Assessment of Uncultivable Soil Microorganisms as a Source of Novel Antibiotic

2020 dissertation for MRes. Multi drug resistance microorganisms are an increasing problem and more than 70% of clinically significant pathogens possesses resistance to currently existing antibiotics. Most antibiotics in clinical use were discovered by screening cultivable soil microorganisms and of these only 1% are cultivable using laboratory media and rest are uncultivable. Soil still offers a great potential for antibiotic discovery. In this study, iChip in situ environmental cultivation technology was used to cultivate previously uncultivated microorganisms and screened for antagonistic organisms. Soil samples were collected from various geographic locations in UK based on soil characteristics. Two to three samples were collected from each site and screened for antagonistic microorganisms using iChip-based technology along with soil supplemented nutrient agar which enabled the growth of previously uncultivated microorganisms. This study found an antagonistic microorganism and identified as Bacillus pumilus. Cell free supernatant of B. pumilus fermented broth showed 12 mm zone of inhibition against Staphylococcus aureus ATCC 25922 and butanol extract and compound-3 showed 9 mm and 8 mm zone of inhibition respectively. Bacillus pumilus has been previously described to produce antibacterial and antifungal compounds. Mueller Hinton broth with addition of 5% glucose was used for B. pumilus fermentation to assess secondary metabolites. Fermented broth supernatant and extracts produced zone of inhibition (ZI) ranging from 8-12mm against S. aureus but could not determine minimum inhibitory concentration. Fermented broth was extracted sequentially with ethyl acetate, n-butanol and methanol. n-Butanol extract in thin layer chromatography showed three compounds, and of these only Compound 3 showed inhibitory effect on S. aureus. Nuclear magnetic resonance (both 1D and 2D) analysis revealed compound 3 as a mixture of possible 3 compounds or their fragments but could not identify these compounds. However, compounds fragments had chemical structures containing peptide bonds and aliphatic chains which are also found in known peptide antibiotic, for example, vancomycin. Further studies are needed to fully characterise this compound-3.