| Summary: | Endophytic <i>Aspergillus</i> species represent an inexhaustible source for many medicinally important secondary metabolites. The current study isolated the endophytic <i>Aspergillus niger</i> (OL519514) fungus from <i>Opuntia ficus-indica</i> fruit peels. The antibacterial activities were reported for both <i>Aspergillus</i> species and <i>Opuntia ficus-indica</i> fruit peel extract. Extraction of the endophytic fungal metabolites using ethyl acetate and fractionation was performed, yielding dihydroauroglaucin (C1), isotetrahydroauroglaucin (C2), and cristatumin B (C3). Resistant bacterial strains were used to investigate the efficiency of the total fungal ethyl acetate extract (FEA) and the isolated compounds. FEA showed promising wide spectrum activity. (C3) showed excellent activity against selected Gram-negative resistant bacteria; However, (C2) exhibited tremendous activity against the tested Gram-positive resistant strains; conversely, (C1) possessed the lowest antibacterial activity compared to the two other compounds. An in silico virtual molecular docking demonstrated that cristatumin B was the most active antimicrobial compound against the selected protein targets. In conclusion, the active metabolites newly isolated from the endophytic fungus <i>Aspergillus niger</i> (OL519514) and present in plants’ waste can be a promising antimicrobial agent against multidrug-resistant bacteria.
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