Chemical composition, antibacterial and antioxidant activities of some essential oils against multidrug resistant bacteria

Journal Pre-proof Chemical composition, antibacterial and antioxidant activities of some essential oils against multidrug resistant bacteria Nait Irahal Imane (Conceptualization) (Writing - original draft), Hmimid Fouzia (Conceptualization) (Methodology), Lahlou Fatima azzahra (Conceptualization) (Visualization), Errami Ahmed (Conceptualization) (Methodology), Guenaou Ismail (Conceptualization) (Formal analysis), Diawara Idrissa (Conceptualization) (Investigation), Kettani-Halabi Mohamed (Conceptualization) (Resources), Fahde Sirine (Conceptualization) (Validation), Ouafik L’Houcine (Conceptualization) (Data curation), Bourhim Noureddine (Conceptualization) (Supervision)

PII:

S1876-3820(19)31413-1

DOI:

https://doi.org/10.1016/j.eujim.2020.101074

Reference:

EUJIM 101074

To appear in:

European Journal of Integrative Medicine

Received Date:

19 December 2019

Revised Date:

20 February 2020

Accepted Date:

21 February 2020

Please cite this article as: Imane NI, Fouzia H, azzahra LF, Ahmed E, Ismail G, Idrissa D, Mohamed K-Halabi, Sirine F, L’Houcine O, Noureddine B, Chemical composition, antibacterial and antioxidant activities of some essential oils against multidrug resistant bacteria, European Journal of Integrative Medicine (2020), doi: https://doi.org/10.1016/j.eujim.2020.101074

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Chemical composition, antibacterial and antioxidant activities of some essential oils against multidrug resistant bacteria

Nait Irahal Imane1, Hmimid Fouzia1,2, Lahlou Fatima azzahra1,3,6, Errami Ahmed4, Guenaou Ismail1, Diawara Idrissa5,6, Kettani-Halabi Mohamed6, Fahde Sirine1, Ouafik L’Houcine7,8 &

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Bourhim Noureddine1*

Laboratoire Santé et Environnement, Faculté des Sciences, Université Hassan II-Ain Chock,

Biotechnologie, Environnement et Santé, Faculté des Sciences El Jadida, Université

Chouaïb Doukkali, El Jadida, Maroc.

Laboratoire National de Référence. Université Mohammed VI des Sciences de la Santé

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Casablanca, Maroc.

Faculté de Médecine, Casablanca, Maroc.

National Institute of Forensic Science of the Police, Casablanca, Morocco.

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Service de Microbiologie, CHU Ibn Rochd, B.P 2698, Casablanca, Maroc.

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Mohammed VI University of Health Sciences (UM6SS), Casablanca, Morocco.

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Université Aix Marseille, CNRS, INP, Inst Neurophysiopathol, Marseille, France.

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Université Aix Marseille, APHM, CHU Nord, Service de Transfert d’Oncologie Biologique,

Marseille, France.

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*Corresponding Author: Tel: + 212 667125761 Email: [email protected] Fax: 00 212 522 23 06 74

Abstract Introduction: Antibiotic resistance is a serious threat to both human and environmental ecosystems. Alternatives to conventional antimicrobial therapy such as essential oils (EOs) are needed. The aim of the present study is to

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investigate the chemical composition, antibacterial and antioxidant activities of some commercial EOs. Methods: In this study, six selected EOs were screened against four bacterial strains identified as resistant to antibiotics: Staphylococcus aureus, Escherichia coli, Enterococus faecalis and Klebsiella pneumonia, as well as two sensitive bacterial strains: Staphylococcus aureus and Escherichia coli. Isolated bacterial strains were characterized and

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identified by morphological and biochemical tests. Commercially EOs of Rosmarinus officinalis L., Zingiber officinale Roscoe., Melaleuca alternifolia Cheel., Cymbopogon winterianus, Salvia sclarea L. and Syzygium

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aromaticum were evaluated for their antibacterial and antioxidant activities. A GC/MS was carried out to analyze the composition of the EOs investigated. Results: From this study, the results obtained reveal that no bacterial

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strain tested was resistant to any of the studied EOs. The EOs obtained from rosemary contained high amount of β-Pinene, ginger oil was rich in Zingiberene and the tea tree oil was dominated by α-Carene. Linalool was the

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major component in lemon and clary sage. Clove oil contained high amount of 3-Allylguaiacol. The antioxidant results showed a noticeable antioxidant activity in -carotene-linoleic acid system in all EOs. While, in DPPH method, only clove showed activity. Conclusion: These antibacterial and antioxidant activities support further

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studies to discover new chemical structures that can inhibit the growth of multiresistant bacteria.

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Keywords: Antibiotic resistance; Antioxidant; Active compounds; Essential oils; Multidrug resistant bacteria; Terpenes.

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1. Introduction Antimicrobials are substances with the capacity to selectively inhibit or kill microorganisms [1]. Unfortunately, humans can develop bacterial multiresistance to antibiotic treatments that were originally effective for the treatment of infection caused by that microorganism. Misuse of antibiotics has resulted in the emergence of resistance against them, which is another problem affecting public health [2, 3]. Bacteria have a remarkable ability to adapt to adverse environmental conditions [4] that lead to the emergence of resistant bacteria, which is recognized as a major problem in the treatment of microbial infections in hospitals and in the community [5]. Resistance to antibiotics is time-consuming generating a problem of global public health. Many studies show that

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pathogenic bacteria are increasing and becoming multiresistant. Therefore, the search for new preventive measures to slow down this process is necessary to overcome this public health problem [6].

An alternative to antibiotics commonly used in medicine may be natural products of plant origin widely distributed in nature [7]. Over the last few year, scientific interest in essential oils (EOs) has increased due to their potential

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employment as products in food and cosmetic industries and as well as an alternatives for treating multi-resistant bacteria diseases [8]. Several EOs have shown promising microbiostatic and microbiocidal activities against

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different strains [9-11] and this can be attributed to the presence of terpenes which are the main components of the EOs from medicinal plants [12]. In addition to their antimicrobial potential, essential oils have the capacity to

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modulate bacterial resistance and can be used as an adjuvant therapy against antibiotic resistance [13]. In the Mediterranean region, Rosmarinus officinalis and Salvia sclarea (Lamiaceae), Zingiber officinale

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(zingiberaceae), Melaleuca alternifolia and Syzygium aromaticum (Myrtaceae) and Cymbopogon winterianus (Poaceae) have been studied extensively for their anti-inflammatory, anticancer, anticholinesterase and radical

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scavenging activities [14-16]

The aim of this work was to isolate and identify novel clinical strains of bacteria isolated from Moroccan patients

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in Hospital University Cheik Khalifa in Casablanca. Then, investigate the antibacterial and antioxidant properties of different EOs: Rosmarinus officinalis L., Zingiber officinale Roscoe, Melaleuca alternifolia Cheel, Cymbopogon

winterianus,

Salvia

sclarea

L.

and

Syzygium

aromaticum,

characterized

by

gas

chromatography/mass Spectrophotometry (GC/MS) against these isolates.

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2. Materials and Methods 2.1. Isolation Clinical isolates including Gram-positive organisms Staphylococcus aureus and Enterococus faecalis, and the Gram-negative bacteria Escherichia coli were obtained from the bacteria bank of the laboratory of Microbiology of the University Hospital Center of Casablanca, Morocco. 2.2. Morphological and biochemical testing of the isolates The bacterial strains were identified to the genus level based on the colony morphology (appearance, size, margin, form, elevation), microscopic examination (Gram’s staining), physiological tests (Kligler test, mannitol motility,

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and growth at different NaCl concentrations, temperature and pH) and biochemical tests (catalase, oxidase, nitrate reduction, starch hydrolysis, casein hydrolysis, Voges Proskauer, citrate utilization, gelatin liquefaction, methyl red) by using API 20E, API-Staph , and API-Strep (bioMerieux) and adopting standard procedures. 2.3. Antibiotic sensitivity test

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The susceptibility test was carried out using the agar diffusion assay following the Clinical and Laboratory Standards Institute (CLSI) guidelines [17]. Susceptibility to the following antibiotics was tested: Penicillin (PC),

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Ampicillin (AM), Cefoxitin (FOX), Amikacin (AK), Gentamicin (CN), Tobramycin (TOB), Erythromycin (ERM), Tetracycline (TET), Fusidic acid (FA), Vancomycin (VAN), Teicoplanin (TEC), Cefixime (CFM5), Amoxicillin

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(AMX), Amoxicillin + Clavulanic acid (AMC), Cefotaxime (CTX), Ciprofloxacin (CIP5), Norfloxacine (NOR), Ceftazidime (CAZ), Trimethoprim-sulphamethoxazole (SXT) and clindamycin (CLI).

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2.4. Essential oils

The EOs used in this work were rosemary (Rosmarinus officinalis L.), ginger (Zingiber officinale Roscoe), tea tree (Melaleuca alternifolia Cheel), lemon (Cymbopogon winterianus), clary sage (Salvia sclarea L.) and clove

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(Syzygium aromaticum) obtained from PHYTOSUN arôms France in pure concentration. Their geographical origin and batch number are shown in table 1.

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2.5. Analysis of the essential oil

The GC/MS analysis of the EOs was performed using Agilent GC-Mass 6890 model gas chromatograph-5973N model mass spectrometer equipped with a 7683 series auto-injector (Agilent, USA). The system is equipped with a DB-5MS column (30 m x 0.25 mm x 0.25 µm film thickness, Agilent, USA). The temperature was programmed from 50°C to 260°C at 15°C min-1 and then held at 260°C for 20 min. Helium gas at a constant flow rate of 1 mL.min-1 was used as a carrier gas. The samples of 1.0 L were manually injected in the splitless mode. MS interface temperature was 230°C. For CG mass detection, an electron ionization system with ionization energy of

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70 eV was used and the scan range was 30 -700 amu. Identification and percentage composition of the compounds were performed using MS library and the NIST 98 spectrometer data bank and by comparison of their relative retention times with those of authentic samples on the DB-5MS column [17-24]. To determine the retention indices (Kovats indices) of the components, a mixture of alkanes (C8–C20) was used under the same conditions before injecting into the GC/MS system 2.6. Antibacterial activity tests 2.6.1.

Disc diffusion assays

A disc-diffusion assay was used to determine the growth inhibition of bacteria by EOs [26]. A single colony from

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an overnight bacterial culture plate was seeded into 5mL of an appropriate growth medium broth (NB). Culture tubes were at 37 °C until the 600 nm absorbance of the growth solution was greater than 1.0. Using a sterile swab, cultures were spread evenly into pre-warmed 37 °C agar plates. Sterile filter paper discs (6 mm in diameter) were gently pressed into the surface of the agar plates, and 10 L of the EOs were then pipetted into the discs. In order

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to enhance the detection limit, the inoculated system is kept at a lower temperature for several hours to increase the inhibition diameter [27]. Plates were then inverted and incubated for approximately 24h at 37 °C and the

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diameter of the inhibition zones was measured in mm, including the diameter of the disc. The sensitivity was

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classified according to [28] as follows: not sensitive for diameter less than 8 mm, sensitive for a diameter of 9–14 mm, very sensitive for a diameter of 15–19 mm, and extremely sensitive for diameter larger than 20 mm. Each test was performed in two replicates.

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Microdilution assay

For the minimum inhibitory concentration (MIC) of the EOs, a modified resazurin microtiter plate assay was used as reported by [29]. The resazurin reagent was obtained as resazurin sodium salt powder. A working solution was

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prepared at a concentration of 0.01 % (w/v) in distilled water and sterilized by filtration through a 0.22 mm membrane [30].

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Nutrient broth (50 L) was added to all the wells of the microtiter plates; 10 L of EOs dissolved in 10% DMSO was then added to all the wells in the row and then serially diluted down the rows from the first row. Bacterial cultures (50 L) 106 CFU mL-1 were added to all the wells and then incubated at 37°C for 24 hrs. A 30 L of 0.01% (w/v) resazurin solution was then added to each well and the samples were incubated for an additional 4 h at 37°C. The MIC, which is the lowest concentration at which no visible microbial growth is seen, was recorded. Each plate had a set of controls:

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A column with all solutions with the exception of the test compound; a column with all solutions with the exception of the bacterial solution adding of nutrient broth instead and a column with DMSO solution as a negative control. The plates were prepared in triplicate and incubated at 37 °C for 24 h. The color change was then assessed visually. The growth was indicated by color changes from purple to pink. The lowest concentration at which color change occurred was taken as the MIC value. 2.6.2.

Minimum bactericidal concentration

The minimum bactericidal concentration (MBC) which is regarded as the lowest concentration of the sample at which inoculated bacterial strains are completely killed, were confirmed by reinoculating 10 L of each culture

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medium from the microtiter plates, which was used for MIC, on nutrient agar plates and incubated at 37 °C for 24 h [31]. 2.7. Antioxidant activity 2.7.1.

DPPH free radical scavenging assay

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The DPPH free radical scavenging activity of the EOs was quantified according to the method [32] with some modifications. Briefly, 50 L of EOs with different concentrations (0.25-4 mg/mL) was added to 1ml of 60 µM

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DPPH ethanol solution. The mixture was shaken vigorously and allowed to stand in the dark for 30 min at room temperature. Absorbance was recorded at 517 nm. The scavenging activity was calculated using the following

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equation:

DPPH scavenging activity (%) = [(A0-At)/A0]x100

2.7.2.

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Where A0 is the absorbance of the control after 30 min and At is the absorbance of each EOs after 30 min. Bleaching of carotene in linoleic acid system

The antioxidant activity of EOs was also evaluated by β-carotene-linoleic acid bleaching according to a previous

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publication [33] with a little modification. A stock was prepared for an emulsion of β-carotene-linoleic acid by dissolving β-carotene at 0.8mg/ml in chloroform, 25 L of linoleic acid and 200mg Tween 80. Then, the

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chloroform was removed under low temperature 40°C, 100 mL of distilled water were added with vigorous shaking. To 2.5 mL of the obtained solution, were added 350 L of the sample at different concentrations (0.254mg/mL), after shaking, the mixture was incubated for 2 h at 50°C. Two controls were prepared, one with the standard BHT (positive control) and the other without samples (blank). Absorbance at 470 nm of each EO was immediately measured at 0 min, 30 min, 60 min, 90 min and 120 min. Relative antioxidant activity was calculated using the following equation:

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Antioxidant activity (%) =

𝐴𝑡 𝐴0

x100

Where At is the absorbance of the sample after 2 h, and A0 is the EO absorbance at the beginning of incubation. Results were expressed as IC50 (concentration required to cause a 50% inhibition: mg/mL). 2.8. Statistical analysis The data are given as means of three experiments ± one standard deviation (SD). Analysis of Variance (ANOVA) using Prism 7 software for Windows (GraphPad Software Inc., San Diego, CA, USA) with post-hoc Tukey HSD test, the probability value of p < 0.05 was considered to denote a statistical significance difference.

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Results

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3.1. Identification of the bacterial strains

The API system was used to identify 6 strains isolated from various clinical sources. The biochemical characteristics of the bacterial strains varied depending on the strain. While the API 20E test was used for gram-

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negative bacteria, the API 20 staph and 20 Strep test was used for gram-positive bacteria. Among the 6 identification results, 4 were identified with the API 20 E. The API 20 Staph system identified two isolates and

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API Strep identified one (table1).

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3.2. Antibiotic sensitivity test

Drug resistance analysis was conducted and the overall result is shown in table 3. The in vitro sensitivity of strains was done against multiple antibiotics.

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3.3. Chemical Constituents

The chemical composition of EOs was analyzed by GC/MS. The percentage composition of the identified

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compounds is presented in table 4 below. Results of the GC/MS analysis of the oil revealed the presence of a total of 35, 49, 27,49, 50 and 30 compounds from rosemary (R. officinalis), ginger (Z. officinale), tea tree (M.

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alternifolia), lemon (C. winterianus), clary sage (S. sclarea) and clove (S. aromaticum), respectively. 3.4. Antibacterial activity of essential oils The in vitro antibacterial activity of six EOs against pathogenic strains (both Gram-positive and Gram-negative bacteria) was assayed using the disc diffusion method by measuring inhibition zone diameter (Table 5). All EOs tested showed significant antibacterial activity. Tea tree (M. alternifolia) EO was extremely effective on all tested bacteria, with inhibition zones ranging from 14.7–35 mm. Clove (S. aromaticum), rosemary (R. officinalis), lemon (C. winterianus) and clary sage (Salvia sclarea) EOs exhibited a degree of bacterial growth inhibition less than

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Tea tree but remain good, while the greatest inhibition observed was caused by clary sage against E. coli ESBL. Ginger (Z. officinale) EO was less active against all bacteria, with inhibition zones ranging from 9.7–11.5 mm. Preliminary screening revealed that all EOs were effective against all tested bacteria; therefore, additional minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays were performed. The results obtained from these assays are shown in Table 10. These antibacterial assays revealed that tea tree has a very strong activity (MIC 0.55-17.6 mg/mL; MBC 2.21-17.6 mg/mL) together with clove (MIC 0.21 mg/mL in all bacteria; MBC 0.21 mg/mL in all bacteria), rosemary (MIC 0.67-10.8 mg/mL; MBC 2.7-21.6 mg/mL), and ginger (MIC 0.15-9.85 mg/mL; MBC 0.61-19.71 mg/mL); while the lemon and clary sage EOs had less

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antibacterial activity. 3.5. Antioxidant activity

The EOs were conducted to screening for their antioxidant activity with two tests; DPPH free radical scavenging and b-carotene-linoleic acid. Only clove (S. aromaticum) is able to consume free radicals, to neutralize the DPPH.

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This oil scavenged the DPPH radical in a dose-dependent manner and IC50 value was 0.26 ± 0.7 mg/mL for the oil and 0.015 ± 0.3 mg/mL for ascorbic acid (Table 7).

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The inhibition of the lipid peroxidation activity was assayed by the -carotene bleaching test, it determines the ability of antioxidants (EOs) to protect target molecules exposed to a free radical source and their capacity to delay

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lipids oxidation. As results from Figures 1 and 2, the activity of the EOs was found to be dose and time-dependent. The more effective an antioxidant the slower is the depletion of color. Initially, the performance of the clove (S. aromaticum) and BHT were very similar and showed lower exhaustion of color than the others. All EOs show an

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efficient antioxidant capacity except lemon (C. winterianus). Antioxidant activities of rosemary (R. officinalis), ginger (Z. officinale), tea tree (M. alternifolia), clary sage (S. sclarea) and clove (S. aromaticum) increased with

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their increasing concentration. Their antioxidant activities were 60.79%, 92.06 %, 82.44%, 70.09% and 98% at 4 mg/mL The absorbance decreased rapidly in the control sample, without the addition of an antioxidant, while in

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the presence of the oil/BHT, they retained their color, and thus absorbance for a longer time.

4.

Discussion

Pathogenic bacteria are increasing and becoming multiresistant because of irregular and unreasonable use of antibacterial agents [34]. From antibiotic resistance profiles determined for a range of antibiotics in this study, most isolates were clearly multidrug-resistant.

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The study of the chemical composition is generally carried out by GC/MS. Our results show that there are complexity and variability within the EOs. For example, rosemary has α-Pinene (13.36%) and β-Pinene (14.04%) as the major chemical components, α-Zingiberene is the major component in Zinger and α-Carene (17.41%), αPinene (13.05%), γ-Terpinene (10.06%) and Terpinen-4-ol (13.17%) are present at high concentrations in tea tree. Linalool is the major component in lemon and other EOs such as clary sage. In the composition of the EO of clove 3-Allylguaiacol (42.64%), Eugenol acetate (15.91%) and Caryophyllene (15.51%) are the main components. Generally, these major components are responsible for the various biological activities of EOs [8, 35-37]. The measured inhibition halos of rosemary (R. officinalis), ginger (Z. officinale), tea tree (M. alternifolia), lemon (C. winterianus), clary sage (S. sclarea) and clove (S. aromaticum) indicated that all of EOs are effective against

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reference strains and clinical isolates. The tea tree used in this study was more effective than the antibiotic chloramphenicol most likely due to the higher concentration of α-Carene, α-Pinene, and Terpinen-4-ol. Clove EO has a high antibacterial activity against Gram-positive and Gram-negative bacteria and showed MIC and MBC

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values of 0.21 mg/mL, indicating that this oil possesses strong bactericidal activity. The results indicated that this activity does not depend on the antibiotic susceptibility pattern, even in bacteria with high antibacterial resistance

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rates including methicillin-resistant S. aureus and extended-spectrum beta-lactamase producers E. coli, possibly due to the higher content of 3-Allylguaiacol, Eugenol acetate, and Caryophyllene. The activity of ginger was only

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limited to S. aureus the reference strain and clinical isolate. The lemon and clary sage EOs showed only bacteriostatic activity. However, the clary sage EO was only active against S. aureus with MIC value of 1.38

composition of this EO

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mg/mL. The low antibacterial activity of clary sage oil may be due to the relatively low content of the major

There are several targets described for EOs, namely, disrupting cell wall and membrane, and membrane-

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permeabilization, targeting drug efflux pumps, targeting R-plasmids and resistance spread, and Targeting microbial communications (quorum sensing and biofilm) [38]. Many studies showed that EOs tend to act more

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strongly on Gram-positive than on Gram-negative bacteria [39, 40], It is probably due to the differences in cell wall composition [41] since Gram-positive bacteria lack an outer membrane (OM), which Gram-negative bacteria have. This, being said the disc-diffusion method, is limited by the hydrophobic nature of EOs, which prevents their diffusibility through the agar medium, and shows a better action in liquid medium as seen in clove (S. aromaticum). However, EOs can also exist in a potentially highly bioactive vapor phase, and some EOs have shown antimicrobial activity that does not require direct contact with the EO [11, 26]. It is also quite interesting

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that no bacterial strain tested (clinical/reference or sensitivity/resistance) was resistant to any of the EOs studied. A list of reports summarized in Table 12 indicates various antibacterial activities possessed by EOs. The -carotene-linoleic acid assay, employs a lipid substrate (linoleic acid), which is closer to the real lipid system occurring in food products and in humans. The EOs are more active on the inhibition of the lipid peroxidation, presumably due to the high specificity of the test for lipophilic compounds, which proved that the clove EO is more potent antioxidant than the others suggesting that the effect depends on the composition of each oil. Ascorbic acid was tested and he had shown a weak activity probably because he’s a polar compound and the low activity of lemon oil can be explained by the abundance of the ineffective compounds. In the DPPH method, only clove

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showed activity with an IC50 of 0.26 ± 0.7 mg/mL. This can and may be attributed to the presence of phenolic compounds and the superiority of the polar compounds. The conclusion showed that clove (S. aromaticum) contains terpenes/terpenoids, with hydrogen-donating capacity and the ability to protect the oil against multiresistance and sensitive bacteria.

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The chemical composition of many EOs can vary according to various factors, including development stage of the plants, the organs harvested, the period, the geographical harvesting area [54] and can be influenced by multiple

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factors, including the biomass used (leaves or leaves plus terminal branchlets), chemotype, and mode of production

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(commercial steam distillation versus preparation by hydrodistillation in the laboratory with a Clevenger‐ type apparatus) [55]. The combination of these various parameters seems to be an explanation for the differences in

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Conclusion

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activity observed between the EOs.

The investigations on antibacterial and antioxidant activity of the six essential oils used in this study against

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multiresistant bacteria confirmed the potential of plant EOs to be used as antioxidant and antibacterial agents, especially with the emergence of antibiotic resistance. Further investigations are necessary to understand the

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mechanisms of the antibacterial activity of these EOs. Author statement

All authors: Conceptualization. Bourhim Noureddine: Supervision. Guenaou Ismail: Formal analysis. Fouzia hmimid and Errami Ahmed: Methodology. Lahlou Fatima azzahra: Visualization. Diawara Idrissa: Investigation. Kettani-Halabi Mohamed: Resources. Ouafik L’Houcine: Data Curation. Fahde Sirine; Validation. Nait Irahal Imane: Writing- Original draft preparation.

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Financial support This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of Competing Interests None.

Acknowledgment

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The authors would like to thank Pr. Abdelaziz Elamrani.

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References

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R. officinalis Z. officinale M. alternifolia C. winteriarus

50

S. sclarea S. aromaticum

ro of

% Inhibition of beta-carotene

100

BHT

Vit.C

0 0

1

2 3 4 Concentration (mg/mL)

5

Jo

ur

na

lP

re

Each value is expressed as mean ± standard deviation (n = 3).

-p

Fig. 1 Antioxidant activity of the EOs, BHT and control measured by -carotene-linoleic acid test.

15

Absorbance at 470nm

0.8

R. officinalis Z. officinale

0.6

M. alternifolia C. winteriarus

0.4

S. sclarea 0.2

S. aromaticum

0

50

100

150

Time (min)

BHT

Jo

ur

na

lP

re

-p

Fig. 2 Absorbance change of -carotene in the presence of EOs.

ro of

Carotene+lin 0.0

16

TABLES Table 1: Geographic origin and batch numbers of essential oils. Essential oil

Part used

Batch number

Collection region

Chemotype

R. officinalis

Twigs

3595894484007

Africa

1,8-cineole/camphre

Z. officinale

Rhizome

3401599424883

Sri Lanka

-Zingiberen

M. alternifolia

Aerial part

3595894883923

Australia

4-terpineol/y-terpinen

C. winteriarus

Aerial part

3401351481

Sri Lanka

Geraniol/Citronellal

S. sclarea

Aerial part

3595894468465

France

Acetate de lanlyle/linalool, gemacrene D, sclareol

Clove buds

3595890239540

Madagascar

Table 2: Bacterial strains used in this study. Identification Bacterial strain system

API 20 STREP

Gram-positive : Staphylococcus aureus Staphylococcus aureus NCTC 12493 Enterococcus faecalis Gram-negative: Escherichia coli ATCC 25922 Klebsiella pneumoniae ATCC 700603 Escherichia coli

Clinical isolates Reference strain Clinical isolates Reference strain Reference strain Clinical isolates

na

lP

re

API 20

E

Source

-p

API 20S

Eugenol, acetate d’eugenyl

ro of

S. aromaticum

Jo

ur

Table 3: Resistance of studied strains to antibacterial agents. Strain Resistance profile Staphylococcus aureus PC, AM, FOX, AK, CN, TOB, ERM, TET, FA, Staphylococcus aureus MRSA NCTC 12493 VAN, TEC Escherichia coli ATCC 25922 AM, AMC, CA, CTX, NOR, CN, TOB, STX Klebsiella pneumoniae CRK ATCC 700603 AMX, AMC, CTX, CFMS, CIPS, STX Escherichia coli ESBL VAN Enterococcus faecalis ERV

17

Table 4: Percentage compositions of six essential oils.

706

702

Chavibetol

750

3-Hexen-1-ol

760

769

1.4-Cyclohexadiene.1methyl-4-

770

773

Furfural

831

828

Cyclopentane. 1-methyl-3-(1-methylethyl)-

862

856

2-Heptanone

886

889

Cyclofenchene

887

888

Thiophene. 3-ethyl-

909

912

α-Thujene

921

2.6-Octadien-1-ol. 3.7-dimethyl-. acetate

926

α-Pinene

929

Camphene

945

β-Thujene

0.18

0.23

1.78

S. sclarea (Area%)

0.37 0.16 0.93 0.12 0.29 0.53 0.21 0.13

13.36

1.53

0.58

13.05

1.15

1.17

0.76

0.54

2.31 0.65 2.59

960

969

2.01

0.17

0.99

0.14

971

974

0.27

6.86

2.49

2.10

982

988

983

985

985

987

Isoaromadendrene epoxide

990

994

3-Carene

1005

1008

1007

1001

α-Phellandrene

1008

1002

Limonene

β-Myrcene Cis-2.6-Dimethyl-2.6-octadiene

na l

946

β-Pinene

α-Carene

Jo ur

Cyclohexene, 4-methylene-1-(1-methylethyl)

1020

1024

1,8-Cineole

1023

1026

p-Cymene

1025

1020

S. aromaticum (Area%)

0.67

924 932

C. winterianus (Area%)

f

Furan. α-ethyl-

oo

658

M. alternifolia (Area%)

pr

656

Z. officinale (Area%)

e-

Isovaleraldehyde

R. officinalis (Area%)c 0.54

Pr

Benzene

Kováts index KIExpa KILitb 654 654

Compounds

14.06

0.37

0.57 2.29 0.24

0.04 0.46 0.90

1.66

0.25

17.41 0.37

1.91 1.18

6.15

1.63

1.54

0.21

3.49

1.88

3.13

0.21

0.08

0.06

18

1030

1024

Ocimene

1040

1032

Ethanone. 1-(1-methyl-2-cyclopenten-1-yl)-

1043

γ-Terpinene

1057

1054

Linalool oxide

1066

1067

Camphenilone

1075

1078

Cyclooctane. 1.4-dimethyl-. cis-

1079

1081

α-Terpinolene

1085

1086

Linalool

1090

1095

3.53

α-Pinene oxide

1096

1099

0.44

β-Thujone

1103

1101

0.33

p-Mentha-1.3.8-triene

1112

1108

p-Menth-2-en-1-ol

1120

p-Menth-2-en-1-ol. trans

1133

Isopulegol

1140

Camphor

1147

0.31

0.40

0.39

e-

Pr 1141

7.12

1151

1155

5-Decen-3-yne. 2.2-dimethyl-. (Z)-

1152

1155

Borneol

1159

1165

1160

1164

cis-Linalool oxide

1167

1170

Terpinen-4-ol

1172

1174

α-Terpineol

1185

1186

2.51

Myrtenol

1189

1194

2.50

β-Terpineol

1189

1186

0.55

Dodecane

1199

1202

(R)-(+)-β-Citronellol

1221

1223

3-Allylguaiacol

1223

Neral

1240

Jo ur

0.07 1.41 0.07

0.37

0.04 1.43

0.48 10.97

11.99

0.52

1.55

0.08

0.16

0.11 0.35

1145 1153

α-Thujol

0.01

0.49

1136

1149

Isoborneol

10.06

pr

0.14

oo

1.71

1118

na l

(R)-(+)-Citronellal

3.83

f

D-Limonene

3.07 0.18 7.69 0.26 0.19

2.65

0.73

1.30 0.89 0.51

0.36

13.17

2.06

0.35

2.65

1.48

0.27

0.04 0.17

1.57

2.65

0.08

6.50 42.64

1235

1.12

19

1249

1247

Linalyl acetate

1263

1264

Bicyclo[3.1.1]hept-2-ene.3

1269

Thymol

1285

1289

p-Cymen-7-ol

1286

1289

E.Z-3-Ethylidenecyclohexene

1294

Geranyl formate

1297

1298

β-Cubebene

1339

1345

2.6-Octadien-1-ol. 3.7-dimethyl-

1350

Eugenol

1352

1356

0.44

α-Cubebene

1354

1345

2.13

Neryl acetate

1355

1359

Isoledene

1370

Cyclosativene

1370

α-Copaene

1373

β-Elemene

1385

Indol-5-ol β-Longipinene Di-epi-α-cedrene

Jo ur

Acetic acid. 1.7.7-trimethylbicyclo[2.2.1]hept-2-yl ester α-Bergamotene

0.13

Pr

e-

pr

0.19

0.14

1374 1369 1374

1.34

1389

1390

1393

1393

1395

1398

1400

1399

1402

1405 1408

1411

Longifolene

1408

1407

α-Santalene

1414

1416

α-Gurjenene

1415

1418

Isocaryophyllene

1415

1411

α-Cedrene

1416

1410

Caryophyllene

1419

1417

Cis-Thujopsene

1426

1429

oo

0.30

0.70

na l

3-Allyl-6-methoxyphenol

0.02

f

p-Chavicol

0.11 0.16 0.50 4.13 0.61

0.34

2.24 2.44

0.52

0.75 0.41 0.50

0.13

1.47

1.59 0.20 0.23

0.14 0.11 2.23 0.30

0.21

0.27 0.36 0.66 0.13 0.16 5.77

0.56

1.84

2.13

15.51

0.05

20

1431

0.71

Alloaromadendrene

1428

1432

1.44

γ-Elemene

1429

1434

β-Humulene

1434

1436

Aromandendrene

1443

1440

α-Himachalene

1451

1449

Humulene

1453

1452

Neoclovene

1456

1453

(E)-β-Famesene

1457

1459

Aromadendrene oxide-(1)

1458

1462

Salicylic acid, butyl ester

1465

1469

α-Curcumene

1476

1479

γ-Muurolene

1480

Germacrene D

1480

δ-Selinene

1488

β-Guaiene

1490

2.87 0.80

e-

pr

0.19

Pr 1478

0.05

1.23 0.81

0.21 0.29

0.59 0.38

1.48 4.26

5.4

0.13

1.66

0.44 1.42

1492

2.47 1.52

1492

0.42

1490

1493

1492

1495

0.62

1494

1496

0.34

1496

1500

1.48

1497

1496

γ-Patchoulene

1498

1502

0.42

γ-Selinene

1499

1492

0.06

β-Bisabolene

1503

1505

5.40

α-Farnesene

1507

1505

1.49

γ-Cadinene

1510

1513

α-Panasinsen

1515

1518

Eugenol acetate

1520

1521

β-Sesquiphellandrene

1524

1521

Calamenene

1526

1528

Cadina-1.4-diene Valencene Ledene

Jo ur

α-Muurolene

3.26

0.30

1484

na l

α-Zingiberene

1.54

f

1428

oo

β-Gurjunene

9.05 0.23 0.19 0.79 1.64

2.09

0.27

0.19

0.29

0.80 15.91 4.01 0.85

0.12

21

δ-Cadinene

1527

1522

Epiglobulol

1528

1530

α-Cadinene

1545

1537

Elemol

1550

1548

Germacrene B

1557

1559

Bicyclo [7.2.0]undec-4-ene. 4.11.11trimethyl-8-methyleneβ-Spathulenol

1564 1574

1577

Caryophyllene oxide

1577

1582

Cedrol

1596

1600

Ledol

1599

1602

Humulene epoxide II

1610

1608

Caryophylla-4(12),8(13)-dien-5-β-ol

1637

Methyl jasmonate

1643

β-Selinenol

1646

Isolongifolol

1712

Trans-Farnesol

1738

1742

1747

1749

1809

1813

0.51

1835

1838

0.63

2005

2002

0.47

Aristolene epoxide

2218

2221

0.99

Sclareol

2226

2222

1.49

2051

2056

0.50

Cyclodecasiloxane. eicosamethyl-

2064

2067

Anthracene, 9-propyl-

2090

2094

Linalyl anthranilate

2154

2157

Platambin Hexahydrofarnesyl acetone

Manool

a

Jo ur

Epimanoyl oxide

0.17

e-

2.61

Pr

0.42

1639

0.50

1648

0.27

1649

2.51

f

pr

0.81

2.17

0.25

oo

0.27

0.41 1.53 0.83 0.24

0.32

1.95 0.39

2.26

3.16

0.15 0.90 0.43 0.85

0.28

1715

0.17 0.09

na l

Carbofuran

1.65

0.21

0.05 0.65 0.31

3.24

0.98

Kováts index experimental. Kováts index literature. c Relative proportions as percentage of the total peak area. b

22

Table 5: Antibacterial activity (inhibition zone measured in mm) of six essential oils against selected strains of bacteria. Microorganisms

R.officinalis

Z.officinale

M.alternifolia

C. winteriarus

1 2 3 4 5 6

12.7 ±3.8b 22.0 ±2.8a 19.5 ±3.5a 19.7 ± 2.4a 19.5 ± 0.7a 19.5 ± 0.7a

10.2 ± 3.1a 11.5 ± 0.7a 11.2 ± 1.0a 7.5 ± 2.1a 10.5 ± 0.7a 9.7 ± 0.3a

31.0 ± 1.4ac 35.0 ± 1.4ab 29.5 ± 1.0c 37.2 ± 0.3b 14.7 ± 0.7e 28.2 ± 1.0c

12.0 10.0 15.0 16.5 14.7 18.5

± 0.7ab ± 0.0a ± 1.4bc ± 2.1bc ± 1.7bc ± 2.8c

S.sclarea

S.aromaticum

Cloramphenicol

9.75 ± 1.06a 8.55 ± 0.0a 16.7 ± 3.18b 10.7 ± 1.06a 31.2 ± 1.7c 9.7 ± 0.35a

18.3 ± 0.4a 16.1 ± 0.2a 19.9 ± 0.4a 15.5 ± 0.7a 15.4 ± 1.2a 17.4 ± 0.2a

26.0 ± 0.5a 24.0 ±1.2a 26 .0 ±0.5a 23.6 ±0.08a 26 .0 ±0.1a 24.6 ±0.5ca

1

S. aureus. 2S. aureus MRSA NCTC 12493. 3E. coli ATCC 25922. 4K. pneumoniae CRK ATCC 700603. E. coli ESBL. 6E. faecalis ERV. Mean ± SD in the same column with different superscript letters differ significantly (P < 0.05) Diameter of inhibition zone (mm) including disc diameter of 6 mm.

ro of

5

Table 6: Minimum inhibitory concentration (MIC; mg/mL) and Minimum bactericidal concentration (MBC; mg/mL) of six essential oils against selected strains of bacteria. Each value is the mean of triplicate assays. - not detected.

1 2 3 4 5 6

CMB 2.70a 2.70a 21.6b 2.70a 7.20c 5.40d

Z. officinale CMI CMB 0.15c 0.61a d 0.30 1.23b 9.85a 4.92b 9.85a 4.92b 19.71c

C. winteriarus

S. sclarea

S. aromaticum

CMI 2.21b 4.42a 17.6c 8.84d 4.42a 0.55e

CMI CMB 8.07a 8.07a 4.03b 5.38c 8.07a 8.07a -

CMI CMB 1.38 c 11.05a 22.11b 44.23d 11.05a 22.11b -

CMI 0.21a 0.21a 0.21a 0.21a 0.21a 0.21a

CMB 4.42a 17.6b 2.21c

CMB 0.21a 0.21a 0.21a 0.21a 0.21a 0.21a

S. aureus. 2S. aureus MRSA NCTC 12493. 3E. coli ATCC 25922. 4K. pneumoniae CRK ATCC 700603. E. coli ESBL. 6E. faecalis ERV. Column with different superscript letters differ significantly (P < 0.05).

lP

1

M. alternifolia

-p

R. officinalis CMI 1.35a 0.67b 10.8c 0.67b 1.85d 1.35a

re

Microorganisms

na

5

IC50 (mg/mL) 0.26 ± 0.7 mg/mL 0.015 ± 0.3 mg/mL -

Jo

ur

Table 7: IC50 values (mg/mL) of the DPPH antioxidant activity Samples S. aromaticum Ascorbic acid (Vit.C) BHT All the values are mean ± SD.

23

Table 8: Antibacterial activity against human pathogens. Plants EOs

Microorganism(s)

R.officinalis

Z.officinale

Determination of growth inhibition zones by disc diffusion.

Determination of MIC and MBC by the microdilution method.

Gram-positive B. cereus, B. subtilis, B. pumilis, S. aureus Gram-negative P. aeruginoa, E. coli [41] Gram-positive S. aureus, L. monocytogenes

M.alternifolia

Gram-positive Clinical S. aureus S. aureus NCTC 12493 Clinical E. faecalis

Gram-positive S. aureus and S. Typhimurium

re

Gram-positive B. subtilis, S. aureus Gram-negative S.paratyphi, S.flexneri and E. coli, [48]

lP

Gram-negative E. coli ATCC 25922 K. pneumoniae ATCC 700603 Clinical E. coli

-p

[46] Gram-negative P.aeruginoa [47] C.winteriarus

Anti-Inflammatory [44] Antioxidant, cardiovascular and neuroprotection, antiobesity, antinausea and antiemetic activities [45] Anti-Inflammatory and antimicrobial activity [16] Antidiarrheal, antimicrobial, antiinflammatory, antimalarial, antimutagenicity [48]

Gram-positive S.aureus ATCC 25923, B. pumilus ATCC 27142 B. subtilis IFO 3457 Gram-negative K.pneumonia ATCC 13883, S. typhimurium B11, P. aeruginosa [49]

Antimicrobial, Antioxidant, and Immunomodulatory [50]

Gram-positive E. coli O157:H7, L. monocitogenes [51]

Treatment of the Oral Pathology, Dental pain [52]

Jo

ur

S.aromaticum

na

S.sclarea

Antioxidant, antiproliferative Antimicrobial [15, 42]

ro of

Gram-negative P.aeruginoa,E.coli [43]

Traditional uses

24