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Spectrum Β-Lactamase-Producing Escherichia Coli Sharifi-Rad et al. Cell. Mol. Biol.2016, 62 (9): 75-82 Cellular and Molecular ISSN: 1165-158X Biology doi: 10.14715/cmb/2016.62.9.12 Original Research Antibacterial activities of essential oils from Iranian medicinal plants on extended- spectrum β-lactamase-producing Escherichia coli J. Sharifi-Rad1, 2, D. Mnayer3, A. Roointan4, F. Shahri5, S. A. M. Ayatollahi6,7, M. Sharifi-Rad8, N. Molaee9, M. Sharifi-Rad10* 1 Zabol Medicinal Plants Research Center, Zabol University of Medical Sciences, Zabol, Iran 2 Department of Pharmacognosy, Faculty of Pharmacy, Zabol University of Medical Sciences, Zabol, Iran 3 Faculty of Agricultural Engineering and Veterinary Medicine, Lebanese University, Dekwaneh, Lebanon 4 Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz university of Medical sciences, Shiraz, Iran 5 Department of Optometry, School of Rehabilitation, Zahedan University of Medical Sciences, Zahedan, Iran 6 Phytochemistry Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran 7 Department of Pharmacognosy, School of Pharmacy, Shahid Beheshti University of Medical Sciences Tehran, Iran 8 Department of Chemistry, Faculty of Science, University of Zabol, Zabol 98615-538, Iran 9 Department of Microbiology and Immunology, Arak University of Medical Sciences, Arak, Iran 10 Zabol University of Medical Sciences, Zabol, Iran Abstract: The extended-spectrum beta-lactamase (ESBL) -producing Escherichia coli strains can lead to various infections particularly uri- nary tract infections. The main objective of this investigation was to evaluate the antibacterial activities of essential oils (EOs) from different Iranian medicinal plants against TEM gene positive ESBL-producing E. coli strains isolated from urine samples of patients with urinary tract infections. EOs were extracted using hydrodistillation method. E. coli strains were isolated by different specific Medias. ESBL-producingE. coli strains were isolated from urine samples of patients with urinary tract infections in Shiraz hospital, Iran. Then, ESBL- producing strains were identified using double disk synergy test, phenotypic disc confirmatory test and polymerase chain reaction (PCR) for TEM gene detection. The antibacterial activity of the EOs from different plants (Achillea wilhelmsii C. Koch, Echinophora platyloba DC., Lallemantia royleana, Nepeta persica Boiss., Pulicaria vulgaris Gaertn., Salvia nemorosa, and Satureja intermedia C.A.Mey) and antibiotics against ESBL-producing strains was studied using the microdilution method for the evaluation of the minimum inhibitory concentration (MIC). The 103 out of 295 E. coli strains with 97 (90.65%) TEM gene distributions were identified as ESBL-producing strains. All of the EOs derived from different plants displayed high inhibitory effects against ESBL-producing E. coli strains. The results of our investigations may propose a good treatment option against resistant infectious bacteria. Key words: Essential oils, Escherichia coli, antibacterial activities. Introduction tabolites which can originate directly or indirectly from plants. It is assumed that the antibacterial effectiveness Escherichia coli is one of the main cause of acquired of plants is due to their secondary metabolites such as infections in human community. For about 30 years, β-- essential oils, flavonoids, tannins, saponins and phenols lactam antibacterial agents were efficiently used for the (6). Since many years ago, medicinal herbs have been treatment of infections caused by Gram-negative bacte- used because of their health benefits. Due to their com- ria especially E. coli isolates (1). Although β-lactam an- plex composition, they are widely used for treatments of tibacterial agents and their extended spectrum types are many human and animal disorders (7, 8). Essential oils the suitable choices for the treatment of such infections, (EOs) are one of the secondary metabolites of plants E. coli isolates produce or gain β-lactamase enzymes which are of great interest because of their safety and and develop resistance to such antibacterial agents (2). economic aspects. These derivatives have a vast appli- Currently, extended-spectrum β-lactamases (ESBLs) cation in cosmetic, flavor and pharmaceutical fields (9). are the prevalent kinds of β-lactamases in E. coli iso- The antibacterial activities of these volatile compounds lates which can transmit among bacteria and have the have been shown in different studies (10-12). Their ability to inactivate various types of β-lactam agents (3). biodegradability and producing non-toxic components Nowadays, the risk of ESBL-producing E. coli strains allowed them to be used safely as antibacterial agents among humans, domesticated animals and wildlife re- (12). presents a great concern in medicine. Therefore their fast discovery and treatments are of outmost importance Received May 11, 2016; Accepted August 23, 2016; Published in these fields (1, 4). September 19, 2016 Natural products are among suitable candidates for * Corresponding author: Mehdi Sharifi-Rad, Zabol University of Medical developing new remedies against resistance bacterial Sciences, Zabol, Iran. E-mail: [email protected] isolates (5). At this time, many researchers focus in their studies on these products and their secondary me- Copyright: © 2016 by the C.M.B. Association. All rights reserved. 75 J. Sharifi-Rad et al. 2016 | Volume 62 | Issue 9 Antibacterial activities of EOs against ESBL-producing E. coli strains. Recently, there is a growing trend in reports of the considered in the zone between Cephalosporin disc and in vitro testing of EOs against bacteria, viruses, proto- Clavulanic acid disc. zoa and fungi in medical and biological literature (13). It is believed that the antibacterial activity of EOs is Phenotypic disc confirmatory test largely caused by several components such as mono- Disks were laid on to the surface of Muller Hinton terpenes, terpenaceous and sesquiterpenes groups (13). agar. ESBL producing organisms were confirmed ac- Therefore, the objective of our study was to assess the in cording to inhibition diameter of 5 mm for Ceftriaxone vitro antibacterial activities of EOs derived from aerial (CE) versus Clavulanic acid (CEC) (20-10 μg) and Cef- parts (leaves, stems and flowers) of some Iranian plants tazidime disks (CA) (30 μg) versus Ceftazidime-Clavu- (Achillea wilhelmsii C. Koch, Echinophora platyloba lanic acid (CAC) (20-10 μg) or Ceftriaxone (CE) (30 DC., Lallemantia royleana, Nepeta persica Boiss., Puli- μg). (E. coli ATTCC 25922 was used as control and the caria vulgaris Gaertn., Salvia nemorosa, and Satureja test was done according to CLSI (Clinical and Labora- intermedia C.A.Mey) against E. coli isolates which tory Standards Institute) (15). were collected from urinary tract of infectious patients attended in Hospitals of Shiraz, Iran. DNA extraction and polymerase chain reaction The whole DNA of isolated ESBL-producing colo- Materials and Methods nies was extracted using boiling method after suspen- ding the isolates in Tris/EDTA. Polymerase chain reac- Plant materials tion (PCR) was done for detection and amplification of Aerial parts (leaves, stems and flowers) of Achillea 857 bp TEM gene by using of (5´- GAGTATTCAA- wilhelmsii C. Koch, Echinophora platyloba DC., Lalle- CATTTCCGTGTC-3´) as the forward primer and (5´ mantia royleana, Nepeta persica Boiss., Pulicaria vul- TAATCAGTGAGGCACCTATCTC-3´) as the reverse garis Gaertn., Salvia nemorosa, and Satureja interme- primer (Gene Gostar- Iran). The PCR reaction was in dia C.A.Mey were collected from different areas in Iran a total amount of 50 μL and contained the below items: at flowering stage (Table 1). The taxonomic identifica- 1 μL DNA sample (3 μg/μL), 10 pmol of each forward tion of plant materials was confirmed by a botanist at and reverse primers, 0.2 mM from each dNTP, 1.5 mM the herbarium affiliated to Shahid Beheshti University MgCl2, and 5 Unit Taq DNA polymerase (CinnaGen Co, of Iran. The collected plant materials were dried in a Iran). PCR amplification program was done according dark place. to the following platform: initial denaturation (94 °C for 120 seconds), (35 cycles of 60 seconds at 94 °C), (30 Essential oils extraction seconds at 52 °C) and (60 seconds at 72 °C) and for For EOs extraction, 100 g of each dried plants were final extension (300 seconds at 72° C) was considered. subjected to hydrodistillation for 3 hours using a Cle- After PCR amplification of TEM gene, the agarose gel venger-type apparatus according to the method out- electrophoresis was used for analysis of products. lined by the British Pharmacopeia (14). The obtained essential oils for each plant were dried over anhydrous Determination of minimum inhibitory concentration sodium sulfate (Sigma-Aldrich, St. Louis, MO, USA) of antibiotic and kept at 4 °C until further tests. For the investigation of antibiotic effects, different concentrations (512, 256, 128, 64, 32, and 2 µg/mL) of Escherichia coli isolates antibiotics (Amikacin, Ceftazidime, Ceftriaxone, Gen- The clinical specimens E. coli isolates were collec- tamicin, ciprofloxacin) (Farabi Pharmaceutical Co, Isfa- ted from urinary tract of infected patients who attended han, Iran) in Nutrient Broth (Merck, Darmstadt, Germa- Hospitals in Shiraz, Iran from January to November ny) with pH= 6.5 were prepared. Nutrient Broth without 2014. Microscopic Gram strain was used for the exami- any antibiotics was used as control media and E. coli nation of samples. Then,
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