HR-LCMS profiling, in vitro and in silico assessment of the antibacterial activities of endophytic Bacillus amyloliquefaciens NWR-14 from Piper chaba W. Hunter

• Antibacterial activity of B. amyloliquefaciens NWR-14 against ESBL-producing K. pneumoniae. • Molecular docking reveals strong binding of 8′-Hydroxydihydroergotamine to CTX-M-15 (−8.8 kcal/mol). • Non-cytotoxic effects on fibroblast cells (>97 % viability at high concentrations). • Moderate antioxidant activity (DPPH IC50: 170.8 µg/mL) in bacterial extracts. • First report of endophytic B. amyloliquefaciens NWR-14 from Piper chaba as a source of novel antimicrobials. The rise of multidrug-resistant (MDR) pathogens such as Klebsiella pneumoniae , particularly those producing extended-spectrum β-lactamases (ESBL), has intensified the search for novel bioactive compounds. Medicinal plants, including Piper chaba , host endophytic bacteria that may serve as promising sources of antimicrobial agents. This study aimed to evaluate the antibacterial, antibiofilm, antioxidant, and cytotoxic properties of secondary metabolites derived from Bacillus amyloliquefaciens NWR-14, an endophyte isolated from P. chaba , with a focus on its activity against ESBL-producing K. pneumoniae . B. amyloliquefaciens NWR-14 was isolated and identified via 16S rRNA gene sequencing. The ethyl acetate extract of this isolate was tested for antibacterial activity using MIC and MBC assays. Antibiofilm potential was assessed using crystal violet staining. Scanning electron microscopy (SEM) was employed to visualize morphological changes in K. pneumoniae . HR-LCMS was used to putatively annotate bioactive compounds. In silico molecular docking was conducted to assess binding affinities to the CTX-M-15 β-lactamase enzyme. Antioxidant activity was evaluated using DPPH, FRAP, and ABTS assays, and cytotoxicity was tested on fibroblast cells. The extract exhibited notable antibacterial activity with a MIC and MBC of 1.56 mg/mL, and showed dose-dependent biofilm inhibition, achieving maximum effect at 6 mg/mL. SEM analysis confirmed significant cellular damage in K. pneumoniae . HR-LCMS analysis putatively annotated 76 compounds, including alkaloids, saponins, and phenolic acids. Molecular docking revealed strong binding affinities of compounds such as 8′-Hydroxydihydroergotamine and Licoricesaponin E2 to CTX-M-15. The extract demonstrated moderate antioxidant activity and low cytotoxicity, with over 97% cell viability in fibroblast assays. B. amyloliquefaciens NWR-14 shows promise as a source of bioactive secondary metabolites with antibacterial, antibiofilm, and antioxidant properties. These findings underscore the potential of endophytic bacteria from medicinal plants as reservoirs of compounds for addressing antimicrobial resistance. Further purification and structural validation of active constituents are recommended to enhance therapeutic applicability.