| Summary: | Piper betle L. has potent of antimicrobial activity and is widely used as a traditional remedy to treat skin infections. However, no clear evidence exists concerning antimicrobial and antibiofilm activity against Staphylococcus pseudintermedius and methicillin-resistant S. pseudintermedius (MRSP) opportunistic pathogens that cause wound infections and pyoderma in canines and zoonotic disease. The antimicrobial and antibiofilm activities of P. betle extract were assessed against S. pseudintermedius and MRSP strains. Ethanol leaf extract of P. betle was investigated for its antibacterial effect on S. pseudintermedius and MRSP by broth microdilution and time-kill assays. Biofilm inhibition and production assays were performed to evaluate antibiofilm and biofilm eradication effects, respectively. Biofilm-associated gene expression was further studied using real-time polymerase chain reaction (PCR). The possible interaction between IcaA and major compounds in P. betle was analyzed by molecular docking. The extract showed minimum inhibitory concentration (MIC) at 250 μg/mL. Growth inhibition of P. betle at 1 MIC against the bacteria was initially observed after treatment for 4 h. All isolates were completely killed after 18 h exposure to the extract. Minimum biofilm inhibitory concentrations (MBICs) of the extract against the tested isolates ranged 1/2 MIC to 1 MIC, while minimum biofilm eradication concentration (MBEC) of P. betle was initialed at 8 MIC. Quantitative inhibition and eradication effects were observed in representative strains. The extract at 1/2 MIC and 1 MIC values inhibited biofilm formation up to 100%, with bacterial biofilm removed at up to 94.21% by 4 MIC of the extract. The extract downregulated the expression of the ica A gene among biofilm-producing isolates. The most abundant compounds, 4-allyl-1,2-diacetoxybenzene and eugenol showed a strong affinity with IcaA protein at −5.65 and −5.31 kcal/mol, respectively. P. betle extract demonstrated the antibacterial, antibiofilm, and biofilm-removal activity against S. pseudintermedius and MRSP. Downregulation of the ica A gene expression and protein interaction were possible modes of action of the extract that impacted biofilm production. This extract showed promise as an alternative treatment for S. pseudintermedius infection, especially drug-resistant and biofilm-associated cases. [Display omitted] • P. betle leaf extract showed antibacterial activity against S. pseudintermedius and MRSP. • P. betle exhibited anti-biolfilm and biofilm-removal activities against S. pseudintermedius and MRSP. • Downregulation of ica A gene expression and protein interaction may be the extract mechanism to reduce biofilm production. • This study hightlighted P. betle leaf extract as a beneficial alternative treatment for S. pseudintermedius infection.
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