Abstract
<jats:p>Propolis, a natural resinous mixture produced by honeybees derived from plant exudates, exhibits promising antibiofilm properties against multidrug-resistant pathogens. This study evaluated the antibiofilm efficacy of methanol propolis extract (MPE) and ethyl acetate propolis extract (EAPE) against Escherichia coli isolated from buffaloes’ mastitis. Crystal violet assays demonstrated concentration dependent biofilm inhibition, with MPE and EAPE achieving 97.9 ± 0.2 % and 95.8 ± 0.3 % inhibition respectively, at 5 mg/mL (mean ± SD, n = 5). Both extracts maintained >85 % inhibition down to 0.312 mg/mL, with MPE showing significantly higher activity at lower concentration (p < 0.05). Scanning electron microscopy (SEM) revealed marked disruption of biofilm architecture and compromised bacterial cell integrity post treatment. A total of 15 bioactive compounds were identified in the propolis extracts using high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) analysis. Two categories of phytochemicals were identified: common (caffeic acid, p-coumaric acid, galangin, chrysin, kaemferol and quercetin) and unique (quinic acid, gallic acid, epicatechin (MPE), apigenin, fumaric acid, vanillic acid, rutin and epigallocatechin (EAPE)). These compounds including major constituents like caffeic acid, p-coumaric acid, galangin, chrysin, kaempferol and quercetin to inhibit bacterial adhesion, disrupt extracellular polymeric substance (EPS) and attenuate quorum sensing. These findings provide a robust phytochemical rationale for the potent antibiofilm activity of Indian propolis, positioning it as a viable natural alternative for managing biofilm associated mastitis infections.</jats:p>