In Vitro Susceptibility of Multidrug Resistant Strains of Salmonella to Essential Oils

Abstract

Antimicrobial resistance has become a global threat to public health. There is a critical need to find new antimicrobial substances from natural sources. The aim of this study was to investigate the antimicrobial activity of essential oils (EOs) obtained from Origanum vulgare, Thymus serpyllum, Thymus vulgaris, and Melaleuca alternifolia against multidrug resistant strains of Salmonella isolated from samples of diverse animal origin. The strains were biochemically identified, serotyped, and characterized for their antimicrobial resistance profiles. The antimicrobial activity of the EOs against the strains was evaluated using the Kirby-Bauer diffusion method, followed by determination of the minimal inhibitory concentration and minimum bactericidal concentrations. The EOs of T. serpyllum and O. vulgare, which contain carvacrol as the main compound, show excellent antimicrobial activity.

Antimicrobial resistance has become a global threat to public health safety.¹ The emergence of multidrug resistant (MDR) pathogens has drawn attention to the use of natural compounds as antibiotic alternatives.² Furthermore, consumers are more inclined to use “green” and more sustainable products. Salmonella, a major causative agent of gastrointestinal illness, was the second leading cause of food-borne disease in Europe, with almost 94 500 human cases recorded in 2016. The most common strains belong to Salmonella ser. Enteritidis and Typhimurium, including the monophasic variant 1,4,[5],12:i:- of emerging interest.³ Multidrug resistant strains of Salmonella are continuously reported. The spread of resistant clones has been exacerbated by the improper use of antimicrobials in humans and animals and the associated selective pressure.⁴

Most Salmonella infections in humans result in mild, self-limiting, gastrointestinal illness and usually do not require antimicrobial treatment. In cases of severe enteric disease or invasive infection, effective antimicrobials are essential for treatment. Infection with Salmonella strains resistant to these antimicrobials may be associated with treatment failure and poor patient outcome. Moreover, the horizontal transmission of resistance genes is the most important factor in the current pandemic of antimicrobial resistance.⁵ The presence of MDR Salmonella strains can promote the emergence of antibiotic resistance in other pathogens that is then transmitted from animals to humans via direct contact or through the food chain or the environment. If excessive antibiotic use can be reduced, the expectation is that the less “fit” resistant bacteria might be replaced by susceptible bacteria.⁶ The reduction of antibiotic use and the development of natural, safe, sustainable intervention strategies, such as the use of essential oils (EOs), for example, could help to stem antibiotic resistance.

Essential oils are aromatic oily liquids obtained from plants. They contain diverse components, in particular phenolic compounds, with antimicrobial activity.⁷ The antimicrobial activity of EOs has been successfully investigated in vitro against various gram-positive and gram-negative pathogens, including Salmonella.^7

-15

The aim of this study was to evaluate the susceptibility of MDR strains of Salmonella to EOs of Origanum vulgare, Thymus serpyllum, Thymus vulgaris, and Melaleuca alternifolia.

The antimicrobial resistance profiles of the isolated strains are presented in Table 1. The strains resulted resistant at least to 2 of the tested antimicrobials. All the strains resulted resistant to triple-sulfa and 23 strains resulted resistant to tetracycline, considered a highly important antimicrobial.¹⁶ Regarding the critically important antimicrobials,¹⁶ 23 of the 25 strains resulted sensitive to colistin, except for 1 Salmonella Saintpaul that resulted resistant and 1 Salmonella Oranienburg that resulted intermediate. In all, 21 strains resulted sensitive to ciprofloxacin, 3 S. Saintpaul and 1 S. Oranienburg resulted intermediate. Furthermore, 14 strains resulted sensitive, 6 intermediate and 5 resistant to ceftazidime; 16 strains resulted sensitive, 5 intermediate and 4 resistant to cefotaxime.

Table 1

Results of Agar Disk Diffusion Testing of Each Salmonella Strain With an Antibiotic.

Strain	Antibiotic class
	A	AMC	C	KF	CTX	CIP	CST	CAZ	ENR	G	K	NAL	S	SSS	SXT	T
S almonella Oranienburg	R	R	S	R	R	I	I	R	R	S	I	R	I	R	S	I
Salmonella Saintpaul (1)	R	R	S	R	R	I	S	R	S	S	R	R	R	R	R	R
Salmonella Saintpaul (2)	R	R	S	R	S	S	S	S	I	R	R	R	R	R	R	R
Salmonella Saintpaul (3)	I	S	R	S	S	S	S	S	R	S	I	R	R	R	R	R
Salmonella Saintpaul (4)	R	S	R	S	I	S	S	I	I	I	R	I	R	R	R	R
Salmonella Saintpaul (5)	R	R	S	R	I	I	R	R	R	I	R	R	R	R	R	R
Salmonella Saintpaul (6)	R	R	S	R	R	I	S	R	R	R	R	R	R	R	R	R
S almonella Typhimurium—1,4,[5],12:i:- (1)	R	R	S	I	S	S	S	S	\	S	\	S	R	R	R	R
S almonella Typhimurium—1,4,[5],12:i:- (2)	R	R	S	S	S	S	S	S	\	S	\	S	R	R	R	R
Salmonella Typhimurium—1,4,[5],12:i:- (3)	R	R	S	R	S	S	S	S	\	S	\	S	R	R	R	R
S almonella Typhimurium—1,4,[5],12:i:- (4)	R	R	S	R	S	S	S	S	\	S	\	R	R	R	R	R
Salmonella Colindale	R	S	S	R	S	S	S	I	I	S	S	R	R	R	R	R
Salmonella Brandenburg	R	I	S	S	I	S	S	I	S	S	I	S	R	R	R	R
S almonella Minnesota	S	S	S	S	S	S	S	S	S	S	S	S	S	R	R	R
Salmonella Coeln (1)	S	I	S	I	S	S	S	S	S	S	S	S	R	R	R	R
S almonella Coeln (2)	I	R	S	S	S	S	S	S	S	S	S	S	S	R	I	S
S almonella Coeln (3)	I	S	S	S	S	S	S	S	S	S	I	S	S	R	R	R
Salmonella salamae (1)	R	S	S	I	S	S	S	S	\	S	\	I	S	R	S	R
Salmonella salamae (2)	S	I	S	R	I	S	S	I	\	S	\	I	I	R	I	R
Salmonella salamae (3)	S	S	S	I	I	S	S	R	\	S	\	S	S	R	I	R
Salmonella salamae (4)	R	S	S	R	R	S	S	S	\	S	\	I	S	R	S	R
Salmonella salamae (5)	S	S	S	I	S	S	S	I	\	S	\	I	S	R	S	R
Salmonella diarizonae (1)	S	I	S	S	S	S	S	I	\	S	\	\	R	R	R	R
Salmonella diarizonae (2)	S	I	\	I	S	S	S	S	S	S	I	S	S	R	R	R
Salmonella diarizonae (3)	I	S	S	S	S	S	S	S	I	I	I	S	R	R	S	R

A, ampicillin (10 µg); AMC, amoxicillin/clavulanic acid 2:1 (30 µg); C, chloramphenicol (30 µg); KF, cefalotin (30 µg); CTX, cefotaxime (5 µg); CIP, cyprofloxacin (5 µg); CST, colistin (10 µg); CAZ, ceftazidime (10 µg); ENR, enrofloxacin (5 µg); G, gentamicin (10 µg); K, kanamycin (30 µg); NAL, nalidixic acid (30 µg); S, streptomycin (10 µg); SSS, triple-sulfa (250 µg); SXT, trimethoprim-sulfamethoxazole (1.25/23.75 µg); T, tetracycline (30 µg).

Table 2 shows the results of the agar disk diffusion method for testing the antimicrobial activity of the EOs. The antimicrobial activity of EOs of O. vulgare and T. serpyllum was higher as compared with T. vulgaris and M. alternifolia (P-value <0.01). The mean diameter of the bacterial growth inhibition zone was 18.8 mm (±3.5) for O. vulgaris, 19 mm (±4.4) for T. serpyllum, 14.2 mm (±3.2) for T. vulgaris, and 10.8 mm (±2.4) for M. alternifolia. As expected, the negative controls showed no inhibition zone of bacterial growth around the disks.

Table 2

Results of Agar Disk Diffusion Testing of Each Salmonella Strain with 4 Essential Oils.

Strain	Thymus serpyllum	Origanum vulgare	Thymus vulgaris	Melaleuca alternifolia
Salmonella Oranienburg	17.3 (±2.3)	21.7 (±2.9)	15 (±1.7)	10 (±0)
S almonella Saintpaul (1)	19.3 (±3.1)	22.3 (±2.1)	13 (±2.6)	9.7 (±0.6)
S almonella Saintpaul (2)	24.7 (±3.1)	10.3 (±1.5)	8.3 (±0.6)	12.3 (±0.6)
S almonella Saintpaul (3)	21.3 (±1.1)	19 (±0.00)	17.7 (±0.6)	12 (±0)
S almonella Saintpaul (4)	20.7 (±3.1)	16.3 (±0.6)	15.7 (±1.1)	10.3 (±0.6)
S almonella Saintpaul (5)	18.7 (±1.1)	20 (±0)	15.3 (±1.1)	10.7 (±0.6)
S almonella Saintpaul (6)	16 (±3.5)	22 (±2)	15.7 (±0.6)	9.7 (±0.6)
Salmonella Typhimurium—1,4,[5],12:i:- (1)	19.3 (±1.1)	20.7 (±1.1)	15.7 (±1.5)	11.3 (±0.6)
Salmonella Typhimurium—1,4,[5],12:i:- (2)	16 (±3.5)	17.3 (±1.1)	16 (±1.7)	11.3 (±0.6)
Salmonella Typhimurium—1,4,[5],12:i:- (3)	26 (±2)	22.7 (±1.1)	17 (±2.6)	10.7 (±0.6)
Salmonella Typhimurium—1,4,[5],12:i:- (4)	22 (±2)	21.3 (±1.1)	14 (±1.1)	11 (±0)
S almonella Colindale	11.7 (±0.6)	20 (±2)	7 (±1.7)	9.3 (±0.6)
S almonella Branderbourg	18.7 (±1.1)	20 (±2)	16 (±2)	11.7 (±0.6)
S almonella Minnesota	17.3 (±1.1)	17.3 (±1.1)	18.3 (±1.5)	10.3 (±0.6)
S almonella Coeln (1)	24 (±2)	18 (±0)	15.7 (±1.1)	10.3 (±0.6)
S almonella Coeln (2)	14 (±2)	22 (±2)	10.3 (±2.5)	11 (±1)
S almonella Coeln (3)	24.7 (±1.1)	20 (±2)	16.7 (±0.6)	9.7 (±0.6)
Salmonella salamae (1)	20 (±3.5)	15.3 (±4.2)	14.3 (±0.6)	9.7 (±0.6)
Salmonella salamae (2)	22 (±0)	16 (±2)	10.3 (±3.8)	8.7 (±0.6)
Salmonella salamae (3)	14 (±2)	18.7 (±1.1)	12.3 (±3.8)	9 (±0)
Salmonella salamae (4)	20 (±2)	23.3 (±2.3)	13 (±1.7)	10.3 (±1.1)
Salmonella salamae (5)	15.3 (±3.1)	13.3 (±2.3)	14 (±3.5)	9.3 (±0.6)
Salmonella diarizonae (1)	24 (±0)	21.3 (±1.1)	15 (±0)	21.3 (±1.1)
Salmonella diarizonae (2)	10.7 (±2.3)	15.3 (±1.1)	12 (±2)	11.3 (±0.6)
Salmonella diarizonae (3)	16.7 (±2.3)	15.3 (±1.1)	16.3 (±0.6)	10.3 (±0.6)
Mean	19^* (±4.4)	18.8^* (±3.5)	14.2^* (±3.2)	10.8^* (±2.4)

The results are expressed as the mean of the diameters in mm of 3 replicates for each strain (±standard deviation).

^*The differences are statistically different (P-value <0.01).

The minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of the tested EOs against 25 MDR Salmonella strains considered in this study are summarized in Table 3. In general, more effective EOs were O. vulgare (MIC range 0.012%-0.2%, mean 0.045%; MBC range 0.012%-0.2%, mean 0.049) and T. serpyllum (MIC range 0.012%-0.2%, mean 0.056%; MBC range 0.012%-0.2%, mean 0.062%), M. alternifolia (MIC range 0.10%-0.2%, mean 0.2%; MBC 0.2%, mean 0.2%), and T. vulgaris (MIC range 0.05%-0.39%, mean 0.14%; MBC range 0.05%-0.39%, mean 0.15%) showed a moderate efficacy against tested strains; the results are considered statistically significant (P-value <0.01).

Table 3

Minimal Inhibitory Concentration and Minimal Bactericidal Concentration Values (% v/v) of Selected Essential Oils Against Multidrug Resistant Salmonella Strains.

Strain	MIC				MBC
Strain	T hymus serpyllum	O riganum vulgare	T hymus vulgaris	M elaleuca a l ternifolia	T hymus serpyllum	O riganum vulgare	T hymus vulgaris	M elaleuca a l ternifolia
S almonella Oranienburg	0.2	0.1	0.2	0.2	0.2	0.1	0.2	0.2
S almonella Saintpaul (1)	0.2	0.1	0.2	0.2	0.2	0.1	0.2	0.2
S almonella Saintpaul (2)	0.1	0.2	0.39	0.2	0.2	0.2	0.39	0.2
S almonella Saintpaul (3)	0.05	0.05	0.1	0.2	0.05	0.05	0.1	0.2
S almonella Saintpaul (4)	0.05	0.05	0.1	0.2	0.05	0.05	0.1	0.2
S almonella Saintpaul (5)	0.05	0.025	0.1	0.2	0.05	0.025	0.1	0.2
S almonella Saintpaul (6)	0.05	0.025	0.1	0.2	0.05	0.025	0.1	0.2
S almonella Typhimurium—1,4,[5],12:I:- (1)	0.05	0.025	0.1	0.2	0.05	0.025	0.2	0.2
S almonella Typhimurium—1,4,[5],12:I:- (2)	0.05	0.05	0.1	0.2	0.05	0.05	0.1	0.2
S almonella Typhimurium—1,4,[5],12:I:- (3)	0.025	0.025	0.1	0.2	0.05	0.025	0.2	0.2
S almonella Typhimurium—1,4,[5],12:I:- (4)	0.05	0.05	0.2	0.1	0.05	0.1	0.2	0.2
S almonella Colindale	0.1	0.025	0.2	0.2	0.10	0.025	0.2	0.2
S almonella Branderbourg	0.05	0.025	0.1	0.1	0.05	0.025	0.1	0.2
S almonella Minnesota	0.012	0.012	0.05	0.2	0.012	0.025	0.05	0.2
S almonella Coeln (1)	0.05	0.05	0.1	0.2	0.05	0.05	0.1	0.2
S almonella Coeln (2)	0.012	0.025	0.1	0.2	0.012	0.025	0.1	0.2
S almonella Coeln (3)	0.05	0.05	0.1	0.2	0.05	0.05	0.1	0.2
Salmonella salamae (1)	0.025	0.025	0.1	0.2	0.025	0.025	0.1	0.2
Salmonella salamae (2)	0.025	0.05	0.2	0.2	0.05	0.05	0.2	0.2
Salmonella salamae (3)	0.012	0.012	0.1	0.2	0.012	0.012	0.1	0.2
Salmonella salamae (4)	0.05	0.025	0.2	0.2	0.05	0.025	0.2	0.2
Salmonella salamae (5)	0.025	0.05	0.1	0.2	0.025	0.05	0.2	0.2
Salmonella diarizonae (1)	0.05	0.05	0.2	0.2	0.05	0.05	0.2	0.2
Salmonella diarizonae (2)	0.012	0.012	0.1	0.2	0.012	0.012	0.2	0.2
Salmonella diarizonae (3)	0.05	0.05	0.1	0.2	0.05	0.05	0.1	0.2
Mean	0.056^*	0.045^*	0.14^*	0.2^*	0.062^*	0.049^*	0.15^*	0.2^*

MBC, minimal bactericidal concentration; MIC, minimal inhibitory concentration.

^*The differences are statistically different (P-value <0.01).

The present study shows that the EOs of O. vulgare and T. serpyllum, which contain carvacrol as the main constituent, had the greatest antimicrobial effect against MDR strains of Salmonella, whereas the effect was lower for T. vulgaris and M. alternifolia, which contain thymol and 4-terpineol as main constituents, respectively. Several studies have been conducted to test the antimicrobial activity of EOs or their compound against Salmonella spp. in vitro. A review comparing the results of many studies revealed that the EO of O. vulgare, containing carvacrol as main compound, shows the better antimicrobial activity.^9,17 The EOs of O. vulgare and T. serpyllum rich in carvacrol used in our study show a comparable antimicrobial activity, and it is also greater than that obtained using a EO of O. vulgare rich in thymol¹² and a EO of Thyme rich in thymol.¹¹ The antimicrobial activity obtained using O. vulgare and T. serpyllum is also comparable to that obtained using EOs of Origanum heracleoticum and Origanum compactum containing high level of carvacrol and carvacrol tested alone against Salmonella.^9,18

Carvacrol appears to make the cell membrane more permeable.¹⁹ It disintegrates the outer membrane of gram-negative bacteria, which releases lipopolysaccharides and increases the permeability of the cytoplasmic membrane to adenosine triphosphate (ATP).²⁰ Carvacrol showed a good antimicrobial activity against Salmonella Typhimurium in vitro⁹ and other food-borne pathogens. Studies testing carvacrol for its antimicrobial effects in vitro have observed that it can inhibit the growth of Bacillus cereus.²¹ The effect of carvacrol also against Escherichia coli has been investigated. An in vitro study observed antimicrobial activity with negligibledetrimental effects on cells after balancing appropriate doses of the substance.²² Carvacrol at subtherapeutic doses was found to be effective in reducing S. Typhimurium motility and its invasion of porcine epithelial cells.²³

A possible synergic effect of EOs could be due to the interaction between their various components.⁷ p-Cymene, the biological precursor of carvacrol, is hydrophobic and causes swelling of the cytoplasmic membrane to a greater extent than does carvacrol.²⁴ p-Cymene is not an effective antibacterial when used alone,^25

-28 but when combined with carvacrol, synergism has been observed against B. cereus.²⁹ Probably, p-cymene enables carvacrol to be more easily transported into the cell.²⁴ In our study, p-cymene, in relatively high concentrations in O. vulgare and T. serpyllum EOs, probably contributed to the antimicrobial effect of these EOs. Thymus vulgaris has a high percentage of p-cymene but a low percentage of carvacrol; therefore, its antimicrobial activity was less as compared with the other EOs.

Different studies showed that MDR pathogens were sensitive to some EOs, including O. vulgare and T. vulgaris.^8,10,30
-32 This observation is consistent with our findings that there is no correlation between the sensitivity to conventional antibacterial drugs and EOs. It could be interesting to further investigate these natural products for their application in alternative novel therapies.

Moreover, EOs and their compounds might be used as antimicrobial additives for food safety. In fact, many EOs and their individual constituents are considered to be Generally Recognized as Safe at the doses typically used in foods⁷ and have been approved by the U.S. Food and Drug Administration (FDA) for use in edible products. However, their use at high concentrations in food might negatively impact organoleptic qualities, including flavor and odor. A delicate balance must be struck between selecting an effective concentration of these compounds against pathogenic microorganisms and their use in food systems.⁷ The present study demonstrates a great in vitro antimicrobial activity of EOs of O. vulgare and T. serpyllum against MDR strains of Salmonella and provides a contribution of knowledge to allow the use of EOs such as alternative therapy or additives for food safety.

Experimental

Bacterial Strains

A total of 25 Salmonella strains were isolated in the 3-year period from 2014 to 2016 by our institute. Table 4 lists the species, year of isolation, and source of strains used in this study. The strains were isolated according to ISO 6579:2002/COR 1, 2004 (Microbiology of food and animal feeding stuffs: horizontal method for the detection of Salmonella spp.).³³ Serotype identification of the isolated strains was carried out according to ISO/TR 6579-3, 2014 (Microbiology of the food chain—Horizontal method for the detection, enumeration and serotyping of Salmonella—Part 3: Guidelines for serotyping of Salmonella spp.).³⁴

Table 4

Salmonella Sero Var, Source, and Year of Isolation.

Strain	Source	Year of isolation
Salmonella Oranienburg	Chicken	2014
S almonella Saintpaul (1)	Turkey	2016
S almonella Saintpaul (2)	Turkey	2016
S almonella Saintpaul (3)	Turkey	2016
S almonella Saintpaul (4)	Turkey	2016
S almonella Saintpaul (5)	Turkey	2016
S almonella Saintpaul (6)	Turkey	2016
S almonella Typhimurium—1,4,[5],12:i:- (1)	Wild boar (liver)	2014
S almonella Typhimurium—1,4,[5],12:i:- (2)	Wild boar (liver)	2014
S almonella Typhimurium—1,4,[5],12:i:- (3)	Wild boar (liver)	2014
S almonella Typhimurium—1,4,[5],12:i:- (4)	Wild boar (liver)	2014
S almonella Colindale	Chicken	2014
Salmonella Brandenburg	Calf (hamburger)	2014
S almonella Minnesota	Turkey	2014
S almonella Coeln (1)	Wild boar (liver)	2015
S almonella Coeln (2)	Wild boar (liver)	2015
S almonella Coeln (3)	Wild boar (liver)	2016
Salmonella salamae (1)	Wild boar (liver)	2014
Salmonella salamae (2)	Wild boar (liver)	2014
Salmonella salamae (3)	Wild boar (liver)	2014
Salmonella salamae (4)	Wild boar (liver)	2014
Salmonella salamae (5)	Wild boar (liver)	2014
Salmonella diarizonae (1)	Wild boar (liver)	2014
Salmonella diarizonae (2)	Wild boar (liver)	2016
Salmonella diarizonae (3)	Wild boar (liver)	2016

Antimicrobial Susceptibility Test

The Kirby-Bauer disk diffusion test was performed following Clinical and Laboratory Standard Institute (CLSI) guidelines,³⁵ using Mueller-Hinton agar plates (Microbiol, Uta (CA), Italy) with the following antimicrobials and concentrations (μg): ampicillin (A, 10), amoxicillin/clavulanic acid 2:1 (AMC, 30), chloramphenicol (C, 30), ceftazidime (CAZ, 10), cyprofloxacin (CIP, 5), colistin (CST,10), enrofloxacin (ENR,5), triple sulfa (SSS,250), cefotaxime (CTX, 5), gentamicin (G, 10), kanamycin (K, 30), cefalotin (KF, 30), nalidixic acid (NAL, 30), streptomycin (S, 10), trimethoprim-sulfamethoxazole (SXT, 1.25/23.75), and tetracycline (T, 30). Data were analyzed following the CLSI guideline instructions.³⁶

Essential Oils Activity

The antimicrobial activity of 4 EOs was investigated: O. vulgare, T. serpyllum, T. vulgaris, and M. alternifolia, kindly provided by the producer FLORA (Pisa, Italy). Table 5 reports the percentage of each class of component of the EOs, identified using gas chromatography analysis by the producer. The in vitro antibacterial activity of each EO was tested by the Kirby-Bauer agar disk diffusion method following the procedures described in the CLSI guidelines³⁵ with some modifications. Briefly, each isolated strain of Salmonella was cultivated in liquid medium Buffer Peptone Water (BPW) at 37°C for 24 hours. After incubation, a sterile swab was dipped in each tube containing the culture broth (approximately 0.5 McFarland standard) and then used for spread-plating. Each EO was mixed with 1% v/v of Tween 80 and 1 absorbent paper disk was impregnated with 15 µL of each oil, respectively. All EOs were tested in triplicate for each bacterial strain. One Petri dish was used to test each EO against each Salmonella strain. The Petri dishes were closed with a plastic paraffin film (Parafilm M, Sigma-Aldrich) and incubated at 37°C for 24 hours. A negative control plate containing sterile BPW and 3 absorbent paper disks impregnated with Tween 80 alone was also tested. The results are expressed as the diameter of the inhibition of bacterial growth.

Table 5

Relative Percentage of the Main Compounds in Essential Oils.

Component	Percentage
	Thymus serpyllum	Thymus vulgaris	Melaleuca alternifolia	Origanum vulgare
α-Thujene	1	1.9	0.6	1.3
α-Pinene	0.8	0.8	2.8	0.9
Camphene		0.6		0.2
Sabinene	0.3		0.3
β-Pinene			0.6	0.2
β-Myrcene	1.4	1.6	0.6	2.3
α-Phellandrene			0.4	0.2
β-Phellandrene	0.2	0.3	0.6
α-Terpinene	1.2	1.3	9.7	1.2
Limonene	0.2	0.7	1.3	0.2
1.8 Cineol	0.3	0.2	1.5
1.8 Cineol + β-Phellandrene				0.2
γ-Terpinene	4	7.7	20.5	7.9
trans-Sabinene hydrate		0.7
cis-Sabinene hydrate	0.5
p-Cymene	5.2	23.2	2.5	9.8
Terpinolene			3.3	0.1
trans-Thujanol				0.1
Linalool	8.2	1.3	0.1	1.6
Camphor		0.4
Borneol	0.4	0.9		0.2
cis-Thujanol				0.1
1-Octanol				0.1
Terpinen 4-ol	0.6	0.8	40.7	0.5
β-Caryophyllene	0.9	5.5	0.3	2.2
γ-Cadinene		0.4
α-Humulene				0.1
α-Terpineol	0.6		3.8	0.1
α-Gurjunene			0.4
β-Bisabolene				0.2
Thymol	2.6	45		3.7
Carvacrol	68.5	3		64.7
Aromadendrene	0.3		1.5
Viridiflorene
δ-Cadinene			0.8
Globulol			0.4
Viridiflorene + Guaiol			0.3

Significant of bold are reported the main constituent of each oil.

Broth microdilution method was performed to determine the MIC for the EOs according to the Clinical and Laboratory Standards Institute³⁷ with minor modifications. The MIC is defined as the lowest concentration of an antimicrobial agent that prevents visible growth of a microorganism in an agar or broth dilution susceptibility test. Briefly, microtiter plates containing 2-fold serial dilutions of each oil were inoculated with aliquots of 100 µL of bacterial suspensions containing approximately 1.5 × 106 cfu/mL in a final volume of 200 µL. Tween 80 was used as a solubilizer at a concentration of 1% (v/v). The final concentrations of EOs were 25%, 12.5%, 6.25%, 3.13%, 1.56%, 0.78%, 0.39%, 0.20%, 0.10%, 0.05% to 0.025% and 0.012% v/v in final volume; all EOs were tested in triplicate for each bacterial strain. Minimal inhibitory concentrations were determined by spectrophotometric assay to 620 nm (BioRad). Control wells contained broth and broth with bacteria were used, respectively, as negative or sterility control and positive control.

Minimum bactericidal concentrations were determined by inoculating the assay mixtures from the wells showing no microbial growth onto the surface of blood agar. The presence of microbial growth on the medium indicated that the EO possessed bacteriostatic activity, while the absence of the growth implied bactericidal activity of the oil sample. The MBC is identified by determining the lowest concentration of antibacterial agent that reduces the viability of the initial bacterial inoculum by a predetermined reduction such as ≥99.9%.

Statistical Analysis

The results of the antibacterial activity of different EOs were assessed by using one-way analysis of variance (ANOVA). A P-value <0.01 was considered statistically significant. The statistical analysis was performed using the program one-way ANOVA calculator on the website Social Science Statistic (https://www.socscistatistics.com/tests/anova/default2.aspx).

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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