In‐Vitro Antimicrobial Activity and Chemical Composition of <i>Satureja khuzestanica</i> Jamzad Essential Oils Against Multidrug-Resistant <i>Acinetobacter baumannii</i>

Fereshteh Eftekhar; Nastaran Ashoori; Morteza Yousefzadi

doi:10.5812/ajcmi.45601

In‐Vitro Antimicrobial Activity and Chemical Composition of Satureja khuzestanica Jamzad Essential Oils Against Multidrug-Resistant Acinetobacter baumannii

Fereshteh Eftekhar ^{1 , *} , Nastaran Ashoori ¹ and Morteza Yousefzadi ²

Authors Information

¹ Faculty of Biological Sciences, Department of Microbiology, Shahid Beheshti University, G.C., Tehran, IR Iran

² Faculty of Basic Sciences, Department of Marine Biology, Hormozgan University, Bandar Abbas, IR Iran

Article Notes and Dates

Avicenna Journal of Clinical Microbiology and Infection: May 2017, 4 (2); e45601.Published Online: April 24, 2017

Article Type: Research Article; Received : January 11, 2017; Revised : February 22, 2017; Accepted : March 13, 2017
DOI: http://dx.doi.org/10.5812/ajcmi.45601

To Cite : Eftekhar F, Ashoori N, Yousefzadi M. In‐Vitro Antimicrobial Activity and Chemical Composition of Satureja khuzestanica Jamzad Essential Oils Against Multidrug-Resistant Acinetobacter baumannii, Avicenna J Clin Microb Infec. 2017 ;4(2):e45601. doi: 10.5812/ajcmi.45601.

Abstract

Copyright: Copyright © 2017, Avicenna Journal of Clinical Microbiology and Infection. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.

1. Background

Acinetobacter baumannii has become an important cause of nosocomial infections in hospital which outbreaks during the past few decades (1). The target of this organism usually is ill patients in intensive care units (ICU) who resistant to most commonly used antibiotics (2). The remarkable ability of A. baumannii to accumulate diverse antibiotic resistance mechanisms has led to the emergence of strains with a broad range of resistance to all existing antibiotic classes and causes a serious concern in clinical practice (2-4). Among the resistance mechanisms, a number of metallo-beta-lactamases and extended-spectrum beta-lactamases production is well documented in clinical isolates of A. baumannii (5-8) Therefore, investigating for effective and alternative antibacterial agents such as plant products has been increased. Essential oils which contain high levels of monoterpens are particularly interest, since their lipophilic nature allows them in order to pass through the bacterial cell walls and cytoplasmic membranes. As a result, they can cause cellular content leakage, cell death by disruption of the cytoplasmic membrane, or reaching to other intracellular targets for their antimicrobial activity (9, 10).

Satureja khuzestanica Jamzad is an endemic plant from Southwestern Iran. The essential oil of this plant has been reported for a number of biological activities, including: antibacterial, antifungal, anti-leishmanial, anticancer, antioxidant, anti-inflammatory, anti-diabetic, anti-hyperlipidemic and antispasmodic properties (11, 12). The antibacterial activity of S. khuzestanica essential oil has been shown against a number of bacteria including Gram-positive food pathogens, some members of Enterobacteriaceae, as well as Pseudomonas aeruginosa (13, 14). Other recent studies have been shown that carvacrol-rich S. khuzestanica EO has antimicrobial activity against clinical urinary isolates of E. coli, a number of Gram-positive cocci and yeast strains (15, 16). Also the effect of S. khuzestanica EO has been shown on the expression of the bap gene which is involved in biofilm formation and bla_OXA-23gene in antibiotic resistant A. baumannii using polymerase chain reaction (17, 18).

2. Objectives

3. Methods

3.1. Preparation of S. khuzestanica Essential Oil

The aerial parts of S. khuzestanica were collected from Khorramabad (Lorestan province, South of Iran), which is verified by Dr. Sonboli and was deposited with a voucher specimen code of MPH-352 at medicinal plants and drug research institute, Shahid Beheshti University. A Clevenger type apparatus was used for hydro-distillation of the powdered plant aerial parts (250 g) for 3 hours which is recommended by the European Pharmacopoeia. Drying of the obtained essential oil was carried out over anhydrous sodium sulfate before storing at 4°C. The EO was re-suspended with dimethyl sulfoxide (1% DMSO) before using in test assays.

3.2. Essential Oil Components Identification

The analysis of the EO was carried out by gas chromatography-flame ionization detection (GC-FID) using a Finnigan system (Thermoquest, Manchester, UK) by a 60 m × 0.25 mm with 0.25 µm film thickness DB-5 fused silica column (J&W Scientific, Folsom, CA). The carrier gas (nitrogen) was used at a constant flow of 1.1 mL/min with a split ratio of 1:50. The raise of oven temperature is occurred at a rate of 5°C/min from 60°C to 250°C and the temperatures of the injector and detector (FID) were kept at 250°C and 280°C, respectively. Mass spectroscopy (GC-MS) was performed using a Thermoquest Trace GC-MS instrument with the column and temperature program which is described above with the temperature of transfer line at 250°C. The carrier gas (Helium) was used at a flow rate of 1.1 mL/min with a 1:50 split ratio. The components of the EO were identified with their retention indices using same temperature-programmed conditions for n-alkanes and DB-5 column. The EO individual compounds were identified by comparing their mass spectra with reference mass spectra library or authentic compounds, as well as comparing their retention indices with authentic or literature cited compounds. FID area percentages were used in order to obtain semi-quantitative data without using correction factors (12).

3.3. Bacterial Isolates

Twenty one non-repetitive MDR isolates of A. baumannii (11 ICU and 10 burn isolates) was collected in 2014 from Imam Hossein and Shahid Motahari Burn hospitals in Tehran. The ATCC standards (E. coli 25922, K. pneumoniae 10031 and P. aeruginosa 85327) were used as susceptibility controls.

3.4. Antimicrobial Susceptibility

According to the CLSI guidelines the disc diffusion method was used in order to confirm the susceptibility of isolates to aztreonam (30 µg), amikacin (30 µg), gentamicin (10 µg), tobramycin (10 µg), cefepime (30 µg), cefotaxime (30 µg), ceftazidime (30 µg), ciprofloxacin (5 µg), imipenem (10 µg), meropenem (10 µg), piperacillin (100 µg) and piperacillin-tazobactam (110 µg) (MAST, UK) (19). The isolates which were resistant to > 3 antibiotic classes were defined as multidrug-resistant (MDR). Also, the disc method was used in order to determine the susceptibility of the bacterial isolates to the oil using discs containing of 1.64 mg essential oil in 1% DMSO. Negative control discs that only contained DMSO were also included. Minimum inhibitory concentrations (MICs) of the essential oil were determined with broth microdilution as recommended by CLSI (20). Fifty μL of serial two-fold dilutions of each essential oil concentration (0.019 - 5 mg/mL) in Muller Hinton broth (MHB) were added to the wells of 96 well flat bottomed microtiter plates. Fresh bacterial overnight grown were cultured in MHB (50 μL) at 5 × 10⁵/mL. Then, it is added to four wells for each test bacterium, before incubating the microplates at 37°C for 24 hours. The lowest EO concentration which inhibited growth was recorded as MIC. MBC was measured with inoculating 10 μL of the wells contents without bacterial growth on nutrient agar plates before incubation at 37°C for 24 hours. MBC was reported if the colony counts were < 5.

4. Results

Qualitative and quantitative analysis of S. khuzestanica EO are presented in Table 1. Nineteen compounds were presented among which carvacrol was the main component (92.87%) followed by limonene (1.2%) (12).

Table 1. Chemical Composition of the Essential Oil of S. khuzistanica

Component	RI	%	ID Method
α-Thujene	925	0.21	RI, MS
α-Pinene	933	0.18	RI, MS, CO
Myrcene	981	0.16	RI, MS
α-Terpinene	1013	0.40	RI, MS, CO
p-Cymene	1017	0.51	RI, MS, CO
Limonene	1026	1.20	RI, MS, CO
Z-β-Ocimene	1036	0.18	RI, MS
γ-Terpinene	1053	0.52	RI, MS, CO
trans-Sabinene hydrate	1081	0.57	RI, MS
Terpin-4-ol	1163	0.27	RI, MS
α-Terpinole	1175	0.14	RI, MS
Thymol	1266	0.11	RI, MS, CO
Carvacrol	1282	92.87	RI, MS, CO
Thymyl acetate	1329	t	RI, MS
β-Caryophyllene	1425	0.40	RI, MS, CO
α-Humulene	1427	0.22	RI, MS
β-Bisabolene	1501	0.14	RI, MS
trans-β-Bisabolene	1522	t	RI, MS
TOTAL		98.08

Abbreviations: CO, co-injection with authentic compounds; MS, mass spectroscopy; RI, retention indices relative to C6-C24 n-alkanes on a DB-5 column; t, trace i.e., < 0.1 % by MS, < 0.05% by CO.

Antibiotic disc susceptibility results showed that all test isolates were multidrug resistant (Figure 1). All of isolates were resistant to ceftazidime, piperacillin, piperacillin-tazobactam, cefepime, cefotaxime, azetronam, imipenem, gentamicin and ciprofloxacin. The susceptibility to other antibiotics was: amikacin, 76%; meropenem, 38%; and tobramycin, 14%. As shown, tobramycin and meopenem had the highest antibacterial activity against our MDR isolates of A. baumannii.

Figure 1. Antibiotic Susceptibility of Twenty One Acinetobacter baumannii Clinical Isolates

Aztreonam (ATM), amikacin (AK), gentamicin (GM), tobramycin (TN), cefepime (CPM), cefotaxime (CTX), ceftazidime (CAZ), ciprofloxacin (CIP), imipenem (IMP), meropenem (MEM), piperacillin (PRL) and piperacillin-tazobactam (PTZ).

The disc diffusion results for S. khuzistanica essential oil is revealed inhibition zones of 29 - 42 mm (Table 2). Both of ICU and burn isolates were similarly susceptible to the essential oil. The ATCC standards were also susceptible to the essential oil with the highest inhibition zone for P. aeruginosa (43 mm) followed by E. coli (26 mm) and K. pneumoniae (20 mm). The MIC values for the essential oil were 0.31 mg/mL for all test isolates and 0.62 mg/mL for ATCC susceptible standards. The MBCs were 0.31 - 0.62 mg/mL for A. baumannii test isolates and 0.62 mg/mL for ATCC standards showing the bactericidal activity of the essential oil (Table 1).

Table 2. Susceptibility of MDR Clinical Isolates of A. baumannii to S. khuzestanica EO Measured by Disc Diffusion, MIC and MBC Determinations^a

A. baumannii Isolates	Disc Inhibition Zone, mm	MIC, mg/mL	MBC, mg/mL
1 NB	37 ± 5.6	0.31	0.31
2 NB	30 ± 0.0	0.31	0.62
5 NB	30 ± 4.2	0.31	0.62
7 NB	31 ± 0.7	0.31	0.31
12 NB	29 ± 1.4	0.31	0.62
13 NB	31 ± 2.1	0.31	0.31
14 NB	30 ± 1.4	0.31	0.62
15 NB	31 ± 2.8	0.31	0.31
35 NB	33 ± 1.4	0.31	0.31
36 NB	37 ± 4.2	0.31	0.31
37 NB	32 ± 4.2	0.31	0.31
26 B	32 ± 2.1	0.31	0.31
27 B	33 ± 1.4	0.31	0.62
28 B	32 ± 1.4	0.31	0.31
29 B	31 ± 1.4	0.31	0.62
31 B	33 ± 2.1	0.31	0.31
33 B	34 ± 0.7	0.31	0.62
47 B	42 ± 0.7	0.31	0.31
54 B	37 ± 4.2	0.31	0.62
55 B	40 ± 0.0	0.31	0.62
58 B	36 ± 2.1	0.31	0.31
E. coli^b	26 ± 1.4	0.62	0.62
K. pneumoniae^b	20 ± 0.0	0.62	0.62
P. aeruginosa^b	43 ± 1.4	0.62	0.62

Abbreviations: B, burn; NB, non-burn ICU isolates.

^aThe numbers assigned to each isolate represents the sequence in which they were obtained from patients.

^bATCC standards.

5. Discussion

The antibacterial activity of S. khuzistanica essential oil has been shown against a number of bacterial pathogens. Akbari-Shahabi observed that S. khuzistanica essential oil was active against Listeria monocytogenes, an important food pathogen (14). Abbasi and coworkers showed the antibacterial activity of S. khuzistanica essential oil against MDR Pseudomonas aeruginosa, an important opportunistic pathogen which is responsible for outbreaks of nosocomial infections (13). Ghodrati et al. has been demonstrated the activity of S. khuzistanica essential oil against Escherichia coli, Staphylococcus aureus, Bacillus cereus, Staphylococcus epidermidis and Candida albicans (11). In literature search, recent report showing that the antibacterial activity of S. khuzistanica essential oil against A. baumannii was related to reduce the bap gene that involved in biofilm formation by the organism (17). Another recent study showed that Satureja khuzestanica Jamzad EO has inhibitory effects on the expression of bla_OXA-23gene in drug-resistant A. baumannii (18). We believe that our results present the first report on the susceptibility of drug-resistant A. baumannii nosocomial strains to S. khuzistanica EO. Our results are important since in the recent decades, which A. baumannii infections have become a major cause of mortality in hospitalized patients, especially in ICUs and burn wards (21, 22). Azimi et al. has been reported 12% mortality rate in Motahari Burn hospital in 2011, where at least 1 positive A. baumannii culture was recovered from all patients (23).

In this study, the essential oil of S. khuzestanica contained almost 93% carvacrol as the major component. Also other investigators have reported over 90% carvacrol content in the S. khuzestanica essential oils which is collected from different areas of Iran (11, 15, 24, 25). The antimicrobial activity of carvacrol, a phenolic monoterpene has been shown that be related to the presence of a free hydroxyl group and their hydrophobic nature essential for damaging cell membranes (26). Xu et al showed the carvacrol and thymol had the ability in order to permeabilize and depolarize the cytoplasmic membrane of E. coli (27). Cristani et al reported that among four tested monoterpnes, carvacrol caused a gross perturbation of the lipid fraction in bacterial cytoplasmic membranes, depending on the lipid composition and the net surface charge of membrane (28). Ultee and coworkers showed that carvacrol changes the permeability of B. cereus membrane by dissipation of H⁺ and K⁺ ion gradients that leads to cell death (29). Di Pasqua et al. reported that carvacrol along with some other monoterpenes, leads to decrease the unsaturated fatty acids in the membrane of treated cells and exert their antimicrobial activities by alterations of the cell envelope (30).

The strong antibacterial activity of S. khuzistanica Jamzad essential oil is most probably related to its carvacrol content. Meanwhile limonene, the other major component of our S. khuzistanica EO (1.2%), has been reported to have weak or no antibacterial activity against bacterial pathogens (31). In conclusion, carvacrol rich essential oil of S. khuzistanica Jamzad showed the strong antibacterial activity against MDR clinical isolates of A. baumannii which can be used for treatment of infections in order to eradicate these pathogens. Further research employing a larger number of bacterial isolates is needed to confirm the effectiveness of S. khuzistanica Jamzad EO as an anti-infective agent.

Footnotes

References

1. Zarrilli R, Giannouli M, Tomasone F, Triassi M, Tsakris A. Carbapenem resistance in Acinetobacter baumannii: the molecular epidemic features of an emerging problem in health care facilities. J Infect Dev Ctries. 2009; 3(5): 335-41[PubMed]
2. Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol Rev. 2008; 21(3): 538-82[DOI][PubMed]
3. Maragakis LL, Perl TM. Acinetobacter baumannii: epidemiology, antimicrobial resistance, and treatment options. Clin Infect Dis. 2008; 46(8): 1254-63[DOI][PubMed]
4. Manchanda V, Sanchaita S, Singh N. Multidrug resistant acinetobacter. J Glob Infect Dis. 2010; 2(3): 291-304[DOI][PubMed]
5. Safari M, Saidijam M, Bahador A, Jafari R, Alikhani MY. High prevalence of multidrug resistance and metallo-beta-lactamase (MbetaL) producing Acinetobacter baumannii isolated from patients in ICU wards, Hamadan, Iran. J Res Health Sci. 2013; 13(2): 162-7[PubMed]
6. Farajnia S, Azhari F, Alikhani MY, Hosseini MK, Peymani A, Sohrabi N. Prevalence of PER and VEB Type Extended Spectrum Betalactamases among Multidrug Resistant Acinetobacter baumannii Isolates in North-West of Iran. Iran J Basic Med Sci. 2013; 16(6): 751-5[PubMed]
7. Safari M, Mozaffari Nejad AS, Bahador A, Jafari R, Alikhani MY. Prevalence of ESBL and MBL encoding genes in Acinetobacter baumannii strains isolated from patients of intensive care units (ICU). Saudi J Biol Sci. 2015; 22(4): 424-9[DOI][PubMed]
8. Owlia P, Azimi L, Gholami A, Asghari B, Lari AR. ESBL- and MBL-mediated resistance in Acinetobacter baumannii: a global threat to burn patients. Infez Med. 2012; 20(3): 182-7[PubMed]
9. Saad NY, Muller CD, Lobstein A. Major bioactivities and mechanism of action of essential oils and their components. Flavour Fragrance J. 2013; 28(5): 269-79[DOI]
10. Trombetta D, Castelli F, Sarpietro MG, Venuti V, Cristani M, Daniele C, et al. Mechanisms of antibacterial action of three monoterpenes. Antimicrob Agents Chemother. 2005; 49(6): 2474-8[DOI][PubMed]
11. Ghodrati L, Alizadeh A, Ketabchi S. Essential oil constituents and antimicrobial activities of Iranian Satureja khuzistanica Jamzad. Int J Biosci. 2015; 6(3): 249-57
12. Yousefzadi M, Riahi-Madvar A, Hadian J, Rezaee F, Rafiee R, Biniaz M. Toxicity of essential oil of Satureja khuzistanica: in vitro cytotoxicity and anti-microbial activity. J Immunotoxicol. 2014; 11(1): 50-5[DOI][PubMed]
13. Abbasi A, Bahador A, Esmaeili D, Mahbubi A, Amiri M, Amiri M. The study of inhibitory effects of satureja khuzestanica against MDR isolates of pseudomonas aeruginosa. Int J Curr Microbiol App Sci. 2014; 3(2): 614-8
14. Akbari-Shahabi, S. , Assmar M, Massiha A, Ghaemi N, Issazadeh K, Shokri-Fashtali S. Evaluation of antibacterial activity of Satureja khuzestanica J. essential oil against standard and isolated strains of Listeria monocytogenes. Zahedan Med Sci J. 2014; 16(10): 38-41
15. Mahboubi M, Kazempour N. The Antibacterial Activity of Satureja khuzestanica Essential Oil Against Clinical Isolates of E. coli. Jundishapur J Nat Pharm Prod. 2016; 11(2)[DOI]
16. Zomorodian K, Ghadiri P, Saharkhiz MJ, Moein MR, Mehriar P, Bahrani F, et al. Antimicrobial activity of seven essential oils from Iranian aromatic plants against common causes of oral infections. Jundishapur J Microbiol. 2015; 8(2)[DOI][PubMed]
17. Bahador A, Saghii H, Ataee R, Esmaeili D. The Study of inhibition effects Satureja khuzestaniea essence against gene expression bap acinetobacter baumannii with real time PCR technique. Iran J Med Microbiol. 2015; 9(1): 42-9
18. Saghi H, Dastjerdi FA, Zahedi B, Pour MM, Khorrami M, Efati M, et al. The Study of the Inhibition Effects of Satureja khuzestaniea Essence on the Gene Expression of bla-OXA-23 in Multidrug-Resistant Strains of Acinetobacter baumannii. Avicenna J Clin Microbiol Infect. 2016; 3(3)
19. Wayne P. Clinical and Laboratory Standards Institute (CLSI) performance standards for antimicrobial disk diffusion susceptibility tests. 2009;
20. Wayne P. Clinical and Laboratory Standards Institute (CLSI) Method for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically. 2009;
21. Albrecht MC, Griffith ME, Murray CK, Chung KK, Horvath EE, Ward JA, et al. Impact of Acinetobacter infection on the mortality of burn patients. J Am Coll Surg. 2006; 203(4): 546-50[DOI][PubMed]
22. Dijkshoorn L, Nemec A, Seifert H. An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii. Nat Rev Microbiol. 2007; 5(12): 939-51[DOI][PubMed]
23. Azimi L, Motevallian A, Ebrahimzadeh Namvar A, Asghari B, Lari AR. Nosocomial infections in burned patients in motahari hospital, tehran, iran. Dermatol Res Pract. 2011; 2011
24. Saidi M. Antioxidant Activities and Chemical Composition of Essential Oils fromSatureja khuzestanica,Oliveria decumbensandThymus daenensis. J Essential Oil Bearing Plants. 2014; 17(3): 513-21[DOI]
25. Ghasemi Pirbalouti A, Moalem E. Variation in antibacterial activity of different ecotypes of Satureja khuzestanica Jamzad, as an Iranian endemic plant. Ind J Tradit Know. 2013; 12: 623-9
26. Ben Arfa A, Combes S, Preziosi-Belloy L, Gontard N, Chalier P. Antimicrobial activity of carvacrol related to its chemical structure. Lett Appl Microbiol. 2006; 43(2): 149-54[DOI][PubMed]
27. Xu J, Zhou F, Ji BP, Pei RS, Xu N. The antibacterial mechanism of carvacrol and thymol against Escherichia coli. Lett Appl Microbiol. 2008; 47(3): 174-9[DOI][PubMed]
28. Cristani M, D'Arrigo M, Mandalari G, Castelli F, Sarpietro MG, Micieli D, et al. Interaction of four monoterpenes contained in essential oils with model membranes: implications for their antibacterial activity. J Agric Food Chem. 2007; 55(15): 6300-8[DOI][PubMed]
29. Ultee A, Kets EP, Smid EJ. Mechanisms of action of carvacrol on the food-borne pathogen Bacillus cereus. Appl Environ Microbiol. 1999; 65(10): 4606-10[PubMed]
30. Di Pasqua R, Betts G, Hoskins N, Edwards M, Ercolini D, Mauriello G. Membrane toxicity of antimicrobial compounds from essential oils. J Agric Food Chem. 2007; 55(12): 4863-70[DOI][PubMed]
31. Srisukh V, Tribuddharat C, Nukoolkarn V, Bunyapraphatsara N, Chokephaibulkit K, Phoomniyom S, et al. Antibacterial activity of essential oils from Citrus hystrix (makrut lime) against respiratory tract pathogens. Sci Asia. 2012; 38(2): 212[DOI]

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