Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley

Bacillus species are one of the predominant soil bacteria that are able to produce essential secondary metabolites that have antagonistic effects on other microorganisms. They are Gram-positive, endospore-forming, chemoheterotrophic, aerobic or facultative anaerobic rods usually consisting of peritrichous flagella for motility. The major aim of this study was to isolate the antimicrobials producing Bacillus spp. from soil samples of different parts of the Kathmandu Valley, identify them and to assess their antimicrobial activity against different pathogenic bacteria. The test organisms used were Staphylococcus aureus (ATCC 25923), E. coli (ATCC 25922), Pseudomonas spp., Salmonella spp., methicillin-resistant Staphylococcus aureus (MRSA) and extended spectrum beta-lactamase (ESBL) producing E. coli. Twenty four isolates from 9 soil samples identified as Bacillus spp. showed the zone of inhibition around their growth on Nutrient agar during isolation. These 24 isolates were chosen for primary screening of production of antimicrobial by perpendicular streaking method using four test organisms. . Of these 24 isolates, six isolates showing a significant zone of inhibition (≥1mm) against two or more test organisms from the primary screening were chosen for secondary screening which was further tested with six test organisms including ESBL E.coli and MRSA. They were further characterized through different physiological and biochemical tests. All 6 isolates showed inhibitory action against MRSA and the largest zone of inhibition (30mm) was shown by isolate U6. Isolate U3 was found to have broad spectrum antimicrobial activity with inhibitory effect against gram negative organisms- Pseudomonas and Salmonella and gram positive organism S. aureus (ATCC 25923). Isolate U5 showed a zone of inhibition of about 25mm against S. aureus which was comparable to that of erythromycin. Hence, this study determines the soil in Kathmandu Valley as a potential source of antimicrobial producing Bacillus spp. and recommends isolation and further characterization of Bacillus isolates as a possible source of novel drug to combat with the emergence of multidrug resistant strains

Isolation, Characterization, and Screening of Antimicrobial-Producing Actinomycetes from Soil Samples

Actinomycetes are Gram-positive, facultative anaerobic fungus-like filamentous bacteria which remain on the top of the natural antibiotic producers. Due to the climatic and geographical diversity of Nepal, a wide range of microorganisms with potent source of antimicrobials are available. The objective of this study was to isolate, identify, and screen the potential antimicrobial-producing actinomycetes from soils covering different altitude range of Nepal. Forty-one isolates of actinomycetes were isolated from 11 soil samples collected from different locations in Nepal with altitude ranging from 1500 to 4380 meters. The isolates were identified on the basis of morphological study, different sugar utilization, protein utilization, and hydrolysis tests. They were also characterized on the basis of temperature and pH. Primary screening for antimicrobial activity was carried out against several test organisms: Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 700603), and Pseudomonas aeruginosa (ATCC 27853) by the perpendicular streaking method, and secondary screening was carried out by the agar well diffusion method using ethyl acetate for solvent extraction. 70.7% of the isolates were identified as Streptomyces spp., 19.5% as Nocardia spp., and 9.5% as Micromonospora spp. 43.34% of actinomycete isolates was found to be potent antimicrobial producers from the primary screening among which 46.34% were effective against Gram-positive and 12.19% against Gram-negative test organisms. Isolate C7 (Micromonospora spp.) showed the best broad-spectrum antimicrobial activity during secondary screening. A total of 11 different types of pigments were observed to be produced by different isolates, of which, the yellow pigment was the most prominent. The association between elevation, pH, and pigment with the antimicrobial production was found to be insignificant. This finding can be of importance for further investigation towards obtaining broad-spectrum antibiotics for therapeutic purpose