Synergistic Effect Between Some Antimicrobial Agents and Rosemary (Rosmarinus Officinalis) Toward Staphylococcus Aureus

BENHA VETERINARY MEDICAL JOURNAL, VOL. 28, NO. 2:195‐201, JUNE 2015

Synergistic effect between some antimicrobial agents and rosemary (rosmarinus officinalis) toward staphylococcus aureus – in-vitro 1Ashraf, A. Abd-El Tawab; 1Fatma, I. El-Hofy; 2Elham, A. Mobarez; 2Hesham, S. Taha and 3Nancy, Y. Tawkol 1Bacteriology, Immunology and Mycology Dep., Faculty of Vet. Med., Benha Uni., 2Animal Health Research Institute Dokki, Giza.3Veterinarian

A B S T R A C T

The present study was carried out to evaluate the antimicrobial activity of ethanol plant extract of rosemary (Rosmarinus officinalis) and / with five antimicrobial agents of different mechanisms (oxytetracycline Hcl, amoxicillin, cefquinome, sulphaquinoxaline and danofloxacin) against field strain of S. aureus. By using agar well diffusion method, the mean zone of inhibition (mm) of ethanol extract of Rosemary were 21.67±0.33, 20.33±0.33, 19.67±0.17, 18.83±0.17, 18.17±0.17, 16.33±0.33 and 11.5±0.29 mm at different concentrations. While the mean zone of inhibition (mm) of different antimicrobial agents at different concentrations for amoxacillin was 24±0.29, 20.67±0.33, 17.67±0.33 and 14.17±0.17mm and for cefquinome was 20.83±0.17, 18±0.29 and 13.5±0.29 mm. no inhibition zones were detected with danofloxacin, sulphaquinoxalline and oxytetracycline Hcl. By macrodilution method, the MIC of rosemary was 0.048mg/ml and the MICs for the aforementioned antimicrobial agents were 0.015, 0.25, 1.0, 8.0 and 16 µg/ml respectively. The synergistic effects were recorded by FIC index between 0.006 and 0.00038 mg/ml of rosemary with 0.125 and 4.0 µg/ml of cefquinome and sulphaquinoxalline respectively. Keywords: Rosemary, Antimicrobial agents, MICs, S.aureus

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1. INTRODUCTION

The incorrect and overuse of existing biological activities. Accordingly, antimicrobials is becoming a formidable secondary products have both a defensive threat in the fight against disease due to the role against herbivory, pathogen attack, emergence of multi-drug resistant strains. and inter-plant competition. They also Thus, alternatives to the standard have an attractant role toward beneficial treatments with single agents become organisms such as pollinators or symbionts necessary. In clinical practice, (Briskin, 2000 and Mhanna and Adwan, combination antibiotic therapy is being 2008). Combinations of antimicrobials that used in an attempt to broaden the bacterial demonstrate an in vitro synergism against spectrum and to avoid the emergence of infecting strains are more likely to result in resistant strains (Lambert, 2000 and van successful therapeutic protocols. Thus, Vuuren et al., 2009). Furthermore, to avoid evidence of in vitro synergism could be the undesirable toxic effects of useful in selecting most favorable antimicrobial therapy, the combination combinations of antimicrobials for the with herbal products may be an innovative practical therapy of serious bacterial alternative to prescriptive treatment infections (Hooton et al., 1984 and protocols (Harris, 2002). Higher plants Aiyegoro and Okoh, 2009). Drug produce hundreds to thousands of diverse synergism between known antimicrobial chemical compounds with different agents and bioactive plant extracts is a

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Abd-El Tawab et al. (2015) novel concept and has been recently at 4°C until use, the resulted deposit was reported (Nascimento et al., 2000, Aqil et dissolved in distilled water to prepare the al., 2005, Betoni et al., 2006 and Mhanna different concentrations. (Parish and and Adwan, 2008). The current study was Davidson, 1993). planned to assess the antimicrobial activity 2.3.Media used of ethanol extract of rosemary and some antimicrobials agents such as Mullar Hinton agar and broth (Himedia) oxytetracycline HCl; amoxicillin; were used for evaluation of antibacterial cephquinome; sulphaquinoxaline sodium activity by agar well diffusion method and danofloxacin. Both agar gel diffusion (NCCLS, 1993) and determination of MICs method against S.aureus strain and by macrodilution method (Hindler, 1995) estimation of MICs by using macrodilution respectively. method were applied to evaluate in-vitro 2.4.Antimicrobial agents interaction between the used plant extract and antimicrobial agents. Different antimicrobial agents as amoxicillin, sulphaquinoxaline and 2. MATERIAL AND METHODS oxytetracycline HCl (El–Nasr Pharma- ceutical Chemical Co.,), cefquinome 2.1. Bacterial strain (Internet International GmbH) and The examined strain of coagulase positive danofloxacin (Adwia). S.aureus from rabbits was provided from 2.5. FIC index calculation Microbiology Dep., Faculty of Veterinary Medicine, Benha University. The bacterial Antimicrobial agents were used at suspension was adjusted to be the proper concentration (1/2 MIC) in combination of density 1.5X106 bacteria / ml. (Golshani rosemary. Type of interaction between plant and Sharifzadeh, 2013). extract of rosemary and antibiotics was determined through calculation of FIC 2.2. Plant sample Index. The FIC (Fractional Inhibitory 2.2.1. Rosmary Concentration) value for each agent was calculated using the formula (El-Kalek and Rosemary (Rosmarinus officinalis) is a Mohamed 2012). FIC (antibiotic) = MIC of woody, perennial herb with fragrant antibiotic in combination / MIC of evergreen needle-like leaves. It is a member antibiotic alone. FIC (extract) = MIC of of the mint Family: Lamiaceae, Genus: extract in combination / MIC of extract Rosemarinus. The leaves are 2-4 cm long alone. FIC Index = FIC (antibiotic) + FIC and 2-5 mm broad, green above, and white (extract). Synergistic: if the FIC indices below with dense short woolly hairs. The were <1. Additive: if the FIC indices were flowers are variable in color, being white, = 1. Indifferent: if the FIC indices were pink, purple, or blue (Fritzweiss and between 1 and 2. Antagonistic: if the FIC Fintelmann, 2000 and Mhanna and Adwan, indices were >2 2008). 2.2.2. Plant Extract preparation 3. RESULTS Ten grams of dried ground plant material 3.1. Antibacterial activity of ethanol was added to 500 ml conical flasks plus150 extract of rosemary ml of solvent (ethanol 99.9%). Ratio (1 : 15) Agar well diffusion method was used to of the plant material and solvent with slow determine the mean inhibition zones of mixing , the extract solution was filtered the Rosemary which were 21.67±0.33, solvent was removed using rotary 20.33±0.33, 19.67±0.17, 18.83±0.17, evaporator at 40 °C to obtain the crude 18.17±0.17, 16.33±0.33, 11.5±0.29 and extract and then it was kept in sterile bottles zero mm at different concentrations

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(100,50, 25,12.5, 6.25, 3.125, 1.56 and 3.3. Determination of MIC of rosemary 0.78) against field isolate of S.aureus ethanol extract respectively ( Table, 1). The result of MIC of rosemary ethanol 3.2. Determination of antibacterial extract was 0.048 mg/ml by using activity of antimicrobial agents macrodilution method. Mean zones of inhibition of five 3.4. Determination of MICs of Antimicrobial agents (amoxicillin, antimicrobial agents cefquinome, danofloxacin, sulpha- The MICs of antibacterial agents were quinoxaline and oxytetracycline) were 0.015, 0.25, 1.0, 8.0 and 16.0µg/ml for the determined against field isolate of S.aureus different antimicrobial agents (amoxicillin, using agar well diffusion method. cefquinome, danofloxacin, amoxicillin and cefquinome showed high sulphquinoxaline and oxytetracycline inhibitory effect but danofloxacin, respectively). sulphaquinoxaline and oxytetracycline

HCL showed no inhibitory effect (Table 2).

Table (1): Inhibition zones (mm) of rosemary ethanol extract.

Zones of inhibition(mm) rosemary Experiment Experiment concentrations(mg/ml) Experiment 1 Mean ± *SE 2 3 100 22 21 22 21.67±0.33 50 20 20 21 20.33±0.33 25 19.5 19.5 20 19.67±0.17 12.5 19 18.5 19 18.83±0.17 6.25 18.5 18 18 18.17±0.17 3.125 16 17 16 16.33±0.33 1.56 12 11.5 11 11.5 ±0.29 0.78 *- *- *- *-

*- = no inhibition zone. *SE = Standard Error.

Table (2): Inhibition zones (mm) of Amoxicillin and Cefquinome.

Zones of inhibition (mm) Conc. Amoxacillin Cefquinome µg/ml Mean Exp. 1 Exp. 2 Exp. 3 Mean± SD Exp. 1 Exp. 2 Exp. 3 ± SD 8.0 24 23.5 24.5 24 ±0.29 21 20.5 21 20.83±0.17 4.0 21 20 21 20.67±0.33 17.5 18 18.5 18 ±0.29 2.0 17 18 18 17.67±0.33 13.5 14 13 13.5 ±0.29 1.0 14 14.5 14 14.17±0.17 *- *- *- *- 0.5 *- *- *- *- *- *- *- *- 0.25 *- *- *- *- *- *- *- *- 0.125 *- *- *- *- *- *- *- *- 0.0625 *- *- *- *- *- *- *- *- *- = no inhibition zone. *SE = Standard Error.

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Table (3): Types of interactions between rosemary and different antimicrobial agents

Antimicrobial agent FIC of rosemary FIC index Type of Interaction amoxicillin 2.0 2.5 Antagonism cefquinome 0.125 0.625 Synergism danofloxacin 2.0 2.5 Antagonism sulphaquinoxaline 0.008 0.508 Synergism oxytetracycline 2.0 2.5 Antagonism

3.5.Determination of synergistic effect using macrodilution method was 0.015 between rosemary and antimicrobial µg/ml. These results were inaccordance agents. with Idamokoro et al. (2013) who stated The synergistic interaction was determined that amoxicillin (0.01 µg/ml) against between rosemary and cefquinome and S.aureus showed inhibition zone of 23 ±1.5mm using agar well diffusion method between rosemary and sulphquinoxaline, -4 but rosemary showed antagonistic and MIC of 6.25x10 mg/ml using the interaction with amoxacillin, danofloxacin broth microdilution method. Similar and oxytetracycline (Table, 3). findings were recorded by Palaksha et al., (2013) who claimed that the mean inhibitory zone was 14.5±0.2887 mg/ml 4. DISCUSSION and Nnamani et al., (2012) who added that MIC of amoxicillin against S.aureus was The current results indicated that ethanol 0.025±0.002 mg/ml. On the other hand extract of rosemary leaves with Ghannadi et al., (2012) stated that concentrations between 1.56 - 100 mg/ml amoxicillin (25 µg/disc) against S.aureus has the ability to inhibit the growth of using disc diffusion method achieved S.aureus on Muller Hinton agar with inhibition zone 39.5±0.1 mm. Nazima et al., inhibition zones from 11.5±0.29 – (2010) recorded no inhibition against S. 21.67±0.33 mm. These results were in aureus using disc diffusion method for accordance with those recorded by Rasheed amoxacillin. The results indicated that et al., (2010), Al Laham and Al Fadel cefquinome had inhibitory effect on Muller (2013) and Golshani and Sharifzadeh Hinton agar medium against S.aureus at (2013). The MIC for rosemary in this study concentrations 2.0-8.0 µg/ml with was reported as 0.048 mg/ml while Rasheed inhibition zone between 13.5±0.29– et al., (2010) and Golshani and Sharifzadeh 20.83±0.17 mm. This was in agreement (2013) reported MIC for rosemary as 0.5 with Al-Khafaji (2004) who recorded the mg/ml and 6.25 mg/ml respectively. The inhibitory zone as 19.2 mm. concentrations wide difference between the of 0.0625 – 1.0 µg/ml showed no inhibitory aforementioned results might be attributed effect on S.aureus. Minimum Inhibitory to the method of extraction and the use of Concentration (MIC) of cefquinome using relatively higher temperature, which macrodilution method was 0.25 µg/ml. decrease the rate of destruction of effective Quan-chao et al., (2009) stated that MIC90 herbal compound. The present study of cefquinome on S. aureus using the revealed that amoxicillin had inhibitory microdilution method was 0.125 µg/ml. The effect on Muller Hinton agar medium results indicated that danofloxacin, against S.aureus at concentrations 1.0– sulphaquinoxaline and oxytetracycline had 8.0µg/ml with inhibition zone between no inhibitory effect on Muller Hinton agar 14.17±0.17- 24±0.29 mm. Other medium against S.aureus at concentrations concentrations which ranged between of 0.0625 – 8.0µg/ml. MIC of danofloxacin, 0.0625 - 0.5 µg/ml showed no inhibitory sulphaquinoxaline and oxytetracycline effect on S.aureus. MIC of amoxicillin

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Synergistic effect between some antimicrobial agents and rosemary using macrodilution method were 1.0,8.0 when combination of rosemary essential oil and16.0µg/ml respectively. Mhanna and and gentamycin antibiotic discs (10µg) Adwan (2008) who studied the antibacterial were applied. While there was no effect of activity of antibiotics at concentration 100 rosemary with erythromycin (15µg). An µg/ml against 4 strains of S.aureus using increase in antibacterial activity of agar well diffusion method and minimum rosemary ethanol extract when combined inhibitory concentration using micro with gentamycin or tetracycline was dilution method. The inhibition zones were reported by Thorria (2013). In contrast a 16.6-28.3, 27-30 and 8.3-9.3mm for decrease in its antibacterial activity when oxytetracycline HCl, enrofloxacin and combined with ampicillin but no difference sulphadimethoxine sodium respectively. with amoxicillin against S.aureus. There MICs were 0.78-6.25, 0.39-0.195 and >100 was an increase in inhibition zone in its mg/ml respectively. Moreover, Adeshina et combination with cephalothin by using disc al.,(2011) studied the antibiotic sensitivity diffusion method. pattern of S.aureus to ofloxacin (30 µg) and tetracycline (30 µg). They stated that 5. CONCLUSION S.aureus was sensitive to ofloxacin and resistant to tetracycline. The present results Ethanol plant extract of rosemary has indicated that cefquinome and antimicrobial activity against local strain of sulphaquinoxaline showed synergism with S. aureus and has a synergistic effect with rosemary ethanol extract against S.aureus. cefquinome and sulphquinoxalline. These But showed antagonism with amoxicillin, results signify that rosemary extract danofloxacin and oxytetracycline. These potentiates the antimicrobial action of results were in agreement with Mhanna and antibiotics, suggesting a possible utilization Adwan (2008) who evaluated the of this herb in combination therapy against interaction between water extract of staphylococcal infections in vivo. rosemary and known antimicrobial agents of different mechanisms (oxytetracycline 6. REFERENCES HCl, gentamicin sulfate, penicillin G, cephalexin; sulfadimethoxine sodium and Adeshina, G.O., Jibo, S., Agu, V.E., enrofloxacin) using microdilution method Ehinmidu, J.O. 2011. Antibacterial against five S.aureus isolates resmbled activity of fresh juices of Allium cepa synergistic effects. On the other, hand van and Zingiber officinale against Vuuren et al., (2009) reported that multidrug resistant bacteria. interaction of the essential oils (Mentha International Journal of Pharma. and piperita and Rosmarinus officinalis) with BioSciences, 2(2):289-295. ciprofloxacin against S.aureus indicated Aiyegoro, O. A., Okoh, A. I. 2009. Use of mainly antagonistic profiles. The current bioactive plant products in combination results were in accordance with Jarrar et al., with standard antibiotics: Implications (2010) who recorded that there was in antimicrobial chemotherapy. Journal synergistic effect between ethanol extract of of Medicinal Plants Research, rosemary and cefuroxime against 3(13):1147-1152. methicillin-resistant Staphylococcus aureus Al-Khafaji, B.A. 2004. In vitro evaluation and Toroglu (2011) who recorded of some antibacterial drugs against antagonism for the combination of Staphylococcus aureus isolated from Rosemary essential oil and gentamycin recurrent boil. Medical Journal of antibiotic discs (10 µg), but synergism Babylon, 1 (3 & 4):308-311. with cephalothin. Shaaban et al., (2013) Al Laham, S.H., Al Fadel, F. 2013. stated that a synergistic effect was seen in Antibacterial effectiveness of many S.aureus by using disc diffusion method plants extracts against the resistant

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