Thin Layer Chromatography-Bioautography Assay for Antibacterial Compounds from Streptomyces sp. TBRC 8912, a Newly Isolated Actinomycin D Producer

Abstract

Based on a primary screening for antibacterial agents using the agar disc diffusion method, among one hundred and sixty-one actinobacterial strains isolated from herbicide sprayed soil in agricultural areas, a newly isolated Streptomyces sp. TBRC 8912 exhibited antibacterial activity against Xanthomonas axonopodis pv. citri (Xac) a pathogen that causes canker disease in kaffir lime. The strain TBRC 8912 was cultivated in the International Streptomyces Project (ISP) 2 and modified Wickerhams Antibiotic Test Medium (WATM) in liquid and solid fermentations. Ethyl acetate crude extracts from ISP2 and WATM cultures showed antibacterial activity against Xac by causing an inhibition zone diameter with a range of 15.75±0.48-17.38±0.24 mm on bioassay plates when tested using the paper disc diffusion method. The ethyl acetate extract obtained from the WATM liquid culture, was separated by column chromatography over Sephadex LH-20 (dichloromethane/methanol, 1:1) and silica gel (dichloromethane/methanol). Subsequently, bioassay-guided fractionation was used to select active fractions, and then a bioautographic technique was employed to isolate a mixture of compound 1 and 2 (10 mg). The active fraction was further investigated using liquid chromatography-electrospray ionization-quadrupole-time-of-flight mass spectrometry (LC-ESI-Q-TOF-MS). ESI-MS of the isolated compounds showed pseudomolecular ion peaks at m/z 1255.6 [M+H]+ and 1277.6 [M+Na]+ at tR 28.44 and 28.84 min corresponded to the molecular mass of 1254 which matches to actinomycin D and its isomer. The 16S rDNA gene sequence of the strain TBRC 8912, exhibited 99.11 % identity with the Streptomyces panaciradicis strain 1MR-8T. This is the first report of a representative of Streptomyces panaciradicis capable of producing actinomycins. Further study is recommended for evaluation of the persistence and biodegradation of plant tissue and soils in a greenhouse, before application in the environment.