Antimicrobial Efficacy and Chemical Properties of Caryophyllus aromaticus and Origanum majorana Essential Oils Against Foodborne Bacteria Alone and in Combination

© 2018 The Author(s); Published by Alborz University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Waterborne and foodborne diseases are examples of the most important public health problems. It is estimated that over 200 microbial, chemical, and physical agents contribute to foodborne illnesses.1 Approximately one in ten people experience food poisoning caused by bacteria each year in the United States.2 Centers for Disease Control and Prevention (CDC) estimates that 76 million people get sick, more than 300 000 are hospitalized, and 5000 die from foodborne illnesses each year.3 Furthermore, spoilage microorganisms can shorten the shelf-life of food, causing a remarkable concern.4 Some of the most popular methods for food preservation are drying, freezing, irradiation, refrigeration, thermal

processing, adding antimicrobial agents, and modified atmosphere packaging. 5There are many antimicrobial agents, however consumers' tendency to use synthetic additives is drastically decreasing due to their side effects and also the emergence of multidrug resistant microorganisms. 6Therefore, food industry is seeking natural, safe, and effective alternatives. 7Spices and herbs are generally recognized as safe (GRAS) and have been used as flavouring agents and food preservatives for thousands of years. 80][11][12] The addition of high concentrations of EOs to foods, however, in order to achieve antimicrobial activity has proven some adverse effects on food sensory acceptability including alteration in the taste, color, odor, and texture of food.One efficient procedure in order to reduce the required concentration of each EO is to benefit from combinational effects of EOs. 6,13][16] Caryophyllus aromaticus (clove) belongs to Myrtaceae family. 17Clove EO contains antimicrobial constituents which have been used as traditional antimicrobial additives and flavouring agents in food and dental medicine. 18Eugenol is the most important component of clove EO which exerts strong biological effects such as antimicrobial, insecticidal, and antioxidant activities.Clove EO has also remedial effects, including antiseptic, anti-vomiting, anti-carminative, analgesic, antispasmodic, and anti-phlogistic. 18,19Previous studies have reported antimicrobial activity of clove EO against Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, Micrococcus luteus, Escherichia coli O157:H7, and Pseudomonas aeruginosa. 10ome researchers have proved the synergistic effect of clove and cinnamon EOs against L. monocytogenes, B. cereus and Y. enterocolitica. 16,17riganum majorana (marjoram) belongs to Lamiaceae family whose EO is used as a food flavour. 20Marjoram has also been used in cosmetics and medications. 21Major constituents of marjoram EO are terpinen-4-ol, (+)-cissabinene hydrate, γ-terpinene, and terpinolene. 22,23Some studies have reported antimicrobial and antifungal activities of O. majorana EO. 21,23,24 Based on a report from Gutierrez et al, 15 the combination of thyme and marjoram shows additive effect against E. cloacae.

Objectives
The objectives of this study were to assess chemical compositions and to evaluate antimicrobial activities of C. aromaticus and O. majorana EOs against some foodborne gram-positive (L.monocytogenes ATCC 7644, S. aureus ATCC 65138, B. subtilis ATCC 11778) and gram-negative (E. coli O157:H7 ATCC 43895 and Salmonella typhimurium ATCC 14028) bacteria alone and in combination.

Materials and Methods
Plant Material and Extraction Procedure Leaves of C. aromaticus and O. majorana were purchased from Pakan Bazr Company (Isfahan, Iran) and transferred to the Pharmacognosy Department, Faculty of Pharmacy, University of Tehran, Tehran, Iran for approval.The plants were placed in a Clevenger-type apparatus (Tajhizyar, Tehran, Iran) for hydrodistillation at 100ºC for 5 hours.The EOs were isolated and dried over anhydrous sodium sulfate (Merck, Darmstadt, Germany) and then stored in dark glass bottles at 4ºC until required.

Gas Chromatography-Mass Spectrometry Analysis
The EOs were analyzed by gas chromatography-mass spectrometry (GC-MS) (Thermoquest 2000, Manchester, UK).The chromatograph was equipped with DB5 capillary column (30 m × 0.25 mm ID× 0.25 µm film thickness) and the data were acquired under the following conditions: initial temperature 50°C, program rate 2.5°C, final temperature 265°C, and injector temperature 250°C.The carrier gas was helium and the split ratio was 1:120.The mass spectrum (MS) was run in the electron ionization mode, using an ionization energy of 70 eV.The components of EOs were identified tentatively by comparing their retention indices and mass spectra with those of Wiley 275 Registry of Mass Spectral Data and literature citations. 25,26cterial Strain and Inoculum Preparation The frequently-reported foodborne Gram positive (L.monocytogenes ATCC 7644, S. aureus ATCC 65138, B. subtilis ATCC 11778) and gram-negative (E. coli O157:H7 ATCC 43895 and S. typhimurium ATCC 14028) bacteria were supplied from Faculty of Veterinary Medicine, Amol University of Special Modern Technologies, Amol, Iran.Bacterial strains were cultured in sterile brain heart infusion (BHI) broth (Merck, Darmstadt, Germany) at 37°C for 18 hours.Then a loopful of the bacterial suspension was inoculated in sterile BHI broth and incubated at 37°C for 18 hours.The bacterial broth culture was placed in sterile cuvette and its optical density (OD) was adjusted to absorbance 0.1 at 600 nm, using T80+ UV/VIS Spectronic spectrophotometer (PG Instruments Ltd, Leicestershire, UK).The number of cells in the suspension was estimated by replica plating from 10-fold serial dilutions on BHI agar (Merck, Darmstadt, Germany) and counting the colonies after 18-hour incubation at 37°C. 27

Disk Diffusion Assay
Antibacterial activities of C. aromaticus and O. majorana EOs against foodborne bacteria were assessed using disc diffusion method.The OD of bacterial strains was adjusted to absorbance 0.1 at 600 nm, and according to the results of the previous section on colony counting, the bacterial suspensions were diluted to obtain10 5 ×1 colony forming units per mL (CFU/mL).One hundred microliters of bacterial suspension containing 1×10 5 CFU/mL were spread onto BHI agar containing 10% dimethyl sulfoxide (DMSO).The inoculated plates were left at room temperature for 5 minutes to dry.Then sterile paper disks inoculated with 10 µL of EO were placed on BHI plates containing chloramphenicol and streptomycin disks as positive controls and blank discs as negative controls.The plates were left at room temperature for 15 minutes to allow the diffusion of the EOs, and then were incubated at 37°C for 24 hours.In the end, the diameter of the clear zone around each disk was measured with a ruler and expressed in millimeters as its antimicrobial activity.EOs showed antimicrobial activities if inhibition zone was above 12 mm. 28,29

Determination of Minimum Inhibitory Concentration and Minimum Bactericidal Concentration
The minimum inhibitory concentration (MIC) values of the EOs were determined by microdilution broth method based on the document M7-A6 of CLSI 30 against foodborne bacteria.Sterile 96-well microplates were used for the assay.Stock solutions of the EOs were prepared in 10% (v/v) DMSO.Dilution series of EOs were prepared from 0.0031% to 1% (v/v) in BHI broth.Two hundred microliters of each dilution was transferred into 96well microliter plate, followed by the addition of 20 μL of respected standardized microorganism suspension containing 1×10 5 CFU/mL.Growth control consisted of BHI broth, 10% (v/v) DMSO, and bacterial suspension.After incubation at 37°C for 24 hours, the lowest concentrations without visible growth were defined as the concentrations that completely inhibited bacterial growth (MICs).The minimum bactericidal concentration (MBC) values of the EOs were determined according to the MIC values, based on Celiktas et al. 31 Ten microliters of each well that showed complete absence of growth was transferred to BHI agar plates and incubated at 37°C for 24 hours.The lowest concentrations of EOs in which no viable bacteria were identified were the MBCs.

Checkerboard Assay
Checkerboard synergy testing was performed to determine the fractional inhibitory concentration index (FICI).Checkerboard assay was done by the microdilution broth method.In brief, serial double dilutions of the EOs from 2 MIC to 1.64 MIC were prepared.One hundred microliter of C. aromaticus dilutions were added to the rows of a 96-well microtiter plate in diminishing concentrations and 100 μL of O. majorana dilutions were also added to the columns in diminishing concentrations.A 20-μL suspension of the bacterial strains adjusted to 1×10 5 CFU/mL was added to each well and incubated at 37°C for 24 hours, shaking at 125 rpm.The MIC of the EOs in combination was determined as described above.Each experiment was repeated 2 times.FICI was calculated as follows 32 : .( . ) ( . )

Time-Kill Assay
EOs used in time-kill assay had concentrations equivalent to 1×MIC.The final concentration of the bacterial suspension in BHI tubes was adjusted to 1×10 5 CFU/ mL.A growth control without EO was included.The suspensions were incubated at 37°C for 24 hours, shaking at 125 rpm.Each suspension was cultured on BHI agar after 0, 3, 6, and 24 hours of incubation and was then again incubated at 37°C for 24 hours.Time-kill curves were drawn based on the bacterial population (CFU/ mL) against time (h). 13Experiments were carried out in duplicate.

Statistical Analysis
Data from disk diffusion assay were subjected to Kruskal-Wallis test in SPSS software version 22.0.The Kruskal-Wallis test was also applied for the comparison of MIC and MBC of each EO on the evaluated bacteria.Furthermore, the Mann-Whitney U test was applied for the comparison of MIC and MBC of EOs on each evaluated bacterium.For all analyses, P < 0.05 was considered statistically significant.

Agar Disk Diffusion Assay
Antimicrobial activities of C. aromaticus and O. majorana EOs against foodborne bacteria were evaluated by disk diffusion method (Table 2).The inhibition zones of the evaluated EOs against all the tested bacteria were greater than 12 mm, so the EOs showed remarkable antimicrobial effects.Range of inhibition zone for O. majorana EO (22.96-33.16mm) was greater than that for C. aromaticus EO (14.16-18.66mm).C. aromaticus EO was more effective than streptomycin against all the tested bacteria except for B. subtilis, but less effective than chloramphenicol against all the bacteria under study (P > 0.05).O. majorana EO showed significantly greater antimicrobial effect against B. subtilis than C. aromaticus EO (P < 0.05).The inhibition zone of O. majorana EO against all the tested bacteria except for B. subtilis was even significantly greater than that of streptomycin (P < 0.05).Likewise, the inhibition zone of O. majorana EO against all the foodborne bacteria was greater than that of chloramphenicol (P > 0.05).Furthermore, the most sensitive foodborne bacterium to both evaluated EOs was B. subtilis.On the contrary, the least sensitive foodborne bacteria to C. aromaticus and O. majorana EOs were E. coli O157:H7 and S. typhimurium, respectively (P > 0.05).

Discussion
The major constituents of C. aromaticus EO in the current study were eugenol and carvacrol, and those of O.  14 Clove EO had high eugenol (70%-90%) content. 9Barbosa et al 33 found the major constituents of C. aromaticus EO to be eugenol (75.85%) and eugenyl acetate (16.38%).Daferera et al 34 reported the main constituents of O. majorana EO to be thymol, 3-carene, and terpinen-4-ol.Contrary to the results of the current study, the major constituents of O. majorana EO were linalool, terpinen-4-o1, and p-cymene in another study. 35ifferences in the chemical composition of EOs are the results of different factors including plant species, age of the plant, plant part used for extraction, geographic area, soil, weather conditions, harvest season, and extraction technique. 36,37Culture conditions, crop processing, and post-harvest processing can be other explanations for variations in chemical composition of EOs. 38aryophyllus aromaticus and O. majorana EOs showed antimicrobial effects against all foodborne bacteria as was evident from the large inhibition zones (inhibition zone ˃12 mm).C. aromaticus EO was more effective than streptomycin against all the tested bacteria except for B. subtilis, but less effective than chloramphenicol against all the bacteria under study (P > 0.05).The inhibition zone of O. majorana EO against all the tested bacteria except for B. subtilis was significantly greater than that of streptomycin (P < 0.05).According to the results of agar disc diffusion assay, C. aromaticus and O. majorana EOs were more effective against gram-positive bacteria (the most sensitive bacterium to both EOs was B. subtilis) than gram-negative ones (the least sensitive foodborne bacteria to both EOs were E. coli O157:H7 and S. typhimurium, respectively).In the study of Hoque et al, 9 clove EO showed antimicrobial effect against L. monocytogenes, S. aureus, E. coli O157:H7, Salmonella enteritidis, and B. cereus.Eugenol is the main constituent of C. aromaticus EO whose antimicrobial activity against foodborne pathogens and drug resistant microorganisms has been proved in other studies. 39Mith et al 12 found that O. majorana EO had antimicrobial activity against E. coli O157:H7, S. typhimurium, L. monocytogenes, Brochothrix thermosphacta, and Pseudomonas fluorescens.Carvacrol as the main constituent of O. majorana EO, holding antimicrobial effects, has been recognized GRAS by the Food and Drug Administration (FDA). 40EOs exert their antimicrobial effects through a number of mechanisms including inhibition of nucleic acid synthesis, disturbance of the cytoplasmic membrane, and energy metabolism. 33n this study, it was found that the antimicrobial activity of C. aromaticus EO against all the tested bacteria was weaker than that of chloramphenicol.Similar result was found by Hoque et al 9 in that antimicrobial effect of clove EO against L. monocytogenes, E. coli O157:H7, S. aureus, B. cereus, and S. enteritidis was weaker than that of gentamicin.Mith et al 11 showed that the antimicrobial activity of thymol was greater than that of eugenol, thereby the antimicrobial activity of O. majorana being greater than that of C. aromaticus.Similarly, clove EO produced the largest inhibition zone against Gram positive bacteria especially Bacillus sp.(24 mm) and B. subtilis (19.5 mm) 10,14 ; this can be attributed to the outer membrane structure having hydrophilic lipopolysaccharide and lipoproteins which form a barrier to restrict the entrance of hydrophobic compounds to gram-negative bacteria. 9,10,12,41n microdilution broth test, the MIC value of C. aromaticus and O. majorana EOs against all foodborne bacteria was 0.1% except for O. majorana EO against B. Subtilis (0.3%).MBC values of C. aromaticus and O. majorana EOs against foodborne bacteria in the current study ranged from 0.5% to 1.0 % (v/v) and 0.3% to 0.5% (v/v), respectively.E. coli O157:H7 was the most resistant microorganism to bactericidal effects of both the EOs (P > 0.05).Similar to the results of the previous section, O. majorana EO showed stronger bactericidal effect than C. aromaticus EO against foodborne bacteria (P > 0.05).In two similar studies, the MIC value of clove EO against S. aureus was reported to be 0.09% (v/v). 42,43Fu et al 14 found the MIC and MBC values of clove EO against foodborne microorganisms as 0.06%-0.50%(v/v) and 0.12%-0.50%(v/v), respectively.Contrary to the results of the present study, the lowest MIC and MBC values of clove EO against foodborne pathogens and spoilage bacteria were found to be1.25% and 2.5% (v/v) in the study of Hoque et al, respectively. 9Gutierrez et al 13 showed that MIC value of marjoram against Listeria innocua was 0.5% (v/v).Similar to the results of the previous section, Gram negative bacteria were less sensitive to bactericidal effect of C. aromaticus EO than Gram positives ones; this has been proved by other researchers. 24,41,44,45ntimicrobial combinational effect of C. aromaticus and O. majorana EOs against foodborne bacteria was studied for the first time.The present study revealed that the MIC value of each EO against L. monocytogenes was higher than the MIC value in combination.The combination of C. aromaticus and O. majorana EOs showed partial synergistic effect against B. subtilis, and additive effect against S. typhimurium.It was indifferent against S. aureus and E. coli O 157 H 7 .Goñi et al, 16 Probst et al, 17 and Nascimento et al 46 14 Gutierrez et al 15 reported that combination of marjoram and oregano exerted additive effect against B. cereus and E. coli O157:H7, while it was indifferent against L. monocytogenes and P. aeruginosa.The combinations of marjoram and rosemary EOs, sage and basil showed additive effects against L. monocytogenes.Marjoram in combination with lemon balm and thyme was indifferent against L. monocytogenes. 15Goñi et al 16 demonstrated that the combination of marjoram and oregano EOs can be used for controlling the growth of Gram positive and gram-negative bacteria.The synergistic or additive effects of EOs may decrease the need for usage of chemical additives, limit their adverse effects and antibiotic resistance, and also may reduce required doses and expand the spectrum of activity. 6,32Furthermore, by the use of combinations of EOs, various chemical compounds of EOs could simultaneously inhibit microorganisms, and accordingly the combinational antimicrobial activity could be improved. 17ime-kill curves of C. aromaticus and O. majorana EOs showed strong bactericidal effects against all foodborne bacteria at 6 and 24 hours either alone or in combination.O. majorana EO was the only EO that showed bactericidal effect against B. subtilis at 3 hours.Contradictory results were reported by Fu et al 14 who studied the antimicrobial activity of clove and rosemary EOs against S. epidermidis, E. coli O157:H7, and Candida albicans at 1×MIC, and colony counts remained near the initial starting concentration after 30 hours.Barbosa et al 33 found that clove EO had bactericidal effect against S. aureus and bacteriostatic effect against E. coli O157:H7.Zu et al 47 studied the antimicrobial effect of 10 EOs (mint, ginger, rose, cinnamon, grapefruit, jasmine, lavender, chamomile, thyme, and lemon) at 0.25% v/v against Propionibacterium acnes and showed bactericidal effects at 30 minutes except for jasmine.Similar to the results of the current study, Probst et al 17 reported that the MIC value was bactericidal for clove EO against S. aureus and E. coli O157:H7 at 6 hours.The combination of C. aromaticus and O. majorana EOs in the current study reduced the colony count of L. monocytogenes at 24 hours in comparison with C. aromaticus and O. majorana EOs alone by 4 and 3 log, respectively.The results of time-kill assay proved the results of the previous section on the synergistic effect of C. aromaticus and O. majorana EOs against L. monocytogenes.Probst et al 17 showed that the combination of clove with cinnamon EOs reduced the colony count of E. coli O157:H7 by 1 log at 1.5 hours in comparison to clove EO alone.
According to the results of the current study, C. aromaticus and O. majorana EOs showed promising antimicrobial activities against the most important foodborne pathogens and spoilage bacteria.Gram positive microorganisms were more sensitive to C. aromaticus and O. majorana EOs than Gram negative ones.The combination of C. aromaticus and O. majorana EOs exhibited synergistic, partial synergistic and additive antibacterial activities depending on the corresponding microorganism.These interactions strengthen the antimicrobial activity, expand the spectrum of activity, reduce the concentrations required, decrease the side effects, and prevent the alteration of organoleptic properties of food.
The combination of C. aromaticus and O. majorana EOs can be used as a potential alternative for synthetic additives, as food preservatives, and also in the production of herbal medicines.Further studies are required on the interaction of these EOs with food ingredients and their modes of action.To the best of our knowledge, this was the first study on the antimicrobial effects of C. aromaticus and O. majorana EOs in combination.

Table 1 .
Inhibitory Concentration and Minimum Bactericidal Concentration MIC and MBC values of C. aromaticus and O. majorana EOs against foodborne bacteria are shown in Table 3. MIC value of the EOs against foodborne bacteria was 0.1% except for that of O. majorana EO against B. Subtilis Chemical Composition (%) of Caryophyllus aromaticus and Origanum majorana EOs Determined by GC-MS

Table 2 .
Antimicrobial Activities (mm) of Caryophyllus aromaticus and Origanum majorana EOs Against Foodborne Bacteria Determined by Agar Disk Diffusion Assay The inhibition zones of the EOs and antibiotics including 6 mm disc diameter are presented.Results are presented as mean ± SD of 2 replicates.Within the columns, significant differences are presented by different superscript capital letters (P<0.05).Within the rows, significant differences are represented by different superscript small letters (P<0.05).
*Checkerboard AssayThe results of checkerboard synergy analysis of C. aromaticus and O. majorana EOs against foodborne bacteria are shown in Table4.FICI values for C. aromaticus EO plus O. majorana EO ranged from 0.500 to 2.000 against foodborne bacteria.The combination of C. aromaticus and O. majorana EOs showed synergistic interaction (FICI = 0.500) against L. monocytogenes.The MIC values of C. aromaticus and O. majorana EOs alone against L. monocytogenes were lowered from 0.100 to 0.025 in combination.The combination of C. aromaticus and O. majorana EOs showed partial synergistic activity (FICI = 0.583) against B. subtilis and additive effect (FICI = 1.000) against S. typhimurium.E. coli O157:H7 and S. aureus were indifferent against C. aromaticus plus O. majorana EOs.No antagonistic effect was observed for the EOs.monocytogenes, and E. coli O157:H7 was observed at 24 hours.O. majorana EO showed bactericidal effect against B. subtilis at 3 hours, while the same effect was seen against S. aureus and L. monocytogenes at 6 hours.Accordingly, O. majorana EO showed bactericidal effect against S. typhimurium and E. coli O17:H7 at 24 hours.The combination of C. aromaticus and O. majorana EOs reduced the colony count of B. subtilis and S. aureus in comparison to C. aromaticus EO by 1 and 2 log, respectively at 3 hours.In the same way, the combination of C. aromaticus and O. majorana EOs reduced the colony count of L. monocytogenes and E. coli O157:H7 in comparison with C. aromaticus EO by 3 log at 6 hours.Furthermore, the combinational effect of C. aromaticus and O. majorana EOs caused a reduction in the colony count of S. aureus in comparison with O. majorana EO by 1 log at 3 hours.Additionally, the combination of both EOs reduced the colony count of L. monocytogenes and E. coli O157: H7 in comparison with O. majorana EO by 2 log at 6 hours.Similarly, the combination of C. aromaticus and O. majorana EOs diminished the colony count of L. monocytogenes in comparison with either of EOs by 4 and 3 log, respectively at 24 hours.

Table 3 .
MIC and MBC Values (% v/v) of Caryophyllus aromaticus and Origanum majorana EOs Against Foodborne Bacteria Determined by Microdilution Broth Method

Table 4 .
Combinational Effects of Caryophyllus aromaticus and Origanum majorana EOs Against Foodborne Bacteria Using Checkerboard Assay majorana EO were carvacrol, thymol, trans-caryophyllene, and cymene.Similar findings have been reported by other investigators.
assessed the antimicrobial activity of C. aromaticus EO in combination with other antimicrobial agents.Clove plus rosemary EOs had additive antimicrobial effects against S. epidermidis, S. aureus, B. subtilis, E. coli O 157 H 7 , Proteus vulgaris, and P. aeruginosa.