Activity of Six Essential Oils Extracted from Tunisian ...

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Comparative evaluation of the antimicrobial activity of 19 essential oils Article in Advances in Experimental Medicine and Biology · November 2015 DOI: 10.1007/5584_2015_5011

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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)

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Activity of Six Essential Oils Extracted from Tunisian Plants against Legionella pneumophila by Naouel Chaftar* a ) b ) c ), Marion Girardot a ), Nathalie Quellard d ), J¦rüme Labanowski e ), Tawfik Ghrairi b ), Khaled Hani b ), Jacques FrÀre a ), and Christine Imbert a ) a

) Universit¦ de Poitiers, EBI, UMR CNRS 7267, Bt. D1, 6 rue de la Mil¦trie, TSA 51115, FR-86073 Poitiers (phone: þ 33-2-35146454; e-mail: [email protected]) b ) Facult¦ de M¦decine, UR08 – 45, D¦partement de Biochimie, Avenue Mohamed Karoui, 4002 Sousse, Tunisie c ) Universit¦ de Rouen, LMSM - EA 4312, UFR des Sciences et Techniques, FR-76821 Mont-SaintAignan (current address) d ) CHU de Poitiers, Service de Microscopie, 2 rue de la Mil¦trie, FR-86021 Poitiers e ) Universit¦ de Poitiers, IC2 MP, UMR CNRS 7285, 4 rue Michel Brunet, FR-86022 Poitiers

The aim of this study was to investigate the composition of six essential oils extracted from Tunisian plants, i.e., Artemisia herba-alba Asso, Citrus sinensis (L.) Osbeck, Juniperus phoenicea L., Rosmarinus officinalis L., Ruta graveolens L., and Thymus vulgaris L., and to evaluate their activity against Legionella pneumophila (microdilution assays). Eight Legionella pneumophila strains were studied, including the two well-known serogroup 1 Lens and Paris strains as controls and six environmental strains isolated from Tunisian spas belonging to serogroups 1, 4, 5, 6, and 8. The essential oils were generally active against L. pneumophila. The activities of the A. herba-alba, C. sinensis, and R. officinalis essential oils were strain-dependent, whereas those of the J. phoenicea and T. vulgaris oils, showing the highest anti-Legionella activities, with minimum inhibitory concentrations (MICs) lower than 0.03 and lower than or equal to 0.07 mg/ml, respectively, were independent of the strainsÏ serogroup. Moreover, the microorganisms treated with T. vulgaris essential oil were shorter, swollen, and less electron-dense compared to the untreated controls. Isoborneol (20.91%), (1S)-a-pinene (18.30%) b-phellandrene (8.08%), a-campholenal (7.91%), and a-phellandrene (7.58%) were the major components isolated from the J. phoenicea oil, while carvacrol (88.50%) was the main compound of the T. vulgaris oil, followed by p-cymene (7.86%). This study highlighted the potential interest of some essential oils extracted from Tunisian plants as biocides to prevent the Legionella risk.

Introduction. – The Legionella genus consists of ca. 56 species/subspecies of Gramnegative bacteria belonging to over 70 serogroups [1]. Legionella pneumophila is responsible for two clinical forms of diseases, Pontiac fever and LegionnairesÏ disease, which is a severe form of pneumonia. Legionella bacteria are associated with aquatic environments, such as hot spring water, air-conditioners, cooling towers, and showers, and are able to survive to temperatures up to 608 [2]. This thermal resistance enables Legionella bacteria to easily survive and possibly proliferate; hence, it may represent an infection risk for patients, tourists, and staff in public health resorts and thermal spas, where people go for therapeutic vacations as well as rest periods. Numerous thermal springs are found in Tunisia; most of them are used to supply water for public baths (hammam), swimming pools, as well as thermal establishments. Public awareness of the risks of containing Legionella started emerging in recent years. Indeed, some studies Õ 2015 Verlag Helvetica Chimica Acta AG, Zîrich

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reported cases of legionellosis related to hot spring-water facilities [3] [4]. L. pneumophila strains belonging to serogroup 1 were the most often isolated in case of legionellosis [5] [6]. To prevent the risk of human contamination by Legionella bacteria, many chemical treatments as well as biocides may be applied to cooling towers and drinking-water systems [7]. However, such molecules are not allowed to be used for spa treatments, because they may alter the initial composition of the water; for example, the French legislation is based on the ÐD¦cret 89 – 369 – 06Ï of June 1989, related to natural mineral water and packaged drinking water. It is essential to find both alternative and effective treatments to prevent infection risks associated with Legionella survival and proliferation. Essential oils (EOs), which are complex mixtures of components including terpenic derivatives, are already well-known for their aromatic properties. The antimicrobial interest of numerous EOs was also demonstrated [8] [9]. EOs from Mediterranean plants, such as oregano and thyme, and terpenes, such as carvacrol and thymol, have already demonstrated antimicrobial activity against many Gram-positive and Gramnegative bacterial species [10] [11]. However, the anti-Legionella activity of EOs has been poorly investigated up to now. Only EOs from Cinnamomum osmophloeum, Cryptomeria japonica, Chamaecyparis obtuse, and Melaleuca alternifolia were shown to be active against L. pneumophila, and these activities were demonstrated against only one or a few strains belonging to different serogroups (1, 3, 6, and 8) and having a clinical or an environmental origin [12 – 14]. EOs are characterized by their hydrophobic properties, which may be an obstacle to their application as water treatment. However, their use as micellar solutions may overcome this issue [15] [16]. The aim of this study was to investigate the anti-Legionella activity of six EOs extracted from selected Tunisian plants against a relatively large number of strains belonging to different serogroups, to develop innovative strategies to fight the Legionella risk in recreational water-related facilities. In addition, the chemical composition of these EOs was investigated, to establish a potential link between antiLegionella activity and specific compounds. Results and Discussion. – Composition of EOs. The compositions of the six extracted EOs are shown in Table 1. The compounds were listed according to their retention time (tR ) on a Varian VF-5 MS column. The GC/MS analyses showed the presence of a total of 12, 17, 28, and 17 compounds for the A. herba-alba, C. sinensis, J. phoenicea, and R. graveolens EOs, respectively. Only six and eight compounds were obtained for the R. officinalis and T. vulgaris EOs, respectively, representing over 90% of the total oil compositions. Most of the studied oils were mainly composed of monoterpenes ( > 90%), oxygen-containing monoterpenes being the main compounds in the A. herba-alba, C. sinensis, R. officinalis, and T. vulgaris EOs. The A. herba-alba EO was composed to 96.68% of oxygenated monoterpenes, mainly a-thujone (36.38%), b-thujone (22.24%), camphor (19.12%), and chrysanthenone (10.88%), which is in agreement with the study of Aouadi et al. [17]. The EO extracted from C. sinensis leaves was mainly composed of cis-linalool oxide (24.90%), linalool (16.50%), trans-linalool oxide (15.82%), and b-myrcene (6.69%). Little data is available in the literature regarding the composition of EOs obtained


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Table 1. Chemical Composition of the Six Essential Oils (EOs) Obtained from Tunisian Plants Compound name and class a )

tR [min] b )

KI c )

Tricyclene (1S )-a-Pinene (1R )-a-Pinene Camphene b-Pinene b-Myrcene ( Z )-2,6-Dimethylocta-1,6-diene a-Phellandrene p-Menth-1-ene b-Phellandrene p-Cymene p-Menth-3-ene d-Limonene 1,8-Cineole trans-b-Ocimene g-Terpinene Nonan-2-one cis-Linalool oxide trans-Linalool oxide Terpinolene Linalool a-Thujone b-Thujone Chrysanthenone Lilac alcohol A a-Campholenal cis-Limonene oxide Camphor cis-Chrysanthenol Isoborneol Nonanol Borneol Pinocamphone Decan-2-one a-Terpineol trans-Carveol Fenchyl acetate Linalyl acetate exo-2-Hydroxycineole Tridecanol Linalyl butyrate cis-Chrysanthenyl acetate Isobornyl acetate Thymol Undecan-2-one Carvacrol Tridecane Mirtenyl acetate

6.06 6.33 6.73 6.78 7.52 7.80 8.21 8.35 8.53 8.82 8.83 8.92 8.94 9.06 9.42 9.77 10.70 10.16 10.62 10.73 10.97 11.22 11.55 11.66 11.79 11.80 11.98 12.42 12.85 12.99 13.05 13.13 13.25 13.63 13.76 13.93 14.44 14.64 14.65 14.72 15.20 15.44 16.21 16.40 16.42 16.63 16.83 16.86

930 941 957 958 985 994 1009 1015 1022 1033 1033 1036 1037 1041 1054 1066 1096 1079 1093 1097 1105 1115 1127 1131 1136 1136 1142 1158 1172 1177 1178 1181 1185 1197 1201 1208 1227 1235 1235 1237 1255 1263 1289 1296 1296 1304 1312 1313

Content [%] d ) A. h-a

C. sin

J. pho

R. off

R. gra

T. vul

– e) – – – – – – – – – 2.03 – – 3.58 – – – – – – 1.37 36.38 22.24 10.88 – – – 19.12 0.79 – – 1.33 – – – – – – – – – 0.60 0.39 – – – – –

– 3.00 – – – 6.69 2.86 – 0.38 – – 1.80 1.66 2.52 1.58 – – 24.90 15.82 – 16.50 2.98 – – 2.43 – – – – – 6.04 – – – 3.48 – – – 0.99 – 5.66 – – – – – – –

0.15 18.30 0.53 0.67 0.25 1.04 – 7.58 – 8.08 – – 3.52 – – 2.36 – – – 2.71 – – – – – 7.91 0.73 – – 20.91 – – 0.91 – – – 3.03 1.77 – – – – 0.19 – – – – 3.11

– – – – – – – – – – 3.31 – – 59.18 – – – – – – – – – – – – – 21.60 – – – 11.95 – – – 2.70 – – – – – – 1.23 – – – – –

– – – – – – – – – – 0.32 – – 0.20 – – 4.85 – – – – 1.49 0.65 0.36 – – – 1.68 – – – 0.99 – 1.39 – – – – – 4.19 – – 0.33 – 60.63 – 0.46 –

– – – – – – – – – – 7.86 – – 0.93 – – – 0.26 – – – 0.82 – – – – – 0.52 – – – 0.24 – – – – – – – – – – – 0.42 – 88.50 – –

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Table 1 (cont.) Compound name and class a )

tR [min] b )

KI c )

Content [%] d ) A. h-a

a-Longipinene d-Elemene a-Cubebene trans-Carvyl acetate a-Terpinyl acetate Dodecan-2-one b-Caryophyllene Dodecan-1-ol Humulene Tridecan-2-one Germacrene D g-Cadinene Cedrol Total identified Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Others

17.18 17.56 17.70 17.87 18.18 18.31 19.72 19.93 20.61 21.67 22.12 22.31 24.47

1326 1340 1345 1352 1363 1368 1421 1430 1457 1497 1516 1524 1614

1.25 – – – – – – – – – – – – 99.96 2.03 96.68 1.25 – –

C. sin – – – – – – – – – – – – – 99.29 17.97 75.28 – – 6.04

J. pho – 1.48 1.21 1.63 3.53 – 0.32 – 0.38 – 0.39 0.75 3.68 97.12 45.19 43.72 4.53 3.68 –

R. off – – – – – – – – – – – – – 99.97 3.31 96.66 – – –

R. gra – – – – – 3.74 – 7.90 – 2.43 – – 1.68 93.29 0.32 5.70 – 1.68 85.59

T. vul – – – – – – – – – – – – – 99.55 7.86 91.69 – – –

a

) Compounds with contents of at least 0.2% are listed in order of their elution from a Varian VF-5 MS column. b ) tR : Retention time. c ) KI: Kovats index; for experimental details, cf. Exper. Part. d ) Content expressed as percentage of the total oil composition in the six EOs: A. h-a, Artemisia herba-alba; C. sin, Citrus sinensis; J. pho, Juniperus phoenicea; R. off, Rosmarinus officinalis; R. gra, Ruta graveolens; T. vul, Thymus vulgaris. e ) –: Not detected.

from C. sinensis leaves, as they are usually extracted from peels. Nevertheless, EOs extracted from the leaves of Nigerian C. sinensis (L.) Osbeck were studied by Kasali et al. [18] and contained 54 compounds, with sabinene and d-car-3-ene being the major compounds. Blanco Tirado et al. [19] investigated the composition of an EO obtained from leaves of Colombian C. sinensis and also showed that sabinene (47.68%) was the major component, but followed by trans-b-ocimene (8.31%). Hence, the composition of C. sinensis leaf oils appears both variable and complex. Regarding the J. phoenicea EO, the contents of the monoterpene hydrocarbon and the oxygenated monoterpene fractions were balanced (45.19 and 43.72%, resp.), with isoborneol (20.91%) and (1S)-a-pinene (18.30%) being the major components. AitOuazzou et al. [20] recently reported a quite different chemical composition for an EO from Moroccan J. phoenicea; this oil was dominated by the monoterpenoids a-pinene (24.90%), b-phellandrene (24.40%), and a-terpinyl acetate (12.90%) [20]. Another study focusing on EOs from French J. phoenicea showed a variable composition depending on the studied EO sample, and the main difference was based on the apinene content [21]. The R. officinalis EO was mainly composed of oxygenated monoterpenes (96.66%); 1,8-cineole (59.18%), camphor (21.60%), and borneol (11.95%) were its


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major components. Again, according to the literature, the composition of this EO might be influenced by various factors, as the variety of Rosmarinus, bioclimatic conditions, or the extraction method [22 – 24]. The R. graveolens EO was mainly composed of fatty acid derivatives ( > 85%), undecan-2-one being the predominant component (60.63%), as previously observed by De Feo et al. [25]. Finally, the EO of T. vulgaris was mainly composed of carvacrol (88.50%) and to a lower extent of p-cymene (7.86%); thymol was poorly present (0.42%). This composition may be specific of the studied Tunisian EO, in so far as Dimitrijevic et al. [26] found quite balanced concentrations of thymol, carvacrol, and p-cymene (12.0, 10.0, and 17.4%) in an EO of T. vulgaris from Serbia. Antimicrobial Activity of EOs. The antimicrobial activity of the Tunisian EOs was assessed against L. pneumophila strains belonging to various serogroups (1, 4, 5/8, 6, and 8), to evaluate both strain and serogroup influences. The obtained minimum inhibitory concentrations (MICs) are shown in Table 2. Two peptides highly active against L. pneumophila, viz., warnericin RK [27] and surfactin, were included in this study as positive controls. Their MICs against the strain L. pneumophila Lens were 3 10 ¢ 3 and 4 10 ¢ 3 mg/ml, respectively. The EOs of A. herba-alba and C. sinensis showed the poorest anti-Legionella activity, with MICs mainly 0.90 mg/ml. Only strains GC-4 (serogroup 5/8), GC-5 (serogroup 4), and Paris (serogroup 1) were sensitive to these EOs (0.11 mg/ml MIC 0.45 mg/ml). Thus, their activity would be strain- and/or serogroup- dependent. Mighri et al. [28] [29] showed that an EO of Tunisian A. herba-alba containing balanced amounts of a- and b-thujone (ca. 24%) displayed a high activity against Gram-positive bacteria [28]. Our results supplemented these available data by demonstrating the low activity of this EO, containing more a-thujone (36.38%) than b-thujone (22.24%), against a Gram-negative bacterium. Concerning C. sinensis, the poorly known activities of oxidized forms of linalool, which are the main compounds of its EO, are completed by our study, suggesting a poor anti-Legionella activity. The EO of R. officinalis was efficient against L. pneumophila strains GC-4, GC-5, GC-6, GC-11, and Paris (MICs 0.55 mg/ml) and less active against strains GC-3 and Table 2. Minimal Inhibitory Concentrations ( MICs) of the Six Tunisian Essential Oils ( EOs) against Eight Strains of Legionella pneumophila Strain GC-3 GC-4 GC-5 GC-6 GC-11 GC-13 Lens Paris a

Serogroup 5/8 5/8 4 8 1 6 1 1

MIC [mg/ml] A. h-a a )

C. sin

J. pho

R. off

R. gra

T. vul

> 0.90 > 0.90 0.23 > 0.90 > 0.90 > 0.90 > 0.90 0.23

> 0.90 0.45 0.11 0.90 > 0.90 0.90 > 0.90 0.23

< 0.03 < 0.03 < 0.03 < 0.03 < 0.03 < 0.03 < 0.03 < 0.03

> 1.10 0.28 0.07 0.55 0.55 1.10 > 1.10 0.13

0.05 < 0.02 < 0.02 0.40 < 0.02 0.06 < 0.02 < 0.02

< 0.03 < 0.03 < 0.03 < 0.03 < 0.03 0.07 0.07 < 0.03

) For the abbreviations of the EOs, cf. Table 1.

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GC-13 (MICs 1.10 mg/ml). This EO was more active on the Paris strain (MIC ¼ 0.13 mg/ml) than on the Lens strain (MIC > 1.10 mg/ml), both belonging to serogroup 1, suggesting that this heterogeneous activity may not be directly linked to the serogroup. To the best of our knowledge, this is the first study focusing on the antiLegionella activity of R. officinalis EO. However, some R. officinalis EOs, with a specific origin and composition, have previously been shown to significantly inhibit the growth of other Gram-negative bacteria [30 – 33]. The R. graveolens EO demonstrated a strong anti-Legionella activity (MICs 0.06 mg/ml) against all strains except GC-6 (MIC ¼ 0.40 mg/ml). To the best of our knowledge, the anti-Legionella interest of R. graveolens EO has not been investigated yet. According to its high content in this oil ( > 60%), undecan-2-one may possibly be responsible, at least to some extent, for the observed high anti-Legionella activity. Finally, the EOs of both J. phoenicea and T. vulgaris were the most active against all the tested strains, with MICs > 0.03 and 0.07 mg/ml, respectively, regardless of the strain and serogroup. This activity of the J. phoenicea EO does not support the results of Angioni et al. [34] and Ait-Ouazzou et al. [20], which suggested that J. phoenicea EOs were not active against Gram-negative bacteria (E. coli, P. aeruginosa, etc.); however, bacteria of the Legionella genus were not included in these studies. This observation confirmed the singularity of the Legionella genus. Regardless of their composition, literature data suggests that EOs of Thymus species would have significant antimicrobial activities [11] [26], which would be related to their high content of phenolic compounds [35]. However, the anti-Legionella activity of thyme EOs has not been investigated yet. Carvacrol, the main component of the present T. vulgaris EO, is well-known for its wide spectrum of antibacterial properties [11] [36]. Electron-Microscopic Observations. The effect of the T. vulgaris EO on the bacterial structure was analyzed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The SEM experiments (data not shown) highlighted the morphological alteration of L. pneumophila cells treated with T. vulgaris EO at doses of 0.03 or 0.15 mg/ml, confirming the interest of this oil as antiLegionella biocide. Indeed, the treated L. pneumophila cells of the Paris strain were shorter, flattened, and expanded, compared to the cells of the untreated controls. Similar morphological modifications were observed studying the Lens strain. The TEM experiments demonstrated ultrastructural differences of the external membrane and inner cellular components between the cells of the Paris and Lens strains treated with T. vulgaris EO at doses of 0.03 and 0.07 mg/ml and untreated cells, respectively. The treated Legionella cells were less homogeneous and electron-dense than the untreated controls, as shown for the Lens strain in the Figure, highly suggesting a membrane disruption. The hypothesis that thymol, which was identified in a small amount in the present T. vulgaris EO (content of 0.42%), might act by altering the cell membrane was already suggested by previous studies [37] [38]. Moreover, carvacrol, constituting 88.5% of the present oil, has also been shown to alter the fluidity and the permeability of cell membranes, due to its lipophilic properties [38]. Carvacrol might destabilize the cytoplasmic membrane and act as a proton exchanger. The pH gradient across the membrane might therefore be reduced, inducing a collapse of the protonmotive force, and the resulting depletion of the ATP pool might lead to cell death [37].


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Figure. Transmission-electron micrographs of a) untreated control cells of L. pneumophila (Lens strain) and b) the same cells treated with Thymus vulgaris EO at the MIC (0.070 mg/ml). Legionella cells treated with T. vulgaris EO were significantly less homogeneous and electron-dense, compared with the untreated control cells, suggesting a membrane disruption.

Conclusions. – The compositions of the EOs isolated from six Tunisian plants were characterized. These EOs were mainly composed of oxygenated compounds. Some of these EOs displayed predominant components, whereas others were composed of several or numerous compounds present in balanced concentrations. It was difficult to make a direct connection between the anti-Legionella activity levels and the chemical composition of the EOs, as the antimicrobial activity could depend not only on the major components, but also on its less abundant components. Moreover, some components may display a synergistic activity. The activities of the EOs were evaluated against different L. pneumophila strains, taking into account the serogroup of the latter. The J. phoenicea and T. vulgaris EOs were the most active, with MICs 0.07 mg/ml against all the tested strains, showing for the first time their potential interest as L. pneumophila biocides. The present results suggested that J. phoenicea and T. vulgaris from Tunisia may be useful to control the legionellosis risks associated with water used for recreational purposes. This work was partially funded by grants from the CMCU (09G824), PHC Utique, and the Ministry of Higher Education and Scientific Research of Tunisia. The authors are grateful to Mr. Emile Bere and Ms. B¦atrice Fernandez for their helpful assistance in microscopy and Ms. Deborah Bell for the English revision.

Experimental Part Plant Material. The plant material was gathered on private lands belonging to Tunisian co-authors authorizing the collection. This study did not involve endangered or protected plant species. Aerial parts of Artemisia herba-alba Asso, Juniperus phoenicea L., Rosmarinus officinalis L., Ruta graveolens L., and Thymus vulgaris L. and leaves of Citrus sinensis (L.) Osbeck were collected twice from March to May, in 2010 and 2012. These harvesting periods corresponded to the presence of flowers for T. vulgaris, cones for J. phoenicea, and fruits for C. sinensis or happened just before the florescence of R. graveolens, A. herbaalba, and R. officinalis. A. herba-alba, C. sinensis, R. graveolens, and T. vulgaris were collected from the Sahel region (Sousse, Tunisia), R. officinalis from the Tunisian northeast (region of Zaghouan), and J. phoenicea from the Tunisian northwest (An Drahem). A voucher specimen of each plant has been deposited with the Herbarium of the School of Pharmacy at the University of Poitiers (France).

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Essential-Oil Extraction. After two weeks of drying in the shade, the EOs were extracted from the dried plants (200 g) by hydrodistillation for 4 h using a modified Clevenger-type apparatus, according to the procedure described in the European Pharmacopoeia [39]. The oils were then collected and stored at 48 in tight vials in the dark, until analysis. GC/MS Analysis. The GC/MS analyses were performed using a HP 6890 series chromatograph coupled to a HP 5973 mass selective detector and equipped with a Varian VF-5 MS cap. column (30 m 0.25 mm i.d., film thickness 0.25 mm). The oven temp. was programmed isothermal at 508 for 1 min, rising from 50 to 1808 at 58/min, isothermal at 1808 for 2 min, rising from 180 to 2508 at 108/min, and finally isothermal at 2508 for 1 min. Samples were diluted and injected automatically, and the injector was working in pulsed splitless mode (25 psi, 05 min); injector temp., 2508; detector temp., 2308; carrier gas, He (1 ml/min); ionization voltage, 70 eV. The identification of the components was based on i) the comparison of both their retention times (tR ) and their linear retention indices (KIs), determined rel. to the tR of a homologous series of n-alkanes (Kovats retention index), with those of reference samples and ii) computer matching of the mass spectra against those of a commercial mass spectral library (NIST Mass Spectral Search Program for the NIST/ EPA/NIH Mass Spectral Library database version 2.0 d, December 02, 2005) and mass-spectral literature data [18 – 22] [24 – 28]. Each component was quantified by integrating the peak area of the chromatograms. Bacterial Strains. The antibacterial activity of the EOs was evaluated against six strains of Legionella pneumophila isolated from three Tunisian spas located in the north of Tunisia (Korbous, Hammam Bourguiba, and Djebel el Oust) [40] and two reference strains of L. pneumophila, the Paris and Lens strains, both belonging to serogroup 1 and having been shown to be sensitive to erythromycin and rifampicin [41] [42]. Each strain was first cultured in buffered charcoal-yeast extract (BCYE) agar medium for 3 d at 378. One colony of this 72-h agar culture was then suspended in buffered yeast extract (BYE) medium to obtain a bacterial suspension at 106 CFU/ml measured by optical density (OD) at 600 nm (1 OD corresponding to ca. 109 CFU/ml). Antibacterial Test. Preliminary tests were performed, to evaluate the minimal concentration of MeCN required to solubilize the EOs and to assure that this concentration was not inhibiting bacterial growth (data not shown). The six EOs were diluted in 50% (v/v) MeCN. The tested concentrations of EOs ranged from 1.1 to 0.02 mg/ml. Minimum inhibitory concentrations (MICs) were determined as previously described [27]. Aliquots of 100 ml of BYE medium supplemented with the tested strain (106 CFU/ml) were added in the wells of 96-well microplates, followed by the addition of 10 ml of each EO. Growth controls without EO, MeCN controls, and negative controls (culture medium alone) were prepared. The peptide warnericin RK, which was previously isolated in our laboratory, was included as a positive control, as it has been shown to highly inhibit L. pneumophila (MIC for the L. pneumophila strain Lens ¼ 3 10 ¢ 3 mg/ml) [27]. Surfactin (Sigma) was the second positive control used (MIC for the L. pneumophila strain Lens ¼ 4 10 ¢ 3 mg/ml). Microplates were incubated for 3 d at 378 in a 5% CO2 atmosphere. MICs, determined using a microplate analyzer (Tecan-sunrise), were defined as the lowest concentration of EOs inhibiting growth of the tested strain. Two separate experiments were performed and analyzed in triplicate. Electron-Microscopic Analysis. Strains were first grown in BYE medium for 2 d with gentle shaking, and then the EO was added at the tested concentrations. After 24 h of incubation at 378 with gentle shaking, the cell suspension was centrifuged (20 min at 3000g). The culture medium was eliminated and bacteria were fixed with 3% glutaraldehyde in phosphate buffer (0.1 m, pH 7.4). The samples for the transmission electron microscopy (TEM) and the scanning electron microscopy (SEM) observations were prepared according to previously described protocols [43] [44].

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