Streptomycetes are known to produce metabolites that act as antimicrobials and they also produce indole derivatives as antimicrobials (17). An ecologically diverse country Nepal, 49th in the whole world (18), built through the rising of seabed from tectonic movement (19) could harbor different Streptomycetes including marine which could produce potent secondary metabolites including antimicrobials. Thus, soil samples from 24 different parts of Nepal were collected for screening of Streptomyces species.
The isolates were cultured as described in materials and methods. The cell free culture media was further extracted using ethyl acetate as has been performed to extract metabolites produced by Streptomycetes (20) to obtain polar and non-polar secondary metabolites and preferred also because of its minimal toxicity to test organisms (21). Ethyl acetate was evaporated and the extract was concentrated as described to test its anti-microbial potential.
The insignificant inhibitory zones during agar diffusion antimicrobial susceptibility tests could indicate that there were some antimicrobial compounds whose concentrations were either low or, the insolubility in agar due to polarity (22) could have hindered the movement and efficacy of the potential antibacterial molecules in the medium.
Moreover, in the present study the search of novel and effective antimicrobials were pursued based on color forming isolates because colored indole derivatives, indirubin and isatin, have been reported as the major constituents of plant and microbial extracts with potent antimicrobial properties (23,24,25).
Since the world is in desperate need of novel antibiotics (26), even the extracts with small amount of antimicrobials should be considered as the source of new drug candidate as these could be novel and could function against the emerging resistance. Thus, effective and robust assay system should be developed where factors like polarity of the compound should not be stumbling block in antimicrobial assay. A robust and faster screening protocol for detecting antimicrobials is thus of dire need for which Resazurin based microbial viability assay could be modified for antimicrobial screening.
During antimicrobial potency screening, the antioxidant property of the crude extract could be of concern. Since most antibiotics at present are known to produce reactive oxygen species (ROS) in killing the bacterial cells (27) (28) and if the mechanism of the inhibition could be through ROS then the antioxidant properties of the extract could hinder antimicrobial potential (29). Thus, antioxidant properties of these extracts were also investigated. Extracts from KH8 that had the lowest antimicrobial potential had the strongest antioxidant property among all the extracts tested (results not shown). Thus antimicrobial potency of the compound included in the extract could have been affected if the bactericidal effect was due to ROS formation. However this needs further validation. Moreover, in future screening of potential antimicrobials, the ROS quenching effect of the compound or extract should also be taken in consideration.
Some of the compounds reported in GCMS analysis were non-polar and their movement in agar could have been hindered (30). Thus, when screening for antimicrobials, one should consider the polarity of the compounds where resazurin based assay could be more potent. In addition, the assay could be done in 384 plates and robotics could be used to screen vast library of compounds or multiple extracts (31) (32).
Moreover, the extract contained a mixture of antioxidant compounds as well and could have impacted in antimicrobial potency of the compound(s) in the extract by mitigating ROS produced by the antimicrobials. In addition to other known antimicrobial compounds, the extract also contained pyrrolo (1, 2- a) pyrazine –1, 4-dione, hexahydro–3-(2-methylpropyl) (PPDHMP) that has not yet been reported as an antimicrobial agent to the best of our knowledge. The pyrrolo pyrazine scaffold in the ring structure (Figure 3) that could make it a probable antimicrobial agent by inhibiting the activities of S-adenosyl methionine utilizing enzymes (essential proteins) since it is an anticancer agent and most probable function could be due to the kinase inhibition and warrants further study. Furthermore, compound purification and antimicrobial tests against drug resistant strains could be done to access their inhibitory potential against increasing resistant strains.