Isolation, Characterization, and Optimization of Antimicrobial Proteinaceous Compounds Produced by Bacillus safensis Isolated From Rhizosphere Soil Samples in Bangladesh.

Bacillus species are widely distributed in nature and are recognized for their capacity to synthesize a wide range of antimicrobial compounds effective against multidrug-resistant bacterial and fungal pathogens, highlighting their potential as alternatives to conventional antibiotics. This study focused on the isolation of Bacillus spp. from rhizosphere soil capable of producing antimicrobial compounds effective against both gram-positive and gram-negative bacteria, and the optimization of different parameters to enhance their production. Of rhizosphere-soil samples collected from eight different sites in Dhaka city, 96 bacterial isolates were selected to evaluate their antimicrobial activity. Twenty-seven isolates were preliminarily identified as Bacillus spp. based on their morphological characteristics and biochemical reactions. Five isolates were found to produce inhibitory compounds having antimicrobial properties. The highest antimicrobial compound production was observed at pH 7 and 30°C, rather than at alkaline or acidic pH (p value = 0.04, < 0.05). Sequence analysis of the 16S rRNA gene of three significant isolates producing antimicrobial compounds revealed that isolates S02b and S03b had 100% sequence similarity to Bacillus safensis, and S04b had 100% similarity with Bacillus pseudomycoides. Whole-genome sequencing revealed that isolate S02b harbors biosynthetic gene clusters (BGCs) similar to schizokinen (60%), fengycin (53%), bacilysin (85%), bacillibactin (80%), and lichenysin (92%), indicating specialization in peptide-based secondary metabolite production. It also identified one RiPP and three putative bacteriocin clusters, with area of interest (AOI) 1 matching sactipeptide synthetase (BmbF), amylocyclicin, and UviB, and AOI 2 matching UviB. Thin layer chromatography (R f values: 0.45-0.52), the absorbance peak in UV spectra (210-220 nm), and protease sensitivity confirmed the proteinaceous nature of the extracted antimicrobial compounds. The antimicrobial proteinaceous compound (APC) from isolate S02b was nonhemolytic and stable at heat, pH, surfactant, food-grade metal salt solvent, and organic acid. The scanning electron microscopy image revealed that APCs exhibited significant antimicrobial activity against Staphylococcus aureus, suggesting that APCs could be a promising candidate for new antibiotics.