Molecular identification and the effect of abiotic stress on bioactive metabolite production for endophytic Bacillus isolates from Solanum nigrum

Bacterial endophytes isolated from medicinal and wild plant species have recently gained significant attention for their medicinal properties, often closely linked to those of their plant hosts. In this study, two endophytic Bacillus isolates were identified and taxonomically characterized using 16S rRNA sequencing and multi-locus sequence typing (MLST). We also investigated the impact of sublethal concentrations (0.5 mg/mL) of cadmium and hydrogen peroxide on metabolite production and bioactivity. Phytochemical testing, along with antimicrobial and antioxidant assays, revealed shifts in metabolite production under stress conditions. According to 16S rRNA-based similarity searchs, both isolates share a close relationship with Bacillus cereus complex; however, phylogeny and MLST failed to resolve their species identity. Phytochemical screening of methanolic crude extracts from both isolates tested positive for alkaloids, flavonoids, and saponins. Notably, tannins were detected only after cadmium treatment, while steroids were present following exposure to both cadmium and H₂O₂. LC-MS fingerprinting confirmed the presence of several tannins and steroids in treated samples. The untreated crude extracts exhibited an IC₅₀ of ~ 3 mg/mL in the DPPH assay, which decreased to ~ 1.5 mg/mL after treatment with cadmium or H₂O₂, indicating a significant increase in radical scavenging activity. Additionally, extracts from both treated and untreated bacteria displayed antimicrobial activity against selected bacterial pathogens, with MIC values ranging from 62.5 µg/mL to 125 µg/mL. LC-MS analysis identified various antimicrobial and antioxidant metabolites, including phenoxomethylpenicilloyl, maculosin, (S,R,S)-alpha-tocopherol, 3-indoleacrylate, procyanidin A2, cis-11-eicosenamide, 3-hydroxy-3-phenacyloxindole, and 9-octadecenamide.