The Effect of Aqueous Extract and Essential Oils of the Leaves of Eucalyptus incrassate on β- Lactam Resistant Staphylococcus aureus Ehab D. Salman Dept. of Biotechnology/ College of Science / University of Baghdad. Abbas J. H. AL-Saedi Amel Gh. M. AL-Kazzaz Suhad S. Yahya Dept. of Biology/ College of Education for Pure Science (Ibn AL –Haitham) / University of Baghdad. Received in : 23 September 2013 , Accepted in : 19 February 2014 Abstract According to the extraction procedure , Eucalyptus incrassata leave sample yielded 5% and 2% w/w(Based on dry leaves ) of the aqueous extract and essential oils respectively. Disk diffusion method was used to determine the antimicrobial activity of aqueous extract and essential oils of E . incrassata leaves against eight isolates of multidrug- resistant of Staphylococcus aureus ( S. aureus) . It was found that aqueous extract and essential oils have variable antimicrobial activity(the inhibition zone diameter ranged from 7 to 14 mm respectively ) , while essential oils showed more effect than aqueous extract . It was noticed that values of Minimal Inhibitory Concentration ( MIC ) for aqueous extract is different from MIC of essential oils and these values were ranged from12.5 to 75mg/ml . It was also found that isolates collected from wound infections showed high resistance against aqueous extract and essential oils with MIC ranged from 50-75mg/ml, these variation in the MIC are probably due to some virulence factors which have a unique genetic determinant . The results indicated that the potential usefulness of the aqueous extract and essential oils of Eucalyptus incrassata as an antimicrobial agent can be used in the treatment of multidrug- resistant S. aureus infectious . Key words: Eucalyptus incrassate , Staphylococcus aureus , β- lactam , aqueous extract , essential oils . 76 | Biology @a@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹127@@ÖÜ»€a@I1@‚b«@H2014 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 27 (1) 2014 Introduction Staphylococcus aureus ( S. aureus) is a Gram positive spherical bacterium that colonizes mainly the nasal passages. It causes a serious infections in human such as pneumonia, mastitis, meningitis ,urinary tract infections, osteomyelitis and endocarditis. S. aureus is a major cause of hospital acquired (nosocomial) infection of surgical wound [1,2,3] . These infections can lead to substantial morbidity and mortality, as well as high healthcare costs[4]. This situation has been exacerbated by the rising incidence of strains that are less susceptible to a variety of antibiotics, making treatment of these infections more difficult [5,6]. Multidrug-resistant S. aureus cause major problem in clinical human healthcare . This problem is related to the increasing consumption and misuse of antibiotics [7]. It was reported in many studies that antibiotic abuse in treatment of S. aureus infection is the main cause of multidrug-resistant of this bacterium [6,7]. During the past four decades, resistance of S. aureus has continued to develop, with decreasing susceptibility to first-line agents such as penicillin, amoxicillin ,oxacillin , and this was followed by rapid spread appearance of methicillin resistant of S. aureus (MRSA) strains[4,8] .It was found that frequency resistance of S. aureus to β-lactam is increased worldwide, as strains of bacteria that produce β- lactomase have become more common[9,10] . These beta-lactamase enzymes make many, if not all, of the penicillins and cephalosporins ineffective as therapy[10,11] . As a result, for the reasons mentioned in the above , the necessitated need to search for new sources of antimicrobial agents, and among the potential sources of the new agents are the medicinal plants, which have been considered as alternative source for antimicrobial drugs[12]. The medicinal plants are known to contain substances that can be used for therapeutic purposes or as precursors for the synthesis of useful drugs [13]. One of the most important ethnomedicinal plant is Eucalyptus camaldulensis which belongs to the family of Myrtaceae. It is used as a remedy for sore throat and other bacterial infection of the respiratory and urinary tracts[3].It was reported in many studies [12,14] that the leaves of the Eucalyptus camaldulensis contain essential oil which is used as an antiseptic and have efficiency in killing bacteria. It was also found[14,15] that the phytochemical analysis of the crude extract of the leaves revealed the presence of saponin, saponin glycosides, steroid, cardiac glycoside, tannins, volatile oils, phenols and balsam (gum) , and this extract have the ability to inhibit the growth of S. aureus . The main objective of this study is to examine the effect of the crude extracts of the leaves of E. camaldulensis on β- lactam resistant S. aureus . Materials and Method Collection of plant materials The leaves of Eucalyptus incrassate (E. incraaaata ) was collected from the garden of College of Education for Pure Science / Ibn AL –Haitham and other region of Baghdad city in April, 2012. The plant was classified at the Department of Biology, College of Education for pure Science / Ibn Al -Haitham . Dry matter percentage was determined in the leaves . Preparation of plant materials and extract The green leaves were picked from stem and dried at 40 ˚C over a period of four days, the dried leaves were milled using an electric grinder and samples were taken to determine the dry matter percentage. The aqueous solution of leaves was prepared according to [16] and the extraction of essential oils from the leaves was performed by hydrodistillation in a Clevenger type apparatus and according to[17]. A stock of 10% (w/v) concentration for aqueous extract and essential oils were prepared according [16]. 77 | Biology @a@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹127@@ÖÜ»€a@I1@‚b«@H2014 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 27 (1) 2014 Bacterial isolates: Eight local isolates of S. aureus were marked (ST1-ST8) as shown in table( 1) . All isolates were maintained in screw-caped universal tubes containing nutrient agar(Acumedia,U.S.A.) and store at 4οC. Antibiotic Susceptibility Testing: Disk diffusion method was used as described by [18] Sterilized filter papers discs (6 mm diameter) with concentration 2.5mg/disc from each of aqueous extract and essential oils were used . Five antibiotic disks were used ( table 2) to detect antibiotic susceptibility of eight isolates of S. aureus . The plates cultures were observed after 18h of incubation at 35 οC . All tests were performed in duplicate and results recorded as mean values. The zones of growth inhibition were measured in mm and interpreted as sensitive or intermediate or resistant according to[19]. Minimal inhibitory concentration (MIC) test: The MIC of aqueous extract and essential oils against eight resistant of S.aureus were determined by an agar dilution method according to[20] .The final concentration for each of aqueous extract and essential oils in the medium were 12.5,25 ,50,75 and 100 mg/ml .The plates cultures were incubated at 35 οC for 18 h and the MIC was defined as the lowest concentration for each of aqueous extract and essential oil inhibiting the visible growth. All determinations were performed in duplicate and results recorded as mean values. Growth control consisting of Muller Hinton Agar was included. Results and Discussion A re-test was carried out by using mannitol salt ager as a selective and differential medium for Staphylococcus aureus . It was found that all isolates, which were used in this study displayed a positive reaction and that mean that the mannitol positive isolates were Staphylococcus aureus [21 ].Another test was conducted to confirm a susceptibility of isolates under investigation toward five types of antibiotics which belong to β- lactam group .The result showed that all isolates displayed multidrug-resistant toward these types of antibiotics (table 3) . Extraction procedures yielded 5% and 2% w/w(Based on dry leaves ) of the aqueous extract and essential oils respectively, the color of the aqueous extract was brown and have a sticky texture with unique smell. The antimicrobial activity of the aqueous extract and essential oils was assayed against eight isolates of S. aureus . It was found that aqueous extract and essential oils have different antimicrobial activity (the values of inhibition zone range from 7- 14 mm) on multidrug- resistant S. aureus isolates (table 4 ) . These differences in the level of resistance are probably due to several factors, including isolation source, genetic determinants and some virulence factor like biofilm formation [ 22,23,24]. The results of MIC for aqueous extract and essential oils of E . incrassata leaves are listed in table( 5). These results revealed that MIC for essential oils was less than aqueous extract which means that all isolates were more sensitive toward essential oils compared with aqueous extract. The variation in antimicrobial activity of aqueous extract and essential oils may be due to several factors including chemical constituents of the extract, drying method of the plant material, and extraction procedures(25,26,27). It was noticed that the isolates ST5 and ST6, which were isolated from wounds infection displayed more resistance to aqueous extract and essential oils of E . incrassata leaves compared with other isolates, where the values of MIC for these isolates ranged from 50-75mg/ml, these variations in the MIC are probably due to some virulence factors and unique genetic determinants [22, 24]. In conclusion, the aqueous extract and essential oils obtained from E . incrassata leaves have potential as antimicrobial agents in the treatment of infectious multidrug-resistant S. aureus . Further investigations of the active components of the plant for the exact mechanism of action and synergistic interaction of plant extracts with some type of antibiotics ,will contribute to the development of new pharmaceuticals. 78 | Biology @a@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹127@@ÖÜ»€a@I1@‚b«@H2014 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 27 (1) 2014 References 1. Oliveira, D.; Thomasz, A. and deLencastre, H. (2001). The evolution of pandemic clones of methicillin resistant Staphylococcus aureus: Identification of two ancestral genetic backgrounds and the associated mec elements. Microbial Drug Resistance. 7:49-61. 2. Robinson, D. A. and Enright, M. C. (2003). Evolutionary models of the emergence of methicillin-resistant Staphylococcus aureus. Antimicrobial Agents Chemotherapy 47: 3926-3934. 3. Musa, D. A.; Nwodo, F. O.C. and Ojogbane, E.(2011). Phytochemical, antibacterial and toxicity studies of the aqueous extract of Euclayptus camaldulensis Dehnh. Asian Journal of Plant Science and Research. 1 (3) : 1-10 4. Diekema DJ; Pfaller MA and Schmitz FJ.( 2001). Survey of infections due to Staphylococcus species: frequency of occurrence and antimicrobial susceptibility of isolates collected in the United States, Canada, Latin America, Europe, and the Western Pacific region for the SENTRY Antimicrobial Surveillance Program, 1997–1999. Clin Infect Dis. 32(2): 114–132. 5. Khan, F., Shukla, I. and Rivzi, M.(2011). The role of non-β-lactam antimicrobials and screening for vancomycin resistance in methicillin resistant Staphylococcus aureus. Mal. J. of Microbio.. 7(2). 66-70. 6. Layton, M. C.; Hierholzer, W. J. and Patterson, J. E. (1995). The evolving epidemiology of methicillin resisitant Staphylococcus aureus at a university hospital. Infection Control and Hospital Epidemiology. 16: 12-17. 7. World Health Organization. Global strategy for containment of antimicrobial resistance. http://www.who.int/ rugresistance/en/WHO_Global_Strategy_English.pdf. 8. Woodford N.(2005). Biological counterstrike: antibiotic resistance mechanisms of Gram- positive cocci. Clin Microbiol Infect. 11(3): 2–21. 9. Lowy, FD.(2003) Antimicrobial resistance: the example of Staphylococcus aureus. J. Clin. Invest. 111: 1265–1273. 10. Onanuga, A.; Oyi, A. R., Olayinka, B.O. and Onaolapo, J. A.(2005). Prevalence of community-associated multi-resistant Staphylococcus aureus among healthy women in Abuja, Nigeria. Afr. J. Biotechnol.4(9):942-945. 11. Olayinka, BO.; Olonitola, OS; Olayinka, AT. and Raji, B. (2004). Antibiotic susceptibility pattern and multiple antibiotic resistance index of Staphylococcus aureus isolates in Zaria, Nigeria. J. Trop. Biosci. 4: 51-54. 12. Ghalem, B. R. and Mohamed B. (2008). Antibacterial activity of leaf essential oils of Eucalyptusglobulus and Eucalyptus camaldulensis. African Journal of Pharmacy and Pharmacology.2(10):211-215. 13. Cowan, MM. (1999). Plant products as antimicrobial agents. Clin.Microbiol. Rev. 12: 564-582 . 14. Mehani, M, and Ladjel, S.(2012). Antimicrobial effect of essential oils of the plant Eucalyptus camaldulensis on some pathogenic bacteria. International Journal of Environmental Science and Development.3(2):86-88. 15. Jouk,M. and Kazaei,N.(2010). The Antimicrobial activities of methanolic extracts of Eucalyptus camaldulensis against Bacillus subtilis, Staphylococcus aureus and Escherichia coli . Journal of Research in Agricultural Science . 6: 63- 67. 16. AL-Sammaraiae,T.S.M.(2011).Evaluation of the aqueous and alcoholic extract and essential oil of the leaves of Euclayptus camaldulensis toward some biological properties of Saprolegnia hypogyna and Saprolegnia ferax .MSc.Thesis. Baghdad University- College of Education Ibn AL- Haitham. Baghdad-Iraq.(In Arabic). 17. Bankole, S. A. and Joda, A.O. (2004) . Effect of lemon grass( Cypbopogon citrates staff ) powder and essential oil on mould deterioration and aflatoxin contamination of melon seed ( Colocynthis citrullus L.). African Journal of Biotechnology.3(1):52-59. 79 | Biology @a@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹127@@ÖÜ»€a@I1@‚b«@H2014 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 27 (1) 2014 18. Bauer, A. W.; Kirby, W. M. M; Sherris, J. C.; and Turck, M. (1966) .Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45(4): 493- 496. 19. National Committee for Clinical Laboratory Standards (NCCLS).(2002). Performance Standards for Antimicrobial Susceptibility Testing; Twelfth Informational Supplement. Vol. 22(1). 20. Morello, J.A.; Mizer, H.E. and Granato, P.A. (2006) .Laboratory Manual and Workbook in Microbiology. 8th ed. McGraw – Hill Companies, New York. 21. Gerhardt, P.; Murray, R. G. E.; Wood, W. A. and Krieg , N. R.( 1981). Methods for general and molecular bacteriology. ASM Press, Washington, D.C. 22. Frana, T.S.; Beahm, A.R.; Hanson, B.M.; Kinyon, J.M.; Layman, L.L.; Karriker, L., A., Ramirez, A., and Smith, T. C. (2013). Isolation and characterization of methicillin-resistant Staphylococcus aureus from pork farms and visiting veterinary students. PLoS ONE 8(1): e53738. 23. Lowy F.D.( 2003 ) Antimicrobial resistance: the example of Staphylococcus aureus. J. Clin. Invest. 111: 1265–1273. 24. Otto, M.(2008).Staphylococcal Biofilms. Curr Top Microbiol Immunol. 322: 207–228. 25. Coppen, J. W. (2002). Eucalyptus the Genus Eucalyptus, Taylor and Francis Inc., London and New York. 26. Suhr, K. I. and Nielsen, P. V. (2003). Antifungal activity of essential oils evaluated by two different application techniques against rye bread spoilage fungi, J. Appl. Microbiol. 94: 665-674. 27. Siramon, p.; Ohtani,Y. and Ichiura, H.(2013). Chemical composition and antifungal property of Eucalyptus camaldulensis leaf oils from Thailand. Rec. Nat. Prod. 7(1) : 49-53. Table No. ( 1) . Staphylococcus aureus isolates and Sources Bacterial Isolates Sources and Characteristics Reference ST1 ST2 ST3 ST4 ST5 ST6 ST7 ST8 Isolated from sputum patients. Resistance to penicillin G , Oxacillin, Cephalothin. Isolated from wounds infection. Resistance to penicillin G, Oxacillin, Cephalothin. Isolated from gastrointestinal disorder patients. Resistance to penicillin G , Oxacillin, Cephalothin. Biotechnology Department-College of Science – Baghdad University. Table No. ( 2). Types of Antibiotics disks and sources of production Antibiotic Disk Symbol Disk Content / di k Company (O i i ) Penicillin G PG 10 BioAnalyse (Turke) Ampicillin AM 10 Oxacillin OX 1 Cephalothin KF 30 Cefotaxime CTX 30 80 | Biology @a@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹127@@ÖÜ»€a@I1@‚b«@H2014 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 27 (1) 2014 Table No. ( 3). Antibiogram results of S. aureus isolates Antibiotic * Inhibition zone diameter( mm ) ST1 ST2 ST3 ST4 ST5 ST6 ST7 ST8 Penicillin G Ampicillin Oxacillin Cephalothin Cefatoxime 15 10 7 11 13 17 8 8 10 11 14 8 7 11 11 17 10 7 10 12 10 7 7 9 10 12 8 6 8 8 18 10 9 12 12 16 10 8 10 11 * According to [20], the inhibition zone diameter for all isolates revealed that these isolates are resistant toward all these type of antibiotics. Table No. ( 4). Disc diffusion test for aqueous solution and essential oils of E . incrassata leaves. Extracts Inhibition zone diameter( mm ) ST1 ST2 ST3 ST4 ST5 ST6 ST7 ST8 Aqueous extract 12 11 12 12 8 7 10 11 Essential oils 14 12 13 14 9 8 11 11 Table No. (5). Minimum inhibitory concentration (MIC) of S. aureus for aqueous extract and essential oils of E . incrassata leaves. Extracts MIC ( mg/ml ) ST1 ST2 ST3 ST4 ST5 ST6 ST7 ST8 Aqueous extract 25.00 50.00 25.00 25.00 75.00 75.00 50.00 50.00 Essential oils 12.500 25.00 12.50 12.5 50.00 75.00 25.00 25.00 81 | Biology @a@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹127@@ÖÜ»€a@I1@‚b«@H2014 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 27 (1) 2014 Eucalyptus الطیارة ألوراق الیوكالبتوستأثیر المستخلص المائي والزیوت incrassate الذھبیة ةالمكورات العنقودی فيStaphylococcus aureus لمجموعة مضادات بیتا الكتام المتعددة المقاومة ایھاب داود سلمان قسم التقنیات األحیائیة / كلیة العلوم / جامعة بغداد. باس جاسم حسین الساعديع زازالق محمودأمل غانم سھاد سعد یحیى جامعة بغداد / )بن الھیثمإ( لعلوم الصرفةالتربیة لكلیة / قسم علوم الحیاة 19/2/2014، قبل البحث في : 23/9/2013استلم البحث في : الخالصة لألوراق) لكل منس المادة الجافة % ( على أسا2، 5أعتمادا على طریقة األستخالص ، تم الحصول على . Eucalyptus incrassataالمستخلص المائي والزیوت الطیارة من أوراق نبات الیوكالبتوس ثمانیة عزل خلص المائي والزیوت الطیارة أتجاه أستعملت طریقة األقراص المنتشرة للتحري عن الفاعلیة المیكروبیة للمست كورا ت العنقودیات الذھبیة ذات المقاومة المتعددة لمجموعة البیتا الكتام . لقد وجد أن لكل من المستخلص المائي ممن ال ملم ) ، وقد أظھرت الزیوت الطیارة أكثر فعالیة 14 -7( تراوح قطر التثبیط من جرثومي مختلف تأثیر والزیوت الطیارة من المستخلص المائي . 75 -12.5قیم التراوحت ھذه ذ، إم التركیز المثبط األدنى لكل من الزیوت الطیارة والمستخلص المائي لوحظ تباین قی كما الجروح لكل منھما على التوالي . كما أظھرت النتائج المقاومة العالیة لعزالت البكتریا المعزولة من التھابات ملیلترملغم/ لكل منھما یلترملغم /مل 75- 50تراوحت قیمة التركیز المثبط األدنى تجاه كل من الزیوت الطیارة والمستخلص المائي ، وإ وعلى التوالي . ستفادة من المستخلص المائي والزیوت الطیارة ألوراق الیوكالبتوس لعالج األصابات یر النتائج أعاله الى أمكانیة اإلتش الذھبیة . ةكورات العنقودیمالناتجة من بكتریا ال م ،المستخلص المائي ، الزیوت االمفتاحیة : الیوكالبتوس ، المكورات العنقودیة الذھبیة ، مجموعة البیتا الكتالكلمات الطیارة 82 | Biology @a@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹127@@ÖÜ»€a@I1@‚b«@H2014 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 27 (1) 2014