Bactericidal Activities of Nanoemulsion Containing Piper betle L. Leaf and Hydroxychavicol Against Avian Pathogenic Escherichia coli and Modelling Simulation of Hydroxychavicol Against Bacterial Cell Division Proteins

<b>Background</b>: Avian pathogenic <i>Escherichia coli</i> (APEC) is a leading cause of colibacillosis in poultry. <i>Piper betle</i> L. is a medicinal plant rich in bioactive compounds including hydroxychavicol that possess potent antibacterial activity. This study aimed to investigate the efficacy of a <i>P. betle</i> L. leaf nanoemulsion (NEPE) and hydroxychavicol against multidrug-resistant APEC isolates. <b>Methods</b>: In vitro and in silico analysis of NEPE and hydroxychavicol against APEC were determined. <b>Results</b>: The nanoemulsion exhibited potent antibacterial activity, with MIC and MBC values of 0.06-0.25% <i>v/v</i> and 0.125-0.25% <i>v/v</i>, respectively. The MIC and MBC values of hydroxychavicol against isolates ranged from 0.25 to 1.0 mg/mL. A time-kill assays revealed rapid bactericidal effects of both compounds, achieving a ≥3-log reduction within 4 h at 4 × MIC. Scanning electron microscopy demonstrated that APEC cells treated with hydroxychavicol exhibited filamentous cells with incomplete septa. Molecular docking and dynamics simulations of hydroxychavicol against APEC cell division proteins were investigated. According to the binding energy, hydroxychavicol exhibited the highest affinity with ZapE, FtsW, FtsX, FtsZ, and FtsA, respectively. However, the FtsA protein showed the least protein conformational change throughout the 5000 ns simulation, reflecting a highly stable conformation. <b>Conclusions</b>: These confirm the potential stability of protein and ligand, as supported by molecular dynamics simulation. The results suggested the potential of NEPE and hydroxychavicol, which may have promising antibacterial potential that can be used to inhibit APEC growth.