Antibiotic Activity of Streptomyces sp.

G againts Staphylococcus aureus ATCC 6538 and Escherichia coli ATCC 8739 using Contact Bioautography Technique
Ifah Yulistyani*, Isnaeni, Juniar Soerjono, Achmad Toto Poernomo
AirlanggaUniversity

Faculty of Pharmacy Airlangga University, Surabaya, Indonesia * Corresponding author: Faculty of Pharmacy Airlangga University, Surabaya, Indonesia, Email: gif.myangel@gmail.com

Introduction
Microbial natural products remain the most promising source of novel antibiotics. About 66% of all the bioactive microbial metabolites were isolated from actinomycetes especially from Streptomyces spp. [1]. Ifah et al. (2010) have done the isolation and primary screening of Streptomyces sp. from soil of Rumah Kompos Bratang Surabaya. From these studies was known Streptomyces sp. G has a relatively strong antimicrobial activity [2]. Accordingly, we are investigating the potential of Streptomyces sp. G as a resource for new antibiotic. To investigate the active compounds of Streptomyces sp. G, we have used contact bioautography technique.

Method
The plates were run in duplicate Prepare TLC plates silica gel 60 F254 10 l ethyl acetate extracts of Streptomyces sp. G were deposited as spots developed in a toluene:ethanol mixture (8:2)

Put down at refrigerator temperature for 2 h The areas of inhibition were marked the plates were incubated in 37oC for 24 h TLC plates were removed from the surface with the help of sterile forceps

viewing under UV at 254 & 365 nm

The TLC plates were then placed aseptically upon Nutrient agar which inoculated with test organism

Rf value measured

Active compound stock solution was tested for heat stability by keeping samples at 60oC for 30 min and at 100oC for 5 min. After heat treatment, samples were used for assay of bioautography again [3].

Results
On the thin layer chromatograms, the extracts were separated into one spot by viewing under ultraviolet at 254 nm (Rf value 0.54) and two spots (Rf value 0.53 and 0.64) at 365 nm. This spot gave purple colours under ultraviolet (Figure 1). In the bioautography studies only one (Rf value 0.53) showed antibacterial activity (Figure 2). These results may suggest that this compound, after its purification and identification, could be used for the development of the valuable pharmaceuticals. In the case of heat treatment, active component was found as heat-stable (Figure 3).

254 nm

365 nm
10 l 20 l
20 l 10 l

S.aureus

E. coli

Figure 1. TLC Plates of Ethyl Acetate Extracts of Streptomyces sp. G Under UV at 254 and 365 nm

Figure 2. Bioautography of Ethyl Acetate Extracts of Streptomyces sp. G Againts Staphylococcus aureus ATCC 6538 And Escherichia coli ATCC 8739 (A, spot which Rf value 0.64; B, spot which Rf value 0.53)

Conclusion
Ethyl acetate extract of Streptomyces sp. G has one spot (Rf value 0.53) showed antibacterial activity.

References:
Figure 3. Heat Treatment of Ethyl Acetate Extracts of Streptomyces sp. G (EA, Ethyl acetate; St, standard, Tetracycline; 100, 100oC; KLT, TLC plate; 0, not heat treatment; 60, 60oC)

1. Hopwood, D.A., Buttner, M.J., Bibb, M.J., Kieser, T., Charter, K.F., 2000. Antibiotic Production by Streptomyces. Practical Streptomyces Genetics. 1, 1-42. 2. Y, Ifah., Olivia, N., Setyani, D.S., Septyanto, T.D., dan Saraswati, M.D., 2010. Laporan Akhir PKMP Pemberdayaan Tanah Rumah Kompos Bratang Surabaya sebagai Sumber Streptomyces sp. Penghasil Antibiotik. Fakultas Farmasi Universitas Airlangga. 3. Yucel, S., and Yamac, M., 2010, Selection of Streptomyces Isolates from Turkish Karstic Caves Against Antibiotic Resistant Microorganisms, J. Pharm. Sci., Vol. 23, No,1, pp. 1-6.