Metagenomic Nanopore Sequencing and phenotypic screening for exploring the nature of antimicrobial metabolites of Bacillus haynesii Metagenome

Multidrug-resistant pathogens are a rising global health worry that imposes an urgent need for the discovery of novel antibiotics particularly those of natural origin. In this context, we aimed to use conventional phenotypic screening coupled with metagenomic nanopore sequence analysis of soil microbiota and genomic analysis for identifying the antimicrobial metabolites produced by promising soil isolate(s). In thus study. whole metagenome analysis of the soil sample(s) was performed using MinION (Oxford Nanopore Technologies). Aligning and analysis of sequences for probable secondary metabolite gene clusters were extracted and analyzed using the antiSMASH version 2. Results of the metagenomic analysis showed the most abundant taxa were Bifidobacterium, Burkholderia, and Nocardiaceae (99.21%, followed by Sphingomonadaceae (82.03%) and B. haynesii (34%). Phenotypic screening of the respective soil samples has resulted in a promising Bacillus isolate that exhibited broad-spectrum antibacterial activities against various MDR pathogens. It was identified using microscopical, cultural, and molecular methods as Bacillus (B.) haynesii isolate MZ922052. Genomic analysis revealed the conservation of seven biosynthetic gene clusters of antibacterial metabolites, namely, siderophore lichenicidin VK21-A1/A2 (95% identity), lichenysin (100%), fengycin (53%), terpenes (100%), bacteriocin (100%), Lasso peptide (95%) and bacillibactin (53%). In conclusion, metagenomic nanopore sequence analysis of soil samples coupled with conventional screening helped in identifying B. haynesii isolate MZ922052 harboring seven biosynthetic gene clusters of promising antimicrobial metabolites. This is the first report for the identification of the bacteriocin, lichenysin, and fengycin biosynthetic gene clusters in B. haynesii MZ922052.