Isolation and Characterization of Antibiotic Producers Streptomyces bacillaris and Streptomyces lavendulae

The emergence of multi-drug resistance in Staphylococcus aureus and Escherichia coli, which are commonly found in the human flora, has triggered an urgent need for new antibiotics and novel antibiotic combinations against both Gram-positive and Gram-negative pathogens. Actinobacteria, in particular the genus Streptomyces, produce almost two-thirds of the clinically useful antibiotics that we use today. The objective of this study is to isolate actinobacteria from local soil samples in Southern California and investigate their potential as antibiotic producers against Staphylococcus aureus and/or Escherichia coli by using direct overlay assays. Ninety-five soil samples were air dried, resuspended in water, and plated on ISP2 and ISP4 media with or without antibiotics. Actinobacteria colonies were examined for antibiotic activity using direct overlay assays with E. coli ATCC 25922 and S. aureus ATCC 25923. A neomycin producer Streptomyces fradiae ATCC 10745 was used as positive control. Inhibition zones surrounding actinobacteria colonies were measured after overnight incubation at 35^oC. Active compounds were extracted using ethyl acetate. The extracts were separated by thin-layer chromatography (TLC) using different ratios of methanol-dichloromethane. The TLC plates were overlaid with S. aureus and/or E. coli to identify compounds with antibiotic activity. A disk diffusion assay was used to confirm the TLC’s result. Species identification was done by 16S rRNA gene sequencing analysis. We isolated more than 400 actinobacteria and observed a high diversity in color and morphology. We discovered 21 that produced antibiotics against both E. coli and S. aureus either on ISP2 and ISP4. In ISP2, 100% (n=21) of these actinobacteria showed antibiotic activities against S. aureus and 80.95% (n=17) showed antibiotic activities against E. coli. In ISP4 the amount of antibiotic activity was significantly less compared to ISP2. Fifty five additional colonies were discovered to only produce antibiotics against S. aureus. Two of these actinobacteria isolates, AC1358 and AC1359 showed a possible synergistic interaction against S. aureus. The compounds from AC1358 and AC1359 were obtained by using ethyl acetate extraction. Both the AC1358 (Rf=0.47) and AC1359 (Rf=0.30) extracts were separated using methanol-dichloromethane with the ratio of 1:9. 16S rRNA gene analysis showed that AC1358 has a 100% identity to Streptomyces bacillaris and AC1359 has a 100% identity to Streptomyces lavendulae. Both S. bacillaris AC1358 and S. lavendulae AC1359 have been found to produce an active compound that acts as antibiotic against S. aureus. Further investigation is necessary to characterize the active compounds and to determine if we have discovered a novel antibiotic or antibiotic combination against Gram-positive bacteria.