Mode of action of Lippia graveolens essential oil on Salmonellaenterica subsp. enterica serovar Typhimurium

The essential oils are a powerful natural resource with antibacterial activity. This work aims at the mode of action of Lippia graveolens Kunth essential oil of plants growing in Cuba on Salmonella enterica subsp. enterica serovar Typhimurium. The effects of this oil on cell integrity were determined by time-kill, bacteriolysis and loss of 260 and 280-nm-absorbing material assays and total proteins leakage. Also, depolarization of the membrane by essential oil was monitored and intracellular and extracellular ATP was measured. The transmission electron microscopy (TEM) was used for observed morphologic change. Minimum inhibitory concentration (MIC) of L. graveolens essential oil and minimum bactericide concentration (MBC) were 0.4 and 0.8 mg/mL respectively. This essential oil showed a bactericidal action against over S . Typhimurium in a few minutes. After treatment, the cell lysis was not occurred, but little intracellular material and total proteins leakage were observed. This essential oil depolarizes the cell membrane, disturb metabolic processes and changes the structure of cytoplasmic membrane. These results suggest as primary mode of action of L. graveolens essential oil over S . Typhimurium an increased permeability of the membrane and depolarization of inside membrane, inhibition of ATPase or disturbance in proton motive force that nally provokes death of cells. L. graveolens essential oil is a botanic resource can be used for the control of the salmonellosis, foodborne disease.

Essential oils are aromatic and volatile liquids, which have a complex composition and are obtained by different methods of extraction as hydrodistillation, steam distillation and cold-press techniques fom fresh and dry plant material, which includes owers, roots, barks, leaves, seeds, husks, fruits, wood and whole plants (Aziz et al., 2018;Preedy, 2015). Essential oils stand out among natural products of plant origin for their versatility in biological properties. Antibacterial activity as broad-spectrum substances against Gram-positive and Gram-negative bacteria, including activity on antibiotic-resistant strains, is one of their main advantages (Guinoiseau et al., 2015;Leyva-López, Gutiérrez-Grijalva, Vazquez-Olivo, & Heredia, 2017).
Plants of the Verbenaceae family are known by the capacity for essential oils production with diversity uses. The Lippia genus includes more than 100 species of plants, which have been used in traditional Latin American medicine (Almeida et al., 2018). One of the major commercial species is Lippia graveolens Kunth known as Mexican oregano, an aromatic plant native of Southern North America, it is used in folk medicine mainly in dermatological, gastrointestinal and respiratory affections (Preedy, 2015) and as culinary seasoning (Pascual, Slowing, Carretero, Sánchez Mata, & Villar, 2001). The essential oil of this plant exhibits antibacterial activity against Gram-positive and Gram-negative bacteria, which is related to major components as thymol, carvacrol, p-cymene (Hernández et al., 2009).
Studies on the antimicrobial activity of essential oils are abundant throughout the world, however, in terms of understanding the mode of action of these natural products as antimicrobials, more depth is needed with an integrative vision. This article is aimed at the mode of action of the essential oil of L. graveolens of plants grown in Cuba on strain of Salmonella enterica subsp. enterica serovar Typhimurium.

Essential Oil
The essential oil of L. graveolens was supplied by the Chemical Ecology Laboratory of National Center of Planta and Animal Health of Cuba. The plant material was collected in the town of Jaruco, Mayabeque, Cuba, located at 23. 076573 and -81. 964266. The essential oil was obtained by the method of hydrodistillation for three hours, using a Clevenger equipment (Benachour, Ramdani, Lograda, Chalard, & Figueredo, 2020). This essential oil was characterized and analyzed its chemical composition analyzed by GC-MS in previous work. The majority compounds include thymol (42.7%), carvacrol (22.2%), pcymene (6.5%).

Bacterial Strains and Growth Conditions
Salmonella enterica subsp. enterica serovar Typhimurium ATCC 14028 (CIP 104115) strain was purchased from the Collection of Institute Pasteur (CIP, Paris, France). Before each experiment, the strain was routinely grown at 37˚C on Mueller-Hinton 2 agar (MHA, Oxoid).

Disc Diffusion Assays
The agar diffusion method was used for the determination of antibacterial activities (CLSI, 2018).
Inoculum were prepared by diluting overnight cultures in Mueller-Hinton broth (MHB, Oxoid) medium to approximately 10 6 CFU/mL. Filter paper discs (6 mm diameter, Dominique Dutscher) were placed onto the inoculated Petri dishes containing Mueller-Hinton 2 agar (MHA, Oxoid) and was applied 15 µL of the tested products in the paper discs. After keeping at room temperature for 1 h, plates were incubated at 37˚C for 24 h. Diameters of inhibition zones were measured (mm) and recorded as the mean ± standard deviation (SD). Each test was performed in triplicate on at least three separate experiments. Cipro oxacin discs (5 µg, Bio-Rad) was used as positive control.

Minimum Inhibitory Concentration and Minimum Bactericidal Concentration Assays
The minimum inhibitory concentration (MIC) assays were performed by a rapid INT (piodonitroterazolium chloride, Sigma-Aldrich) colorimetric assay (Guinoiseau et al., 2015). The L. graveolens oil was serially twofold diluted in dimethylsulfoxide (DMSO, Sigma-Aldrich). The DMSO was previously tested for antibacterial activity and no detrimental effect on bacterial growth has been observed at the concentration used. The solutions obtained were then added (10 µL) to a 96-well microplate containing 190 µL of MHB (1:20, v/v) inoculated with 10 6 CFU/mL. The microplates were incubated at 37˚C for 24 h. The MICs of the samples were then detected following addition (50 µL) of INT (0.2 µg/mL). Viable bacteria reduced the yellow dye to pink. The MIC is de ned as the lowest sample concentration that prevents this change and results in the inhibition of bacterial growth. All determinations were performed in triplicate and a negative control, consisting of MHB with DMSO (5%, v/v), was systematically included. An inoculation loop was introduced in each well and seeded on a Muller Hinton agar plate, free of the antimicrobial agent to determine the minimum bactericidal concentration (MBC), which is de ned as the lowest concentration of the oil that resulted in a negative subculture.

Time-Kill Studies
Time-kill procedure was performed according to the method described by Klepser et al (Klepser, Ernst, Lewis, Ernst, & Pfaller, 1998) and modi ed by Viljoen et al. (Viljoen et al., 2003). The antibacterial activity of L. graveolens essential oil used at their MIC were evaluated against S. Typhimurium by measuring the reduction in the number of CFU (Colony Forming Units) per milliliter at 0,15,30,45,60,120,180,240 minutes and 24 h of incubation at 37°C with agitation. The tested product was applied at MHB with DMSO (0.1%) inoculated with 10 6 CFU/mL. The inoculated medium containing DMSO and without essential oil was used as control. At each evaluation time, aliquot of 100 µL was taken and serially diluted in MHB; from each serial dilution step, 100 µL were transferred to two MHA plates in numbered sections and incubated at 37°C for 24 h. Colony-forming units were counted after incubation. This assay was performed in triplicate.

Cell Integrity Studies
Bacteriolysis assay was carried out according to the standard method described by Carson et al. (Carson, Mee, & Riley, 2002). A bacterial suspension was prepared by inoculating two colonies of S. Typhimurium from overnight cultures on MHA into 40 ml of MHB, which was incubated at 37˚C for 24 h with shaking. After incubation, the bacteria were separated from the growth medium by centrifugation at 10,472 rad/s for 12 min at 4˚C, washed twice with phosphate-buffered saline (PBS, pH 7.4) and resuspended in PBS supplemented with 0.01% Tween 80 (PBS-T, v/v). The bacterial suspension was adjusted so that the optical density (OD) at 550 nm of a 1 in 100 dilution was 0.310 (∼3 × 10 8 CFU/mL). L. graveolens essential oil was added to the bacterial suspension at the MIC. PBS-T was added to the control suspension. The suspensions obtained were mixed for 20 s with a Vortex mixer. Samples (1 mL) were taken in duplicate every 30 minutes from 0 h to 2 h. They were centrifuged and the pellet was resuspended in 1mL of PBS-T. The optical density at 620 nm was measured immediately (Jasco UVisco UV-1200 spectrophotometer). This assay was performed on three independent experiments. The results were expressed as a ratio (in percent) of the OD620 at each time point versus the OD620 at 0 min.

Loss of Cytoplasmic Material
The release of 260-nm and 280-nm absorbing materials from S. Typhimurium cells treated with L.
graveolens essential oil at MIC, was performed on the bacterial suspension (10 8 CFU/mL) in PBS supplemented with 0.01% Tween 80 (PBS-T, v/v). Suspension without essential oil was used as a control. The samples were incubated at 37˚C with shaking. They were taken at time 0, 30, 60, 90 and 120 min, and centrifuged at 10,472 rad/s for 12 minutes. The absorbance of the obtained supernatant was measured at 260 and 280 nm using a spectrophotometer (Jasco UVisco UV-1200). Each test was performed on three independent experiments. The results were expressed as a difference of the OD260 or OD280 at each time point versus the OD260 or OD280 at time 0.

Determination of released proteins
Total protein losses released by the action of the L. graveolens essential oil over S. Typhimurium were determined by Lowry's method (Lowry, Rosebrough, Farr, & Randall, 1951). The preparation of the inoculum and the samples was performed equal to the loss of material. Also, 1% sodium lauryl sulfate (SDS) and bovine serum albumin (BSA) were used as a standard. After treatment with the MIC, the samples were taken at time 0, and 120 minutes, and centrifuged at 10,472 rad/s for 12 minutes. Suspension without essential oil was used as negative control. Lowry's method was applied and the optical density was measured at 730 nm in the Jasco UVisco UV-1200 spectrophotometer. Each sample was prepared in triplicate and three independent experiments were performed. The results were expressed as mean ± standard error.

Measurement of Intra-and Extra-Cellular Adenosine 5'-Triphosphate (ATP) Concentrations
To determine the action of L. graveolens essential oil on energetic molecules, the intracellular and extracellular ATP concentrations were measured as described by Gill and Holley (Gill & Holley, 2006), with modi cations of Turgis et al. (Turgis, Han, Caillet, & Lacroix, 2009). The overnight cultures of S. Typhimurium were centrifuged for 10 minutes at 10,472 rad/s and the supernatants were removed. The cell pellets were washed two times with 20 mM of phosphate potassium buffer (PPB, pH 7.0) and then cells were collected by centrifugation under the same conditions. A cell suspension (10 8 CFU/mL) was prepared in PPB (20 mM; pH 7.4) with glucose (50 mM) and DMSO (0,1%). The essential oil was applied at the MIC. 30 mM of polymyxin B (PMB) was used as positive control and the suspension without essential oil as the negative control. The treatments were incubated at 37°C for 7 min with agitation. Then the samples were centrifuged at 10,472 rad/s for 12 minutes and the supernatant was separated from the pellet.
For ATP extra-cellular, the supernatants were put quickly in ice, and 50 µL of the samples were added to 96 well black plate for duplicate. Then, 50 µL of MIXED kit for ATP (ATP-kit, Sigma) was applied in each well, and the plate was incubated for 30 min in ice in darkness. Fluorescence was measured using FP-83000 Jasco spectro uorophotometer (excitation wavelength (λ ex ) = 535 nm and excitation wavelength (λ em ) = 587 nm). Each test was performed on three independent experiments. The results were expressed as a ratio (in percent) of the relative uorescence unit (RFU) at each time point versus the RFU of the DMSO control.
For ATP intra-cellular, the pellets were resuspended in 1 mL of NaCl (0,85%) and centrifugated at the same previous conditions. The cells were resuspended in 200 µL of ATP buffer assays (ATP-kit, Sigma). After, 5 µL of 15% ammonium bromide (CTAB, Sigma) were added in each treatment and incubated at room temperature for 15 min. The samples were centrifuged and 50 µL of the supernatant was taken and applied to 96 well black plate for duplicate. Then this experiment was continued like for ATP extra-cellular assay.

Membrane depolarization assay
The cytoplasmic membrane depolarization activity of L. graveolens essential oil over S. Typhimurium was measured by using 3,3'-dipropylthiadicarbocyanine iodide (DiSC 3, Sigma) (Chehimi, Pons, Sablé, Hajlaoui, & Limam, 2010). A S. Typhimurium overnight culture in MHB was centrifuged at 10,472 rad/s for 12 minutes at 4˚C, washed twice with phosphate-buffered saline (PBS, pH 7.4), and resuspended in buffer solution (10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) with glucose 50 mM at pH 7.0, and DMSO 0.1%). The bacterial suspension was adjusted at 10 8 CFU/mL and 99 µL was applied to 96 well black plate for duplicate. The cells were incubated with 1.68 µM DiSC 3 at room temperature with shaking for 1h. Then KCl was added to a nal concentration of 0.1 M to equilibrate the cytoplasmic and external K + and incubated for 5 min. An aliquot of 1µL of L. graveolens essential oil was applied at MIC and, as positive controls, 30 mM of polymyxin B (PMB) and 4 mM CTAB. Suspension without essential oil was used as the negative control. Fluorescence was monitored with an FP-83000 Jasco spectro uorophotometer (excitation wavelength (λ ex ) = 622 nm and excitation wavelength (λ em ) = 670 nm). The results were expressed at relative uorescence unit (RFU). This assay was performed on three independent experiments.

Transmission Electron Microscopy (TEM)
A suspension of S. Typhimurium in the exponential phase of growth was prepared by inoculating then incubating 80 mL of MHB at 37˚C for 24 h with shaking. The bacterial suspension was adjusted so that the optical density at 620 nm (OD620) of a 1 in 100 dilution in MHB was 0.200 (10 8 CFU/mL) with Tween 80 (0.1%, v/v). The cells of S. Typhimurium were treated with the MIC of L. graveolens essential oil for 7 min. The negative control was the suspension without essential oil. After centrifugation at 10,472 rad/s for 10 minutes, the pellets were rst xed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 1 h at room temperature and then post-xed in 2% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.2) for 1 h at room temperature. The post xed microbial pellets were processed in graded ethyl alcohol, propylene oxide, Spurr resin and cured for 24 h at 45˚C. Ultrathin sections were stained with uranyl acetate followed by lead citrate and then examined with a transmission electron microscope (HITACHI H-7650) at an accelerating voltage of 80 kV.

Susceptibility of Salmonella Typhimurium to Lippia graveolens essential oil
The antibacterial activity of L. graveolens essential oil over S. Typhimurium strain is presented in Table 1. A great zone of inhibition was revealed by the action of this essential oil and It is correspondence with a strong inhibition zone (> 20.1 mm) (Mazzarrino et al., 2015). The results amount the essential oils and the antibiotic control showed signi cant differences, with larger value for the natural product. The MIC value was 0.4 mg/mL and double value of MIC was the MBC.

Time-Kill Studies
The effect of L. graveolens essential oil on the growth of S. Typhimurium demonstrated a negative kinetic with reduced viability at MIC (Fig. 1). This essential oil had a bactericidal end-point (99.9% or ≥ 2 log10 of inhibition) in only 15 minutes of treatment, time that was taken into account for the remainder of the experiments. The total inhibition was at 2 h of treatment and was remained for 24 h (data not shown). The control grew up in the time.

Cell Integrity Studies
The cells of S. Typhimurium not showed a change in OD at 620 nm after 2h of treatment with L. graveolens essential oil at MIC (Figure 2). In all times, the samples had more than 80% of cells integrity indicating that cell lysis did not occur.

Loss of Cytoplasmic Material
The L. graveolens essential oil at MIC induced the releasing of 260-nm and 280-nm absorbing materials from S. Typhimurium (Figure 3). The loss of absorbent material at both 260 and 280 nm was observed signi cantly (p < 0.05) after 60 min of exposure to the oil. The maximum proportion obtained at 2h of treatment; however, was less than 20 %.

Determination of released proteins
To corroborate the release of cellular components by the action of essential oil, total proteins were determined. This test was carried out at time 0 and 2 h, time of maximum release of cellular content demonstrated in the cytoplasmic material loss test. No signi cant changes were shown between oil treatment and the control at the time of exposure. Nevertheless, the results of total proteins leakage of S. Typhimurium for the action of the essential oil (0,35 ± 0,27 mg/mL) revealed larger concentration than the control (0,04 ± 0,02 mg/mL) (p < 0.05).

Membrane depolarization assay
After treatment with L. graveolens essential oil, S. Typhimurium cells have disturbances in the membrane ( Figure.4). The change of value of the membrane potential was similar to the positive control, polymyxin B, which is known to induce the formation of pores in the membrane (Poirel, Jayol, & Nordmanna, 2017).

Measurement of Intra-and Extra-Cellular Adenosine 5'-Triphosphate (ATP) Concentration
Measurements of ATP intracellular and extracellular were determined in S. Typhimurium cells after 7 minutes treatment with L. graveolens essential oil at MIC ( Figure 5). Intracellular ATP was reduced signi cantly (p < 0.05) by the essential oil (7.8% of RFU), in the same way as the antibiotic polymyxin B (8.6% of RFU). The concentration of extracellular ATP was very low (less than 10% of RFU) whatever the treatment but the highest value was observed in presence of the essential oil.

Transmission Electron Microscopy (TEM)
The essential oil effect over S. Typhimurium was observed by transmission electron microscopy ( Figure  6). L. graveolens affected the cellular integrity of Salmonella cells after only 7 minutes of exposure. The majority of cells conserved the external morphology. However, these cells presented a longer distance between the outside and plasmic membrane, which was observed as more periplasmic space. The plasmic material was conserved inside of the plasmic membrane, but a change in density was observed. The cells untreated were observed with the typical structure of gram-negative.

Discussion
The Lippia graveolens essential oil of plants cultivated in Cuba is a natural product with a phenolic group as majority compounds for 64% of total. Thymol, carvacrol and p-cimene have been reported as majoritarian compounds of essential oil of L. graveolens cultivated in Mexico (thymol 10.43%, carvacrol 43.7%, p-cimene 6,4) (Preedy, 2015) and Rio de la Virgen-Jutiapa in Guatemala (carvacrol 44.8%, pcimene 21.8%, thymol 7.4%) (Salgueiro, Cavaleiro, Gonçalves, & Proença Da Cunha, 2003), but the Cuban cultivar has more quantities of this structurestogether. This composition assures a powerful activity over Salmonella Typhimurium. Results from disc diffusion assay and MIC (0.4 mg/mL) and MBC (0.8 mg/mL) values illustrated that it has a strong and consistent inhibitory effect against this foodborne pathogen.
Irreversible damage at viability cellular was con rmed in a short time (15 minutes) of treatment. Similar results were obtained by Kumar and Chul in 2014, using thymol over this strain (Chauhan & Kang, 2014). Thymol at 0.750 mg/mL reduced the number of cells after 20 min of exposition. The membrane cellular is vital to cell structure, because it intervenes in many processes such as energy conversion, nutrient processing, synthesis of structural macromolecules and secretion of growth regulators (Swamy, Sayeed Akhtar, Sinniah, Akhtar, & Sinniah, 2016). For this, plasmic membrane is considerate as an important site of action, and indeed, many essential oil constituents as thymol and carvacrol, have been described to possess activity on this particular structure (Hyldgaard, Mygind, & Meyer, 2012).
The hydrophobic nature of essential oils can interact with the lipid membrane of bacterial pathogens, resulting in the leakage of the inner cell components, damage in potassium ion re ux, and nally leading to cell death (Saad, Muller, & Lobstein, 2013). Membrane disrupts, affecting the structural stability of the membrane or change in the permeability are principal modi cations over the membrane for action of essential oil (Hyldgaard et al., 2012). Generally, phenolic compounds are responsible of major bactericide effect against food-borne pathogenic bacteria. The phenolic compounds disrupt the cell membrane as well as effectively inhibit the functional properties of the cell, and eventually leaking the inner materials of the cell (Bajpai, Baek, & Kang, 2012).
However, L. graveolens essential oil did not induce abrupt cell lysis on S. Typhimurium within two hours of treatment, and was coherent with the out ow of little absorbent cytoplasmic matrix at 260 nm and 280 nm. The total proteins released showed a slow change in the permeability membrane. These results suggest that the initial action is not the total destruction of the membrane. Conversely, thymol severely disrupted the membrane of S. Typhimurium, resulting in the release of intracellular components such as deoxyribonucleic acid (DNA) in the cell supernatant (Chauhan & Kang, 2014). These differences in results are consistent with the research approach in which has been worked and highlights the need to study the mode of action of essential oils as an integral product and not focus on the study of an only component.
On the other hand, effects in the cellular activity were monitored by messing the intracellular and extracellular ATP concentrations. Indeed, in cells treated with L. graveolens no ATPin, was detected. This drastic decrease is not correlated to the release of ATP in the extracellular medium. The depletion of the internal ATP pool was associated with a change in membrane potential. These results were similar to those demonstrated by Ultee et al in 2002 (Ultee, Bennik, & Moezelaar, 2002). More, those results are also associated to change in membrane potential. So, L. graveolens essential oil, like as carvacrol, does not enhance the membrane permeability for ATP and the depletion of the internal ATP pool results suggest the reduction of ATP synthesis and/or increase ATP hydrolysis. Also, this depletion of the ATP pool following the addition of lipophilic components was observed in other studies. The alcohol fraction extracts of Cistus ladaniferus essential oil have the capacity to vanish the ATPin on Staphylococcus aureus cells, without growing the ATP out (Guinoiseau et al., 2015). More, components like carvacrol, eugenol and cinnamaldehyde inhibit the ATPase activity of Escherichia coli and Listeria monocytogenes (Gill & Holley, 2006).
Adenosine triphosphate (ATP) is used in vital cell processes that require energy as respiration, survival, growth and replication. Other functions of ATP include signaling function, participation in storing and supplying energy in metabolism and enzymatic reactions. In Salmonella, is known that ATP regulate virulence gene mgtC (Mempin et al., 2013). Cellular respiration depends on the respiratory chain in the plasma membrane. This converts redox energy into an electrochemical gradient of protons (protonmotive force) which subsequently drives ATP formation from ADP and phosphate by ATP synthase. The membrane potential and the transmembrane proton gradient are the two parameters of respiratory chain in the plasma membrane. Nevertheless, ATP synthase in bacteria is dependent on the potential component (Dimroth, Kaim, & Matthey, 2000). So, L. graveolens essential oil change the potential of membrane of S. Typhimurium and it can affect the ATP synthase, with damage in the respiratory chain of cellular respiration.
TEM was used to observe the effects of L. graveolens essential oil inside of de S. Typhimurium cells.
Outwardly, the cell remains morphologically intact changes occur inside of the bacteria: (i) outside membrane separates from the plasmic membrane; (ii) differences in the electron-dense structure of cytoplasmic material. This phenomenon can be related to the capacity of essential oil diffuse, penetrate and disorganize the lipid tail region of the membrane (Stevens, 2004). The total volume of the membrane is roughly constant, but membrane thinning results in lateral expansion, affecting the mechanical properties of the membrane. As the area per lipid grows the surface tension increases and the bending modulus decreases dramatically, implying membrane deformation (Stevens, 2004).
The expansion of the membrane also results in a reduction in the packing of the lipid molecules leading to the formation of a large number of cavities, signi cantly reducing the translocation free energy of water molecules across the lipid tail region. As a result, a large number of water translocations occur, and the membrane becomes leaky with the colleapse of the transmembrane potential and additional membrane dysfunction, such as inhibition of ATP production and loss of proton motive force and rapid death of the bacterium (Dimroth et al., 2000;Li et al., 2017).
Diverse researches have already highlighted changes in the morphology external of Salmonella strains by the action of essential oils. Raybaudi-Massilia et al. (Raybaudi-Massilia, Mosqueda-Melgar, & Martín-Belloso, 2006) described damages to the cellular membrane of Salmonella Enteritidis by action of lemongrass essential oil at 5 µL/mL in 24 h, including its disruption of the same and leakage of cell content. Zataria multi ora Boiss. essential oil at 0.3 µg/mL in 1h trigger important morphological damages in Salmonella Typhimurium, such as the increase permeabilization and disruption of membranes which allowed to the dye and the contents of the cells appeared depleted and amorphous (Moosavy et al., 2008). Also, cinnamaldehyde at 0.4% in one hour of treatment causes severe damage in Salmonella MT 2195 (Yossa et al., 2014).
Studies on antimicrobial activity of essential oils are abundant worldwide. However, the subject of the mode of action of these natural products as antimicrobials needs more depth (Carson et al., 2002), and an integral comprehension. This work shows a mode of action of L graveolens essential oil on S. typhimurium, as antimicrobial alternative with an action over the membrane plasmatic and the metabolic process. Lipophilic and proteins studies can be realized in the future researches, for understand the molecular speci c change in the membrane after treatment with the essential oil. Also, the action over metabolic process can be better comprehend with studies over particular enzymes, transcriptome, proteome or production of toxins.
The mechanisms of action are key to avoid antimicrobial resistance to antimicrobials. These studies create the basis for preventing the use of compounds with the same mode of action. The complexity of the chemical composition of essential oils allows different antimicrobial mode of action not only at a particular location but also at different cell sites. Synthetic antibiotic only has one mechanism of action over one target site: cell-wall biosynthesis, protein synthesis (subunit 30S or 50S of ribosome), DNA replication and repair (RNA polymerase, DNA gyrase), folic acid metabolism, membrane structure or Lipid A biosynthesis. Therefore, there is a reemerging interest on essential oils as antibiotic alternative.

Conclusion
This work shows a natural option for treatment Salmonella strains, without risk of antimicrobial resistance. Lippia graveolens essential oil increases permeability of the membrane, disturb the inside membrane, metabolic energy depletionand nally provokes the death of cells in a few minutes. These results suggest the action of the essential oil on the cell membrane and over metabolic energetic process. In depth studies are need to fully understand if another structure or speci c metabolic pathway have been damaged by this essential oil. Lippia graveolens essential oil is a resource with several modes of action as antibacterial with possibility for the preservation of food safety.  Depolarization of the cytoplasmic membrane of cells of S. Typhimurium treated with Lippia graveolens essential oil at MIC (squares), with 30 mM PMB (diamond) and 4 mM CTAB (triangle) monitored by uorescence intensity change. Untreated cells used as control negative (circles). Mean values of triplicate independent experiments and standard deviation are shown (n=6). The treatment was added after two minutes of equilibration (dashed line).

Figure 5
See manuscript for full gure caption.

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