Chemical composition and antibacterial activity of Origanum ramonense essential oil on the β -lactamase and extended- spectrum β -lactamase urinary tract isolates

The aim of this study was to evaluate the antibacterial activity of Origanum ramonense essential oil extracted from the air-dried leaves against β -lactamase and extended-spectrum β -lactamase obtained from the patients with urinary tract infection. The essential oil was extracted by hydrodistillation and analyzed by GC – MS. In vitro antibacterial activity was studied using disc diffusion and micro-dilution methods. Twenty compounds were identified representing 97.8% of the total oil. The major components were carvacrol (84.6%), p-cymene (4.3%) and γ -terpinene (3.3%). The oil showed a broad spectrum of antibacterial activity against all tested isolates. Staphylococcus aureus , S. epidermidis , Klebsiella pneumoniae and Enterobacter aerogenes had the lowest minimum inhibitory concentration values (0.015 µg/mL) followed by Escherichia coli (0.14 µg/mL). The lowest susceptible strains to oil were Pseudomonas aeruginosa, Proteus mirabilis, E. coli 25922 and P. aeruginosa 10145. The bacteriostatic and bactericidal effects at concentrations as low as 0.015 µg/mL indicated the potent antibacterial activity of O. ramonense . ramonense on the β lactamase extended-spectrum urinary Bangladesh


Introduction
Urinary tract infection is one of the most common and highly recurrent bacterial infections. The most prevailing bacteria which are responsible is the Escherichia coli. Many other urinary tract infection-causing genera are also isolated from the patients with a variable degree of infection such as Klebsiella, Enterobacter, Proteus, Serratia and Pseudomonas aeruginosa (Al-Asoufi et al., 2017).
However, a significant number of infections caused by multidrug-resistant bacteria among urinary tract infection patients, such as the extended β-lactamaseresistant bacteria, have been reported (Bartoletti et al., 2016). For instance, antimicrobial resistance survey on cystitis showed that a high percentage of E. coli strains were ampicillin, trimethoprim/sulfamethoxazole and nalidixic acid resistant strains while K. pneumoniae strains were mecillinam, fosfomycin, cefuroxime and nitrofurantoin resistant (Schito et al., 2009). In addition, 12% of patient with UPEC urinary tract infection had fluoroquinolone-resistant isolates (Van der Starre et al., 2010). The increasing prevalence of urinary tract infection isolates that were resistant to routinely used antibiotics, particularly to beta-lactams or extendedspectrum beta-lactamase, has stimulated interest in finding new and effective antimicrobial agents from other sources such as plants.
Essential oils have been applied for centuries in pharmaceuticals and alternative medicine. They are effective as antibacterial, antifungal, antiviral, anti-inflammatory, anti-oxidant, and as anti-cancer (Bakkali et al., 2008;Tarawneh et al., 2010;Benelli et al., 2017;Burt, 2004;Martinelli et al., 2017). Essential oils are secondary metabolites produced by aromatic plant families such as Apiaceae and Lamiaceae. They are the mixture of

Abstract
The aim of this study was to evaluate the antibacterial activity of Origanum ramonense essential oil extracted from the air-dried leaves against β-lactamase and extended-spectrum β-lactamase obtained from the patients with urinary tract infection. The essential oil was extracted by hydrodistillation and analyzed by GC-MS. In vitro antibacterial activity was studied using disc diffusion and micro-dilution methods. Twenty compounds were identified representing 97.8% of the total oil. The major components were carvacrol (84.6%), p-cymene (4.3%) and γ-terpinene (3.3%). The oil showed a broad spectrum of antibacterial activity against all tested isolates. Staphylococcus aureus, S. epidermidis, Klebsiella pneumoniae and Enterobacter aerogenes had the lowest minimum inhibitory concentration values (0.015 µg/mL) followed by Escherichia coli (0.14 µg/mL). The lowest susceptible strains to oil were Pseudomonas aeruginosa, Proteus mirabilis, E. coli 25922 and P. aeruginosa 10145. The bacteriostatic and bactericidal effects at concentrations as low as 0.015 µg/mL indicated the potent antibacterial activity of O. ramonense.
Origanum is a genus that includes 54 species in the family Lamiaceae. Traditionally, decoction of leaves and stems as well as their essential oils are widely used as remedies of upper respiratory and digestive complaints such as cough, sore throat and gastric spasm. Generally, the ability of Origanum species to inhibit the growth of pathogenic bacteria has been investigated in numerous studies. Reports showed that Origanum essential oil possesses potential antibacterial activity with low minimum inhibitory concentration and minimum bactericidal concentration (Bassanetti et al., 2017;Carezzano et al., 2017;Habbadi et al., 2017;Özkan et al., 2017). Therefore, they can be a powerful tool to reduce the impact of bacterial drug resistance issue.
To the best of our knowledge, this is the first report on the antibacterial effect and the second one about the chemical composition of O. ramonense essential oil. Thus, the aims of this study are to evaluate the antibacterial activity of essential oil extract from the O. ramonense leaves against β-lactamase and extendedspectrum β-lactamase obtained from patients with urinary tract infection symptoms and to survey the chemical constituents of the essential oil extract of Jordan habitat O. ramonense dried leaves.

Plant materials
Areal parts of O. ramonense were collected from Aye region, Al-Karak Province, south of Jordan during March to April 2017. The plant was identified by Dr. Feryal Al-Khresat (Department of Biology, Mu'tah University, Al-Karak, Jordan) according to flora Palaestina, part 3. Voucher specimen had been deposited in the Department of Medical Laboratory Sciences, Faculty of Science, Mu'tah University, Al-Karak, Jordan.
The freshly collected materials were air dried at room temperature in the shade and then the leaves were separated from the stem and subjected to essential oil extraction.

Essential oil extraction
Sample of 150 g of the dried leaves was subjected to hydrodistillation using simple Clevenger apparatus for 4 hours. The oil was extracted from the aqueous phase using n-hexane and dried over anhydrous sodium sulfate (Na2SO4). Finally, the extracted oil was stored as aliquots at 4°C.

Gas chromatography-mass spectrometry (GC-MS) analysis
GC-MS analysis was performed using Varian chrompack CP-3800 GC-MS-MS-200 equipped with split-splitless injector, DB-5 GC column and the mass detector was set to scan ions between 40-400 m/z using full scan mode and electron impact (EI, 70 eV). The parameters and conditions used in this test were similar to that used by (Tawaha et al., 2015). A hydrocarbon mixture of n-alkanes (C8-C20) was analyzed separately by GC-MS using the same column (DB-5) and under the same chromatographic conditions. The compounds were identified by comparison of their retention time to n-alkaline retention times and their similarities to mass spectra database (NIST library) and published reports.

Bacterial strains
Seven clinical isolates were used in this study including five Gram negative strains: Extended-spectrum βlactamase producing E. coli, extended-spectrum βlactamase producing Proteus mirabilis, β-lactamase producing K. pneumoniae, β-lactamase producing Pseudomonas aeruginosa and E. aerogenes, and two Gram positive strains: β-lactamase producing Staphylococcus aureus and S. epidermidis.
The clinical isolates were obtained from the Al-Salam Specialty Hospital (Al-Karak, Jordan) and Al Bashir Hospital (Amman, Jordan). These strains were isolated from patients with urinary tract infection symptoms and they were characterized by BIOMÉRIEUX VITEK® 2 SYSTEM or by Enterosystem 18 R (Liofilchem). In addition, two reference strains: E. coli 25922 and P. aeruginosa 10145 provided by Dr. Wael Al-Zereini (Department of Biology, Mu'tah University, Al-Karak, Jordan) were used.

Disc diffusion method
The disc diffusion method was performed as previously described (Qaralleh et al., 2010) with some modifications. Briefly, 100 µL of a bacterial suspension containing 1.5 x 10 8 CFU/mL (0.5 McFarland's standard) was spread using a sterile swab on Mueller-Hinton agar plates. Then, sterile blank discs containing 3 µL essential oil or ciprofloxacin (5 µg) were placed onto the inoculated plates. The plates were incubated at 37°C for 24 hours. Each test was performed in triplicate and the zone of inhibition was measured as millimeter diameter.

Minimum inhibitory concentration and minimum bactericidal concentration
The minimum inhibitory concentration was measured using the microdilution method with some modifications (Qaralleh et al., 2010). Three-fold dilution was prepared using 96 well plats from a stock solution of 10 µg essential oil in 10% DMSO solvent. Then 10 µL of bacterial suspension containing 1.5 x 10 8 CFU/mL (0.5 McFarland's standard) was inoculated into each well. The same test was carried out with 10% DMSO as a control. Each test was performed in triplicate. The lowest concentration of essential oil needed to inhibit the visible growth of the tested microbes after 24 hours was considered as the minimum inhibitory concentration value.
The minimum bactericidal concentration was determined by sub-culturing and spreading the wells content with concentrations of equal to and higher than the minimum inhibitory concentration value on agar plate. The lowest concentration of essential oil that exhibited no growth on the agar plate after incubation at 37°C for 24 hours was reported as the minimum bactericidal concentration.

Results
Based on the dry weight of the plant, the obtained yield of the essential oil was determined as 0.9% (v/w).
The essential oil clearly showed inhibition activity against all tested bacteria (Table II). The results of disc diffusion method using O. ramonense essential oil showed that S. aureus, S. epidermidis and E. coli were the most sensitive clinical isolates with inhibition zones of 28.3, 26.2 and 24.5 mm, respectively followed by E. aerogenes, P. aeruginosa and K. pneumoniae (21.3, 20.7 and 20.2 mm, respectively). While P. mirabilis exhibited the smallest inhibition zone (16.7 mm). The minimum inhibitory concentrations and minimum bactericidal concentrations of O. ramonense essential oil for the tested bacteria indicated that all tested bacterial strains were highly susceptible to O. ramonense essential oil. S. aureus, S. epidermidis, K. pneumoniae and E. aerogenes had the lowest minimum inhibitory concentration values (0.015 µg/mL) followed by E. coli (0.14 µg/mL; Table III). The lowest susceptible strains to essential oil were P. mirabilis (1.2 µg/mL) and E. coli 25922 (11.1 µg/ mL) P. aeruginosa and the reference strain P. aeruginosa 10145 exhibited similar minimum inhibitory concentration values with 11.1 µg/mL. In general, the minimum bactericidal concentration and minimum inhibitory concentration values were observed to be equal or close for most of the investigated strains. The exceptions of obtained data for minimum bactericidal concentration were that of P. mirabilis and E. aerogenes in which the minimum bactericidal concentration values are 3-fold and 9-fold of the minimum inhibitory

Discussion
In this study, the GC-MS analysis of the O. ramonense essential oil pointed out that ooxygenated monoterpenes (86.9%) as the major component with carvacrol as predominate component. Previous analysis of the essential oil from the O. ramonense (Danin et al., 1997) led to the identification of most dominated compounds of O. ramonense obtained in this study. These were oxygenated monoterpenes although they were represented by α-terpineol (41.5%), terpinen-4-o1 (16.8%), cis-sabinene hydrate (13.2%) and eugenol (3.6%). In this study, GC-MS analysis showed a lack of α-terpineol and eugenol while terpinen-4-o1 and cissabinene hydrate were indicated at low concentrations. Furthermore, the major component monitored in this study carvacrol (84.6%) whereas too lower concentration (0.1%) was reported (Danin et al., 1997).
Three white-flowered biotypes of O. vulgare studied were growing wild in different locations of Italy (De Falco et al., 2014). Their essential oil was rich with either carvacrol or thymol whereas phenols content was the lowest among them. Factors such as genetic makeup, harvesting seasons, geographical distribution and the extraction methods collectively might affect the chemical composition of essential oil (Ozdemir et al., 2017). Chemical analysis of the essential oil from these plants showed a high variability in the essential oil composition and content, indicating that various compounds may account for the different extents of the antimicrobial activity of plants In this study, our results revealed the potential broad spectrum antibacterial activity of O. ramonense essential oil. This potent activity could be because of the presence of its main component carvacrol. The antibacterial potency of carvacrol and its isomer thymol, has been reported with extremely low minimum inhibitory concentration and minimum bactericidal concentration values (Qaralleh et al., 2009;Althunibat et al., 2016). Carvacrol is considered as one of the most bactericidal essential oil components against both Gram positive and Gram negative bacteria (Hammer et al., 1999;Nieto, 2017). Mihajilov-Kristev et al., showed that essential oil (Satureja hortensis) rich in carvacrol (67%) and γ-terpinene (15%) are strong bacteriostatic and bactericidal agent against both Gram positive and Gram negative bacteria with low minimum inhibitory concentration and minimum bactericidal concentration values (0.025-0.78 μL/mL) (Mihajilov-Krstev et al., 2010). Other compounds such as p-cymene and gamma terpinene are also known for their antibacterial activity. Nevertheless, the antibacterial mechanism at the cellular and molecular level of oregano essential oil against several bacterial strains were previously reported. For instance, the treatment of E. coli O157:H7 with O. vulgare essential oil caused an increment in the cell membrane permeability and leakage of cell contents (Burt and Reinders 2003). Pseudomonas aeruginosa and S. aureus treated with O. compactum essential oil suffered from membrane damage (Bouhdid et al., 2009). In addition, oregano EOs and carvacrol have been shown to down-regulate the expression of virulence genes in enterohaemorrhagic Escherichia coli (EHEC) O157:H7 (Mith et al., 2015). The essential oil concentration is largely related issue to species, season of gathering the plant, geographical place, the plant part that is used, and the method of oil extraction (Sakkas and Papadopoulou, 2017 Susanti et al., 2013). In contrast, essential oil extract of Oregano is not traditionally used to treat urinary tract infection and information about Oregano utilization in treating or controlling urinary tract infection in the literature are rare. Therefore, the results of this study clearly show that O. ramonense essential oil can be used to treat the urinary tract infection patients infected with such urinary tract infection causing agents.

Conclusion
The bacteriostatic and bactericidal effect at concentration as low as 0.015 µg/mL indicated the potent antibacterial activity of O. ramonense essential.

Financial Support
Self-funded

Conflict of Interest
Authors declare no conflict of interest