Rosmarinus Officinalis Essential Oil and Eucalyptol Act as Efflux Pumps Inhibitors and Increase Ciprofloxacin Efficiency against Pseudomonas Aeruginosa and Acinetobacter Baumannii MDR Strains

Romanian Biotechnological Letters Copyright © 2016 University of Bucharest Vol. 21, No. 4, 2016 Printed in Romania. All rights reserved ORIGINAL PAPER Rosmarinus Officinalis Essential Oil and Eucalyptol Act as Efflux Pumps Inhibitors and Increase Ciprofloxacin Efficiency against Pseudomonas Aeruginosa and Acinetobacter Baumannii MDR Strains Received for publication, October 13, 2015 Accepted, February 29, 2016 CRINA SAVIUC1, 2, IRINA GHEORGHE1, SORINA COBAN1, VERONICA DRUMEA2, MARIANA-CARMEN CHIFIRIUC*,1, OTILIA BANU3, EUGENIA BEZIRTZOGLOU4, VERONICA LAZĂR1 1 University of Bucharest, Faculty of Biology, Department of Botany-Microbiology, Research Institute of the University of Bucharest-ICUB, Splaiul Independenţei 91-95 Sector 5, cod 76201, Bucureşti, Romania, Phone: 004-021-318.15.66, 004-021.310.85.03; 2 S.C. Biotehnos S.A. – Gorunului Str. 3-5, 075100, Otopeni, Ilfov, Romania, Phone: 40-317102402, fax: 40-31-7102400; 3 Emergency Institute of Cardiovascular Diseases “Prof. Dr. C.C. Iliescu”, Fundeni R., No. 258, Sector 2, Bucharest, Romania; 4 Department of Agricultural Development; Democritus University of Thrace, Greece; *Address correspondence to: University of Bucharest, Department of Biochemistry and Molecular Biology, 91-95 Spl. Independentei, 050095, Bucharest 5th District, Romania.Tel.: +4021 318 15 75; Email: carmen_balotescu@yahoo.com Abstract In this study, our aim was to assess the antimicrobial activity of Rosmarinus officinalis essential oil (EO) and eucalyptol (EU) against multiresistant strains of Acinetobacter baumannii and Pseudomonas aeruginosa. The resistance mechanisms of the used bacterial strains were characterized by phenotypic and genotypic methods. Essential oil was extracted and performed by hydrodistilation and characterized by GC-MS analysis. The intrinsic antimicrobial activity, as well as the synergic effect with ciprofloxacin was assessed by quantitative methods, while the potential mechanisms of the antimicrobial action were investigated by flow citometry.The selected P. aeruginosa strains and A. baumannii strains exhibited MDR phenotypes. The screening of beta-lactams resistance genes proved the presence of blaTEM genes in 60% of the A. baumannii strains and in 37.5% of the P. aeruginosa strains, while blaOXA-23 was evidenced in 80% of the A. baumannii strains.The GC-MS analysis of the R. officinalis essential oil allowed the identification of 28 volatile compounds, the eucalyptol content being 5.49%. The MIC values for Rosmarinus officinalis EO ranged from 13.07 to 52.26 mg/mL, while for EU from 1.86 ÷ 41.86 mg/mL. Both EU and EO exhibited a synergic effect with ciprofloxacin. The mechanisms of action revealed by the flow cytometry analysis were the inhibition of efflux pumps activity and the cellular wall permeabilization. These results demonstrate that rosemary EO and its majoritary compound EU can be used as efflux pump inhibitors, to restore the efficiency of current antibiotics against MDR strains or even suppress the emergence of such resistance phenotypes. Keywords: antibiotic resistance, essential oil, antimicrobial combinations, flow cytometry 1. Introduction Resistance to antibiotics is used by microbial strains as a survival, competitive and adaptive mechanism to the selective pressure imposed by biotic or abiotic factors. The widespread and inappropriate use of antibiotics in medicine for the prophylaxis and treatment 11782 Romanian Biotechnological Letters, Vol. 21, No. 4, 2016 Rosmarinus Officinalis Essential Oil and Eucalyptol Act as Efflux Pumps Inhibitors and Increase Ciprofloxacin Efficiency against Pseudomonas Aeruginosa and Acinetobacter Baumannii MDR Strains of infectious diseases, agriculture and animal breeding led to the permanent increase of the number of bacterial strains resistant to more and more classes of antibiotics. In addition, the horizontal gene transfer contributed to the selection of multiple-drug (MDR), extended-drug (XDR) and pan-drug (PDR) resistance phenotypes, with major impact for the global public health, due to the severe outcome of nosocomial infections, with significant increase in morbidity, mortality and hospital costs. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter sp.) are representing a significant and increasing percent of the resistant clinical isolates involved in nosocomial infections [1, 2]. Plants and plant extracts have been used for centuries in traditional medicine for their different therapeutic effects by 80% of the world population. Also, many medications contain compounds directly isolated from plants, or modified versions of natural products [3, 4]. Nowadays, plant extracts and essential oils (EOs), or their fractions/ isolated compounds are under investigation, representing viable solutions for developing new anti-infective strategies. In this respect, our aim was to assess the intrinsic antimicrobial activity, mechanisms of action and synergic effect with antibiotics of Rosmarinus officinalis essential oil and one of its majoritary compounds, eucalyptol, against multiresistant strains of Acinetobacter baumannii and Pseudomonas aeruginosa. 2. Materials and Methods 2.1. Bacterial strains We have selected for this study eight P. aeruginosa and six A. baumannii clinical isolates collected from The Institute of Cardiovascular Diseases Prof. C.C. Iliescu, Bucharest in September 2014 - March 2015, as well as one P. aeruginosa ATCC 27853 reference strain (table 1). The identification of bacterial strains was performed using the automated VITEK 2 Compact system. 2.2. Phenotypic and genotypic characterization of antibiotic resistance profiles of the tested strain Evaluation of the antibiotic susceptibility of P. aeruginosa and Acinetobacter baumannii strains was performed by disk diffusion method recommended by CLSI [5].The β-lactamase producer phenotypes (metallo-β-lactamase-MBL, extended spectrum β-lactamase, inducible cephalosporinase) were highlighted by double disk diffusion. Several multiplex or simplex PCR assays were performed to detect the MBL genes - blaVIM, blaIMP and the extended spectrum β-lactamase ESBL genes – blaTEM, blaCTX-Min P. aeruginosa and the MBL genes blaVIM, blaIMP, the extended spectrum β-lactamase genes – blaTEM, blaCTX-M and the oxacillinase genes blaOXA-23 and blaOXA-24 in A. baumannii strains. 2.3. Essential oil (EO) extraction Four samples (200g each) of dry plant material were hydrodistillated in a Neo-Clevengertype apparatus for 4 hours according to European Pharmacopoeia 7th ed. The extraction yield was considered as the medium yield of the four experiments, expressed as the EO volume per plant material weight (v/w). The EOs portions were mixed, dried with anhydrous magnesium sulfate and stored in brown vessels at 4oC prior to the physico-chemical analysis and antimicrobial properties assessment. Romanian Biotechnological Letters, Vol. 21, No. 4, 2016 11783 CRINA SAVIUC, IRINA GHEORGHE, SORINA COBAN, VERONICA DRUMEA, MARIANA-CARMEN CHIFIRIUC, OTILIA BANU, EUGENIA BEZIRTZOGLOU, VERONICA LAZĂR 2.4. Physico-chemical characterization of the volatile oils extracted fromRosmarinus officinalis herba For the GC-MS analysis a 1:50 dilution of the EO sample in hexane was made. Chemical components identification was conducted on a GC-MS system, 7890A coupled with 5975C MSD (Agilent Techonologies, Santa Clara, CA). A DB-5 MS capillary column (60 m x 0.25 mm x 0.25 µm) was used. The acquisition was conducted with different oven temperature ramps, ranging between 90o and 280oC. Split mode injection was selected and the split ratio was 1:300. The injection volume was set up at 1 µl and the flow rate at 1.3 mL/min. MS acquisition parameters were scan mode; relative EMV mode; scanning range (50 ÷ 400) a.m.u.; MS source 230oC; MS quad 150oC. The compounds’ identification was made by using the NIST mass spectral library. 2.5. Qualitative screening for the antimicrobial activity of EO and eucalyptol (EU) An adapted diffusion method was used for the qualitative screening of the antimicrobial activity. The Rosmarinus officinalis EO and EU diluted in dimethyl sulfoxide (DMSO) 1:1 (10 µl) were used for spotting the plates previously seeded as recommended by CLSI. The plates were incubated for 24 h at 37oC and the results were quantified according to the growth inhibition diameters. 2.6. Quantitative assay of the antimicrobial activity of EO and EU The quantitative assay of the antimicrobial activity was performed by serial binary micro dilution (ranging from 1.86 to 52.26mg/mL) method in Müller-Hinton liquid medium distributed in 96-well plates. Dimethyl sulfoxide (DMSO) was used to facilitate mixing the essential oil and EU with the broth in a 1:1 ratio. 2.7. Flow citometry assessment of the bacterial efflux pump activity for the natural compounds and antimicrobial combinations of natural compounds and antibiotics Flow citometry was carried out in order to evaluate the possible mechanisms of the antimicrobial activity of the natural compounds and of antimicrobial mixtures. For the cell staining two intercalant fluorochromes with affinity for DNA were used: propidium iodide (PI 10µg/mL) and ethidium bromide (EB 5µg/mL). PI was used for the cellular viability’s determination, as living cells are impermeable to this dye, due to their intact membranes. EB was used for the detection and quantification of bacterial efflux activity. The study was conducted in two different experiments: (1) for the EO/EU and (2) for the combinations EO/EU: antibiotic (i.e. ciprofloxacin – 2µg/mL). Two concentrations of natural compounds, corresponding to the MIC and MIC/8 values were tested. Staining procedures were applied to the harvested cells grown in the presence of the tested compounds. The cells were centrifuged at 13000 rpm for 3 minutes, washed 2 times, resuspended in phosphate buffered saline (PBS), stained with 10μL PI or EB and incubated for 10 minutes at 4oC in dark. Heat-treated cells for 30 minutes at 100ºC were used as positive controls and viable cells were used as negative controls. The samples were analyzed with a FACS Calibur instrument equipped with a 488 nm Argon laser, using 670 nm long pass filter for the samples stained with PI and (585±42) nm band pass filter for the samples stained with EB. The measured parameters were forward scatter FSC, side scatter SSC and fluorescence and the back gating procedure was used, excluding all non-fluorescent particles. Typical photomultiplier tube voltages were SSC 550 V (log scale), 670 nm long pass filter (log scale) and (585±42) nm band pass filter 550V (log scale). 10000 events were collected in all runs. 11784 Romanian Biotechnological Letters, Vol. 21, No. 4, 2016 Rosmarinus Officinalis Essential Oil and Eucalyptol Act as Efflux Pumps Inhibitors and Increase Ciprofloxacin Efficiency against Pseudomonas Aeruginosa and Acinetobacter Baumannii MDR Strains 3. Results and Conclusions 3.1. Phenotypic and genotypic characterization of antibiotic resistance profiles of the tested strains The A. baumannii and P. aeruginosa strains exhibited resistance to the main classes of antibiotics used to treat infections caused by nonfermentative Gram-negative bacteria (i.e., βlactam antibiotics, aminoglycosides and quinolones) [6, 7-11]. Thus, in P. aeruginosa there were identified the following β-lactam resistance phenotypes: constitutive cephalosporinases (2 strains), carbapenemases or another resistance mechanism to carbepenems (6 strains), 1 strain resistant to all the tested β-lactams, 1 strain resistant to the β-lactamase inhibitors, and two strains isolated from tracheal secretions presented a MDR phenotype. All A. baumannii strains exhibited a MDR phenotype, being resistant to the majority of the tested antibiotics, excepting for tobramycin and polymyxin B. The results of PCR assays for beta-lactams resistance genes proved the presence of blaTEM genes in 60% A. baumannii strains and in 37.5% P. aeruginosa strains and the presence of blaOXA-23 in 80% A. baumannii strains (Table 1). As the carbapenems resistance in Gram-negative non-fermentative bacteria is often associated with mutations in porins, which induce decreased outer membrane permeability, the presence of oprD gene was tested in P. aeruginosa strains. The OprD porin allows the passive uptake across the outer membrane of basic amino acids and dipeptides with a basic residue and forms pores that are permeable for carbapenems. The external loops 2 and 3 were identified as entrances for basic amino acids and binding sites for imipenem. Any substitution or deletion within loop 2 or 3 induces changes in conformation and can cause imipenem resistance, as well as the loss of the OprD porin [12].The PCR assay results proved the absence of this gene in all analyzed P. aeruginosa strains. Table 1 –The resistance profiles of the selected strains No. Bacterial strain 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Pseudomonas aeruginosa 49 (Ps49) Pseudomonas aeruginosa 147 (Ps147) Pseudomonas aeruginosa 156 (Ps156) Pseudomonas aeruginosa 157 (Ps157) Pseudomonas aeruginosa 160 (Ps160) Pseudomonas aeruginosa 164(Ps164) Pseudomonas aeruginosa 165 (Ps165) Pseudomonas aeruginosa 234 (Ps234) Acinetobacter baumannii 64 (Ac64) Acinetobacter baumannii 110 (Ac110) Acinetobacter baumannii 230 (Ac230) Acinetobacter baumannii 286 (Ac286) Acinetobacter baumannii 288 (Ac288) 15 Pseudomonas aeruginosa ATCC 27853 Acinetobacter baumannii 388 (Ac388) Clinical specimen stool tracheal secretion tracheal secretion tracheal secretion urinary tract infection tracheal secretion tracheal secretion urinary tract infection catheter tracheal secretion urinary tract infection nasal exudate nasal exudate pharyngeal exudate Resistance phenotype C+ MDR MDR Resistance genes blaTEM blaTEM blaTEM C+ β-lactam resistance C+ MDR MDR MDR MDR MDR MDR blaOXA-23 blaTEM, blaOXA-23 blaTEM, blaOXA-23 blaTEM, blaOXA-23 reference strain C+ - carbapenemase production 3.2. Extraction andphysico-chemical characterization of the EO The extraction yield considered as the medium yield of the four experiments was 0.987% (V/w). Our results in extraction yield are in accordance with the literature data, taking into account the extraction time and vegetal material grounding state [13].The GC-MS analysis of the EO extracted from R. officinalis herba allowed the identification of around 28 volatile Romanian Biotechnological Letters, Vol. 21, No. 4, 2016 11785 CRINA SAVIUC, IRINA GHEORGHE, SORINA COBAN, VERONICA DRUMEA, MARIANA-CARMEN CHIFIRIUC, OTILIA BANU, EUGENIA BEZIRTZOGLOU, VERONICA LAZĂR compounds accounting for about 94.934% of the total compounds (table 2, fig. 1). The major compound was α-pinene, with 19.01% of the relative aria, followed by the 1,8-cineole (EU) (5.49%), L-camphor (5.71%), verbenone (5.42%), camfene (4.67%) and carvacrol (4.71%). These results are in accordance with other literature data [14]. 3.3. Qualitative screening for the antimicrobial activity of EO and EU The results of the qualitative testing of the antimicrobial activity were analyzed following the algorithm suggested by Singh et al., 2014, i.e: less than 8mm of growth inhibition zone (no antimicrobial activity), 9-14mm (susceptible), 15-19mm (very susceptible) and > 20mm (extremely susceptible) [15]. 75% of the P. aeruginosa strains proved to be susceptible to EO and 50% to EU, exhibiting growth inhibition zones between 10 and 14 mm. All A. baumannii strains were susceptible to EO and EU, with growth inhibition zones between 12 and 19 mm. 3.4. Quantitative assay of the antimicrobial activity of EO and EU Both rosemary EO and EU exhibited good antimicrobial activity against the Gramnegative non-fermentative bacterial strains P. aeruginosa and A. baumannii as seen in figure 2.The MIC values for Rosmarinus officinalis E Oranged from 13.07 to 52.26 mg/mL, while for EU between 1.86 and 41.86 mg/mL. Synergistic relationships between EO/ EU: CIP have been proved, except for the A. baumannii 286 (MIC EU – 1.86 mg/ mL and MIC EU: CIP 8.86 mg/ mL, suggesting a possible antagonism of action between the two components), by calculating the fractional inhibitory concentration index (data not shown). Table 2 – Compositionof Rosmarinus officinalis EO No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 11786 Compound α-pinene camfene 3-octanone mircene β-pinene d-limonene 1,8-cineol α-terpinene terpinolene d-linalool crisantenone cis-verbenol L-camfor borneol 4-terpineol α-terpineol 3-ciclopenten-1-etanol verbenone 5-caranol bornyl acetate carvacrol butyl carbinol acetate geranyl isovalerate valeric acid 2 ethyl-hexyl esther caryophyllene 2-methyl decane hexadecane γ-sitosterol Total Retention time (min) 7.501 7.779 7.997 8.081 8.165 8.832 8.934 9.234 9.683 9.748 10.291 10.664 10.796 11.145 11.221 11.398 11.466 11.685 12.356 12.852 12.914 13.500 14.401 14.650 15.477 16.127 17.533 28.131 % of relative aria 19.094 4.672 1.433 1.620 1.935 1.935 5.486 0.516 0.690 1.274 0.592 0.619 5.713 3.238 1.644 0.956 0.896 5.418 1.099 4.962 4.707 0.575 1.160 5.553 1.706 2.338 1.737 13.366 94.934 Romanian Biotechnological Letters, Vol. 21, No. 4, 2016 Rosmarinus Officinalis Essential Oil and Eucalyptol Act as Efflux Pumps Inhibitors and Increase Ciprofloxacin Efficiency against Pseudomonas Aeruginosa and Acinetobacter Baumannii MDR Strains Figure 1 – Chromatogram of the Rosmarinus officinalis EO – separation details and retention times of the identified compounds 3.5. Investigation of the underlying mechanisms of the antimicrobial activity of EO and EU by flow cytometry A promising approach in fighting the multidrug resistance issue by improving the clinical performance of the antibiotics is represented by efflux pump inhibitors (EPIs).The targeting of the efflux pump activity with an inhibitor could restore the microbial susceptibility to a certain antibiotic. Ideally such an inhibitor should facilitate the intracellular accumulation of an antibiotic and therefore reduce the minimum inhibitory concentration [16].It is already known that the antimicrobial/EPI synergy is a general mechanism of plant defense against Gram-negative bacteria and fungal infections [17]. It has been also shown that several Berberis species (B. repens, B. aquifolia and B. fremontii) synthesize an inhibitor of the Staphylococcus aureus NorA MEP, identified as 59-methoxyhydnocarpin (59-MHC). Antimicrobial activity of Rosmarinus officinalis essential oil and 1,8-cineole (eucalyptol) 50 40 (mg/mL) minimuminhibitory concetration 60 30 20 10 Ps .a er ug in os a 49 Ps .a er ug in os a 14 7 Ps .a er ug in os a 15 6 Ps .a er ug in os a 15 Ps 7 .a er ug in os a 16 0 Ps .a er ug in os a 16 Ps 4 .a er ug in os a 23 4 A .b au m an ni i6 4 A .b au m an ni i1 10 A .b au m an ni i2 86 A .b au m an ni i2 88 A .b au m an ni i3 88 Ps .a er ug in os a A T C C 27 85 3 0 bacterial strains rosemary essential oil 1,8-cineole (eucalyptol) Figure 2 – Antimicrobial activity of Rosmarinus officinalis essential oil and 1,8-cineole (EU) against P. aeruginosa and A. baumannii strains Considering these aspects, we have investigated the potential of rosemary EO and EU to modulate the efflux pumps activity in the selected MDR strains. EB is used for detection and quantification of bacterial efflux pumps activity, and the intensity of the EB treated samples could be associated with the ratio between the net influx of EB due to the permeabilization of the bacterial cell wall and its extracellular elimination through the efflux pump activity [18]. Measuring the fluorescence intensity (FI) in samples stained with PI and EB and combining the results, the EPI activity as well as the cell coatings permeabilisation could be assessed as potential mechanisms of the proposed antimicrobial Romanian Biotechnological Letters, Vol. 21, No. 4, 2016 11787 CRINA SAVIUC, IRINA GHEORGHE, SORINA COBAN, VERONICA DRUMEA, MARIANA-CARMEN CHIFIRIUC, OTILIA BANU, EUGENIA BEZIRTZOGLOU, VERONICA LAZĂR combinations. Typical histograms of the fluorescence intensity (MFI) measurements median are presented in figure 3. In our experiments most of the tested variants induced a drastic decrease in the viable P. aeruginosa bacterial population, when tested at the MIC concentration (fig. 3b), but not of the A. baumannii strains, in whose case there were registered higher MFI values for the cells labeled with PI (fig. 3 c, d). In our experiments, EU at subinhibitory concentrations corresponding to MIC/8 induced cell wall permeabilization demonstrated by an increased of PI MFI only in case of P. aeruginosa (fig. 4 a, c). Also, EO and EU in subinhibitory concentration exhibited both a cellular wall permeabilization and an EPI effect both on P. aeruginosa and A. baumannii strains (fig. 4 b, d). The increased PI uptake indicating a membrane damage was previously reported after treatment with EOs, such as clove, oregano, thyme, tea-tree oils, or cinnamon oil [19]. The combinations between EO/ EU and ciprofloxacin exhibited an EPI effect on the tested strains (fig. 5 a-d), with increased MFI values for the EB labeled samples comparing with the viable cells control. The EPI effect was more intensive for EU as compared to EO. 4. Conclusions The results of the present study demonstrate that Rosmarinus officinalis essential oil and one of its majoritary compounds, 1,8-cineole (eucalyptol) exhibited very good antimicrobial activity against P. aeruginosa and A. baumannii MDR strains and a synergic activity with ciprofloxacin, a 2nd generation quinolone. The flow cytometry analysis demonstrated that the tested natural compounds act by inducing cellular wall permeabilization and efflux pumps inhibitory activity, suggesting their potential use for the restoration of antibiotics efficiency on resistant strains harboring an efflux mechanism. This study could be extended at molecular level in order to assess the vegetal extracts’ influence on the expression of some antibiotic resistance genes. Figure 3 – a - overlay of the histogram for the median of the fluorescence intensity, viable cells control (green) and heat treated cells control (red); b, c, d - typical aspects of the histograms obtained at MIC concentration values for P. aeruginosa (b) and A. baumannii (c, d) strains 11788 Romanian Biotechnological Letters, Vol. 21, No. 4, 2016 Rosmarinus Officinalis Essential Oil and Eucalyptol Act as Efflux Pumps Inhibitors and Increase Ciprofloxacin Efficiency against Pseudomonas Aeruginosa and Acinetobacter Baumannii MDR Strains Figure 4 – a, b – median of fluorescence intensity measurements obtained for the P. aeruginosa samples treated with eucalyptol and rosmary EO respectively; c, d - median of fluorescence intensity measurements obtained for the A. baumannii samples treated with eucalyptol and rosmary EO respectively in subinhibitory concentrations, corresponding to MIC/8 Figure 5 – a, b – median of fluorescence intensity measurements obtained for the P. aeruginosa samples treated with eucalyptol:ciprofloxacin combinations and rosmary EO:ciprofloxacin combinations, respectively; c, d - median of fluorescence intensity measurements obtained for the A. baumannii samples treated with eucalyptol:ciprofloxacin combinations and rosmary:EO ciprofloxacin combinations, respectively – all the results are normalised to the MFI of the ciprofloxacin treated cells 4. 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