed by exposing the test strains tosublethal OVEO concentrations in broth for different time intervals, followed by adetermination of the MIC values for the same stressing agent. For this assay, 4 mL ofBHI broth was inoculated with 1 mL of bacterial suspension (c.a. 107 cfumL−1); thus, OVEO was added at the appropriate amount to obtain thedesired final concentration (1/2 MIC or 1/4 MIC), followed by static incubation at 37°C. An aliquot of each system was taken after 24, 48 and 72 h of incubation (andstandardized again to OD660 values of 0.1, c.a. 107 cfumL−1 of habituated cells) and used as inoculum (10 μL) to determine theMIC of OVEO by using the same microtiter plate assay before described (McMahon et al., 2008). Theinduction of direct tolerance in the bacteria was assessed by comparing the MIC ofOVEO against those of the tested strains before and after the habituation treatmentwith the same stressing agent. Control systems without exposure to OVEO were assayedsimilarly (by non-habituation treatment).
Assaying the induction of cross-tolerance
The induction of bacterial cross-tolerance was performed by exposing the test strainsto sublethal amounts of OVEO in broth for different time intervals, followed bydetermination of MIC values of the assayed heterologous stressing agents (NaCl, KCl,AA and LA). For this assessment, 4 mL of BHI broth was inoculated with 1 mL ofbacterial suspension (c.a. 107 cfu mL−1); thus, the OVEO wasadded at an appropriate amount to obtain the desired final concentration (1/2 MIC or1/4 MIC), followed by static incubation at 37 °C. After 24, 48 and 72 h ofincubation, an aliquot of each system was taken (standardized again toOD660 values of 0.1, c.a. 107 cfu mL−1 ofhabituated cells) and used as an inoculum (10 μL) to determine the MIC of the NaCl,KCl, AA and LA by using the same microtiter plate assay before described (McMahon et al., 2008). Theinduction of bacterial cross-tolerance was assessed by comparing the MIC values ofNaCl, KCl, AA and LA against the tested strains before and after the habituationtreatment with sublethal amounts of OVEO. Control systems without OVEO exposure wereassayed similarly (non-habituation treatment).
The assays were performed in triplicate on three separate experiments, and theresults were expressed as modal or median values; where the values were the same,only the modal values were presented (McMahonet al., 2008).
Results and Discussion
The habituation effects of some enterotoxigenic S. aureus strains onthe development of bacterial direct-tolerance and cross-tolerance after differentintervals of exposure to sublethal concentrations of OVEO with regards to the modulationof MIC values were assessed in this study. The MIC values of OVEO against the teststrains ranged from 2.5 to 10 μL mL−1 (Table1). NaCl, KCl, AA and LA yielded MIC values of 200 mg mL−1, 300 mgmL−1, 2.5 μL mL−1 and 10 μL mL−1, respectively,against all the assayed strains.
The minimum inhibitory concentration of the essential oil from O.vulgare L. against different enterotoxigenic strains of S.aureusthat were isolated from food7s
| Strains | MIC of OVEO (μLmL−1) |
|---|---|
| S. aureus FRI-S-6 | 2.5 |
| S. aureus FRI-196-E | 2.5 |
| S. aureus FRI-326 | 10 |
| S. aureus ATCC 13565 | 10 |
MIC: Minimum Inhibitory Concentration; OVEO: Origanum vulgareL. essential oil.
The OVEO MIC values against the habituated cells were maintained or decreased up tofive-fold when compared with the previously determined MIC values (10 μL mL−1to 0.6 μL mL−1) (Table 2), indicatingthat there was no induction of direct-tolerance in these cells following OVEOhabituation up to 72 h. The decreased MIC of OVEO against habituated enterotoxigenicS. aureus cells was related to time of exposure to the sublethalconcentrations of this substance because the smaller MIC values were generally foundagainst cells that were pre-exposed to OVEO for 72 h, when compared with non-habituatedcells (control assay). During all of the assessed time intervals, the MIC values of OVEOagainst non-habituated cells ranged from 5 to 10 μL mL−1.
The minimum inhibitory concentration of the essential oil from O.vulgare L. against different enterotoxigenic strains of S.aureusthat were isolated from foods, with or without habituation tothe same stressing agent up to 72 h
| Strains | Treatment | MIC (μLmL−1) | ||
|---|---|---|---|---|
| 24 h* | 48 h* | 72 h* | ||
| S. aureus FRI-S-6 | Control (0 μL OVEOmL−1) | 5.0 | 5.0 | 2.5 |
| 1/2 MIC OVEO (1.25 μLOVEO mL−1) | 2.5 | 1.25 | 0.6 | |
| 1/4 MIC OVEO (0.6 μL OVEOmL−1) | 2.5 | 1.25 | 0.6 | |
| S. aureus FRI-196-E | Control (0 μL OVEOmL−1) | 5.0 | 2.5 | 2.5 |
| 1/2 MIC OVEO (1.25 μLOVEO mL−1) | 0.6 | 0.6 | 0.6 | |
| 1/4 MIC OVEO (0.6 μL OVEOmL−1) | 0.6 | 0.3 | 0.6 | |
| S. aureus FRI-326 | Control (0 μL OVEOmL−1) | 10 | 5 | 5 |
| 1/2 MIC OVEO (5 μL OVEOmL−1) | 1.25 | 0.6 | 0.6 | |
| 1/4 MIC OVEO (2.5 μL OVEOmL−1) | 0.6 | 0.6 | 0.6 | |
| S. aureus ATCC 13565 | Control (0 μL OVEOmL−1) | 10 | 5 | 5 |
| 1/2 MIC OVEO (5 μL OVEOmL−1) | 1.25 | 0.6 | 0.6 | |
| 1/4 MIC OVEO (2.5 μL OVEOmL−1) | 1.25 | 0.6 | 0.6 | |
*hours of previous habituation or not in the assayed sublethal concentrations ofO. vulgare L. essential oil;
MIC: Minimum Inhibitory Concentration; OVEO: O. vulgare L.essential oil.
This lack of direct-tolerance induction in the test strains following different OVEOhabituation times is interesting; previous studies showed that S.aureus was able to develop tolerance after being exposed to other sublethalenvironmental conditions. The habituation of S. aureus CECT 4459 from 5min to 2 h to stress conditions caused by acid (hydrochloric acid pH 2.5), alkali(sodium hydroxide pH 12.0), hydrogen peroxide (50 mM) and heat (58 °C) in tryptone soybroth resulted in increased direct-tolerance to all tested antimicrobial agents when thesurvivor/death curves (viable cell counts) were observed. The development of bacterialcross-tolerance to hydrogen peroxide and acid after submitting the cells to heat shock,in addition to their increased tolerance to heat and hydrogen peroxide after acid shock,was already reported (Cebrián et al.,2010).
Existing literature on the development of tolerance by S. aureus whenexposed to sublethal amounts of essential oils regarding the modulation of MIC values isscarce, making any extensive comparative discussion of the results difficult. Thesusceptibility of methicillin-resistant/-sensitive S. aureus isolatesto tea tree (Melaleuca alternifolia) essential oil (TTEO) and toantibiotic were determined by modulating the MIC values following a 72 h habituation tosublethal TTEO concentrations in Luria-Bertani broth. This habituation led tostress-hardening with a subsequent increase in the MIC values (≥ 2-fold increase) ofTTEO and of different clinically important antibiotics (mupirocin, chloramphenicol,linezolid and vancomycin) (McMahon etal., 2008). Another study assessed the increased resistance (byemploying viable cell counts) of four enterotoxigenic strains of S.aureus (CECT 976, CECT 4459, CECT 4465 and CECT 4466 that produced SEA, B, Cand D, respectively) after habituating to a high temperature (58 °C) in McIlvainecitrate phosphate buffer, and the development of heat tolerance was observed upon theentry of cells into the stationary phase of growth (Cebrián et al., 2007).
In accordance with the direct-tolerance results, the MIC values for NaCl, KCl, AA and LAagainst the OVEO-habituated cells were the same or decreased (two- to six-fold) in eachassessed exposure time interval when compared with MIC values against non-habituatedcells (control cells) (Table 3). However, formost of the assessed time intervals, the MIC values remained the same. There was noclear effect of the time-of-habituation with OVEO in relation to the sensitivity ofhabituated cells to NaCl, KCl and LA. Otherwise, the decrease in the MIC values of AAagainst habituated-cells always occurred after 48 h (S. aureus ATCC13565) or 72 h (S. aureus FRI-S-6) of exposure to sublethal amounts ofOVEO.
The minimum inhibitory concentrations of sodium chloride, potassium chloride,acetic acid and lactic acid against enterotoxigenic strains of S.aureus that were isolated from foods, with or without habituation tothe essential oil from O. vulgare L. up to 72 h
| Strains | Treatment | Sodium chloride MIC (mgmL−1) | Potassium chloride MIC(mg mL−1) | Acetic acid MIC (μLmL−1) | Lactic acid MIC (μLmL−1) | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 24 h* | 48 h* | 72 h* | 24 h* | 48 h* | 72 h* | 24 h* | 48 h* | 72 h* | 24 h* | 48 h* | 72 h* | ||
| S. aureus FRI-S-6 | Control (0 μL OVEOmL−1) | 200 | 200 | 200 | 300 | 200 | 300 | 2.5 | 2.5 | 2.5 | 10 | 5 | 5 |
| 1/2 MIC OVEO (1.25 μLOVEO mL−1) | 150 | 75 | 100 | 200 | 200 | 300 | 2.5 | 2.5 | 1.25 | 10 | 5 | 5 | |
| 1/4 MIC OVEO (0.6 μL OVEOmL−1) | 150 | 75 | 75 | 200 | 300 | 300 | 2.5 | 2.5 | 1.25 | 10 | 5 | 5 | |
| S. aureus FRI-196-E | Control (0 μL OVEOmL−1) | 200 | 200 | 150 | 300 | 300 | 300 | 2.5 | 2.5 | 2.5 | 10 | 5 | 5 |
| 1/2 MIC OVEO (1.25 μLOVEO mL−1) | 150 | 150 | 75 | 300 | 300 | 150 | 2.5 | 2.5 | 2.5 | 10 | 5 | 5 | |
| 1/4 MIC OVEO (0.6 μL OVEOmL−1) | 150 | 200 | 150 | 300 | 300 | 300 | 2.5 | 2.5 | 2.5 | 10 | 5 | 5 | |
| S. aureus FRI-326 | Control (0 μL OVEOmL−1) | 200 | 150 | 150 | 300 | 300 | 300 | 2.5 | 2.5 | 2.5 | 10 | 5 | 5 |
| 1/2 MIC OVEO (5 μL OVEOmL−1) | 50 | 50 | 100 | 50 | 50 | 100 | 2.5 | 2.5 | 2.5 | 5 | 5 | 5 | |
| 1/4 MIC OVEO (2.5 μL OVEOmL−1) | 100 | 150 | 100 | 200 | 200 | 200 | 2.5 | 2.5 | 2.5 | 10 | 5 | 5 | |
| S. aureus ATCC 13565 | Control (0 μL OVEOmL−1) | 150 | 150 | 200 | 300 | 300 | 300 | 2.5 | 2.5 | 2.5 | 10 | 5 | 5 |
| 1/2 MIC OVEO (5 μL OVEOmL−1) | 50 | 50 | 100 | 50 | 50 | 50 | 2.5 | 1.25 | 1.25 | 5 | 5 | 5 | |
| 1/4 MIC OVEO (2.5 μL OVEOmL−1) | 100 | 100 | 150 | 150 | 200 | 200 | 2.5 | 1.25 | 1.25 | 10 | 5 | 5 | |
*hours of previous habituation (or not) to O. vulgare L.essential oil at the assayed sublethal concentrations;
MIC: Minimum Inhibitory Concentration; OVEO: O. vulgare L.essential oil.
The overnight cultivation of S. aureus ATCC 6538 in meat brothcontaining the essential oil from Rosmarinus officinalis L. (ROEO), andits majority compound 1,8-cineole (CIN), at sublethal amounts (ROEO 10 and 5 μLmL−1; CIN 20 and 10 μL mL−1), induced no direct-tolerance orcross-tolerance (NaCl 100 g l−1; lactic acid pH 5.2; high temperature 45 °C)in the tested bacte-ria when assessed by viable cell count and growth/sur-vivalbehavior. The cells subjected to pre-habituation with ROEO or CIN revealed an increasedsensitivity to LA, high temperature and NaCl when compared with the non-habituatedcells. The repeated exposure of S. aureus cells to amounts of essentialoils (or related compounds) lower than their MICs could cause an imbalance between theanabolism and catabolism that was sufficient to stop growth and cause the cells to beunable to maintain their viability (Gomes Neto et al., 2010).
The sublethal injury caused by phenolic compounds in essential oils, such as thecarvacrol or thymol present in OVEO (Barros etal., 2009a; Luz etal., 2013), can result in a damaged bacterial cell membrane, withchanges in its structure and permeability (Espinaet al., 2013). Furthermore, an injury of the microbialcell membrane provided by sublethal concentrations of antimicrobial compounds may affectthe ability of the membrane to osmoregulate the cell adequately or to exclude toxicmaterials (Carson et al., 2002),and consequently, the decreased tolerance to salts or acids caused by OVEO may berelated to membrane damage in sublethally injured bacteria. The cultivation ofS. aureus strains isolated from foods in nutrient broth containingsublethal concentrations of OVEO (0.3 and 0.15 μL mL−1) for 24 h interferedwith the metabolic activity of the assayed strains, inhibiting the activity of theenzymes lipase and coagulase and enterotoxin production (Barros et al., 2009b). The ability of OVEO to suppressenzyme synthesis and/or activity in S. aureus result in blocked proteinsynthesis (Nostro et al., 2001;Oliveira et al., 2010; Gomes Neto et al., 2012), and thisaction could also be related to the difficulty of the different enterotoxigenic strainsof S. aureus in developing direct-tolerance or cross-tolerance underthe conditions used in this study.
The results from this study confirm that OVEO is an effective anti-staphylococcalsubstance because exposing enterotoxigenic S. aureus strains tosublethal amounts of OVEO caused no direct-tolerance and cross-tolerance induction tostressing agents, such as NaCl, KCl, LA an AA. Exposing the test strains to sublethalconcentrations of OVEO maintained or increased susceptibility to the same stressingagent and to the assayed heterologous stressing agents, suggesting that OVEO had noimpact on the induction of tolerance in enterotoxigenic strains of S.aureus as assessed by the modulation of MIC values. Thesefindings reinforce the possible rational use of OVEO by food industry to control thegrowth and survival of enterotoxigenic S. aureus in foods whenconsidered their efficacy to inhibit the growth of this bacterium besides the lowcapacity to induce bacterial tolerance.
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