Phytomedicine 21 (2014) 1666–1674

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Synergistic effect of Myrtus communis L. essential oils and conventional antibiotics against multi-drug resistant Acinetobacter baumannii wound isolates Verica Aleksic a , Neda Mimica-Dukic b , Natasa Simin b , Natasa Stankovic Nedeljkovic c , Petar Knezevic a,∗ a

Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovica 2, 21 000 Novi Sad, Vojvodina, Serbia Department of Chemistry, Biochemistry and environmental protection, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovica 3, 21 000 Novi Sad, Vojvodina, Serbia c Health Center “Aleksinac”, Hygienic and Epidemiological Service, Momcila Popovica 114, 18220 Aleksinac, Serbia b

a r t i c l e

i n f o

Article history: Received 26 June 2014 Received in revised form 29 July 2014 Accepted 24 August 2014 Keywords: Essential oils/Myrtus communis Antimicrobial agents Synergism Acinetobacter baumannii Multi-drug resistant

a b s t r a c t Acinetobacter baumannii is a rapidly emerging, highly resistant clinical pathogen with increasing prevalence. In recent years, the limited number of antimicrobial agents available for treatment of infections with multi-drug resistant (MDR) strains reinforced tendency for discovery of novel antimicrobial agents or treatment strategies. The aim of the study was to determine antimicrobial effectiveness of three Myrtus communis L. essential oils, both alone and in combination with conventional antibiotics, against MDR A. baumannii wound isolates. The results obtained highlighted the occurrence of good antibacterial effect of myrtle oils when administered alone. Using checkerboard method, the combinations of subinhibitory concentrations of myrtle essential oils and conventional antibiotics, i.e. polymixin B and ciprofloxacine were examined. The results proved synergism among M. communis L. essential oils and both antibiotics against MDR A. baumannii wound isolates, with a FIC index under or equal 0.50. Combination of subinhibitory concentrations of essential oils and ciprofloxacin most frequently reduced bacterial growth in synergistic manner. The similar has been shown for combination with polymyxin B; furthermore, the myrtle essential oil resulted in re-sensitization of the MDR wound isolates, i.e. MICs used in combination were below the cut off for the sensitivity to the antibiotic. Time-kill curve method confirmed efficacy of myrtle essential oil and polymyxin B combination, with complete reduction of bacterial count after 6 h. The detected synergy offers an opportunity for future development of treatment strategies for potentially lethal wound infections caused by MDR A. baumannii. © 2014 Elsevier GmbH. All rights reserved.

Introduction Acinetobacter baumannii is a Gram negative pleomorphic, nonmotil, and nonfermentative bacterium, which is an etiological agent of various infections, including bacteremia, pneumonia, meningitis, urinary tract and wound infections (Maragakis and Perl, 2008; Peleg et al., 2008). The wound infections are of particular interest, since skin and deep soft tissue infections can lead to osteomyelitis, bacteriemia and other complications (Davis et al., 2005; Guerrero et al., 2010). In wound infections, A. baumannii was reported to be the most common Gram negative bacillus recovered from traumatic

∗ Corresponding author. Tel.: +381 214852681; fax: +381 21450620. E-mail address: [email protected] (P. Knezevic). http://dx.doi.org/10.1016/j.phymed.2014.08.013 0944-7113/© 2014 Elsevier GmbH. All rights reserved.

injuries to extremities and from patients who suffered traumatic injuries, particularly those obtained during emergency situations (for instance wars and earthquakes) (Tong, 1972; Arabi, 1987; Oncul et al., 2002; Heath et al., 2003). Beside the environment, the other important sources of the bacterium are healthcare settings, where it is nosocomially transmitted/acquired during wound care procedures (Murray et al., 2006; Maragakis and Perl, 2008). Taking into account these facts, A. baumannii has been characterized as a novel and a rapidly emerging clinical pathogen whose prevalence continues to increase (Guerrero et al., 2010). It has been proven that A. baumannii possesses variety of antimicrobial resistance mechanisms (Perez et al., 2007; Poirel and Nordmann, 2006) and publishing A. baumannii genome sequences confirmed existence of a wide array of resistance markers (Fournier et al., 2006; Park et al., 2011). Critical skills that

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have brought A. baumannii increasing antimicrobial resistance are the genetic flexibility of this pathogen, up-regulation of innate resistance mechanisms, acquisition of foreign resistance determinants and the potential to respond swiftly under selective environmental pressure (Peleg et al., 2008). Due to increasing antibiotic resistance rates, multi-drug (MDR) and pan-resistant A. baumannii have become a serious threat particularly to immunocompromised persons (Lockhart et al., 2007; Valencia et al., 2009). The number of currently available treatment options, i.e. fully active antibiotics for combating multi-drug resistant A. baumannii infections is extremely limited (Tan et al., 2011). The problems regarding application of conventional antibiotics, except antimicrobial resistance, include side effects (toxic, teratogenic and/or mutagenic; reaction of hypersensitivity etc.), high costs and environmental problems. These problems reinforced a tendency for finding new solutions, i.e. alternative treatments or strategies (Gortzi et al., 2006). Plant based products are among the alternative agents examined in order to replace conventional antibiotics and synthetic antimicrobials (Harikrishnan et al., 2003; Immanuel et al., 2004). To develop safer drugs, many studies of the herbal extracts and essential oils antimicrobial activity were carried out. One of the plants whose essential oils have been extensively examined is Myrtus communis L. (synonyms: Common myrtle, True myrtle). It is an evergreen shrub and a common part of typical Mediterranean flora, belonging to the Myrtaceae family (Snow et al., 2011; Bruna et al., 2007). M. communis is one of the important aromatic and medicinal species from this family with high essential oil content in its leaf, flower and fruit glands. Although in vitro and in vivo biological activity of myrtle essential oils has been confirmed and reviewed in literature (Akin et al., 2010; Berka-Zougali et al., 2012; MimicaDukic´ et al., 2010; Aleksic and Knezevic, 2014), there is still a lack of data on its activity against A. baumannii. Furthermore, myrtle essential oils has proven to be safe for use (Nassar et al., 2010; Tavassoli et al., 2011). Although there are studies focusing on the synergistic effect of essential oils in combination with antibiotics (e.g. Giordani et al., 2001; Rosato et al., 2007), there are no reports on the synergistic effect of antibiotics and Myrtus communis L. essential oil against MDR A. baumannii strains. Accordingly, the aim of this study was to evaluate in vitro antimicrobial effect of three myrtle essential oils, both alone and in combination with conventional antimicrobial agents, against MDR A. baumannii wound isolates.

Materials and methods Plant-derived materials and essential oil extraction Myrtus communis L. leaves were collected from three different coastal areas of the Montenegro territory (Bar, Kotor and Herceg Novi). The voucher specimens were prepared and identified by Goran Anaˇckov, PhD, and deposited at the Herbarium of the Department of Biology and Ecology (nos. 2-1819; 2-1821; 2-1823, BUNS Herbarium), University of Novi Sad Faculty of Sciences. Air-dried and ground leaves of collected plants underwent hydrodistillation according to the Ph. EUR. IV (European Pharmacopeia, 2002), with n-hexane as recipient solvent. According to the procedure, 100 g of plant material was topped with 1000 ml of distilled water in a round-bottomed glass balloon and distilled for 3 h. The obtained essential oils were dried with anhydrous sodium sulfate, filtered, and n-hexane was removed under reduced pressure. The obtained tree myrtle essential oils originating from Herceg Novi (MyHN), Kotor (MyK) and Bar (MyB) were stored at −20 ◦ C prior to analysis.

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Gas chromatography–mass spectrometry (GC–MS) analysis Composition of essential oils diluted with n-hexane (1 nl/ml) was analyzed by gas chromatography with mass-selective detector (Agilent Technologies 6890N + 5975B). The amount of 1 ␮l of the sample was injected into a split/splitless inlet at 250 ◦ C, with a split ratio 1:10. Helium (purity 99.999%) was used as a carrier, with a constant flow of 1.0 ml/min. The separation was achieved on a 30 m × 0.25 mm × 0.25 ␮m semipolar HP-5ms capillary column (Agilent Technologies) made of polydimethylsiloxane with 5% of phenyl groups, using the following temperature program: start at 50 ◦ C, 8 ◦ C/min to 120 ◦ C, 15 ◦ C/min to 230 ◦ C, 20 ◦ C/min to 270 ◦ C and hold for 16.9 min (total run time 35 min). Elute was delivered to the mass spectrometer via a transfer line held at 280 ◦ C. Ion source temperature was 230 ◦ C, electron energy 70 eV, and quadrupole temperature 150 ◦ C. Data were acquired in Scan mode (m/z range 35–400). Data were analyzed by Agilent MSD ChemStation software and AMDIS (Automated Mass Spectral Deconvolution and Identification System) in conjunction with NIST MS Search software. The compounds were identified by mass spectra comparison with libraries (Wiley Registry of Mass Spectral Data 7th ed. (McLafferty, 2005), and NIST/EPA/NIH Mass Spectral Library 05 (NIST/EPA/NIH, 2005) and confirmed by comparison of Kovats retention indices (KI) with literature data (Adams, 2001). Diesel oil, consisting of a mixture of C8 –C28 n-alkanes corresponding to 800–2800 KI, was used as a standard for determination of retention indices. Relative amounts of components, expressed in percentages, were calculated by normalization measurement according to peak area in total ion chromatogram.

Bacterial strains Two reference Acinetobacter baumannii strains from American Type Culture Collection (ATCC19606 and ATCC BAA747, Rockville, MD, USA) and twenty A. baumannii outpatient and clinical wound isolates (Table 2) were used to test the antibacterial activity of essential oils. The strains were identified according to Gram reaction, morphological, cultural, as well as physiological properties, and/or using VITEK2 system (Biomerieux, France). Identification was confirmed using molecular methods, i.e. multiplex PCR technique where reference strains were used as positive control. The bacterial DNA was extracted using GeneJET Genomic DNA Purification Kit (Thermo Scientific, Pittsburgh, US) following the protocol recommended by the manufacturer for Gram negative bacteria. The isolates were identified to the genomic species level by ITS sequence analysis, based on the method of Chen et al. (2007). Briefly, a multiple PCR was carried out using primers PrA1 and P-rA2, which target a highly conserved 425-bp region of the recA gene of genus Acinetobacter, and a second pair of primers P-Ab-ITSF and P-Ab-ITSB, used to amplify an internal 208-bp fragment from the ITS region of A. baumannii genomic species. The multiplex PCR was performed as described previously and amplicons were analysed by electrophoresis on agarose gels (1.5% w/v) amended with ethidium bromide. The gels were visualized using gel documentation system and analyzed by corresponding software (BioDocAnalyse, Biometra GmbH, Goettingen, Germany). Identified A. baumannii wound isolates were stored in Luria Bertani broth (LB) containing glycerol (v/v 10%) at −70 ◦ C. For the experiments, they were inoculated in LB and incubated overnight at 37 ◦ C. For all experiments, Muller Hinton agar or broth were used, unless stated otherwise.

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Determination of the antimicrobial agents MICs and MBCs values The antimicrobial agents evaluated in the study included three Myrtus communis L. essential oils (EO): MyHN, MyK and MyB, and eight antibiotics (AB): ceftriaxone, gentamicin, ciprofloxacin (CIP), kanamycine, amikacin, tetracycline, chloramphenicol, and polymyxin B (PMB) (Sigma, USA). The minimal inhibitory concentration (MIC) of the A. baumannii strains were determined by broth microdilution susceptibility testing method according to Eloff (2004). The modification for assessment of essential oils activity in all tests was made by incorporating a final concentration of ≤0.8% DMSO (Sigma, USA) (v/v) into the broth medium to enhance oil solubility. The concentrations of examined essential oils were two-fold serial dilutions ranging from 8 to 0.0625 ␮l ml−1 and for antibiotics two-fold serial dilutions ranging from 256 to 0.25 ␮g ml−1 . For minimal bactericidal concentration (MBC), 10 ␮l from wells without obvious bacterial growth was subcultured on nutrient agar plates to determine if the inhibition was reversible or permanent. MBC was determined as the highest dilution (lowest concentration) at which >99.9% reduction of initial CFU was obtained. Each experiment was performed in triplicate and three independent experiments, with Escherichia coli ATCC 25922 and S. aureus ATCC 25923 used as internal quality control strains for antibiotic susceptibility test. The results are represented as geometrical means of replications. Synergism testing Broth microdilution checkerboard method In order to determine synergism between essential oils and antibiotics, beside a reference strain (ATCC 19606), three strains of MDR A. baumannii (Aba-6637, Aba-4914 and Aba-5055) were selected. The combination of antibiotics and strains were selected according to the MICs which were above breakpoints, but under maximal examined concentration (256 ␮g ml−1 ). Accordingly, the following combinations were examined: CIP vs. Aba-6673 (MIC was 45.25 ␮g ml−1 ); CIP and PMB vs. Aba-4914, as well vs.. Aba5055 (MICs for both strains were the same, 64 and 4 ␮g ml−1 , respectively). The strains and range of concentrations was determined according to the previously assessed MIC of each antibiotic (AB) and essential oil (EO) for each of examined isolate. The concentrations of essential oils and antibiotics were prepared in range 0.03 × MICEO to 1 × MICEO and 0.03 × MICAB to 4 × MICAB , respectively. Using these concentrations, we tried to re-sensitize the strains combining antibiotics with myrtle oils, in order to obtain therapeutically acceptable concentrations in corresponding combinations. The activity of the antimicrobial combinations was determined using synergy method, previously described by Wagner and Ulrich-Merzenich (2009). The synergy method was combined with calculation of fractional inhibitory concentration indexes (FICI) to assess the antimicrobial effects of essential oil and antibiotic combinations. The FICI was calculated as FICEO + FICAB , where FICEO = MICEO of the combination/MICEO alone, and FICAB = MICAB of the combination/MICAB alone. The results were interpreted as a synergistic effect if FICI < 0.5; as an additive or indifferent effect if 0.5 ≤ FICI ≤ 4 and as an antagonistic effect if FICI > 4 (Eucast, 2000). All experiments carried out in triplicate as at least two independent experiments and the calculated FIC indexes were averaged and expressed with corresponding standard errors (mean ± SE). The FIC combination of the antibiotics and myrtle essential oil are shown graphically as isobolograms (Berenbaum, 1989). Time-kill assay The effectiveness of the myrtle essential oil MyHN and PMB combinations against reference ATCC 19606 and MDR strain

Aba-4914 was determined by the time-kill curve assay (Verma, 2007), to confirm results obtained by broth microdilution checkerboard method. Changes in bacterial count during incubation period were monitored parallel in four test tubes containing the following: (1) only bacteria (1 × 108 CFU ml−1 ); (2) bacteria and a sub-inhibitory concentration of antibiotics that showed synergistic effect in combination with EO; (3) bacteria and sub-inhibitory concentration of essential oil that showed synergistic effect in combination with AB; and (4) bacteria, a sub-inhibitory concentration of antibiotics and essential oils. Final volume of each tube was 10 ml and they were incubated at 37 ◦ C for 24 h. The bacterial counts were determined after 0, 3, 6, 9, 12, 15 and 24 h of incubation by spreading appropriate dilutions on Muller Hinton agar (detection limit was 102 CFU ml−1 ). The plates were incubated at 37 ◦ C overnight and bacterial colonies were counted. The results from the experiments, carried out in triplicate an in at least two independent occasions, were averaged and expressed as logarithms with corresponding standard errors (mean ± SE). The interaction was considered to be effective and synergistic sensu stricto if the starting bacterial count (CFU ml−1 ) decreased after 24 h of incubation by Table 1 Chemical composition (%) of Myrtus communis L. essential oil collected from three coastal areas of the Montenegro territory – Kotor (MyK), Herceg Novi (MyHN) and Bar (MyB). Components

R.I.a

MyK

MyHN

MyB

Isobutyl isobutyrate ␣-Pinene p-Cymene Limonene 1,8-Cineole trans-Linalool oxide (furanoid) Linalool Dehydro linalool 4-Terpineol p-Cymene-8-ol Cryptone ␣-Terpineol Myrtenol Nerol Linalyl-acetate n.i.b Pinocarvyl acetate Myrtenyl acetate Acetoxy eucaliptol ␣-Terpinyl acetate Neryl acetate n.i. Geranyl acetate Methyl eugenol trans-Caryophyllene ␣-Humulene n.i. n.i. Caryophyllene oxide n.i. n.i. n.i. n.i. n.i. n.i. n.i.

909 933 1024 1029 1033 1075 1105 1108 1192 1199 1202 1206 1212 1240 1266 1273 1313 1338 1352 1358 1367 1373 1385 1406 1432 1465 1522 1589 1597 1613 1625 1647 1660 1693 1728 1802

0.215 6.820 0.536 0.738 13.250 – 22.276 – 0.549 0.324 – 7.021 0.913 0.494 5.358 – 0.319 16.561 0.239 1.947 1.628 0.321 6.520 2.615 1.407 2.785 2.536 0.523 0.824 0.196 0.938 0.472 0.224 0.390 0.872 0.191

0.455 7.873 0.864 1.299 16.878 – 18.320 – 0.678 0.311 0.288 6.163 1.066 0.371 5.291 0.274 0.355 18.009 0.217 1.948 1.123 – 6.589 2.300 1.117 2.859 1.609 1.148 0.581 – 0.707 0.401 – 0.348 0.557 –

– 3.403 0.523 1.073 15.762 0.224 26.591 0.246 0.738 0.446 – 7.565 1.353 0.605 4.666 0.380 0.378 18.489 0.202 1.918 0.959 – 6.515 2.200 0.662 1.504 0.869 0.325 0.560 – 0.714 0.288 – 0.300 0.369 0.172

8.094 77.720 4.192 0.824 2.615 6.663 93.34

10.036 77.881 3.976 0.581 2.300 5.004 94.96

4.999 87.037 2.166 0.560 2.200 3.417 96.58

Chemical clases Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Phenylpropanoids Others Total identified compounds a

R.I. – Retention indices relative to C9 –C24 n-alkanes on the HP-5MS column. n.i. – precise identification was not possible because of the low intensity and/or the absence of similar spectra in the databases used for identification. b

Table 2 MICs and MBCs values of convetional antimicrobial drugs against A. baumannii wound isolates. A. baumannii strains

Antimicrobial drugs Amikacin

Outpatient isolates

Reference strains E. coli ATCC25922 S. aureus ATCC 25923 *

Aba-2572 Aba-2793 Aba-4156 Aba-4727 Aba-4779 Aba-4803 Aba-4804 Aba-4890 Aba-4914 Aba-5055 Aba-5074 Aba-5081 Aba-5372 Aba-6673 Aba-7860 Aba-34963 Aba-40100 Aba-8255 Aba-8781 Aba-8833 ATTC19606 ATCCBAA747

Ciprofloxacin

Chloramphenicol

Gentamicin

Kanamycin

Polymyxin B

Tetracycline

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

1.41* 2* >256 8* 11.31* 22.63 22.63 >256 22.63 4* 16 8* 0.35* 2.83* 32 5.66* 2.83* 1* 256 >256 4 16 16 1 4 32 16 45.25 2 256 >256 >256 >256 >256 256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 22.63 16 45.25 45.25 0.25 256 >256 >256 >256 >256 256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 45.25 45.25 45.25 90.51 – –

0.71* 0.25* 45.25 32 45.25 45.25 32 32 64 64 32 45.25 0.5* 45.25 32 16 32 0.5* 0.125* 0.125* 0.35* 0.35* 256 90.51 64 128 >256 128 256 – –

22.63 256 >256 16 2.83* 64 45.25 >256 >256 16 >256 16 0.35* 64 >256 128 128 1.41* 0.5* 0.5* 16 0.5* 0.5 0.25

90.51 256 >256 256 2.83 64 64 >256 >256 16 >256 45.25 0.35 64 >256 128 128 5.66 0.5 0.5 16 0.5 – –

>256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 2.83* >256 4* 2* 2 2

>256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 2.83 >256 4 2 – –

4 0.71* 4 0.71* 1.41* 0.71* 2 0.71* 4 4 0.5* 0.71* 0.5* 0.71* 2 1* 0.5* 0.71* 0.5* 0.5* 0.5* 0.5* 1 0.5

4 1 4 2 2 1 5.66 1 8 16 1 1 0.5 1 16 1 11.31 2 0.5 0.5 0.5 0.5 – –

128 22.63 >256 >256 256 256 256 >256 256 >256 >256 >256 >256 >256 >256 128 8 >256 2* 0.71* 2* 5.66 1 1

>256 64 >256 >256 >256 256 >256 >256 256 >256 >256 >256 >256 >256 >256 128 32 >256 16 22.63 4 11.31 – –

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Clinical isolate

Ceftriaxone

Strains sensitive to antibiotics according to MIC (␮g ml−1 ) interpretative standard (Clinical and Laboratory Standards Institute, 2007).

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≥2 log for the antibiotic-essential oil (AB-EO) combination in comparison to the more active single agent (essential oil or antibiotic) (Knezevic et al., 2013). Results

Table 3 Minimum inhibitory concentrations (MICs)a and minimum bactericidal concentrations (MBCs)b of M. communis essential oils against A. baumannii wound isolates. A. baumannii isolates

MIC and MBC values of antibiotics and essential oils Activities of conventional antibiotics varied among the strains, and most of them were not efficient in therapeutic doses against A. baumannii (Table 2). The most efficient antibiotic was polymyxin B, while the chloramphenicol and ceftriaxone had the lowest effect, showing no activity against any strain. The activity of kanamicin, tetracycline, gentamicin, ciprofloxacin and amikacin was also low, since less than a half of the strains were susceptible. All three examined myrtle essential oils showed considerable and mutually similar antibacterial activity. As alternative antimicrobial agents, myrtle essential oils exhibited bacteriostatic and bactericidal effect against MDR A. baumannii wound isolates. In general, myrtle essential oils were effective against A. baumannii strains with MIC in the range from 0.25 to 4 ␮l ml−1 , with a mediane 2 ␮l ml−1 for MyHN and MyK essential oils, while for MyB the mediane value was 1.41 ␮l ml−1 . More precisely, MICs values for essential oil MyHN ranged from 1.41 to 4 ␮l ml−1 , for MyB these values were 0.25–4 ␮l ml−1 and for MyK were in range 0.71–4 ␮l ml−1 . MBCs values were the same as MICs or one fold greater (Table 3). The most sensitive isolates to all examined myrtle essential oils, excluding reference strains, was outpatient wound isolate Aba8833. Synergism of antibiotics and myrtle essential oils Synergistic effect of selected conventional antibiotics and myrtle essential oils according to the calculated FIC indexes are presented on Figs. 1 and 2. Among three selected MDR A. baumannii wound isolates, synergistic activity was detected for most of the AB-EO combinations. When the interaction of MyHN with CIP was tested, the synergy was detected against all three strains (Aba4914, Aba-5055 and Aba-6673), so as in combinations with PMB against strains Aba-4914, and Aba-5055 (Fig. 2). The combination against strain Aba-6673 was not performed because this strain was sensitive when antibiotic was administered alone. Synergy was also

MyHN MIC

Chemical composition of Myrtus communis L. essential oils The results obtained by GC–MS analysis are listed in Table 1 in order of their elution time from a HP-5 column. In M. communis L. essential oils originating from Kotor, Herceg Novi and Bar, 36 components were detected, representing 93.34%, 94.96%, and 96.58% of the total essential oil, respectively. Among all detected components, 25 were identified, while the identification of remaining eleven components was not possible because of the low signal intensity and/or the absence of similar spectra in the databases used for the identification. The most dominant of all identified compounds were linalool (18–26.5%), myrtenyl acetate (16.5–18.5%) and 1,8-cineole (13–16.8%), followed by ␣-pinene (3.4–7.9%), ␣terpineol (6–7.5%) and geranyl acetate (6.5%). Bradesi et al. (1997) proposed classification of M. communis L. plants in two chemotypes: CT1 with high content of 1,8-cineole and ␣-pinene and CT2 with high content of myrtenyl acetate and low content of ␣-pinene. In line with that, the three investigated myrtle plants from Montenegro can be classified in the chemotype CT2. The most dominant chemical class in examined essential oils was oxygenated monoterpenes (77.7–87.04%), while the distribution of other classes (monoterpene and sesquiterpenes hydrocarbons) varied.

M. communis essential oils

ATTC 19606 ATCC BAA747 Aba-2572 Aba-2793 Aba-4156 Aba-4727 Aba-4779 Aba-4803 Aba-4804 Aba-4890 Aba-4914 Aba-5055 Aba-5074 Aba-5081 Aba-5372 Aba-6673 Aba-7860 Aba-8255 Aba-8781 Aba-8833 Aba-34963 Aba-40100 E. coli ATCC 25922 S. aureus ATCC 25923

a

1.41 1.41 4 2 2 2 2.83 2.83 2.83 4 4 1.41 2 2 2 2 2 2 2 1.41 1.41 2 2 2

MyB MBC 4 2 4 4 4 4 4 2.83 4 4 4 2 2 2 4 4 4 4 2 1.41 2 2 2 2

b

MyK

MIC

MBC

MIC

MBC

2 2 1.41 1 0.71 1 1 1 0.25 1.41 4 1.41 2 2.83 1.41 2 2 2 1.41 1.41 1.41 2 1 1

2.83 2 2 2 1.41 1.41 2 1 0.71 4 4 2.83 4 2.83 4 2.83 2.83 2 8 2.83 8 8 1 1

4 2 4 2 2 2 2 2 2 2.83 2 2 2.83 2 1.41 2 2 2 2.83 0.71 2 2 2 1

4 4 4 4 2 4 2 2 2 4 2 4 4 2 2 2 2 2 4 0.71 4 2 2 2

a MIC, minimum inhibitory concentration, values given as ␮l ml−1 from the geometrical means of triplicate experiments. b MBC, minimum bactericidal concentration, values given as ␮l ml−1 from the geometrical means of triplicate experiments.

detected in combination of MyK with CIP against Aba-4914, and with CIP and PMB against strain Aba-5055 (Fig. 1). Although the reference strain ATCC 19606 was sensitive to conventional antibiotics, the synergy of two essential oils (MyHN and MyK) in combination with PMB was determined against it as a control, and in order to obtain the result repeatability, since it is deposited in ATCC public culture collection. Time effect of AB-EO combination The effect of combination of MyHN essential oil and PMB subinhibitory concentrations against the reference strain ATCC 19606 is shown by time kill curve (Fig. 3). The reduction of bacterial cell count was greater than 3 log compared to initial count for the first 6 h of incubation, but in the next 6 h the count started to increase from approximately 3 log up to 5 log. From the twelfth hour of incubation the cell count reduced again, and final bacterial count was under 4 log. Such bacterial cell count reduction is significant and greater than effect of the same antimicrobials when administrated alone (Fig. 3A). Synergistic effect of the same antimicrobial agent combination against MDR A. baumannii strain Aba-4914 showed significantly higher reduction rate compared to the initial cell count and/or when antimicrobials administrated alone. Combination of MyHN and PMB subinhibitory concentrations reduced MDR A. baumannii count to 0 (Fig. 3B). This is more than 6 log greater reduction rate, compared to the administration of PMB alone. Discussion In this study, three Myrtus communis L. essential oils were analyzed in order to determine their efficacy against MDR strains. GC/MS analysis of essential oils from Herceg Novi, Kotor, and Bar provided their classification in chemotype with high content of myrtenyl acetate. Bradesi et al. (1997) showed that there is a good

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FIC Ciprofloxacin 1

1 Aba-4914

Aba-4914

0,5

0 0

0,5

1

1 Aba-5055

0,5

0

0

0,5

1

1

0

FIC Mytrus communis L. Kotor essential oil

FIC Mytrus communis L. Herceg Novi essential oil

0,5

1

0

0,5 Aba-5055

0,5

0 1

0

0,5

Aba-6673

1 Aba-6673

0,5

0

1

0,5

0

0,5

1

0 0

0,5

1

Fig. 1. Synergistic effect of ciprofloxacin and myrtle essential oils against MDR A. baumannii strains.

correlation between chemotype and geographic origin of the plant. According to our results, myrtle oils originating from Montenegro coastline belong to the same chemotype as Portuguese (Pereira et al., 2009) and Croatian (Jerkovic et al., 2002). In addition, our myrtle essential oils contain considerable amount of linalool and the result is in accordance with a previous report on myrtle essential oils from Montenegro (Mimica-Dukic´ et al., 2010). Although there are only slight differences in chemical composition between the three examined myrtle essential oils, this can affect their antimicrobial activity. These differences probably represent a consequence of biotope factors (distance between locations with different environmental/microenvironmental factors) and plant genetic/epigenetic factors. The results obtained for myrtle essential oils from three Montenegro coastline locations confirmed previously reported antibacterial activity of M. communis L. essential oils (Akin et al., 2010; Berka-Zougali et al., 2012) and indicated considerable oil bacteriostatic and bactericidal effect against MDR A. baumannii wound isolates (Table 3). The activity of the oils was mutually similar. The lowest concentration that had effect on A. baumannii was 0.25 ␮l ml−1 of MyB. The myrtle oil MyB was slightly more active, compared to MyHN and MyK, probably as a consequence of their different chemical composition. Namely, MyB had higher amount of linalool and myrtenyl acetate, and more than twice less amount of ␣-pinene, in comparison to the other two myrtle oils. It has been reported that some essential oils components exhibited

greater antimicrobial activity against Gram negative bacteria, such as 1,8-cineole (1.87 mg ml−1 ) and linalool (0.4 mg ml−1 ) against E. coli ATCC 25922, than ␣-pinene (15 mg ml−1 ) (Sonboli et al., 2006). Moreover, the specific oil composition along with a synergistic interaction among oil constituents (Burt et al., 2005) is probably most responsible for the slightly different myrtle essential oils antibacterial activity. The obtained results cannot be compared to data of other authors regarding M. communis essential oils MICs and MBCs values, not only because essential oil chemical composition is variable and depends on multiple factors, but also because there are no data on myrtle oil effect on A. baumannii. Nevertheless, considering the growing rate of antibiotic resistance and natural origin of essential oils vs. chemical synthetic origin and unfavorable/toxic effects of some drugs, recorded M. communis L. essential oils activity seems to be very significant and promising. The current state of urgency in finding solution for A. baumannii infection treatment led to development and application of combination therapies as an alternative treatment which consequently has become common practice. Many studies confirmed in vitro synergistic effect of antibiotics combinations against MDR A. baumannii. For instance, a synergy was demonstrated when ampicillin–sulbactam was combined with a carbapenem, or between meropenem and polymyxin B (Özseven et al., 2012; Pankey and Ashcraft, 2009). Such combined treatment prolongs the urgency for other solutions for

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FIC Polymyxin B 1

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Aba-4914

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FIC Mytrus communis L. Herceg Novi essential oil

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Fig. 2. Synergistic effect of polymyxin B and myrtle essential oils against MDR A. baumannii strains.

12

et al., 2013). These combinations reduce antibiotic minimum efficient dose and thus can minimize potential antibiotic side effects or prevent the emergence of antibiotics resistance during monotherapy. Here we tested and proved synergistic effect of CIP or PMB with M. communis L. essential oils against three strains

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10 8 6 4 2 0

0

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A. baumannii Aba-4914 count (log CFU mL )

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MDR A. baumannii infection treatment, but there is still necessity for finding alternative solutions. Thus some authors tested combination of conventional antibiotics with some unconventional agents, such as herbal extracts (Basri and Khairon, 2012), essential oils (Rosato et al., 2007) and bacteriophages (Knezevic

5

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25

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Fig. 3. Time-kill curves showing effect of MyHN and polymixin B subinhibitory concentrations combination against (A) A. baumannii reference strain ATCC 19606 and (B) MDR A. baumannii strain Aba-4914; () bacteria without treatment; (䊉) antibiotic; () essential oil; () essential oil and antibiotic.

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exhibiting resistance to the antibiotics when they are administered alone. These AB-EO combinations and their synergistic effect were strain dependent and also dependent on AB and EO mode of action, which should also be taken into consideration when synergistic effect is considered. Namely, PMB and CIP belong to different classes and have different mode of action: CIP inhibits DNA replication targeting DNA gyrase (Zhao et al., 1997) and PMB disturbs membrane structures (Tam et al., 2005). The modes of myrtle essential oil activity mainly concern the cell wall and membrane structures, changing their permeability and leading to release of intracellular contents outside of a cell (Amensour et al., 2010; Bakkali et al., 2008). Thus, affecting cell envelopes as first barrier for antimicrobials, myrtle essential oil probably makes the open path for antibiotics, enhancing entrance and activity of conventional antibiotics, which is probably the case with CIP. The combination of myrtle essential oils and CIP was the most frequent combination giving synergy against three MDR strains in concentration 1/4 MICAB and 1/8 MICEO . Acting on diverse cell levels, these ABEO combinations increase MDR A. baumannii cells sensitivity to conventional antibiotics, i.e. reduced the effective antiobiotic concentrations. This combination, however, was not efficient enough because the MIC value of CIP was not significantly reduced in combination, as MIC was not reduced below A. baumannii break point for this antibiotic. Nevertheless, the myrtle essential oils and PMB combination was more efficient, probably because of their similar mode of action and the same target site. PMB mode of action, which was the most effective agent in the study administered alone, differs among all other examined antibiotics, implying that cell membrane structures should be the target point of MDR A. baumannii strains in order to eradicate persistent infections. This combination with myrtle oil in concentration 1/4 MICEO gave synergistic effect reducing the MIC value of PMB 4 times (to 1/4 MICAB ), making MDR A. baumannii strain Aba-4914 sensitive to this antibiotic. The fluoroquinolone synergy with essential oils have been previously confirmed for Pelargonium graveolens (Rosato et al., 2007), and it was confirmed for Myrtus communis L. oils in the present study. However, the synergistic activity of essential oils and PMB was confirmed here for the first time. This combination reduces concentrations of both agents and even re-sensitivitative of MDR strains, most probably because MIC values for PMB were closer to sensitivity break points than those for CIP. In order to recheck the results obtained calculating FIC index for PMB, which indicated synergy, two strains were selected to examine the effect of PMB/essential oil combination during time against MDR A. baumannii wound isolate Aba-4914 and the reference strain ATCC 19606. The obtained antibacterial effect of combination was confirmed by time kill curve, reducing A. baumannii count for approximately 4 log comparing to the effect of polymixin B as a single agent (Fig. 3A). Further, this combination of agents in concentrations 1/4 MICEO and 1/4 MICAB against MDR wound isolate resulted in very efficient synergy, completely reducing cell count after only 6 h of incubation (Fig. 3B). Interestingly, while 1/4 MICEO did not reduce bacterial count, at both graphs it is obvious that 1/4 MIC of PMB rapidly reduces bacterial count for short period of time, which is followed by gradual regrowth, and finally results in high bacterial count after prolonged incubation. This phenomenon is not previously observed for this antibiotic (Qian et al., 2012). However, when PMB is combined with EO, the regrowth is only temporary or absent, resulting in final significant bacterial count reduction after 24 h, which additionally empasizes the synergism. Although the application of PMB was restricted in the past, since it was considered as nephrotoxic and neurotoxic, recent studies have shown that its nephrotoxicity is less severe and common than it was thought to be, while neurotoxic effect is mild, with no case of neuromuscular blockade and apnea reported in recent literature

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(Falagas and Kasiakou, 2006). However, there are various antimicrobial ointments against wound infections today and the most effective ones against A. baumannii seem to be those containing combination of bacitracin and PMB (Geronemus et al., 1979; Cooper et al., 1991). Similar formulation is used as irrigant of orthopedic surgical wounds in order to prevent their infections – combination of 50 000 U of bacitracin and 50 mg/l of PMB in sterile saline or lactated Ringer solution (Rosemberg et al., 2008). Beside this double antibiotic ointment and irrigant, neomycin is sometimes used in combination with polymyxine and/or bacitracine. However, due to the fact that neomycin causes contact dermatitis, i.e. allergic reaction in some patients, it is excluded from the ointment combination (Fletcher et al., 2007; Dirschl and Wilson, 1991). According to the results of our study, it would be interesting to examine ex vivo effect of myrtle essential oils and polymyxine in combination, as active compounds of an ointment or irrigant for skin wounds and/or superficial injuries. As no new antibiotics will be available in the next few years for MDR Gram negative bacteria, including A. baumannii wound isolates, the results obtained for myrtle essential oils make them promising alternative agents, while their combinations with currently available antimicrobials appear as a promising therapeutic strategy. Also, myrtle essential oils could be incorporated in formulations of some commercial hygiene chemical products, such as sanitation agents, with purpose of reduction bacterial transmission, especially MDR microorganisms causing nosocomial infections. However, additional in vitro and in vivo experiments of Myrtus communis L. essential oil are required prior to the approval of its application as a novel commercial and therapeutic agent. Conflict of interest statement The authors declares no conflict of interest. Acknowledgments This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, grant OI 172058. The authors acknowledge Prof. Ljiljana Knezevic, PhD (Faculty of Sciences, University of Novi Sad) for language revision. References Adams, R.P., 2001. Identification of Essential Oil Components by Gas Chromatography/Quadrupole Mass Spectroscopy. Allured, Carol Stream, IL. Akin, M., Aktumsek, A., Nostro, A., 2010. Antibacterial activity and composition of the essential oils of Eucalyptus camaldulensis Dehn. and Myrtus communis L. growing in Northern Cyprus. Afr. J. Biotechnol. 9 (4), 531–535. Aleksic, V., Knezevic, P., 2014. Antimicrobial and antioxidative activity of extracts and essential oils of Myrtus communis L. Microbiol. Res. 169 (4), 240–254. Amensour, M., Bouhdid, S., Fernandez-Lopez, J., Idaomar, M., Senhaji, N.S., Abrini, J., 2010. Antibacterial activity of extracts of Myrtus communis against food-borne pathogenic and spoilage bacteria. Int. J. Food Prop. 13, 1215–1224. Arabi, B., 1987. Comparative study of bacteriological contamination between primary and secondary exploration of missile head wounds. Neurosurgery 20, 610–616. Bakkali, F., Averbeck, S., Averbeck, D., Idaomar, M., 2008. Biological effects of essential oils – a review. Food Chem. Toxicol. 46, 446–475. Basri, D.F., Khairon, R., 2012. Pharmacodynamic interaction of Quercus infectoria galls extract in combination with vancomycin against MRSA using microdilution checkerboard and time-kill assay. Evidence Based Complement. Altern. Med. 2012, 150–156. Berenbaum, M., 1989. What is synergy? Pharmacol.Rev. 41, 93–141. Berka-Zougali, B., Ferhat, M.A., Hassani, A., Chemat, F., Allaf, K.S., 2012. Comparative study of essential oils extracted from Algerian Myrtus communis L. leaves using microwaves and hydrodistillation. Int. J. Mol. Sci. 13, 4673–4695. Bradesi, P., Tomi, F., Casanova, J., Costa, J., Bernardini, A.F., 1997. Chemical composition of myrtle leaf essential oil from Corsica (France). J. Essent. Oil Res. 9, 283–288. Bruna, S., Portis, E., Cervelli, C., De Benedetti, L., Schiva, T., Mercuri, A., 2007. AFLPbased genetic relationships in the Mediterranean myrtle (Myrtus communis L.). Sci. Hortic. 113, 370–375.

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