Antibiofilm Activity of Agrimonia eupatoria Extracts Against Clinically Relevant Pathogens

Biofilms are surface-attached bacterial communities that contribute significantly to chronic infections. Their altered metabolism promotes antibiotic resistance and makes treatment more difficult. Alternative strategies, such as the use of medicinal plants, are being actively investigated and Agrimonia eupatoria L. is one of them. This study is aimed at evaluating the antibiofilm activity of ethanol, acetone, and ethyl acetate extracts of A. eupatoria against Staphylococcus aureus, Proteus spp., and Pseudomonas aeruginosa strains isolated from human wounds. In addition, the effects of these extracts on bacterial auto-aggregation, surface hydrophobicity, and motility were investigated. The phytochemical analysis included FT-IR spectroscopy and spectrophotometric quantification of the total phenolic compounds. FT-IR analysis confirmed the presence of characteristic functional groups vibrations (O-H at ~3425 cm^-1, C=O at ~1735-1685 cm^-1, and aromatic C=C at ~1618-1515 cm^-1), whereas phytochemical profiling revealed that the ethyl acetate extract contained the highest phenolic acid content (149.65 ± 0.73 mg CAE/g). All tested extracts inhibited initial cell adhesion and biofilm formation, although they were less effective against mature biofilms. Among them, acetone and ethyl acetate extracts exhibited the strongest antibiofilm activity. S. aureus strains S1 and S2 were the most susceptible, with inhibition rates of at least 92% and 85% for the acetone extract, and 78% and 86% for the ethyl acetate extract, respectively. Although auto-aggregation and cell surface hydrophobicity remained unaffected, both swimming and swarming motilities were reduced. Finally, fluorescence microscopy confirmed that the inhibitory effect was dose-dependent. These results suggest that A. eupatoria extracts represent promising natural antibiofilm agents against clinically relevant human pathogens.