A Comparative Study of the Antibacterial Activity of Clove and Rosemary Essential Oils on Multidrug Resistant Bacteria

Baydaa Hameed Abdullah*, Suhad Faisal Hatem, Widad Jumaa

Department of clinical and laboratory sciences, Pharmacy college, Al Mustansiriyah University. Baghdad, Iraq

Received: 20-Nov-2014 , Accepted: 27-Feb-2015

Keywords: Antibacterial, Essential oil clove, Rosemary, MIC

DOI: http://dx.doi.org/10.20510/ukjpb/3/i1/89220

 

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Abstract

The essential oils of Syzygium aromaticum (clove bud) and Rosmarinus officinalis L. (rosemary) were obtained by hydro-distillation. The antimicrobial activity of clove bud oil and rosemary oil was investigated by agar well diffusion method against four multidrug resistant strains namely Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus feacalis as well as two standard strains, Staphylococcus aureusATCC29213 and Pseudomonas aeruginosaATCC27853. Both essential oils exhibited inhibitory effects towards all the test organisms, clove essential oil had antibacterial activity little higher than of rosemary oil, MICs ranged from 0.312% (v/v) to 1.25% (v/v) for all tested bacteria while MICs for rosemary oil ranged from 0.312% (v/v) to 5 % (v/v). Based on this finding, it may be suggested that these essential oils may be used as natural antibacterial agents to treat infections caused by multidrug resistant bacteria.

1 Introduction

Antibiotics provide the main basis for the therapy of microbial (bacterial and fungal) infections. There was a medical belief since antibiotics discovery and their uses chemotherapeutic agents that this would lead to the infectious diseases eradication.  However , the major factor for the dissemination and emergence of muli- drug resistant bacterial strains was the overuse of antibiotics1.The worldwide emergence of Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus feacalis and Acinetobacter baumanni has become a majortherapeutic problem. Multi-drug resistant strains of Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus feacalis are widely distributed in hospitals and are increasingly being isolated from community acquired infections2.Thus, as a result of the rapid global spread of emergence of resistant bacteria and the need to find new antibacterial agents. However even new generations of antibiotics have low effectiveness. For this reason, Natural products provide unlimited opportunities for new drug.  The search for new antibacterial compounds an alternative way for solving this problem so herbal products are increasingly turning the researchers attention to develop better drugs against Multidrug Resistant Bacteria(MDR)3,4,32.

Cloves (Syzygium aromaticum) are belonging to the family Myrtaceae which are dried unopened floral buds of an evergreen tree5. Clove is used as flavoring agent and as spice .It has been known as aromatic, stimulant and carminative, used for dyspepsia and gastric irritations. Antimicrobial and antioxidant properties have been known to clove buds and their essential oils 6.

Clove oil is widely used as a perfume and food flavouring7, as a medicine for the treatment of asthma, rheumatoid arthritis, acne, warts, scars and various allergic disorders8, as an analgesic, antispasmodic, and as a general antiseptic in medical dental practices9.Antimicrobial properties of clove essential oil was tested and showed inhibitory activity to Listeria monocytogenes, Campylobacter jejuni, Salmonella enteritidis, Bacillus cereus, Escherichia coli and Staphylococcus aureus10. Importantly, clove oil might be considered as anti-carcinogenic agent due to its antioxidant properties11.

Rosemary (Rosmarinus officinalis L.)   is a spice and me­dicinal herb widely used around the world. Of the natural antioxidants, rosemary has been widely accepted as one of the spices with the highest antioxidant activity11. Rosemary essential oil is also used as an antibacte­rial, antifungal13,14,15 and anticancer agent16.

The aim of the present study was to investigate the antibacterial effects of the oil extracts of Syzygium aromaticum and Rosmarinus officinalis against multi-drug resistant strains isolated from clinical infection.

2 Materials and Methods

2.1 Collection and identification of plant materials

The leaves of rosemary plant and the flower buds of clove used in this study were purchased from the local market of Baghdad, Iraq.The plants were identified and authenticated at the Department of Pharmacogonosy, Faculty of Pharmacy, and University of AlMustansiriah.

2.2 Extraction of volatile oil by steam distillation

Steam distillation method was used in order to obtain the oil extracts of the dried flower buds of cloves and the dried rosemary by using the Clavenger apparatus as mentioned by Harbone17.Briefly,120g of grinded dried two plants were placed in a round bottom flask and the  added water full approximately three-quarter of the flask. The distillation apparatus was then connected to the flask and by heating  the water boiled, steam carrying volatile condensed by condenser and over anhydrous sodium sulphate the oil was drained and dried.

Determination the density of the oil was carried according to the weight: volume ratio (w/v).

2.3 Bacterial test isolates

Four mulidrug resistant bacteria clinical isolates and two standard strains were selected for this study. The mulidrug resistant bacteria comprised Acinetobacter baumanni, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus feacalis were isolated from different clinical samples(stool, wound, ear, throat and sputum) and the standard strains were Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosaATCC27853.The bacteria were cultured on nutrient agar medium. The agar plates were incubated at 37°C for 24 h.

2.4 Antibacterial assay

Determination the antibacterial activities of each plants oils was carried out by agar well diffusion method18 . Briefly, one hundred microlitres (100µL) of inoculums (106CFU/mL; 0.5 Mac-Farland) of each test bacterium was spread on to a sterile Muller-Hinton Agar plate (Hi Media). The plates were allowed to dry and a sterile cork borer of diameter 6.0mm was used to bore wells in the agar plates. Subsequently, a 50μL volume of each dilution of the oil with DMSO (10% to 0.312% v/v) introduced in triplicate wells into Muller-Hinton Agar plate. Sterile DMSO served as the negative control .The plates were allowed to stand for at least 1h for diffusion to take place and then incubated at (37 ºC for 24h).The antimicrobial activity, indicated by an inhibition zone surrounding the well containing the extract, was recorded if the zone of inhibition was greater than 6mm19.

2.5 Determination of minimum inhibitory concentration (MIC)

The MIC was defined as the lowest concentration that completely inhibited the growth for 24 h20. The MIC for the oils of each plant was determined by the agar well diffusion method. A two-fold serial dilution of the clove oil and rosemary oil was prepared in sterile DMSO to achieve a decreasing concentration range of 10% to 0.039% (v/v). A 50 µL volume of each dilution was added aseptically into the wells in Mueller Hinton agar plates that had been inoculated with standardized inoculums (106 CFU/mL) of the test bacteria. The agar plates were incubated at 37°C for 24 h. All experiments were performed in triplicate. The lowest concentration of clove and rosemary oils showing a clear zone of inhibition was considered as the MIC.

3 Results

The essential oils obtained were yellow in color with a density of 1.04 mg/ml for Syzygium aromaticum flower budsand 1.07 mg/ml and Rosmarinus officinalis L.

In the present investigation, we have tested the essential oil extracts of the two plants for their antimicrobial activity against four multidrug resistant strains. Two ATCC strains,  one of Gram-positive bacterium (Staphylococcus aureus ATCC 29213) and one of Gram-negative bacterium (Pseudomonas aeruginosa ATCC 27853) were also used as control sensitive strains.

The two plants essential oils showed antibacterial activity against all microorganisms tested, as exhibited by an agar diffusion assay.

Acinetobacter baumanni was found to be the most sensitive to rosemary oil at the higher concentration (10% v/v) with an inhibition zone diameter of 35 mm, followed by Pseuomonas aeruginosa (34 mm). Staphylococcus aureus and Enterococcus faecalis were found to be less sensitive to the test oil with an inhibition zone diameter of 30mm and 32 mm, respectively. However the lower concentrations of rosemary oil under (5% v/v) were found to be not effective in all tested multidrug resistant bacteria except Acinetobacter baumanni as shown in table 1.

The growth of the tested bacteria was also inhibited by clove oil ,the widest inhibition zone diameter was produced against Acinetobacter baumanni (28mm) at the higher concentration (10% v/v) followed by Enterococcus faecalis and Pseuomonas aeruginosa produced an inhibition zone diameter  of (25mm) and (17mm) respectively (table 2).Acinetobacter baumanni and Enterococcus faecalis were found not sensitive to the concentration less than (1.25% v/v) and Staphylococcus aureus  was not sensitive to the concentration less than (0.625% v/v).

As it can be seen, both essential oils exhibited inhibitory effects towards all the test organisms. Clove essential oil exhibited a little higher antimicrobial activity than that of rosemary essential oil, which was similar to the results of sensitivity test (Tables 1 and 2). MICs for clove essential oil ranged from 0.312% (v/v) to 1.25% (v/v) for all test microorganisms, while MICs for rosemary oil ranged from 0.312% (v/v) to 5 % (v/v).

4 Discussion

The increasing occurrence, particularly in hospitals, of multidrug resistant bacteria has made therapy more difficult21. The spread control strategies have been proposed and also searched for treatment MDR bacteria, the alternative treatment of these bacteria is by using natural compounds investigations22.

The antibacterial activitywere seen against MDR bacteria. However, the clove oil was more active against  Acinetobacter baumanni and Enterococcus faecalis isolates at concentrations of  0.312% and above while rosemary oil was  active against all bacteria tested  at concentrations of  5% and above except Acinetobacter baumanni which was sensitive at concentrations of  0.312%.

Antibacterial properties of clove and rosemary have already been reported23,24 but in the present study we tested the antibacterial activity of clove and rosemary essential oils on multidrug resistance bacteria. The antibacterial activity of the essential oils of against clove and rosemary  MDR bacteria has not been reported before.

MICs of clove and rosemary essential oils in this study were similar to the known literature6,9,27,28 ,with a little difference, which could be several reasons such as different growing environment of clove and rosemary, different extracting methods of essential oils, andmay be due to the use of MDR bacteria in the present study.

Different essential oils have different antimicrobial activity because of the components in them29. The antimicrobial activity of clove essential oils could be associated with Eugenol (2 methoxy-4 allyl-phenol)25, the main component of clove oil, which is already known to exhibit antibacterial and antifungal activity30.Clove antimicrobial activity also due to high tannin content (10-19%)26.The main compounds present in rosemary essential oil were 1,8-cineole, α-pinene, camphor, etc., which have been evaluated for their antimicrobial effects31,32,33.

Based on these data, further chemical and pharmacological investigations are required for clove and rosemary essential oils. These in vitro results of the present study  provide evidence that the two essential oils represent a potentially rich source for medicine and food preservatives against microorganisms.

5 Conclusion

On comparison of the antibacterial activities of cloveoil and rosemary oil tested against the MDR bacterial   strains, it was finally concluded that clove oil and rosemary oil emerged as the potent agent exhibiting high antibacterial activity in the treatment of the infections caused by multidrug resistant bacteria and the clove oil is more potent than rosemary oil.

6 Competing interests

 Plant extaction, antimicrobial evaluation, multidrug resistance

7 Author’s contributions

BHA carried out literature review, draft the manuscript, data collection and tabular form. BHA and SHF carried out the bacterial isolation and antibacterial evaluation. WJ participated in the plants extraction and data collection. All authors read and approved the final manuscript.

References

  1. Harbottle H, Thakur S, Zhao S, White DG. Genetics of Antimicrobial Resistance. Anim. Biotechnol. 2006; 17: 111-124.
  2. Akram M, Shahid M , Khan A.U. Etiology and Antibiotics Resistance Pattern of Community Acquired Urinary Infections in J N M C Hospital Aligarh India. Ann. Clin. Microbiol. Antimicrob. 2007; 6: 4.
  3. Coates A, Hu Y, Bax R, Page C. The future challenges facing the development of new antimicrobial drugs. Nat. Rev. Drug Discov. 2002; 1: 895-910.
  4. Braga L C, Leite A A M, Xavier K G S , Takahashi J A , Bemquer M P, Chartone-Souza E, Nascimento, A M A. Synergic interaction between pomegranate extracts and antibiotics against Staphylococcus aureus. Can. J. Microbiol. 2005; 51: 541-547.
  5. Shyamala, M. P., Venukumar, M.R. and Latha, M.S. Antioxidant potential of this Syzygium aromaticum(Gaertn.) Linn. (Cloves) in rats fed with high fat diet. Ind. J. Pharmacol.2003; 35: 99-103.
  6. Fu Y, Zu Y, Chen L, Shi X, Wang Z, Sun S. and Efferth T. Antimicrobial activity of clove and rosemary essential oils alone and in combination. Phytother. Res.2007; 21: 989-994.
  7. Kalemba D. and Kunicka A. Antibacterial and antifungal properties of essential oils. Current Medicinal Chemistry.2003; 10: 813-829.
  8. Kim H M, Lee E H, Hong S H, Song H J, Shin M K, Kim, S H and Shin T Y. Effect of Syzygium aromaticum extract on immediate hypersensitivity in rat. J. Ethnopharmacology.1998; 60: 125-131.
  9. Cai, L. and Wu, C.D. Compounds from Syzygiumaromaticum possessing growth inhibitory activity against oral pathogens, J. Natural Products.1996; 59: 987-990.
  10. Cressy H K, Jerrett A R, Osborne C M, Bremer P J. A novel method for the reduction of numbers of Listeria monocytogenes cells by freezing in combination with an essential oil in bacteriological media. J Food Protect.2003; 66:390–395.
  11.  Lee K G and Shibamoto T. Antioxidant property of aroma extract isolated from clove buds (Syzygium aromaticum(L.) Merr. Et Perry), Food Chemistry2002; 74: 443– 448.
  12. Peng Y. Yuan J, Liu F, Ye J. Determination of active components in rosemary by capillary electrophoresis with electrochemical detection. Journal of Pharmaceutical and Biomedical Analysis.2005; 39(3-4): 431-437.
  13. Oluwatuyi M, Kaatz G W, Gibbons S.  Antibacterial and resistance modifying activity of Rosmarinus officinalis. Phytochemistry.2004; 65(24): 3249-3254.
  14. Fernandez-Lopez J, Zhi N, Aleson-Carbonell  L, Perez-  Alvarez J A, Kuri  V. Antioxidant and antibacterial activities of natural extracts: application in beef meatballs. Meat Science. 2005; 69(3):  371-380.
  15. Rezzoug S A, Boutekedjiret C ,Allaf K. Optimization of operating conditions of rosemary essential oil extraction by a fast controlled pressure drop process using response surface methodology. Journal of Food Engineering, 2005; 71(1): 9-17.
  16. Leal P F, Braga M E M, Sato D N, Carvalho J E , Marques M OM., Meireles.M.A.A. Functional Properties of Spice Extracts Obtained via Supercritical Fluid Extraction. Journal of Agricultural and Food Chemistry.2003; 51(9): 2520-2525. 
  17. Harbone JB (1998). Essential oils, In: Phytochemical Methods: A guide to modern techniques in plant analysis, 3rd ed. Chapman & Hall, PA, USA. pp. 110-124.
  18. Okeke M I, Iroegbu C U, Eze E N, Okoli A S,  Esimone C O. Evaluation of extracts of the root of Landolphia owerrience for antibacterial activity. J. Ethnopharmacology.2001; 78: 119-127.
  19. Hammer K A, Carson C F, Riley T V. Antimicrobial activity of essential oils and other plant extracts. J. Applied Microbiology.1999; 86: 985-990.
  20. Thongson, C, Davidson, P M, Mahakarrchanakul, W, Weiss J. Antimicrobial activity of ultrasound – assisted solvent – extracted spices. Letters in Applied Microbiology.2004; 39: 401-406.
  21. Blatnik J, Lesnicar G. Propagation of methicillin-resistant Staphylococcus aureus due to the overloading of medical nurses in intensive care units. J Hosp Infect.2006; 63: 162-166.
  22. Lewis K, Ausubel F M. Prospects of plant derived antibacterials. Nat. Biotechnol.2006; 24:1504-1507.
  23. Ayoola G A, Lawore F M , Adelowotan  T , Aibinu I E, Adenipekun E, Coker H A B, Odugbemi T O. Chemical analysis and antimicrobial activity of the essential oil of Syzygium aromaticum (Clove). Afr. J. Microbiol. Res.2008; 2: 162-166.
  24. Saeed S. and Tariq P. In vitro antibacterial activity of clove against Gram negative bacteria. Pakistan J. Botany.2008; 40(5): 2157-2160.
  25. Gupta C, Garg A P , Uniyal R C. Antibacterial activity of Amchur (dried pulp of unripe Mangiferaindica) extracts on some food borne bacteria. J. Pharm. Res.2008; 1: 54-57.
  26. Namasombat S and Lohasupthawee P. Antibacterial activity of ethanolic extracts and essential oils of spices against Salmonella and other enterobacteria. KMITL,Sci. Tech.J. 2005;  5: 527-538.
  27. Kamal RaiAneja and Radhika Joshi. Antimicrobial Activity of Syzygium aromaticum and Its Bud Oil against Dental Cares Causing Microorganisms. Ethnobotanical Leaflets , 2010;14: 960-75.
  28. Del Campo J, Amiot M J, Nguyen C.The antimicrobial effect of rosemary   extracts. J Food Protect. 2000; 63:1359-1368.
  29. Sulieman A M E, Boshra I M O , El Khalifa E A A. Nutritive value of clove (Syzygium aromaticum) and detection of antimicrobial effect of its bud oil. Research J. Microbiology.2007; 2: 266-271.
  30. Suresh P, Ingle VK, Vijayalakshmi. Antibacterial activity of eugenol in   comparison with other antibiotics. J Food Sci Tech.1992; 29: 256–257.
  31. Viljoen A, Vuuren S V, Ernst E, Klepser M, Demirci B, Baser H, Wyk B E V. Osmitopsis asteriscoides (Asteraceae)- The antimicrobial and essential oil composition of Cape-Dutch remedy. J Ethnopharmacol.2003; 88:137–143.
  32. Purohit P, Bais RT, Singh P, Khan S. Assessment of antibacterial activity and phytochemical screening of Hemidesmus indicus root extracts. UK Journal of Pharmaceutical and Biosciences.2014; 2(6): 67-72.
  33. Gupta AK, Ahirwar NK, Shinde N, Choudhary M, Rajput YS, Singh A. Phytochemical Screening and Antimicrobial Assessment of Leaves of Adhatoda vasica, Azadirachta indica and Datura stramonium. UK Journal of Pharmaceutical  and Biosciences. 2013; 1(1): 42-47.