Accepted Manuscript Antibiofilm formation and anti-adhesive property of three mediterranean essential oils against a foodborne pathogen Salmonella strain Hanene Miladi, Donia Mili, Rihab Ben Slama, Sami Zouari, Emna Ammar, Amina Bakhrouf PII:

S0882-4010(15)30018-8

DOI:

10.1016/j.micpath.2016.01.017

Reference:

YMPAT 1762

To appear in:

Microbial Pathogenesis

Received Date: 12 June 2015 Revised Date:

15 January 2016

Accepted Date: 19 January 2016

Please cite this article as: Miladi H, Mili D, Ben Slama R, Zouari S, Ammar E, Bakhrouf A, Antibiofilm formation and anti-adhesive property of three mediterranean essential oils against a foodborne pathogen Salmonella strain, Microbial Pathogenesis (2016), doi: 10.1016/j.micpath.2016.01.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

ACCEPTED MANUSCRIPT

Antibiofilm formation and anti-adhesive property of three

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mediterranean essential oils against a foodborne pathogen

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Salmonella strain

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Hanene Miladi1,2,*, Donia Mili3, Rihab Ben Slama1, Sami Zouari4, Emna Ammar2, Amina

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Bakhrouf1

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Faculty of Pharmacy, Monastir, Tunisia;

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Engineering School, Sfax, Tunisia

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Laboratory of Analysis, Treatment and Valorisation of Environment Polluants and Products,

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UR Study & Management of Urban and Coastal Environments, LARSEN—National

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Laboratory of Biochemistry, Faculty of Medicine, Monastir, Tunisia

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Range Ecology Laboratory, Arid Land Institute of Medenine, Medenine, Tunisia

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* Corresponding author: Hanene MILADI

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Laboratory of Analysis, Treatment and Valorisation of Environment Polluants and Products,

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Faculty of Pharmacy, Monastir

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E-mail: [email protected]

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Phone: +216 97 776 410; Fax: +216 73 461 830

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Abstract

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Plant extracts, and their essential oils (EOs) are rich in a wide variety of secondary

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metabolites with antimicrobial properties. Our aim was to determine the bioactive compound

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in three mediterranean essential oils belonging to Lamiaceae family, Satureja montana L.,

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Thymus vulgaris L. and Rosmarinus officinalis L., and to assess their antimicrobial,

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antibiofilm and anti-adhesive potentials against a foodborne pathogen Salmonella strain.

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The antibacterial activity of EOs and its biofilm inhibition potencies were investigated on 2

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reference strains Salmonella typhimurium and 12 Salmonella spp. isolated from food. Biofilm

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inhibition were assessed using the 2, 3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-

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tetrazolium-5-carboxanilide (XTT) reduction assay.

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The analytical data indicated that various monoterpene hydrocarbons and phenolic

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monoterpenes constitute the major components of the oils, but their concentrations varied

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greatly among the oils examined. Our results showed that S. montana L. and T. vulgaris L.

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essential oils possess remarkable anti biofilm, anti-adhesive and

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compared to R. officinalis EO. There is an indication that Rosmary EO might inhibit biofilm

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formation at higher concentrations. Therefore, the witer savory and thyme EOs represent a

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source of natural compounds that exhibit potentials for use in food systems to prevent the

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growth of foodborne bacteria and extend the shelf life of the processed food.

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Keywords: Essential oils; chemical composition; antimicrobial activity; biofilm inhibition;

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anti-adhesive property; Salmonella spp.

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bactericidal properties,

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1. Introduction

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Bacteria are traditionally thought to grow only in a planktonic structure. However, they can to

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easily survive in hostile environmental conditions as structure embedded in extracellular

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matrix called biofilm [1, 2]. Pathogenic bacteria develop a number of mechanisms causing

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damage disease in their hosts and they express a wide range of molecules that adhere to host

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cell-surface [3, 4]. Adhesion of pathogenic bacteria to host cell surface is a crucial event in

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colonisation and infection [5]. A biofilm is a microbial-derived sessile community

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characterised by cells that attach to an interface, embedded in a matrix. Depending on the

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species involved, the microcolony may be composed of 10–25% cells and 75–90%

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extracellular polymeric substances (EPS) matrix [6]. Elhariry et al. (2014), showed that some

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food-related pathogens such as Bacillus spp. and Pseudomonas spp. are recognised to form a

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stable biofilms in food-processing environments [3]. The presence of biofilms in food

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processing environments is a potential source of contamination that may lead to food spoilage

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[7, 8] and allow them to trap nutrients and withstand hostile environmental conditions [1]. It is

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believed that 99% of bacteria in nature exist in a biofilm [9, 10]. This structure is a very

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important step in the pathogenicity and drug resistance resistance to conventional antibiotics

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as well as to host immune system [11, 12]. Biofilm provides protection for the bacteria against

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antimicrobials and other living cells so that cells in biofilm are about 1000 times more

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resistant to antimicrobial agents [13, 14]. It is believed that about 60% of microbial infections

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are caused by biofilms [15].

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Salmonella infection constitutes a major public health problem in many countries and millions

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of cases of salmonellosis are noticed worldwide [16]. It has been reported that biofilm may

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confer bacterial resistance against several environmental stresses, antibiotics, disinfectants

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and the host immune system [17].

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ACCEPTED MANUSCRIPT For this reason, Food preservation has become a complex problem. New food products are

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frequently being introduced onto the market. Generally, they require a longer shelf life and

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greater assurance of freedom from foodborne pathogenic organisms [18]. There is therefore

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still a need for new methods of reducing or eliminating foodborne pathogens [19]. In this

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respect, essential oils and other extracts of plants have evoked interest as sources of natural

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products [20, 21]. They have been screened for their potential uses as alternative food

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additives in order to prevent the growth of foodborne pathogens or to delay the onset of food

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spoilage [22, 23, 24, 25]. The compounds are widely accepted because of the perception that

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they are safe and have a long history of use in folk medicine for the prevention and treatment

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of diseases and infections [26, 27].

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In this context, essential oils derived from plants of Satureja montana L., Thymus vulgaris

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L. and Rosmarinus officinalis L. which are a common household plant grown in many parts of

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the mediterranean region and belongs to the Lamiaceae family [25] have been used in food

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industry as flavouring agents, antioxidants and antimicrobials as well as in cosmetics industri

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[18, 28]. They are a rich source of biologically active compounds mainly monoterpenes,

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sesquiterpenes, and their oxygenated derivatives such as alcohols, aldehydes, esters, ethers,

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ketones, and phenols which may be involved in its physiological and biological activities [29,

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30].

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This study was therefore undertaken to determine the bioactive compounds in some

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commonly used dietary and medicinal plants and evaluate they antimicrobial and antibiofilm

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effect against some adherents Salmonella spp. where blocking bacterial adhesion to host

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surfaces provides potential approach to control the microbial infections.

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2. Materials and methods

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2.1. Microorganisms and condition for cultivation

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Salmonella typhimurium ATCC 1408; Salmonella typhimurium LT2 DT104 and 12

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Salmonella spp. isolated from food were used in this study which was kindly provided by

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Prof. Rhim Amel from the Regional Laboratory of Public Health of Monastir (Tunisia).

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Bacteria were cultured in tryptic soy broth (TSB) (Biorad, France). Inocula were prepared by

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adjusting the turbidity of the medium to match the 0.5 McFarland Standard Dilutions of this

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suspension in 0.1 % peptone (w/v) solution in sterile water inoculated on TSB to check the

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viability of the preparation. The bacterial cultures were maintained in their appropriate agar

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slants at 4 °C throughout the study and used as stock cultures.

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2.2. Plant material and essential oil extraction

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Aerial parts of Satureja montana L., Thymus vulgaris L. and Rosmarinus officinalis L.,

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belonging to the Lamiaceae family, were freshly collected in 2011 during the period of full

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flowering on the mountain in the south of France (Mediterranean climate country and

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mountainous region). A voucher specimens of evert plant were taxonomically identified

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according to the forester flora of France [31]. Dry aerial materials of plants were subjected to

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hydrodistillation for 3 h with 500 mL distilled water using a Clevenger-type apparatus

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according to the European Pharmacopoeia [32]. The EO was collected and dried over

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anhydrous sodium sulfate and then stored in sealed glass vials in darkness at 4 °C prior to

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analysis. EOs yield was calculated based on dryweight.

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2.3. Essential Oil Analyses

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2.3.1. Gas Chromatography/Mass Spectrometry (GC/MS)

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The volatile compounds isolated by hydrodistillition were analysed using gas

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chromatography–mass spectrometry (GC–MS). The GC–MS analyses were carried out using

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an Agilent Technologies 6890N GC. The fused HP-5MS capillary column (the same as that 8

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Packard, Palo Alto, CA, USA). The oven temperature was programmed as previously (50 °C

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for 1 min, then 7 °C/min to 250 °C, and then left at 250 °C for 5 min). The injection port

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temperature was 250 °C and that of the detector was 280 °C (split ratio: 1/100). The carrier

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gas was helium (99.995% purity) with a flow rate of 1.2 ml/min. The MS conditions were as

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follow: ionization voltage, 70 eV; ion source temperature, 150 °C; electron ionization mass

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spectra were acquired over the mass range 50 to 550 m/z.

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2.3.2.Volatile Compounds Identification

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Components were identified on the basis of gas chromatographic retention indices, mass

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spectra from Wiley 275 mass spectra libraries (software, D.03.00). The relative amounts of

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each individual component of the essential oils was expressed as the percentage of the peak

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area relative to total peak area. Kovats retention indices were calculated for each seperate

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component against n-alkanes mixture (C8–C26) [33] and to those previously reported in the

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literature [34, 35].

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2.4.Antimicrobial Activity

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2.4.1.Determination of minimal inhibitory concentrations

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The minimal inhibitory concentration (MIC) and minimum bactericidal concentration

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(MBC) of EOs) on planktonic cells were determined in Mueller–Hinton Broth (MHB) using a

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microtitre broth dilution method as recommended by the Clinical and Laboratory Standards

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Institute [36]. The inoculums of the bacterial strains were prepared from 12 h broth cultures,

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and suspensions were adjusted to 0.5McFarland standard turbidity. EOs were dissolved in

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10% dimethylsulfoxide (DMSO) and then with MHB as required. Cell suspensions (200 µL)

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were inoculated into the wells of 96-well microtitre plates to get the final concentration

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ranging from 0.0488 to 50 mg/mL for S. montana L. and T. vulgaris L. EOs and from 0.1953

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to 200 mg/mL for R. officinalis L. The wells containing only MHB and MHB with inoculum

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37°C for 18 h - 24 h. The lowest concentration of the tested samples, which did not show any

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visual growth of tested organisms after macroscopic evaluation, was determined as MIC,

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which was expressed in mg/ml. Each assay was performed in triplicate for all bacteria.

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2.4.2. Determination of Minimum bactericidal concentration

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In order to determine the minimum bactericidal concentration (MBC) values, 10 µL of each

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well medium with no visible growth was removed and inoculated in Mueller–Hinton plates

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(MH). After 24 h of incubation at 37°C, the number of surviving organisms was determined.

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MBC was defined as the lowest concentration at which 99% of the bacteria were killed. Each

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experiment was repeated in triplicate [37].

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2.5. Biofilm inhibition assays

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2.5.1. Assessment of biofilm metabolic activity using XTT reduction assay

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The metabolic activity of cells in biofilm was assessed using the XTT [2, 3-bis (2-methyloxy

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4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay according to methods

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described previously [1, 38] which measures the reduction of a tetrazolium salt by

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metabolically active cells to a coloured water soluble formazan derivative that can be easily

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quantified colorimetrically.

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Each EOs was tested for its potential to prevent biofilm formation of all strains. The EOs were

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added to the growth medium at the time of inoculation and the cells were allowed to form

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biofilms [1]. Prevention of biofilm formation by every EO was examined by microdilution,

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similar to the MIC assay for planktonic cells. A two-fold serial dilution of EOs (final

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concentrations from 0 to 25 mg/mL for S. montana L.and T. vulgaris L. and from 0 to 200

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mg/mL for R. officinalis L.) was prepared in 96-well polystyrene tissue culture plates

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containing TSB broth with 2% glucose (w/v).

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blank control.

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XTT (Sigma-Aldrich, Switzerland) solution (1 mg/ml) was prepared in PBS, filter sterilized

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and stored at -80 °C. Menadione (Sigma-Aldrich, Switzerland) solution (1 mM) was prepared

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in acetone and sterilized immediately before each assay.

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Aliquots of bacterial suspension (10 µL) were inoculated in tissue culture plate wells (5.104

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cfu/mL, final concentration). Following incubation at 37 °C for 24h, culture supernatants

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from each well were decanted and planktonic cells were removed by washing three times with

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phosphate-buffered saline (7 mM Na2HPO4, 3 mM NaH2PO4 and 130 mM NaCl at pH 7.4).

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Then 100 µl PBS and 12 µl XTT-menadione solution (12.5:1 v/v) were added to each of the

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prewashed wells and the control wells. The plate was then incubated for 3 h in the dark at

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37°C. Following incubation, 100 µl of the solution was transferred to fresh wells, and the

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colour change in the solution was measured with a multiskan reader at 492 nm. The

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absorbance values for the controls were then subtracted from the values of the tested wells to

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eliminate spurious results due to background interference. The percentage of biofilm

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inhibition was calculated using the equation [(OD growth control - OD sample)/ OD growth

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control] × 100. Each assay was repeated three times.

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The minimum biofilm inhibition concentration (MBIC50) was defined as the lowest

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concentration of each EO tested that showed 50% inhibition on the biofilm formation.

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2.5.2. Microscopic techniques

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Prevention of biofilm formation by each EO was confirmed by microscopic technique of

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Salmonella typhimurium ATCC 1408 and food isolated Salmonella spp. strain (S1: 6554).

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Briefly, strains were allowed to grow on round covers glass slides (diameter 1 cm) placed in

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24-well polystyrene plates (Greiner Bio-One, France) supplemented with every EO extracted

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from S. montana L., T. vulgaris L. and R. officinalis L. (0, MIC/2, MIC, 2 × MIC), incubated

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ACCEPTED MANUSCRIPT for 24 h at 37°C and stained with 1/20 Giemsa (Sigma, Switzerland) solution (v/v) for 20 min

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at room temperature. Stained glass pieces were placed on slides with the biofilm pointing up

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and were inspected by light microscopy at magnifications ×40 [39].

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2.5.3. Anti-adhesive property

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Adhesion test was carried out using A549 (human lung adenocarcinoma epithelial cell line).

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For the assay, a mixture of 100 µl of 107 cells/ml and A549 cell was treated with every EO

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extracted from S. montana L. by DL50 and 4 × DL50 concentration (0.4 and 1.6 mg/mL),

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from T. vulgaris L. by DL50 and 4 × DL50 concentration (0.3 and 1.2 mg/mL) for their 48 h

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cytotoxic activity and from R. officinalis L. EO by DL50, 4 × DL50 and 8 × DL50

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concentrations (0.08, 0.32 and 0.64 mg/mL) for their 48 h cytotoxic activity previously

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decribed [40, 41]. The 24- well plates were incubated at 37°C for 24 h in 5% CO2. After

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being inspected by optical inversion microscope at magnifications ×40.

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2.6. Statistical analysis

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Statistical analysis was performed on SPSS v.17.0 statistics software. Statistical differences

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and significance were assessed by one-way ANOVA test and Wilcoxon signed ranks test, as

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appropriate, to evaluate the antimicrobial activity and the biofilm inhibition according the

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type of strains and the EOs supplementation. A P value < 0.05 was considered significant.

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3. Results and discussion

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3.1. Chemical Composition of Essential Oil

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To determine the chemical composition of the essential oils of S. montana L., T. vulgaris L.

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and R. officinalis L., conventional GC-MS analyses were performed. The identified

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components, their percentages, their calculated RI are listed in Table 1, according to their

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elution order on a HP-5MS column. These analyses revealed that the most important feature

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of the oils was the presence of a high percentage of oxygenated monoterpenes which varies

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from 50 to 60% approximately, followed by the monoterpene hydrocarbons by 33.2, 39.85

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ACCEPTED MANUSCRIPT and 42.03 % for S. montana L., T. vulgaris L. and R. officinalis L. EOs respectively. Other

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classes that are sesquiterpene hydrocarbons and oxygenated sesquiterpenes were also present

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in small quantities in different oils.

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In the EO extracted from S. montana L., a single compound, carvacrol, accounted for 53.35 %

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of the EO, although 32 compounds were identified. ??-terpinene and p-cymene were also

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found in the EO of S. montana L. as major components (Table 1). Thus, this EO also showed

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a high content of oxygenated monoterpenes (59.11%) whereas no sesquiterpene compounds

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were found at quantifiable levels.

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Satureja montana L. oil showed a large variations in the relative concentration of major

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components: carvacrol, linalool, γ- terpinene, p-cymene and β-caryophyllene, depending on

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their geographic origin [42]. Moreover, the essential oil extracted from S. montana grown in

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Croatia contained Linalool, p-cymene, and γ -terpinene as the main components [43].

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In contrast, previous work on the phytochemical content of the essential oil of S. montana L.

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[43] (sample obtained from different country, same maturation stage) has identified linalool as

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the major component (61.5%). Furthermore, they reported that only a small amount of

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monoterpene phenols (carvacrol, 0.1%) is present, presumably because their samples have

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been derived from different chemotypes, and environmental conditions seem to have a

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significant influence on the relative amounts of EO components of S. montana [44].

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Moreover, 31 compounds were identified in the EO of T. vulgaris L. This EO consisted

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mostly of monoterpenes with both nonoxygenated (39.85%) and oxygenated structures

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(50.88%) and lower contents of sesquiterpene hydrocarbons (5.9%) and oxygenated

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sesquiterpenes (0.70%). The elevated Thymol content (41.33%) of this EO was highly

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significant. This constituent, conjointly to p-cymene (18.08%), and γ-terpinene (13.12%), p-

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Cymene-3-ol (5.24%) and and β-caryophyllene (5.05%) were the major components,

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representing 82.82% of the oil. Thyme oil was also characterised by the presence of a number

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of other constituents at concentrations ranging from 0.1% to 2.5% (Table 1). These results are

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in accordance with previous observations concerning several other thyme populations of the

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same taxon [19]. Regarding R. officinalis EO, 37 compounds, corresponding to 99.38% of the chemical

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components in the EO, were identified. Among these, 42.03% were monoterpene

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hydrocarbons and 52.04% were oxygenated monoterpenes, and it also contained 1.94%

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sesquiterpene hydrocarbons and 0.79% oxygenated sesquiterpenes. The major constituents of

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R. officinalis L. EO were 1,8-cineole (24.1%), camphor (19.87%), α-pinene (19.49%),

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camphene (8.65%), and p-cymene (3.79%), representing 75.9% of the EO. Chemical

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composition of rosemary oil have already been described. A study carried out by Wang et al.,

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(2008) [45] confirmed that 1,8-cineole (27.23%), α-pinene (19.43%), camphor (14.26%),

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camphene (11.52%), and β-pinene (6.71%) were the main constituents of rosemary oil in

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concentrations. Comparably, the rosemary oil examined in the present study contained large

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quantities of 1,8-cineole, camphor, and α-pinene, followed by β-pinene (4.84%) and

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camphene (3.82%). These results are all in agreement with Pintore et al., (2002) [46] who

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reported that rosemary oil with more than 40% 1,8-cineole was characteristic of plants found

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in Morocco, Tunisia, Turkey, Greece, Yugoslavia, Italy and France.

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The observed diversity in chemical composition of the various oils, when compared with

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those reported in previous studies, could be due to a number of factors. Such factors may

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include differences in climatic conditions, geographical location, season at the time of

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collection, stage of development, processing of plant materials before extraction of the oils,

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and occurrence of chemotypes.

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3.2. Variation of the antimicrobial activity

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The bacteriostatic and bactericidal effectiveness of the different EOs estimated by minimum

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inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) respectively

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belonging to Salmonella genus. The Results indicate a high variation of MICs and MBCs

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among the three mediterranean EOs and Salmonella spp. strains. Different essential oils

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showed a significant anti-bacterial activity against all tested strains that confirms previous

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findings [47, 48]. Differences in chemical composition may also explain the variation

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observed in antimicrobial activity of the three EOs evaluated, with the winter savory (S.

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montana L.) EO exhibited the highest anti-microbial activity than that of thyme EO and

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thereafter followed by the anti-microbial activity of rosemary essential oil.

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For S. montana L., MIC values were ranging from 0.39 to 0.78 mg/mL. The MBC values

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were also important, and low concentration of S. montana L. EO was sufficient to eliminate

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the growth of reference strains S. typhimurium (MBC: 0.78 mg/mL). It has shown also that

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0.39 mg/mL of this EO was sufficient to stop the growth of several pathogenic food isolated

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strains of Salmonella species (Table 2).

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T. vulgaris L. EO demonstrated a significant antimicrobial property. As presented in table 2, it

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exhibited bactericidal activity on all tested strains with MIC and MBC values ranging from

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0.78 to 1.56 mg/mL and 1.56 to 3.12. mg/mL, respectively. The major activity of essential

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oils of S. montana L. and T. vulgaris L. is due to their richness in phenolic compounds

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(carvacrol and thymol). Most antimicrobial compounds are phenols (carvacrol, thymol,

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eugenol), followed by alcohol (cineole, linalool ...) and to a lesser extent alkenes (p-cymene,

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pinene, terpinene ...) [19, 49]. Indeed, several studies have shown that high antimicrobial

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power of essential oils of several species of Satureja and thyme is attributed to their high

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phenolic compounds (carvacrol and thymol) [18, 50, 51, 52, 53]. One of the main

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characteristics of EOs is their hydrophobicity, which enables their incorporation in to the cell

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membrane [54]. Previous studies reported that EOs contained phenolic compounds with

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antimicrobial activity against a large number of bacterial strains and exhibits this role through

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ACCEPTED MANUSCRIPT the disruption of the cytoplasmic membrane [55]. The membrane loses its structure and

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becomes more permeable to ions [56].

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The antimicrobial activity of rosemary essential oil against a foodborne pathogen Salmonella

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spp. Strains was less than against the other EOs (Table 2). Oils from R. officinalis L. showed a

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low bactericidal effect (MICs = 12.5 to 25 mg/mL). However, MBC values of rosemary oil,

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were even higher than the corresponding MIC values (MBC = 25 to 50 mg/mL). We noted

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also that the MBC values of rosemary oil were 2-4 times higher than the MICs values.

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However, R. officinalis EO analysed in this study, even with a high content of 1,8-cineole,

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showed less antibacterial activity. Actually, this EOs has been previously reported to possess

305

moderate antibacterial activity [57, 58].

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The Turkish R. officinalis oil possesses a moderate antibacterial activity attributed to the high

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content of 1.8-cineole, the low content of camphor and verbenone, respectively [59]. A weak

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activity was reported for samples from Sardinia dominated by α -pinene, camphene,

309

verbenone, bornyl-acetate, camphor and borneol tested against several pathogenic bacteria

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[57, 58]. Our work showed that the variation of antibacterial activity in french R. officinalis

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differed according to the quantitative variation of essential oil compounds attributed to both

312

varietal and populational effects corroborating previous works [60, 61]. Considering the high

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number of the identified compounds in the analysed oils, it would be difficult to attribute the

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antibacterial activity differences to specific compounds [62].

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3.3. Inhibition of biofilm formation

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3.3.1. Effect of EOs on biofilm metabolic activity using XTT reduction assay

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In the presence of EOs, the metabolic activity of cells in biofilms was distinctly reduced after

318

24 h of incubation (Table 2). Our data also provides preliminary evidence that EO affect the

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oxidative activity of all the tested strains compared to the non treated biofilm (Table 3). EOs

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showed a significant inhibitory effect (P < 0.05) on biofilm formation of reference and food

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S. montana L. and T. vulgaris L. EOs. The results indicate that in addition to reducing

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biomass, most of the EO had an effect on the metabolic activity (Table 3).

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For S. montana L. EO, the BIC50 was observed with concentration about 0.113 and 0.22

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mg/mL for S. typhimurium ATCC 1408 and S. typhimurium LT2 DT104 respectively (Table

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3). For the food isolated strains Salmonella spp., the BIC50 of S. montana L. EO ranges from

327

0.12 to 0.499 mg/mL. We noted also that the two reference strains were more susceptible to

328

the winter savory EO than the others strains. Moreover, his BIC90 varies between from 0.6 to

329

6.45 mg/mL suggesting the ability of S. montana L. EO to prevention of biofilm formation.

330

Prevention of biofilm formation by T. vulgaris L. EO was also confirmed using XTT assay.

331

Thyme oil supplementation between 0.106 and 0.725 mg/mL, can inhibited 50% of biofilm

332

formation of all tested strains market by BIC50 values (Table 3). In addition we showed that

333

BIC90 was obtained after 2.78 and 3.5 mg/mL of T. vulgaris L. EO supplementation for the

334

two reference strains and was more in food isolated strains by 3.12 and 7.25 mg/mL (Table 3).

335

For R. officinalis L. EO, our study showed that rosomary oil might has an inhibitory effect on

336

the biofilm formation at higher concentrations than another EOs which were used in this study

337

(Table 3).

338

A statistical significant difference in prevention of biofilm formation between the treated

339

strains with each EO and control was found (P < 0.001). These results indicated that the

340

different EOs used in this study have an effect on the metabolic activity of cells embedded in

341

biofilm. Inhibition of growth in a preformed biofilm was also successful; however, the extent

342

of inhibition was much less compared to initial attachment. The reduced antibiofilm activity

343

towards a preformed biofilm is evidence that cells in a biofilm are more resistant to

344

antimicrobial agents compared to free-floating cells. Morover, most antibiotics are up to

345

1000-times less efficient against bacteria in biofilm than in suspension [63], which makes EO

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ACCEPTED MANUSCRIPT a very promising treatment alternative. Several author reported that the slower growth rate in

347

biofilms compared to planktonic cells as a result of reduced nutrient and oxygen supply has

348

been reported as an important factor attributed to the resistance in biofilms [14, 64]. Many

349

antimicrobial agents have been shown to be effective against microorganisms that are rapidly

350

growing and dividing, with only a few exceptions known. As a consequence, the slow growth

351

rate observed in biofilms makes them less susceptible to antimicrobial agents that are

352

normally effective against metabolically active cells [15].

353

The increased sensitivity observed in the reference isolate compared to the food isolate

354

confirms previous reports where, food isolates strain were able to withstand harsh

355

environmental conditions compared to type strains [65]. The resistance of clinical isolates in

356

both Gram-positive and Gram-negative bacteria to antibiotics is well known [66]. This has

357

been attributed to prior exposure to antimicrobial agents during therapy or other disinfection

358

processes resulting in secondary ⁄ acquired resistance [67].

359

3.3.2. Prevention of biofilm formation on glass microscope slide covers

360

The effect of three essential oils on biofilm formation of S. typhimurium ATCC 1408 was

361

confirmed by microscopic visualization. As shown in figure 1, a moderate reduction of

362

biofilm formation was observed with each EO supplementation (1/2 MIC) on the strong

363

biofilm formers (S. typhimurium ATCC 1408) whereas the biofilm former was relatively

364

inhibited with MIC from each EO and significantly inhibited with 2 × MIC EO

365

supplementation.

366

For a food isolated strain Salmonella spp. strain (S1), the microscopic visualization (Figure 2)

367

showed that 2 × MIC of the winter savory and Thyme EOs decreased the biofilm formation

368

on glass slides covers but not wholly suppressed. Moreover, we noted that adhesion of food

369

isolated strain on glass slides was completely inhibited by MIC R. officinalis L. EO

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ACCEPTED MANUSCRIPT supplementation. As described above, we showed that the food isolated strain was more

371

resistant that the reference strain.

372

Our results revealed that each of the three EOs efficiently kills Salmonella in suspension and

373

prevent biofilms formation. This effect on biofilm formation was confirmed by microscopic

374

analysis of strains grown on the surface of glass slides covers. Statistical analysis revealed a

375

significant difference between the percentage of biofilm inhibition obtained after EO

376

supplementation (2 × MIC) between treated cells and non treated ones (P < 0.001).

377

3.3.3. Anti-adhesive effect (on A545) of EOs

378

Adhesion of S. typhimurium ATCC 1408 and a food isolated strain Salmonella spp. strain

379

(S1) cells has been studied using cell lines of human origin in culture as in vitro models for

380

human respiratory epithelium. In the present study, adhesion of reference and food isolated

381

strains to A549 cells in absence and presence of 48 h of cytotoxic dose, which has been

382

recorded against A549, was investigated. In the absence of EO, S. typhimurium ATCC 1408

383

and S1 showed an important adhesive ability to A549 cells as presented in figure 3 and 4

384

respectively. They described the anti-adhesive activity of different EOs and shown that

385

DL50×4 EO of each one can attenuate the adhesive ability of S. typhimurium ATCC 1408 and

386

S1 to A549. We also noted that S montana L. EO was more effective for inhibiting bacterial

387

cell adherencethan thyme and rosemary EO, where is used at a concetration of DL50×4. On

388

the other hand, figure 4 showed that the food isolated strain (S1) was less susceptible to EOs

389

which can adhere to A549 celles after exposure to DL50×8 R. officinalis L. EO. So, S1 may

390

acquire some characteristics as a result of their presence in biofilm layers in the environment,

391

where the exposure to environmental stresses leads to alteration of their surface properties

392

[68]. This may explain the obtained results which showed clearly that the biofilm-forming

393

strains of S1 displayed high resistance to the anti-adhesive effect of studied EOs compared

394

with the reference strains (Figure 4).

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ACCEPTED MANUSCRIPT Significant antimicrobial, antibiofilm and anti-adhesive activities of EOs were demonstrated

396

against Salmonella spp. Practically, as anti-adhesion therapy may become feasible for

397

attenuating infectious disease. The winter savory, thyme and rosemary EO can be developed

398

as an effective inhibitor to prevent bacterial cell adhesion to biotic or abiotic surfaces and

399

subsequently inhibiting biofilm formation. This may provide natural protection from the

400

infection by some pathogenic bacteria [69].

401

4. Conclusion

402

Our study showed a potential antibacterial and antibiofilm activity for three mediterranean

403

EOs used as they are able to inhibit the initial stage of biofilm formation and subsequent

404

growth. Although most EOs were able to inhibit cell attachment. Isolation and identification

405

of the constituents that exhibit antibiofilm properties might be essential to include these as

406

alternatives in the control of biofilms. In view of their broad activity, these EOs may find

407

industrial applications as natural preservatives and conservation agents in food industries and

408

as active ingredients in medical preparations.

411 412 413 414 415

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ACCEPTED MANUSCRIPT 420

5. ACKNOWLEDGEMENTS

421

We are grateful to Prof. Rhim Amel from the Regional Laboratory of Public Health of

422

Monastir (Tunisia) for her help to collect the microorganisms.

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Table 1 : Chemical composition of Satureja montana L., Thymus vulgaris L. and Rosmarinus officinalis L. essential oils. Compounda

Retention index (RIb)

Percentage

RI PT

Peak number

S. montana L.

EP

SC

̶ 1.14 0.73 0.31 ̶ ̶ 0.15 0.86 ̶ 1.3 ̶ 0. 22 ̶ 1.76 13.03 0.73 ̶ 0.42 13.54 0.87 ̶ ̶ 1.81 ̶ ̶ ̶ ̶ 1.14 ̶ 0.5 ̶ 0.13 ̶

M AN U

921 927 934 949 954 974 978 981 987 992 1005 1006 1011 1018 1027 1030 1033 1033 1060 1070 1088 1089 1101 1107 1143 1148 1161 1170 1177 1181 1189 1194 1198

TE D

Tricyclene α-thujene α-pinene Camphene Verbenene Sabinene β-Pinene 1-Octen-3-ol 3-Octanone β-myrcene β-terpinene α-phellandrene δ-3-Carene α-terpinene p-cymene Limonene 1,8-cineole Eucalyptol γ-terpinene trans-sabinene hydrate α-Terpinolene Terpinolene Linalool 2,3-Dimethyl-2,3 Dihydropyridine Pinocarveol Camphor Isoborneol Borneol Isopinocamphone Terpinen-4-ol Cuminol α-terpineol Endo-isocamphonone

AC C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

T. vulgaris L. ̶ 1.69 0.85 0.60 ̶ 0.09 0.24 0.39 ̶ 1.7 ̶ 0.27 0.11 2.02 18.08 0.85 0.34 ̶ 13.12 0.43 ̶ 0.23 2.44 ̶ ̶ ̶ ̶ 1.35 ̶ 0.96 ̶ 0.14 ̶

R. officinalis L. 0.15 0.07 19.49 8.65 0.11 ̶ 3.34 0.12 0.26 2.28 0.09 ̶ 0.39 ̶ 3.79 3.41 24.10 ̶ 0.07 ̶ 0.19 ̶ 1.08 0.15 0.26 19.87 0.30 2.91 0.11 0.42 0.18 1.86 0.15

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a

̶ 0.19 ̶ 0.89 ̶ 53.35 ̶ 0.19 0.15 2.23 ̶ ̶ ̶ 0.3 0.25 0.48 0.15 0.43 ̶ ̶

̶ ̶ 0.10 ̶ 0.33 41.33 5.24 ̶ ̶ ̶ ̶ 5.05 ̶ 0.15 0.14 0.45 0.25 ̶ ̶ 0.40 ̶ 0.30

0.21 0.80 ̶ 0.10 1.57 ̶ ̶ ̶ 0.17 ̶ ̶ 1.27 0.36 0.31 ̶ ̶ ̶ ̶ ̶ 0.70 0.09 ̶

99.85

99.64

99.38

33.2 59.11 5.72 0.58 1.24

39.85 50.88 5.9 0.7 0.96

42.03 52.04 1.94 0.79 2.37

̶

SC

RI PT

̶

TE D

M AN U

1200 1213 1236 1255 1289 1299 1308 1309 1351 1374 1390 1427 1446 1461 1473 1521 1521 1529 1588 1594 1621 1651

EP

Total identified Grouped components (%) Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Others

α-terpinene Verbenone Thymol methyl ether Linalyl acetate Bornyl acetate Thymol p-Cymene-3-ol Carvacrol α-Terpinyl acetate Carvacrol acetate β-Bourbonene β-caryophyllene Aromadendrene α-Humulene Lavandulyl acetate α-amorphene γ-cadinene δ-cadinene Spathulenol Caryophyllene oxide Humulene epoxide T-Cadinol

AC C

34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55

, Compounds are listed in order of their elution from the HP-5MS column. , Retention index calculated against C8–C26 n-alkanes mixture on HP-5MS column.

b

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Table 2 : Antibacterial activity of three essential oils against foodborne pathogen Salmonella spp. strains S. montana a

MIC

MBC

MIC

MBC

0.78 0.78

0.78 0.78

1.56 1.56

3.12 1.56

25 25

50 50

0.39 0.39 0.39 0.39 0.39 0.39 0.39 0.39 0.39 0.39 0.39 0.39

0.39 0.39 0.39 0.39 0.39 0.39 0.39 0.78 0.39 0.39 0.39 0.78

3.12 1.56 1.56 3.12 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56

12.5 12.5 12.5 25 25 12.5 12.5 12.5 12.5 12.5 12.5 25

25 25 50 50 50 25 50 25 25 25 25 50

1.56 1.56 1.56 1.56 1.56 1.56 0.78 1.56 0.78 1.56 1.56 0.78

AC C

EP

Salmonella spp. strains Samples (date) S. spp (S1: 6554) Merguez (07-05-2012) S. spp (S2: 6877) Sandwich (16-05-2012) S. spp (S3: 6907) Salad (06-06-2012) S. spp (S4: 7215) Tajine (23-07-2012) S. spp (S5: 7466) Cheese (29-08-2012) S. spp (S6: 7643) Cheese (14-09-2012) S. spp (S7: 7945) Merguez (21-09-2012) S. spp (S8: 9487) Dinde (21-06-2013) S. spp (S9: 9340) Cooked meat (16-07-2013) S. spp (S10: 9681) Dinde (12-08-2013) S. spp (S11: 9812) Sandwich (28-08-2013) S. spp (S12: 9983) Merguez (30-09-2013) a , Minimum inhibitory concentration. b , Minimum bactericidal concentration.

R. officinalis

MBC

M AN U

ATCC 1408 LT2 DT104

b

TE D

Bacterial strains Salmonella typhimurium Salmonella typhimurium

MIC

RI PT

Antimicrobial susceptibility T. vulgaris

Origin

SC

Strains

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Table 3 : Antibiofilm effect of three essential oils against Salmonella spp. strains Strains

Inhibition of biofilm development (%) b

BIC50 (mg/ml)

RI PT

a

S. montana 1.15 0.85 4.5 0.68 0.75 0.6 3.125 3.00 5.375 6.00 6.45 6.25 5.50 5.50

BIC90 (mg/ml) T. vulgaris 2.78 3.50 7.25 6.60 6.00 5.51 3.12 6.11 6.21 5.51 4.62 6.89 7.51 7.00

R. officinalis 29.35 30.06 55.01 72.20 30.62 49.45 57.42 31.25 35.14 52.51 82.51 48.78 87.54 60.00

AC C

EP

TE D

M AN U

SC

S. montana T. vulgaris R. officinalis 2.31 0.106 S. typhimurium ATCC 1408 0.113 3.10 0.113 S. typhimurium LT2 DT104 0.22 3.51 0.725 S. spp (6554) 0.325 3.31 0.475 S. spp (6877) 0.195 0.462 2.94 S. spp (6907) 0.13 6.00 0.387 S. spp (7215) 0.24 3.44 S. spp (7466) 0.225 0.344 3.75 0.212 S. spp (7643) 0.121 2.06 0.205 S. spp (7945) 0.31 2.44 S. spp (9487) 0.565 0.197 6.25 0.112 S. spp (9340) 0.458 0.307 4.74 S. spp (9681) 0.499 6.75 S. spp (9812) 0.45 0.437 S. spp (9983) 0.21 0.19 5.37 a , minimum biofilm inhibition concentration of EOs that showed 50% inhibition on the biofilm formation. b , minimum biofilm inhibition concentration of EOs that showed 90% inhibition on the biofilm formation.

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Figure captions

RI PT

Figure 1 : Microscopic visualization of the effect of three essential oils on biofilm formation of Salmonella typhimurium ATCC 1408 cultured on glass slides covers. NC, Negative control ; PC, positive control (non treated slides). For Satureja montana L. EO S1, cells supplemented with EO MIC/2 ; S2, cells supplemented with EO MIC ; S3, cells supplemented with EO 2 × MIC. For Thymus vulgaris L. EO T1, cells supplemented with EO MIC/2 ; T2, cells supplemented with EO MIC ; T3, cells

SC

supplemented with EO 2 × MIC. For Rosmarinus officinalis L. EO R1, cells supplemented with EO MIC/2 ; R2,

M AN U

cells supplemented with EO MIC ; R3, cells supplemented with EO 2 × MIC.

Figure 2 : Microscopic visualization of the effect of three essential oils on biofilm formation of food isolated Salmonella spp. strain (S1) cultured on glass slides covers. NC, Negative control ; PC, positive control (non treated slides). For Satureja montana L. EO S1’, cells supplemented with EO MIC/2 ; S2’, cells supplemented with EO MIC ; S3’, cells supplemented with EO 2 × MIC. For Thymus vulgaris L. EO T1’, cells supplemented with EO MIC/2 ; T2’, cells supplemented with EO MIC ; T3’, cells

TE D

supplemented with EO 2 × MIC. For Rosmarinus officinalis L. EO R1’, cells supplemented with EO MIC/2 ; R2’, cells supplemented with EO MIC ; R3’, cells supplemented with EO 2 × MIC.

EP

Figure 3: Microscopic visualization of the effect of three essential oils on adhesion ability of Salmonella typhimurium ATCC 1408 to A549 cells. NC, negative control (A549 cells); PC, positives control (bacteria + A549 cells); For Satureja montana L. EO S1, cells supplemented with DL50 EO;

AC C

S2, cells supplemented with DL50×4 EO; For Thymus vulgaris L. EO T1, cells supplemented with DL50 EO; T2, cells supplemented with DL50×4 EO; For Rosmarinus officinalis L. EO R1, cells supplemented with DL50 EO; R2, cells supplemented with DL50×4 EO.

Figure 4: Microscopic visualization of the effect of three essential oils on adhesion ability of food isolated Salmonella spp. strain (S1) to A549 cells. NC, negative control (A549 cells); PC, positives control (S1 + A549 cells); For Satureja montana L. EO S1’, cells supplemented with DL50 EO; S2’, cells supplemented with DL50×4 EO; For Thymus vulgaris L. EO T1’, cells supplemented with DL50 EO; T2’, cells supplemented with DL50×4 EO; For Rosmarinus officinalis L. EO R1’, cells supplemented with DL50 EO; R2’, cells supplemented with DL50×4 EO; R3’, cells supplemented with DL50×8 EO.

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M AN U

SC

RI PT

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AC C

EP

Figure 1

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TE D

M AN U

SC

RI PT

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AC C

EP

Figure 2

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AC C

EP

TE D

M AN U

Figure 3

SC

RI PT

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AC C

EP

TE D

M AN U

Figure 4

SC

RI PT

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Highlights

1- Chemical composition of three medeterranien essential oils (EOs)

RI PT

2- EOs rich in phenolic compounds (carvacrol and thymol) had excellent anti-bacterial activities.

SC

3- Inhibition of biofilm formation to biotic and abiotic surfaces of different EOs

M AN U

4- The food isolated Salmonella spp. strains was more resistant that the reference strain to EOs activity

5- EOs had the potential to be used as food preservatives and may provide natural protection

AC C

EP

TE D

from the infection by some pathogenic bacteria

3