Int. J. Pharm. Sci. Rev. Res., 30(1), January – February 2015; Article No. 03, Pages: 11-17 ISSN 0976 – 044X International Journal of Pharmaceutical Sciences Review and Research Available online at www.globalresearchonline.net © Copyright protected. Unauthorised republication, reproduction, distribution, dissemination and copying of this document in whole or in part is strictly prohibited. © Copyright pro 11 Siti Junaidah Ahmad 1 , Hing Hiang Lian 2 , Dayang Fredalina Basri 2 , Noraziah M ohamad Zin 2* 1 School of Health Sciences, Faculty of M edicines and Health Sciences, Universiti Sultan Zainal Abidin, Jalan Sultan M ahmud, Kuala Terengganu, Terengganu, M alaysia. 2 School of Diagnostic and Applied Health Sciences , Faculty of Health Sciences, Universiti Kebangsaan M alaysia(UKM ), Jalan Raja M uda A. Aziz, Kuala Lumpur, M alaysia. * Corresponding author’s E-mail: noraziah.zin@ukm.edu.my Accepted on: 05-10-2014; Finalized on: 31-12-2014. ABSTRACT Bioactive compound from endophytic Streptomyces sp. has been claimed as a source of antibiotic. This study focused on the investigation of pharmacodynamic pattern and visualization of the mechanism of SUK 25 extracts against MRSA. The pharmacodynamic characteristic of the extract against MRSA 43300 was determined using time-kill assay. Then, the mode of action of the extracts was observed through biochemical assay and transmission electron microscopy. The SUK 25 extracts displayed bacteriostatic mode of action and concentration-dependent manner. The action of SUK 25 extracts against MRSA ATCC 43300 caused irregular shape of cells, which affected changes in Crystal Violet uptake by the cells. The release of UV absorbing materials and protein from the cells was caused by cell lysis. In conclusion, the action of the SUK 25 extracts against MRSA ATCC 43300 led to the internal change in cells, through which permeability of cells was altered by a decrease in the Crystal Violet uptake, shape of cell changes, and in turn brought about the lysis of cell. Keywords: Mode of action, endophytic Streptomyces, M RSA INTRODUCTION treptomyces sp. was classified as filamentous Gram- positive bacteria and naturally lives in plants, soils and marine environments. Its bioactive secondary metabolites have proved to exhibit antibiotic, anticancer, anti-inflammation and anti-viral activities. 3,29 For example, peptide antibiotic isolated from Streptomyces sp. in Monstera tree, Peru displayed antifungal property against Cryptococcus neoformans, and anti-malarial activity against Plasmodium falciparum. 12 Infectious disease caused by resistant bacteria, such as Methicillin Resistance Staphylococcus aureus (M RSA), has emerged as virulent pathogen in public and clinical settings. Vancomycin is the last resort of available antibiotic, but the current development of VRSA is making the treatment to become more difficult. It also has some side effects administered, such as nephrotoxicity. 14 Nowadays, alternative medicine from natural sources is an important solution to overcome the side effects of synthetic drugs. The purpose of this study is to determine the pharmacodynamic pattern of SUK 25 extract treatments against M RSA ATCC 43300, and to visualize the mechanism of action of SUK 25 extracts against MRSA. M ATERIALS AND M ETHODS Culture Condition M RSA ATCC 43300 and SUK (UKM Strain) 25 were obtained from Novel Antibiotic Laboratory, UKM. The SUK 25 was isolated from Zingiber spectabile root. The overnight culture of MRSA ATCC 43300 at 37 ˚C on Muller Hinton Agar (MHA), (Merck, USA) supplement with 2% sodium chloride (Sigma-Aldrich, USA) was performed before testing. The mature spore of 14-day culture of SUK 25 at 28 ˚C on International Streptomyces Project (ISP) 2 Agar was used for extraction and testing. Both MRSA ATCC 43300 and SUK 25 were cultured in the 20% glycerol (Merck, USA) solution and placed at -80 ˚C for prolonged storage. 8,33 Secondary M etabolite Extraction SUK 25 culture in Thornton media was extracted for exploitation of its secondary metabolites. Ethyl acetate (R & M Chemical, Malaysia) extraction was carried out by extracting the culture filtrates with three half-volumes of ethyl acetate. The extracts were collected (from upper layer) using a separating funnel (Pyrex, USA). The solvent phase was dried through the rotating evaporator (Buchi, Switzerland) at 40 °C with 240 mbar. The extracts ( in solvent phase) were dried up, weighed and tested as anti- M RSA agent s. 32 Anti-M RSA Activity The MIC (Minimum Inhibitory Concentration) of the dried extracts of SUK 25 was determined against M RSA ATCC 43300 8 . The crude extracts of SUK 25 were dissolved in 10% methanol (R & M Chemical, Malaysia) and two-fold dilution technique w as applied to prepare the concentration of 0.488 µg/mL to 1000 µg/mL in a 96-well microtiter plate (Thermo-Scientific, USA). Then, 50 µl of 1 x 10 6 CFU/mL of bacterial inoculums was added to Mueller Hinton Broth (MHB) to make a total volume of 100 µl. Vancomycin (Sigma-Aldrich, USA) was used as positive control. The plate was incubated at 37 °C overnight. This assay was carried out three times to obtain consistent readings. M ode of action of Endophytic Streptomyces sp., SUK 25 extracts Against M RSA; M icroscopic, Biochemical and Time-Kill Analysis S Research Article