412 ANTIMICROBIAL ACTIVITY OF SOME ESSENTIAL OILS ALONE AND IN COMBINATION WITH AMIKACIN AGAINST ACINETOBACTER SP Mohaddese Mahboubi 1 *, Mohammad Mehdi Feizabadi 2 Address(es): Dr. Mohaddese Mahboubi, 1 Microbiology Department, Medicinal Plant Research Center of Barij, Kashan, Iran. 2 Department of Microbiology, Faculty of Medical Sciences, University of Tehran, Iran. *Corresponding author: mahboubi@barijessence.com, mahboubi1357@yahoo.com ABSTRACT Keywords: Acinetobacter sp., essential oil, synergistic effect, amikacin INTRODUCTION Acinetobacter sp. isolates are problematic pathogens in intensive-care units and other hospital units in recent years. They are the causes of health care associated pneumonia, surgical site infections, bloodstream infections, urinary tract infections (Tolbat et al., 2006). Acinetobacter sp. isolates with multi drug resistance (MDR) are markedly increasing and treatment of Acinetobacter sp. infections have been limited to few broad spectrum antibiotics, including carbapenems, amikacin, doxycycline, minocycline, and ampicillin/sulbactam (Van Looveren and Guossens, 2004). As resistance to antibiotics has emerged, the mortality rates in Acinetobacter sp. infected patients have increased. Therefore, the popularity of natural essential oils as alternative treatment has increased (Sienkiewicz et al., 2011; Mikaili et al., 2011; Candan et al., 2003; Damjanovic- Vratnica et al., 2011). In this research, we isolated 35 clinical isolates of Acinetobacter sp. and determined the sensitivity of these isolates to different antibiotics; then we evaluate the anti Acinetobacter sp. activity of ten essential oils alone against clinical isolates of Acinetobacter sp. The combination of ten different essential oils with amikacin (AMI) was evaluated against one AMI resistant isolates by measuring the FIC and FIC indexes. MATERIAL AND METHODS Essential oils and their analysis 10 different essential oils including Cymbopogon olivieri, Heracleum persicum, Juniperus comminus, Azillia eryngioides, Dacus carrota, Ferula gummosa, Acorus calamus, Mentha pulegium, Achillea biebersteinii and Chaerophyllum macropodum were prepared from Barij Essence Pharmaceutical Company. The essential oils were analyzed using GC-FID and GC-MS. The GC-FID and GC- MS apparatus were conducted on an HP 6890 GC system coupled with 5973 network mass selective detectors with a capillary column of HP-5MS (30 m × 0.25 mm, film thickness 0.25 μm). The oven temperature program was initiated at 60 °C, held for 1 min, then raised up to 245 °C at a rate of 3 °C/min held for 10 min. Helium was used as the carrier gas at a flow rate 1.5 ml/min. The detector and injector temperatures were 250 and 230 °C, respectively. The compounds of the essential oil were identified by comparison of their retention indices (RI), mass spectral fragmentation with those in the stored Wiley 7n.1 mass computer library (Adams, 2001). Antibiotics The antibiotic discs that were used in this study including ciprofloxacine (CIPR 5 µg), cefepime (FEP 30 µg), ceftazidime (CAZ 30 µg), levofloxacin (LEVOF 5 µg), amikacin (AMI 30 µg), amoxicillin (AMOXY 30 µg), Imipenem (IMI 10 µg), tobramycin (TOB 10 µg), cefotaxim (CTX 30 µg), norfloxacin (NOR 10 µg), ampicillin+sulbactam (SAM 20 µg (10+10)), meropenem (MRP 10 µg), gentamicin (GEN 10 µg), piperacillin+tazobactum (PI 100+ IZ 10 µg), amoxicillin+clavulonate (AMC 30 µg; (20+10)) were purchased from Rosco (Diagnostica A/S, Taastrupgaardsvej 30 DK-2630 Taastrup). Acinetobacter isolates and antimicrobial susceptibility testing A total of 35 clinical isolates cultured from different samples of wounds, trachea, blood, CSF, catheter and other samples of patients at hospitals from Tehran were the subject of this investigation. Antimicrobial susceptibility testing was evaluated using disc diffusion (NCCLS, 2012) and micro broth (CLSI, 2009) dilution assays. This inoculate of microorganism was adjusted to 0.5 McFarland (1×10 7 -1×10 8 CFU/ml) and using a sterile cotton swab, the microbial suspensions were cultured on appropriate media. Subsequently, sterile blank discs (6 mm in diameter) were saturated with 0.5, 1 and 2 µl of essential oil and were put on the cultured media. The plates were incubated at 37 °C for 24 h. The inhibition zones (IZ) diameters were measured in millimeters (mm) and average of IZ was recorded as means ± SD (Standard Deviation). The minimal inhibitory concentration (MIC) and minimal Bactericidal Concentration (MBC) values of essential oils were determined by micro broth dilution assay. The essential oil was twofold serially diluted (8 - 0.0125 µl/ml of essential oil). Cation adjusted Muller Hinton broth was used as broth media. After shaking, 100 µl of essential oil was added to each well. The above microbial suspensions were diluted to 1×10 6 and then 100 µl were added to each well and incubated at 35±2 °C. MIC was defined as the lowest concentration of essential oil that inhibits bacteria after 24 h. MBC value was the first well that Acinetobacter sp. as gram negative bacilli is one of the most problematic bacteria in hospital environments. The emergence of multi- drug resistant isolates of Acinetobacter sp. encourages the scientists to find the new antimicrobial agent with less side effects. The aim of this study was to evaluate the antibacterial activity of Cymbopogon olivieri, Heracleum persicum, Juniperus comminus, Azillia eryngioides, Dacus carrota, Ferula gummosa, Acorus calamus, Mentha pulegium, Achillea biebersteinii, and Chaerophyllum macropodum essential oils against clinical trials of Acinetobacter sp. by disc diffusion and micro broth dilution assays. The synergistic effect of these essential oils and amikacin (AMI) were determined. The higher inhibition zone diameters were for 2 µl of C. macropodum (15.3±0.48 mm). The lower MIC and MBC values were for C. olivieri (1.4 and 1.9 µl/ml) and J. comminus (1.9 and 2.6 µl/ml), followed by C. macropodum (2.01 and 3.2 µl/ml), D. carrota (2.1 and 3.8 µl/ml), A. eryngioides (2.3 and 3.1 µl/ml) essential oils and F. gummosa (2.4 and 4 µl/ml). AMI showed synergistic effect with all of the essential oils. D. carrota and A. eryngioides showed the best synergistic effect with AMI, followed by C. macropodum, A. biebersteinii, J. comminus and F. gummosa essential oils. ARTICLE INFO Received 6. 5. 2014 Revised 2. 12. 2015 Accepted 12. 1. 2016 Published 1. 4. 2016 Regular article doi: 10.15414/jmbfs.2016.5.5.412-415