Detection of anti-bacterial activity of Medicinal plant Quercus infectoria against MRSA isolates in clinical samples

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1 Available online at Research article JPBMS ISSN NO CODEN JPBSCT JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL SCIENCES Detection of anti-bacterial activity of Medicinal plant Quercus infectoria against MRSA in clinical samples *G. Sucilathangam 1, S. Nithya Gomatheswari 2, G. Velvizhi 3, C. Pauline Vincent 4, N. Palaniappan 5. 1 M. D., Tirunelveli Medical College, Tirunelveli , Tamil Nadu, India. 2 M. D., Chengalpet Medical College, Chengalpet, Tamil Nadu, India. 3 M. D., Tirunelveli Medical College, Tirunelveli , Tamil Nadu, India. 4 M. D., (S), Govt. Siddha College, Tirunelveli , Tamil Nadu, India. 5M. D., Tirunelveli Medical College, Tirunelveli , Tamil Nadu, India. Abstract: Background:- Methicillin-resistant Staphylococcus aureus (MRSA) has been well-documented as a major cause of hospital infections. The aim of the present work was to evaluate the antibacterial potential of aqueous and ethanol s of nutgalls of Quercus infectoria by disc diffusion method, determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values. Materials and Methods: A total of 103 clinical of Staphylococcus aureus were collected from Tirunelveli Medical College Hospital. The isolated strains of S. aureus were subjected to the following phenotypic methods viz., Oxacillin screen agar and Cefoxitin disc diffusion test to screen and confirm MRSA. The nutgalls of Quercus infectoria were ed with 95% ethanol and water. The antibacterial activity of aqueous and ethanol of Quercus infectoria was determined against Staphylococcus aureus 25923, MRSA and clinical by disc diffusion method, MIC and MBC. The MIC of the s were then determined using the twofold serial micro dilution technique at a concentration ranging from 50 to 0.4. The MBC values were finally obtained from the MIC micro titre wells which showed no turbidity after 24 hrs of incubation by sub culturing method. Results: Ethanolic and aqueous s of Quercus infectoria demonstrated significant antibacterial activities against all strains of methicillin-resistant Staphylococcus aureus (MRSA). Inhibition zones of ethanolic and aqueous s of Quercus infectoria for clinical of MRSA strains were in the range mm. Staphylococcus aureus 25923, MRSA demonstrated similar results. The MIC values of aqueous for Staphylococcus aureus 25923, MRSA and clinical of MRSA strains were , and respectively and minimum bactericidal concentration (MBC) values were 3.2, 6.3 and 6.3, respectively. The MIC values of ethanolic for Staphylococcus aureus 25923, MRSA and clinical of MRSA strains were , and respectively and MBC values were , 0.4 and respectively. Conclusion: The present study demonstrated that the ethanolic of Quercus infectoria had significant antibacterial activities against all strains of Methicillin Resistant Staphylococcus aureus (MRSA). This finding provides an insight into the usage of the nutgalls of Q. infectoria in traditional treatment of wounds or burns associated with MRSA infections. Key-words: Medicinal plant - Quercus infectoria - Methicillin Resistant Staphylococcus aureus- Minimum Inhibitory Concentration- Minimum Bactericidal Concentration-Aqueous -Ethanolic MRSA infections are limited to very few and expensive drugs like Teicoplanin, Vancomycin and Linezolid. [2] Thus, control of MRSA is essential to curtail the introduction and spread of infection. The continuing emergence and development of resistance to existing antibacterial agents by bacteria has created the need for new antibacterial compounds that exhibit activity against these resistant strains. [3,4] Introduction: Nosocomial infections account for morbidity and mortality of millions of patients annually, worldwide. Staphylococcus aureus especially Methicillin Resistant Staphylococcus aureus (MRSA) is relatively ubiquitous and is the cause of many community, endemic and epidemic nosocomial colonization and infections. MRSA is of concern not only because of its resistance to Methicillin but also because it is generally resistant to many other chemotherapeutic agents. Since, MRSA strains are also resistant to multiple antibiotics there is possibility of extensive outbreaks, which may be difficult to conclude. [1] Accurate detection of MRSA is an important prerequisite for appropriate therapy and epidemiological assessment of nosocomial infections caused by this strains. Currently, the treatment options for Natural products and their derivatives have continued to be the most significant sources of new leads into the development of new pharmaceutical agents. Quercus infectoria Olivier (Fagaceae) is a small tree native of Greece, Asia Minor and Iran. The galls arise on young branches of this tree as a result of attack by the gall-wasp Adleria gallae-tinctoria.[5] The galls are locally known as 1 Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 14, Issue 14

2 Machika or Oak galls. The galls of Q. infectoria have also been pharmacologically documented to possess astringent, antidiabetic, antitremorine, local anaesthetic, antiviral, antibacterial, antifungal, larvicidal and anti-inflammatory activities. [6-12] The main constituents found in the galls of Q. infectoria are tannin (50-70%) and small amount of free gallic acid and ellagic acid. [13-15] Some work has also clearly indicated that various s from the nutgalls exhibited high activity against MRSA. [16] The antibiotic resistance mechanism and the prevalence of MRSA from clinical samples are essential because of its public health importance and the threat posed by MRSA infection. Methicillin resistant is useful marker in selecting appropriate antimicrobial agents for treatment of infection caused by S. aureus and to detect the changing pattern of resistance of S. aureus. Timely detection of methicillin resistant strain will help in prevention of hospital-acquired infections. Hence, the present study was carried out to study the prevalence and sensitivity pattern of MRSA in clinical samples in a tertiary care hospital and to evaluate the antibacterial potential of aqueous and ethanol s of galls of Quercus infectoria by disc diffusion method, determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values. Materials and Methods: Study subjects: After obtaining Institutional Ethical Committees approval, the clinical specimens like urine, pus. blood and sputum were inoculated on three different media namely Nutrient agar, Blood agar and Mannitol Salt agar. The isolated S. aureus from clinical specimens were subjected to morphological and biochemical studies such as microscopic examination, Gram s staining, motility, catalase, slide and tube coagulase, oxidase, indole, methyl red, Voges-Proskauer, urease, citrate utilization test to confirm the identification by using standard methods Then the of S. aureus were subjected to antibiotic susceptibility test by adopting Kirby-Bauer disc diffusion method with standard antibiotic discs like Ampicillin (10 µg), Amikacin, Ciprofloxacin (5 µg), Erythromycin (15 µg), Clindamycin (2 µg), Gentamicin (10 µg), Oxacillin (1 µg), and Vancomycin (30 µg). MRSA detection: The isolated strains of S. aureus were subjected to the following phenotypic methods to screen and confirm MRSA viz., Oxacillin screen agar and Cefoxitin disc diffusion test. Oxacillin Screen Agar: Spot of a colony suspension of 0.5 McFarland standard was inoculated on the agar with a 1 μl loop. Then the plates were incubated for 24 hrs at 37 0 C.Oxacillin resistance was detected by the appearance of even a single colony or film of growth. Positive control (MRSA) and negative control (S. aureus ) were inoculated for quality control. Cefoxitin Disc Diffusion Test: All the were subjected to cefoxitin disc diffusion test using a 30 µg disc. Lawn culture was done from a 0.5 Sucilathangam. G. et al. / JPBMS, 2012, 14 (08) Mc Farland standard suspension of the isolate on MHA plate. Plates were incubated at 37 C for 18 hrs and the zone diameters were measured. An inhibition zone diameter of 19 mm was reported as oxacillin resistant and 20 mm was considered as oxacillin sensitive. Plant materials: The galls of Q. infectoria used in this study were obtained from the local market and were identified based on its physical characteristics. The galls were crushed to small pieces using pestle and mortar and powdered in an electric grinder. Preparation of aqueous : In the preparation of aqueous, the powdered material was dissolved in distilled water for 24 hrs at 45 C and centrifuged at 3,000 rpm at 4 C. The supernatant was then filtered and the whole process repeated using the remaining residue with 300 ml distilled water. The filtrates were combined and freeze-dried at -50 C under vacuum for 12 h to produce a fine crystal-like crude aqueous. The s were stored in air-tight jars at 4 C until further use. Preparation of ethanolic : In the preparation of ethanolic, the powdered material was dried and ed by using 95% ethanol. The was weighed and stored in closed vessel at 4ºC in refrigerator for further use. Preparation of solution: The s were dissolved in sterile distilled water to a final concentration of 10 for disc diffusion assay and a 5 concentration for broth micro dilution technique. All the s were sterilized by passing through a 0.45 μm membrane filter. Microorganisms: The bacterial strains used in this study were Staphylococcus aureus 25923, MRSA 43300, clinical of Staphylococcus (103). All the bacterial strains were grown and maintained on nutrient agar slants.the inoculum size of each test strain was 10 8 bacteria/ml for disc diffusion assay which was standardized by adjusting the optical density of the bacterial suspension to a turbidity corresponding to spectrophotometric absorbance at 620 nm. Screening for antibacterial activity: The disc diffusion method was used to evaluate the antibacterial activity. Mueller Hinton agar was prepared in the plates as the media for the test microorganisms. Sterile filter paper discs (Whatman No.1,6 mm) were impregnated with100μl of each of the s (10 ) to give a final concentration of 1 mg/disc and left to dry under the laminar flow cabinet overnight. Different concentration of discs (2.5, 5, 7.5, 10 mg) were prepared The bacterial inoculum was spread evenly onto the surface of the Mueller Hinton agar plates using a sterile cotton bud before the discs were positioned on the inoculated agar surface. Each was assayed in triplicate. Sterile distilled water served as negative control. All the plates 2 Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 14, Issue 14

3 were incubated for 24 hr at 37 C. The antibacterial activity was interpreted from the size of the diameter of zone inhibition measured to the nearest millimetre (mm) as observed from the clear zones surrounding the discs. Determination of MIC and MBC values: The minimum inhibitory concentration (MIC) of the s of the Quercus infectoria was determined for S. aureus, MRSA and clinical using broth micro dilution method. [17] MIC was determined by using the two-fold serial micro dilution method at a final concentration ranging from 50 to 0.4. The tested s were added to sterile Mueller Hinton broth (MHB; Himedia) into 96-well sterile micro titre plates before the diluted bacterial suspension (final inoculum of 10 5 bacteria/ml) were added. Each was assayed in triplicate. The bacterial suspensions were used as positive control and s in broth were used as negative control. The MIC values were taken as the lowest concentration of the s in the wells of the micro titre plate that showed no turbidity after 24 hours of incubation at 37 C. The turbidity of the wells in the micro titre plate was interpreted as visible growth of the microorganisms. The minimum bactericidal concentration (MBC) was determined by subculture of the well showing no apparent growth in a sterile agar plate. The least concentration showing no visible growth on agar subculture was taken as MBC value. Results: A total of 103 Staphylococcus aureus were obtained from various clinical samples. Out of 103 clinical, 74 (71.8%) were MRSA and 29 (28.2%) were MSSA. Disc diffusion method: Antibacterial activity of aqueous and ethanolic was evaluated by measuring zone of inhibition. In aqueous of Quercus infectoria, the zone of inhibition of standard strain of Methicillin Resistant Staphylococcus aureus (MRSA), Staphylococcus aureus and clinical were in the range 10 to 14 mm, 16 to18 mm and 12 to16mm.in ethanolic of Q. infectoria the zone of inhibition of standard strain of Methicillin Resistant Staphylococcus aureus (MRSA), Staphylococcus aureus and clinical were in the range 11to 12 mm,14 to16 mm and 11-17mm. (Table 1) MIC: In Aqueous, MIC for staphylococcus aureus, MRSA and clinical were in the range , and In Ethanolic, MIC for Staphylococcus aureus, MRSA and clinical were in the range , and The MIC values of both the s against clinical of MRSA showed similar values. The MIC values of the aqueous and ethanolic s from the galls of Q. infectoria against S. aureus are shown in Table 2. MBC: Table 3 shows the result of MBC of the aqueous and acetone s from the galls of Sucilathangam. G. et al. / JPBMS, 2012, 14 (08) Q. infectoria against S. aureus. In aqueous, MBC for staphylococcus aureus, MRSA and was 3.2, 6.3 and 6.3 respectively. In Ethanolic, MBC for staphylococcus aureus, MRSA was 0.4, respectively. MBC for clinical ranges between Table 1: Zone of inhibition of the aqueous and ethanolic s from the galls of Q. infectoria against S. aureus, MRSA, in Disc diffusion method Type of s Aqueous Ethanolic MRSA Staphylococcus aureus(25923) 10 to 14 mm 16 to18mm 12 to 16mm 11to 12 mm 14 to16 mm 11-17mm Table2: MIC values of the aqueous and ethanolic s from the galls of Q. infectoria against S. aureus, MRSA, Type of s MRSA Aqueous Ethanolic Staphylococ cus aureus (25923) Table 3: MBC values of the aqueous and ethanolic s from the galls of Q. infectoria against S. aureus, MRSA, Type of s Aqueous Ethanolic MRSA Staphylococcus aureus (25923) Discussion: Methicillin-resistant Staphylococcus aureus (MRSA) has become increasingly widespread as a major cause of both nosocomial and community infections. The recent increase in the methicillin-resistant and multiple resistant strains at large hospitals have started to pose a great difficulty in selecting antimicrobial agents for the management of the infections that they cause. Hence, an accurate and rapid detection of methicillin resistance in Staphylococci is therefore important, not only for choosing the appropriate antibiotic therapy for the individual patient, but also for the control of the endemicity of the MRSA. This situation has placed limits on our options to treat infections by this organism. Glycopeptide derivatives, such as vancomycin and teicoplanin, are now considered to be agents of last resort for the treatment of MRSA infections. However, there are increasing numbers of reports indicating the emergence of vancomycin-resistant S. aureus (VRSA) strains exhibiting two different resistance mechanisms. Therefore, before therapy with vancomycin and teicoplanin fails completely, we need to find some alternative antibacterial agents against MRSA infections. 3 Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 14, Issue 14

4 Natural products and their derivatives have continued to be the most significant sources of new leads into the development of new pharmaceutical agents. Herbal medicines are a valuable and readily available resource for primary health care and complementary health care system undoubtedly, the plant kingdom still holds many species of plants containing substances of medicinal value that had to be discovered. In our study while investigating the anti-mrsa activity of crude s of Q. infectoria by disc diffusion method, we found that both aqueous and ethanolic s of Q. infectoria showed antibacterial activities against all strains of MRSA. The inhibition zones ranged from mm. The ethanolic demonstrated the largest inhibition zone. Similar result was observed in a study conducted by Chusri and Voravuthikunchai. [18] Most of the MRSA strains treated by the ethanolic of Q. infectoria exhibited the MIC and MBC values at and respectively. Staphylococcus aureus 25923, a reference strain also showed similar results. The MIC and MBC values of ethanolic were lower than that of aqueous of the nutgalls suggesting that ethanolic of galls of Q. infectoria showed potential antibacterial effect against clinical of MRSA. Studies conducted by Voravuthikunchai et al. and Chusri and Voravuthikunchai also showed that ethanolic of Q. Infectoria was active against hospital of MRSA. [16,18] In addition, Basri and Fan showed that water and acetone s from galls of Q. infectoria have a high potential antibacterial activity, which seemed to depend on the presence of tannins. Our findings were also supported by other researchers who reported that the crude powder of the galls of Q. infectoria was found to be active against S. aureus and B. subtilis while both the methanol and aqueous s were active against S. epidermidis. [19] In conclusion, ethanol of Quercus infectoria showed better antibacterial activity against clinical of MRSA isolated from Tirunelveli. This finding provides an insight into the usage of the galls of Quercus infectoria in traditional treatment of wounds or burns associated with bacterial infections. Further investigations on the isolation and identification of bioactive components on the plant would help to ascertain its potency. This could be further exploited by in vivo study systems to increase the overall activity. References: 1. Herold BC, Immergluck LC, Maranan MC, Lauderdale DS, Gaskin RE, Boyle-Vavra S, Community-acquired methicillin resistant Staphylococcus aureus in children with no identified predisposing risk. JAMA 1998; 279: Ferrara, A.M. Treatment of hospital-acquired pneumonia caused by methicillin-resistant Staphylococcus aureus. Int J Antimicrob Agents 2007;30: Levy, S.B. and Marshall, B. Antibacterial resistance worldwide: causes, challenges and responses. Nat Med 2004:10;S 122 S Norrby, S.R., Nord, C.E. and Finch, R.Lack of development of new antimicrobial drugs: a potential serious threat to public health. Lancet Infect Dis 2005;5: Samuelsson G. Drugs of Natural Origin. 4th Ed. Sweden: Swedish Pharmaceutical Press; Hwang JK, Kong TW, Baek NI, Pyun YR. -Glycosidase Inhibitory Activity of hexagalloylglucose from the galls of Quercus infectoria. Planta Med 2000; 66: Hussein G, Miyashiro H, Nakamura N, Hattori M, Kakiuchi N, Shimotohno K.Inhibitory effects of Sudanese medicinal plant s on hepatitis C virus protease. Phytother Res 2000; 14: Fatima S, Farooqi AHA, Kumar R, Kumar TRS, Khanuja SPS. Antibacterial activity possessed by medicinal plants used in tooth powders. J Med Aromatic Plant Sci 2001; 22: Digraki M, Alma MH, Ilcim A, Sen S. Antibacterial and antifungal effects of various commercial plant s. Pharm Biol 1999; 37: Redwane A, Lazrek HB, Bouallam S, Markouk M,Amarouch H, Jana M. Larvicidal activity of s from Quercus lusitania var. Infectoria galls (Oliv.). J Ethnopharmacol 2002; 79: Kaur G, Hamid H, Ali A, Alam MS, Athar M. Antiinflammatory evaluation of alcoholic of galls of Quercus infectoria. J Ethnopharmacol 2004; 90: Ikram M, Nowshad F. Constituents of Quercus infectoria. Planta Med 1977; 31: Dar MS., Ikram M., Studies on Quercus infectoria; isolation of syringic acid and determination of its central depressive activity. Planta Med 1979;35: Wiart C, Kumar A. Practical Handbook opharmacognosy. Malaysia: Pearson Education Malaysia Sdn Bhd; Kokate CK Practical Pharmacognosy, New Delhi:Vallabh prakashan, p Voravuthikunchai, S.P. and Kitpipit, L. Activity of medicinal plant s against hospital of methicillin- resistant Staphylococcus aureus. Clin Microbiol Infect 2005;11: and Laboratory Standards Institute Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard, 7 th edn. and Laboratory Standards Institute Document M7-A7. Wayne: and Laboratory Standards Institute; Chusri, S. and Voravuthikunchai, S.P. Quercus infectoria: a candidate for control of methicillin-resistant Staphylococcus aureus infections. Phytother Res 2007;22: D. F. Basri and S. H. Fan, The potential of aqueous and acetone s of galls of Quercus infectoria as antibacterial agents, Indian Journal of Pharmacology 2005;37: Conflict of Interest: - None Source of funding: - Tamil Nadu state Council for Science and Technology (TNSC&ST), Chennai 4 Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 14, Issue 14

5 *Corresponding author: Dr. G. SUCILATHANGAM, M.D., Assistant Professor, Department of Microbiology, Tirunelveli Medical College, Tirunelveli , Tamil Nadu, India. Contact no: Quick Response code (QR- Code) for mobile user to access JPBMS website electronically. Website link:- 5 Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 14, Issue 14