Antimicrobial resistance is a growing global concern exacerbated by the scarcity of new medications and resistance to current antibiotics. Microbes from unexplored habitats are promising sources of natural products to combat this challenge. This study aimed to isolate bacteria producing secondary metabolites and assess their antimicrobial efficacy against human pathogens. Soil and liquid samples were collected from Afar region, Ethiopia. Bacterial isolates were obtained using standard serial dilution techniques. Antimicrobial activity was evaluated using agar plug and well diffusion methods, matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF MS) and whole-genome sequencing (WGS) were conducted for the isolate exhibiting the highest antimicrobial activity. Secondary metabolites were extracted and analyzed using gas chromatography-mass spectra (GC-MS). In this study, 301 bacteria isolates were identified, of which 68 (22.6%) demonstrated antagonistic activity against at least one reference pathogen. Whole-genome sequencing revealed that Sl00103 belongs to the genus Bacillus, designated as Bacillus sp. Sl00103. The extract of Sl00103 showed zones of inhibition ranging between 17.17 ± 0.43 and 26.2 ± 0.4 mm against bacterial pathogens and 19.5 ± 0.44 to 21.0 ± 1.01 mm against Candida albicans. GC-MS analysis of ethyl acetate and n-hexane extracts identified major compounds including (R,R)-butane-2,3-diol; 3-isobutylhexahydropyrrolo[1,2a] pyrazine-1,4-dione; cyclo(L-prolyl-L-valine); and tetradecanoic acid, 12-methyl-, methyl ester; hexadecanoic acid, methyl ester, among others. In conclusion, this study isolated several promising bacterial strains from the Afar region in Ethiopia, with strain Sl00103 (Bacillus sp. Sl00103) demonstrating notable antimicrobial and antioxidant activities and warranting further studies.
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