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Industrial Crops and Products 61 (2014) 370–376

Contents lists available at ScienceDirect

Industrial Crops and Products

jo u r n al homep age: www.elsev ier .com/ locate / indcrop

irst evidence of antibacterial and synergistic effects of Thymusiatarum essential oil with conventional antibiotics

ariam Fadli a,b, Jean-Michel Bollab, Nour-Eddine Mezriouia, Jean-Marie Pagèsb,ahcen Hassania,∗

Laboratory of Biology and Biotechnology of Microorganisms, Faculty of Science, University Cadi Ayyad, Marrakech, MoroccoUMR-MD1, Aix-Marseille University, IRBA, Marseille, France

r t i c l e i n f o

rticle history:eceived 7 April 2014eceived in revised form 6 July 2014ccepted 16 July 2014

eywords:ntibacterial activityfflux pumpsermeabilizing effectynergy. riatarum

a b s t r a c t

The in vitro antibacterial activity of Thymus riatarum essential oil was evaluated in the present study usingdifferent assays toward human pathogenic isolates collected during serious infections.

The phytochemical analysis of T. riatarum essential oil (EO) indicated the presence of 20 components,including mainly borneol (41.67%), terpinen-4-ol (8.65%), and trans-caryophyllene (7.59%). Determina-tion of antibacterial activity showed that the EO possesses a noticeable potential of inhibiting the growthof tested strains with Minimum Inhibitory Concentrations (MICs) ranging from 3.57 mL/L to 7.5 mL/L.However, Pseudomonas aeruginosa showed the least sensitivity and was only inhibited with high con-centrations (MIC > 7.5 mL/L). The release of cytoplasmic material absorbing at 260 nm, measured in thepresence of T. riatarum EO, increased in response to oil concentration.

The ability of T. riatarum EO to block efflux pumps systems of resistant Gram-negative bacteria wasalso studied. The results indicated that the EO activity was significantly enhanced in the presence of anefflux pump inhibitor such as phenylalanine arginyl ß-naphthylamide (PAßN). In addition, the tested EO,

used at MIC/4, was able to increase chloramphenicol susceptibility of several resistant isolates.

These results demonstrated that T. riatarum EO could permeabilize bacterial membrane impairingefflux pump activity and may be used in combination with conventional antibiotics against multidrug-resistant bacteria. To our knowledge, this is the first report about the antibacterial activity and the modeof action of T. riatarum EO.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

The genus Thymus (Family Lamiaceae) comprises about 350pecies that are widely distributed in temperate zones (Asfaw et al.,000). Morocco is rich by about twenty species of thyme withn endemism rate of 57%, represented by twelve endemic speciesBenabid, 2000). Some of these species, like Thymus broussonetiioiss, Thymus maroccanus Ball and Thymus satureioides Coss, areommonly used in Moroccan traditional medicine because of theiredicinal and aromatic properties (Bellakhdar, 1997).The chemical investigation of Thymus genus has allowed

he isolation and identification of several compounds. They are

ainly flavonoids (Watanabe et al., 2005), triterpenes, glyco-

ides (Watanabe et al., 2005) and phenolic acids that characterizehe chemical composition of different species of this genus

∗ Corresponding author. Tel.: +21252430434649; fax: +212524437412.E-mail addresses: lhassani@uca.ma, lahcen.hassani@gmail.com (L. Hassani).

ttp://dx.doi.org/10.1016/j.indcrop.2014.07.029926-6690/© 2014 Elsevier B.V. All rights reserved.

(McGimpsey et al., 1994). Thyme EOs are characterized by avery pronounced chemical polymorphism within the same species(Thompson et al., 2003). However, the majority of the EOs arecharacterized by their high content of monoterpenes, pheno-lic compounds, especially thymol and carvacrol, and of othercompounds more or less biologically active, including eugenol,p-cymene, �-terpinene, linalool, geraniol and bornéol (El Bouzidiet al., 2013; Fadli et al., 2012b; Saad et al., 2010; Stahl-Biskup andSaez, 2002).

Some studies have shown that Thymus species have strongantibacterial activity, insecticide, antioxidant, antifungal, anti-inflammatory, antiviral, and antispasmodic (El Bouzidi et al., 2013;Fadli et al., 2012b; Ismaili, 2004; Jamali et al., 2013; Saad et al.,2010; Stahl-Biskup and Saez, 2002).

Consequently, thyme EOs are classified as the most powerful

natural antimicrobial agents because of their high activity espe-cially against pathogens resistant to antibiotics (El Bouzidi et al.,2013; Fadli et al., 2012b; Manou et al., 1998). In our previouswork, the antimicrobial efficacy of some thyme species has been

M. Fadli et al. / Industrial Crops and Products 61 (2014) 370–376 371

Table 1Bacterial strains used in the study.

Bacteria Strain Origin

Gram-negative bacteriaEnterobacter aerogenes EAEP289, MDR clinical isolate; KanS Pradel and Pagès (2002)Enterobacter aerogenes EAEP294, EAEP289 �acrA::Kanr (pEP755 integration) Pradel and Pagès (2002)Escherichia coli AG100, Wild-type E. coli K-12 Viveiros et al. (2005)Escherichia coli AG100A, AG100 �acrAB:: Kanr Viveiros et al. (2005)Escherichia coli K12W1130 Hamdali et al. (2008)Salmonella sp CCMM B17 (laboratory collection) Bensultana et al. (2010)Enterobacter cloacae Clinical isolate Bensultana et al. (2010)Klebsiella pneumoniae Clinical isolate Bensultana et al. (2010)Pseudomonas aeruginosa Clinical isolate Bensultana et al. (2010)Gram-positive bacteriaBacillus subtilis ATCC 9524 Hamdali et al. (2008)

ion)

dp2

Tf(tia

2

2

-

-

af

2r

iptUs

Bacillus cereus ATCC 14579

Micrococcus luteus ATCC 10240Staphylococcus aureus CCMM B3 (laboratory collect

emonstrated, in combination with usual antibiotics as membraneermeabilizers or as new inhibitors of efflux pumps (Fadli et al.,011, 2012a,b).

The aim of this study was to define the antibacterial effect ofhymus riatarum EO on antibiotic-resistant bacteria, with a specialocus on (i) its permeabilizing effect on the bacterial membrane andii) its capability to restore chloramphenicol efficacy by blockinghe efflux pump expressed in some isolates. To our knowledge, thiss the first demonstration of the antibacterial activity and mode ofction of T. riatarum EO.

. Material and methods

.1. Bacterial strains

The bacterial strains tested in this study are presented in Table 1:

Six Gram-negative bacteria (Escherichia coli, Salmonella sp, Entero-bacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae,Pseudomonas aeruginosa) and four Gram-positive bacteria (Bacil-lus subtilis, Bacillus cereus, Micrococcus luteus, Staphylococcusaureus). The susceptibility of these strains was previously evalu-ated by agar dilution method in Mueller–Hinton agar (Fadli et al.,2012b).

Four multidrug resistant (MDR) isolates expressing efflux mecha-nisms: Two isogenic strains of E. coli, AG100 is a wild-type E. coliK-12 used as control strain and AG100A a kanamycin-resistantderivative strain deleted of AcrAB and hypersusceptible to chlor-amphenicol, tetracycline, ampicillin and nalidixic acid (Viveiroset al., 2005); E. aerogenes EAEP289 is a kanamycin-sensitive MDRisolate that exhibits active efflux of norfloxacin and chloramphen-icol, and EAEP294 is an AcrA− derivative mutant constructedfrom EAEP289 (Pradel and Pagès, 2002).

Strains were preserved at 4 ◦C and were grown on Luria–Bertanigar 24 h prior to any assay. Mueller–Hinton (MH) broth was usedor the antibiotic susceptibility tests.

.2. Plant material: Isolation and chemical characterization of T.iatarum EO

Aerial parts of T. riatarum were collected at pre-flowering stagen April 2009, from Tazzeka-region. The botanical identification of

lant material was confirmed by Prof. Ahmed Ouhammou, a plantaxonomist in the Laboratory of Ecology and Environment of theniversity Cadi Ayyad of Marrakech (Morocco), where the voucher

pecimens were deposited (no. 6287).

De Vries et al. (2004)Bensultana et al. (2010)Bensultana et al. (2010)

Dry shoots of T. riatarum were subjected to hydrodistillation for4 h. The oil recovered was stored in darkness at 4 ◦C. The yield per-centage, calculated as volume (mL) of EO per 100 g of plant drymatter, was 0.26%.

Qualitative and quantitative analyses of the EOs were carried outusing GC–MS (Agilent Technologies, J&W Scientific Products, PaloAlto, CA, USA), equipped with a Agilent Technologies capillary DB-5MS column (30 m length; 0.25 mm i.d.; 0.25 mm film thickness),and coupled to a mass selective detector (MSD5975B, ionizationvoltage 70 eV; all Agilent, Santa Clara, CA). The carrier gas He wasused at 1 mL/min flow rate. The oven temperature program was:1 min at 100 ◦C ramped from 100 to 260 ◦C at 4 ◦C/min and 10 minat 260 ◦C. The chromatograph was equipped with a split/splitlessinjector used in the split mode. The split ratio was 1:100. Iden-tification of components was carried out by matching their massspectra with Wiley and NIST library data, standards of the maincomponents and comparing their Kovats retention indices withreference libraries. The component concentration was obtained bysemi-quantification by peak area integration from GC peaks and byapplying the correction factors.

2.3. Assessment of antibacterial activity

The antibacterial activity of T. riatarum EO was determined usingthe disk diffusion agar method as previously described (Fadli et al.,2012b). Petri plates containing 20 mL of MH agar medium wereseeded with 18–24 h culture of the bacterial strains in nutrientbroth. The inoculum size was adjusted with sterile saline solutionto approximately 108 UFC/mL. Ten microliters of tested EO wereloaded on sterile filter paper disks (Whatman no 1; 6 mm in diam-eter) and placed on the surface of the inoculated plates. Incubationswere carried out for 24 h at 37 ◦C. Antibacterial activity was deter-mined by measuring the diameter of the inhibition zone (in mm)generated around the disc. Discs of ciprofloxacin (5 �g) and gen-tamicin (15 �g), to which tested bacteria are resistant as it hasbeen previously evaluated (Fadli et al., 2012b), were used as pos-itive controls. All the tests were performed in triplicate.

2.4. Determination of minimal inhibitory and minimalbactericidal concentrations

The minimal inhibitory concentration (MIC) was determinedusing the macrodilution broth method as previously described(Fadli et al., 2012b). An overnight culture was appropriately diluted

in MH broth to obtain viable counts of approximately 108 UFC/mL.Concentrations of T. riatarum EO ranging from 0.058 to 7.5 mL/Lwere used. 850 �L of MH broth were added in each tube. One hun-dred microliters aliquots of oil dilution, solvent blank and positive

3 s and Products 61 (2014) 370–376

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Table 2Chemical composition of Thymus riatarum essential oil.

Kovats indices Component Percentage (%)

1017 Octan-3-ol 0.431033 o-Cymen 1.011056 �-Terpinen 0.591142 Camphor 0.941201 Borneol 41.671208 Terpinen-4-ol 8.651225 �-Terpineol 1.981255 Carveol 0.751271 Carvacrol methyl ether 1.291288 Carvenon 1.651312 l-Bornyl acetate 1.831418 ß-Bourbonen 0.401454 trans-Caryophyllene 7.591488 Humulene 0.241516 Germacrene D 2.841542 ß-Bisabolene 2.031552 �-Amorphene 1.161560 �-Cadinene 1.411691 �–Cadinol 1.91

different MDR isolates expressing efflux mechanisms is summa-rized in Table 5. T. riatarum EO showed an important activity againstthe tested bacteria, with MICs ranging from 15 mL/L to 7.5 mL/L forEAEP289 (overexpresses efflux pump) and AG100 (basal expression

Table 3Inhibition zone diameter of T. riatarum essential oil and antibiotics against Gram-positive and Gram-negative bacteria.

Inhibition zone diameter (mm)

T. riatarum(10 �L)

Ciprofloxacin(5 �g)

Gentamicin(15 �g)

Gram-negative bacteriaE. coli 12.3 ± 0.6 35.3 ± 0.6 25.7 ± 0.6S. sp 12.3 ± 0.6 10.3 ± 0.6 20.7 ± 0.6E. cloacae 11.7 ± 0.6 NI 18.3 ± 1.5K. pneumoniae 12 ± 0 NI NIP. aeruginosa 8 ± 0 35.3 ± 0.6 20 ± 1Gram-positive bacteriaB. subtilis 22 ± 1 36 ± 3.5 40 ± 0B. cereus 17.7 ± 0.6 25.3 ± 0.6 42.7 ± 0.6

72 M. Fadli et al. / Industrial Crop

ontrol samples were added in the tubes, separately. Fifty micro-iters of test bacterial suspension were then added. After careful

ixing, the inoculated tubes were incubated at 37 ◦C for 24 h.The MIC was defined as the lowest concentration of the EO

nhibiting the visible growth of tested strain. The minimal bacte-icidal concentration (MBC) was determined by spreading 0.1 mLf clear tubes, which did not show any visible growth after incu-ation during the test, on normal nutrient agar plate. The MBCas defined as the lowest EO concentration killing the bacterial

noculum after a further 24 h of incubation at 37 ◦C.

.5. Release of cytoplasmic material absorbing at 260 nm

The release of cytoplasmic material absorbing at 260 nm waseasured according to the method described previously (Fadli

t al., 2012b). Viable cells in exponential growth phase were col-ected by centrifugation (4000 rpm for 15 min), washed three times,nd resuspended in a saline buffer solution. Three milliliters of celluspension of approximately 108 UFC/mL were incubated, underentle agitation, for 1 h at 37 ◦C in the presence of T. riatarum EOt eight different concentrations, ranging from 2 MIC to MIC/64.fter incubation, cells were centrifuged at 4000 rpm for 20 min,nd the absorbance (260 nm) of the supernatant was determinedsing a Cary 50 Scan UV–vis spectrophotometer (Varian Australia,ty Ltd.). Correction was made for the absorption of the suspendingiquids with buffer saline containing the same concentration of oilltrated after 2 min contact with bacteria. Correction of untreatedells (control) optical density was made using buffer saline. Theumber of viable cells following EO treatment (CFU/mL) was deter-ined in triplicate by plating 100 �L of serial dilutions on nutrient

gar plates.

.6. Activity of EO against various gram-negative resistant strains

MICs were determined by microdilution assay as described pre-iously (Fadli et al., 2011). T. riatarum EO was analyzed using awo-fold dilution series prepared in dimethyl sulfoxide (DMSO)4%). Microwells containing 100 �L of oil dilution were inoculatedith 100 �L of cell suspension prepared by diluting an overnight

ulture in MH broth twice concentrated to obtain viable countsf approximatively 106 CFU/mL at a final DMSO concentration of%. At this concentration, no significant effect was observed onhe bacteria tested as control under these conditions. The inoc-lated microplates were incubated at 37 ◦C for 18–24 h and theIC was determined. Chloramphenicol and norfloxacin, substrates

f AcrAB-TolC efflux pump (Piddock, 2006), were used as usualntibiotics.

The MIC of T. riatarum EO was determined in combination withAßN at a final concentration of 20 mg/L, whereas synergy betweenhloramphenicol and T. riatarum EO was studied using the EO at

low concentration (MIC/4). Assays were repeated three times oneparate days and similar results were obtained in each experiment.

. Results

The chemical composition of T. riatarum EO identified 20 com-ounds (Table 2), representing 78.93% of the EO. It was mainlyomposed by borneol, terpinene-4-ol and trans-caryophyllene rep-esenting, respectively, 41.67%, 8.65% and 7.59%. Results of the diskiffusion experiment (Table 3) revealed that the tested EO had aide antibacterial spectrum. It was active on tested strains by pro-

ucing inhibition zone diameters varying from 8 to 22 mm. Theseiameters were lower than those obtained with usual antibiotics.ram-positive bacteria (B. cereus, B. subtilis and M. luteus) wereenerally found to be more sensitive than the Gram-negative ones

– Linoleic acid 0.56

Total 78.93

(E. coli, E. cloacae, K. pneumoniae), P. aeruginosa being the most resis-tant. The MIC and the MBC values of T. riatarum EO against the testedstrains (Table 4) showed that this EO inhibited Gram-negativebacteria (E. coli, E. cloacae, K. pneumoniae) at a concentration of7.5 mL/L. In contrast, Gram-positive bacteria (B. cereus, B. subtilisand M. luteus) were inhibited with MIC of 3.75 mL/L. This oil showedlow activity on the growth of P. aeruginosa which was only sus-ceptible to high concentration (>7.5 mL/L). It is also important tomention that the MIC was often equivalent to the MBC supportinga bactericidal action of T. riatarum EO.

For both Gram-positive and Gram-negative bacteria, the releaseof cellular content, absorbing at 260 nm, increased in response to EOconcentration (Fig. 1). This release was associated with the measureof cellular mortality. The effect on Gram-positive bacteria (B. cereus)was more pronounced than on Gram-negative bacteria (E. cloacae,K. pneumoniae): for the same EO concentration, i.e., 0.69 mL/L, theleakage value in B. cereus reached 0.93 optical density values com-pared to 0.588 and 0.456 obtained for E. cloacae and K. pneumonia,respectively.

The activity of T. riatarum EO and standard antibiotics against

M. luteus 15.3 ± 1.1 24.3 ± 0.6 26.7 ± 0.6S. aureus 11 ± 1 27.3 ± 1.1 31 ± 1

Inhibition zone includes diameter of disc (6 mm). Values are given asmean ± standard deviation. NI, No inhibition.

M. Fadli et al. / Industrial Crops and Products 61 (2014) 370–376 373

Table 4Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values of T. riatarum essential oil (mL/L) and antibiotics (mg/L).

MIC and MBC of T. riatarum (mL/L) and antibiotics (mg/L)

T. riatarum Ciprofloxacin Gentamicin

MIC MBC MIC MBC MIC MBC

Gram-negative bacteriaE. coli 7.5 7.5 62 62 31 31S. sp 7.5 7.5 31 62 4 15E. cloacae 7.5 7.5 125 >125 >250 >250K. pneumoniae 7.5 7.5 125 >125 >250 >250P. aeruginosa >7.5 >7.5 8 8 125 125Gram-positive bacteriaB. subtilis 3.57 7.5 2 62 2 8B. cereus 3.75 7.5 2 62 2 4M. luteus 3.75 3.75 2 62 2 31S. aureus 7.5 7.5 31 125 8 15

Table 5Minimum inhibitory concentrations (MICs) of T. riatarum essential oil and antibiotics in combination with phenylalanine arginyl ß-naphthylamide (20 mg/L) against testedGram-negative bacteria.

Strains MIC of T. riatarum (mL/L) and of antibiotics (mg/L)

T. riatarum Norfloxacin Chloamphenicol

−PAßN +PAßN −PAßN +PAßN −PAßN +PAßN

E. aerogenesEAEP289 15 1.875 64 64 1024 256EAEP294 0.936 0.936 8 8 64 32E. coliAG100 7.5 1.875 0.06 0.06 8 2AG100A 0.468 0.468 0.015 0.002 0.5 0.25

Table 6Minimum inhibitory concentrations (MICs) of chloramphenicol in combination with T. riatarum EO at MIC/4 against tested Gram-negative bacteria.

Combinations MIC of chloramphenicol (mg/L)

AG100 AG100A EAEP289 EAEP294

Chloramphenicol 8 0.5 1024 64Chloramphenicol + PAßN 2 0.25 256 32Gain 4 2 4 2

oEoc

Aca

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Chloramphenicol + T. riatarum (MIC/4) 2

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f efflux pump) and from 0.93 mL/L to 0.468 mL/L, respectively, forAEP294 and AG100A deleted of AcrAB. Similarly for tested antibi-tics, strains deleted for AcrAB (EAEP294, AG100A) had lower MICsompared to the strains used as control (EAEP289, AG100).

In combination with PAßN, the MIC of T. riatarum EO forG100 and EAEP289 decreased 4–8 times (Table 5), whereas thehemosensitizing effect of PAßN was less important for EAEP294nd AG100A (AcrAB−) with no reduction of the MICs.

The ability of T. riatarum EO, at MIC/4, to reduce chloramphen-col resistance of E. aerogenes (EAEP289 and EAEP294) and E. coliAG100 and AG100A) strains is presented in Table 6. For E. coliG100 used as control strain, T. riatarum decreased the MIC ofhloramphenicol from 8 mg/L to 2 mg/L and a similar gain wasbserved with PAßN. Regarding the strains deleted of efflux pumpsEAEP294 and AG100A), T. riatarum decreased chloramphenicol

IC 1-fold and 2-fold, in comparison with 2-fold obtained withAßN.

. Discussion

This study was aimed to investigate the biological potential ofhe essential oil of a wild growing T. riatarum endemic to Morocco.he phytochemical investigation showed a specific qualitativend quantitative composition of T. riatarum EO. Borneol (41.67%),

0.25 512 322 2 2

terpinene-4-ol (8.65%) and trans-caryophyllene (7.59%) were themost abundant compounds. A very different composition hasbeen reported previously for the same species, the major com-pounds were carvacrol (22.3%), p-cymene (17.5%) and �-terpinene(10.3%) (Iglesias et al., 1991). Whereas, a more recent study ofthis EO revealed a slightly similar chemical composition, with33 compounds identified (87.71%), and borneol (31.3%) as themajor compound (Belmalha et al., 2012). In fact, it is now widelyknown that the chemical composition of the EO can vary withinthe same species depending on the geographical location (Jaafariet al., 2007; Thompson et al., 2003). Other factors may be alsoresponsible for this variability, the most important are the climate,the soil, the harvest period and the method of preservation andextraction (Bakkali et al., 2008; Sivropoulou et al., 1997). Geneticfactors and the vegetative cycle may also influence this variability(Echeverrigaray et al., 2001; Jamali et al., 2013).

T. riatarum EO exhibited a significant antibacterial activityagainst tested strains. A correlation between this antimicrobialactivity and the chemical composition suggests that the activityof tested EO could be associated with the presence of high con-

centration of borneol. A bicyclic monoterpenes found widely inplants and a largely used food and cosmetics additive, borneolhas significant antimicrobial activity that was previously reported(Al-Farhan et al., 2010; Shunying et al., 2005; Tabanca et al., 2001).

374 M. Fadli et al. / Industrial Crops and Products 61 (2014) 370–376

Fig. 1. Leakage of cytoplasmic material in OD at 260 nm (—) and cell concentration in CFU/mL (—) after incubation in the presence of T. riatarum essential oil at differentc

Iawea2

oncentrations.

n addition, other major components could also contribute to thentimicrobial activity of the oil, including Terpinen-4-ol, which

as found to be responsible for bacteriostatic activity against sev-

ral microorganisms (Barel et al., 1991). Trans-beta-caryophyllenelso exhibited antibacterial and antifungal activities (Ozturk et al.,009). The constituents present in the greatest proportions are

not necessarily involved in the total antimicrobial activity; thequalitative involvement of less abundant components should also

be considered (Radulovic et al., 2013; Ultee et al., 2002). T. riatarumEO was found to be generally more effective against Gram-positivebacteria than Gram-negative ones. Gram-negative bacteria pos-sess an outer membrane which restricts diffusion of hydrophobic

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ompounds (Tian et al., 2009) and without this membrane, theell wall of Gram-positive bacteria can be permeated more easilynd external agents can disturb the cytoplasmic membrane (Burt,004; Radulovic et al., 2013).

During the incubation of E. cloacae, K. pneumoniae, and B. cereusith T. riatarum, a release of cellular material absorbing at 260 nmas observed. This release increased in response to EO concen-

ration and was associated with total cellular mortality. Theseesults have led us to hypothesize that a first effect of T. riatarumO is membrane disruption. An hypothesis that fits well withrevious studies demonstrating that some thyme species com-ounds permeabilize and disrupt the cell wall and cytoplasmicembrane and provoke leakage of cytoplasmic constituents (Fadli

t al., 2012a,b). Moreover, numerous studies have shown thatany essential oils, extracted from different plant species, induce

hanges in the cell membrane permeability/integrity (Radulovict al., 2013).

To assess the involvement of efflux pumps in the activity of. riatarum EO, MDR Gram-negative strains and their derivativesere studied. First, the direct activity of the EO on the bacte-

ia showed that the MICs of the strains expressing efflux pumpsAG100 and EAEP289) were greater than the MICs obtained forhe isogenic strains deleted of AcrAB pump (AG100A and EAEP294)Pradel and Pagès, 2002; Viveiros et al., 2005). This demonstrateshat the active compounds of tested EO could be substrates of thefflux pump involved in the antibiotic resistance of these bacte-ia (AcrAB-TolC). These results agree with those recently reportedegarding some natural compounds (Fadli et al., 2011; Kuete et al.,011). In the presence of PAßN, a well-known efflux pump inhibitorLomovskaya and Bostian, 2006; Lorenzi et al., 2009), the antibac-erial activity of T. riatarum EO increased significantly for bacterialtrains that express efflux pumps (EAEP289 and AG100) whereas,o reduction was observed in the MICs of the strains AcrAB−

EAEP294 and AG100A). These findings agree with reports abouthe involvement of efflux pumps in the resistance of Gram-negativeacteria to many natural products (Fadli et al., 2011; Kuete et al.,011; Lorenzi et al., 2009; Nikaido, 2001; Saleem et al., 2010).

To study if T. riatarum EO is able to restore antibiotic efficacyhrough specific interaction with drug efflux pumps, its abilityo reduce resistance to chloramphenicol in certain MDR Gram-egative bacteria was assayed at low concentration (MIC/4) .Ouresults showed that T. riatarum EO increased the susceptibility offflux pump-producer strains (EAEP289 and AG100). This findingupports the hypothesis that some compounds of this EO couldlock the efflux of usual antibiotics (Fadli et al., 2011; Lorenzit al., 2009). These active compounds could restore susceptibilityo chloramphenicol by blocking the drug efflux with a mechanismimilar to 5′-methoxyhydnocarpin that inhibits efflux of hydropho-ic alkaloid berberine in resistant bacteria (Lewis and Ausubel,006; Stermitz et al., 2000). Nevertheless, the results confirm that

nhibition of efflux pumps and the flux competition conferred byheir saturation are not the only mechanisms by which the activeompounds of tested EO act in synergy with chloramphenicol,ince it also increased the MIC of pumps-deleted strains. In fact,here are some generally accepted mechanisms of antimicrobialction of such plant metabolites that produce synergism, includingequential or dose dependent effect on common biochemical path-ays, inhibition of protective enzymes, combination of membrane

ctive agents, and use of membranotropic agents to enhance theiffusion of other antimicrobials (Radulovic et al., 2013; Sokolovat al., 2005).

As it has been demonstrated for other EOs (Langeveld et al.,

013) and for some thyme essential oils (Fadli et al., 2012b), T.iatarum EO can be considered as a good candidate for source ofew drugs, able to restore the antibacterial activity of antibioticsy a chemosensitive and a membranotropic effects.

Products 61 (2014) 370–376 375

Acknowledgements

This work was partially supported by the program Averroès-Erasmus Mundus, University Cadi Ayyad (Marrakech) and Aix-Marseille University. Many thanks to Prof. Rachid Serraj (CGIARFAO, Roma, Italy) for editing the manuscript.

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